CN117843777A

CN117843777A - Novel nanobody for the treatment of inflammatory diseases, and products and methods thereof

Info

Publication number: CN117843777A
Application number: CN202311774242.1A
Authority: CN
Inventors: 刘拥军; 刘广洋; 张晨亮; 苗丽; 王荷蕊; 徐利强
Original assignee: Beijing Beilai Pharmaceutical Co ltd
Current assignee: Beijing Beilai Pharmaceutical Co ltd
Priority date: 2023-12-21
Filing date: 2023-12-21
Publication date: 2024-04-09

Abstract

The invention belongs to the field of antibodies, and particularly relates to a novel nano antibody for treating inflammatory diseases, and a product and a method thereof. The novel nano antibody is a single-domain antibody 1-C3 of IL-17A, and the amino acid sequence of the heavy chain CDR region of the novel nano antibody is SEQ ID NO.1-SEQ ID NO.3. The novel nano antibody provided by the invention is easy to express, has good affinity and blocking effect and strong stability, is expected to be used for diagnosing and treating inflammatory diseases, and has wide application prospect.

Description

Novel nanobody for the treatment of inflammatory diseases, and products and methods thereof

Technical Field

The invention belongs to the field of antibodies, and particularly relates to a novel nano antibody for treating inflammatory diseases, and a product and a method thereof.

Background

The interleukin-17 (IL-17) family is an important class of pro-inflammatory factors that promote the release of inflammatory factors (IL 1, IL6, IL17 and IL 23) by a variety of cells, and promote cell proliferation and angiogenesis. The IL-17 family, an important inflammatory factor involved in chronic inflammation and inflammation-related tumorigenesis and development, is mainly produced by activated CD4+ T helper cells, th cells. It has now been found that the IL-17 family comprises at least 6 family members, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, respectively. The IL-17 family exerts biological effects by binding to the corresponding homo-or heterodimeric receptors (IL-17 RA, IL-17RB, IL-17RC, IL-17RD, and IL-17 RE). IL-17A, known as IL-17, is a major executor of the action of Th17 cells, and promotes the occurrence of inflammatory responses, and plays an important role in autoimmune diseases, inflammation, tumors, and graft rejection.

Where the body is infected or injured, the migrated lymphocytes secrete IL-17A. IL-17A induces the expression of inflammatory factors and chemokines on the one hand, thereby recruiting more immune cells to reach the inflammatory sites to exacerbate the inflammatory response of the body, and on the other hand, IL-17A induces the expression of some tissue repair related factors to accelerate the recovery of the body. Although IL-17A plays a role in expanding immune defenses and protecting the body of the host during anti-infection and tissue repair, IL-17A is highly expressed in many autoimmune and oncological patients, and excessive IL-17A levels play a worsening role in the pathological development of the disease due to the fact that it can induce the expression of many inflammatory factors. In autoimmune disease treatment, the traditional treatment has the defects of slower effect, poor compliance and the like, and IL-17A deficiency or IL-17A neutralization by antibodies can effectively inhibit the pathological degrees of various autoimmune diseases.

Single domain antibodies (singledomain antibody, sdAb), also known as nanobodies (nanobodies), which contain only one heavy chain variable region (VHH) and CH2, CH3 regions, are naturally absent from the light chain of the nanobody, as compared to conventional antibodies, and are the smallest fragments that naturally exist that can bind to an antigen. Nanobodies can bind tightly to targets such as antigens like normal antibodies, but do not bind to each other and aggregate into blocks like single chain antibodies. The molecular weight of the nano antibody based on the heavy chain antibody is only 1/10 of that of the 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.

The single domain antibody has the advantages of small molecular weight and strong penetrability which are not possessed by the traditional antibody, is considered to have great application potential in the fields of disease diagnosis and treatment, pathogen detection, drug residue analysis, environmental monitoring and the like, and has also acquired some new researches in the aspects of genetic engineering and ADC drug research and development, in-vitro diagnosis technology (colloidal gold method, enzyme-linked immunosorbent assay, electrochemiluminescence method), tumor and immunology research and the like.

The patent CN 114920838A uses biological gene engineering technology to screen out the single domain antibody specific to IL17A, has better antibody affinity, can block specific cells from releasing cytokines, has good binding activity through prokaryotic expression and eukaryotic expression, and has certain drug property. At present, therapeutic antibodies aiming at IL-17A are under development, other antibodies still need to be developed for clinical research, and in view of the advantages of the single-domain antibodies, the research and development of the IL17A single-domain antibodies can overcome the defects of the traditional antibodies and has wide application prospect.

The present invention is a patent application filed by the inventors for an anti-IL-17A antibody developed autonomously.

In addition to the antibodies claimed in the present invention, the inventors developed 8 additional antibodies at the same time, each of which was claimed for 9 different antibodies based on the relevant regulations of patent law singleness.

The inventors have also developed 12 tandem antibodies from these 9 antibodies, and separately claimed 12 different tandem antibodies based on the relevant regulations of patent law singleness.

The inventors have also developed a genetically modified stem cell technology based on these 12 tandem antibodies, and separately claimed 3 different genetically modified stem cells and 3 different applications based on the relevant regulations of patent law singleness.

For ease of understanding the invention, reference is optionally made to other series of application text for this project.

Disclosure of Invention

In order to solve the problems, the invention provides novel anti-IL-17A antibody molecules, and a preparation method and application thereof. The single domain antibody is easy to express, good in affinity and blocking effect and strong in stability.

Terminology:

in the present invention, a single domain antibody (nanobody) is an antibody whose complementarity determining region is a part of a single domain polypeptide. Thus, a single domain antibody comprises a single complementarity determining region. Single domain antibodies are heavy chain-only antibodies that naturally do not contain a light chain, single domain antibodies derived from conventional antibodies, and engineered antibodies. The single domain antibodies may be derived from any species including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules may be derived from antibodies provided by camelidae species (e.g. camels, dromedaries, llamas and dromedaries). Like whole antibodies, single domain antibodies are capable of selectively binding to a particular antigen. A single domain antibody may contain only the variable domains of an immunoglobulin chain, which domains have CDR1, CDR2 and CDR3, as well as framework regions.

In the present invention, anti-IL-17A single domain antibodies include not only whole single domain antibodies, but also fragments, derivatives and analogs of the anti-IL-17A single domain antibodies. Wherein fragments, derivatives and analogs are synonymous, all refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the present invention may be a polypeptide having one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, and such substituted amino acid residues may or may not be a polypeptide encoded by the genetic code or having a substituent in one or more amino acid residues, or a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a pro-protein sequence for purification of the polypeptide, or a fusion protein with an Fc tag).

In the present invention, the CDR (complementary-determining regions, CDR) is called a complementarity determining region or complementarity determining region. It is located in the hypervariable region of an immunoglobulin, which is the antigen binding site of an antibody, complementary to the structure of an epitope. Generally including CDR1, CDR2, CDR3. 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.

In the present invention, the variable region refers to a region of the immunoglobulin light chain and heavy chain where the amino acid sequence changes greatly near the N-terminus, and is referred to as a variable region.

