Antibody sequence structure and its application

Abstract

The present invention belongs to the field of biomedicine technology and relates to the sequence structure and application of antibodies. The antibody comprises: a first single-domain antibody, the amino acid sequence of the first single-domain antibody comprises HCDR1, HCDR2, and HCDR3 as 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; a second single-domain antibody, the amino acid sequence of the second single-domain antibody comprises HCDR4, HCDR5, and HCDR6 as shown in SEQ ID NO: 4-6; and / or; an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 4-6. The tandem single-domain antibody has higher affinity, better stability, and better blocking effect, and is of great significance in the clinical treatment of autoimmune diseases and cancers.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the sequence structure of antibodies and applications thereof, and specifically to a tandem single-domain antibody that specifically binds to IL-17A and applications thereof. Background Art

[0002] Since its discovery nearly 30 years ago, IL-17 has become a key cytokine for host protection against mucosal infections, and is also a major pathogenic cytokine and drug target for a variety of 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 includes six members: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, each of which mediates its biological function through the IL-17 receptor (IL-17RA to IL-17RE). Among them, the most studied IL-17 family member is IL-17A, which promotes its biological activity by binding to IL-17RA and IL-17RC.

[0003] It is now recognized that IL-17A evolved to regulate innate immunity in invertebrates that lack an adaptive immune system. However, the inflammatory function of IL-17A was initially described in mouse models of autoimmune disease, where studies focused on IL-17A-secreting CD4+ T cells - T helper 17 (TH17) cells - as key producers of this cytokine. It is now known that CD8+ T cells, γδ T cells, innate lymphoid cells (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. While T cell receptor (TCR) activation is critical for IL-17A production by CD4+ and CD8+ T cells, IL-17A production by innate immune cells is primarily driven by inflammatory cytokines, particularly IL-1β and IL-23.

[0004] Studies in experimental autoimmune encephalomyelitis (EAE) have shown that IL-17A is a key pathogenic cytokine in the pathology of T cell-mediated autoimmune diseases (Langrish, CL et al. 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 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 and other studies detailing the pathological role of IL-17A in human disease ultimately led to the development of monoclonal antibodies (mAbs) against 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, and their efficacy has surpassed traditional nonsteroidal anti-inflammatory drugs and tumor necrosis factor (TNF) blocking drugs (Langley, RGet 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, RB et al. Bimekizumab versus Adalimumab in plaquepsoriasis. N. Engl. J. Med. 385, 130-141 (2021).).

[0006] In recent decades, more and more 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 studies have shown that high expression of IL-17A promotes tumor progression by promoting tumor cell proliferation, cholesterol synthesis, epithelial to mesenchymal cell transition (EMT), MMPs expression, recruitment of inflammatory cells to tumor tissues, and inhibiting cell autophagy and apoptosis (Liao R, Sun J, Wu H, et al. High expression of IL-17and 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 use in detecting human IL-17A. The monoclonal antibody detection kit containing the anti-human IL-17A protein can be used to detect IL-17A in human serum.

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

[0009] Currently, many antibody drugs that block IL-17 have been launched worldwide, including monoclonal antibodies that neutralize IL-17A (Secukinumab and Ixekizumab) or IL-17RA (Brodalumab), and there are more than 50 antibody drugs or small molecules under development in China that target IL-17 and are used to treat autoimmune diseases. Although antibody drugs generally have good efficacy and tolerability in humans compared to chemotherapy drugs, there are still many treatment-related side effects in clinical use, such as: nasopharyngitis, headache, nausea and diarrhea, or more serious such as infection, cardiotoxicity and severe immune response; in addition, the complex structure and relatively poor stability of monoclonal antibodies also require repeated large-dose injections to achieve the best effect.

[0010] Therefore, the art needs more anti-IL-17A antibodies or IL-17A inhibitors with high specificity, high stability and long-acting properties for preventing and / or treating autoimmune diseases and cancer.

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

[0012] In addition to the antibodies for which protection is sought in the present invention, the inventors have also developed eight other antibodies. Based on the relevant provisions of the Patent Law on unity, protection is sought for each of the nine different antibodies.

[0013] The inventor also developed 12 tandem antibodies based on these 9 antibodies, and based on the relevant provisions of the Patent Law on unity, requested protection for the 12 different tandem antibodies separately.

[0014] The inventor also developed gene-modified stem cell technology based on these 12 tandem antibodies, and based on the relevant provisions of the Patent Law on unity, requested protection for three different gene-modified stem cells and three different applications.

[0015] To facilitate understanding of the present invention, it is optional to refer to other series of application texts of this project. Summary of the invention

[0016] Single domain antibody (sdAb), also known as nanobody (Nb), was first discovered in camel blood. Because it has only two heavy chains and no light chain, it is also called heavy chain single domain antibody VHH (variable domain of heavy chain of heavy-chain antibody). The nano antibody crystal is 2.5nm in diameter and 4nm long. It contains only one heavy chain variable region (VHH) and CH2 and CH3 regions. Compared with ordinary antibodies, the light chain of nano antibodies is naturally missing and is the smallest naturally existing fragment that can bind to antigens. Nano antibodies can bind tightly to targets such as antigens like other antibodies, but they do not adhere to each other and aggregate into blocks like single-chain antibodies. Nano antibodies based on this "heavy chain antibody" not only have a molecular weight of only 1 / 10 of ordinary antibodies, making them safer for clinical use, but also have more flexible chemical properties, good stability, high solubility, easy expression and easy coupling to other molecules. Therefore, the development of IL-17A nano antibodies may overcome the defects of traditional antibodies and have broad prospects.

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

[0018] In one aspect, the present invention provides an antibody comprising:

[0019] (1) a first single-domain antibody, wherein the amino acid sequence of the first single-domain antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 1-3; and / or an amino acid sequence having at least 80% sequence identity with the amino acid sequence as shown in SEQ ID NOs: 1-3;

[0020] (2) A second single-domain antibody, wherein the amino acid sequence of the second single-domain antibody comprises HCDR4, HCDR5, and HCDR6 as shown in SEQ ID NOs: 4-6; and / or an amino acid sequence having at least 80% sequence identity with the amino acid sequence as shown in SEQ ID NOs: 4-6.

[0021] 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 on the amino acid sequence shown in SEQ ID NO: 1-6.

[0022] In some embodiments, the amino acid sequence of the antibody comprises: an amino acid sequence having 1, 2, 3, 4 or 5 amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 1-6.

[0023] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably substitutions of one amino acid in the following groups (a) to (e) by another amino acid residue in the same group: (a) small aliphatic, non-polar 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, non-polar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0024] In some specific embodiments, conservative substitutions are as follows: 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.

