CN117843803A

CN117843803A - Novel tandem single domain antibodies and their use in the treatment of disease

Info

Publication number: CN117843803A
Application number: CN202311864358.4A
Authority: CN
Inventors: 刘广洋; 刘拥军; 苗丽; 米一; 徐晓丹; 崔嫄汭
Original assignee: Beijing Beilai Pharmaceutical Co ltd
Current assignee: Beijing Beilai Pharmaceutical Co ltd
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2024-04-09

Abstract

The invention belongs to the technical field of biological medicine, and provides a novel serial single domain antibody and application thereof in disease treatment. The antibody comprises: a first single domain antibody, wherein the amino acid sequence of the first single domain antibody comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO. 1-3; and/or; an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1-3; a second single domain antibody, the amino acid sequence of which comprises HCDR4, HCDR5 and HCDR6 with the sequence shown in SEQ ID NO 4-6; and/or; an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 4-6. The tandem single domain antibody has higher affinity, better blocking effect and good stability, and has important significance in clinic treatment of autoimmune diseases and/or cancers.

Description

Novel tandem single domain antibodies and their use in the treatment of disease

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a novel serial single domain antibody and application thereof in disease treatment.

Background

Since the last 30 years it has been found that IL-17 has become a key cytokine for host protection against mucosal infection, and is also a major causative 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 cytokine receptor.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 functions through the IL-17 receptor (IL-17 RA to IL-17 RE). Among the most studied IL-17 family members is IL-17A, which promotes its biological activity by binding to IL-17RA and IL-17 RC.

It is now recognized that the evolution of IL-17A was aimed at modulating innate immunity in invertebrates lacking an adaptive immune system. However, the inflammatory function of IL-17A was originally described in a mouse model of autoimmune disease, with the focus of the study on CD4 secreting IL-17A ⁺ T cell-T helper 17%T _H 17 Cells-as key producers of such cytokines. Now know CD8 ⁺ T cells, γδ T cells, congenital lymphocytes (ILCs), natural Killer (NK) cells, constant NK T cells (Invariant NK T cells), mucosa-associated constant T cells (Invariant T cells), mast cells (post cells) and Paneth cells may also be sources of IL-17A. Although T Cell Receptor (TCR) activation is CD4 ⁺ And CD8 ⁺ The key to the production of IL-17A by T cells, but the production of IL-17A by innate immune cells is driven primarily by inflammatory cytokines, particularly IL-1. Beta. And IL-23.

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 (Langorish, C.L. et al IL-23drives a pathogenic T cell population that induces autoimmune inflammation.J.Exp.Med.201,233-240 (2005); sutton, C., brereton, C., keogh, B., mills, K.H. & Lavelle, E.C.A crucial role for Interleukin (IL) -1in the induction of IL-17-producing T cells that mediate autoimmune enceflower, J.exp.Med.203,1685-1691 (2006)). Subsequent studies have shown that in EAE, IL-17A is secreted by TH17 cells and by gamma delta T (gamma delta T17) cells that secrete IL-17A (Sutton, C.E. et al. Intereukin-1 and IL-23 gene index IL-17production from gammadelta T cells,amplifying Th17 responses and autoimmunity.Immunity 31,331-341 (2009)).

These studies and others detailing the pathological role of IL-17A in human disease have 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, in particular psoriasis, which have therapeutic efficacy over traditional nonsteroidal anti-inflammatory drugs and Tumor Necrosis Factor (TNF) blocking drugs (Langley, r.g. et al, secure kukuumab in plaque psoriasis-results of two phase three.n. engl.j. Med.371,326-338 (2014); reich, k.et al, bimekizumab versus Secukinumab in plaque psoriaisis.n. engl.j. Med.385,142-152 (2021); warren, r.b. et al, bimekizumab versus Adalimumab in plaque psoriaisis.n.engl.j.med.385, 130-141 (2021).

Over the last decades, an increasing number of studies have found that IL-17A plays an important role in the development of a variety of cancers. IL-17A is highly expressed in prostate, colorectal, gastric, breast, lung and hepatocellular carcinoma (HCC) tissues at levels that are 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 indicate that high expression of IL-17A promotes tumor progression by promoting tumor cell proliferation, cholesterol synthesis, epithelial to mesenchymal cell transformation (EMT), MMPs expression, recruitment of inflammatory cells to tumor tissue, and inhibition of autophagy and apoptosis, respectively (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 Interrukin-17A Plays a Pivotal Role in Chemically Induced Hepatocellular Carcinoma in Mice.Dig Dis Sci.2016;61 (2): 474-488.).

Chinese patent application 202310019497.0 discloses a monoclonal antibody against human IL-17A protein, a kit comprising the antibody and application thereof in detecting human IL-17A. The monoclonal antibody detection kit containing the anti-human IL-17A protein can be used for detecting IL-17A in human serum.

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

A number of IL-17 blocking antibody drugs have been marketed worldwide, including monoclonal antibody drugs that neutralize IL-17A (Secukinumab and Ixekizumab) or IL-17RA (Brodalumab), while more than 50 antibody drugs or small molecules have been developed around the IL-17 target and used for autoimmune disease treatment. Although antibody drugs generally have good therapeutic effects and tolerability in humans compared to chemotherapeutic drugs, there are still many therapeutically relevant 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 monoclonal antibody has complex structure and relatively poor stability, so that repeated large-dose injection is required to achieve the optimal effect.

There is therefore a need in the art 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 cancers.

The present inventors have focused on the development of anti-IL-17A antibodies, further technical development based on the screened 9 single domain antibodies (filed in another application), resulting in 12 single domain antibody combinations with relatively improved affinity, blocking effect and stability, and separately claimed 12 different single domain antibody combinations based on the relevant regulations of patent law singleness.

The present invention is a patent application directed to one of the above 12 single domain antibody combination antibodies.

Based on the above, the inventors have developed a genetically modified stem cell technology and subsequent application technology, and based on the relevant regulations of the patent law singleness, the protection is respectively requested for 3 different genetically modified stem cells and 3 different applications.

For the convenience of understanding the technical solution of the present invention, reference is optionally made to other patent application documents of this project.

Disclosure of Invention

Single domain antibodies (singledomain antibody, sdAb), also known as nanobodies (Nb), were the earliest novel antibodies found in camel blood, and are also known as heavy chain single domain antibodies VHH (variabledomain of heavy chain of heavy-chainantibody) because they have only two heavy chains and no light chains. The nano antibody crystal has the diameter of 2.5nm and the length of 4nm, only comprises a heavy chain variable region (VHH) and CH2 and CH3 regions, and compared with the common antibody, the nano antibody light chain is naturally deleted and is a naturally-occurring minimum fragment capable of combining with antigen. Nanobodies can bind tightly to targets such as antigens like other antibodies, but do not bind to each other to form a mass like single chain antibodies. The molecular weight of the nano antibody based on the heavy chain antibody is only 1/10 of that of a common antibody, the clinical use is safer, the chemical property is more flexible, the stability is good, the solubility is high, the expression is easy, and other molecules are easy to couple, so that the research and development of the IL-17A nano antibody can overcome the defects of the traditional antibody, and has a wide prospect.

In order to achieve the technical purpose, the invention provides the following technical scheme:

in one aspect, the invention provides an antibody comprising:

(1) A first single domain antibody, wherein the amino acid sequence of the first single domain antibody comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO. 1-3; and/or; an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1-3;

(2) A second single domain antibody, the amino acid sequence of which comprises HCDR4, HCDR5 and HCDR6 with the sequence shown in SEQ ID NO 4-6; and/or; an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 4-6.

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.

In some embodiments, the amino acid sequence of the antibody comprises: amino acid sequences having a difference of 1, 2, 3, 4 or 5 amino acids compared to the amino acid sequences shown in SEQ ID NO. 1-6.