In the present invention, sequence homology means the degree to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is generally expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies with identical sequences have 100% homology. In some embodiments, sequences that replace only one or a few amino acids, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, as compared to the preceding sequences, may also achieve the object. These variants include, but are not limited to: deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. In fact, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution would preferably be conservative amino acid substitutions, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combinations in a single domain antibody. The conserved amino acid, which may be generally described as an amino acid substitution of an amino acid residue with another amino acid residue having a similar chemical structure, has little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are common in the art, e.g., conservative amino acid substitutions are those in which one or a few amino acids in the following groups (a) - (d) are substituted for another or a few amino acids in the same group: (a) a polar negatively charged residue and an uncharged amide thereof: asp, asn, glu, gln; (b) a polar positively charged residue: his, arg, lys; (c) aromatic residues: phe, trp, tyr; (d) aliphatic nonpolar or low polar residues: ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are as follows: asp is substituted with Glu; asn is substituted with Gln or His; glu is substituted with Asp; gln is substituted with Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys is substituted by Arg, gln; phe is replaced by Met, leu, tyr; trp is substituted with Tyr; tyr is substituted with Phe, trp; substitution of Ala with Gly or Ser; ser is substituted by Thr; thr is replaced by Ser; substitution of Gly with Ala or Pro; met is substituted with Leu, tyr or Ile; leu is substituted with Ile or Val; lie is substituted with Leu or Val; val is substituted with Ile or Leu; cys is replaced by Ser. In addition, those skilled in the art will recognize that the creativity of single domain antibodies is represented in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable, and that the sequences of FR1-4 may take the form of conservative sequence variants of the sequences disclosed herein.

In the present invention, the Fc fusion antibody refers to a novel recombinant protein produced by fusing a functional protein molecule having biological activity with an Fc fragment using a genetic engineering technique or the like, which not only retains the activity of the functional protein molecule, but also has some antibody properties such as prolonged half-life by binding to a related Fc receptor, induction of antibody-dependent cell-mediated cytotoxicity effects, and the like. Wherein, the Fc fragment refers to a polypeptide composed of CH2 and CH3 domains of IgA, igD and gG, or composed of CH2, CH3 and CH4 domains of IgE and IgM via a hinge region.

In the present invention, a humanized antibody refers to an antibody obtained by fusing a heavy chain variable region of a target antibody (e.g., an animal antibody) to a constant region of a human antibody, or an antibody obtained by grafting a complementarity determining region (CDR 1-3 sequence) of a target antibody into a variable region of a human antibody, or an antibody obtained by mutating an amino acid of a target antibody according to the characteristics of a human antibody framework region (FR 1-4). Humanized antibodies can be synthesized or site-directed mutagenesis.

In the present invention, recombinant protein refers to a protein obtained by applying recombinant DNA or recombinant RNA technology. Recombinant protein engineering uses gene cloning or chemical synthesis technology to obtain target Gene (GOI), connects to proper expression vector, introduces into specific host cell, and uses host cell genetic system to express functional protein molecule. The recombinant protein reagent can be applied to the research and development and the production of biological drugs, cell immunotherapy and diagnostic reagents. The recombinant protein medicine is an important component of biological medicine, and can be applied to the medical field, including cytokines, antibody therapeutic vaccines, hormones, enzymes and the like.

Eukaryotic cells in the present invention refer to cells that contain a eukaryotic cell (a nucleus surrounded by a nuclear membrane). The chromosome number is more than one, and mitosis can be performed. It is also possible to perform both the protoplasm flow and the deformation movement. Photosynthesis and oxidative phosphorylation are performed by chloroplasts and mitochondria, respectively. All animal cells and plant cells, except for bacterial and cyanobacteria cells, are eukaryotic cells.

In the present invention, a prokaryotic cell is a cell constituting a prokaryote. The main characteristics of the cells are that the cells are not provided with nuclei which are bounded by nuclear membranes, are not provided with nucleoli, and are provided with pseudonuclei. The evolutionary position is low. The organelle is ribosomal only, has a cell membrane, and has a composition different from that of eukaryotic cells. The cells are smaller, have no shaped nuclei, have no chromosomes, and the DNA does not bind to proteins.

In the present invention, expression vectors refer to a type of vector in which some expression elements (such as promoters, terminators, etc.) are added to the basic skeleton of a cloning vector, so that the inserted exogenous DNA (target gene) can be replicated, transcribed and translated in a host cell.

In the present invention, the host cell is also called a recipient cell. The receptor cells include prokaryotic receptor cells (most notably E.coli), eukaryotic receptor cells (most notably yeast), animal cells and insect cells (in fact eukaryotic receptor cells as well). Among prokaryotic receptor cells, the most commonly used host cell is E.coli.

In the invention, pharmaceutically acceptable auxiliary materials refer to excipients and additives used in the production of medicines and the preparation of prescriptions; are substances which, apart from the active ingredient, have been reasonably evaluated in terms of safety and are contained in pharmaceutical preparations. The pharmaceutical excipients not only form, serve as carriers and improve stability, but also have important functions of solubilization, dissolution assistance, sustained and controlled release and the like, and are important components which can influence the quality, safety and effectiveness of the medicine.

In one aspect, the invention provides an antibody comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO.1-3 and/or sequences with at least 80% sequence homology with SEQ ID NO. 1-3.

Preferably, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected from the group consisting of SEQ ID NO.1-3 and/or sequences having at least 85% sequence homology with SEQ ID NO. 1-3.

Further preferably, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected from the group consisting of SEQ ID NO.1-3 and/or sequences having at least 90% sequence homology with SEQ ID NO. 1-3.

Still further preferably, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected from the group consisting of SEQ ID NO.1-3 and/or sequences having at least 95% sequence homology with SEQ ID NO. 1-3.

Still further preferably, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected from the group consisting of SEQ ID NO.1-3 and/or sequences having at least 98% sequence homology with SEQ ID NO. 1-3.

Specifically, the amino acid sequence comprises: an amino acid sequence obtainable by one or more of addition, deletion, modification or substitution on the amino acid sequence shown in SEQ ID NO. 1-3.

More specifically, the amino acid sequence comprises an amino acid sequence having a difference of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids compared to the amino acid sequence shown in SEQ ID NO. 1-3.

In yet another aspect, the invention provides an antibody comprising framework regions FR1, FR2, FR3 and FR4; the amino acid sequences of FR1, FR2, FR3 and FR4 are selected from SEQ ID NO.4-7 and/or sequences having at least 80% sequence homology with SEQ ID NO. 4-7.

Specifically, the amino acid sequence comprises: an amino acid sequence obtainable by one or more of addition, deletion, modification or substitution on the amino acid sequence shown in SEQ ID NO. 4-7.

Further specifically, the amino acid sequence comprises: amino acid sequences having a difference of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 amino acids compared to the amino acid sequence shown in SEQ ID No. 4-7.

Specifically, the aforementioned antibodies further comprise a hinge region and a CH region.

More specifically, the amino acid sequence of the hinge region is shown in SEQ ID NO. 10.

More specifically, the amino acid sequence of the CH region is shown as SEQ ID NO. 11.

In yet another aspect, the invention provides an antibody comprising the amino acid sequence as described above.

In yet another aspect, the invention provides an antibody whose amino acid sequence comprises:

FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO.1-3 or functionally active variants of amino acid sequences having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid differences compared to the amino acid sequences shown in SEQ ID NO. 1-3; the amino acid sequences of FR1, FR2, FR3 and FR4 are selected from SEQ ID NO.4-7 or functionally active variants having 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with SEQ ID NO. 4-7.

Specifically, the amino acid sequence of the HCDR1 is shown as SEQ ID NO.1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.2, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3.

Specifically, the amino acid sequence of the FR1 is shown as SEQ ID NO.4, the amino acid sequence of the FR2 is shown as SEQ ID NO.5, the amino acid sequence of the FR3 is shown as SEQ ID NO.6, and the amino acid sequence of the FR4 is shown as SEQ ID NO. 7.

Preferably, the amino acid sequence of the antibody is shown in SEQ ID NO. 8.

Specifically, the antibody is a single domain antibody.

Further specifically, the antibody is an anti-IL-17A antibody.

In particular, the aforementioned antibodies comprise part or all of an antibody heavy chain framework region selected from human, murine, primate or camelid sources or variants 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.

In yet another aspect, the invention provides a polyclonal antibody comprising the aforementioned antibodies.

In yet another aspect, the invention provides a recombinant protein comprising the aforementioned antibody.