[0025] wherein SEQ ID NO: 1 is GFSFSIYS, SEQ ID NO: 2 is ITKGGLK, and SEQ ID NO: 3 is NAGRENGY;

[0026] SEQ ID NO:4 is GFDLDLYT, SEQ ID NO:5 is IDLTSGAT, and SEQ ID NO:6 is NPVAVGGHLY.

[0027] In some embodiments, the first single domain antibody or the second single domain antibody further comprises at least four heavy chain framework regions (FRs) for connecting heavy chain variable regions.

[0028] In some embodiments, the heavy chain framework region comprises part or all of an antibody heavy chain framework region selected from human, mouse, primate or camelid sources or variants thereof.

[0029] In some specific embodiments, the heavy chain framework region comprises part or all of an antibody heavy chain framework region selected from camelid origin or a variant thereof.

[0030] In some specific embodiments, the heavy chain framework region comprises an antibody heavy chain framework region partially or entirely selected from alpaca origin or a variant thereof.

[0031] In another aspect, the present invention provides an antibody comprising:

[0032] (i) a first single domain antibody, wherein the structure of the first single domain antibody is:

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

[0034] (ii) a second single domain antibody, wherein the structure of the second single domain antibody is:

[0035] FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8;

[0036] wherein HCDR1, HCDR2 and HCDR3 are selected from:

[0037] An amino acid sequence as shown in SEQ ID NO: 1-3; or an amino acid sequence having 1, 2 or 3 amino acid differences compared to SEQ ID NO: 1-3;

[0038] HCDR4, HCDR5 and HCDR6 are selected from:

[0039] An amino acid sequence as shown in SEQ ID NO: 4-6; or an amino acid sequence having 1, 2 or 3 amino acid differences compared to SEQ ID NO: 4-6;

[0040] FR1, FR2, FR3 and FR4 are selected from: the amino acid sequences shown in SEQ ID NOs: 7-10; or; amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID NOs: 7-10;

[0041] FR5, FR6, FR7 and FR8 are selected from the group consisting of: amino acid sequences as set forth in SEQ ID NOs: 11-14; or; amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID NOs: 11-14;

[0042] The amino acid difference is achieved by at least one of addition, deletion, modification and / or substitution on the amino acid sequence shown in SEQ ID NO: 1-14.

[0043] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably substitutions of one amino acid in the following groups (a) to (e) by another amino acid residue in the same group: (a) small aliphatic, non-polar 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, non-polar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0044] In some specific embodiments, conservative substitutions are as follows: 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.

[0045] In some embodiments, the HCDR1 has the amino acid sequence shown in SEQ ID NO:1, the HCDR2 has the amino acid sequence shown in SEQ ID NO:2, the HCDR3 has the amino acid sequence shown in SEQ ID NO:3, the HCDR4 has the amino acid sequence shown in SEQ ID NO:4, the HCDR5 has the amino acid sequence shown in SEQ ID NO:5, and the HCDR6 has the amino acid sequence shown in SEQ ID NO:6.

[0046] In some embodiments, the FR1 has the amino acid sequence shown in SEQ ID NO:7, the FR2 has the amino acid sequence shown in SEQ ID NO:8, the FR3 has the amino acid sequence shown in SEQ ID NO:9, the FR4 has the amino acid sequence shown in SEQ ID NO:10, the FR5 has the amino acid sequence shown in SEQ ID NO:11, the FR6 has the amino acid sequence shown in SEQ ID NO:12, the FR7 has the amino acid sequence shown in SEQ ID NO:13, and the FR8 has the amino acid sequence shown in SEQ ID NO:14.

[0047] wherein SEQ ID NO:7 is EVQLVESGGGLVQPGGSLRLSCVAW;

[0048] SEQ ID NO: 8 is VNWYRQAPGKERELVAG;

[0049] SEQ ID NO:9 is DYADSVKGRFTISRDNAKNAVYLRMNMLKPEDTAVYYC;

[0050] SEQ ID NO: 10 is WGQGTQVAVST;

[0051] SEQ ID NO:11 is AVQLVESGGGLVQPGGSLRLSCAAS;

[0052] SEQ ID NO: 12 is IAWFRQAPGKEREFVSL;

[0053] SEQ ID NO:13 is SDAGSMKGRVAISRDKDSNTVSLQLNSLKPEDTAVYYC;

[0054] SEQ ID NO: 14 is WGQGTQVTVSS.

[0055] In some embodiments, the amino acid sequences of the first single-domain antibody and the second single-domain antibody are directly connected; or; indirectly connected through a linker.

[0056] In some embodiments, the linker is (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5 or 6.

[0057] In some specific embodiments, the linker is (GGGGS)3, i.e. GGGGSGGGGSGGGGS.

[0058] In some embodiments, the antibody has the following structure:

[0059] FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4-linker-FR5-HCDR4-FR6-HCDR5FR7-HCDR6-FR8.

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

[0061] In some embodiments, the recombinant protein further comprises a biologically active protein or a functional fragment thereof that assists its expression and / or secretion, or prolongs its half-life in vivo.

[0062] In some embodiments, the biologically active polypeptide or fragment thereof is selected from at least one of an immunoglobulin Fc domain, serum albumin, an albumin binding polypeptide, prealbumin, a carboxyl-terminal peptide, an elastin-like polypeptide, a His tag, a GST tag, an MBP tag, a FLAG tag, and a SUMO tag.

[0063] In some embodiments, the biologically active protein or fragment thereof may be an immunoglobulin Fc domain.

[0064] In some embodiments, the immunoglobulin Fc domain is derived from a human antibody, a murine antibody, a primate antibody, or a camelid antibody, or a variant thereof.

[0065] In some specific embodiments, the immunoglobulin Fc domain is derived from a human IgG antibody, such as an IgG1 Fc, IgG2 Fc, IgG3 Fc or IgG4 Fc domain, preferably an IgG1 Fc domain.

[0066] In some embodiments, one or more amino acid modifications can be introduced into the Fc region or Fc domain of an antibody provided herein to generate an Fc region variant. The Fc region variant can include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution, deletion and insertion) at one or more amino acid positions.

[0067] In some embodiments, the sequence of the IgG1 Fc is as shown in SEQ ID NO: 15:

[0068] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0069] The nucleotide sequence encoding SEQ ID NO:15 is shown in SEQ ID NO:16:

[0070] .

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

[0072] In yet another aspect, the present invention provides an antibody preparation comprising:

[0073] A1: any of the above antibodies and / or recombinant proteins;

[0074] A2: Pharmaceutically acceptable carriers, such as buffer, sterile water or surfactant.

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

[0076] In yet another aspect, the present invention provides a kit, comprising any of the aforementioned antibodies, recombinant proteins and / or antibody preparations;

[0077] Optionally, the kit further comprises a container for holding the antibody preparation.

