CN117820481B - Novel antibody molecules and their pharmaceutical uses - Google Patents

Abstract

The invention belongs to the technical field of cell immunology, and particularly relates to a novel antibody molecule and pharmaceutical application thereof. The antibody provided by the invention comprises a first single-domain antibody and a second single-domain antibody, wherein the amino acid sequence of the first single-domain antibody comprises HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO.2 and HCDR3 shown as SEQ ID NO. 3, and the amino acid sequence of the second single-domain antibody comprises HCDR4 shown as SEQ ID NO. 4, HCDR5 shown as SEQ ID NO. 5 and HCDR6 shown as SEQ ID NO. 6. The antibody of the invention has strong binding capacity, strong blocking capacity and strong stability.

Description

Novel antibody molecules and pharmaceutical uses thereof

Technical Field

The invention belongs to the technical field of cell immunology, and particularly relates to a novel antibody molecule and pharmaceutical application thereof.

Background

The IL-17 cytokine family includes IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, while the IL-17receptor (IL-17 receptor) family includes IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE. Among them, IL-17A and its receptor IL-17RA have been most studied, and IL-17A is a pro-inflammatory cytokine involved in the development of certain autoimmune diseases and verifying the occurrence and progress of the related diseases. IL-17A can promote secretion of inflammatory factors such as IL-8, IL-6, tumor necrosis chair (TNF) -alpha and the like, promote generation of endothelial cell adhesion molecules, and cause obvious inflammatory response. Helper T cell 17 (Th 17) is a T cell positive for the surface antigen cluster 4 (cd4+) that is capable of producing IL-17, and is involved in the development of autoimmune diseases primarily by secreting Interleukin (IL) -17A.

Monoclonal antibodies are produced by B cells, which exist in the form of receptors on the cell surface and bind to and react specifically with only one epitope, so that the same lymphoid system activated by the reaction can only produce immunoglobulins directed against this epitope. Monoclonal antibodies consist essentially of an antigen binding domain (Fab) and a crystallizable region (Fc). Wherein Fab can regulate and control related signal pathways after tumor antigen recognition, and Fc segment and cell expressing Fc receptor after related action mediate antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated phagocytosis, complement-dependent cytotoxicity, etc. are equivalent to participate in anti-tumor immune response. Neutralizing monoclonal antibodies directed against IL-17 have higher specificity and selectivity and fewer side effects and toxicity in the treatment of autoimmune diseases than immunosuppressants or anti-inflammatory drugs.

Chinese patent CN109745559a discloses a liquid formulation of an anti-human IL-17A monoclonal antibody, which provided by the invention comprises an anti-human IL-17A monoclonal antibody, a surfactant, a sugar and an amino acid, which is stable during manufacture, storage and administration, has low aggregates and is suitable for administration in high concentrations (e.g. for subcutaneous administration). The stable liquid preparation of the anti-human IL-17A monoclonal antibody can be effectively applied to the preparation of medicines for treating immune-mediated inflammatory reactions.

Chinese patent CN110582512a discloses a monoclonal antibody against both IL-17A and IL-17F and uses thereof, which binds to both IL-17A and IL-17F and inhibits the activity of both IL-17A and IL-17F, wherein the antibody comprises CDR1H, CDR2H, CDR H, and CDR1L, CDR2L and CDR3L. The invention also provides the use of the antibodies in the manufacture of a medicament for treating a disease associated with IL-17A and/or IL-17F in a subject, and pharmaceutical compositions comprising the antibodies.

At present, monoclonal antibodies are still more applied to the treatment of immune diseases, but the monoclonal antibodies have certain toxicity and side effects which can affect the physical health of a subject, and the monoclonal antibodies are relatively complex to prepare and relatively high in cost due to the structure.

Unlike conventional monoclonal antibodies, single domain antibodies (sdabs) have no light chain, are small in size, oval in shape, and have a relative molecular mass of about 15kDa, and contain 4 framework regions that form an immunoglobulin core structure and 3 complementarity determining regions that bind antigen. The N-terminal of the CDR1 region of the single domain antibody is very variable, the CDR3 region is longer, and the single domain antibody contains 16 amino acids on average, so that the diversity of VHH is increased. The single-domain antibody can recognize and bind to a special antibody epitope, and can further recognize hidden epitopes wrapped by antigen, so that the area which cannot be reached by the conventional antibody is reached. The single domain antibody has stronger temperature and acid-base tolerance, and has obvious advantages compared with the tolerance capability of the traditional antibody under extreme conditions.

In the prior art, less research is carried out on IL-17A single-domain antibodies, so that the development of a single-domain antibody medicament with high stability, strong affinity and low production cost is a technical problem to be solved at present.

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

Terminology and statement of the invention:

1. As used herein, the term "amino acid" is intended to include natural amino acids, synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to natural amino acids. Natural amino acids are amino acids encoded by the genetic code. Amino acid analogs refer to those compounds that have the same basic chemical structure as a naturally occurring amino acid. Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee (Biochemical Nomenclature Commission).

2. The term "antibody" as used herein refers to a polypeptide or fragment thereof that specifically binds to and recognizes an antigen, including the framework regions of immunoglobulin genes. The use of the term antibody is meant to include whole antibodies and antigen-binding fragments thereof. The term antibody encompasses monospecific antibodies, bispecific antibodies, and multispecific antibodies so long as they exhibit the desired biological activity or function.

3. As used herein, the terms "single domain antibody (VHH)", "single domain antibody" (singledomain antibody, sdAb, or nanobody) have the same meaning, referring to the variable region of a cloned antibody heavy chain, a single domain antibody consisting of only one heavy chain variable region is constructed, which is the smallest antigen-binding fragment with complete function. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant region 1 (CH 1), the variable region of the heavy chain of the antibody is cloned, and a single domain antibody (VHH) consisting of only one heavy chain variable region is constructed.

4. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. An epitope typically comprises a chemically active surface group of a molecule, such as an amino acid or sugar side chain, and typically has specific three-dimensional structural features as well as specific charge features. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which may be a "linear" epitope or a "conformational" epitope.

5. As used herein, the term "amino acid sequence" refers to the order in which amino acids are linked to each other to form a peptide chain (or polypeptide), and the amino acid sequence can only be read in one direction.

6. As used herein, the term "nucleic acid molecule" may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The full-length sequence of the nucleotide molecule or a fragment thereof in the present invention can be generally obtained by a PCR amplification method or an artificial synthesis method.