Specifically, the recombinant protein also comprises a bioactive protein or a functional fragment thereof which assists in the expression and/or secretion of the recombinant protein or prolongs the half-life of the recombinant protein in vivo;

Preferably, the biologically active protein 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 or SUMO tag.

In particular, the biologically active protein or functional fragment thereof is a human immunoglobulin Fc domain, preferably an Fc domain of human IgG, such as an Fc domain of human IgG1, igG2, igG3, igG4, more preferably an Fc domain of human IgG 1.

In yet another aspect, the invention provides an antibody preparation comprising an antibody as described above.

Specifically, the antibody preparation further comprises a pharmaceutically acceptable carrier.

Further specifically, the pharmaceutically acceptable carrier includes, but is not limited to: buffers, sterile water or surfactants.

In yet another aspect, the invention provides a kit comprising an antibody or polyclonal antibody or recombinant protein or antibody preparation as described above.

In yet another aspect, the present invention provides an antibody drug conjugate comprising: (1) the aforementioned antibody or polyclonal antibody or recombinant protein; and, (2) a coupling moiety bound to (1).

In yet another aspect, the invention provides an isolated nucleic acid molecule, wherein said nucleic acid molecule encodes an antibody or polyclonal antibody or recombinant protein as described above.

In yet another aspect, the invention provides an expression vector comprising the nucleic acid molecule as described above.

Specifically, the expression vector can be a plasmid, phage or virus.

In yet another aspect, the invention provides a host cell whose genome integrates the nucleic acid molecule described above; or an expression vector comprising the foregoing.

Specifically, the host cell is a eukaryotic cell or a prokaryotic cell.

Preferably, the eukaryotic cell is selected from the group consisting of: pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces and/or trichoderma;

the prokaryotic cell is selected from the group consisting of: coli, bacillus subtilis, lactobacillus, streptomyces and/or proteus mirabilis.

In yet another aspect, the invention provides a pharmaceutical composition comprising an antibody or polyclonal antibody or recombinant protein or antibody preparation or antibody drug conjugate or nucleic acid molecule or expression vector or host cell as described above; preferably, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

Specifically, the pharmaceutically acceptable auxiliary materials are selected from polysorbate, histidine, sucrose, arginine, sodium chloride, methionine, acetate, trehalose, proline, sorbitol, sodium phosphate, poloxamer 188, ethylenediamine tetraacetic acid, citric acid, mannitol, glutamate, glycine, sodium citrate, sodium succinate and/or lactic acid.

Specifically, the pharmaceutical dosage forms include, but are not limited to: liquid solution only, lyophilized powder, prefilled syringe only, prefilled syringe, lyophilized powder, tablet, capsule, granule, spray.

In particular, the routes of administration include, but are not limited to: intraocular injection, intravenous injection, intramuscular injection, subcutaneous injection, nasal inhalation, and oral administration.

In a further aspect, the invention provides the use of an antibody or polyclonal antibody or recombinant protein or antibody preparation or antibody drug conjugate or nucleic acid molecule or expression vector or host cell or pharmaceutical composition as described above, said use being selected from at least one of the following:

i) preparing a detection reagent or a kit;

ii) preparing a medicament for preventing and/or treating autoimmune diseases;

iii) preparing a medicament for preventing and/or treating cancer.

Specifically, the kit further includes, but is not limited to: solid phase carriers, detection labels, detection substrates and/or buffers.

Further specifically, the solid support may be a material having affinity that immobilizes the specific antibody on the surface.

The detection label may be an enzyme label, which is an enzyme capable of binding to an antibody, for detecting binding of the antibody to an antigen.

The detection substrate may be a reaction product of an enzyme label capable of producing a measurable signal under enzymatic catalysis.

In particular, the autoimmune diseases include, but are not limited to: behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic scleroderma, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, perinodular arteritis, aortitis syndrome, 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, cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, igG 4-related diseases, guillain-Barre syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, inflammatory bowel disease 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, barcup's disease, hashimoto's 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, pregnancy herpes, linear IgA bullous dermatoses, acquired bullous epidermolysis, alopecia areata, leukoplakia, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, former disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplantation immunity, familial mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis or inclusion body myositis.

In particular, the cancers include, but are not limited to: basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, breast carcinoma, peritoneal carcinoma, cervical cancer, cholangiocarcinoma, choriocarcinoma, colorectal cancer, connective tissue carcinoma, digestive system cancer, endometrial carcinoma, esophageal carcinoma, eye cancer, head and neck cancer, gastric cancer, glioblastoma, liver cancer, renal carcinoma, laryngeal carcinoma, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland carcinoma, 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, chronic myeloblastic leukemia.

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:

contacting the aforementioned antibodies or polyclonal antibodies or recombinant proteins or antibody preparations or antibody drug conjugates with a sample to be tested;

II) detecting antigen-antibody complexes;

III) interpreting the result.

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 the aforementioned antibody or polyclonal antibody or recombinant protein or antibody preparation or antibody drug conjugate or nucleic acid molecule or expression vector or host cell or pharmaceutical composition.

In particular, the method may also be used to administer other agents for the treatment of autoimmune diseases; such other drugs for the treatment of autoimmune diseases include, but are not limited to: immunosuppressants, glucocorticoids and/or immunomodulators.

Further specifically, the immunosuppressant is a medicament which can regulate the immune system, strengthen the tolerance of the immune system to autoantigens, and thus inhibit the development of autoimmune diseases;

the glucocorticoid can inhibit inflammatory reaction, regulate immune system and relieve symptoms;

the immunomodulator can regulate the function of the immune system and inhibit the development of autoimmune diseases.

Preferably, the immunosuppressant includes, but is not limited to: cyclosporine capsules or azathioprine tablets;

The glucocorticoids include, but are not limited to: dexamethasone acetate tablet or prednisone acetate;

such immunomodulators include, but are not limited to: tripterygium glycosides tablet or compound cyclophosphamide tablet.

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 the aforementioned antibody or polyclonal antibody or recombinant protein or antibody preparation or antibody drug conjugate or nucleic acid molecule or expression vector or host cell or pharmaceutical composition.

In particular, the methods described may also be used to administer other agents for treating cancer simultaneously.

The invention has the technical effects that:

(1) The affinity was good, and the EC50 = 5.277ug/mL for single domain antibodies 1-C3, all with higher binding capacity than the positive antibody (Ixekizumab).

(2) The blocking effect is good, and the single-domain antibody 1-C3 can block the Human IL-17A protein from activating 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc.

(3) The stability of the single domain antibody 1-C3 is stronger than that of the positive antibody (Ixekizumab).

Drawings

FIG. 1 shows the results of SDS-PAGE detection of IL17A recombinant protein Reduced, wherein lane 1 is before and lane 2 is after purification.

FIG. 2 shows the results of an IL-17A protein activation NIH-3T3 cell assay.

FIG. 3 shows the results of experiments for binding IL17A to reporter cell lines.

FIG. 4 shows the results of IL17A activating cell experiments.

FIG. 5 shows the gel electrophoresis patterns of the VHH fragment PCR products, and the gel electrophoresis patterns of the first round, the second round and the third round of PCR products are sequentially shown from left to right.

FIG. 6 shows the results of a flow assay for alpaca library # 2.

FIG. 7 shows the results of a 2++ 3# alpaca pool flow assay.

FIG. 8 shows FACS detection of IL17A target monoclonal binding to target in alpaca # 2.

FIG. 9 shows FACS detection of IL17A target monoclonal binding to target in alpaca # 3.

FIG. 10 is a diagram showing SDS-PAGE results of single domain antibodies 1 to C3.

FIG. 11 is a graph showing the results of ELISA detection of the binding of recombinant antibodies to target proteins.

FIG. 12 is a graph showing the results of blocking function experiments.

FIG. 13 shows the stability results of single domain antibodies 1-C3.

