Antibodies, nucleic acids, pharmaceutical formulations, and methods of treating inflammatory diseases

Abstract

The application belongs to the technical field of biological medicine, and relates to an antibody, a nucleic acid, a pharmaceutical preparation and an inflammatory disease treatment method, in particular to an anti-IL-17A antibody and a recombinant protein and application thereof. The application provides an antibody, wherein the amino acid sequence of the antibody comprises: HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are shown in SEQ ID NO:1-3; and / or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:1-3. The anti-IL-17A single domain antibody has relatively strong IL-17A protein binding activity, and the stability and affinity are superior to those of a positive control antibody.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-IL-17A antibody and its recombinant protein and applications; particularly relating to an anti-IL-17A single-domain antibody and its downstream products and applications. Background Technology

[0002] Since its discovery nearly 30 years ago, IL-17 has become a key cytokine for host protection against mucosal infections, and a major pathogenic cytokine and drug target for various autoimmune and inflammatory diseases (Yao, Z. et al. Herpesvirus Saimiri encodes a new cytokine, IL-17, which binds to a novel cytokinereceptor. Immunity 3, 811-821 (1995).). The IL-17 family comprises six members: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F, each mediating its biological function through IL-17 receptors (IL-17RA to IL-17RE). Among these, IL-17A is the most studied member of the IL-17 family, which enhances its biological activity by binding to IL-17RA and IL-17RC.

[0003] It is now understood that IL-17A evolved to regulate innate immunity in invertebrates lacking an adaptive immune system. However, the inflammatory function of IL-17A was initially described in mouse models of autoimmune diseases, where the focus was on CD4 cells that secrete IL-17A. + T cells - T helper 17 (T cells) H 17) Cells – as key producers of this cytokine. It is now known that CD8… + T cells, γδ T cells, innate lymphocytes (ILCs), natural killer (NK) cells, invariant NK T cells, mucosa-associated invariant T cells, mast cells, and Paneth cells can also be sources of IL-17A. Although T cell receptor (TCR) activation is CD4+... + and CD8 + T cells are key to the production of IL-17A, but the production of IL-17A by innate immune cells is mainly driven by inflammatory cytokines, especially IL-1β and IL-23.

[0004] Studies of experimental autoimmune encephalomyelitis (EAE) have shown that IL-17A is a key pathogenic cytokine in the pathology of T cell-mediated autoimmune diseases (Langrish, C. Letal. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201, 233-240 (2005).; Sutton, C., Brereton, C., Keogh, B., Mills, KH & Lavelle, ECA, crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis. J. Exp. Med. 203, 1685-1691 (2006).). Subsequent studies have shown that in the EAE, IL-17A is secreted by TH17 cells and IL-17A-secreting γδT (γδT17) cells (Sutton, CE et al. Interleukin-1 and IL-23 induce innate IL-17 production from gammadelta T cells, amplifying Th17 responses and autoimmunity. Immunity 31, 331-341 (2009)).

[0005] These studies, along with other research that detailed the pathological role of IL-17A in human diseases, ultimately led to the development of monoclonal antibodies (mAbs) targeting IL-17A (IL-17A and IL-17F, IL-17RA, or IL-23). These monoclonal antibodies have been licensed for the treatment of certain autoimmune diseases, particularly psoriasis, with efficacy exceeding that of traditional nonsteroidal anti-inflammatory drugs (NSAIDs) and tumor necrosis factor (TNF) blockers (Langley, R. Get et al. Secukinumab in plaquepsoriasis - results of two phase 3 trials. N. Engl. J. Med. 371, 326-338 (2014).; Reich, K. et al. Bimekizumab versus Secukinumab in plaque psoriasis. N. Engl. J. Med. 385, 142-152 (2021).; Warren, R. R et al. Bimekizumab versus Adalimumab in plaquepsoriasis. N. Engl. J. Med. 385, 130-141 (2021).).

[0006] In recent decades, an increasing number of studies have found that IL-17A plays an important role in the development of various cancers. IL-17A is highly expressed in prostate cancer, colorectal cancer (CRC), gastric cancer, breast cancer, lung cancer, and hepatocellular carcinoma (HCC) tissues, and its expression level is positively correlated with tumor progression (Kryczek I, Banerjee M, Cheng P, et al. Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments. Blood. 2009; 114(6):1141-1149.). Current research indicates that high expression of IL-17A promotes tumor progression by stimulating tumor cell proliferation, cholesterol synthesis, epithelial-to-mesenchymal transition (EMT), MMP expression, recruitment of inflammatory cells to tumor tissue, and inhibition of autophagy and apoptosis (Liao R, Sun J, Wu H, et al. High expression of IL-17 and IL-17RE associate with poor prognosis of hepatocellular carcinoma. J Exp Clin Cancer Res. 2013; 32(1):3.; Sun C, Kono H, Furuya S, et al. Interleukin-17A Plays a Pivotal Role in Chemically Induced Hepatocellular Carcinoma in Mice. Dig Dis Sci. 2016; 61(2):474-488.).

[0007] Chinese patent application 202310019497.0 discloses a monoclonal antibody against human IL-17A protein, a kit containing the antibody, and its application in the detection of human IL-17A. The detection kit containing the monoclonal antibody against human IL-17A protein can be used to detect IL-17A in human serum.

[0008] Chinese patent application 202180041325.2 discloses a stable pharmaceutical composition of an anti-IL-17A antibody and its pharmaceutical application. The pharmaceutical composition contains an anti-IL-17A antibody or its antigen-binding fragment, a buffer solution, and may also contain at least one stabilizer; optionally, it may also contain a surfactant.

[0009] Currently, several antibody drugs that block IL-17 are available globally, including monoclonal antibodies that neutralize IL-17A (Secukinumab and Ixekizumab) or IL-17RA (Brodalumab). In China, over 50 antibody drugs or small molecules targeting IL-17 for the treatment of autoimmune diseases are under development. Although antibody drugs generally have better efficacy and tolerability in humans compared to chemotherapy drugs, many treatment-related side effects still exist in clinical use, such as nasopharyngitis, headache, nausea, and diarrhea, or more serious side effects such as infection, cardiotoxicity, and severe immune reactions. Furthermore, the complex structure and relatively poor stability of monoclonal antibody drugs necessitate repeated high-dose injections to achieve optimal results.

[0010] Therefore, there is a need in the field for more highly specific, highly stable, and long-acting anti-IL-17A antibodies or IL-17A inhibitors for the prevention and / or treatment of autoimmune diseases and cancer.

[0011] This invention is a patent application filed by the inventor for an independently developed anti-IL-17A antibody.

[0012] In addition to the antibody for which protection is sought in this invention, the inventors have also developed eight other antibodies, and based on the relevant provisions of the patent law on unity of invention, have sought protection for each of the nine different antibodies.

[0013] The inventors also developed 12 tandem antibodies based on these 9 antibodies, and sought protection for each of the 12 different tandem antibodies based on the relevant provisions of the patent law on unity of invention.

[0014] Based on these 12 tandem antibodies, the inventors also developed a gene-modified stem cell technology and, based on the relevant provisions of the patent law on unity of invention, filed separate claims for protection for three different gene-modified stem cells and three different applications.

[0015] For ease of understanding of this invention, you may optionally refer to other application texts in this series. Summary of the Invention

[0016] Single-domain antibodies (sdAbs), also known as nanobodies (Nb), are novel antibodies first discovered in camel blood. Because they have only two heavy chains and no light chains, they are also called heavy-chain single-domain antibodies (VHHs, variable domain of heavy chain of heavy-chain antibody). Nanobody crystals are 2.5 nm in diameter and 4 nm in length, containing only one heavy chain variable region (VHH) and CH2 and CH3 regions. Compared to ordinary antibodies, nanobodies naturally lack light chains, making them the smallest naturally occurring fragments capable of binding to antigens. Nanobodies can bind tightly to targets such as antigens like other antibodies, but unlike single-chain antibodies, they do not aggregate into clumps. Nanobodies based on this "heavy-chain antibody" not only have a molecular weight only 1 / 10 that of ordinary antibodies, making them safer for clinical use, but also exhibit more flexible chemical properties, better stability, higher solubility, easier expression, and easier conjugation to other molecules. Therefore, the development of IL17A nanobodies may overcome the shortcomings of traditional antibodies and has broad prospects.