In some embodiments, the substitution of one or more amino acids may be conservative substitutions of one or more amino acids. Such conservative substitutions are preferably substitutions of one amino acid in the following groups (a) to (e) with 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 (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) an aromatic residue: phe, tyr and Trp.

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; lie to Leu or to Val; leu to Ile or to Val; lys to Arg, to gin, 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.

Wherein SEQ ID NO. 1 is GEDLGYA, SEQ ID NO. 2 is VTSSGSST, and SEQ ID NO. 3 is ASTILLCSDYISAFGT;

SEQ ID NO. 4 is GFSIHIYA, SEQ ID NO. 5 is ITRGGVT, and SEQ ID NO. 6 is NAGGTNGGY.

In some embodiments, the first or second single domain antibody further comprises at least 4 heavy chain framework regions for linking the heavy chain variable regions.

In some embodiments, the heavy chain framework region comprises a portion or all of an antibody heavy chain framework region selected from human, murine, primate, or camelid sources, or a variant thereof.

In some specific embodiments, the heavy chain framework region comprises a portion or all of an antibody heavy chain framework region selected from a camelid source or a variant thereof.

In some specific embodiments, the heavy chain framework region comprises an antibody heavy chain framework region comprising a portion or all selected from alpaca sources or variants thereof.

In another aspect, the invention provides an antibody comprising:

a first single domain antibody of (i), said first single domain antibody having the structure:

FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4；

(ii) a second single domain antibody, said second single domain antibody having the structure:

FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8；

wherein HCDR1, HCDR2 and HCDR3 are selected from the group consisting of:

amino acid sequence shown in SEQ ID NO. 1-3; or; amino acid sequences having a difference of 1, 2 or 3 amino acids compared to SEQ ID NO 1-3;

HCDR4, HCDR5 and HCDR6 are selected from:

amino acid sequence shown as SEQ ID NO. 4-6; or; amino acid sequence having a 1, 2 or 3 amino acid difference compared to SEQ ID NO. 4-6;

FR1, FR2, FR3 and FR4 are selected from: amino acid sequence shown in SEQ ID NO. 7-10; or; an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID NOs 7-10;

FR5, FR6, FR7 and FR8 are selected from: amino acid sequence shown in SEQ ID NO. 11-14; or; an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs 11-14;

The amino acid difference is realized by at least one mode of adding, deleting, modifying and/or substituting on the amino acid sequences shown in SEQ ID NO. 1-14.

In some embodiments, the HCDR1 has an amino acid sequence as set forth in SEQ ID NO. 1, the HCDR2 has an amino acid sequence as set forth in SEQ ID NO. 2, the HCDR3 has an amino acid sequence as set forth in SEQ ID NO. 3, the HCDR4 has an amino acid sequence as set forth in SEQ ID NO. 4, the HCDR5 has an amino acid sequence as set forth in SEQ ID NO. 5, and the HCDR6 has an amino acid sequence as set forth in SEQ ID NO. 6.

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

Wherein SEQ ID NO. 7 is DVQLVESGGGLVEPGESLRLSCAAP;

SEQ ID NO. 8 is IAWFRQAPGKEREVVSC;

SEQ ID NO. 9 is NYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYC;

SEQ ID NO. 10 is WGQGTQVTVAS;

SEQ ID NO. 11 is EVQLVESGGGLVQPGGSLRLSCAAS;

SEQ ID NO. 12 is MGWYRQAPGKQRELVAT;

SEQ ID NO. 13 is NNADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYC;

SEQ ID NO. 14 is WGQGTQVTVSS.

In some embodiments, the amino acid sequences of the first single domain antibody and the second single domain antibody are directly linked; or; indirectly connected through a connector (linker).

In some embodiments, the antibody has the following structure:

FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4-linker-FR5-HCDR4-FR6-HCDR5FR7-HCDR6-FR8。

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

In some embodiments, the linker is (GGGGS) ₃ I.e., GGGGSGGGGSGGGGS.

In some embodiments, the first single domain antibody and the second single domain antibody are linked in an optional order; or; the first single domain antibody and the second single domain antibody are connected in the order from the N end to the C end; or; the first single domain antibody and the second single domain antibody are linked in order from the C-terminus to the N-terminus.

In some embodiments, the antibody is an anti-IL-17A antibody.

In yet another aspect, the invention provides a recombinant protein comprising any of the antibodies described above.

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

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

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

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.

In some specific embodiments, the immunoglobulin Fc domain is derived from a human IgG antibody, e.g., igG1 Fc, igG2 Fc, igG3 Fc, or IgG4 Fc, preferably IgG1 Fc.

In some embodiments, one or more amino acid modifications may be introduced in the Fc region or Fc domain of an antibody provided herein, thereby producing an Fc region variant. An Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, igG2, igG3, or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions, deletions, and insertions) at one or more amino acid positions.

In some embodiments, the recombinant protein may be a monomer, dimer, or multimer.

In yet another aspect, the invention provides an antibody formulation comprising:

a1: any of the foregoing antibodies and/or recombinant proteins;

a2: a pharmaceutically acceptable carrier, such as a buffer, sterile water or a surfactant.

In some embodiments, the surfactant may be an ionic or nonionic active agent.

In yet another aspect, the invention provides a kit comprising any of the antibodies, recombinant proteins and/or antibody preparations described above;

optionally, the kit further comprises a container for loading the antibody preparation.

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

b1: an antibody or recombinant protein of any of the foregoing, and

b2: a coupling moiety coupled to B1.

In some embodiments, the coupling moiety comprises a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.

In yet another aspect, the invention provides an isolated nucleic acid molecule encoding any of the antibodies or recombinant proteins described above.

In a further aspect, the invention provides an expression vector comprising a nucleic acid molecule as hereinbefore described.

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

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

optionally, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

In some embodiments, the adjuvant is selected from solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsifying accelerators, buffers, absorbents, colorants, flavorants, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, or antioxidants.

In a further aspect, the invention provides at least one of any of the antibodies, recombinant proteins, antibody preparations, drug conjugates and/or pharmaceutical compositions described above for use in:

c1: preparing a detection reagent or a kit;

c2: preparing a medicament for preventing and/or treating autoimmune diseases;

And C3: preparing medicine for preventing and/or treating cancer.

In some embodiments, the autoimmune disease 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, perinodular arteritis (polyarteritis nodosa, microscopic polyarteritis), aortositis syndrome (high-ampere arteritis), malignant rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, kalman's disease, sjogren's syndrome, adult Steve's disease, vasculitis, allergic granulomatous vasculitis, allergic vasculitis, rheumatoid vasculitis, macrovasculitis, ANCA-related vasculitis (e.g., wegener granulomatosis and eosinophilic wegener granulomatosis), cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, igG 4-related diseases (e.g., primary sclerosing cholangitis, autoimmune pancreatitis, etc.), guillain barre syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, good-pasture syndrome, acute glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, baster Du Bing (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, herpes gestation, linear IgA bullous skin disease, acquired bullous epidermolysis, alopecia areata, leukoplakia, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, former field disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease (e.g. ulcerative colitis, crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplantation immunity, familial moderate sea fever, eosinophilic chronic rhinomyelitis, cardiomyopathy, cardiomyocyte myositis, systemic mastitis or the like.

In some embodiments, the autoimmune disease is plaque psoriasis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, or lupus nephritis.

In some embodiments, the cancer includes, but is not limited to, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone cancer, breast cancer, peritoneal cancer, cervical cancer, endometrial cancer, cholangiocarcinoma, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma, liver cancer, renal 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), lymphomas (including hodgkin's lymphoma and non-hodgkin's lymphoma), melanomas, myelomas, neuroblastomas, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastomas, rhabdomyosarcomas, rectal cancer, respiratory system cancer, salivary gland cancer, sarcomas, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, B cell lymphoma, chronic Lymphoblastic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and the like.