7. As used herein, the term "nucleotide sequence" refers to the arrangement of bases in DNA or RNA, i.e., A, T, G, C in DNA, or A, U, G, C in mRNA, including rRNA, tRNA, mRNA.

8. As used herein, the term "framework region" is a region of the framework, which varies widely about 110 amino acid sequences near the N-terminus of the H and L chains of an immunoglobulin, and the amino acid sequences of the other parts are relatively constant, whereby the light and heavy chains can be distinguished as variable (V) and constant (C) regions. The variable region comprises the hypervariable region HVR or complementarity determining region CDR and FR framework regions.

9. As used herein, the term "humanized" antibody refers to the antibody in which the variable region (VH or VHH) Fr region portion, the constant region portion (i.e., CH and CL regions) or all of the antibody is encoded by a human antibody gene. Humanized antibodies include chimeric antibodies, diabodies, and fully humanized antibodies.

10. As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin, which is a functional building block consisting of only CH2 and CH3 in the heavy chain constant domain. Fc has no ability to bind antigen, however it has the property of having an extended half-life and has a constant amino acid sequence.

11. As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments of the light and heavy chain variable regions, known as complementarity determining regions or hypervariable regions. Herein, "variable region" is used interchangeably with "complementarity determining region".

12. As used herein, the term "expression vector" is a polynucleotide that is capable of being transcribed and translated into a polypeptide upon introduction into a suitable host cell.

13. 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, natural four-chain antibodies comprise six HVRs or CDRs, three in the VH (H1, H2, H3) and three in the 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 edtion,Public Health Service,National Institutes of Health,Bethesda,MD(1991)). are 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) microglia gene system antibody preparation, map 3) can be defined in the field by a method based on the well-known by a grafting tool, such as the definition of the domain of human antibody based on the mutation domain, the mutation rules of the human domain, such as the region of the antibody of the human fusion domain, the antibody of interest based on the mutation domain of the reference domain: 927-48, 1997) it will be understood by those skilled in the art that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions defined by any one of the above-described known schemes as described herein although the scope of the disclosure is based on the sequences shown by the IMGT definition rules, however, the amino acid sequences corresponding to other CDR definition rules should also fall within the scope of the present invention.

14. The term "EC50" as used herein refers to the half maximal effect concentration (concentration for 50%of maximal effect), i.e. the concentration that causes 50% of the maximal effect (herein "ability to bind IL-17A").

15. The term "IC50" as used herein refers to a half inhibitory concentration (half maximal inhibitory concentration), i.e., a concentration that causes a 50% maximum inhibitory effect (herein, "ability to inhibit IL-17A and its receptor IL-17RA binding").

The technical scheme of the invention is as follows:

in a first aspect, the invention provides an antibody comprising a first single domain antibody and a second single domain antibody;

The amino acid sequence of the first single domain antibody comprises HCDR1 shown as SEQ ID NO.1, HCDR2 shown as SEQ ID NO.2, HCDR3 shown as SEQ ID NO. 3, and/or an amino acid sequence with at least 80% sequence identity with the amino acid sequence shown as any one of SEQ ID NO. 1-3;

the amino acid sequence of the second single domain antibody comprises HCDR4 shown as SEQ ID NO. 4, HCDR5 shown as SEQ ID NO. 5, HCDR6 shown as SEQ ID NO. 6, and/or an amino acid sequence with at least 80% sequence identity with the amino acid sequence shown as SEQ ID NO. 4-6.

Preferably, 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.

Preferably, the amino acid sequence of the antibody comprises an amino acid sequence having a difference of 1,2, 3,4, 5, 6, 7, 8 or 9 amino acids compared to the amino acid sequence shown in SEQ ID NOS: 1-6.

SEQ ID NO. 1 is ESLLRLYA.

SEQ ID NO. 2 is HTTSDTT.

SEQ ID NO. 3 is HVTSMRDSQNY.

SEQ ID NO. 4 is GFSIHIYA.

SEQ ID NO. 5 is ITRGGVT.

SEQ ID NO. 6 is NVGGTNGGY.

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 those in which one amino acid of groups (a) to (e) is substituted by another amino acid residue of the same group (a) small aliphatic, nonpolar or weakly polar residues Ala, ser, thr, pro and Gly, (b) polar, negatively charged residues and their (uncharged) amides Asp, asn, glu and Gln, (c) polar, positively charged residues His, arg and Lys, (d) large aliphatic, nonpolar residues Met, leu, he, val and Cys, and (e) aromatic residues Phe, tyr and Trp.

In some embodiments, conservative substitutions are Ala to Gly or to Ser, arg to Lys, asn to Gln or to His, asp to Glu, cys to Ser, gln to Asn, glu to Asp, gly to Ala or to Pro, his to Asn or to Gln, ile to Leu or to Val, leu to Ile or to Val, lys to Arg, to Gln or to Glu, met to Leu, to Tyr or to Ile, phe to Met, to Leu or to Tyr, ser to Thr, thr to Ser, trp to Tyr, tyr to Trp, and/or Phe to Val, to Ile or to Leu.

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

Further, the heavy chain framework region comprises part 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.

In a second aspect, the invention provides an antibody comprising a first single domain antibody and a second single domain antibody;

The structure of the first single domain antibody is as follows:

FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4;

the structure of the second single domain antibody is as follows:

FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8;

The amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are shown as SEQ ID NO 1-3, or the amino acid sequences with 1,2, 3,4, 5, 6, 7, 8, 9, 10 or 11 amino acid differences compared with the SEQ ID NO 1-3;

The amino acid sequences of the HCDR4, the HCDR5 and the HCDR6 are shown as SEQ ID NO 4-6, or the amino acid sequences with 1,2, 3,4, 5, 6, 7, 8 or 9 amino acid differences compared with the SEQ ID NO 4-6;

the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID NO. 7-10, or the amino acid sequences with at least 80% sequence identity compared with SEQ ID NO. 7-10;

the amino acid sequences of FR5, FR6, FR7 and FR8 are shown as SEQ ID NO. 11-14, or the amino acid sequences with at least 80% sequence identity compared with SEQ ID NO. 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. 1-6.

Specifically, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID NO. 7-10, or amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO. 7-10;

Specifically, the amino acid sequences of FR5, FR6, FR7 and FR8 are shown as SEQ ID NO. 11-14 or amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO. 11-14.