[0017] In order to achieve the above-mentioned technical objectives, the present invention hereby proposes the following technical solution:

[0018] In a first aspect, the present invention provides an antibody, the amino acid sequence of which comprises:

[0019] (i) Amino acid sequences such as HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO:1-3; or;

[0020] (ii) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:1-3.

[0021] In some embodiments, the amino acid differences are achieved by at least one of addition, deletion, modification and / or substitution of the amino acid sequences shown in SEQ ID NO:1-3.

[0022] Preferably, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such a conservative substitution is preferably a substitution in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0023] Particularly preferred are the following conservative substitutions: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0024] Secondly, the present invention provides an antibody, wherein the amino acid sequence of the antibody comprises:

[0025] i) The amino acid sequences are as shown in SEQ ID NO:4-7, FR1, FR2, FR3, FR4; or;

[0026] ii) An amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:4-7.

[0027] In some embodiments, the amino acid sequence of the antibody comprises an amino acid sequence obtained by at least one of addition, deletion, modification and / or substitution of the amino acid sequence shown in SEQ ID NO:4-7.

[0028] In some embodiments, the amino acid sequence of the antibody comprises an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:4-7 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0029] Thirdly, the present invention provides an antibody, wherein the amino acid sequence of the antibody comprises:

[0030] (1) The amino acid sequence of the antibody described in the first aspect; and

[0031] (2) The amino acid sequence of the antibody described in the second aspect.

[0032] Fourthly, the present invention provides an antibody, wherein the amino acid sequence of the antibody comprises:

[0033] FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4;

[0034] HCDR1, HCDR2, and HCDR3 are selected from the following:

[0035] 1) The amino acid sequence as shown in SEQ ID NO:1-3; or

[0036] 2) Functionally active variants of amino acid sequences that differ from 1) by 1, 2, 3, 4, or 5 amino acids;

[0037] FR1, FR2, FR3, and FR4 are selected from the following:

[0038] (a) An amino acid sequence as shown in SEQ ID NO:4-7; or

[0039] (b) Functional active variants of amino acid sequences that have 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with (a).

[0040] In some embodiments, the functionally active variant is a single-domain antibody variant (mutant) with the same or similar affinity or function as the parent antibody from which it originates, such as a variant (mutant) that can specifically bind to IL-17A and block the binding of IL-17A to its receptor.

[0041] In some embodiments, the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3.

[0042] In some embodiments, the amino acid sequence of FR1 is shown in SEQ ID NO:4, the amino acid sequence of FR2 is shown in SEQ ID NO:5, the amino acid sequence of FR3 is shown in SEQ ID NO:6, and the amino acid sequence of FR4 is shown in SEQ ID NO:7.

[0043] In some embodiments, the amino acid sequence of the antibody is shown in SEQ ID NO:8.

[0044] In some implementations, the antibody is a single-domain antibody.

[0045] In some implementations, the antibody is an anti-IL-17A antibody.

[0046] In some embodiments, the antibody comprises a heavy chain framework region or a variant thereof selected from human, mouse, primate, or camelid sources.

[0047] In some preferred embodiments, the antibody comprises part or all of an antibody heavy chain framework region or a variant thereof derived from camelids.

[0048] In some preferred embodiments, the antibody comprises part or all of an antibody heavy chain framework region or a variant thereof selected from alpaca sources.

[0049] SEQ ID NO:1 is GFSFSIYS;

[0050] SEQ ID NO:2 is ITKGGLK;

[0051] SEQ ID NO:3 is NAGRENGY;

[0052] SEQ ID NO:4 is EVQLVESGGGLVQPGGSLRLSCVAW;

[0053] SEQ ID NO:5 is VNWYRQAPGKERELVAG;

[0054] SEQ ID NO:6 is DYADSVKGRFTISRDNAKNAVYLRMNMLKPEDTAVYYC;

[0055] SEQ ID NO:7 is WGQGTQVAVST;

[0056] SEQ ID NO:8:

[0057] The nucleotide sequence encoding SEQ ID NO:8 is shown in SEQ ID NO:9: EVQLVESGGGLVQPGGSLRLSCVAWGFSFSIYSVNWYRQAPGKERELVAGITKGGLK DYADSVKGRFTISRDNAKNAVYLRMNMLKPEDTAVYYCNAGRENGYWGQGTQVAVST

[0058] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTGGGGATTTAGCTTCAGTATCTATTCGGTGAACTGGTACCGCCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCAGGTATTACGAAAGGTGGTCTAAAA GACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACGCGGTGTATCTGCGAATGAACATGTTGAAAACCTGAAGACACGGCCGTCTACTACTGTAATGCAGGCCGGGAGAATGGTTACTGGGGCCAGGGGACCCAGGTCGCCGTCTCTACC.

[0059] Fifthly, the present invention provides a polyclonal antibody comprising any of the aforementioned antibodies.

[0060] In a sixth aspect, the present invention provides a recombinant protein comprising any of the aforementioned antibodies.

[0061] In some implementations, the recombinant protein may be a fusion protein.

[0062] In some embodiments, the recombinant protein further comprises a bioactive protein or a functional fragment thereof that assists in its expression and / or secretion, or prolongs its half-life in vivo.

[0063] In some embodiments, the bioactive protein (which may also include polypeptides) or a functional fragment thereof is selected from at least one of the following: immunoglobulin Fc domain, serum albumin (e.g., human serum albumin (HSA)), albumin-binding polypeptide (e.g., HAS-binding polypeptide), prealbumin (also known as transthyretin), carboxyl-terminal peptide (e.g., human chorionic gonadotropin β subunit (CTP)), elastin-like polypeptide (ELP), His tag (preferably 6×His), GST (glutathione thiotransferase) tag, MBP (maltose-binding protein) tag, FLAG tag, SUMO (ubiquitin-like modified protein) tag, etc.

[0064] Preferably, the bioactive protein or its functional fragment can be the Fc domain of human immunoglobulin, more preferably the Fc domain of human IgG, such as the Fc domains of human IgG1, IgG2, IgG3, and IgG4, and more preferably the Fc domain of human IgG1.

[0065] In some embodiments, the amino acid sequence of the human IgG1 Fc is shown in SEQ ID NO:10.

[0066] SEQ ID NO:10:

[0067] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0068] The nucleotide sequence encoding SEQ ID NO:10 is shown in SEQ ID NO:11:

[0069] .

[0070] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to produce an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) with amino acid modifications (e.g., substitutions, deletions, and insertions) at one or more amino acid positions.

[0071] In some implementations, the recombinant protein may be a monomer, dimer, or polymer.

[0072] In a seventh aspect, the present invention provides an antibody preparation comprising:

[0073] a) Any of the aforementioned antibodies; and

[0074] b) Pharmaceutically acceptable carriers, such as buffers, sterile water, or surfactants.

[0075] In some embodiments, the surfactant may be an ionic surfactant or a nonionic surfactant.

[0076] Eighthly, the present invention provides a kit comprising any of the aforementioned antibodies, any of the aforementioned polyclonal antibodies, any of the aforementioned recombinant proteins, and / or any of the aforementioned antibody preparations;

[0077] Optionally, it also includes a container for loading the antibody preparation.

[0078] Ninthly, the present invention provides an antibody-drug conjugate comprising:

[0079] (I) Any of the aforementioned antibodies, polyclonal antibodies, or recombinant proteins; and

[0080] (II) The coupled part of (I) is coupled with (II).

[0081] In some implementations, the coupling portion includes a detectable marker, drug, toxin, cytokine, radionuclide, and / or enzyme.

[0082] In a tenth aspect, the present invention provides an isolated nucleic acid molecule that encodes any of the aforementioned antibodies, polyclonal antibodies, or recombinant proteins.

[0083] In the eleventh aspect, the present invention provides an expression vector comprising any of the aforementioned nucleic acid molecules.

[0084] In some implementations, the expression vector can be a eukaryotic expression vector or a prokaryotic expression vector, with a eukaryotic expression vector being preferred.