In yet another aspect, the invention provides a method for in vitro detection of IL-17A in a sample for non-diagnostic purposes, the method comprising the steps of:

d1: contacting any of the foregoing antibodies, recombinant proteins, antibody preparations and/or drug conjugates with a sample to be tested;

d2: detecting the antigen-antibody complex;

d3: judging a result;

if antigen-antibody complexes are formed, the sample to be tested contains IL-17A protein.

In some embodiments, the sample to be tested is a sample not derived from a living human or animal body.

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

administering to a subject in need thereof a therapeutically effective amount of any one of the antibodies, recombinant proteins, antibody preparations and/or drug conjugates described above.

In yet another aspect, the present invention provides a method of preventing and/or treating cancer, the method comprising:

administering to a subject in need thereof a therapeutically effective amount of any of the foregoing tandem single domain antibodies, recombinant proteins, antibody preparations, and/or drug conjugates.

Compared with the prior art, the affinity of the tandem single domain antibody provided by the invention is obviously better than that of a positive control antibody in ELISA detection; IC (integrated circuit) ₅₀ The blocking effect on IL-17A is obviously better than that of positive control anti-tumor agent at 1.356 mu g/mLA body; the stability is obviously better than that of a positive control antibody, and the thermal stability of the serial antibody 1-C3-H10 is obviously better than that of the positive control antibody, especially the aggregation temperature T _agg The increase was about 16 ℃ relative to the control antibody.

Drawings

FIG. 1 shows SDS-PAGE results of IL-17A recombinant proteins. Wherein M is a protein marker, lane 1 is a sample before IL-17A recombinant protein is refined, and lane 2 is a sample after IL-17A recombinant protein is refined.

Fig. 2-5 show the results of alpaca stock flow detection.

FIG. 6 shows the results of yeast monoclonal FACS detection.

FIG. 7 shows the results of a yeast library sequencing analysis.

FIG. 8 shows the SDS-PAGE results of the tandem single domain antibody 1-C3-3-H10.

Figure 9 shows the results of an ELISA assay for detecting tandem single domain antibody affinity.

Fig. 10 shows the experimental results of ELISA detection of positive control antibody affinity.

Fig. 11 shows the experimental results of ELISA detection of negative control affinity.

Fig. 12 shows the experimental results of BLI detection of tandem single domain antibody affinity.

Fig. 13 shows the experimental results of BLI detection of positive control antibody affinity.

FIGS. 14 and 15 show activation of reporter cell lines by IL-17A recombinant proteins.

FIG. 16 shows the experimental results of tandem single domain antibody blocking function detection.

Fig. 17 shows the experimental results of the positive control antibody blocking function detection.

FIG. 18 shows the results of thermostability assays for tandem single domain antibodies.

Figure 19 shows the results of the thermal stability detection of the positive control antibodies.

Detailed Description

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.

The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.

The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.

The terms "include" or "comprising" as used herein mean "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 preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more limiting terms "consisting of … …" and "consisting essentially of … …". In one embodiment, the term "comprising" as used throughout the application, and in particular in the claims, may be replaced by the term "consisting of … …". The amino acid three-letter codes and one-letter codes used herein are as known to those skilled in the art, or as described in J biol. Chem,243, p3558 (1968).

The terms "optional," "any," or "any" used herein mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1 antibody heavy chain variable region" means that an antibody heavy chain variable region of a particular sequence may be, but is not required to be, present.

The term "and/or" as used herein is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

The term "IL-17A", "interleukin-17A" or "IL-17" as used herein refers to a cytokine belonging to the interleukin 17 family, produced by T cells and other types of immune cells, and playing an important role in the immune system. IL-17A is produced primarily by Th17 cells, other cells including CD8 ⁺ T cells, γδ T cells, NK cells, neutrophils, mast cells and macrophages also express IL-17A. It acts primarily on immune cells, such as macrophages, neutrophils and endothelial cells, inducing an inflammatory response. In some examples, the term includes variants, homologs, orthologs, and paralogs. For example, antibodies specific for human IL-17A may in some cases cross-react with IL-17A protein of another species, such as a monkey. In other embodiments, antibodies specific for human IL-17A protein may be completely specific for 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.

The term "anti-IL-17A single domain (nanobody," IL-17A single domain (nanobody) "or" tandem single domain (nanobody) that specifically binds IL-17A, as used herein, refers to an antibody that specifically binds IL-17A and partially or completely neutralizes, inhibits or attenuates IL-17A activity, and/or inactivates IL-17A, prevents IL-17A responses, or downstream pathways mediated by IL-17A or other IL-17A mediated functions.

The term "antibody" as used herein refers to a glycoprotein comprising a heavy chain (H) and a light chain (L) interconnected by disulfide bonds (S-S). 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 3 domains, CH1, CH2 and CH 3. 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 fall into two categories, kappa-type light chains and lambda-type light chains, respectively (e.g., light chain constant regions Ckappa/lambda in the present invention mean that the light chain constant regions are kappa-type light chains or lambda-type light chains). The VH and VL regions may be further subdivided into hypervariable regions (also known as Complementarity Determining Regions (CDRs)) with more conserved framework or Framework Regions (FR) interposed therebetween. Each VH and VL consists of three CDRs and 4 FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies include monospecific antibodies, bispecific antibodies, and multispecific antibodies so long as they exhibit the desired biological activity or function.

The terms "hypervariable region," "complementarity determining region," "HVR," or "CDR," as used herein, refer to regions that are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops") in an antibody variable domain region. Typically, a natural four-chain antibody comprises six HVRs or CDRs: three are present in VH (H1, H2, H3) and three are present in VL (L1, L2, L3). Based on the Chothia definition rules, exemplary CDRs (LCDR 1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR 3) 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 (LCDR 1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR 3) 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 ofImmunological Interest, the fifth edition, public Health Service, national Institutes of Health, bethesda, MD (1991)). Based on IMGT definition rules, exemplary CDRs (LCDR 1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR 3) 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, F.W. (1995) Immunoglobulin genes.academic Press pp.3-443). It is well known to those skilled in the art that the CDRs of an antibody can be defined in a variety of ways, such as Kabat definition rules based on sequence variability, chothia definition rules based on structural loop region positions, and reference tools for humanized design of antibodies based on CDR grafting (see J Mol biol.273:927-48, 1997). It will be appreciated 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) are to be understood as encompassing complementarity determining regions defined in any of the above known schemes as described by the present invention. Although the scope of the present disclosure is based on the sequences shown by IMGT definition rules, amino acid sequences corresponding to other CDR definition rules shall also fall within the scope of the present invention.

The term "single domain antibody" (sdAb) or "nanobody" as used herein has its ordinary meaning in the art and refers to an antibody fragment having a molecular weight of only 12-15kDa, which consists of a single monomeric variable antibody domain derived from a heavy chain. Such single domain antibodies (designated VHH) can be found in camelidae mammals and naturally lack the light chain. 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 1989Oct 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 framework regions or "FR", which are referred to in the art as "framework region 1" or "FR1", respectively; "frame region 2" or "FR2"; is "frame region 3" or "FR3"; "frame region 4" or "FR4"; the framework regions are separated by three complementarity determining regions or "CDRs", referred to in the art as "complementarity determining region 1" or "CDR1", respectively; "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". Thus, a single domain antibody can be defined as an amino acid sequence having the general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1-FR4 refers to framework regions 1-4, respectively, and wherein CDR1-CDR3 refers to complementarity 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 general numbering of the VH domains given by amino acid numbering International ImMunoGeneTics information system (http:// imgt. Cmes. Fr /).