[0078] In yet another aspect, the present invention provides a drug conjugate, comprising:

[0079] B1: any of the above antibodies or recombinant proteins, and

[0080] B2: coupling moiety coupled to B1.

[0081] In some embodiments, the conjugated moiety comprises a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0082] In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding any of the aforementioned antibodies or recombinant proteins.

[0083] In yet another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid molecule.

[0084] In some embodiments, the expression vector may be a eukaryotic expression vector or a prokaryotic expression vector, preferably a eukaryotic expression vector.

[0085] In yet another aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned antibodies, recombinant proteins, antibody preparations, drug conjugates, nucleic acid molecules, and / or expression vectors;

[0086] Optionally, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0087] In some embodiments, the excipient is selected from solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavors, sweeteners, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

[0088] In another aspect, the present invention provides at least one of the following uses of any of the aforementioned antibodies, recombinant proteins, antibody preparations, drug conjugates and / or pharmaceutical compositions:

[0089] C1: Preparation of detection reagents or kits;

[0090] C2: Preparation of drugs for preventing and / or treating autoimmune diseases;

[0091] C3: Preparation of drugs for preventing and / or treating cancer.

[0092] In some embodiments, the cancer includes, but is not limited to, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, breast cancer, peritoneal cancer, cervical cancer, bile duct cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, esophageal cancer, eye cancer, head and neck cancer, stomach 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 lung squamous cell carcinoma), lymphoma (including Hodgkin lymphoma and non-Hodgkin lymphoma), Cancer of the pancreas, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, colorectal 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 myeloblastic leukemia, etc.

[0093] In yet another aspect, the present invention provides a method for detecting IL-17A in a sample in vitro for non-diagnostic purposes, the method comprising the following steps:

[0094] D1: contacting any of the aforementioned tandem single domain antibodies, recombinant protein antibody preparations and / or drug conjugates with a sample to be tested;

[0095] D2: Detection of antigen-antibody complex;

[0096] D3: Interpretation of results;

[0097] If an antigen-antibody complex is formed, the test sample contains IL-17A protein.

[0098] In yet another aspect, the present invention provides a method for preventing and / or treating an autoimmune disease, the method comprising:

[0099] A therapeutically effective amount of any of the aforementioned antibodies, recombinant proteins, antibody preparations and / or drug conjugates is administered to a subject in need thereof.

[0100] In yet another aspect, the present invention provides a method for preventing and / or treating cancer, the method comprising:

[0101] A therapeutically effective amount of any of the aforementioned antibodies, recombinant proteins, antibody preparations and / or drug conjugates is administered to a subject in need thereof.

[0102] The present invention obtained a series of anti-IL-17A single-domain antibodies through yeast library screening. In order to enhance the therapeutic effect, the inventors selected two of the single-domain antibodies 1-G12 and 1-H9 for series connection to obtain the series single-domain antibody 1-G12-1-H9.

[0103] Compared with the prior art, the tandem single-domain antibody 1-G12-1-H9 provided by the present invention has an affinity EC of 50 The blocking effect was better; the stability was better, which was significantly better than the positive control antibody. The aggregation temperature T agg The increase was about 12°C relative to the positive control antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 The SDS-PAGE results of IL-17A recombinant protein are shown, wherein M is a protein marker, lane 1 is a sample before IL-17A recombinant protein purification, and lane 2 is a sample after IL-17A recombinant protein purification.

[0105] Figure 2-Figure 5 The results of flow cytometry detection of the alpaca yeast library are shown.

[0106] Figure 6 Shown are the results of yeast monoclonal FACS detection.

[0107] Figure 7 The results of yeast library sequencing analysis are shown.

[0108] Figure 8 The SDS-PAGE results of the tandem single-domain antibody 1-G12-1-H9 are shown.

[0109] Fig. 9 The results of the ELISA test for the affinity of tandem single domain antibodies are shown.

[0110] Fig.10 The results of the ELISA test for the affinity of the positive control antibody are shown.

[0111] Fig.11 The results of the ELISA test for negative control affinity are shown.

[0112] Fig.12 and Fig.13 The activation of reporter gene cell lines by IL-17A recombinant protein is shown.

[0113] Fig.14 The experimental results of the blocking function test of the tandem single-domain antibody 1-G12-1-H9 are shown.

[0114] Fig.15The experimental results of positive control antibody blocking function detection are shown.

[0115] Fig.16 The results of thermal stability testing of tandem single domain antibodies are shown.

[0116] Fig.17 The results of thermal stability testing of the positive control antibody are shown. DETAILED DESCRIPTION

[0117] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0118] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0119] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0120] Numerical ranges used herein should be understood to include all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0121] As used herein, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any 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. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" as used throughout the application, especially in the claims, may be replaced by the term "consisting of".

[0122] The terms "optionally", "any", "arbitrarily" or "any" as used herein mean that the event or circumstance described subsequently may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur. For example, "optionally comprising an antibody heavy chain variable region" means that an antibody heavy chain variable region of a specific sequence may but need not be present. As used herein, "a" and "an" are used in the present invention to refer to one or more than one grammatical object.

[0123] The term "and / or" used herein should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0124] The terms "IL-17A", "interleukin-17A" or "IL-17" as used herein refer to a cytokine that belongs to the interleukin 17 family, is produced by T cells and other types of immune cells, and plays an important role in the immune system. IL-17A is mainly produced by Th17 cells, and other cells including CD8+T cells, γδT cells, NK cells, and neutrophils, mast cells and macrophages also express IL-17A. It mainly acts on immune cells such as macrophages, neutrophils and endothelial cells to induce inflammatory responses. In some instances, the term includes variants, homologs, orthologs and paralogs. For example, an antibody specific for human IL-17A may cross-react with an IL-17A protein of another species, such as a monkey, in some cases. In other embodiments, an antibody specific for a human IL-17A protein may be completely specific for a human IL-17A protein without cross-reacting with other species or other types of proteins, or may cross-react with IL-17A proteins of some other species but not all other species.

[0125] The terms "anti-IL-17A single domain (nano) antibody", "IL-17A single domain (nano) antibody" or "tandem single domain (nano) antibody that specifically binds to IL-17A" as used herein 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 response or downstream pathways mediated by IL-17A or other IL-17A-mediated functions.

[0126] The term "antibody" as used herein refers to a glycoprotein comprising a heavy chain (H) and a light chain (L) interconnected by disulfide bonds (SS). Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as VH). The light chain constant region consists of one domain CL. Light chains are divided into two categories, namely kappa-type light chains and lambda-type light chains (for example, the light chain constant region Cκ / λ in the present invention indicates that the light chain constant region is a kappa-type light chain or a lambda-type light chain). The VH region and the VL region can be further divided into hypervariable regions (also called complementary determining regions (CDRs)), with more conservative framework regions or framework regions (FRs) inserted therebetween. 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.