[0085] Preferably, the eukaryotic expression vector is selected from yeast expression vectors, insect expression vectors, or mammalian expression vectors;

[0086] More preferably, the mammalian expression vector is selected from retroviral expression vectors, lentiviral expression vectors, adenovirus expression vectors, and adeno-associated virus expression vectors.

[0087] In a twelfth aspect, the present invention provides a host cell whose genome integrates any of the aforementioned nucleic acid molecules or contains any of the aforementioned expression vectors.

[0088] In a thirteenth aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned antibodies, polyclonal antibodies, recombinant proteins, antibody preparations, antibody conjugates, nucleic acid molecules, expression vectors and / or host cells;

[0089] Optionally, the pharmaceutical composition further includes at least one pharmaceutically acceptable excipient.

[0090] In one embodiment, the excipients are selected from solvents, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, adhesives, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavorings, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, or antioxidants.

[0091] In a fourteenth aspect, the present invention provides use of any of the foregoing antibodies, polyclonal antibodies, recombinant proteins, antibody preparations, antibody-drug conjugates, and / or pharmaceutical compositions, said use being selected from at least one of the following:

[0092] I) Preparation of detection reagents or kits;

[0093] II) Prepare drugs for the prevention and / or treatment of autoimmune diseases;

[0094] III) Prepare drugs for the prevention and / or treatment of cancer.

[0095] In some implementations, the autoimmune diseases include, but are not limited to, Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic scleroderma, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa, microscopic polyangiitis), aortitis syndrome (high-pressure arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Kassman's disease, Sjögren's syndrome, adult-onset Still's disease, vasculitis, allergic granulomatous vasculitis, and allergic angiopathy. Inflammation, rheumatoid vasculitis, large vessel vasculitis, ANCA-associated vasculitis (e.g., Wegener's granulomatosis and eosinophilic Wegener's granulomatosis), Cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related diseases (e.g., primary sclerosing cholangitis, autoimmune pancreatitis, etc.), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, Good-Pasture syndrome, rapidly progressive glomerulonephritis Lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Bartholomew's disease (Graves' disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, Addison's disease (chronic adrenocortical insufficiency), idiopathic Addison's disease, type 1 diabetes mellitus, slowly progressive type 1 diabetes mellitus (adult latent autoimmune diabetes mellitus), focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous skin diseases, acquired epidermolysis bullosa, alopecia areata, vitiligo, common vitiligo, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent miscarriage, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, familial Mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis or inclusion body myositis, etc.

[0096] In some implementations, the cancers include, but are not limited to, basal cell carcinoma, bile duct carcinoma; bladder cancer; bone cancer; breast cancer; peritoneal cancer; cervical cancer; bile duct carcinoma; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; glioblastoma; liver cancer; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma); lymphoma (including Hodgkin's lymphoma and...). Non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; testicular cancer; thyroid cancer; uterine cancer; urinary system cancer; B-cell lymphoma; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia, etc.

[0097] In a fifteenth aspect, the present invention provides a method for in vitro detection of IL-17A in a sample for non-diagnostic purposes, the method comprising the following steps:

[0098] I) Contact the sample to be tested with any of the aforementioned antibodies, polyclonal antibodies, recombinant proteins, antibody preparations and / or antibody-drug conjugates;

[0099] II) Detection of antigen-antibody complexes;

[0100] Ⅲ) Interpretation results.

[0101] In a sixteenth aspect, the present invention provides a method for preventing and / or treating autoimmune diseases, the method comprising:

[0102] Administer a therapeutically effective amount of any of the aforementioned antibodies, polyclonal antibodies, recombinant proteins, antibody preparations, antibody-drug conjugates, nucleic acid molecules, expression vectors, or host cells or drug compositions to a subject in need.

[0103] In a seventeenth aspect, the present invention provides a method for preventing and / or treating cancer, the method comprising:

[0104] Administer a therapeutically effective amount of any of the aforementioned antibodies, polyclonal antibodies, recombinant proteins, antibody preparations, antibody-drug conjugates, nucleic acid molecules, expression vectors, or host cells or drug compositions to a subject in need.

[0105] Compared with existing technologies, the anti-IL-17A single-domain antibody provided by this invention can effectively inhibit the binding of IL-17A protein to its membrane receptor and inhibit intracellular NFκB signaling, exhibiting a dose-effect relationship; it also has strong binding activity to IL-17A protein, EC50... 50It can reach 4.7 μg / mL, which is much higher than the 10.06 μg / mL of the positive control antibody; its stability and affinity are both superior to the positive control antibody. Among them, the single-domain antibody 1-G12 has significantly better thermal stability than the positive control antibody, and its aggregation temperature Tg is lower. agg The temperature was increased by approximately 20°C compared to the positive control antibody. Attached Figure Description

[0106] Figure 1 The purity test results for Human IL-17A protein are shown.

[0107] Figures 2-4 The results of PCR cloning and enzyme digestion of the VHH fragment are shown.

[0108] Figures 5-8 The results of flow cytometry analysis of the alpaca yeast library are shown.

[0109] Figure 9 The FACS test results for the yeast library are shown.

[0110] Figure 10 The sequencing analysis results of the yeast library are shown.

[0111] Figure 11 The SDS-PAGE assay results for single-domain antibody 1-G12 are shown.

[0112] Figure 12 The experimental results of ELISA detection of affinity for single-domain antibody 1-G12 are shown.

[0113] Figure 13 The experimental results of ELISA detection of the affinity of the positive control antibody are shown.

[0114] Figure 14 and Figure 15 The illustration shows the cell line in which the IL-17A recombinant protein activates the reporter gene.

[0115] Figure 16 The experimental results of detecting the blocking function of the single-domain antibody 1-G12 are shown.

[0116] Figure 17 The experimental results of the positive control antibody blocking function test are shown.

[0117] Figure 18 The results of thermostability testing for single-domain antibody 1-G12 are shown.

[0118] Figure 19 The results of the thermostability test for the positive control antibody are shown. Detailed Implementation

[0119] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0120] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0121] As used herein, the terms “comprises” or “comprising” mean “including, but not limited to”. This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term “comprising” includes the more restrictive terms “consisting of” and “substantially consisting of”. In one embodiment, the term “comprising” as used throughout the application, particularly in the claims, may be replaced by the term “consisting of”. The amino acid three-letter and single-letter codes used herein are as known to those skilled in the art, or as described in J Biol. Chem, 243, p3558 (1968).

[0122] As used herein, the terms “optional,” “any,” “arbitrary,” or “any one” mean that the event or circumstance described below may, but does not have to, occur, including the circumstances in which the event or circumstance may or may not occur. For example, “optionally containing one antibody heavy chain variable region” means that the antibody heavy chain variable region of a particular sequence may, but does not have to, be present.

[0123] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0124] The term “and / or” as used herein should be understood to mean any one of the options or any combination of two or more of the options.

[0125] As used herein, the term "IL-17A" or "interleukin-17A" refers to a cytokine belonging to the interleukin-17 family, produced by T cells and other types of immune cells, and playing a crucial role in the immune system. IL-17A is primarily produced by Th17 cells, but other cells, including CD8+ T cells, γδ T cells, NK cells, neutrophils, mast cells, and macrophages, also express IL-17A. It primarily acts on immune cells, such as macrophages, neutrophils, and endothelial cells, inducing an inflammatory response. In some instances, the term includes variants, homologs, orthologs, and paralogs. For example, antibodies specific to human IL-17A may cross-react with IL-17A proteins from another species, such as monkeys, under certain conditions. In other embodiments, antibodies specific to human IL-17A proteins may be completely specific to human IL-17A proteins without cross-reacting with proteins from other species or other types, or may cross-react with IL-17A proteins from some other species but not all others.