SEQ ID NO. 7 is DVQLVESGGGLVQPGGSLRLSCAAS.

SEQ ID NO. 8 is MGWYRQLPGQEREWVAI.

SEQ ID NO. 9 is NYRDSVKGRFTLSRDVATNTIYLQMTSLKPEDTAVYYC.

SEQ ID NO. 10 is WGQGTQVTVSS.

SEQ ID NO. 11 is EVQLVESGGGLVQPGGSLRLSCAAS.

SEQ ID NO. 12 is MGWYRQAPGKQRELVAT.

SEQ ID NO. 13 is:

NNADSVKGRFTISRDNAKNTAYLQMNSLKPGDTAVYYC。

SEQ ID NO. 14 is WGQGTQVTVSS.

Preferably, the HCDR1 has an amino acid sequence as shown in SEQ ID NO. 1, the HCDR2 has an amino acid sequence as shown in SEQ ID NO. 2, the HCDR3 has an amino acid sequence as shown in SEQ ID NO. 3, the HCDR4 has an amino acid sequence as shown in SEQ ID NO. 4, the HCDR5 has an amino acid sequence as shown in SEQ ID NO. 5, and the HCDR6 has an amino acid sequence as shown in SEQ ID NO. 6.

Preferably, the amino acid sequences of the first single domain antibody and the second single domain antibody are indirectly linked via a linker, or alternatively, are directly linked.

Further, the linker is (GGGGS) n, n is selected from 1, 2, 3,4, 5 or 6, preferably (GGGGS) 3.

Preferably, 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 linked in an order from the C terminal to the N terminal, or the first single domain antibody and the second single domain antibody are linked in an order from the N terminal to the C terminal.

Preferably, the antibody is an anti-IL-17A antibody.

In a third aspect, the present invention provides a recombinant protein comprising an antibody according to any one of the first or second aspects above.

Preferably, 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.

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

In particular, the immunoglobulin Fc domain is derived from a human antibody, a murine antibody, a primate antibody or a 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.

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 a fourth aspect, the present invention provides a drug conjugate comprising:

(1) The antibody or the recombinant protein according to any one of the above first or second aspects, and;

(2) A coupling moiety coupled to (1).

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

In a fifth aspect, the present invention provides an antibody preparation comprising:

(1) The antibody or the recombinant protein according to any one of the above first or second aspects, and;

(2) A pharmaceutically acceptable carrier.

In particular, the pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration.

Further, the pharmaceutically acceptable carrier is selected from one or more of excipient, buffer, emulsifier, stabilizer, diluent, binder, preservative and lubricant.

In a sixth aspect, the present invention provides a kit comprising an antibody, recombinant protein or antibody preparation according to any one of the first or second aspects above;

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

In a seventh aspect, the present invention provides an isolated nucleic acid molecule encoding an antibody according to any one of the first or second aspects above or a recombinant protein according to any one of the first or second aspects above.

Specifically, the nucleic acid may be RNA, DNA or cDNA.

For some embodiments, the nucleic acids of the invention may also be in the form of a vector, may be present in and/or may be part of a vector, such as a plasmid, cosmid, or YAC. The vector may be an expression vector, which typically comprises at least one nucleic acid of the invention operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.).

In an eighth aspect, the invention provides an expression vector comprising the nucleic acid molecule.

Preferably, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, or combinations thereof.

In a ninth aspect, the present invention provides a pharmaceutical composition comprising an antibody, the recombinant protein, the drug conjugate, the antibody preparation, the nucleic acid molecule or the expression vector of any one of the first or second aspects above;

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

Specifically, the pharmaceutically acceptable auxiliary materials are selected from one or more of diluents, isotonic agents, buffering agents, emulsifying agents, thickening agents, sweeteners, disintegrating agents, preservatives, absorbents, dispersants, stabilizers, suspending agents, solvents, precipitation inhibitors, surfactants, glidants, binders, lubricants, hydration agents, emulsification accelerators, colorants, flavoring agents, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

In a tenth aspect, the invention provides the use of an antibody, recombinant protein, drug conjugate, antibody preparation or pharmaceutical composition according to any of the first or second aspects of the invention, wherein the use is at least one of the following:

(1) Preparing a detection reagent or a kit;

(2) Preparing a medicament for preventing and/or treating autoimmune diseases;

(3) Preparing medicine for preventing and/or treating cancer.

In particular, the method comprises the steps of, the autoimmune diseases include focal scleroderma, polymyositis, multiple sclerosis, systemic scleroderma, progressive systemic sclerosis, dermatomyositis, psoriasis, allergic vasculitis, idiopathic azoospermia, scleroderma, habitual abortion, fibromyalgia, polymyalgia rheumatica, paeder's disease, chronic discoid lupus erythematosus, autoimmune hemolytic anemia, behcet's disease, adult Steve's disease, systemic lupus erythematosus, macrovasculitis, addison's disease, rheumatoid arthritis, myasthenia gravis, kalman's disease, idiopathic thrombocytopenic purpura, chronic atrophic gastritis, malignant rheumatoid arthritis, vasculitis, autoimmune hepatitis, alopecia areata, leukoplakia, spondyloarthritis, lupus nephritis, bullous pemphigoid, pernicious anemia, psoriatic arthritis, autoimmune diseases idiopathic Addison's disease, inflammatory bowel disease, sarcoidosis, herpes gestation, rheumatoid vasculitis, neuromyelitis optica, periarteritis nodosa, aortitis syndrome, juvenile idiopathic arthritis, mixed connective tissue disease, sjogren's syndrome, allergic granulomatous vasculitis, igG 4-related diseases, ANCA-related vasculitis, good-pasture syndrome, cogan's syndrome, nonalcoholic steatohepatitis, autoimmune neutropenia, linear IgA bullous dermatoses, hashimoto's disease, slowly progressive type I diabetes, RS3PE syndrome, temporal arteritis, antiphospholipid antibody syndrome, eosinophilic fasciitis, guillain Barre syndrome, primary biliary cirrhosis, acute glomerulonephritis, megaloblastic anemia, autoimmune adrenocortical hypofunction, primary thyroid function

Hypofunction, pemphigoid, acquired epidermolysis bullosa, vitiligo vulgaris, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, giant cell arteritis, amyotrophic lateral sclerosis, orthofield disease, autoimmune optic neuropathy, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplantation immunity, familial mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis or inclusion body myositis;

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

In particular, the cancer comprises oral cancer, respiratory cancer, eye cancer, skin cancer, head and neck cancer, laryngeal cancer, esophageal cancer, lymphoma, gastric cancer, bone cancer, digestive system cancer, liver cancer, lung cancer, kidney cancer, neuroblastoma, leukemia, glioblastoma, cholangiocarcinoma, bladder cancer, endometrial cancer, breast cancer, peritoneal cancer, cervical cancer, cholangiocarcinoma, choriocarcinoma, colorectal cancer, connective tissue cancer, melanoma, myeloma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, basal cell carcinoma, rectal cancer, salivary gland carcinoma, sarcoma, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, B-cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia or chronic myeloblastic leukemia.