[0127] The term "single domain antibody" (sdAb) or "nanoantibody" as used herein has its general meaning in the art and refers to an antibody fragment with a molecular weight of only 12-15 kDa, which is composed of a single monomeric variable antibody domain derived from a heavy chain. Such single domain antibodies (named VHH) can be found in camelid mammals and naturally lack light chains. For a general description of (single) domain antibodies, reference is also made to the above-mentioned prior art and EP 0368684, Ward et al. (Nature 1989 Oct12; 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 framework regions or "FRs", which are referred to in the art as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4", respectively; the framework regions are separated by three complementary determining regions or "CDRs", which are 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", respectively. Therefore, a single domain antibody can be defined as an amino acid sequence having the following general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1-FR4 refer to framework regions 1-4, respectively, and wherein CDR1-CDR3 refer to complementary determining regions 1-3. In the context of the present disclosure, the amino acid residues of a single domain antibody are numbered according to the common numbering scheme for VH domains given by the International ImMunoGeneTics information system amino acid numbering (http: / / imgt.cmes.fr / ).

[0128] The terms "hypervariable region", "hypervariable region", "complementarity determining region", "HVR" or "CDR" as used herein refer to regions in the variable domain region of an antibody that are highly variable in sequence and / or form structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs or CDRs: three present in VH (H1, H2, H3) and three present in VL (L1, L2, L3). Based on the Chothia definition rules, 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 the Kabat 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 edtion, 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 the CDRs of antibodies can be defined in the art by a variety of methods, such as the Kabat definition rules based on sequence variability, the Chothia definition rules based on the position of the structural loop region and the reference tool for antibody humanization design based on CDR transplantation (see J Mol Biol. 273: 927-48, 1997).It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass the complementarity determining region defined by any of the above-mentioned known schemes described in the present invention. Although the scope of protection claimed in the present disclosure is based on the sequence shown in the IMGT definition rules, the amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0129] As used herein, "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. As used herein, "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, as used herein, "amino acid" or "peptide residue" means naturally occurring and synthetic amino acids. For example, for the purposes of the present invention, homophenylalanine, citrulline and norleucine are considered 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.

[0130] As used herein, the terms "activity", "functional activity" or "biological activity", or the terms "biological property" or "biological characteristic" are used interchangeably herein 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, IC 50 , the in vivo stability of the antibody and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., usually with non-human homologs of the targeted peptide, or with cross-reactivity of 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 different sources (including humans, primates or any other source).

[0131] The term "Fc" or "Fc region" or "Fc fragment" as used herein refers to a polypeptide consisting of the CH2 and CH3 domains of IgA, IgD and IgG, or the CH2, CH3 and CH4 domains of IgE and IgM through a hinge region. Although the decomposition of the Fc fragment is variable, the heavy chain Fc fragment of human IgG generally refers to the polypeptide from A231 to its carboxyl terminus.

[0132] The term "epitope" as used herein refers to a protein determinant that is capable of specific binding to an antibody. An epitope is usually composed of molecules, such as amino acids or sugar side chains, that are clustered on the surface, and usually has specific three-dimensional structural characteristics, as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that the binding to the former, but not the latter, is lost in the presence of a denaturing solvent. An epitope may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked or covered by a specific antigen-binding peptide (in other words, the amino acid residue is within the footprint of the specific antigen-binding peptide).

[0133] The term "affinity" or "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is a function of the dissociation rate constant and the association rate constant (KD, respectively). off and K on ). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0134] The term "high affinity" or "high avidity" as used herein refers to an IgG antibody with a KD of 1.0 × 10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or less, 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or less. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype is defined as a KD of 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0135] The terms "drug conjugate" or "antibody drug conjugate" used herein refer to a substance obtained by connecting a bioactive compound fragment to an antibody or its antigen-binding fragment. The bioactive compound fragment and the targeting portion can be connected through a linker. The linker can be broken in a specific environment (such as a low pH environment in the cell) or under a specific action (such as the action of a lysosomal protease), thereby separating the bioactive compound fragment from the targeting portion or the antibody or its antigen-binding fragment. The linker contains a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. The bioactive compound fragment is directly connected to the targeting portion or the antibody or its antigen-binding fragment through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.

[0136] The term "nucleic acid" or "polynucleotide" as used herein refers 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 reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8278036 belonging to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer, Nucleic Acid Res. 19:5081 (1991); Ohtsuka, J. Biol. Chem. 260:2605-2608 (1985); Rossolini, Mol. Cell. Probes 8:91-98 (1994)).

[0137] As used herein, the term "isolated nucleic acid" is a nucleic acid that has been identified and separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0138] The term "construct" as used herein refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of integrating with a genome or autonomously replicating, constituting a polynucleotide molecule in which one or more polynucleotide molecules have been linked (i.e., operably linked) in a functionally operational manner. Recombinant constructs will typically include a polynucleotide of the present invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the present invention.

[0139] The term "vector" as used herein refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). After introduction into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors".

[0140] The term "expression vector" as used herein refers to a nucleic acid molecule that can replicate and express a target gene when transformed, transfected or transduced into a host cell. The expression vector comprises one or more phenotypic selection markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host if necessary.

[0141] As used herein, the terms "cell" and "cell line" are used interchangeably, and all such names include their progeny. 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 E. coli, fungal cells such as yeast cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0142] As used herein, the term "pharmaceutical composition" generally refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective, and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition would be administered. The composition is sterile.

[0143] The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, and are commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. As used herein, the term "pharmaceutically acceptable carrier, excipient and / or diluent" refers to a carrier that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, media, encapsulating materials, manufacturing aids or solvent encapsulating materials, which are related to maintaining the stability, solubility or activity of the antibodies or antigen-binding fragments thereof of the present disclosure, and include but are not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives. For example, pH regulators include but are not limited to phosphate buffers. Surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Preservatives include but are not limited to various antibacterial agents and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents that maintain osmotic pressure include but are not limited to sugars, NaCl and the like. Agents that delay absorption include but are not limited to monostearate 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, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium 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.

[0144] The term "subject" as used herein includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0145] The terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" as used herein refer to an amount of an anti-IL-17A antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents to a cell, tissue or subject, that is effective to prevent or improve the symptoms of one or more diseases or conditions or the development of the disease or condition. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to cause symptom improvement, such as an amount to treat, cure, prevent or improve a related medical condition or to increase the rate of treatment, cure, prevention or improvement of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of active ingredients that cause a therapeutic effect, whether administered in combination, sequentially or simultaneously. An effective amount of a therapeutic agent will result in an increase in a diagnostic criterion or parameter by at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%.