[0126] As used herein, the terms “hypervariant region,” “hypervariant region,” “complementarity-determining region,” “HVR,” or “CDR” refer to regions in the variable domain region of an antibody that are highly variable in sequence and / or form structurally defined loops (“hypervariant loops”). Typically, a natural tetrachain antibody contains six HVRs or CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Based on Chothia’s definition, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L26–L32 (L1), L50–L52 (L2), L91–L96 (L3), H26–H32 (H1), H52–H56 (H2), and H96–H101 (H3) (Chothia et al., J. Mol. Biol. 196:901–917 (1987)). Based on Kabat's definition rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L24-L34 (L1), L50-L56 (L2), L89-L97 (L3), H31-H35 (H1), H50-H65 (H2), and H95-H102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, the fifth edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Based on the IMGT definition rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L27-L32 (L1), L50-L51 (L2), L89-L97 (L3), H26-H33 (H1), H51-H56 (H2), and H93-H102 (H3) (Honjo, T. and Alt, FW (1995) Immunoglobulin genes. Academic Press pp. 3-443). It is well known to those skilled in the art that antibody CDRs can be defined using various methods, such as the Kabat definition rule based on sequence variability, the Chothia definition rule based on the location of structural loop regions, and reference tools for antibody humanization design based on CDR transplantation (see J MolBiol. 273:927-48, 1997).Those skilled in the art will understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described above by any of the known schemes described in this invention. While the scope of this disclosure is based on the sequence defined by the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0127] As used herein, the terms “anti-IL-17A (single-domain or nano) antibody” or “IL-17A (single-domain or nano) antibody” refer to antibodies that specifically bind to IL-17A and partially or completely neutralize, inhibit, or weaken IL-17A activity, and / or inactivate IL-17A, prevent IL-17A responses or downstream pathways mediated by IL-17A, or other IL-17A-mediated functions.

[0128] As used herein, the term "antibody" refers to a glycoprotein comprising a heavy chain (H) and a light chain (L) linked together by disulfide bonds (SS). 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 three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (VH). The light chain constant region consists of one domain: CL. Light chains are classified into two types: kappa-type light chains and lambda-type light chains (e.g., in this invention, the light chain constant region Cκ / λ indicates that the light chain constant region is either a kappa-type light chain or a lambda-type light chain). The VH and VL regions can be further subdivided into hypervariable regions (also called complementarity-determining regions (CDRs)), interspersed with more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies include monospecific antibodies, bispecific antibodies, and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0129] The term “single-domain antibody” (sdAb) or “nanobody” has its general meaning in the art and refers to an antibody fragment with a molecular weight of only 12-15 kDa, which consists of a single monomeric variable antibody domain derived from the heavy chain. Such single-domain antibodies (named VHH) can be found in camel mammals and are naturally lacking in the light chain. For a general description of (single)domain antibodies, reference is also made to the prior art described above and to EP 0368684, Ward et al. (Nature 1989 Oct 12; 341(6242):544-6), Holt et al, Trends Biotechnol, 2003, 21(11):484-490; and WO 06 / 030220, WO 06 / 003388. The amino acid sequence and structure of a single-domain antibody can be considered to consist of four frame regions, or "FRWs," referred to in the art as "Frame Region 1" or "FRW1"; "Frame Region 2" or "FRW2"; "Frame Region 3" or "FRW3"; and "Frame Region 4" or "FRW4." The frame regions are separated by three complementarity-determining regions, or "CDRs," referred to in the art as "Complementarity-determining Region 1" or "CDR1"; "Complementarity-determining Region 2" or "CDR2"; and "Complementarity-determining Region 3" or "CDR3." Therefore, a single-domain antibody can be defined as having the following general amino acid sequence: FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, where FRW1-FRW4 refer to frame regions 1-4, and CDR1-CDR3 refer to complementarity-determining regions 1-3. In the context of this disclosure, the amino acid residues of the single-domain antibody are numbered according to the general numbering method for the VH domain as given by the International ImMunoGeneTics information system (http: / / imgt.cmes.fr / ).

[0130] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids—that is, carbon bound to hydrogen, carboxyl, amino, and R groups—such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are compounds whose structure differs from the general chemical structure of amino acids but function in a manner similar to that of naturally occurring amino acids.

[0131] The terms “activity,” “functional activity,” or “biological activity,” or “biological property” or “biological characteristic” as used herein, are used interchangeably and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize IL-17A activity in vivo or in vitro, IC50, etc. 50 The in vivo stability and immunogenic properties of antibodies. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics are observed, measured, or evaluated using techniques known in the art, including but not limited to ELISA, FACS, or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from various sources (including human, primate, or any other source).

[0132] As used herein, the term "Fc," "Fc region," or "Fc fragment" refers to a polypeptide composed of the CH2 and CH3 domains of IgA, IgD, and IgG, or the CH2, CH3, and CH4 domains of IgE and IgM via a hinge region. Although the breakdown of Fc fragments is variable, the Fc fragment of the heavy chain of human IgG typically refers to the polypeptide segment from A231 to its carboxyl terminus.

[0133] As used herein, the term "epitaph" refers to a protein determinant that specifically binds to antibodies. Epitopes are typically composed of surface clusters of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. Conformational and non-conformational epitopes differ in that binding to the former, but not the latter, is lost in the presence of denaturing solvents. Epitopes may contain amino acid residues that directly participate in binding and other amino acid residues that do not directly participate in binding, such as amino acid residues that are effectively blocked or covered by the specific antigen-binding peptide (in other words, the amino acid residues are within the footprint of the specific antigen-binding peptide).

[0134] The term "affinity" or "binding affinity" used in this paper refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can usually be represented by the equilibrium dissociation constant (KD), which is the dissociation rate constant and the binding rate constant (KD = KD). off and K on The ratio of affinity to kinetic binding affinity. Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay described in this paper.

[0135] The term "high affinity" or "high affinity" used in this article, for IgG antibodies, refers to a KD of 1.0 × 10⁻⁶ for the antigen. -6 M or lower, preferably 5.0 × 10 -8 M or lower, more preferably 1.0 × 10 -8 M or lower, 5.0×10 -9 M or lower, more preferably 1.0 × 10 -9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding for the IgM subtype refers to a KD of 10. -6 M or lower, preferably 10 -7 M or lower, preferably 10 -8 M or lower.

[0136] The term "antibody-drug conjugate" refers to a substance obtained by linking a bioactive compound fragment to an antibody or its antigen-binding fragment. The bioactive compound fragment and the target moiety can be linked via a linker. This linker can cleave under specific conditions (e.g., intracellular low pH) or under specific actions (e.g., lysosomal proteases), thereby separating the bioactive compound fragment from the target moiety or the antibody or its antigen-binding fragment. The linker contains cleavable or non-cleavable units, such as peptides or disulfide bonds. Alternatively, the bioactive compound fragment can be directly linked to the target moiety or the antibody or its antigen-binding fragment via a covalent bond, which can cleave under specific conditions or actions, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.

[0137] As used herein, the terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides (see U.S. Patent No. 8,278,036, belonging to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (Batzer, Nucleic Acid Res. 19:5081 (1991); Ohtsuka, J. Biol. Chem. 260:2605-2608 (1985); Rossolini, Mol. Cell. Probes 8:91-98 (1994)).

[0138] As used in this article, "isolated nucleic acid" refers to a nucleic acid that has been identified and isolated from components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations other than their natural chromosomal locations.

[0139] As used herein, the term "construct" refers to any recombinant polynucleotide molecule (such as plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecules), derived from any source, capable of integrating into or autonomously replicating with the genome, constituting a polynucleotide molecule in which one or more polynucleotide molecules are functionally linked (i.e., operably linked). Recombinant constructs typically contain polynucleotides of the present invention operably linked to transcription initiation regulatory sequences that guide transcription of the polynucleotide in the host cell. Expression of the nucleic acids of the present invention can be guided using both heterologous and non-heterologous (i.e., endogenous) promoters.

[0140] As used herein, the term "vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into host cells). One type of vector is the "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be ligated. Another type of vector is the viral vector, into which an additional DNA segment can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). After introduction into the host cell, other vectors (e.g., non-free-living mammalian vectors) integrate into the host cell's genome and thus replicate along with the host genome. Furthermore, some vectors can guide the expression of operatively linked genes. Such vectors are referred to herein as "expression vectors."

[0141] As used in this article, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a target gene when transformed, transfected, or transduced into host cells. Expression vectors contain one or more phenotypic selection markers and origins of replication to ensure the maintenance of the vector and to provide amplification within the host when needed.