FIG. 14 shows the stability of the positive control Ixekizumab.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

The main instrument of the invention:

an electrotransport device (Eppendorf Multiporator);

centrifuge (Thermo FRESCO-17);

constant temperature incubator (Shanghai extract macro DNP-9052);

constant temperature shake incubator (refined Qi CO-O6U);

ultra clean bench (Sujing Antai SW-CJ-1 FD);

PCR instrument (Applied Biosystems ABI 2720);

biosafety cabinet (halr, HR40-IIA 2);

a flow cytometer (Thermo Attune Nxt flow cytometer);

Thermo 3111CO ₂ an incubator;

ForteBio OCTET R2。

the main reagent of the invention:

SfiI(NEB,CAT#:R0123L)；

T4 DNA ligase(TaKaRa,CAT#:2011A)；

PrimeScript ^TM II 1st Strand cDNA Synthesis Kit(TaKaRa,CAT#:6210B)；

NuHi power mix (new sea creature, cat#: NH 9303);

3M sodium acetate (pH 5.2-6) (Sigma, CAT#: 126-96-5);

DNA fragment recovery kit (TakaRa, CAT#: 9761);

glue recovery kit (Qiagen, CAT#: 28706);

the Tiangen plasmid large drawing kit (Tiangen, CAT#: DP 117);

HRP-Anti-M13(iCarTab)；

PE-anti-Human IgG(eBioscience,Cat#:12-4998-82)；

PE-Streptavidin(Biolegend,405204)；

Rabbit anti-Llama IgG(H+L)Secondary Antibody[HRP](Novus，CAT#NBP1-75095)；

SS320 competence (iCarTab);

BL21 competence (Biomed, BC 201-02);

pComF phage display vector (iCarTab);

NHS-biotin(APExBIO,CAT#:A8002)；

HRP-Streptavidin(Boster,CAT#:BA1088)；

HRP-ProteinA(Boster，BA1080)；

ProA Biosensors(Sartorius,CAT#:18-5010)；

PBS(Gbico,CAT#14190-250)；

DMEM(Gbico,CAT#41965-062)；

RPMI1640(Gbico,CAT#61870044)；

FBS(VivaCell,CAT#C04001-500)；

Genomic DNA Purification Kit(Lifetech，CAT#K0512)；

Mouse-IL-17A-His(ACRO，CT8-M5240)；

Bright-Lite Luciferase Assay System(Vazyme,CAT#DD1204-01)；

NHS-biotin(APExBIO,CAT#:A8002)。

the primers used for screening, cloning VHH fragments and constructing nanobodies in the invention 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 antigen

(a) Adding a 6xHis tag to the C end of an IL-17 antigen (SEQ ID NO. 6), performing gene synthesis according to prokaryotic codon optimization, and subcloning the gene into a pET28a vector; after being verified by Sanger sequencing, the plasmid is extracted;

(b) Transforming the recombinant plasmid into BL21 competent, inducing overnight with 0.5mM IPTG, and collecting bacterial liquid for cleavage; purifying the recombinant protein using a nickel column;

(c) Purity of the target protein was checked by SDS-PAGE.

As shown in FIG. 1, the IL-17A antigen protein is purified to a purity of more than 90%.

1.2 preparation of positive control antibody Ixekizumab

1. Synthesizing Ixekizumab heavy chain variable region and light chain variable region (the sequence of the heavy chain variable region is shown as SEQ ID NO. 12; the sequence of the light chain variable region is shown as SEQ ID NO. 13), subcloning the heavy chain variable region gene into pcDNA3.4-hIgG4 (the amino acid sequence of the IgG4 constant region is shown as SEQ ID NO. 18) vector, subcloning the light chain variable region into pcDNA3.4-hIgKc (the amino acid sequence of hIgKc is shown as SEQ ID NO. 14) vector; after verification by Sanger sequencing, the plasmid megapump kit is used for preparing the endotoxin-removing plasmid for standby.

2. 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.

3. 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.

4. 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 Protein A.

SDS-PAGE detects purity of target antibody protein, purity >95%;

SEQ ID NO.12：

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEW MGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARY DYFTGTGVYWGQGTLVTVSS；

SEQ ID NO.13：

DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQ LLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFG QGTKLEIK；

SEQ ID NO.14：

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC；

SEQ ID NO.18：

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

1.3 detection of ELISA binding Activity of recombinant Human IL-17A protein (antigen) and control antibody

(1) The IL-17A recombinant protein (IL-17A antigen protein prepared in step (1)) was diluted with sterile CBS to a final concentration of 2. Mu.g/mL. Taking a new 96-well ELISA plate, adding 100 mu L/well recombinant protein diluent, and coating at 4 ℃ overnight;

(2) The antigen coating was removed and washed 3 times with PBST (0.5% tween 20);

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

(4) After removal of the blocking buffer, the well plate was washed 3 times with PBST;

(5) Positive control antibody Ixekizumab was diluted to 10 μg/mL with PBS, 5-fold gradient diluted to 7 spots, added to the elisa plate at 100 μl/well, incubated for 1 hour at room temperature with PBS as control well;

(6) Remove the liquid in the wells and wash 3 times with PBST;

(7) Adding secondary antibody HRP-ProteinA 1:10000 for dilution, adding into an ELISA plate according to 100 mu L/hole, and incubating for 1 hour at room temperature;

(8) After removing the liquid from the wells, the well plate was washed 3 times with PBST;

(9) Adding 100 mu L/hole TMB color development liquid;

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

(11) Add 50. Mu.L/Kong Zhongzhi solution (2M HCl);

(12) OD450 values within wells were read using a microplate reader.

In the method, 2 mug/mL of IL-17A recombinant protein is used for coating an ELISA plate, a positive control antibody Ixekizumab, HRP-ProteinA secondary antibody diluted in a 0.156 mug/mL-to-mL ratio and TMB chromogenic liquid are added into the ELISA plate for incubation, the binding capacity of the IL-17A recombinant protein and the positive antibody is detected, and the detection result of OD450 values is shown in table 1: the positive antibody is well combined with IL-17A antigen protein, and can be used for subsequent immunization.

TABLE 1

1.4Human IL-17A recombinant protein Activity detection

The detection was performed using an IL-17A protein activated NIH-3T3 cell assay.

(1) Resuscitating NIH-3T3 cells from liquid nitrogen, serial subculturing to allow the cells to be in logarithmic growth phase, counting cells, and then culturing according to 2×10 ⁵ Cell mass/well to 96-well plates;

(2) IL-17A (control, ACRO, cat#ILA-H5118) and recombinant IL-17A protein prepared at different concentrations of 100. Mu.L were added to each well at final concentrations of 0,0.00001,0.0001,0.001,0.01,0.1,1, 10. Mu.g/mL, respectively;

(3)37℃，5％ CO ₂ After culturing for 48 hours, the 96-well plate is gently taken out, culture medium supernatant is collected by centrifugation, and secretion of IL6 is detected by using a mouse IL6 ELISA kit;

(4) Data were processed using PRISM GraphPad, plotted, and EC50 values calculated.

NIH-3T3 cells were treated with recombinant IL-17A (TEST) and purchased IL-17A (Acro), respectively, and supernatants were collected 72 hours later to detect secretion of mIL-6, and the results are shown in FIG. 2; according to the detection result, the IL-17A antigen protein has the activity of activating NIH-3T3 cells to express mIL-6, and the IL-17A (TEST, hereinafter referred to as IL-17A recombinant protein) activity is positively related to the concentration thereof, so that the protein can be used for immunization.