[0146] The term "EC50" as used herein refers to the concentration for 50% of maximal effect, ie, the concentration that can induce 50% of the maximal effect (hereinafter, "IL-17A binding ability").

[0147] The term "IC50" as used herein refers to half maximal inhibitory concentration, that is, the concentration that can cause 50% of the maximal inhibitory effect (herein, "the ability to inhibit the binding of IL-17A and its receptor IL-17RA").

[0148] The term "cancer" or "tumor" as used herein refers to a physiological disorder in mammals that is generally characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, breast cancer, peritoneal cancer, cervical cancer, endometrial cancer, bile duct cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma, liver cancer, kidney cancer, laryngeal cancer, leukemia, hepatoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), lymphoma (including Hodgkin's lymphoma and non-Hodgkin's lymphoma) and other diseases. ), 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 myeloblastic leukemia, etc.

[0149] The term "autoimmune disease" or "autoimmune disease" as used herein refers to a disease caused by the immune system's reduced, lowered and / or destroyed immune tolerance to its own components due to certain reasons, resulting in autoantibodies and / or sensitized lymphocytes damaging its own organs and tissues, which is specifically manifested as dysfunction of the corresponding tissues and organs. Including but not limited to Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic sclerosis, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa, microscopic polyangiitis), aortitis syndrome (Takayasu's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Castleman's disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, allergic vasculitis, rheumatoid vasculitis, large vessel blood 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 insulitis, etc.), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, Good-pasture syndrome, rapidly progressive glomerulonephritis, lupus nephritis, megaloblastic Red cell anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Bassetau disease (Graves' disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, Addison's disease (chronic adrenocortical insufficiency), idiopathic Addison's disease, type I diabetes, slowly progressive type I diabetes (latent autoimmune diabetes in adults), focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA Bullous dermatoses, epidermolysis bullosa acquisita, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent spontaneous abortions, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplantation immunization, familial Mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis, or inclusion body myositis.

[0150] To make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples. If the specific conditions are not specified in the examples, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in many publications, such as "Molecular Cloning Experiment Guide (Fourth 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 the present invention are shown in Table 1.

[0152] Table 1

[0153]

[0154]

[0155]

[0156] The primers used in the present invention for screening, cloning VHH fragments and constructing nanobodies were designed with reference to the following documents:

[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 AlpacaVHH phagelibrary and high throughput sequencing to construct Fab antibody purificationsystem (http: / / hdl.handle.net / 10232 / 00030916).

[0160] Example 1 IL-17 Nanobody Screening Method

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

[0162] The sequence information of Human IL-17A (Q16552-1) (AA Gly 24-Ala155, as shown in SEQ ID NO: 17) 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 prokaryotic codon optimization; 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 solution was collected and lysed; the recombinant protein was purified using a nickel column.

[0164] The purity of the target protein was detected by SDS-PAGE, and the results showed that the purity was >90% ( Figure 1 ).

[0165] SEQ ID NO:17:

[0166] MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTN PKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEIL VLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA.

[0167] 1.2 Alpaca Immunity

[0168] The prepared recombinant antigen was used to immunize two alpacas (Alpaca) by subcutaneous multi-point immunization for a total of 6 times with an interval of 21 days. Peripheral blood was collected 10 days after the last immunization, and the immune titer was detected by ELISA.

[0169] After 6 rounds of immunization, the immune titers of alpacas all reached the requirements (see Table 2).

[0170] Table 2 Immune titer test results

[0171]

[0172]

[0173] 1.3 Construction of antibody yeast library

[0174] (1) PBMC isolation and VHH antibody fragment cloning:

[0175] Collect 100 mL of anticoagulated peripheral blood sample and separate PBMC cells using lymphocyte separation solution;

[0176] Extract RNA using PrimeScript TM II 1st Strand cDNA Synthesis Kit for reverse transcription and preparation of cDNA;

[0177] PCR amplification of VHH fragments.

[0178] (2) Construction of single domain antibody yeast display library

[0179] 1.4 Yeast display library selection and screening:

[0180] The prepared IL-17A antigen was used for incubation with streptavidin magnetic beads, and the yeast solution was added to the magnetic beads bound to the antigen, and the yeast display library was rotated and incubated at 4°C for 60 minutes. The constructed yeast display library was subjected to two rounds of magnetic sorting using streptavidin magnetic beads. After sorting, the yeast solution was coated on SDCAA plates, and a single clone was picked for culture. After induction of expression for 48 hours, flow analysis was performed. Incubate with Biotin-IL-17A-His for 1 hour, use PE Streptavidin as the secondary antibody, and perform flow detection after incubation.

[0181] According to the flow cytometry results ( Figure 2-Figure 5 ), after the second magnetic sorting, the yeast positive rate was 37.9%, and the positive clones were significantly enriched. The sorting products were directly coated on SDCAA plates, and single clones were selected for flow cytometry detection.

[0182] 1.5 FACS screening

[0183] After sorting, the yeast liquid was coated on SDCAA plates, and a single clone was picked for culture. After induction of expression for 48 hours, it was incubated with Biotin-antigen, and PE-Streptavidin was used as the secondary antibody. After incubation, flow cytometry detection was performed.

[0184] The results are as follows Figure 6 As shown, FACS detected the binding of IL17A target monoclonal to the target; the amino acid sequences of the candidate single-domain antibodies obtained by sequencing were compared, and the candidate antibodies with different amino acid sequences in the CDR region were selected to construct eukaryotic expression vectors.

[0185] 1.6 Antibody sequence identification

[0186] Enrich the positive clones; select the enriched single clones, perform Phage ELISA identification, and perform sequencing analysis on the clones to obtain the nucleic acid and amino acid sequence information of the candidate single domain antibody. Figure 7 As shown, 20 monoclones were randomly selected for sequencing analysis, and the sequences were highly different, with good library diversity. Based on the amino acid sequence information of the candidate single-domain antibody CDR region, the In silico method was used to analyze possible post-translational modification sites.

[0187] 1.7 Antibody expression and purification

[0188] According to the ELISA test results of the candidate antibodies, positive clones were selected, and the obtained VHH antibody sequences were synthesized separately, and subcloned in tandem with human IgG1Fc into the expression vector pcDNA3.4-hIgG1-Fc. After the vector was verified by sequencing, the Qiagen plasmid extraction kit was used to prepare the endotoxin-free plasmid for use.

[0189] Take out LVTransm transfection reagent and single-chain antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out PBS buffer and warm it to room temperature. Take 2mL PBS to one well of a 6-well plate, add 130μg antibody expression vector respectively, pipette up and down to mix thoroughly, add 400μL LVTransm, and immediately pipette up and down to mix thoroughly, and let stand at room temperature for 10 minutes.