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

(1) Contacting the antibody, the recombinant protein, the drug conjugate, or the antibody preparation of any one of the first or second aspects with a sample to be tested;

(2) The antigen-antibody complex is detected and the results are interpreted.

In a twelfth 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 an antibody, recombinant protein, drug conjugate, or antibody preparation of any one of the above first or second aspects.

In a thirteenth 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 an antibody, recombinant protein, drug conjugate, or antibody preparation of any one of the above first or second aspects.

The invention has the technical effects that:

(1) The tandem antibody 1-C11+1-F12 has good affinity, can be combined with target protein, and has obviously better combining ability than a positive antibody.

(2) The blocking effect is good, the tandem antibody 1-C11+1-F12 can block the Human IL-17A protein from activating 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc, and the blocking effect of the 1-C11+1-F12 is obviously better than that of a positive control antibody.

(3) The stability is strong, the thermal denaturation Tm value and Tagg are higher, the Tm value of 1-C11+1-F12 is 65.07, the Tagg value is 65.19, and the antibody is superior to a positive 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 shows the results of alpaca stock flow assay.

FIG. 3 shows the results of the yeast monoclonal FACS assay.

FIG. 4 shows SDS-PAGE results of tandem single domain antibodies 1-C11+1-F12, wherein the left band is marker and the right band is 1-C11+1-F12 in FIG. 4.

FIG. 5 shows the results of ELISA assays for tandem single domain antibody affinity.

FIG. 6 shows the results of ELISA assays for positive control antibody affinity.

FIG. 7 shows the detection of binding of IL-17A recombinant protein to reporter cell lines by FACS.

FIG. 8 shows the experimental results of IL-17A recombinant protein activation reporter cell lines.

FIG. 9 shows the results of an experiment for blocking IL-17A recombinant protein by the positive antibody Ixekizumab.

FIG. 10 shows the experimental results of blocking function detection of tandem single domain antibodies.

FIG. 11 shows the results of the test for blocking function of the positive control antibody.

FIG. 12 shows the results of thermostability assays for tandem single domain antibodies.

FIG. 13 shows the results of the thermal stability test of the positive control antibody.

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 experimental reagent of the invention:

Agar (Sigma, CAT#A1296), peptone (Sigma, CAT# 93926), yeast extract (OXOID, CAT#: LP 0021), sodium chloride (Allatin, CAT#: C111533), potassium chloride (Allatin, CAT#: P112133), magnesium sulfate (Guozhong, CAT#: 10013018), magnesium chloride (Guozhong, CAT#: 10012818), glucose (Sanken ,CAT#:GT1991);SfiI(NEB,CAT#:R0123L);T4 DNA ligase(TaKaRa,CAT#:2011A);PrimeScript^TMII 1st Strand cDNA Synthesis Kit(TaKaRa,CAT#:6210B);NuHi power mix(, CAT#: NH 9303), 3M sodium acetate (pH5.2-6) (Sigma, CAT#: 126-96-5), DNA fragment recovery kit (TakaRa, CAT#: 9761), gel recovery kit (Qiagen, CAT# 28706), radiculine plasmid megadrawer kit (radicle ,CAT#:DP117);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, 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).

Experiment consumable:

50mL Falcon centrifuge tubes (Corning, CAT# 352070), electric stumps (Bio-Rad 0.2 cm), RNASE FREE 1.5.5 mL EP tubes (QSP, CAT#: 509-GRD-Q), 200 μ L RNASE FREE PCR tubes (Axygen, PCR-02D-C), T125 shake flasks (Corning, CAT# 431143), 15mL Falcon centrifuge tubes (Corning, CAT# 430052), 6 well plates (Corning, CAT#3516), 96 well plates (Corning, CAT#3365), 96 Kong Heiban (F-BOTTOM (CHIMNEY WELL) BLACK).

Experimental facilities:

Electrotransport apparatus (Eppendorf Multiporator), centrifuge (Thermo FRESCO-17), constant temperature incubator (Shanghai Jinghong DNP-9052), constant temperature shake incubator (Ji Qi CO-O6U), ultra clean bench (Sujingan' ai SW-CJ-1 FD), PCR apparatus (Applied Biosystems ABI 2720), biosafety cabinet (sea, HR40-IIA 2), flow cytometer (Thermo Attune Nxt flow cytometer), thermo 3111CO ₂ incubator, forte Bio OCTET R2.

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).JImmunol 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) recombinant proteins (antigens)

The sequence information of Human IL-17A (Q16552-1) is retrieved from the UniProt database (AA Gly 24-Ala155, shown as SEQ ID NO: 15), a 6 XHis tag is added at the C end, gene synthesis is carried out after prokaryotic codon optimization and subcloning is carried out into a pET28a vector, and plasmid extraction is carried out after Sanger sequencing verification.

BL21 was competent to transform the recombinant plasmid, induced overnight with 0.5mM IPTG, bacterial liquid was collected for lysis, and 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:

MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNI HNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINAD GNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA.

1.2 Alpaca immunization

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

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

TABLE 1 results of immunotiter assays

1.3 Construction of antibody Yeast libraries

(1) PBMC isolation and VHH antibody fragment cloning:

Collecting 100mL of peripheral blood anticoagulation sample, and separating PBMC cells by using lymphocyte separation liquid;

RNA was extracted and reverse transcription was performed using PRIMESCRIPT ^TM II 1st Strand cDNA Synthesis Kit 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 antigen-bound magnetic beads, and rotating 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 STREPTAVIDIN for secondary antibody, and performing flow detection after incubation.