1.5 construction and verification of IL-17A reporter Gene cell Strain

Constructing a lentiviral expression vector according to amino acid sequence information of IL-17RA (UniProtKB: Q96F 46) and IL-17RC (UniProtKB: Q8NAC 3), packaging lentivirus, co-infecting 293 cells, screening recombinant 293 cells which simultaneously overexpress the two receptors, further stably transforming NFKB-luciferases (the amino acid sequence of which is shown as SEQ ID NO.15, the nucleotide sequence of which is coded as SEQ ID NO. 16) and ACT1 genes (SEQ ID NO. 17), and constructing IL-17A reporter gene cell lines

293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc。

And adding IL-17A protein for activation, adding a positive control antibody Ixekizumab for detection of a blocking experiment, calculating an EC50 value of the blocking experiment, and establishing a candidate antibody in vitro pharmacodynamics evaluation cell strain targeting IL-17A. FACS results found: the constructed IL-17A receptor over-expression cell strain can be combined with IL-17A, and the positive rate is more than 90%. See fig. 3.

The results of activating 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc with IL-17A recombinant protein are shown in FIG. 4: the IL17A recombinant protein can effectively activate luciferase expression in 293F-IL17Ra/IL17 Rc-NFkB-Luc reporter gene cell strain.

The positive control antibody Ixekizumab and the IL-17A recombinant protein are added into 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc cells together, and the positive control antibody Ixekizumab can inhibit the combination of IL17A protein and a membrane receptor thereof and inhibit the signal of intracellular NFkB and present a dose effect.

SEQ ID NO.15：

MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHS GDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV；

SEQ ID NO.16：

atggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtg；

SEQ ID NO.17：

atgccacctcagttgcaggaaactcggatgaatagaagcatccccgtggaagtggacgagagcgagccgtaccctagtcagctgctgaagccgatccctgagtactccccggaagaggaatccgaaccaccagcccccaacattcgcaatatggcccccaatagcttgtccgcaccaacaatgctgcacaactcttctggcgacttctctcaggcccactccaccctgaaactggcgaatcaccagcggcctgtatcccggcaggtgacctgtctgagaactcaggtgcttgaagactccgaggactctttctgtaggc

ggcatccaggtttgggcaaggcgtttccgtccggctgttccgcggtttcagagcccgcttccgaaagtgtcgtgggcgccc

tgccagccgagcaccagttctccttcatggaaaagcggaaccagtggctggtcagtcagctgagcgccgcgtcacctgat

acaggtcacgattccgacaagtctgaccagtctctgcccaatgcgtcagccgatagtctcgggggctcccaggagatggt

gcagagaccacagccgcacagaaaccgggccgggcttgatctgcccaccattgatacaggctacgattcccagccccag

gacgtccttggcattcgccagctggaaaggcctctgcccttgacctccgtgtgttacccccaggacctgccccgccctttga

gaagccgggagtttccccagtttgagccccaacgataccctgcctgtgctcagatgctgcctccgaacctgagcccacacg

ctccctggaactaccactatcactgtcccggcagccccgatcaccaggtgccttatggacacgactacccgcgggctgcat

accagcaggtcatacagcctgccttgccgggtcagccgctgcccggagcttctgtgcgcggcctgcaccccgttcagaaa

gtgatcctgaactatccaagcccatgggaccatgaagagagaccagcccaaagagattgctcttttcctgggttgcctagac

accaagaccagcctcaccaccagcctcccaatcgggcaggcgccccaggcgaaagtctcgagtgccccgccgaactca

gaccacaggtccctcagcccccttcccccgcggcagtacccagacccccctctaacccacccgcccggggaacgctcaa

gacttcaaatctcccagaagagctgcgcaaagtgttcataacctacagcatggacaccgctatggaggtggttaagttcgtc

aacttcctgctggtcaatgggttccagactgcaatcgacatttttgaggatagaattcggggaatcgacatcatcaagtggat

ggagagatacctgcgggataagacagtgatgattatcgtggccattagtcccaagtacaagcaagatgtggagggcgcag

aatcacagttggacgaagacgagcacggactccatacaaaatatatccacaggatgatgcagatcgagttcattaaacaag

gctccatgaatttccgcttcataccggtcctgtttccaaacgcaaaaaaagagcatgtacccacttggctccagaatacccat

gtctactcctggcccaagaacaagaagaatatcctgctgcgcttgctcagagaagaagagtatgtcgcccctccaagggggcccctccccacactccaagtagtgccactt。

1.6 animal immunization Process

1.6.1 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.

1.6.2 immunotiter detection

The experimental procedure was as follows:

(1) Collecting 5mL of peripheral blood, placing a centrifuge tube with a blood sample collected in a 37 ℃ incubator for 1 hour; the blood samples were then transferred to 4 ℃ overnight.

(2) Placing the centrifuge tube with the collected blood sample in a centrifuge, and centrifuging at 5000rpm for 20min; the upper serum was separated, transferred to a new sterile centrifuge tube, and the immune serum was collected.

(3) The IL-17A recombinant protein was diluted to a final concentration of 1. Mu.g/mL using sterile CBS (carbonate buffer). A new 96-well ELISA plate was taken and coated overnight at 4℃with 100. Mu.L/well.

(4) The antigen coating was removed and washed 5 times with PBST (0.05% tween 20).

(5) 3% MPBS was added at 200. Mu.L/well and blocked at 37℃for 2 hours.

(6) After removal of the blocking buffer, the well plate was washed 5 times with PBST.

(7) 100. Mu.L of gradient diluted serum (100. Mu.L/well) was added and incubated for 1 hour at room temperature, with PBS as control well.

(8) The liquid in the wells was removed and washed 5 times with PBST.

(9) mu.L of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) was added and incubated for 1 hour at room temperature.

(10) After removing the liquid from the wells, the well plate was washed 5 times with PBST.

(11) 100. Mu.L/well TMB color development solution was added.

(12) Incubate at room temperature for 10-15 min in the dark.

(13) 50. Mu.L/Kong Zhongzhi of the solution was added.

(14) OD450 values within wells were read using a microplate reader.

The immune titers of alpaca after 6 rounds of immunization all meet the requirements (see table 1).

TABLE 1 results of immunotiter assays

1.7 antibody Yeast library construction procedure

1.7.1PBMC isolation and VHH antibody fragment cloning

The experimental procedure was as follows:

1. 100mL of peripheral blood anticoagulation sample was collected, and PBMC cells were isolated using lymphocyte separation fluid.

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

The following reaction mixtures (Table 2) were prepared in 200. Mu.L-sized PCR tubes:

TABLE 2

(II) after 5min of heat preservation at 65 ℃, the mixture is rapidly cooled on ice.

(III) the following reaction solutions (Table 3) were prepared in the PCR tubes:

TABLE 3 Table 3

(IV) after blowing and mixing uniformly, split charging 80 mu L/tube, placing in a PCR instrument at 42 ℃ for 1 hour, heat-inactivating at 70 ℃ for 15 minutes, and finally placing cDNA sample on ice or storing at-20 ℃ for a long time.

3. Amplifying the VHH fragment by PCR;

(1) The first round PCR reaction system (50. Mu.L/tube) was configured as shown in Table 4:

TABLE 4 Table 4

After the PCR reaction system was configured, the PCR instrument was set up according to the following procedure, see table 5:

TABLE 5

(2) Agarose electrophoresis of PCR products

The PCR product was analyzed by electrophoresis using 1% agarose, and a fragment having a molecular weight of about 750bp was isolated. The PCR products were recovered using a gel recovery kit and the concentration was determined using NanoDrop.

(3) Two rounds of PCR reaction (50. Mu.L/tube) were configured as shown in Table 6:

TABLE 6

After the PCR reaction system was configured, the PCR instrument was set up according to the following procedure, see table 7:

(4) Agarose electrophoresis analysis of two rounds of PCR products

The PCR products were analyzed by electrophoresis using 1% agarose, and VHH fragments having a molecular weight of about 400bp were isolated. VHH PCR products were recovered using a gel recovery kit and concentration was determined using NanoDrop.