[0142] The terms “cell” and “cell line” used in this article are used interchangeably, and all such names include their descendants. The term “host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0143] As used herein, the term "pharmaceutical composition" generally refers to a formulation which is present in a form that allows for the effective biological activity of the active ingredient and does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition will be administered. The composition is sterile.

[0144] The term "pharmaceutically acceptable" means compounds, materials, compositions, and / or dosage forms that are suitable for use in human and animal tissue contact without excessive toxicity, irritation, allergic response, or other problems or complications, to the extent of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio. As used herein, the term "pharmaceutically acceptable carrier, excipient, and / or diluent" means a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable materials, compositions, or media, such as liquid or solid fillers, diluents, excipients, solvents, media, encapsulating materials, manufacturing aids, or solvent encapsulating materials, which relate to maintaining the stability, solubility, or activity of the antibody or its antigen-binding fragment disclosed herein, and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and their analogues. Agents for delaying absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which stabilize the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0145] The term “subject” as used in this article includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc.

[0146] As used herein, the terms "therapeutic effective amount," "therapeutic effective dose," and "effective amount" refer to the amount of the anti-IL-17A antibody or its antigen-binding fragment of the present invention, when administered alone or in combination with other therapeutic agents to cells, tissues, or subjects, that effectively prevents or improves the symptoms of one or more diseases or conditions, or the development of such diseases or conditions. Therapeutic effective dose also refers to an amount of antibody or its antigen-binding fragment sufficient to cause symptom improvement, such as an amount that treats, cures, prevents, or improves the associated medical condition, or accelerates the treatment, cure, prevention, or improvement of such conditions. When administered to an individual as a single active ingredient, the therapeutic effective dose refers only to that ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that cause the therapeutic effect, whether administered in combination, sequentially, or simultaneously. The effective amount of the therapeutic agent will result in an increase of at least 10% in diagnostic criteria or parameters; typically at least 20%; preferably at least about 30%; more preferably at least 40%; and most preferably at least 50%.

[0147] The term “EC” used in this article 50 "Concentration for 50% of maximal effect" refers to the concentration that can cause 50% of the maximum effect.

[0148] As used herein, the term "cancer" or "tumor" refers to a physiological disorder in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Examples of cancers include, but are not limited to, basal cell carcinoma, cholangiocarcinoma; bladder cancer; bone cancer; breast cancer; peritoneal cancer; cervical cancer; cholangiocarcinoma; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; glioblastoma; liver cancer; kidney cancer; laryngeal cancer; leukemia; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma); lymphoma, including Hodgkin lymphoma and non-Hodgkin lymphoma. Tumors; melanoma; myeloma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; B-cell lymphoma; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia, etc.

[0149] The term "autoimmune disease" or "autoimmune disease" as used in this article refers to diseases caused by a decrease, reduction, and / or destruction of the immune system's tolerance to its own components, resulting in damage to the body's own organs and tissues by autoantibodies and / or sensitized lymphocytes. These diseases manifest as dysfunction of the corresponding organs and tissues. This includes, but is not limited to, Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic scleroderma, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa, microscopic polyangiitis), aortitis syndrome (high-pressure arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Kassman's disease, Sjögren's syndrome, adult-onset Still's disease, vasculitis, allergic granulomatous vasculitis, allergic vasculitis, rheumatoid vasculitis, and large vessel vasculitis. Vasculitis, ANCA-associated vasculitis (e.g., Wegener's granulomatosis and eosinophilic Wegener's granulomatosis), Cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid syndrome, eosinophilic fasciitis, IgG4-related diseases (e.g., primary sclerosing cholangitis, autoimmune pancreatitis, etc.), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, Good-Pasture syndrome, rapidly progressive glomerulonephritis, lupus nephritis, megaloblastic disease Erythrocytic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Bartholomew's disease (Graves' disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, Addison's disease (chronic adrenocortical insufficiency), idiopathic Addison's disease, type 1 diabetes, slowly progressive type 1 diabetes (adult latent autoimmune diabetes), focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, herpes gestationis, linear IgA... Herpetic dermatitis, acquired epidermolysis bullosa, alopecia areata, vitiligo, common vitiligo, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent miscarriage, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplantation immunosuppression, familial Mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis, or inclusion body myositis.

[0150] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques are also described in many publications, such as *Molecular Cloning: A Laboratory Manual (4th Edition)* (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al., *Current Protocols in Molecular Biology*, Greene Publishing Assoc., and Wiley-Interscience.

[0151] The main reagents, materials and equipment involved in this invention are shown in Table 1.

[0152] Table 1. Main reagents, materials and equipment involved in this invention.

[0153]

[0154]

[0155]

[0156] The primers used in this invention for screening, cloning the VHH fragment, and constructing nanobodies were all designed according to the following literature:

[0157] Maass DR, Sepulveda J, Pernthaner A, Shoemaker CB. Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs). JImmunol Methods. 2007; 324 (1-2): 13-25.

[0158] Lin,J,Gu,Y,Xu,Y et al.Characterization and applications of nanobodiesagainst Pseudomonas aeruginosa exotoxin a selected from single alpaca Bcells.Biotechnol Biotechnol Equip 2020;34:1028-37.

[0159] Studies on design of singledomain antibodies by Alpaca VHH phagelibrary and high throughput sequencing to construct Fab antibody purificationsystem (http: / / hdl.handle.net / 10232 / 00030916).

[0160] Example 1: Preparation of anti-IL-17A single-domain antibody

[0161] 1.1 Preparation of IL-17A (Human and Mouse) recombinant protein (antigen)

[0162] The sequence information of Human IL-17A (Q16552-1) (AA Gly 24-Ala155, as shown in SEQ ID NO:12) was retrieved from the UniProt database. A 6×His tag was added to the C-terminus, and the gene was synthesized and subcloned into the pET28a vector after optimization according to the prokaryotic codons. After verification by Sanger sequencing, plasmid extraction was performed.

[0163] The recombinant plasmid was transformed into BL21 competent cells, induced overnight with 0.5 mM IPTG, and the bacterial culture was collected for lysis. The recombinant protein was purified using a nickel column.

[0164] SDS-PAGE analysis of the target protein purity showed a purity >90%. Figure 1 ).

[0165] SEQ ID NO:12:

[0166] MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA.

[0167] 1.2 Animal Immunization

[0168] Two alpacas were immunized with the prepared recombinant antigen by subcutaneous multi-site immunization, for a total of 6 immunizations at 21-day intervals. Peripheral blood was collected 10 days after the last immunization, and the immunogenicity was detected by ELISA.

[0169] 1.3 Immunotiter Detection

[0170] The experimental steps are as follows:

[0171] (1) Collect 5 mL of peripheral blood, place the centrifuge tube containing the blood sample in a 37°C incubator for 1 hour; then transfer the blood sample to 4°C overnight.

[0172] (2) Place the centrifuge tube containing the blood sample in a centrifuge and centrifuge at 5000 rpm for 20 min; separate the upper serum layer and transfer the serum to a new sterile centrifuge tube to collect the immune serum.

[0173] (3) Dilute the IL-17A recombinant protein to a final concentration of 1 μg / mL using sterile CBS (carbonate buffer). Take a new 96-well microplate, add 100 μL / well, and coat overnight at 4°C.

[0174] (4) Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20).

[0175] (5) Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours.

[0176] (6) After removing the blocking buffer, wash the plate 5 times with PBST.

[0177] (7) Add 100 μL of serially diluted serum (100 μL / well), incubate at room temperature for 1 hour, and use PBS for control wells.

[0178] (8) Remove the liquid from the well and wash 5 times with PBST.

[0179] (9) Add 100 μL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour.

[0180] (10) After removing the liquid from the wells, wash the plate 5 times with PBST.

[0181] (11) Add 100 μL / well TMB colorimetric solution.

[0182] (12) Incubate at room temperature in the dark for 10-15 minutes.

[0183] (13) Add 50 μL of stop solution per well.

[0184] (14) Use an ELISA reader to read the OD450 value in the well.

[0185] As shown in Table 2, after 6 rounds of immunization, the alpacas' immune titers all met the requirements.