[0190] Add the above DNA / LVTransm complex to 30 mL of 293F cells and gently shake to mix thoroughly. Place the cells at 37°C and 5% CO 2 After culturing in an incubator at 130 rpm for 6-8 hours, 50 mL of fresh 293 cell culture medium was added and the cells were returned to the incubator for continued cultivation.

[0191] After 7 days of continuous culture, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using Protein A.

[0192] The steps for protein A purification of antibodies are as follows:

[0193] 1) Add the sample containing the target antibody into the EP tube and gently invert the tube to mix.

[0194] 2) Incubate the EP tubes at room temperature (1-4 hours or overnight) or mix on a rotator. 100 mM PMSF may be added to prevent protein degradation.

[0195] 3) Collect the beads using a magnetic separation rack and discard the supernatant. If necessary, retain the supernatant for analysis.

[0196] 4) Add 1 mL of binding / washing buffer to the EP tube and mix thoroughly. Collect the beads using a magnetic stand and discard the supernatant. Repeat the washing step three times.

[0197] 5) Add 500 μL of elution buffer to the EP tube, quickly resuspend by pipetting or vortexing, and then incubate for 5 minutes at room temperature (about 25° C.) in an inverting mixer or by gently inverting the EP tube manually.

[0198] 6) Collect the magnetic beads using a magnetic separation stand and transfer the supernatant containing the eluted antibody to a clean EP tube.

[0199] 7) Repeat steps 1) and 2) twice.

[0200] 8) Add 1 / 10 of the neutralization buffer to each 500 μl of the eluate to neutralize the pH to maintain the biological activity of the antibody and avoid antibody inactivation. If necessary, buffer exchange can be performed by dialysis or desalting.

[0201] 9) Binding / washing buffer: 1× PBS, pH 7.0.

[0202] Elution buffer: 0.1 M glycine, pH 2-3; 0.1 M NaAc-HAc, pH 3.6.

[0203] Neutralization buffer: 1 M Tris, pH 8.5.

[0204] Magnetic bead regeneration buffer: 0.1M NaOH.

[0205] Example 2 Preparation of tandem single domain antibodies

[0206] 2.1 Screening of anti-IL-17A single domain antibodies

[0207] After screening with immune alpacas and yeast libraries, 14 anti-IL-17A single domain antibodies were finally obtained. In the antibody blocking activity test, it was found that although some single domain antibodies can block the activation of downstream target proteins by Human IL-17A protein, the blocking effect is weaker than that of the positive antibody Ixekizumab. Therefore, the present invention connects two of the anti-IL-17A single domain antibodies in series to form a bivalent antibody to enhance its blocking effect. The two anti-IL-17A single domain antibodies are 1-G12 and 1-H9.

[0208] The amino acid sequence of the single-domain antibody 1-G12 is shown in SEQ ID NO: 18:

[0209] EVQLVESGGGLVQPGGSLRLSCVAWGFSFSIYSVNWYRQAPGKERELVAGITKGGLK DYADSVKGRFTISRDNAKNAVYLRMNMLKPEDTAVYYCNAGRENGYWGQGTQVAVST.

[0210] The nucleotide sequence encoding SEQ ID NO:18 is shown in SEQ ID NO:19:

[0211] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTGGGGATTTAGCTTCAGTATCTATTCGGTGAACTGGTACCGCCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCAGGTATTACGAAAGGTGGTCTAAAAGACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACGCGGTGTATCTGCGAATGAACATGTTGAAACCTGAAGACACGGCCGTCTACTACTGTAATGCAGGCCGGGAGAATGGTTACTGGGGCCAGGGGACCCAGGTCGCCGTCTCTACC。

[0212] The amino acid sequence of single-domain antibody 1-H9 is shown in SEQ ID NO:20:

[0213] AVQLVESGGGLVQPGGSLRLSCAASGFDLDLYTIAWFRQAPGKEREFVSLIDLTSGAT SDAGSMKGRVAISRDKDSNTVSLQLNSLKPEDTAVYYCNPVAVGGHLYWGQGTQVTVSS。

[0214] The nucleotide sequence encoding SEQ ID NO:20 is shown in SEQ ID NO:21:

[0215] GCGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCGATTTGGATTTGTATACCATAGCCTGGTTCCGCCAGGCTCCGGGGAAGGAGCGCGAGTTTGTGTCACTCATAGATTTGACTAGTGGTGCCACATCCGACGCGGGCTCCATGAAGGGCCGAGTCGCCATCTCCAGAGACAAAGACAGCAACACGGTGTCGCTGCAATTGAATAGCCTGAAACCTGAAGATACGGCCGTCTACTACTGTAATCCAGTCGCGGTGGGAGGACACTTGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTC。

[0216] 2.2 Preparation of tandem single domain antibody 1-G12-1-H9

[0217] Candidate antibodies with different epitopes were classified according to VHH-(GGGGS) 3 -VHH-IgG1 Fc (the amino acid sequence of IgG1 Fc is shown in SEQ ID NO: 15) to construct a bivalent single-domain antibody, and purify it using Protein A magnetic beads. The specific steps are as follows:

[0218] 1) The above two anti-IL-17A single-domain antibody sequences were synthesized and subcloned in tandem with human IgG1 Fc into the expression vector pcDNA3.4-hIgG1-Fc. After the vector was verified by sequencing, the Qiagen plasmid extraction kit was used to prepare the endotoxin-free plasmid for use;

[0219] 2) Take out the LVTransm transfection reagent and single-chain antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out the PBS buffer and warm it to room temperature. Take 2mL PBS to one well of a 6-well plate, add 130μg of antibody expression vector respectively, pipette up and down to mix thoroughly, add 400μL LVTransm, and immediately pipette up and down to mix thoroughly, and let stand at room temperature for 10 minutes.

[0220] 3) Add the above DNA / LVTransm complex to 30 mL of 293F cells and gently shake to mix thoroughly. Place the cells at 37°C and 5% CO 2 After culturing in an incubator at 130 rpm for 6-8 hours, 50 mL of fresh 293 cell culture medium was added and the cells were returned to the incubator for continued cultivation.

[0221] 4) After 7 days of continuous culture, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube. The antibody was purified using a Protein A column to finally obtain the tandem single-domain antibody 1-G12-1-H9.

[0222] The SDS-PAGE results of the tandem single domain antibody 1-G12-1-H9 are as follows Figure 8 shown.