As a result, as shown in FIG. 5, about 1000bp and about 750bp PCR bands were obtained in the first round of PCR, and a 750bp fragment was recovered as a template for the second round of PCR. The second round of PCR gave a band of about 400bp as VHH fragment and the column was recovered as template for three rounds of PCR. The third round of PCR obtains a band of about 500bp, adds a homology arm, and then carries out homologous recombination into a yeast display vector pDISPLAY.

1.7.2 construction of Single-Domain antibody Yeast display libraries

1. The yeast display vector pDisplay was linearized and the cleavage system is shown in table 8:

TABLE 8

(1) Cutting a pDISPLAY vector by using SfiI, subpackaging with 100 mu L/tube, and cutting overnight at 50 ℃;

(2) pDISPLAY vector fragments were separated using 1% agarose gel, 5000bp vector fragments were excised for gel recovery and concentration was determined using NanoDrop;

(3) The recovered pDISPLAY enzyme-digested products were sub-packed in 200. Mu.L of 1.5mL centrifuge tubes, 1/10 volume (20. Mu.L) of 3M sodium acetate was added, 1. Mu.g/. Mu.L Glycogen (Glycogen) was blown-in and mixed well, 880. Mu.L of absolute ethanol was added, and the mixture was inverted and mixed well at-80 ℃.

2. Construction of Yeast display library by electric transformation

(1) Streaking the frozen yeast competent strain at-80 ℃ onto YPD solid culture medium plates, and activating at 30 ℃ for 3-5 days;

(2) Inoculating single colony yeast to 50mL YPD culture medium, shaking and culturing at 30deg.C at 250rpm for 1-2 days;

(3) Yeast competent strains were prepared. Mixing the linearized carrier segment and the PCR product, and then adding the mixture into a electric shock cup for electric shock; shake-culturing in a yeast competent transfection culture flask at 220rpm and 30deg.C for 1 hr;

(4) Taking 20 mu L of heavy suspension, diluting with SDCAA 5000 times, sucking 100 mu L, coating an SDCAA plate, culturing for 2-3 days, calculating the storage capacity, and continuously culturing the rest bacterial liquid for 24 hours;

(5) And (5) preserving bacteria. The remaining bacterial fluid was collected in a 50mL centrifuge tube, centrifuged for 5min at 3000g, the supernatant discarded, and 10mL of SDCAA was added to resuspend with 50% glycerol: resuspension = 1:1 mix, cryopreserved at-80 ℃.

1.8 Yeast display library panning and screening Process

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. 6-7), 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.

1.9FACS screening procedure

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.

Results the binding of IL17A target monoclonal to the target was detected by FACS as shown in FIGS. 8-9; 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.

1.10 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. 20 monoclonals are randomly selected for sequencing analysis, the sequence difference is large, and the library diversity is good. 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.

According to the result of the monoclonal flow detection of the yeast, positive clones combined with IL-17A-His are selected to extract genome DNA, and the antibody sequence is obtained by PCR. According to the sequencing result of the PCR product, differential clone is selected to carry out overlap PCR amplification, and the specific steps are as follows:

First round PCR: amplification of CMV, VHH and FC

(a) The configuration of the PCR reaction system (50. Mu.L system/reaction) is shown in Table 9.

( CMV fragment amplification primers: yCMV-F and yCMV-R; VHH fragment amplification primers: yVHH-F and yVHH-R; FC fragment amplification primers: yFc-F and yFc-WR )

TABLE 9

The PCR reaction procedure is shown in Table 10:

table 10

(b) mu.L of the PCR product was taken, 1/10 volume of 10×loading buffer was added, electrophoresis analysis was performed using 1% agarose, the CMV band size was about 750bp, the Fc band size was about 1400bp, and the VHH band size was about 500 bp.

(c) The band of interest is excised from the gel and the PCR product purified and the concentration determined by NanoDrop (e.g., too high a concentration, which can be diluted for subsequent reactions).

Second round PCR: overlap Extension PCR CMV, VHH and FC are connected

(a) The configuration PCR reaction system is shown in Table 11:

TABLE 11

The PCR reaction procedure is shown in Table 12:

table 12

2uL of each primer CMV-F and PGK-R are added

The PCR reaction procedure is shown in Table 13:

TABLE 13

(b) The overlay PCR product was purified using a TakaRa DNA fragment recovery kit and the concentration was determined using NanoDrop, requiring at least 10. Mu.g of the PCR product. For subsequent cell transfection verification.

(c) The transfection procedure is identical to the transfection of eukaryotic expression vectors.

Adding a signal peptide to the N end of VHH, adding IgG1-FC to the C end, and transiently transfecting HEK293 cells with the PCR product; ELISA detection was performed on the expressed antibody supernatants: 100uL of transfection supernatant was incubated in a 96-well plate pre-coated with IL-17A recombinant antibody, ELISA detection was performed using HRP-Protein A as the secondary antibody, and the results are shown in Table 14: all clones were able to bind to the IL-17A-His antigen.

TABLE 14 ELISA Binding assay results for candidate clone transfected supernatants

1.11 antibody expression purification Process

(1) According to ELISA detection results of candidate antibodies, selecting positive clones, synthesizing SEQ ID NO.7 by genes, and subcloning the positive clones into an expression vector pcDNA3.4-hIgG1-Fc in series with human IgG1Fc (the amino acid sequence of the IgG1 constant region is shown as SEQ ID NO. 19). After the vector is verified by sequencing, the Qiagen plasmid megapump kit is used for preparing the endotoxin-removing plasmid for standby.

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

SEQ ID NO.19：

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

The procedure for purifying antibodies by Protein A 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: (1) 0.1M glycine, pH 2-3; (2) 0.1M NaAc-HAc, pH 3.6.

Neutralization buffer: 1M Tris, pH 8.5.

Magnetic bead regeneration buffer: 0.1M NaOH.

Results: the SDS-PAGE results of single domain antibodies 1-C3 are shown in FIG. 10, with antibody purity > 90%.

The amino acid sequence of the heavy chain CDR region of the single domain antibody 1-C3 is SEQ ID NO.1-3, the amino acid sequence of the FR region is SEQ ID NO.4-7, the amino acid sequence of the hinge region is SEQ ID NO.10, and the amino acid sequence of the CH region is SEQ ID NO.11; the amino acid sequence of the single domain antibody 1-C3 is SEQ ID NO.8; the nucleic acid sequence of the single domain antibody 1-C3 is SEQ ID NO.9.

SEQ ID NO.1：GEDLGYYA；

SEQ ID NO.2：VTSSGSST；

SEQ ID NO.3：ASTILLCSDYISAFGT；

SEQ ID NO.4：DVQLVESGGGLVEPGESLRLSCAAP；

SEQ ID NO.5：IAWFRQAPGKEREVVSC；

SEQ ID NO.6：NYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYC；

SEQ ID NO.7：WGQGTQVTVAS；

SEQ ID NO.8：

DVQLVESGGGLVEPGESLRLSCAAPGEDLGYYAIAWFRQAPGKEREVVSCVTSSGSSTNYLSSVKDRFTISIDNAKNTVYLQMNSLKPEDTAVYYCASTILLCSDYISAFGTWGQGTQVTVASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；

SEQ ID NO.9：

gatgtgcagctggtggagtctgggggaggcttggtcgagcctggggaatctctgaggctctcctgtgcagcccctggagaggatttgggttattacgccatagcctggttccgccaggccccagggaaggagcgtgaggtagtctcatgtgtcacaagtagtggtagtagcacaaactatttaagttccgtgaaggaccgattcaccatctccatagacaacgccaagaacacggtatatctgcaaatgaacagcctgaaacctgaggacacagccgtttattactgtgcgtccactattctcctctgttcagattatatctctgcctttggcacctggggccaggggacccaggtcaccgtcgcctcggacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcacgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaataa；