The term "amino acid" as used herein 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 naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon bound to hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to compounds that differ in structure from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

The terms "activity", "functional activity" or "biological activity", or the terms "biological property" or "biological feature" 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, in vivo stability of antibodies, 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 targeting peptide in general, or with other proteins or tissues), and the ability to maintain high levels of expression of the protein in mammalian cells. The aforementioned properties or characteristics are observed, assayed or assessed using techniques well 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, cytokine or growth factor production and/or secretion, signal transduction, and immunohistochemistry of tissue sections of different origin (including human, primate or any other source).

The term "Fc" or "Fc region", "Fc domain" 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 breakdown of the Fc fragment is variable, the heavy chain Fc fragment of human IgG generally refers to the polypeptide from A231 to its carboxy terminus.

The term "epitope" as used herein refers to a protein determinant capable of specific binding to an antibody. Epitopes are typically composed of surface-clustered molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural features, as well as specific charge features. 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 can 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, amino acid residues are within the footprint of a specific antigen binding peptide).

The term "affinity" or "binding affinity" as used herein refers to an inherent binding affinity that reflects interactions 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 the dissociation rate constant and the binding rate constant (K, respectively _off And K _on ) Is a ratio of (2). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

The term "high affinity" or "high affinity" as used herein, with respect to IgG antibodies, refers to KD of 1.0 x 10 for antigen ^-6 M or less, preferably 5.0X10 ^-8 M or less, more preferably 1.0X10 ^-8 M or less, 5.0X10 s ^-9 M or less, more preferably 1.0X10 ^-9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of IgM subtype refers to KD of 10 ^-6 M or less, preferably 10 ^-7 M or less, more preferably 10 ^-8 M or lower.

The term "drug conjugate" or "antibody drug conjugate" as used herein refers to a substance obtained by linking a biologically active compound fragment to an antibody or antigen binding fragment portion thereof. The biologically active compound fragment and the targeting moiety may be linked by a linker. The linker is capable of cleaving in a specific environment (e.g., an intracellular low pH environment) or under a specific action (e.g., action of a lysosomal protease) to thereby separate the biologically active compound fragment from the targeting moiety or antibody or antigen binding fragment thereof. The linker comprises a cleavable or non-cleavable unit, such as a peptide or disulfide bond. The biologically active compound fragment is directly linked to the targeting moiety or antibody or antigen binding fragment thereof by a covalent bond that is capable of cleavage under a specific environment or action, thereby separating the biologically active compound fragment from the antibody or antigen binding fragment portion thereof.

The term "nucleic acid" or "polynucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single or double stranded form. Unless specifically limited, the term includes nucleic acids containing known analogues of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No.8278036 to Kariko et al, which discloses mRNA molecules with uridine replaced by pseudouridine, methods of synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases 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)).

The term "isolated nucleic acid" as used herein is a nucleic acid that has been identified and isolated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that typically contains the nucleic acid molecule, but the nucleic acid molecule is present at a chromosomal location that is extrachromosomal or different from its natural chromosomal location.

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

The term "vector" as used herein refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., the introduction of 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 may be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Upon introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell and thereby replicate together with the host genome. In addition, certain vectors are capable of directing the expression of genes that are operably linked. Such vectors are referred to herein as "expression vectors".

The term "expression vector" as used herein refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell. The expression vector contains one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host if desired.

The terms "cell", "cell line" are used interchangeably herein and all such designations include progeny thereof. The term "host cell" refers to a cell that can be used to introduce a vector, and includes, but is not limited to, a prokaryotic cell such as E.coli, a fungal cell such as a yeast cell, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, heLa cell, BHK cell, HEK 293 cell, or human cell.

The term "pharmaceutical composition" as used herein generally refers to a formulation that exists in a form that allows for the biological activity of the active ingredient to be effective and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the composition is to be administered. The composition is sterile.

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. As used herein, the term "pharmaceutically acceptable carrier, excipient and/or diluent" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and active ingredient, as is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995). A pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid or solvent encapsulating material, which is involved in maintaining the stability, solubility or activity of an antibody or antigen binding fragment thereof of the present disclosure, and includes, but is not limited to: pH modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, 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, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugar, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in a medicament, including but not limited to sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like.

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, and the like.

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 invention that is effective to prevent or ameliorate a symptom of one or more diseases or conditions, or the development of the disease or condition, when administered to a cell, tissue, or subject, alone or in combination with other therapeutic agents. A therapeutically effective dose also refers to an amount of an antibody or antigen binding fragment thereof sufficient to result in an improvement in symptoms, e.g., to treat, cure, prevent, or ameliorate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When an active ingredient is administered to an individual, a therapeutically effective dose refers to that ingredient alone. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered in combination, sequentially or simultaneously. An effective amount of the therapeutic agent will result in an increase in the diagnostic criteria or parameter of at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, most preferably at least 50%.

The term "as used herein"EC ₅₀ "refers to half maximal effect concentration (concentration for 50%of maximal effect), i.e., a concentration that causes 50% of the maximal effect (herein" ability to bind IL-17A ").

The term "IC" as used herein ₅₀ "refers to a half inhibitory concentration (half maximal inhibitory concentration), i.e., a concentration that causes 50% of the maximum inhibitory effect (herein," ability to inhibit IL-17A and its receptor IL-17RA binding ").

The term "cancer" or "tumor" as used herein refers to a physiological condition characterized by unregulated cell growth in a mammal. Included within this definition are benign and malignant cancers, and dormant tumors or micrometastases. Examples of cancers include, but are not limited to, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, breast carcinoma, peritoneal carcinoma, cervical cancer, endometrial carcinoma, cholangiocarcinoma, choriocarcinoma, colorectal cancer, connective tissue carcinoma, digestive system cancer, esophageal carcinoma, eye cancer, head and neck cancer, gastric cancer, glioblastoma, liver cancer, renal cancer, laryngeal carcinoma, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lymphomas (including hodgkin's lymphoma and non-hodgkin's lymphoma), melanomas, myelomas, neuroblastomas, oral cancers, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastomas, rhabdomyosarcomas, rectal cancer, respiratory system cancers, salivary gland cancers, sarcomas, skin cancers, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, B cell lymphomas, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and the like.

The term "autoimmune disease" or "autoimmune disease" as used herein refers to a disease caused by the fact that the immune system has a reduced, decreased and/or destroyed immune tolerance to its own components for some reason, resulting in damage of the tissues of the organs by autoantibodies and/or sensitized lymphocytes, and is 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 scleroderma, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa, polyarteritis microscopically), aorticoresis syndrome (Gao' an arteritis), malignant rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, kaschmann's disease, sjogren's syndrome, adult Steve's disease, vasculitis, allergic granulomatosis vasculitis, allergic vasculitis, rheumatoid vasculitis, macrovasculitis, ANCA-related vasculitis (e.g., wegener granulomatosis and eosinophilic wegener granulomatosis), cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, igG 4-related diseases (e.g., primary sclerosing cholangitis, autoimmune insulitis, etc.), guillain barre syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, good-pasture syndrome, aggressive glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, baster Du Bing (hyperthyroidism), hashimoto's disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, additism (chronic adrenocortical insufficiency), idiopathic Addison's disease, type I diabetes, slowly progressing type I diabetes (latent autoimmune diabetes in adults), focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pregnancy herpes, linear IgA bullous dermatoses, acquired epidermolysis bullosa, alopecia areata, leukoplakia, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, former disease, autoimmune optic neuropathy, idiopathic azoospermia, urticaria, inflammatory bowel disease (e.g., ulcerative colitis, crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic IgA graft immunity, familial moderate sea fever, eosinophilic chronic inflammation, dilated cardiomyopathy, systemic mastocytosis or inclusion myositis.

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention. Such structures and techniques are also described in a number of publications.

The main reagents, materials and equipment to which the present invention relates are shown in table 1.