[0186] Table 2 Results of Immunotiter Detection

[0187]

[0188]

[0189] 1.4 Antibody yeast library construction process

[0190] 1.4.1 PBMC isolation and VHH antibody fragment cloning

[0191] 100 mL of anticoagulated peripheral blood was collected, and PBMCs were isolated using lymphocyte separation medium.

[0192] RNA extraction using PrimeScript TM II. The 1st Strand cDNA Synthesis Kit is used for reverse transcription to prepare cDNA, which includes the following steps:

[0193] (1) Prepare the following reaction mixture Mix1 (Table 3) in 200 μL of PCR:

[0194] Table 3

[0195]

[0196] (2) After holding at 65℃ for 5 minutes, cool rapidly on ice.

[0197] (3) Prepare the following reaction solutions in the above PCR tubes (Table 4):

[0198] Table 4

[0199]

[0200] (4) After mixing by pipetting, dispense 80 μL / tube and place in a PCR instrument at 42°C for 1 hour, then heat-inactivate at 70°C for 15 minutes. Finally, place the cDNA sample on ice or at -20°C for long-term storage.

[0201] PCR amplification of the VHH fragment:

[0202] 1) Prepare the first-round PCR reaction system (50 μL / tube), see Table 5:

[0203] Table 5

[0204]

[0205]

[0206] After configuring the PCR reaction system, set up the PCR instrument according to the following procedure, as shown in Table 6:

[0207] Table 6

[0208]

[0209] 2) Agarose gel electrophoresis of PCR products

[0210] PCR products were analyzed by electrophoresis using 1% agarose gel to separate fragments with a molecular weight of approximately 750 bp. PCR products were recovered using a gel extraction kit, and their concentrations were determined using NanoDrop.

[0211] 3) Prepare the two-round PCR reaction system (50 μL / tube), see Table 7:

[0212] Table 7

[0213]

[0214] After configuring the PCR reaction system, set up the PCR instrument according to the following procedure, as shown in Table 8:

[0215] Table 8

[0216]

[0217] 4) Agarose gel electrophoresis analysis of the second round of PCR products

[0218] PCR products were analyzed by electrophoresis using 1% agarose gel to isolate the VHH fragment with a molecular weight of approximately 400 bp. The VHH PCR product was recovered using a gel extraction kit, and its concentration was determined using NanoDrop.

[0219] The results showed that the first round of PCR yielded PCR bands of approximately 1000bp and 750bp, respectively. The 750bp fragment was recovered from the gel and used as a template for the second round of PCR. Figure 2 The second round of PCR yielded a band of approximately 400 bp, which was the VHH fragment. This fragment was recovered from the column and used as a template for the third round of PCR. Figure 3 The third round of PCR yielded a band of approximately 500 bp. A homologous arm was added, and subsequent homologous recombination was performed into the yeast display vector pDisplay. Figure 4 ).

[0220] 1.4.2 Construction of a single-domain antibody yeast display library

[0221] (a) Linearization of the yeast display vector pDisplay, enzyme digestion system is shown in Table 9:

[0222] Table 9

[0223]

[0224] The pDisplay vector was digested with SfiI enzyme, 100 μL / tube, and digested overnight at 50°C.

[0225] The pDisplay vector fragment was separated using a 1% agarose gel, a 5000bp fragment was excised and recovered using the gel, and the concentration was determined using NanoDrop.

[0226] Aliquot 200 μL of the recovered pDisplay enzyme digestion product into each 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3M sodium acetate and 1 μg / μL of glycogen, mix by pipetting and aspiration, add 880 μL of anhydrous ethanol, mix by inversion, and incubate at -80℃.

[0227] (b) Electroconversion to construct a yeast display library

[0228] Straw the competent yeast strains frozen at -80℃ onto YPD solid medium plates and activate them at 30℃ for 3-5 days.

[0229] Inoculate single colony yeast competent cells into 50 mL of YPD medium and incubate at 250 rpm and 30 °C for 1-2 days.

[0230] Prepare competent yeast strains. The linearized vector fragment and PCR product were mixed and added to an electroporation cuvette for electroporation. The electroporated competent yeast cells were then transfected into culture flasks and incubated at 220 rpm and 30°C for 1 h.

[0231] Take 20 μL of resuspension, dilute it 5000 times with SDCAA, take 100 μL, spread it on an SDCAA plate, incubate for 2-3 days, calculate the volume of the culture, and continue to incubate the remaining bacterial culture for 24 days.

[0232] Preservation of bacteria. Collect the remaining bacterial culture into a 50 mL centrifuge tube, centrifuge at 3000 g for 5 min, discard the supernatant, add 10 mL of SDCAA to resuspend, mix with 50% glycerol: resuspension = 1:1, and freeze at -80℃.

[0233] 1.5 Yeast Display Library Selection and Screening Process

[0234] Using the prepared IL-17A antigen, yeast culture was incubated with streptavidin magnetic beads. The yeast culture was then added to the antigen-bound magnetic beads and incubated at 4°C for 60 minutes using a rotating incubator. The constructed yeast display library underwent two rounds of magnetic sorting using streptavidin beads. After sorting, the yeast culture was plated on SDCAA plates, and single colonies were picked and cultured. After induced expression for 48 hours, flow cytometry analysis was performed. The culture was incubated with Biotin-IL-17A-His for 1 hour, using PE Streptavidin as the secondary antibody. Flow cytometry detection was performed after incubation.

[0235] Based on the flow cytometry results ( Figures 5-8 After the second magnetic separation, yeast positive clones were significantly enriched. The separated products were directly plated on SDCAA plates, and single clones were selected for flow cytometry detection.

[0236] 1.6 FACS Screening Process

[0237] After sorting, the yeast culture was plated on SDCAA plates, single clones were picked and cultured, and expression was induced for 48 h. Then, it was incubated with Biotin-antigen, and PE-Streptavidin was used as the secondary antibody. After incubation, flow cytometry was performed.

[0238] The results are as follows Figure 9 The FACS test was used to detect the binding of IL-17A target monoclonal antibodies to the target. The amino acid sequences of candidate single-domain antibodies obtained by sequencing were compared, and candidate antibodies with different amino acid sequences in the CDR region were selected to construct eukaryotic expression vectors.

[0239] 1.7 Antibody Sequence Identification

[0240] Positive clones were enriched; enriched monoclonal antibodies were selected for Phage ELISA identification, and the clones were sequenced to obtain the nucleic acid and amino acid sequence information of candidate single-domain antibodies. Twenty single clones were randomly selected for sequencing analysis; the sequences showed significant differences and good library diversity. Figure 10 Based on the amino acid sequence information of the CDR region of candidate single-domain antibodies, the Insilico method was used to analyze possible post-translational modification sites.

[0241] Based on the flow cytometry results of yeast monoclonal antibodies, genomic DNA was extracted from positive clones that bound to IL-17A-His, and antibody sequences were obtained by PCR. Based on the sequencing results of the PCR products, differentially expressed clones were selected for overlap PCR amplification, with the specific steps as follows:

[0242] First round of PCR: Amplification of CMV, VHH, and Fc:

[0243] a) The PCR reaction system (50 μL system / reaction) is shown in Table 10.

[0244] Table 10

[0245]

[0246] The PCR reaction procedure is shown in Table 11:

[0247] Table 11

[0248]

[0249]

[0250] b) Take 50 μL of PCR product, add 1 / 10 volume of 10× loading buffer, and perform electrophoresis analysis using 1% agarose. The band size of CMV is about 750 bp, the band size of Fc is about 1400 bp, and the band size of VHH is about 500 bp.

[0251] c) Remove the target band from the gel, purify the PCR product, and determine the concentration using NanoDrop (if the concentration is too high, dilute it for subsequent reactions).

[0252] Second round of PCR: Overlap Extension PCR ligation of CMV, VHH and FC.

[0253] d) The PCR reaction system is shown in Table 12:

[0254] Table 12

[0255]

[0256] The PCR reaction procedure is shown in Table 13:

[0257] Table 13

[0258]

[0259] Add 2 μL each of primers CMV-F and PGK-R.

[0260] The PCR reaction procedure is shown in Table 14:

[0261] Table 14

[0262]

[0263]

[0264] e) Purify the overlap PCR product using TakaRa's DNA fragment recovery kit and determine the concentration using NanoDrop. At least 10 μg of PCR product is required for subsequent cell transfection validation.