[0223] Example 3 Tandem Single Domain Antibody Affinity Detection

[0224] 3.1 Preparation of Positive Control Antibody Ixekizumab

[0225] The heavy chain and light chain variable regions of ixekizumab were gene synthesized (the sequence of the light chain variable region is shown in SEQ ID NO: 22; the sequence of the heavy chain variable region is shown in SEQ ID NO: 23), the heavy chain variable region was subcloned into the pcDNA3.4-hIgG4 vector (the amino acid sequence of IgG4 is shown in SEQ ID NO: 24), and the light chain variable region was subcloned into the pcDNA3.4-hIgG1 Kc vector (the amino acid sequence of IgG1 Kc is shown in SEQ ID NO: 25); after verification by Sanger sequencing, an endotoxin-free plasmid was prepared using a plasmid extraction kit for use.

[0226] Take out LVTransm transfection reagent and heavy chain and light chain expression vectors from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out PBS buffer and warm it to room temperature. Take 2mL PBS to one well of a 6-well plate, add 50μg of heavy chain and light chain expression vectors respectively, mix thoroughly by pipetting up and down, then add 300μL LVTransm, immediately mix thoroughly by pipetting up and down, and let stand at room temperature for 10 minutes.

[0227] Add the above DNA / LVTransm complex to 100 mL of 293F cells, gently shake to mix thoroughly, and place the cells at 37°C, 5% CO 2 Incubator, continue culturing at 130RPM.

[0228] After continuous culture for 5-7 days, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using Protein A.

[0229] The purity of the target antibody protein was detected by SDS-PAGE, and the results showed that the protein purity was >95%.

[0230] SEQ ID NO:22:

[0231] DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKVSNR FIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIK.

[0232] SEQ ID NO:23:

[0233] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYG TTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVT VSS.

[0234] SEQ ID NO:24:

[0235] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0236] SEQ ID NO:25:

[0237] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0238] 3.2 Detection of binding activity of human IL-17A recombinant protein and control antibody by ELISA

[0239] Use sterile CBS to dilute the IL-17A recombinant protein to a final concentration of 2 μg / mL. Take a new 96-well plate and add 100 μL / well to coat overnight at 4°C;

[0240] The antigen coating solution was removed and the cells were washed three times with PBST (containing 0.5% Tween);

[0241] Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours;

[0242] After removing the blocking buffer, wash the plate three times with PBST;

[0243] The positive control antibody Ixekizumab was diluted to 10 μg / ml with PBS, 5-fold diluted to 7 points, added to the ELISA plate at 100 μL / well, and incubated at room temperature for 1 hour. The control wells were PBS;

[0244] Remove the liquid in the wells and wash three times with PBST;

[0245] Secondary antibody HRP-ProteinA (Boster, BA1080) was added at a dilution of 1:10000, added to the ELISA plate at 100 μL / well, and incubated at room temperature for 1 hour;

[0246] After removing the liquid in the wells, wash the plate three times with PBST;

[0247] Add 100 μL / well TMB colorimetric solution;

[0248] Incubate at room temperature in the dark for 15 minutes;

[0249] Add 50 μL / well stop solution (2 M HCL);

[0250] The OD450 value in the well was read using an ELISA reader. The results are shown in Table 3. The positive antibody binds well to the IL-17A antigen protein and can be used for immunization.

[0251] Table 3 Detection of binding activity of Human IL17A and positive antibodies

[0252]

[0253] 3.3ELISA to detect affinity of tandem single domain antibodies

[0254] The purified single-domain antibody was coated on a 96-well ELISA plate at 2 μg / mL, and Biotin-IL-17A-His was added at a starting concentration of 10 μg / mL, with 5-fold gradient dilution at 7 points. ELISA was performed using HRP-Streptavdin. The results showed that the EC of 1-G12-1-H9 50 1.772 μg / mL ( Fig. 9 ), which is much lower than 10.06μg / mL of the positive control antibody and 76.15μg / mL of the negative control ( Fig.10 and Fig.11 ), which is also better than the 4.716μg / mL of single-domain antibody 1-G12 and 21.08μg / mL of single-domain antibody 1-H9. Therefore, the tandem single-domain antibody can bind to the target protein with a higher binding ability than the positive antibody and a single single-domain antibody.

[0255] Example 4 Detection of blocking function of tandem single domain antibodies

[0256] 4.1 Construction of IL-17A reporter gene cell line

[0257] According to the amino acid sequence information of IL-17RA (UniProtKB: Q96F46) and IL-17RC (UniProtKB: Q8NAC3), a lentiviral expression vector was constructed and packaged with lentivirus, which was co-infected with 293 cells, and recombinant 293 cells that simultaneously overexpressed these two receptors were screened. NFKB-Luciferase (the amino acid sequence is shown in SEQ ID NO: 26, and the nucleotide sequence encoding it is shown in SEQ ID NO: 27) and ACT1 gene (nucleotide sequence is shown in SEQ ID NO: 28) were further stably transfected to construct the IL-17A reporter gene cell line 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc.

[0258] SEQ ID NO:26:

[0259] .

[0260] SEQ ID NO:27:

[0261]

[0262] SEQ ID NO:28:

[0263]

[0264] 4.2 Binding of IL-17A recombinant protein to reporter gene cell lines

[0265] The 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cell line was revived from liquid nitrogen and the cells were adjusted to the logarithmic growth phase;

[0266] The cells were divided into several portions, with the number of cells in each portion being 2×10 5 cells;

[0267] The IL-17A-His protein was incubated with the target cells, mixed thoroughly, and incubated at room temperature for 1 hour;

[0268] Centrifuge at 800 × g for 3 min at room temperature, remove the supernatant containing the antibody, and wash the cells three times with PBS;

[0269] Add the secondary antibody APC-His (1:500 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 minutes;

[0270] Centrifuge at 800 × g for 3 min at room temperature, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS;

[0271] Resuspend the cells in 500 μL PBS and perform flow cytometry analysis.

[0272] FACS results showed that the constructed IL-17A receptor overexpressing cell line could bind to IL-17A, with a positive rate of more than 90% ( Fig.12 ).

[0273] IL-17A recombinant protein was used to activate 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc. The results showed that IL-17A recombinant protein can effectively activate the expression of luciferase in the 293F-IL17Ra / IL17Rc-NFκB-Luc reporter gene cell line ( Fig.13 ).

[0274] 4.3 Functional experiment of ixekizumab blocking the binding of IL-17A recombinant protein to reporter gene cell line

[0275] In this example, the positive antibody Ixekizumab was used to detect the effectiveness of the fluorescent reporter system. The positive control antibody Ixekizumab was added to 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cells together with the IL-17A recombinant protein. The results showed that the positive control antibody Ixekizumab could inhibit the binding of IL-17A protein to its membrane receptor and inhibit the intracellular NFκB signal, showing a dose effect.