TABLE 1

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The primers used for screening and cloning VHH fragments and constructing nanobodies are designed by referring to the following documents:

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

Lin,J,Gu,Y,Xu,Y et al.Characterization and applications of nanobodies against Pseudomonas aeruginosa exotoxin a selected from single alpaca B cells.Biotechnol Biotechnol Equip 2020；34:1028-37.

Studies on design of singledomain antibodies by AlpacaVHH phage library and high throughput sequencing toconstruct Fab antibody purification system(http://hdl.handle.net/10232/00030916).

EXAMPLE 1IL-17 nanobody screening method

1.1 preparation of IL-17A (Human and Mouse) recombinant proteins (antigens)

Retrieving the sequence information (AA Gly 24-Ala 155, shown as SEQ ID NO: 15) of Human IL-17A (Q16552-1) from a UniProt database, adding a 6 XHis tag at the C end, performing gene synthesis according to prokaryotic codon optimization, and subcloning into a pET28a vector; after verification of Sanger sequencing, plasmid extraction was performed.

Transforming the recombinant plasmid into BL21 competent, inducing overnight with 0.5mM IPTG, and collecting bacterial liquid for cleavage; the recombinant protein was purified using a nickel column.

SDS-PAGE detects the purity of the target protein, and the results show that the purity is >90% (FIG. 1).

SEQ ID NO:15：

MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA。

1.2 alpaca immunization

2 Alpaca (Alpaca) are immunized by the prepared recombinant antigen in a subcutaneous multipoint immunization mode, 6 times are immunized at intervals of 21 days, and after 10 days of last immunization, peripheral blood is collected and ELISA detection of immunization titer is carried out.

The immunological titers of alpaca after 6 rounds of immunization all meet the requirements (see table 2).

TABLE 2 results of immunotiter assays

1.3 construction of antibody Yeast libraries

(1) PBMC isolation and VHH antibody fragment cloning:

100mL of peripheral blood anticoagulation sample was collected, and PBMC cells were isolated using lymphocyte separation fluid.

RNA extraction using PrimeScript ^TM II 1st Strand cDNA Synthesis Kit reverse transcription was performed to prepare cDNA.

The VHH fragment was PCR amplified.

(2) Construction of Single-Domain antibody Yeast display library

1.4 Yeast display library panning and screening:

using the prepared IL-17A antigen, incubation with streptavidin magnetic beads, adding yeast liquid to the antigen-bound magnetic beads, and spin incubation at 4 ℃ for 60 minutes for 2 rounds of magnetic sorting of the constructed yeast display library using streptavidin magnetic beads. After sorting, the saccharomycete liquid is coated on an SDCAA plate, monoclonal culture is selected, and flow analysis is carried out after 48h of induced expression. Incubation with Biotin-IL-17A-His for 1h, using PE strepitavidin for the secondary antibody, and flow detection after incubation was completed.

According to the flow detection result (fig. 2-5), after the second magnetic separation, the yeast positive rate is 37.9%, positive clones are remarkably enriched, the separated products are directly coated on an SDCAA plate, and single clones are selected for flow detection.

1.5FACS Screen

After sorting, the saccharomycete liquid is coated on an SDCAA plate, monoclonal culture is selected, after induction expression is carried out for 48 hours, the monoclonal antibody is incubated with Biotin-antigen, and after incubation is completed, flow detection is carried out by using PE-strepitavidin for the secondary antibody.

The results are shown in FIG. 6, and the binding of IL17A target monoclonal and target is detected by FACS; and comparing the amino acid sequences of the candidate single-domain antibodies obtained by sequencing, and selecting candidate antibodies with different CDR region amino acid sequences to construct eukaryotic expression vectors.

1.6 identification of antibody sequences

Enriching positive clones; and selecting the enriched single gram drop, performing Phage ELISA identification, and performing sequencing analysis on clones to obtain the nucleic acid and amino acid sequence information of the candidate single domain antibody. As shown in FIG. 7, 20 monoclonals were randomly picked for sequencing analysis, with large sequence differences and good library diversity. The potential post-translational modification sites were analyzed by the In silico method against the amino acid sequence information of the CDR regions of the candidate single domain antibodies.

1.7 antibody expression purification

According to ELISA detection results of candidate antibodies, positive clones are selected, the obtained VHH antibody sequences are respectively subjected to gene synthesis, and subcloned into an expression vector pcDNA3.4-hIgG1-Fc in series with human IgG1 Fc. After the vector is verified by sequencing, the Qiagen plasmid megapump kit is used for preparing the endotoxin-removing plasmid for standby.

And taking out the LVTransm transfection reagent and the single-chain antibody expression vector from the refrigerator, thawing at room temperature, and blowing up and down by a pipetting gun to completely mix uniformly. The PBS buffer was removed and warmed to room temperature. Taking 2mL of PBS to one hole of a 6-hole plate, respectively adding 130 mug antibody expression vector, blowing up and down by a pipette, fully and uniformly mixing, adding 400 mug LVTransm, immediately blowing up and down by the pipette, uniformly mixing, and standing for 10 minutes at room temperature.

The DNA/LVTransm complex was added to 30mL of 293F cells, and the mixture was thoroughly mixed with gentle shaking. The cells were exposed to 5% CO at 37 ℃ ₂ After culturing for 6-8 hours at 130rpm in the incubator, 50mL of fresh 293 cell culture medium was added and the cells were returned to the incubator for continued culturing.

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 a Protein A column.

The procedure for purifying antibodies on Protein A column is as follows:

1) Samples containing the target antibodies were added to the EP tube and mixed by gently inverting the tube.

2) EP tubes were mixed at room temperature or incubated on a rotator, (1-4 hours or overnight) and 100mM PMSF was added to prevent protein degradation.

3) The magnetic beads were collected using a magnetic separation rack and the supernatant was discarded. The supernatant was retained for analysis, if necessary.

4) To the EP tube, 1mL of binding/washing buffer was added and thoroughly mixed, the beads were collected using a magnetic rack and the supernatant was discarded, and the washing step was repeated three times.

5) To the EP tube, 500. Mu.L of elution buffer was added, and resuspended rapidly with pipetting or vortexing, and then incubated at room temperature (about 25 ℃) for 5 minutes either in a tumble mixer or by manually gently tumbling the EP tube.

6) Magnetic beads were collected using a magnetic separation rack and the supernatant containing the eluted antibodies was transferred to a clean EP tube.

7) Steps 1) and 2) were repeated twice.

8) To each 500. Mu.L of eluate, 1/10 of a neutralization buffer was added to neutralize the pH in order to maintain the biological activity of the antibody and avoid inactivation of the antibody. Buffer exchange can be performed by dialysis or desalting, if desired.

9) Binding/washing buffer: 1 XPBS, pH 7.0.

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

Neutralization buffer: 1M Tris, pH 8.5.

Magnetic bead regeneration buffer: 0.1M NaOH.

Example 2 preparation of tandem Single Domain antibodies

2.1 screening of anti-IL-17A Single-Domain antibodies

The inventor finally obtains 14 anti-IL-17A single domain antibodies through screening of immune alpaca and yeast library. In the detection of the blocking activity of the antibody, the blocking effect of a part of single domain antibodies is weaker than that of a positive antibody Ixekizumab although the single domain antibodies can block the Human IL-17A protein from activating a downstream target protein. Thus, the inventors formed bivalent antibodies by tandem two of the anti-IL-17A single domain antibodies to enhance their blocking effect. Two anti-IL-17A single domain antibodies were 1-C3 and 3-H10.