[0265] f) The transfection steps are the same as those for eukaryotic expression vectors.

[0266] A signal peptide was added to the N-terminus of VHH, and IgG1-Fc was added to the C-terminus. The PCR product was transiently transfected into HEK293 cells. The supernatant of the expressed antibody was used for ELISA detection: 100 μL of transfection supernatant was added to a 96-well plate pre-coated with IL-17A recombinant antibody and incubated. HRP-Protein A was used as the secondary antibody for ELISA detection. The results are shown in Table 15.

[0267] Table 15

[0268]

[0269] 1.8 Antibody Expression and Purification Process

[0270] S1: Based on the ELISA results of the candidate antibodies, positive clones were selected. The obtained VHH antibody sequences were synthesized and tandemly subcloned with human IgG1 Fc (SEQ ID NO:10) into the expression vector pcDNA3.4-hIgG1Fc. After the vector was verified by sequencing, endotoxin-free plasmids were prepared using the Qiagen plasmid desorption kit for later use.

[0271] S2: Remove the LVTransm transfection reagent and single-chain antibody expression vector from the freezer. After thawing at room temperature, mix thoroughly by pipetting. Remove the PBS buffer and warm it to room temperature. Add 2 mL of PBS to one well of a 6-well plate, add 130 μg of antibody expression vector, mix thoroughly by pipetting, then add 400 μL of LVTransm, immediately mix by pipetting, and let stand at room temperature for 10 minutes.

[0272] S3: Add the above DNA / LVTransm complex to 30 mL of 293F cells and gently shake to mix thoroughly. Place the cells in a 37°C, 5% CO2 incubator and incubate at 130 rpm for 6-8 hours. Then add 50 mL of fresh 293 cell culture medium and return the cells to the incubator to continue culturing.

[0273] S4: After continuous culture for 7 days, the supernatant of the culture medium was collected by centrifugation, filtered through a 0.45μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube and purified using a Protein A column.

[0274] The steps for purifying antibodies using a Protein A column are as follows:

[0275] 1) Add the sample containing the target antibody into the EP tube and gently invert the tube to mix.

[0276] 2) Mix the EP tubes at room temperature or incubate them on a vortex mixer (1-4 hours or overnight), and add 100mM PMSF to prevent protein degradation.

[0277] 3) Collect the magnetic beads using a magnetic separator and discard the supernatant. If necessary, retain the supernatant for analysis.

[0278] 4) Add 1 mL of binding / washing buffer to the EP tube and mix thoroughly. Use a magnetic rack to collect the magnetic beads and discard the supernatant. Repeat the washing step three times.

[0279] 5) Add 500 μL of elution buffer to the EP tube, quickly resuspend it by pipetting or vortexing, and then incubate it at room temperature (about 25°C) in a reverse mixer or by gently inverting the EP tube manually for 5 minutes.

[0280] 6) Use a magnetic separator to collect the magnetic beads and transfer the supernatant containing the eluted antibodies into a clean EP tube.

[0281] 7) Repeat steps 1) and 2) twice.

[0282] 8) Add 1 / 10 of the neutralization buffer to each 500 μL of elution buffer to neutralize the pH, in order to maintain the biological activity of the antibody and prevent antibody inactivation. If necessary, buffer exchange can be performed by dialysis or desalting.

[0283] 9) Binding / washing buffer: 1×PBS, pH 7.0.

[0284] Elution buffer: 0.1M glycine, pH 2-3; 0.1M NaAc-HAc, pH 3.6.

[0285] Neutralization buffer: 1M Tris, pH 8.5.

[0286] Magnetic bead regeneration buffer: 0.1M NaOH.

[0287] The SDS-PAGE results of the 1-G12 antibody are as follows: Figure 11 .

[0288] Example 2: ELISA detection of the affinity between recombinant antibody and target protein

[0289] 2.1 Preparation of positive control antibody Ixekizumab

[0290] The Ixekizumab heavy and light chain variable regions were synthesized (light chain sequence as shown in SEQ ID NO:13; heavy chain sequence as shown in SEQ ID NO:14). The heavy chain variable region was subcloned into the pcDNA3.4-hIgG4 vector (amino acid sequence of IgG4 Fc as shown in SEQ ID NO:15), and the light chain variable region was subcloned into the pcDNA3.4-hIgG1 Kc vector (amino acid sequence of IgG1 Kc as shown in SEQ ID NO:16). After verification by Sanger sequencing, endotoxin-free plasmids were prepared using a plasmid extraction kit for later use.

[0291] Remove the LVTransm transfection reagent and heavy and light chain expression vectors from the freezer. After thawing at room temperature, mix thoroughly by pipetting. Remove the PBS buffer and warm it to room temperature. Add 2 mL of PBS to one well of a 6-well plate, and add 50 μg of heavy and light chain expression vectors respectively. Mix thoroughly by pipetting, then add 300 μL of LVTransm. Immediately mix by pipetting and let stand at room temperature for 10 minutes.

[0292] Add the DNA / LVTransm complex to 100 mL of 293F cells, gently shake to mix thoroughly, and place the cells in a 37°C, 5% CO2 incubator for further culture at 130 RPM.

[0293] After continuous culture for 5-7 days, the supernatant of the culture medium is collected by centrifugation, filtered through a 0.45μm filter membrane, and the filtrate is transferred to a sterile centrifuge tube for antibody purification using a Protein A column.

[0294] SDS-PAGE analysis of the target antibody protein showed a purity of >95%.

[0295] SEQ ID NO:13:

[0296] DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKVSNR FIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIK.

[0297] SEQ ID NO:14:

[0298] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYG TTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVT VSS.

[0299] SEQ ID NO:15:

[0300] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0301] SEQ ID NO:16:

[0302] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0303] 2.2 Detection of the binding activity of recombinant Human IL-17A protein to control antibody by ELISA

[0304] Dilute IL-17A recombinant protein to a final concentration of 2 μg / mL using sterile CBS. Take a new 96-well plate, add 100 μL / well, and coat overnight at 4°C.

[0305] Remove the antigen coating solution and wash three times with PBST (containing 0.5% Tween);

[0306] Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours;

[0307] After removing the blocking buffer, wash the plate three times with PBST;

[0308] The positive control antibody Ixekizumab was diluted to 10 μg / mL with PBS, and then diluted 5 times for 7 spots. 100 μL was added to each well of the microplate and incubated at room temperature for 1 hour. The control wells were prepared with PBS.

[0309] Remove the liquid from the well and wash three times with PBST;

[0310] Add secondary antibody HRP-Protein A (Boster, BA1080) diluted 1:10000, add 100 μL / well to the microplate, and incubate at room temperature for 1 hour;

[0311] After removing the liquid from the wells, wash the plate three times with PBST.

[0312] Add 100 μL / well of TMB colorimetric solution;

[0313] Incubate at room temperature in the dark for 15 minutes;

[0314] Add 50 μL / well stop solution (2M HCl);

[0315] The OD450 values ​​in the wells were read using an ELISA reader. The results are shown in Table 16. The positive antibody binds well to the IL-17A antigen protein and can be used for immunization.

[0316] Table 16 Detection of Human IL17A Binding Activity with Positive Antibodies

[0317]

[0318] 2.3 ELISA detection of the binding of recombinant antibody to target protein

[0319] Dilute the recombinant protein to a final concentration of 2 μg / mL using sterile CBS. Take a new 96-well microplate, add 100 μL / well, and coat overnight at 4°C.

[0320] Remove the antigen coating solution and wash five times with PBST (containing 0.05% Tween 20).

[0321] Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours;

[0322] After removing the blocking buffer, wash the plate 5 times with PBST;

[0323] Add purified single-domain antibody at an initial concentration of 10 μg / mL, serially dilute 5-fold to 7 spots (100 μL / well), incubate at room temperature for 1 hour, and use PBS as the control well;

[0324] Remove the liquid from the well and wash 5 times with PBST;

[0325] Add 100 μL / well of HRP-Protein A antibody (1:50000 dilution) and incubate at room temperature for 1 hour;

[0326] After removing the liquid from the wells, wash the plate 5 times with PBST.