SEQ ID NO.10：DKTHTCP；

SEQ ID NO.11：

PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

EXAMPLE 2ELISA detection of the binding procedure of recombinant antibodies to target proteins

2.1 affinity detection methods and results

2.1.1 detection methods

(1) The recombinant protein was diluted using sterile CBS to a final concentration of 2. Mu.g/mL. Taking a new 96-well ELISA plate, adding 100 mu L/well and coating at 4 ℃ overnight;

(2) The antigen coating was removed and washed 5 times with PBST (0.05% tween 20);

(3) 3% MPBS added at 200. Mu.L/well was blocked for 2 hours at 37 ℃;

(4) After removal of the blocking buffer, the well plate was washed 5 times with PBST;

(5) Adding purified single domain antibody, wherein the initial concentration is 10 mug/mL, 7 points (100 mug/hole) are diluted in a 5-time gradient, incubating for 1 hour at room temperature, and the control hole is PBS;

(6) Remove the liquid in the wells and wash 5 times with PBST;

(7) Add 100. Mu.L/well HRP-Protein A antibody (1:50000 dilution), incubate at room temperature for 1 hour;

(8) After removing the liquid from the wells, the well plate was washed 5 times with PBST;

(9) Adding 100 mu L/hole TMB color development liquid;

(10) Incubating for 10-15 minutes at room temperature in a dark place;

(11) Adding 50 mu L/Kong Zhongzhi liquid;

(12) OD450 values within wells were read using a microplate reader.

2.1.2 results

The results showed that the EC 50= 5.277ug/mL of the single domain antibody 1-C3 and the EC 50=10.06 ug/mL of the positive antibody (Lxekizumab) demonstrate that the single domain antibody 1-C3 has better binding activity to the Human IL-17A protein and higher binding capacity than the positive antibody (Ixekizumab) as shown in fig. 11.

Example 3 Single Domain antibody blocking function assay

3.1 Experimental procedure

The antibody to be detected was serially diluted 10 times at a final concentration of 100. Mu.g/mL, 10. Mu.g/mL, 1. Mu.g/mL, 0.1. Mu.g/mL, 0.01. Mu.g/mL, 0.001. Mu.g/mL, 0.0001. Mu.g/mL, 0.00001. Mu.g/mL, 0.000001. Mu.g/mL, 0.0000001. Mu.g/mL, 0. Mu.g/mL, and 50. Mu.L of the diluted antibody was added to the 96-well plate, 2 multiplex wells per gradient. Then 50. Mu.L of IL-17A protein (0.4. Mu.g/mL final concentration) was added to the corresponding wells. After mixing, the mixture was placed in an incubator at 37℃and incubated for 1 hour. 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc (A3) cells cultured to logarithmic growth phase were aspirated into 96-well plates, each well was seeded with 2X 10 ⁴ Individual cells. After 18h of co-cultivation, 20. Mu.L of Bright-GloTM assay reagent was added to each well and the luciferase activity values in the wells were measured using a Tecan M1000pro microplate reader.

3.2 results

The positive control Ixekizumab can block Human IL-17A protein from activating 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc. The single domain antibody 1-C3 can also block the Human IL-17A protein from activating 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc, and the blocking effect of the single domain antibody 1-C3 is stronger than that of a positive control, as shown in figure 12.

Example 4 stability experiment

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 ) 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 Tm value, T _onset And T _agg Higher indicates that the antibody protein is more stable.

5.1 Experimental procedure

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.

5.2 results

The stability results of single domain antibodies 1-C3 and positive control Ixekizumab are shown in FIGS. 13-14, which show T for 1-C3 _m1 At 60.25 ℃, T _m3 82.10 ℃, T _onset 49.09 ℃, T _agg 67.32 ℃; t of positive control Ixekizumab _m1 At 56.10 ℃, T _m2 79.84℃T _onset 47.50 ℃, T _agg 61.86 ℃. The results show that the stability of the single domain antibody 1-C3 is obviously better than that of the positive control Ixekizumab.

Application example 1: single-domain antibody-containing 1-C3 pharmaceutical composition

A pharmaceutical composition comprising: (1) a buffer; and (2) single domain antibodies 1-C3 or antigen binding fragments thereof.

In some embodiments, the single domain antibody 1-C3 or antigen binding fragment thereof is 1-300mg/L; preferably 5-100mg/L; more preferably 5-50mg/L.

In some embodiments, the buffer is selected from at least one of acetate buffer, histidine buffer phosphate buffer, citrate buffer, carbonate buffer, or Tris; preferably, the buffer is histidine buffer; more preferably, the histidine buffer is histidine-histidine hydrochloride buffer.

In some embodiments, the buffer is at a concentration of 3 to 60mM; preferably 5-20mM; the pH of the buffer is 4.0-7.0, preferably 5.0-6.0.

In some embodiments, the pharmaceutical composition further comprises a stabilizer selected from at least one of arginine salt, sodium chloride, mannitol, sorbitol, sucrose, glycine, and trehalose; preferably, the concentration of the stabilizing agent in the pharmaceutical composition is 50-400mM, preferably 100-200mM, more preferably 100-150mM.

In some embodiments, the stabilizer is sucrose, at a concentration of 100-300mM, preferably 150-200mM; or the stabilizer is a combination of sucrose and arginine hydrochloride, wherein the concentration of sucrose is 50-200mM, the concentration of arginine hydrochloride is 10-100mM, preferably, the concentration of sucrose is 100-150mM, and the concentration of arginine hydrochloride is 30-50mM.

In some embodiments, the pharmaceutical composition further comprises a surfactant selected from at least one of polysorbate 80, polysorbate 20, and poloxamer 188.

In some embodiments, the pharmaceutical composition further comprises an additional agent for treating an immune disorder or an anti-tumor agent.

Application example 2: drug conjugate containing single domain antibody 1-C3

The invention provides a preparation method of a drug conjugate containing single domain antibody 1-C3, which comprises the following steps:

(1) Reacting the single domain antibodies 1-C3 or antigen binding fragments thereof with a reducing agent in a buffer;

(2) Reacting the linker-payload with the thiol-bearing antibody or antigen-binding fragment thereof obtained in step (1);

in some embodiments, the amount ratio of reducing agent to substance of the antibody or antigen binding fragment thereof is from 1:1 to 5:1; preferably 1:1 to 4:1; further preferably 1:1 to 3:1; even more preferably 2:1 to 3:1.

In some embodiments, the reducing agent is selected from tris (2-carboxyethyl) phosphine (TCEP) or a salt thereof, dithiothreitol, or 2-mercaptoethanol. In some embodiments the reducing agent is tris (2-carboxyethyl) phosphine or a salt thereof, preferably tris (2-carboxyethyl) phosphine hydrochloride (tcep·hcl).

In some embodiments, the buffer is selected from HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid) buffer, histidine buffer, phosphate buffer, borate buffer, or acetate buffer; preferably histidine buffer; further preferred is histidine-hydrochloric acid buffer; the concentration of the buffer is 1mM-30mM, such as 5mM, 10mM, 15mM, 20mM, 25mM or 30mM, preferably 20mM.

In some embodiments, the ratio of the amount of linker-payload to the amount of substance of the antibody or antigen binding fragment thereof is from 2:1 to 10:1; preferably, the ratio of the amount of linker-payload to the amount of substance of the antibody or antigen binding fragment thereof is from 3:1 to 9:1; further preferred, the ratio of the amount of linker-payload to the amount of substance of the antibody or antigen binding fragment thereof is 4:1 to 7:1; further preferably, the ratio of the amount of linker-payload to the amount of substance of the antibody or antigen binding fragment thereof is from 5:1 to 7:1.