According to the flow detection result (figure 2), 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 figure 3, the binding condition of IL17A target monoclonal and target is detected by FACS, the amino acid sequences of the candidate single domain antibodies obtained by sequencing are compared, and the candidate antibodies with different CDR region amino acid sequences are selected to construct eukaryotic expression vectors.

1.6 Identification of antibody sequences

Enriching positive clone, selecting enriched single gram drop, carrying out PHAGE ELISA identification, and carrying out sequencing analysis on clone to obtain nucleic acid and amino acid sequence information of 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.

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.

Taking out LVTransm transfection reagent and single-chain antibody expression vector from refrigerator, thawing at room temperature, and blowing with pipetting gun. 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 L 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 mixed thoroughly with gentle shaking. After placing the cells in a 37℃5% CO ₂ incubator at 130rpm for 6-8 hours, 50mL of fresh 293 cell medium was added and the cells were returned to the incubator for continued culture.

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.

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 on 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.

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.

Neutralizing buffer 1M Tris, pH 8.5.

Magnetic bead regeneration buffer solution, 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 have tandem-enhanced blocking effects with two of the anti-IL-17A single domain antibodies. Two anti-IL-17A single domain antibodies were 1-C11 and 1-F12.

The amino acid sequence of the first single domain antibody 1-C11 is shown as SEQ ID NO. 16.

SEQ ID NO:16:

DVQLVESGGGLVQPGGSLRLSCAASESLLRLYAMGWYRQLPGQEREWV AIHTTSDTTNYRDSVKGRFTLSRDVATNTIYLQMTSLKPEDTAVYYCHVTSM RDSQNYWGQGTQVTVSS.

The nucleotide sequence of the coded single-domain antibody 1-C11 is shown as SEQ ID NO. 17.

SEQ ID NO:17:

GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGAAAGCCTCCTCAGGTTGTATGCCATGGGCTGGTACCGCCAACTTCCAGGGCAGGAGCGCGAGTGGGTCGCAATACACACTACTAGTGACACCACTAATTATAGAGACTCCGTGAAGGGCCGATTCACGCTCTCCAGAGACGTCGCCACGAACACGATTTATCTCCAAATGACCAGCCTCAAACCTGAAGACACGGCCGTCTATTATTGTCATGTTACTTCCATGAGAGATTCACAAAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCG.

The amino acid sequence of the second single domain antibody 1-F12 is shown as SEQ ID NO. 18.

SEQ ID NO:18:

EVQLVESGGGLVQPGGSLRLSCAASGFSIHIYAMGWYRQAPGKQRELVA TITRGGVTNNADSVKGRFTISRDNAKNTAYLQMNSLKPGDTAVYYCNVGGT NGGYWGQGTQVTVSS.

The nucleotide sequence of the coded single domain antibody 1-F12 is shown as SEQ ID NO. 19.

SEQ ID NO:19:

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCGGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTTAGTATCCACATCTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGCTGGTCGCAACTATTACTAGAGGTGGTGTAACAAATAATGCAGACTCCGTGAAGGGGCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGCGTATCTGCAAATGAACAGCCTGAAACCTGGGGACACGGCCGTCTATTACTGTAATGTAGGTGGGACGAACGGGGGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

2.2 Preparation of tandem Single domain antibody 1-C11+1-F12

The candidate antibody is constructed into bivalent single domain antibody according to VHH- (GGGGS) ₃ -VHH-IgG1 Fc form, and adopts ProteinA magnetic beads for purification, and the amino acid sequence of the IgG1 Fc is shown as SEQ ID NO. 20.

SEQ ID NO:20:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The nucleotide sequence of the coding SEQ ID NO. 20 is shown as SEQ ID NO. 21.

SEQ ID NO:21:

GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAtaa.

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) Taking out LVTransm transfection reagent and single-chain antibody expression vector from refrigerator, thawing at room temperature, and blowing with pipetting gun. 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 L 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 mixed thoroughly with gentle shaking. After placing the cells in a 37℃5% CO ₂ incubator at 130rpm for 6-8 hours, 50mL of fresh 293 cell medium was added and the cells were returned to the incubator for continued culture.

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 a Protein A column, to finally obtain the tandem single domain antibody 1-C11+1-F12.

SDS-PAGE results of the tandem single domain antibody 1-C11+1-F12 are shown in FIG. 4, and the results show that the molecular weight of the candidate antibody is in accordance with the expected molecular weight and the purity of the candidate antibody can be used for the next experiment.

Example 3 tandem single domain antibody affinity assay

1 Preparation of positive control antibody Ixekizumab

Gene synthesis Ixekizumab heavy chain and light chain variable regions (the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:22, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 23), subcloning the heavy chain variable region into pcDNA3.4-hIgG4 (the amino acid sequence of IgG4 is shown as SEQ ID NO: 24) vector, subcloning the light chain variable region into pcDNA3.4-hIgKc vector (the amino acid sequence of IgG KC is shown as SEQ ID NO: 25), and preparing endotoxin-removing plasmid by using plasmid large pumping kit after Sanger sequencing verification.

Taking the LVTransm transfection reagent and the heavy chain and light chain expression vector out of 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 heavy chain and light chain expression vectors, fully and uniformly mixing the mixture up and down by a pipetting gun, adding 300 mug L LVTRANSM, immediately and uniformly mixing the mixture up and down by a pipetting device, and standing for 10 minutes at room temperature.

The DNA/LVTransm complex was added to 100mL of 293F cells, gently swirled and thoroughly mixed, and the cells were placed in a 37℃5% CO ₂ incubator at 130RPM for continued 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:

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEW MGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARY DYFTGTGVYWGQGTLVTVSS.

SEQ ID NO:23:

DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQL LIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQ GTKLEIK.

SEQ ID NO:24:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

SEQ ID NO:25:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC.

2ELISA detection of Human IL-17A recombinant protein and reference antibody binding Activity

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

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

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

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

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

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

(7) Adding secondary antibody HRP-ProteinA (Boster, BA 1080) for 1:10000 dilution, adding into enzyme label 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 of liquid (2M HCl);

(12) OD450 values within wells were read using a microplate reader. As shown in Table 2, the positive antibodies bind well to IL-17A antigen protein and can be used for immunization.