[0327] Add 100 μL / well of TMB colorimetric solution;

[0328] Incubate at room temperature in the dark for 10-15 minutes;

[0329] Add 50 μL of stop solution per well;

[0330] Use an ELISA reader to read the OD450 value in the well.

[0331] The results are shown in Table 17. Except for 1-B11, which did not bind to the IL-17A-His antigen, all 14 clones bound. The 14 differential clones were used to construct eukaryotic expression vectors.

[0332] Table 17 ELISA Detection of Recombinant Antibody Binding to Target Protein

[0333]

[0334] Example 3: ELISA detection of anti-IL17A single-domain antibody

[0335] The purified single-domain antibody (2 μg / mL) was used to coat the ELISA plate, and Biotin-IL-17A-His was added at an initial concentration of 10 μg / mL. The plate was serially diluted 5-fold to 7 spots, and HRP-Streptavdin was used for ELISA detection.

[0336] The results showed that antibody 1-G12 had good binding activity to Human IL-17A protein. EC50 of antibody 1-G12... 50 The concentration was 4.716 μg / mL, and the binding activity was much higher than that of the positive control antibody (10.06 μg / mL). Figure 12 and Figure 13 ).

[0337] Example 4: Anti-IL17A Single-Domain Antibody Blocking Function Experiment

[0338] 4.1 Construction of IL-17A reporter gene cell line

[0339] Based on the amino acid sequence information of IL-17RA (UniProtKB: Q96F46) and IL-17RC (UniProtKB: Q8NAC3), a lentiviral expression vector was constructed and lentivirus was packaged. This lentivirus was then used to co-infect 293 cells. Recombinant 293 cells that simultaneously overexpressed both receptors were screened. NFKB-Luciferase (amino acid sequence shown in SEQ ID NO:17, nucleotide sequence encoding it shown in SEQ ID NO:18) and the ACT1 gene (nucleotide sequence shown in SEQ ID NO:19) were then stably transfected to construct the IL-17A reporter gene cell line 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc.

[0340] SEQ ID NO:17:

[0341] .

[0342] SEQ ID NO:18:

[0343]

[0344] SEQ ID NO:19:

[0345]

[0346] 4.2 Binding of recombinant IL-17A protein to reporter gene cell lines

[0347] The 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cell line was revived in liquid nitrogen and the cell state was adjusted to the logarithmic growth phase.

[0348] The cells were divided into several groups, with each group containing 2 × 10 cells. 5 One cell;

[0349] Incubate the IL-17A-His protein with the target cells, mix thoroughly, and incubate at room temperature for 1 hour.

[0350] Centrifuge at 800×g at room temperature for 3 minutes, remove the supernatant containing antibodies, and wash the cells 3 times with PBS;

[0351] Add secondary antibody APC-His (1:500 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 minutes.

[0352] Centrifuge at 800×g at room temperature for 3 minutes, remove the supernatant containing the secondary antibody, and wash the cells 3 times with PBS;

[0353] Cells were resuspended in 500 μL PBS for flow cytometry analysis.

[0354] FACS results showed that the constructed IL-17A receptor overexpressing cell line could bind to IL-17A, with a positive rate greater than 90%. Figure 14 ).

[0355] The recombinant IL-17A protein was used to activate luciferase expression in the 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cell line. The results showed that the recombinant IL-17A protein effectively activated luciferase expression in the 293F-IL17Ra / IL17Rc-NFκB-Luc reporter gene cell line. Figure 15 ).

[0356] 4.3 Functional experiment of Ixekizumab blocking the binding of recombinant IL-17A protein to reporter gene cell lines

[0357] Add serially diluted detection antibodies (positive antibody: Ixekizumab; antibody to be detected) to 96-well plates, performing 10-fold serial dilutions to obtain 10 final concentrations: 100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, 0.001 μg / mL, 0.0001 μg / mL, 0.00001 μg / mL, 0.000001 μg / mL, 0.0000001 μg / mL, and 0 μg / mL. Add 50 μL of each diluted antibody to a well of the 96-well plate, with two replicates for each dilution. Then, add 50 μL of 0.4 μg / mL IL-17A protein (final concentration 0.1 μg / mL) to each well. Mix well and incubate at 37°C for 1 hour. 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cells cultured to the logarithmic growth phase were seeded into 96-well plates, with 2 × 10⁶ cells per well. 4 Cells were co-cultured for 18 hours. 20 μL of Bright-Glo™ assay reagent was added to each well, and the luciferase activity in the wells was measured using a Tecan M1000pro microplate reader.

[0358] The results showed that the positive control antibody Ixekizumab could inhibit the binding of IL17A protein to its membrane receptor and inhibit intracellular NFκB signaling, exhibiting a dose-response effect.

[0359] 4.4 Single-domain antibody blocking function

[0360] The blocking activity of the single-domain antibody 1-G12 was detected using the 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc reporter gene cell line. The results showed that the IC50 of 1-G12 was [missing information]. 50 The concentration was 6.923e-005 μg / mL, and the IC50 of the positive control antibody was... 50 It is 1.356 μg / mL ( Figure 16 and Figure 17 Therefore, both 1-G12 and the positive control antibody can block the activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc by Human IL-17A protein, but 1-G12 has a significantly better blocking effect than the positive control antibody.

[0361] Example 5: Stability Detection of Anti-IL-17A Single-Domain Antibody

[0362] Nano-Differential Scanning Fluorescence (nanoDSF) technology can be used to detect fluorescence changes, enabling the detection of protein thermal and chemical denaturation under natural conditions and accurately determining the temperature at which 50% of the protein is in its unfolded state (T0). m), the temperature at which folding begins (T) onset ) and the temperature at which aggregation begins (T) agg ); Thermal denaturation T m Value, T onset and T agg A higher value indicates a more stable antibody protein.

[0363] Take 100 μL of the candidate antibody prepared in the previous project and Lxekizumab (sample concentration greater than 200 μg / mL), centrifuge at 12000×g for 10 min at 4℃, and then use a capillary tube to aspirate the sample. Prepare two capillary tubes for each sample as parallel controls, and place them in the corresponding slots in sequence to ensure that the capillary tube is filled with sample and free of air bubbles before detection and analysis.

[0364] The results showed that T of 1-G12 m1 The temperature was 61.32℃, T m3 The temperature was 81.49℃, T onset The temperature was 54.70℃, T agg It is 81.92℃ ( Figure 18 ); T positive control antibody m1 The temperature was 56.10℃, T m2 The temperature was 79.84℃, T onset The temperature was 47.50℃, T agg It is 61.86℃ ( Figure 19 The above results indicate that the single-domain antibody 1-G12 has better thermal stability than the positive control antibody, with the aggregation temperature T... agg The temperature was increased by approximately 20°C compared to the positive control antibody.

[0365] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A single-domain antibody against IL-17A, characterized in that, The amino acid sequence of the anti-IL-17A single-domain antibody is shown in SEQ ID NO:

8.

2. A recombinant protein, characterized in that, The recombinant protein is composed of the anti-IL-17A single-domain antibody as described in claim 1 and the human immunoglobulin Fc domain.

3. The recombinant protein according to claim 2, characterized in that, The human immunoglobulin Fc domain is the Fc domain of human IgG1.

4. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-IL-17A single-domain antibody of claim 1 or the recombinant protein of any one of claims 2-3.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4.

6. A host cell, characterized in that, The host cell genome integration includes the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. A pharmaceutical composition, characterized in that, The drug combination comprises: The anti-IL-17A single-domain antibody of claim 1, the recombinant protein of any one of claims 2-3, the nucleic acid molecule of claim 4, the expression vector of claim 5 or the host cell of claim 6, and pharmaceutically acceptable excipients.

8. Use of the antibody of claim 1, the recombinant protein of any one of claims 2-3, or the pharmaceutical composition of claim 7 in the preparation of a medicament for treating psoriasis.

9. A method for in vitro detection of IL-17A in samples for non-diagnostic purposes, characterized in that, Includes the following steps: (A) Contact the anti-IL-17A single-domain antibody of claim 1 or the recombinant protein of any one of claims 2-3 with the sample to be tested; (B) Detection of antigen-antibody complexes; (C) Interpretation results.

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