Wherein the amino acid sequence of the single domain antibody 1-C3 is shown as SEQ ID NO. 16:

DVQLVESGGGLVEPGESLRLSCAAPGEDLGYYAIAWFRQAPGKEREVVSCVTSSGSST NYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYCASTILLCSDYISAFGTWGQGTQV TVAS。

the nucleotide sequence of the coded single-domain antibody 1-C3 is shown as SEQ ID NO. 17:

GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCGAGCCTGGGGAATCTCTGAGGCTCTCCTGTGCAGCCCCTGGAGAGGATTTGGGTTATTACGCCATAGCCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGTAGTCTCATGTGTCACAAGTAGTGGTAGTAGCACAAACTATTTAAGTTCCGTGAAGGACCGATTCACCATCTCCATAGACAACGCCAAGAACACGGTATATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCGTCCACTATTCTCCTCTGTTCAGATTATATCTCTGCCTTTGGCACCTGGGGCCAGGGGACCCAGGTCACCGTCGCCTCG。

the amino acid sequence of the single domain antibody 3-H10 is shown as SEQ ID NO. 18:

EVQLVESGGGLVQPGGSLRLSCAASGFSIHIYAMGWYRQAPGKQRELVATITRGGVT NNADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCNAGGTNGGYWGQGTQVTVSS。

the nucleotide sequence of the coded single domain antibody 3-H10 is shown as SEQ ID NO. 19:

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCGGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTTAGTATCCACATCTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGCTGGTCGCAACTATTACTAGAGGTGGTGTAACAAATAATGCAGACTCCGTGAAGGGGCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGCGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCAGGTGGGACGAACGGGGGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。

2.2 preparation of tandem Single domain antibody 1-C3-3-H10

Candidate antibodies of different epitopes are subjected to VHH- (GGGGS) ₃ The form of the-VHH-IgG 1 Fc is constructed into a bivalent single-domain antibody, and the single-domain antibody is purified by using magnetic beads of protein A, and the amino acid sequence of the IgG1 Fc is shown as SEQ ID NO. 20:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

The nucleotide sequence for encoding SEQ ID NO. 20 is shown as SEQ ID NO. 21:

GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA。

the method comprises the following specific steps:

1) The two anti-IL-17A single domain antibody sequences are subjected to gene synthesis and subcloned into an expression vector pcDNA3.4-hIgG1-Fc in series with human IgG1 Fc. After the vector is verified to be correct by sequencing, preparing endotoxin-removing plasmids for later use by using a Qiagen plasmid large-pump kit;

2) And taking out the LVTransm transfection reagent and the single-chain antibody expression vector from the refrigerator, thawing at room temperature, and blowing up and down by a pipetting gun to completely mix uniformly. The PBS buffer was removed and warmed to room temperature. Taking 2mL of PBS to one hole of a 6-hole plate, respectively adding 130 mug antibody expression vector, blowing up and down by a pipette, fully and uniformly mixing, adding 400 mug LVTransm, immediately blowing up and down by the pipette, uniformly mixing, and standing for 10 minutes at room temperature.

3) The DNA/LVTransm complex was added to 30mL of 293F cells, and the mixture was thoroughly mixed with gentle shaking. The cells were exposed to 5% CO at 37 ℃ ₂ After culturing for 6-8 hours at 130rpm in the incubator, 50mL of fresh 293 cell culture medium was added and the cells were returned to the incubator for continued culturing.

4) After continuous culture for 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 a Protein A column, to finally obtain the tandem single domain antibody 1-C3-3-H10.

SDS-PAGE results of the tandem single domain antibody 1-C3-3-H10 are shown in FIG. 8.

Example 3 tandem single domain antibody affinity assay

3.1 preparation of positive control antibody Ixekizumab

Gene synthesis of Ixekizumab heavy chain and light chain variable region (the sequence of the light chain variable region is shown as SEQ ID NO: 22; the sequence of the heavy chain variable region is shown as SEQ ID NO: 23), subcloning the heavy chain variable region into pcDNA3.4-hIgG4 vector (the amino acid sequence of IgG4 is shown as SEQ ID NO: 24), and subcloning the light chain variable region into pcDNA3.4-hIgG1 Kc (the amino acid sequence of IgG1 Kc is shown as SEQ ID NO: 25); after verification by Sanger sequencing, the plasmid megapump kit is used for preparing the endotoxin-removing plasmid for standby.

And taking out the LVTransm transfection reagent and the heavy chain and light chain expression vector from the refrigerator, thawing at room temperature, and blowing up and down by a pipetting gun to mix completely. The PBS buffer was removed and warmed to room temperature. Taking 2mL of PBS to one hole of a 6-hole plate, respectively adding 50 mug of heavy chain and light chain expression vectors, fully and uniformly mixing the heavy chain and the light chain expression vectors by up-and-down blowing of a pipette, adding 300 mug of LVTransm, immediately and uniformly mixing the LVTransm by up-and-down blowing of the pipette, and standing for 10 minutes at room temperature.

Adding the above DNA/LVTransm complex into 100mL of 293F cells, gently shaking, mixing, and placing the cells at 37deg.C and 5% CO ₂ Incubator, 130RPM continued to culture.

After continuous cultivation 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 a Protein A column.

SDS-PAGE detects the purity of the target antibody protein, and the result shows that the protein purity is >95%.

SEQ ID NO:22：

DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKVSNR FIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIK。

SEQ ID NO:23：

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYG TTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVT VSS。

SEQ ID NO:24:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

SEQ ID NO:25：

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

3.2 detection of ELISA binding Activity of Human IL-17A recombinant protein and control antibody

The IL-17A recombinant protein was diluted with sterile CBS to a final concentration of 2. Mu.g/mL. A new 96-well plate is taken, 100 mu L/well is added for coating overnight at 4 ℃;

the antigen coating was removed and washed 3 times with PBST (0.5% tween);

blocking was performed at 37℃for 2 hours with the addition of 200. Mu.L/well of 3% MPBS;

after removal of the blocking buffer, the well plate was washed 3 times with PBST;

the positive control antibody Ixekizumab is diluted to 10 mug/mL by PBS, 7 points are diluted 5 times, 100 mug/hole is added into an ELISA plate, and the incubation is carried out for 1 hour at room temperature, and the control hole is PBS;

remove the liquid in the wells and wash 3 times with PBST;

secondary anti-HRP-protein a (Boster, BA 1080) 1 was added: 10000 diluted, adding into the ELISA plate according to 100 mu L/hole, and incubating for 1 hour at room temperature;

after removing the liquid from the wells, the well plate was washed 3 times with PBST;

adding 100 mu L/hole TMB color development liquid;

Incubating for 15 minutes at room temperature in a dark place;

add 50. Mu.L/Kong Zhongzhi solution (2M HCL);

OD450 values within wells were read using a microplate reader. As shown in Table 3, the positive antibodies bind well to IL-17A antigen protein and can be used for immunization.

TABLE 3 detection of binding Activity of human IL17A to Positive antibodies

3.3ELISA detection of tandem Single Domain antibody affinity

Coating the purified single domain antibody with 2 mug/mL of a 96-well ELISA plate, adding Biotin-IL-17A-His, diluting 7 points with a 5-fold gradient at an initial concentration of 10 mug/mL, and performing ELISA detection by using HRP-strepitavdin. The results showed that the EC50 of 1-C3-3-H10 was 1.134. Mu.g/mL (FIG. 9), well below 10.06. Mu.g/mL for the positive control antibody and 76.15. Mu.g/mL for the negative control (FIGS. 10 and 11), and also superior to 5.277. Mu.g/mL for the single domain antibody 1-C3 and 1.224. Mu.g/mL for the single domain antibody 3-H10. Thus, the tandem single domain antibody can bind to the target protein with a higher binding capacity than the positive antibody and the single domain antibody.

3.4BLI detection tandem Single Domain antibody affinity

Antibody affinity determination using ForteBio OCTET R2 instrument, HIS1K sensorProA Biosensors) solidified IL7A-His at a concentration of 5 μg/mL.