In some embodiments, a solvent for dissolving the linker-payload is also included; the solvent comprises an organic solvent; the organic solvent may be selected from aqueous acetone (e.g., 50% aqueous acetone), aqueous ethanol (e.g., 80% aqueous ethanol), aqueous methanol (e.g., 80% aqueous methanol), aqueous isopropanol (e.g., 80% aqueous isopropanol), aqueous dimethyl sulfoxide (e.g., 80% aqueous dimethyl sulfoxide), acetone, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), or N-methyl-2-pyrrolidone (NMP); preferably an aqueous acetone or DMSO, preferably 50% aqueous acetone.

Application example 3: kit for detecting IL-17A

The kit comprises the following components:

(1) Solid phase carriers (immunoadsorbents) coated with antigen or antibody;

(2) An enzyme-labeled antigen or antibody (conjugate);

(3) A substrate for an enzyme;

(4) Negative and positive controls (in qualitative assays), reference standards and control serum (in quantitative assays);

(5) A conjugate and a dilution of the specimen;

(6) Washing solution, in plate ELISA, common diluent containing 0.05% Tween-20 phosphate buffer saline;

(7) The enzyme reaction stopping solution, commonly used HRP reaction stopping solution is sulfuric acid, the concentration of which varies according to the addition amount and the final volume of the colorimetric solution, and 2mol/L is generally adopted in plate ELISA.

In some embodiments, the coating concentration is 0.3-5 μg/mL; preferably, the coating concentration is 2-3 mug/mL.

In some embodiments, the enzyme is selected from one of horseradish peroxidase, alkaline phosphatase, glucose oxidase, or β -galactosidase; preferably horseradish peroxidase; the substrate is selected from one of o-phenylenediamine, tetramethyl benzidine or aminosalicylic acid; tetramethyl benzidine is preferred.

Claims

1. An antibody comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO.1-3 and/or sequences with at least 80% sequence homology with SEQ ID NO. 1-3.

2. The antibody of claim 1, wherein the amino acid sequence comprises: an amino acid sequence obtainable by one or more of addition, deletion, modification or substitution on the amino acid sequence shown in SEQ ID NO. 1-3.

3. The antibody of claim 2, wherein the amino acid sequence comprises: amino acid sequences having a difference of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids compared to the amino acid sequence shown in SEQ ID NO. 1-3.

4. An antibody comprising framework regions FR1, FR2, FR3 and FR4; the amino acid sequences of FR1, FR2, FR3 and FR4 are selected from SEQ ID NO.4-7 and/or sequences having at least 80% sequence homology with SEQ ID NO. 4-7.

5. The antibody of claim 4, wherein the amino acid sequence comprises: an amino acid sequence obtainable by one or more of addition, deletion, modification or substitution on the amino acid sequence shown in SEQ ID NO. 4-7.

6. The antibody of claim 5, wherein the amino acid sequence comprises: amino acid sequences having a difference of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 amino acids compared to the amino acid sequence shown in SEQ ID No. 4-7.

7. An antibody comprising the amino acid sequence of any one of claims 1-3 and the amino acid sequence of any one of claims 4-6.

8. An antibody, wherein the amino acid sequence of the antibody comprises:

9. The antibody of claim 8, wherein the amino acid sequence of HCDR1 is shown in SEQ ID No.1, the amino acid sequence of HCDR2 is shown in SEQ ID No.2, and the amino acid sequence of HCDR3 is shown in SEQ ID No. 3.

10. The antibody of claim 8 or 9, wherein the amino acid sequence of FR1 is shown in SEQ ID No.4, the amino acid sequence of FR2 is shown in SEQ ID No.5, the amino acid sequence of FR3 is shown in SEQ ID No.6, and the amino acid sequence of FR4 is shown in SEQ ID No. 7.

11. The antibody of claim 10, wherein the amino acid sequence of said antibody is as set forth in SEQ ID No. 8.

12. The antibody of any one of claims 1-11, wherein the antibody is a single domain antibody.

13. The antibody of any one of claims 1-12, wherein the antibody is an anti-IL-17A antibody.

14. An antibody according to any one of claims 1 to 14, comprising part or all of an antibody heavy chain framework region selected from human, murine, primate or camelid origin or a variant thereof;

15. A polyclonal antibody comprising the antibody of claims 1-14.

16. A recombinant protein comprising the antibody of claims 1-14.

17. The recombinant protein according to claim 16, further comprising a biologically active protein or functional fragment thereof that assists in its expression and/or secretion, or that extends its half-life in vivo;

18. Recombinant protein according to claim 17, characterized in that said biologically active protein or functional fragment thereof is a human immunoglobulin Fc domain, preferably an Fc domain of human IgG, such as an Fc domain of human IgG1, igG2, igG3, igG4, more preferably an Fc domain of human IgG 1.

19. An antibody preparation comprising the antibody of claims 1-14.

20. The antibody formulation of claim 19, further comprising a pharmaceutically acceptable carrier.

21. A kit comprising the antibody of any one of claims 1-14 or the polyclonal antibody of claim 15 or the recombinant protein of any one of claims 16-18 or the antibody preparation of any one of claims 19-20.

22. An antibody drug conjugate, comprising: (1) The antibody of any one of claims 1-14 or the polyclonal antibody of claim 15 or the recombinant protein of any one of claims 16-18; and

(2) A coupling moiety that binds to (1).

23. An isolated nucleic acid molecule encoding the antibody of any one of claims 1-14 or the polyclonal antibody of claim 15 or the recombinant protein of any one of claims 16-18.

24. An expression vector comprising the nucleic acid molecule of claim 23.

25. A host cell having the nucleic acid molecule of claim 22 integrated into its genome; or an expression vector according to claim 24.

26. A pharmaceutical composition characterized by: the pharmaceutical combination comprising the antibody of any one of claims 1-14 or the polyclonal antibody of claim 15 or the recombinant protein of any one of claims 16-18 or the antibody preparation of any one of claims 19-20 or the antibody drug conjugate of claim 22 or the nucleic acid molecule of claim 23 or the expression vector of claim 24 or the host cell of claim 25; preferably, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

27. Use of the antibody of any one of claims 1-14 or the polyclonal antibody of claim 15 or the recombinant protein of any one of claims 16-18 or the antibody preparation of any one of claims 19-20 or the antibody drug conjugate of claim 22 or the nucleic acid molecule of claim 23 or the expression vector of claim 24 or the host cell of claim 25 or the pharmaceutical composition of claim 26, selected from at least one of the following:

I) preparing a detection reagent or a kit;

ii) preparing a medicament for preventing and/or treating autoimmune diseases;

iii) preparing a medicament for preventing and/or treating cancer.

28. The use according to claim 27, 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.

29. The use according to claim 27, wherein the cancer comprises basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, 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, kidney 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, cancer of the urinary system, B-cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic myeloblastic leukemia.

30. A method for in vitro detection of IL-17A in a sample for non-diagnostic purposes, the method comprising the steps of:

contacting the antibody of any one of claims 1 to 14 or the polyclonal antibody of claim 15 or the recombinant protein of any one of claims 16 to 18 or the antibody preparation of any one of claims 19 to 20 or the antibody drug conjugate of claim 22 with a sample to be tested;

II) detecting antigen-antibody complexes;

III) interpreting the result.

31. 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 1-14 or the polyclonal antibody of claim 15 or the recombinant protein of any one of claims 16-18 or the antibody preparation of any one of claims 19-20 or the antibody drug conjugate of claim 22 or the nucleic acid molecule of claim 23 or the expression vector of claim 24 or the host cell of claim 25 or the pharmaceutical composition of claim 26.

32. A method of preventing and/or treating cancer, the method comprising: administering to a subject in need thereof a therapeutically effective amount of the antibody of any one of claims 1-14 or the polyclonal antibody of claim 15 or the recombinant protein of any one of claims 16-18 or the antibody preparation of any one of claims 19-20 or the antibody drug conjugate of claim 22 or the nucleic acid molecule of claim 23 or the expression vector of claim 24 or the host cell of claim 25 or the pharmaceutical composition of claim 26.