Table 2 detection of binding Activity of human IL17A to Positive antibodies

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-STREPTAVDIN. The results showed that the EC50 of 1-C11+1-F12 was 1.681 (FIG. 5), well below 10.06 of the positive control antibody (FIG. 6). Therefore, the tandem single domain antibody can be combined with target protein, and the combination capacity is obviously higher than that of positive antibody.

Example 4 tandem single domain antibody blocking function detection

Construction of 1IL-17A reporter cell line

Based on the amino acid sequence information of IL-17RA (UniProtKB: Q96F46, SEQ ID NO: 26) and IL-17RC (UniProtKB: Q8NAC3, SEQ ID NO: 27), a lentiviral expression vector is constructed and lentivirus is packaged, 293 cells are co-infected, recombinant 293 cells which simultaneously overexpress the two receptors are screened, NFKB-Luciferase (the amino acid sequence of which is shown as SEQ ID NO:28, the nucleotide sequence of which is shown as SEQ ID NO: 29) and ACT1 gene (the nucleotide sequence of which is shown as SEQ ID NO: 30) are further stably transformed, and an IL-17A reporter cell strain 293F-IL-17 RA-17 Rc-ACT 1-NFkB-Luc is constructed.

SEQ ID NO:26:

MGAARSPPSAVPGPLLGLLLLLLGVLAPGGASLRLLDHRALVCSQPGLNCTVKNSTCLDDSWIHPRNLTPSSPKDLQIQLHFAHTQQGDLFPVAHIEWTLQTDASILYLEGAELSVLQLNTNERLCVRFEFLSKLRHHHRRWRFTFSHFVVDPDQEYEVTVHHLPKPIPDGDPNHQSKNFLVPDCEHARMKVTTPCMSSGSLWDPNITVETLEAHQLRVSFTLWNESTHYQILLTSFPHMENHSCFEHMHHIPAPRPEEFHQRSNVTLTLRNLKGCCRHQVQIQPFFSSCLNDCLRHSATVSCPEMPDTPEPIPDYMPLWVYWFITGISILLVGSVILLIVCMTWRLAGPGSEKYSDDTKYTDGLPAADLIPPPLKPRKVWIIYSADHPLYVDVVLKFAQFLLTACGTEVALDLLEEQAISEAGVMTWVGRQKQEMVESNSKIIVLCSRGTRAKWQALLGRGAPVRLRCDHGKPVGDLFTAAMNMILPDFKRPACFGTYVVCYFSEVSCDGDVPDLFGAAPRYPLMDRFEEVYFRIQDLEMFQPGRMHRVGELSGDNYLRSPGGRQLRAALDRFRDWQVRCPDWFECENLYSADDQDAPSLDEEVFEEPLLPPGTGIVKRAPLVREPGSQACLAIDPLVGEEGGAAVAKLEPHLQPRGQPAPQPLHTLVLAAEEGALVAAVEPGPLADGAAVRLALAGEGEACPLLGSPGAGRNSVLFLPVDPEDSPLGSSTPMASPDLLPEDVREHLEGLMLSLFEQSLSCQAQGGCSRPAMVLTDPHTPYEEEQRQSVQSDQGYISRSSPQPPEGLTEMEEEEEEEQDPGKPALPLSPEDLESLRSLQRQLLFRQLQKNSGWDTMGSESEGPSA.

SEQ ID NO:27:

MPVPWFLLSLALGRSPVVLSLERLVGPQDATHCSPVSLEPWGDEERLRVQFLAQQSLSLAPVTAATARTALSGLSGADGRREERGRGKSWVCLSLGGSGNTEPQKKGLSCRLWDSDILCLPGDIVPAPGPVLAPTHLQTELVLRCQKETDCDLCLRVAVHLAVHGHWEEPEDEEKFGGAADSGVEEPRNASLQAQVVLSFQAYPTARCVLLEVQVPAALVQFGQSVGSVVYDCFEAALGSEVRIWSYTQPRYEKELNHTQQLPDCRGLEVWNSIPSCWALPWLNVSADGDNVHLVLNVSEEQHFGLSLYWNQVQGPPKPRWHKNLTGPQIITLNHTDLVPCLCIQVWPLEPDSVRTNICPFREDPRAHQNLWQAARLQLLTLQSWLLDAPCSLPAEAALCWRAPGGDPCQPLVPPLSWENVTVDKVLEFPLLKGHPNLCVQVNSSEKLQLQECLWADSLGPLKDDVLLLETRGPQDNRSLCALEPSGCTSLPSKASTRAARLGEYLLQDLQSGQCLQLWDDDLGALWACPMDKYIHKRWALVWLACLLFAAALSLILLLKKDHAKGWLRLLKQDVRSGAAARGRAALLLYSADDSGFERLVGALASALCQLPLRVAVDLWSRRELSAQGPVAWFHAQRRQTLQEGGVVVLLFSPGAVALCSEWLQDGVSGPGAHGPHDAFRASLSCVLPDFLQGRAPGSYVGACFDRLLHPDAVPALFRTVPVFTLPSQLPDFLGALQQPRAPRSGRLQERAEQVSRALQPALDSYFHPPGTPAPGRGVGPGAGPGAGDGT.

SEQ ID NO:28:

MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV.

SEQ ID NO:29:

atggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtg.

SEQ ID NO:30:

ATGCCACCTCAGTTGCAGGAAACTCGGATGAATAGAAGCATCCCCGTGGAAGTGGACGAGAGCGAGCCGTACCCTAGTCAGCTGCTGAAGCCGATCCCTGAGTACTCCCCGGAAGAGGAATCCGAACCACCAGCCCCCAACATTCGCAATATGGCCCCCAATAGCTTGTCCGCACCAACAATGCTGCACAACTCTTCTGGCGACTTCTCTCAGGCCCACTCCACCCTGAAACTGGCGAATCACCAGCGGCCTGTATCCCGGCAGGTGACCTGTCTGAGAACTCAGGTGCTTGAAGACTCCGAGGACTCTTTCTGTAGGCGGCATCCAGGTTTGGGCAAGGCGTTTCCGTCCGGCTGTTCCGCGGTTTCAGAGCCCGCTTCCGAAAGTGTCGTGGGCGCCCTGCCAGCCGAGCACCAGTTCTCCTTCATGGAAAAGCGGAACCAGTGGCTGGTCAGTCAGCTGAGCGCCGCGTCACCTGATACAGGTCACGATTCCGACAAGTCTGACCAGTCTCTGCCCAATGCGTCAGCCGATAGTCTCGGGGGCTCCCAGGAGATGGTGCAGAGACCACAGCCGCACAGAAACCGGGCCGGGCTTGATCTGCCCACCATTGATACAGGCTACGATTCCCAGCCCCAGGACGTCCTTGGCATTCGCCAGCTGGAAAGGCCTCTGCCCTTGACCTCCGTGTGTTACCCCCAGGACCTGCCCCGCCCTTTGAGAAGCCGGGAGTTTCCCCAGTTTGAGCCCCAACGATACCCTGCCTGTGCTCAGATGCTGCCTCCGAACCTGAGCCCACACGCTCCCTGGAACTACCACTATCACTGTCCCGGCAGCCCCGATCACCAGGTGCCTTATGGACACGACTACCCGCGGGCTGCATACCAGCAGGTCATACAGCCTGCCTTGCCGGGTCAGCCGCTGCCCGGAGCTTCTGTGCGCGGCCTGCACCCCGTTCAGAAAGTGATCCTGAACTATCCAAGCCCATGGGACCATGAAGAGAGACCAGCCCAAAGAGATTGCTCTTTTCCTGGGTTGCCTAGACACCAAGACCAGCCTCACCACCAGCCTCCCAATCGGGCAGGCGCCCCAGGCGAAAGTCTCGAGTGCCCCGCCGAACTCAGACCACAGGTCCCTCAGCCCCCTTCCCCCGCGGCAGTACCCAGACCCCCCTCTAACCCACCCGCCCGGGGAACGCTCAAGACTTCAAATCTCCCAGAAGAGCTGCGCAAAGTGTTCATAACCTACAGCATGGACACCGCTATGGAGGTGGTTAAGTTCGTCAACTTCCTGCTGGTCAATGGGTTCCAGACTGCAATCGACATTTTTGAGGATAGAATTCGGGGAATCGACATCATCAAGTGGATGGAGAGATACCTGCGGGATAAGACAGTGATGATTATCGTGGCCATTAGTCCCAAGTACAAGCAAGATGTGGAGGGCGCAGAATCACAGTTGGACGAAGACGAGCACGGACTCCATACAAAATATATCCACAGGATGATGCAGATCGAGTTCATTAAACAAGGCTCCATGAATTTCCGCTTCATACCGGTCCTGTTTCCAAACGCAAAAAAAGAGCATGTACCCACTTGGCTCCAGAATACCCATGTCTACTCCTGGCCCAAGAACAAGAAGAATATCCTGCTGCGCTTGCTCAGAGAAGAAGAGTATGTCGCCCCTCCAAGGGGGCCCCTCCCCACACTCCAAGTAGTGCCACTT.

Binding of 2IL-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 a plurality of parts, wherein the number of each part of cells is 2×10 ⁵ cells;

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

centrifuging at 800 Xg at room temperature for 3 min, removing supernatant containing antibody, washing cells with PBS for 3 times, adding secondary antibody APC-His (1:500 dilution), mixing, and incubating at room temperature in dark place for 30 min;

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 showed that the constructed IL-17A receptor overexpressing cell lines could bind IL-17A with a positive rate of more than 90% (FIG. 7).

The IL-17A recombinant protein is adopted to activate 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc. The results showed that the IL17A recombinant protein was effective in activating luciferase expression in 293F-IL17Ra/IL17 Rc-NFkB-Luc reporter cell lines (FIG. 8).

Functional experiments of 3Ixekizumab blocking IL-17A recombinant protein

The present example uses the positive antibody Ixekizumab to detect the validity of the fluorescence reporting system. Positive control antibody Ixekizumab was added to 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc cells along with IL-17A recombinant protein, and as a result, positive control antibody Ixekizumab inhibited IL17A protein binding to its membrane receptor and intracellular NFkB signaling, exhibiting a dose effect (FIG. 9).

Blocking function of 4 tandem single domain antibody

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 showed that IC50 of 1-C11+1-F12 was 0.7807. Mu.g/mL and that of the positive control antibody was 1.356. Mu.g/mL (FIGS. 10 and 11). Therefore, both the 1-C11+1-F12 and the positive control antibody can block the Human IL-17A protein from activating 293F-IL-17RA-IL-17Rc-ACT 1-NFkB-Luc, and the blocking effect of the 1-C11+1-F12 is obviously better than that of the positive control antibody.

Example 5 tandem Single Domain antibody stability detection

The thermal denaturation and chemical denaturation of the protein can be detected under natural conditions by detecting the fluorescence change through a micro-differential scanning fluorescence technology (nanoDSF), the temperature (T _m) when 50% of the protein is in an unfolded state and the temperature (T _agg) when aggregation begins to occur are precisely determined, and the higher the thermal denaturation T _m value and the higher the T _agg value are, the more stable the antibody protein is.

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.

Experimental results:

As shown in fig. 12-13, the stability of the tandem antibody 1-c11+1-F12 was strong, better than the positive control, tm=65.07, tagg= 65.19, tm=56.1, tagg=61.86 for the positive control antibody.

Application example 1 an antibody preparation

The antibody preparation comprises serial single domain antibody 1-C11+1-F12, buffer solution, surfactant, amino acid, tonicity agent and the like.

In one embodiment of the invention, the preparation of the antibody preparation comprises weighing each substance, adding water for dissolving and uniformly mixing, and regulating each component to the following concentration to obtain (100-200) mg/ml serial single domain antibody 1-C11+1-F12, (1-10) mM citrate buffer, (0.1-1%w/v) Tween 80, (100-200) mM arginine and (1-10)%sucrose, wherein the pH of the preparation is 5.0-8.0.

Application example 2A kit

The kit comprises serial single domain antibodies 1-C11+1-F12, recombinant proteins, antibody preparations and/or polyclonal antibodies, containers for loading the antibody preparations, buffers and the like.

In one embodiment of the invention, the kit comprises (100-200) mg/ml of the tandem single domain antibody 1-C11+1-F12, buffer at pH 5.0-8.0.

Application example 3 antibody drug conjugate

The antibody drug conjugate comprises serial single domain antibodies 1-C11+1-F12, recombinant proteins, antibody preparations and/or polyclonal antibodies, wherein the drug is a physiologically active substance (such as nucleic acid and the like) and a linker (the linker comprises maleimide linker, val-Cit linker, SS linker and DMSS linker) for connecting the antibody and the drug.