[0276] 4.4 Tandem single domain antibody blocking function

[0277] The blocking activity of tandem single-domain antibodies was tested using the 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc reporter gene cell line. The results showed that the IC 50 The IC value of the positive control antibody was 1.082 μg / mL. 50 1.356 μg / mL ( Fig.14 and Fig.15 ). Therefore, both 1-G12-1-H9 and the positive control antibody can block the activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc by Human IL-17A protein, and the IC of 1-G12-1-H9 50 The lower, the better the blocking effect.

[0278] Example 5 Stability test of tandem single domain antibodies

[0279] NanoDSF technology can be used to detect fluorescence changes, detect thermal and chemical denaturation of proteins under native conditions, and accurately determine the temperature (T) at which 50% of the protein is in an unfolded state. m ), the temperature at which unfolding begins (T onset ) and the temperature at which aggregation begins (T agg ); thermal denaturation T m Value, T onset and the aggregation temperature T agg The higher the value, the more stable the antibody protein.

[0280] Take 100 μL of tandem single-domain antibody and Ixekizumab (sample concentration greater than 200 μg / mL), centrifuge at 4°C, 12,000 × g for 10 min, and then aspirate the sample with a capillary. Prepare two capillaries for each sample as parallel controls and place them in the corresponding card slots in order to ensure that the capillaries are full of samples without bubbles for detection and analysis.

[0281] The results showed that the T of 1-G12-1-H9 m1 is 58.61℃, T m2 is 82.49℃, T onset is 51.51℃, T agg 73.57℃( Fig.16 ); T of positive control antibody m1 is 56.10℃, T m2 is 79.84℃, T onset is 47.50℃, T agg 61.86℃( Fig.17), it can be seen that the thermal stability of the tandem antibody 1-G12-1-H9 is better than that of the positive control antibody, and the aggregation temperature T agg The increase was about 12°C relative to the positive control antibody.

[0282] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An anti-IL-17A antibody, characterized in that The anti-IL-17A antibody comprises: (i) A first single domain antibody, wherein the structure of the first single domain antibody is: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; (ii) a second single domain antibody, wherein the structure of the second single domain antibody is: FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8; The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR4 is shown in SEQ ID NO: 4, the amino acid sequence of the HCDR5 is shown in SEQ ID NO: 5, the amino acid sequence of the HCDR6 is shown in SEQ ID NO: 6, The amino acid sequence of the FR1 is shown in SEQ ID NO: 7, the amino acid sequence of the FR2 is shown in SEQ ID NO: 8, the amino acid sequence of the FR3 is shown in SEQ ID NO: 9, the amino acid sequence of the FR4 is shown in SEQ ID NO: 10, the amino acid sequence of the FR5 is shown in SEQ ID NO: 11, the amino acid sequence of the FR6 is shown in SEQ ID NO: 12, the amino acid sequence of the FR7 is shown in SEQ ID NO: 13, the amino acid sequence of the FR8 is shown in SEQ ID NO: 14, The first single domain antibody and the second single domain antibody are connected via a linker having an amino acid sequence of (GGGGS)3, and the first single domain antibody and the second single domain antibody are connected in a sequence from N-terminus to C-terminus.

2. The anti-IL-17A antibody according to claim 1, characterized in that The first single domain antibody or the second single domain antibody further comprises at least four heavy chain framework regions for connecting heavy chain variable regions.

3. The anti-IL-17A antibody according to claim 2, characterized in that The heavy chain framework region comprises part or all of an antibody heavy chain framework region selected from human, mouse, primate or camelid sources or variants thereof.

4. The anti-IL-17A antibody according to claim 3, characterized in that The heavy chain framework region comprises part or all of an antibody heavy chain framework region selected from camelid origin or a variant thereof.

5. The anti-IL-17A antibody according to claim 4, characterized in that The heavy chain framework region comprises a portion or all of an antibody heavy chain framework region selected from alpaca sources or a variant thereof.

6. A recombinant protein, characterized in that The recombinant protein consists of the anti-IL-17A antibody according to any one of claims 1 to 5 and a biologically active protein or a functional fragment thereof that assists its expression and / or secretion or prolongs its half-life in vivo.

7. The recombinant protein according to claim 6, characterized in that The biologically active protein or its functional fragment is selected from at least one of an immunoglobulin Fc domain, serum albumin, an albumin binding polypeptide, prealbumin, a carboxyl terminal peptide, an elastin-like polypeptide, a His tag, a GST tag, an MBP tag, a FLAG tag and a SUMO tag.

8. The recombinant protein according to claim 7, characterized in that The immunoglobulin Fc domain is derived from human antibody, mouse antibody, primate antibody or camelid antibody, or variants thereof.

9. The recombinant protein according to claim 8, characterized in that The immunoglobulin Fc domain is derived from human IgG antibody.

10. The recombinant protein according to claim 9, characterized in that The immunoglobulin Fc domain is derived from IgG1 Fc, IgG2 Fc, IgG3 Fc or IgG4 Fc.

11. The recombinant protein according to claim 10, characterized in that The immunoglobulin Fc domain is derived from IgG1 Fc.

12. An antibody preparation, characterized in that The antibody preparation comprises: A1: the anti-IL-17A antibody according to any one of claims 1 to 5 or the recombinant protein according to any one of claims 6 to 11; and A2: Pharmaceutically acceptable carrier.

13. A kit, characterized in that: The kit comprises the anti-IL-17A antibody according to any one of claims 1 to 5, the recombinant protein according to any one of claims 6 to 11, or the antibody preparation according to claim 12.

14. The kit according to claim 13, characterized in that The kit also includes a container for holding the antibody preparation.

15. A drug conjugate, characterized in that: The drug conjugate comprises: B1: the anti-IL-17A antibody of any one of claims 1 to 5 or the recombinant protein of any one of claims 6 to 11; and B2: coupling moiety coupled to B1.

16. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-IL-17A antibody according to any one of claims 1 to 5 or the recombinant protein according to any one of claims 6 to 11.

17. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule according to claim 16.

18. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-IL-17A antibody according to any one of claims 1 to 5, the recombinant protein according to any one of claims 6 to 11, the antibody preparation according to claim 12, the drug conjugate according to claim 15, the nucleic acid molecule according to claim 16 or the expression vector according to claim 17.

19. The pharmaceutical composition according to claim 18, characterized in that The pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

20. A method for detecting IL-17A in a sample in vitro for non-diagnostic purposes, characterized in that: The method comprises the following steps: D1: contacting the anti-IL-17A antibody according to any one of claims 1 to 5, the recombinant protein according to any one of claims 6 to 11, the antibody preparation according to claim 12, or the drug conjugate according to claim 15 with a sample to be tested; D2: Detection of antigen-antibody complex; D3: Interpret the results.

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