The buffer was PBST (PBS+0.02% tween 20), and the candidate antibody samples were diluted to 50, 25, 12.5, 6.25,

3.13、0nM。

Affinity detection: equilibrium is carried out for 60s, binding for 180s, dissociation for 180s, and detection temperature is 25 ℃.

Kinetic characterization analysis was performed using the ForteBio OCTET R2 system.

The results show that K is 1-C3-3-H10 _D The value was 1.317×10 ^-9 M, K of positive control antibody _D The value is 0.3910 multiplied by 10 ^-9 M, indicating that the affinities were comparable (FIGS. 12 and 13).

Example 4 tandem single domain antibody blocking function detection

4.1 construction of IL-17A reporter Gene cell lines

According to the amino acid sequence information of IL-17RA (UniProtKB: Q96F 46) and IL-17RC (UniProtKB: Q8NAC 3), constructing a lentiviral expression vector, packaging lentivirus, co-infecting 293 cells, screening recombinant 293 cells which simultaneously overexpress the two receptors, and further stably transferring NFKB-Luciferase (the amino acid sequence of which is shown as SEQ ID NO:26, the nucleotide sequence of which is shown as SEQ ID NO: 27) and ACT1 gene (the nucleotide sequence of which is shown as SEQ ID NO: 28), thereby constructing an IL-17A reporter cell strain 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc.

SEQ ID NO:26：

MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV。

SEQ ID NO:27：

atggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtg。

SEQ ID NO:28：

ATGCCACCTCAGTTGCAGGAAACTCGGATGAATAGAAGCATCCCCGTGGAAGTGGACGAGAGCGAGCCGTACCCTAGTCAGCTGCTGAAGCCGATCCCTGAGTACTCCCCGGAAGAGGAATCCGAACCACCAGCCCCCAACATTCGCAATATGGCCCCCAATAGCTTGTCCGCACCAACAATGCTGCACAACTCTTCTGGCGACTTCTCTCAGGCCCACTCCACCCTGAAACTGGCGAATCACCAGCGGCCTGTATCCCGGCAGGTGACCTGTCTGAGAACTCAGGTGCTTGAAGACTCCGAGGACTCTTTCTGTAGGCGGCATCCAGGTTTGGGCAAGGCGTTTCCGTCCGGCTGTTCCGCGGTTTCAGAGCCCGCTTCCGAAAGTGTCGTGGGCGCCCTGCCAGCCGAGCACCAGTTCTCCTTCATGGAAAAGCGGAACCAGTGGCTGGTCAGTCAGCTGAGCGCCGCGTCACCTGATACAGGTCACGATTCCGACAAGTCTGACCAGTCTCTGCCCAATGCGTCAGCCGATAGTCTCGGGGGCTCCCAGGAGATGGTGCAGAGACCACAGCCGCACAGAAACCGGGCCGGGCTTGATCTGCCCACCATTGATACAGGCTACGATTCCCAGCCCCAGGACGTCCTTGGCATTCGCCAGCTGGAAAGGCCTCTGCCCTTGACCTCCGTGTGTTACCCCCAGGACCTGCCCCGCCCTTTGAGAAGCCGGGAGTTTCCCCAGTTTGAGCCCCAACGATACCCTGCCTGTGCTCAGATGCTGCCTCCGAACCTGAGCCCACACGCTCCCTGGAACTACCACTATCACTGTCCCGGCAGCCCCGATCACCAGGTGCCTTATGGACACGACTACCCGCGGGCTGCATACCAGCAGGTCATACAGCCTGCCTTGCCGGGTCAGCCGCTGCCCGGAGCTTCTGTGCGCGGCCTGCACCCCGTTCAGAAAGTGATCCTGAACTATCCAAGCCCATGGGACCATGAAGAGAGACCAGCCCAAAGAGATTGCTCTTTTCCTGGGTTGCCTAGACACCAAGACCAGCCTCACCACCAGCCTCCCAATCGGGCAGGCGCCCCAGGCGAAAGTCTCGAGTGCCCCGCCGAACTCAGACCACAGGTCCCTCAGCCCCCTTCCCCCGCGGCAGTACCCAGACCCCCCTCTAACCCACCCGCCCGGGGAACGCTCAAGACTTCAAATCTCCCAGAAGAGCTGCGCAAAGTGTTCATAACCTACAGCATGGACACCGCTATGGAGGTGGTTAAGTTCGTCAACTTCCTGCTGGTCAATGGGTTCCAGACTGCAATCGACATTTTTGAGGATAGAATTCGGGGAATCGACATCATCAAGTGGATGGAGAGATACCTGCGGGATAAGACAGTGATGATTATCGTGGCCATTAGTCCCAAGTACAAGCAAGATGTGGAGGGCGCAGAATCACAGTTGGACGAAGACGAGCACGGACTCCATACAAAATATATCCACAGGATGATGCAGATCGAGTTCATTAAACAAGGCTCCATGAATTTCCGCTTCATACCGGTCCTGTTTCCAAACGCAAAAAAAGAGCATGTACCCACTTGGCTCCAGAATACCCATGTCTACTCCTGGCCCAAGAACAAGAAGAATATCCTGCTGCGCTTGCTCAGAGAAGAAGAGTATGTCGCCCCTCCAAGGGGGCCCCTCCCCACACTCCAAGTAGTGCCACTT。

4.2 binding of IL-17A recombinant proteins to reporter cell lines

Resuscitating 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc cell lines from liquid nitrogen, and adjusting the cell state to the logarithmic growth phase;

dividing the cells into several parts, each cell number being 2×10 ⁵ A cell;

incubating IL-17A-His protein and target cells, and incubating for 1 hour at room temperature after fully mixing;

centrifugation at 800 Xg for 3 min at room temperature, removal of antibody-containing supernatant, washing of cells 3 times with PBS;

adding secondary antibody APC-His (1:500 dilution), fully mixing, and incubating for 30 minutes at room temperature in a dark place;

centrifugation at 800 Xg for 3 min at room temperature, removal of the secondary antibody containing supernatant, washing the cells 3 times with PBS;

flow assays were performed using 500 μl PBS to resuspend cells.

FACS results show: the constructed IL-17A receptor over-expression cell strain can be combined with IL-17A, and the positive rate is more than 90 percent (figure 14).

The IL-17A recombinant protein is adopted to activate 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc. The results showed that the IL-17A recombinant protein was effective in activating luciferase expression in 293F-IL17Ra/IL17 Rc-NFkB-Luc reporter cell lines (FIG. 15).

4.3 functional experiments of Ixekizumab blocking IL-17A recombinant proteins

The present example uses the positive antibody Ixekizumab to detect the validity of the fluorescence reporting system. The positive control antibody Ixekizumab was added to 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc cells together with the IL-17A recombinant protein, and the results showed that: the positive control antibody Ixekizumab can inhibit the binding of IL17A protein and a membrane receptor thereof, and inhibit the signal of intracellular NF- κB, and has a dose effect.

4.4 tandem single domain antibody blocking function

Tandem single domain antibody blocking Activity assays were performed using the 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc reporter cell line. The results show that the IC of 1-C3-3-H10 ₅₀ IC of the positive control antibody at 0.6304nM ₅₀ 2.235nM (FIGS. 16 and 17). Therefore, both the 1-C3-3-H10 and the positive control antibody can block the Human IL-17A protein from activating 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc, but the blocking effect of the 1-C3-3-H10 is obviously better than that of the positive control antibody.

Example 5 tandem Single Domain antibody stability detection

By detecting fluorescence change by differential scanning fluorescence (nanoDSF) technique, thermal denaturation and chemical denaturation of proteins can be detected under natural conditions, and the temperature (T) at which 50% of proteins are in an unfolded state can be precisely determined _m ) Temperature at which unfolding starts (T _onset ) The temperature at which aggregation starts to occur (T _agg ) The method comprises the steps of carrying out a first treatment on the surface of the Thermal denaturation T _m Value, T _onset And aggregation temperature T _agg Higher indicates that the antibody protein is more stable.