In one embodiment of the invention, the serial single domain antibody 1-C11+1-F12 is connected with the drug through an SS joint, and (100-200) mM aqueous solution is added and mixed uniformly at room temperature, and the joint reaction is terminated, thus obtaining the antibody drug conjugate.

Application example 4 pharmaceutical composition

The pharmaceutical composition comprises serial single domain antibodies 1-C11+1-F12, recombinant proteins, antibody preparations, polyclonal antibodies, nucleic acid molecules, biological expression vectors and/or host cells, and pharmaceutically acceptable auxiliary materials.

In one embodiment of the invention, the preparation of the pharmaceutical composition comprises the steps of preparing serial single domain antibody 1-C11+1-F12 with the concentration of (100-200) mg/ml, and adding sucrose (1-20 w/v), histidine (10-300) mM and Tween 80 (0.1-10)% to obtain the pharmaceutical composition.

Claims (22)

1. A tandem single-domain antibody against IL-17A, characterized in that: the tandem single-domain antibody comprises a first single-domain antibody and a second single-domain antibody; The structure of the first single domain antibody is: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; The structure of the second single domain antibody is: FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8; The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR4 is shown in SEQ ID NO: 4, the amino acid sequence of the HCDR5 is shown in SEQ ID NO: 5, and the amino acid sequence of the HCDR6 is shown in SEQ ID NO: 6. 2. The tandem single-domain antibody according to claim 1, characterized in that the heavy chain framework region comprises part or all of an antibody heavy chain framework region or a variant thereof selected from human, mouse, primate or camelid sources. 3. The tandem single-domain antibody according to claim 2, characterized in that the heavy chain framework region comprises part or all of an antibody heavy chain framework region selected from camelid origin or a variant thereof. 4. The tandem single-domain antibody according to claim 3 is characterized in that it comprises part or all of an antibody heavy chain framework region selected from alpaca or a variant thereof. 5. A tandem single domain antibody against IL-17A, characterized in that: the antibody comprises a first single domain antibody and a second single domain antibody; The structure of the first single domain antibody is: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; The structure of the second single domain antibody is: FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8; The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR4 is shown in SEQ ID NO: 4, the amino acid sequence of the HCDR5 is shown in SEQ ID NO: 5, and the amino acid sequence of the HCDR6 is shown in SEQ ID NO: 6; The amino acid sequence of FR1 is shown in SEQ ID NO:7, the amino acid sequence of FR2 is shown in SEQ ID NO:8, the amino acid sequence of FR3 is shown in SEQ ID NO:9, the amino acid sequence of FR4 is shown in SEQ ID NO:10, the amino acid sequence of FR5 is shown in SEQ ID NO:11, the amino acid sequence of FR6 is shown in SEQ ID NO:12, the amino acid sequence of FR7 is shown in SEQ ID NO:13, and the amino acid sequence of FR8 is shown in SEQ ID NO:14. 6. The antibody according to any one of claims 1 to 5, characterized in that: the amino acid sequences of the first single-domain antibody and the second single-domain antibody are indirectly connected through a linker; or; directly connected. The antibody according to claim 6, characterized in that the linker is (GGGGS) _n , where n is selected from 1, 2, 3, 4, 5 or 6. 8. The antibody according to claim 7, characterized in that the linker is (GGGGS) ₃ . 9. A recombinant protein, characterized in that: the recombinant protein comprises the antibody according to any one of claims 1 to 8; the recombinant protein also comprises a biologically active protein or a functional fragment thereof that assists its expression and/or secretion, or prolongs its half-life in vivo. 10. The recombinant protein according to claim 9, characterized in that the biologically active protein or its functional fragment is at least one selected from serum albumin, His tag, GST tag, albumin binding polypeptide, prealbumin, carboxyl terminal peptide, elastin-like polypeptide, immunoglobulin Fc domain, MBP tag, FLAG tag and SUMO tag. 11. The recombinant protein according to claim 10, characterized in that the immunoglobulin Fc domain is derived from human antibody, mouse antibody, primate antibody or camelid antibody, or variants thereof. 12 . The recombinant protein according to claim 11 , wherein the immunoglobulin Fc domain is derived from human IgG antibody, including IgG1 Fc, IgG2 Fc, IgG3 Fc or IgG4 Fc. 13 . The recombinant protein according to claim 12 , wherein the immunoglobulin Fc domain is IgG1Fc. 14. An antibody preparation, characterized in that: the antibody preparation comprises: (1) the antibody according to any one of claims 1 to 8 or the recombinant protein according to any one of claims 9 to 13; and; (2) A pharmaceutically acceptable carrier. 15. A kit, characterized in that the kit comprises the antibody according to any one of claims 1 to 8, the recombinant protein according to any one of claims 9 to 13 or the antibody preparation according to claim 14. 16. The kit according to claim 15, characterized in that the kit further comprises a container for loading the antibody preparation. 17. An isolated nucleic acid molecule, characterized in that the nucleic acid molecule encodes the antibody according to any one of claims 1 to 8 or the recombinant protein according to any one of claims 9 to 13. 18. An expression vector, characterized in that: the expression vector comprises the nucleic acid molecule according to claim 17. 19. A pharmaceutical composition, characterized in that: the pharmaceutical composition comprises the antibody according to any one of claims 1 to 8, the recombinant protein according to any one of claims 9 to 13, the antibody preparation according to claim 14, the nucleic acid molecule according to claim 17 or the expression vector according to claim 18. 20. The pharmaceutical composition according to claim 19, characterized in that the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient. 21. Use of the antibody according to any one of claims 1 to 8, the recombinant protein according to any one of claims 9 to 13, the antibody preparation according to claim 14 or the pharmaceutical composition according to any one of claims 19 to 20, characterized in that the use is at least one of the following uses: (1) Preparing reagents or kits for detecting IL-17A; (2) Preparation of drugs for treating autoimmune diseases, wherein the autoimmune diseases are plaque psoriasis, rheumatoid arthritis, and psoriatic arthritis. 22. A method for detecting IL-17A in a sample in vitro for non-diagnostic purposes, characterized in that the method comprises the following steps: (1) contacting the antibody according to any one of claims 1 to 8, the recombinant protein according to any one of claims 9 to 13, or the antibody preparation according to claim 14 with a sample to be tested; (2) Detect antigen-antibody complexes and interpret the results.