Taking 100 mu L of candidate antibody prepared in the earlier stage and Ixekizumab (the concentration of a sample is greater than 200 mu g/mL), centrifuging at 4 ℃ and 12000 Xg for 10min, sucking the sample by using a capillary tube, preparing two capillaries for each sample, taking the capillaries as parallel control, putting the capillaries into corresponding clamping grooves in sequence, ensuring that the capillaries are full of the sample, and carrying out detection analysis.

The results show that T is 1-C3-H10 _m1 56.36 ℃, T _m3 81.61 ℃, T _onset 49.32 ℃, T _agg 77.67 ℃ (fig. 18); t of positive control antibody _m1 At 56.10 ℃, T _m2 79.84℃T _onset 47.50 ℃, T _agg 61.86 ℃ (fig. 19). From this, the thermostability of the tandem antibody 1-C3-H10 is significantly better than that of the positive control antibody, especially the aggregation temperature T _agg The increase was about 16 ℃ relative to the control antibody.

Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An antibody, said antibody comprising:

2. The antibody of claim 1, wherein 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.

3. The antibody of claim 1 or 2, wherein the amino acid sequence of the antibody comprises: amino acid sequences having a difference of 1, 2, 3, 4 or 5 amino acids compared to the amino acid sequences shown in SEQ ID NO. 1-6.

4. The antibody of any one of claims 1-3, wherein the first single domain antibody or the second single domain antibody further comprises at least 4 heavy chain framework regions for linking heavy chain variable regions.

5. The antibody of claim 4, wherein the heavy chain framework region comprises a portion or all of an antibody heavy chain framework region selected from human, murine, primate, or camelid sources or a variant thereof;

preferably, the antibody heavy chain framework regions or variants thereof comprise part or all of a source selected from camelids;

more preferably, the heavy chain framework regions of the antibodies or variants thereof are comprised in part or in whole selected from alpaca sources.

6. An antibody, said antibody comprising:

FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4；

FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8；

wherein HCDR1, HCDR2 and HCDR3 are selected from the group consisting of:

HCDR4, HCDR5 and HCDR6 are selected from:

7. The antibody of claim 6, wherein said HCDR1 has an amino acid sequence as set forth in SEQ ID NO. 1, said HCDR2 has an amino acid sequence as set forth in SEQ ID NO. 2, said HCDR3 has an amino acid sequence as set forth in SEQ ID NO. 3, said HCDR4 has an amino acid sequence as set forth in SEQ ID NO. 4, said HCDR5 has an amino acid sequence as set forth in SEQ ID NO. 5, and said HCDR6 has an amino acid sequence as set forth in SEQ ID NO. 6.

8. The antibody of any one of claims 1-7, wherein the amino acid sequences of the first single domain antibody and the second single domain antibody are directly linked; or; indirectly connected through a linker.

9. The antibody of claim 8, wherein the linker is (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5 or 6; preferably, the linker is (GGGGS) ₃ 。

10. The antibody of any one of claims 1-9, wherein the first single domain antibody and the second single domain antibody are linked in an optional order; or; the first single domain antibody and the second single domain antibody are connected in the order from the N end to the C end; or; the first single domain antibody and the second single domain antibody are linked in order from the C-terminus to the N-terminus.

11. The antibody of any one of claims 1-10, wherein the antibody is an anti-IL-17A antibody.

12. A recombinant protein comprising the antibody of any one of claims 1-11.

13. The recombinant protein according to claim 12, wherein said recombinant protein further comprises a biologically active protein or functional fragment thereof that aids in its expression and/or secretion, or that extends its half-life in vivo.

14. The recombinant protein according to claim 13, wherein said biologically active polypeptide or functional fragment thereof is selected from at least one of an immunoglobulin Fc domain, serum albumin, albumin binding polypeptide, prealbumin, carboxy terminal peptide, elastin-like polypeptide, his tag, GST tag, MBP tag, FLAG tag, and SUMO tag.

15. The recombinant protein according to claim 14, wherein said Fc domain is derived from a human, murine, primate or camelid antibody, or a variant thereof;

preferably, the immunoglobulin Fc domain is derived from a human IgG antibody, such as an IgG1 Fc, an IgG2 Fc, an IgG3 Fc or an IgG4 Fc, preferably an IgG1 Fc.

16. An antibody preparation, comprising:

a1: the antibody of any one of claims 1-11 or the recombinant protein of any one of claims 12-15; and

a2: a pharmaceutically acceptable carrier.

17. A kit comprising the antibody of any one of claims 1-11, the recombinant protein of any one of claims 12-15, or the antibody preparation of claim 16;

18. A drug conjugate, the drug conjugate comprising:

b1: the antibody of any one of claims 1-11 or the recombinant protein of any one of claims 12-15; and

b2: a coupling moiety coupled to B1.

19. An isolated nucleic acid molecule encoding the antibody of any one of claims 1-11 or the recombinant protein of any one of claims 12-15.

20. An expression vector comprising the nucleic acid molecule of claim 19.

21. A pharmaceutical composition comprising the antibody of any one of claims 1-11, the recombinant protein of any one of claims 12-15, the antibody preparation of claim 16, the drug conjugate of claim 18, the nucleic acid molecule of claim 19, or the expression vector of claim 20;

22. The antibody of any one of claims 1-11, the recombinant protein of any one of claims 12-15, the antibody preparation of claim 16, the drug conjugate of claim 18, or the pharmaceutical composition of claim 21 for at least one of the following uses:

c1: preparing a detection reagent or a kit;

c2: preparing a medicament for preventing and/or treating autoimmune diseases;

and C3: preparing medicine for preventing and/or treating cancer.

23. The use according to claim 22, characterized in that, the autoimmune disease includes Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic scleroderma, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, perinodular arteritis, aortitis syndrome, malignant rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, kasmann's disease, sjogren's syndrome, adult Steve's disease, vasculitis, allergic granulomatous vasculitis, allergic vasculitis, rheumatoid vasculitis, macrovasculitis, ANCA-related vasculitis, cogan's syndrome, RS3PE syndrome, temporal arteritis, polymyositis rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, igG 4-related diseases, guillain Barre syndrome myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, good-pasture syndrome, acute glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, bazedol's disease, crohn's disease bridge disease, autoimmune adrenocortical insufficiency, primary hypothyroidism, addison's disease, idiopathic Addison's disease, type I diabetes, slowly progressive type I diabetes, focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, herpes gestation, linear IgA bullous dermatoses, acquired epidermolysis bullosa, alopecia areata, leukoplakia, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, former field disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, familial mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis or inclusion body myositis;

Preferably, the autoimmune disease is plaque psoriasis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis or lupus nephritis.

24. The use according to claim 22 or 23, wherein the cancer comprises basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, peritoneal cancer, cervical cancer, cholangiocarcinoma, choriocarcinoma, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma, liver cancer, renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, B-cell lymphoma, chronic lymphoblastic leukemia, acute lymphoblastic leukemia, hairy cell leukemia or chronic myelogenous leukemia.

25. A method for in vitro detection of IL-17A in a sample for non-diagnostic purposes, characterized in that the method comprises the steps of:

D1: the antibody of any one of claims 1-11, the recombinant protein of any one of claims 12-15, the antibody preparation of claim 16, or the drug conjugate of claim 18, in contact with a sample to be tested;

d2: detecting the antigen-antibody complex;

d3: and judging the result.

26. A method of preventing and/or treating an autoimmune disease, the method comprising:

administering to a subject in need thereof a therapeutically effective amount of the antibody of any one of claims 1-11, the recombinant protein of any one of claims 12-15, the antibody preparation of claim 16, or the drug conjugate of claim 18.

27. A method of preventing and/or treating cancer, the method comprising: