Novel anti-IL-17A single-domain antibody as well as preparation and application thereof

Abstract

The invention provides a novel anti-IL-17A single-domain antibody as well as preparation and application thereof, and belongs to the technical field of cellular immunology. The amino acid sequences of the antibody provided by the invention comprise: (1) the amino acid sequences of HCDR1, HCDR2 and HCDR3 respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; and / or; and (2) an amino acid sequence having at least 80% of sequence consistency with the amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The antibody has the activity or performance of strong binding capacity and stability, good blocking effect and the like. The invention also provides application of the antibody in preparation of drugs for treating autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of cellular immunology, and particularly relates to a novel anti-IL-17A single-domain antibody, its preparation and application. Background Art

[0002] IL-17A is secreted by TH17 cells. IL-17A interacts with various mediators such as GM-CSF, IFN-γ, IL-22, IL-1β and TNF-α to exert its pro-inflammatory function. The downstream signaling pathway mediated by IL-17A further induces the production of chemokines, cytokines, AMPs and remodeling proteins, thus playing an important role in cell trafficking, immune regulation, host defense and tissue repair. IL-17A induces the production of various cytokines and chemokines, thus playing an important role in autoimmune diseases and various types of tumors.

[0003] Studies have found that specifically blocking the binding of IL-17A to its receptor can block the signal transduction of inflammatory factors, thereby achieving the purpose of treating diseases such as psoriasis. Currently, several antibody drugs that block IL-17 have been marketed globally, including monoclonal antibody drugs that neutralize IL-17A (Secukinumab and Ixekizumab) or IL-17RA (Brodalumab), and more than 50 antibody drugs or small molecules targeting the IL-17 target and used for the treatment of autoimmune diseases are under research in China. Although antibody drugs usually have good efficacy and tolerance in the human body compared with chemotherapy drugs, there are still many treatment-related side effects in clinical use, such as nasopharyngitis, headache, nausea and diarrhea, or more serious ones such as infection, cardiac toxicity and severe immune reactions; in addition, the complex structure and relatively poor stability of monoclonal antibody drugs also lead to the need for repeated large-dose injections to achieve the best effect.

[0004] Nanobodies (Nb) are derived from the variable region fragments of the heavy chains of antibodies in camelids. It has only two heavy chains, lacks the light chain, and its heavy chain lacks the constant heavy chain (CH1) domain. The average molecular weight of nanobodies is about 12-15 kDa, which is the smallest single polypeptide chain that can bind to antigens found in natural antibodies. The conserved region of nanobodies shows a high degree of sequence and structural homology with the VH domain of human monoclonal antibodies. Therefore, nanobodies have immunogenicity comparable to that of human antibodies. Compared with traditional monoclonal antibodies, nanobodies have a higher expression level, which can reduce the production cost. Nanobodies have excellent biophysical and biochemical properties, have high thermal stability, and have a higher affinity compared with traditional monoclonal antibodies, and have broad application prospects.

[0005] Currently, there is still a lack of products of anti-IL-17A single-domain antibodies with strong affinity, good blocking effect, and strong stability in the prior art.

[0006] This invention is a patent application filed by the inventor for the self-developed anti-IL-17A antibody.

[0007] In addition to the antibody claimed in this invention, the inventor has also developed 8 other antibodies. Based on the relevant provisions of the unity of invention in the Patent Law, protection is separately claimed for 9 different antibodies.

[0008] The inventor has also developed 12 tandem antibodies based on these 9 antibodies. Based on the relevant provisions of the unity of invention in the Patent Law, protection is separately claimed for 12 different tandem antibodies.

[0009] The inventor has also developed a gene-modified stem cell technology based on these 12 tandem antibodies. Based on the relevant provisions of the unity of invention in the Patent Law, protection is separately claimed for 3 different gene-modified stem cells and 3 different applications.

[0010] For ease of understanding the technical solution of this invention, optionally refer to other series of application texts of this project. Summary of the Invention

[0011] Terms and declarations of this invention:

[0012] 1. As used herein, the term "amino acid": includes natural amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that act 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 having the same basic chemical structure as the naturally occurring amino acids. Amino acids can be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the Biochemical Nomenclature Commission of IUPAC-IUB.

[0013] 2. As used herein, the term "antibody": refers to a polypeptide or a fragment thereof that specifically binds and recognizes an antigen and includes the framework regions of immunoglobulin genes. The use of the term antibody means including whole antibodies and their antigen-binding fragments. The term antibody includes monospecific antibodies, bispecific antibodies, and multispecific antibodies as long as they exhibit the desired biological activity or function.

[0014] 3. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigenic site. In addition, compared to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.

[0015] 4. As used herein, the terms "single-domain antibody (VHH)", "single-domain antibody" (singledomain antibody, sdAb, or nanobody) have the same meaning and refer to the variable region of a cloned antibody heavy chain, and a single-domain antibody composed of only one heavy-chain variable region is constructed, which is the smallest antigen-binding fragment with complete function. Usually, after obtaining an antibody that is naturally lacking the light chain and the first constant region of the heavy chain (CH1), the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) composed of only one heavy-chain variable region.

[0016] 5. As used herein, the terms "homology" or "identity" refer to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence, and for the purpose of comparison, these positions may be aligned. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position.

[0017] 6. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Antigenic determinants usually contain the chemically reactive surface groups of a molecule, such as amino acids or sugar side chains, and usually have specific three-dimensional structural features as well as specific charge characteristics. For example, an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation, which can be a "linear" epitope or a "conformational" epitope.

[0018] 7. As used herein, the term "amino acid sequence" refers to the order in which amino acids are linked together to form a peptide chain (or polypeptide), and the amino acid sequence can only be read in one direction.

[0019] 8. As used herein, the term "nucleotide sequence" refers to the arrangement order of bases in DNA or RNA, i.e., the arrangement order of A, T, G, C in DNA, or the arrangement order of A, U, G, C in mRNA, and also includes the arrangement order of bases in rRNA, tRNA, and mRNA.

[0020] 9. As used herein, the term "framework region", also known as the "scaffold region", refers to a region of approximately 110 amino acids at the near N-terminus of the H and L chains of immunoglobulins that varies greatly, while the amino acid sequences of other parts are relatively constant. Based on this, the light and heavy chains can be divided into variable regions (V) and constant regions (C). The variable region contains hypervariable regions (HVRs), also known as complementarity-determining regions (CDRs), and framework regions (FRs).

[0021] 10. As used herein, the term "humanized" antibody refers to an antibody in which the Fr region of the variable region (VH or VHH), the constant region (i.e., the CH and CL regions), or the entire antibody is encoded by human antibody genes. Humanized antibodies include chimeric antibodies, reshaped antibodies, and fully humanized antibodies, among others.

[0022] 11. As used herein, the terms "hypervariable region", "complementary determining region", "HVR", or "CDR" refer to regions in the antibody variable domain region that are highly variable in sequence and / or form structurally defined loops ("hypervariable loops"). Typically, a native four-chain antibody contains six HVRs or CDRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Based on the Chothia definition rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L26-L32 (L1), L50-L52 (L2), L91-L96 (L3), H26-H32 (H1), H52-H56 (H2), and H96-H101 (H3) (Chothia et al., J. Mol. Biol. 196:901-917 (1987)). Based on the Kabat definition rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L24-L34 (L1), L50-L56 (L2), L89-L97 (L3), H31-H35 (H1), H50-H65 (H2), and H95-H102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, the fifth edtion, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Based on the IMGT definition rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L27-L32 (L1), L50-L51 (L2), L89-L97 (L3), H26-H33 (H1), H51-H56 (H2), and H93-H102 (H3) (Honjo, T. and Alt, F.W. (1995) Immunoglobulin genes. Academic Press pp. 3-443). It is well known to those skilled in the art that in the art, CDRs of antibodies can be defined by various methods, such as the Kabat definition rules based on sequence variability, the Chothia definition rules based on the position of structural loop regions, and reference tools for antibody humanization design based on CDR grafting (see J Mol Biol. 273:927-48, 1997).Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementary determining region" of a given antibody or its region (such as the variable region) should be understood to cover the complementary determining regions defined by any of the above-known schemes described in the present invention. Although the scope of protection claimed in the present disclosure is based on the sequences shown by the IMGT definition rules, the amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0023] 12. As used herein, the term "comprises" or "comprising" means "including but not limited to". This term is intended to be open-ended, specifying the presence of any of the stated features, elements, integers, steps, or components, but not excluding the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application, particularly in the claims, may be replaced by the term "consisting of". The amino acid three-letter codes and single-letter codes used herein are known to those skilled in the art or as described in J Biol.Chem, 243, p3558 (1968).

[0024] 13. As used herein, the terms "optionally", "any one", "any", or "any item" mean that the subsequently described event or circumstance may but need not occur, and this description includes the instances where the event or circumstance occurs or does not occur. For example, "optionally comprising one variable region of an antibody heavy chain" means that a variable region of an antibody heavy chain of a specific sequence may but need not be present.

[0025] 14. As used herein, the term "about" represents a range of ±20% of the value that follows. In some embodiments, the term "about" represents a range of ±10% of the value that follows. In some embodiments, the term "about" represents a range of ±5% of the value that follows.

[0026] 15. As used herein, the term "and / or" should be understood to mean any one of the alternatives or any combination of any two or more of the alternatives.

[0027] 16. As used herein, the term "IL-17A" or "interleukin-17A" refers to a cytokine belonging to the interleukin-17 family, which is produced by T cells and other types of immune cells and plays an important role in the immune system. IL-17A is mainly produced by Th17 cells, and other cells including CD8+ T cells, γδ T cells, NK cells, neutrophils, mast cells and macrophages also express IL-17A. It mainly acts on immune cells such as macrophages, neutrophils and endothelial cells to induce an inflammatory response. In some instances, the term includes variants, homologs, orthologs and paralogs. For example, an antibody specific for human IL-17A can cross-react with IL-17A protein of another species such as a monkey in certain cases. In other embodiments, an antibody specific for human IL-17A protein can be completely specific for human IL-17A protein without cross-reacting with IL-17A proteins of other species or other types of proteins, or can cross-react with IL-17A proteins of some other species but not all other species.

[0028] 17. As used herein, the term "anti-IL-17A (single-domain or nanobody) antibody" or "IL-17A (single-domain or nanobody) antibody" refers to an antibody that specifically binds to IL-17A and partially or completely neutralizes, inhibits or impairs the activity of IL-17A, and / or inactivates IL-17A, blocks the IL-17A response or downstream pathways mediated by IL-17A or other IL-17A-mediated functions.

[0029] 18. As used herein, the term "antibody" refers to a glycoprotein comprising a heavy chain (H) and a light chain (L) linked to each other by disulfide bonds (S-S). Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as VH). The light chain constant region consists of one domain CL. Light chains are divided into two types, namely kappa-type light chains and lambda-type light chains (for example, in the present invention, the light chain constant region Cκ / λ represents that the light chain constant region is a kappa-type light chain or a lambda-type light chain). The VH region and the VL region can be further divided into hypervariable regions (also called complementarity-determining regions (CDRs)), which are interspersed with relatively conserved framework regions or FRs. Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies include monospecific antibodies, bispecific antibodies and multispecific antibodies as long as they exhibit the desired biological activity or function.

[0030] 19. As used herein, the terms "active", "functionally active" or "biologically active", or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize IL-17A activity in vivo or in vitro, IC50, the in vivo stability of the antibody and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity typically with non-human homologues of the targeted peptide, or with other proteins or tissues), and the ability to maintain high expression levels of proteins in mammalian cells. The foregoing properties or characteristics are observed, measured or evaluated using techniques known in the art, including but not limited to ELISA, FACS or surface plasmon resonance analysis such as BIACORE, unrestricted in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections from various sources (including human, primate or any other source).

[0031] 20. As used herein, the term "Fc" or "Fc region" or "Fc fragment" refers to a polypeptide composed of the CH2 and CH3 domains of IgA, IgD and IgG, or the CH2, CH3 and CH4 domains of IgE and IgM through the hinge region. Although the breakdown of the Fc fragment is variable, the Fc fragment of the human IgG heavy chain generally refers to the polypeptide from A231 to its carboxyl terminus.

[0032] 21. As used herein, the term "affinity" or "binding affinity" refers to the inherent binding affinity reflecting the interaction between the members of a binding pair. The affinity of molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (Koff and Kon, respectively). Affinity can be measured by common methods known in the art. A specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0033] 22. As used herein, the term "high affinity" or "high avidity", for IgG antibodies, refers to a KD for the antigen of 1.0×10 -6 M or lower, preferably 5.0×10-8 M or lower, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or lower, more preferably 1.0×10 -9 M or lower. For other antibody isotypes, "high affinity" binding may vary. For example, "high affinity" binding for the IgM isotype refers to a KD of 10 -6 M or lower, preferably 10 -7 M or lower, more preferably 10-8 M or lower.

[0034] 23. The term "antibody-drug conjugate" refers to a substance obtained by linking a bioactive compound fragment to an antibody or an antigen-binding fragment thereof. The bioactive compound fragment and the targeting moiety may be linked by a linker. The linker is capable of cleavage in a specific environment (e.g., an intracellular low pH environment) or under a specific action (e.g., the action of lysosomal proteases), so that the bioactive compound fragment is separated from the targeting moiety or the antibody or its antigen-binding fragment. The linker contains cleavable or non-cleavable units, such as peptides or disulfide bonds. The bioactive compound fragment is directly linked to the targeting moiety or the antibody or its antigen-binding fragment by a covalent bond, and the covalent bond is capable of cleavage in a specific environment or under a specific action, so that the bioactive compound fragment is separated from the antibody or its antigen-binding fragment.

[0035] 24. As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly restricted, the term includes nucleic acids containing analogs of known natural nucleotides that have binding properties similar to those of the reference nucleic acid and are metabolized in a manner similar to that of natural nucleotides (see, e.g., U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing such mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequence. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer, Nucleic Acid Res. 19:5081 (1991); Ohtsuka, J. Biol. Chem. 260:2605-2608 (1985); Rossolini, Mol. Cell. Probes 8:91-98 (1994)).

[0036] 25. The term "isolated nucleic acid" as used herein is a nucleic acid that has been identified and separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain nucleic acid molecules, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0037] 26. As used herein, the term "vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated to the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). After being introduced into a host cell, other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate with the host genome. In addition, certain vectors are capable of directing the expression of genes operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0038] 27. As used herein, the term "expression vector" refers to a nucleic acid molecule that is capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell. An expression vector contains one or more phenotypic selection markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host if desired.

[0039] 28. As used herein, the terms "cell", "cell line" are used interchangeably and all such names include their progeny. The term "host cell" refers to a cell into which a vector can be introduced, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0040] 29. As used herein, the term "pharmaceutical composition" generally refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition is to be administered. The composition is sterile.

[0041] 30. The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0042] 31. As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc.

[0043] 32. As used herein, the terms "therapeutically effective amount", "therapeutically effective dose", and "effective amount" refer to an amount of the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention that, when administered alone or in combination with other therapeutic agents to cells, tissues, or a subject, is effective in preventing or ameliorating the symptoms of one or more diseases or conditions or the progression of the disease or condition. A therapeutically effective dose also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to result in symptom amelioration, such as an amount that treats, cures, prevents, or ameliorates a related medical condition or increases the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, the therapeutically effective dose refers only to that ingredient. When administered in combination, the therapeutically effective dose refers to the combined amount of the active ingredients that elicits a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an increase of at least 10% in a diagnostic criterion or parameter; typically at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%.

[0044] 33. As used herein, the term "EC50" refers to the concentration for 50% of maximal effect, i.e., the concentration that elicits 50% of the maximal effect.

[0045] 34. As used herein, the terms "cancer" or "tumor" refer to a physiological disorder in mammals typically characterized by unregulated cell growth. This definition includes benign and malignant cancers as well as dormant tumors or micrometastases.

[0046] 35. As used herein, the terms "autoimmune disease" or "autoimmune disorder" refer to a disease caused by a decrease, reduction, and / or disruption of immune tolerance of the immune system to its own components for some reason, resulting in damage to its own organ tissues by autoantibodies and / or sensitized lymphocytes, and specifically manifested as dysfunction of the corresponding tissues and organs.

[0047] The technical solution of the present invention is as follows:

[0048] In a first aspect, the present invention provides an antibody, the amino acid sequence of which comprises:

[0049] (1) HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and / or;

[0050] (2) amino acid sequences having at least 80% sequence identity with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0051] SEQ ID NO:1 is GFSIHIYA.

[0052] SEQ ID NO:2 is ITRGGVT.

[0053] SEQ ID NO:3 is AGGTNGGY.

[0054] According to some embodiments of the present invention, 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 sequences shown in SEQ ID NOs: 1-3.

[0055] According to some embodiments of the present invention, the amino acid sequence of the antibody comprises: an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid differences compared to the amino acid sequences shown in SEQ ID NOs: 1-3.

[0056] Specifically, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably substitutions in which one amino acid residue in the following groups (a) to (e) is replaced by another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0057] Furthermore, the conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln, or to Glu; Met to Leu, to Tyr, or to Ile; Phe to Met, to Leu, or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile, or to Leu.

[0058] According to some embodiments of the present invention, the functional active variant is a single-domain antibody variant (mutant) having the same or similar affinity or function as the anti-IL-17A single-domain antibody described in (1), for example, a variant (mutant) that can specifically bind to IL-17A and block the binding of IL-17A to its receptor.

[0059] In a second aspect, the present invention provides an antibody, the amino acid sequence of which comprises:

[0060] (1) FR1, FR2, FR3, and FR4 with amino acid sequences as shown in SEQ ID NOs: 4 - 7; and / or;

[0061] (2) An amino acid sequence having at least 80% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4 - 7.

[0062] Furthermore, 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 sequences shown in SEQ ID NOs: 4 - 7.

[0063] FR1 shown in SEQ ID NO: 4, FR2 shown in SEQ ID NO: 5, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 7.

[0064] SEQ ID NO: 4 is EVQLVESGGGLVQPGGSLRLSCAAS.

[0065] SEQ ID NO: 5 is MGWYRQAPGKQRELVAT.

[0066] SEQ ID NO: 6 is

[0067] NNADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCN.

[0068] SEQ ID NO: 7 is WGQGTQVTVSS.

[0069] Furthermore, the amino acid sequence of the antibody comprises: an amino acid sequence having 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,

[0070] 34, 35, 36, 37, 38, or 39 amino acid differences compared with the amino acid sequences shown in SEQ ID NOs: 4 - 7.

[0071] In a third aspect, the present invention provides an antibody, the amino acid sequence of which comprises:

[0072] (1) The amino acid sequence of the antibody in the first aspect; and

[0073] (2) The amino acid sequence of the antibody described in the second aspect.

[0074] In a fourth aspect, the present invention provides an antibody, the amino acid sequence of which comprises:

[0075] FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4;

[0076] wherein HCDR1, HCDR2, and HCDR3 are selected from the following:

[0077] (1) The amino acid sequences shown in SEQ ID NO: 1-3; or

[0078] (2) Functional active variants of the amino acid sequences having 1, 2, 3, 4, or 5 amino acid differences compared to (1).

[0079] FR1, FR2, FR3, and FR4 are selected from the following:

[0080] 1) The amino acid sequences shown in SEQ ID NO: 4-7; or

[0081] 2) Functional active variants of the amino acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to 1).

[0082] Preferably, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 2, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 3.

[0083] Preferably, the amino acid sequence of the FR1 is as shown in SEQ ID NO: 4, the amino acid sequence of the FR2 is as shown in SEQ ID NO: 5, the amino acid sequence of the FR3 is as shown in SEQ ID NO: 6, and the amino acid sequence of the FR4 is as shown in SEQ ID NO: 7.

[0084] Preferably, the amino acid sequence of the antibody is as shown in SEQ ID NO: 8.

[0085] SEQ ID NO: 8 is EVQLVESGGGLVQPGGSLRLSCAAS GFSIHIYA MGWYRQAPGKQRELVATITRGGVTNNADSVKGRFTISRDNAKNTAYLQMN SLKPEDTAVYYCNAGGTNGGYWGQGTQVTVSS.

[0086] Furthermore, the antibodies described in the first to fourth aspects above are single-domain antibodies.

[0087] Furthermore, the antibody is an anti-IL-17A antibody.

[0088] Preferably, the antibody comprises an antibody heavy chain framework region or a variant thereof partially or wholly selected from human, murine, primate or camelid sources;

[0089] Furthermore, it comprises an antibody heavy chain framework region or a variant thereof partially or wholly selected from camelid sources;

[0090] Even further, it comprises an antibody heavy chain framework region or a variant thereof partially or wholly selected from alpaca sources.

[0091] In a fifth aspect, the present invention provides a recombinant protein comprising the antibody according to any one of the first to fourth aspects above.

[0092] Preferably, the recombinant protein further comprises a bioactive protein or a functional fragment thereof that assists in its expression and / or secretion, or prolongs its half-life in vivo;

[0093] Furthermore, the bioactive protein or the functional fragment thereof is selected from at least one of an immunoglobulin Fc domain, serum albumin, albumin-binding polypeptide, prealbumin, carboxy-terminal peptide, elastin-like polypeptide, His tag, GST tag, MBP tag, FLAG tag and SUMO tag.

[0094] Even further, the bioactive protein or the functional fragment thereof is a human immunoglobulin Fc domain, preferably the Fc domain of human IgG, such as the Fc domains of human IgG1, IgG2, IgG3, IgG4, and more preferably the Fc domain of human IgG1.

[0095] According to some embodiments of the present invention, the amino acid sequence of the human IgG1 Fc is as shown in SEQ ID NO:10.

[0096] SEQ ID NO:10:

[0097] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0098] Specifically, the nucleotide sequence encoding SEQ ID NO:10 is as shown in SEQ ID NO:11.

[0099] SEQ ID NO:11:

[0100] GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA.

[0101] According to some embodiments of the present invention, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants. The Fc region variants can comprise a human Fc region sequence (such as a human IgG1, IgG2, IgG3 or IgG4 Fc region) that contains amino acid modifications (such as substitutions, deletions and insertions) at one or more amino acid positions.

[0102] According to some embodiments of the present invention, the recombinant protein can be a monomer, dimer or multimer.

[0103] In a sixth aspect, the present invention provides an antibody preparation, which comprises the antibody according to any one of the first to fourth aspects described above and a pharmaceutically acceptable carrier.

[0104] Specifically, the "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration.

[0105] In a seventh aspect, the present invention provides a polyclonal antibody, which comprises the antibody according to any one of the first to fourth aspects described above.

[0106] In an eighth aspect, the present invention provides a kit, which comprises:

[0107] (1) The antibody, the recombinant protein, the antibody preparation or the polyclonal antibody according to any one of the first to fourth aspects; and;

[0108] (2) A container for loading the antibody preparation.

[0109] Preferably, the kit may further include a container, a buffer solution, an antibody that recognizes the IL-17A protein, a detection substrate, etc.

[0110] In a ninth aspect, the present invention provides an antibody-drug conjugate, which comprises:

[0111] (1) The antibody, the recombinant protein, the antibody preparation or the polyclonal antibody according to any one of the first to fourth aspects; and;

[0112] (2) A conjugate moiety that binds to (1).

[0113] According to some embodiments of the present invention, the conjugate moiety includes a detectable label, a drug, a toxin, a cytokine, a radionuclide and / or an enzyme.

[0114] In a tenth aspect, the present invention provides a nucleic acid molecule, which encodes the antibody, the recombinant protein or the polyclonal antibody according to any one of the first to fourth aspects described above.

[0115] According to some embodiments of the present invention, the nucleic acid can be RNA, DNA or cDNA.

[0116] According to some embodiments of the present invention, the nucleic acids of the present invention can also be in the form of a vector, can be present in a vector and / or can be part of a vector, such as a plasmid, a cosmid or a YAC. The vector can be an expression vector, which usually contains at least one nucleic acid of the present invention, operably linked to one or more suitable expression regulatory elements (such as promoters, enhancers, terminators, etc.).

[0117] Specifically, the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:8 has the sequence shown in SEQ ID NO:9.

[0118] SEQ ID NO:9:

[0119] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCGGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTTAGTATCCACATCTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGCTGGTCGCAACTATTACTAGAGGTGGTGTAACAAATAATGCAGACTCCGTGAAGGGGCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGCGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCAGGTGGGACGAACGGGGGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。

[0120] In the eleventh aspect, the present invention provides a biological expression vector, which contains the nucleic acid molecule.

[0121] According to some embodiments of the present invention, the biological expression vector can be a eukaryotic expression vector or a prokaryotic expression vector, preferably a eukaryotic expression vector.

[0122] Preferably, the eukaryotic expression vector is selected from yeast expression vectors, insect expression vectors or mammalian expression vectors;

[0123] More preferably, the mammalian expression vector is selected from retroviral expression vectors, lentiviral expression vectors, adenoviral expression vectors, adeno-associated viral expression vectors.

[0124] In the twelfth aspect, the present invention provides a host cell, the genome of which is integrated with the nucleic acid molecule; or; contains the biological expression vector.

[0125] Preferably, the host cell is a bacterial cell, a fungal cell or a mammalian cell.

[0126] Further, the bacterial cell includes cells of Gram-negative bacterial strains and Gram-positive bacterial strains.

[0127] Still further, the Gram-negative bacterial strains include Escherichia coli strains, Pseudomonas strains and Proteus strains.

[0128] Still further, the Gram-positive bacterial strains include Streptomyces strains, Bacillus strains and Staphylococcus strains.

[0129] Further, the fungal cell includes cells of species of Trichoderma, Neurospora and Aspergillus.

[0130] Further, the mammalian cell includes HEK293 cells, HeLa cells, CHO cells and COS cells.

[0131] Still further, the mammalian cell is HEK293 cell.

[0132] In a thirteenth aspect, the present invention provides a pharmaceutical composition, which comprises: the antibody, the recombinant protein, the antibody preparation, the polyclonal antibody, the antibody-drug conjugate, the nucleic acid molecule, the biological expression vector or the host cell according to any one of the first aspect to the fourth aspect; and;

[0133] Optionally, the pharmaceutical composition further includes at least one pharmaceutically acceptable excipient.

[0134] Specifically, the pharmaceutically acceptable excipient is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, demolding agents, coating agents, taste correctors, antioxidants.

[0135] In a fourteenth aspect, the present invention provides the use of the antibody, the recombinant protein, the antibody preparation, the polyclonal antibody, the kit, the antibody-drug conjugate, the nucleic acid molecule, the biological expression vector or the host cell according to any one of the first aspect to the fourth aspect, and the use is selected from at least one of the following:

[0136] (1) Preparing a drug for preventing and / or treating autoimmune diseases;

[0137] (2) Preparing a drug for preventing and / or treating cancer;

[0138] (3) Preparing a detection reagent or kit.

[0139] Specifically, the autoimmune diseases include Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic scleroderma, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, aortitis syndrome, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Castleman's disease, Sjogren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, allergic vasculitis, rheumatoid vasculitis, large vessel vasculitis, ANCA-associated vasculitis, Cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disease, Guillain-Barre syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic fatty liver disease, primary biliary cirrhosis, Good-pasture syndrome, rapidly progressive glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, autoimmune adrenocortical hypofunction, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type I diabetes mellitus, slowly progressive type I diabetes mellitus, localized scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vulgar vitiligo, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada's 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 rhinosinusitis, dilated cardiomyopathy, systemic mastocytosis or inclusion body myositis.

[0140] Specifically, the cancers include basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, peritoneal cancer, cervical cancer, cholangiocarcinoma, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, 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 system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, B-cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic myelogenous leukemia.

[0141] In a fifteenth aspect, the present invention provides a method for in vitro detecting IL-17A in a sample for non-diagnostic purposes, the method comprising the following steps:

[0142] (1) Binding the antibody, the recombinant protein, the antibody preparation, the polyclonal antibody, the antibody-drug conjugate described in any one of the first to fourth aspects to the test sample;

[0143] (2) Detecting the antigen-antibody complex and interpreting the results.

[0144] In a sixteenth aspect, the present invention provides a method for preventing and / or treating an autoimmune disease, the method comprising: administering to a subject a therapeutically effective amount of the antibody, the recombinant protein, the antibody preparation, the polyclonal antibody, the antibody-drug conjugate, the nucleic acid molecule, the biological expression vector, the host cell or the pharmaceutical composition described in any one of the first to fourth aspects.

[0145] In a seventeenth aspect, the present invention provides a method for preventing and / or treating cancer, the method comprising:

[0146] Administering to a subject a therapeutically effective amount of the antibody, the recombinant protein, the antibody preparation, the polyclonal antibody, the antibody-drug conjugate, the nucleic acid molecule, the biological expression vector, the host cell or the pharmaceutical composition described in any one of the first to fourth aspects.

[0147] The beneficial effects of the present invention include:

[0148] The single-domain antibody 1-F8 of the present invention has good binding ability. The EC50 value of the single-domain antibody 1-F8 is 1.614, and the EC50 of the positive antibody (Lxekizumab) is 10.06. The single-domain antibody 1-F8 has good binding activity with the Human IL-17A protein.

[0149] The IL-17A single-domain antibody 1-F8 can block the activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc by the Human IL-17A protein, showing a good blocking effect.

[0150] The single-domain antibody 1-F8 has strong stability, with Tm = 62.57 and Tagg = 80.09. Description of the Drawings

[0151] Figure 1 It is a SDS-PAGE test result diagram of the IL-17A recombinant protein.

[0152] Figure 2 It is a test result diagram of the NIH-3T3 cell activation experiment with the purchased IL-17A (Acro) protein.

[0153] Figure 3 It is a test result diagram of the NIH-3T3 cell activation experiment with the recombinant IL-17A (TEST) protein.

[0154] Figure 4 It is a test result diagram of the binding experiment between IL17A and the reporter gene cell line.

[0155] Figure 5 It is the test result of the IL17A-activated cell experiment.

[0156] Figure 6 It is the agarose gel electrophoresis diagram of alpaca #2.

[0157] Figure 7 It is the agarose gel electrophoresis diagram of alpacas #2 + #3.

[0158] Figure 8 It is the flow cytometry test result diagram of the yeast library of alpaca #2. A: NC group: primary antibody: not added, secondary antibody: PE-streptavidin; B: original library experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, AlexaFluor 647-V5; C: 1MACS experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, AlexaFluor 647-V5; D: 2MACS experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, AlexaFluor 647-V5.

[0159] Figure 9Flow cytometry detection results of the yeast library for Alpaca 2#+3#. A: NC group: primary antibody: not added, secondary antibody: PE-streptavidin; B: original library experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, AlexaFluor 647-V5; C: 1 MACS experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, AlexaFluor 647-V5; D: 2 MACS experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, AlexaFluor 647-V5.

[0160] Figure 10 FACS detection of the binding of monoclonal antibodies against the IL17A target of Alpaca 2#.

[0161] Figure 11 FACS detection of the binding of monoclonal antibodies against the IL17A target of Alpaca 2#+3#.

[0162] Figure 12 SDS-PAGE results of antibody purification. The left lane is the marker, and the right lane is 1-F8.

[0163] Figure 13 ELISA Binding experimental results of the candidate antibody.

[0164] Figure 14 Blocking function experimental results of the candidate antibody.

[0165] Figure 15 Thermal stability detection results of the single-domain antibody 1-F8.

[0166] Figure 16 Thermal stability detection results of the positive control antibody. Detailed implementation

[0167] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the following specific embodiments are used to further clarify the present invention. However, the following embodiments are only the preferred embodiments of the present invention, not all. Based on the embodiments in the implementation manner, other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operation methods used are conventional operation methods, the equipment used is conventional equipment, and the equipment materials used in each embodiment are the same.

[0168] Experimental reagents:

[0169] Agar (Sigma, CAT#A1296); Peptone (Sigma, CAT#93926); Yeast extract (OXOID, CAT#:LP0021); Sodium chloride (Aladdin, CAT#:C111533); Potassium chloride (Aladdin, CAT#:P112133); Magnesium sulfate (Sinopharm, CAT#:10013018); Magnesium chloride (Sinopharm, CAT#:10012818); Glucose (Sangon Biotech, CAT#:GT1991); SfiI (NEB, CAT#:R0123L); T4 DNA ligase (TaKaRa, CAT#:2011A); PrimeScript TM II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#:6210B); NuHi power mix (Xinhai Biotech, CAT#:NH9303); 3M Sodium acetate (pH5.2 - 6) (Sigma, CAT#:126 - 96 - 5); DNA Fragment Recovery Kit (TakaRa, CAT#:9761); Gel Extraction Kit (Qiagen, CAT#:28706); Tiangen Plasmid Maxi Kit (Tiangen, 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 Competent Cells (iCarTab); BL21 Competent Cells (Biomed, BC201-02); 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).

[0170] Experimental consumables:

[0171] 50 mL Falcon centrifuge tubes (Corning, CAT#352070); Electroporation cuvettes (Bio-Rad 0.2 cm); RNase-free 1.5 mL EP tubes (QSP, CAT#: 509-GRD-Q); 200 μL RNase-free PCR tubes (Axygen, PCR-02D-C); T125 shake flasks (Corning, CAT#431143); 15 mL Falcon centrifuge tubes (Corning, CAT#430052); 6-well plates (Corning, CAT#3516); 96-well plates (Corning, CAT#3365); 96-well black plates (F-BOTTOM(CHIMNEY WELL)BLACK).

[0172] Experimental equipment:

[0173] Electroporator (Eppendorf Multiporator); Centrifuge (Thermo FRESCO-17); Incubator (Shanghai Jinghong DNP-9052); Thermostatic shaking incubator (Jingqi CO-O6U); Laminar flow hood (Sujing Antai SW-CJ-1FD); PCR instrument (Applied Biosystems ABI2720); Biological safety cabinet (Haier, HR40-IIA2); Flow cytometer (Thermo Attune Nxt flow cytometer); Thermo 3111 CO2 incubator; ForteBio OCTET R2.

[0174] The primers used for screening, cloning VHH fragments, and constructing nanobodies in the following examples were designed with reference to the following literature:

[0175] 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.

[0176] 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.

[0177] Studies on design of single domain antibodies by AlpacaVHH phage library and high throughput sequencing to construct Fab antibody purification system (http: / / hdl.handle.net / 10232 / 00030916).

[0178] Example 1

[0179] 1. Preparation of Recombinant Protein IL-17A (Human)

[0180] Retrieve the sequence information of Human IL-17A (Q16552-1) (SEQ ID NO: 12) from the UniProt database, add a 6xHis tag at the C-terminus, perform gene synthesis after prokaryotic codon optimization, and subclone it into the pET28a vector; after verification by Sanger sequencing, extract the plasmid.

[0181] Transform the recombinant plasmid into BL21 competent cells, induce overnight with 0.5 mM IPTG, collect the bacterial solution and lyse it; purify the recombinant protein using a nickel column.

[0182] The purity of the target protein was detected by SDS-PAGE, and the results are as Figure 1 shown. After purification, the purity of the IL-17A antigen protein was greater than 90%.

[0183] SEQ ID NO:12:

[0184] MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNI HNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINAD GNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA.

[0185] 2. Preparation of the positive control antibody Ixekizumab

[0186] (1) Synthesize the variable regions of the heavy and light chains of Ixekizumab (the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14). Subclone the heavy chain variable region into the pcDNA3.4-hIgG4 vector (the amino acid sequence of IgG4 is as shown in SEQ ID NO:15), and subclone the light chain variable region into the pcDNA3.4-hIgKc vector (the amino acid sequence of IgG KC is as shown in SEQ ID NO:16). After verification by Sanger sequencing, use a plasmid large-scale extraction kit to prepare endotoxin-free plasmids for standby.

[0187] (2) Take out the LVTransm transfection reagent, heavy chain expression vector, and light chain expression vector from the refrigerator, thaw them at room temperature, and mix them thoroughly by pipetting up and down. Take out the PBS buffer and warm it to room temperature. Add 2 mL of PBS to one well of a 6-well plate, add 50 μg of the heavy chain expression vector and light chain expression vector respectively, mix well by pipetting up and down, then add 300 μL of LVTransm, and immediately mix well by pipetting. Let it stand at room temperature for 10 minutes.

[0188] (3) Add the above DNA / LVTransm complex to 100 mL of 293F cells, gently shake to mix well, and place the cells in an incubator at 37°C and 5% CO2, and continue to culture at 130 RPM.

[0189] (4) After continuous culture for 5 - 7 days, centrifuge to collect the culture medium supernatant, filter it through a 0.45 μm filter membrane, transfer the filtrate to a sterile centrifuge tube, and purify the antibody using a Protein A column.

[0190] The purity of the target antibody protein was detected by SDS-PAGE, and the purity > 95%.

[0191] SEQ ID NO:13:

[0192] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEW MGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARY DYFTGTGVYWGQGTLVTVSS。

[0193] SEQ ID NO:14:

[0194] DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQ LLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFG QGTKLEIK。

[0195] SEQ ID NO:15:

[0196] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0197] SEQ ID NO:16:

[0198] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC。

[0199] 3. ELISA Detection of the Binding Activity of Recombinant Human IL-17A Protein with Control Antibody

[0200] (1) Dilute the recombinant IL-17A protein with sterile CBS to a final concentration of 2 μg / mL. Take a new 96-well plate and add 100 μL / well for coating overnight at 4°C.

[0201] (2) Remove the antigen coating solution and wash 3 times with PBST (containing 0.5% Tween).

[0202] (3) Add 200 μL / well of 3% MPBS and incubate at 37°C for 2 hours for blocking.

[0203] (4) After removing the blocking buffer, wash the plate 3 times with PBST.

[0204] (5) Dilute the positive control antibody Ixekizumab to 10 μg / ml with PBS, make 7-fold serial dilutions 5 times, add 100 μL / well to the ELISA plate, and incubate at room temperature for 1 hour. The control well is PBS.

[0205] (6) Remove the liquid in the wells and wash 3 times with PBST.

[0206] (7) Dilute the secondary antibody HRP-ProteinA (Boster, BA1080) 1:10000 and add 100 μL / well to the ELISA plate, then incubate at room temperature for 1 hour.

[0207] (8) After removing the liquid in the wells, wash the plate 3 times with PBST.

[0208] (9) Add 100 μL / well of TMB chromogenic solution.

[0209] (10) Incubate at room temperature in the dark for 15 minutes.

[0210] (11) Add 50 μL / well of stop solution (2M HCl).

[0211] (12) Read the OD450 value in the wells using an ELISA reader.

[0212] The results of the binding ability of recombinant IL-17A protein with the positive antibody are shown in the following table. It can be seen from the table that the positive antibody binds well with the IL-17A antigen protein and can be used for immunization.

[0213] Table 1.

[0214]

[0215] 4. Detection of the Activity of Recombinant Human IL-17A Protein

[0216] Experimental procedures:

[0217] (1) Resuscitate NIH-3T3 cells from liquid nitrogen, perform continuous passage culture to make the cells in the logarithmic growth phase. After cell counting, inoculate the cells into a 96-well plate at a cell density of 2×10 5 / well.

[0218] (2) Add 100 μl of IL-17A at different concentrations (ACRO, Cat#ILA-H5118) and the prepared recombinant IL-17A protein (the protein from ACRO as a control) to each well, with final concentrations of 0 μg / ml, 0.00001 μg / ml, 0.0001 μg / ml, 0.001 μg / ml, 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml, and 10 μg / ml respectively.

[0219] (3) Incubate at 37 °C and 5% CO2 for 48 hours. After the culture is completed, gently take out the 96-well plate, centrifuge to collect the culture medium supernatant, and detect the secretion of IL-6 using a mouse IL-6 ELISA kit.

[0220] (4) Process the data using PRISM GraphPad, draw a curve graph, and calculate the EC50 value.

[0221] Experimental results:

[0222] The results are as Figures 2 - 3 shown. According to the detection results, the IL-17A antigen protein has the activity of activating NIH-3T3 cells to express mIL-6, and the activity of IL-17A (TEST, hereinafter referred to as the recombinant IL-17A protein) is positively correlated with its concentration and can be used for immunization.

[0223] 5. Construction of IL-17A reporter gene cell line

[0224] Experimental steps:

[0225] According to the amino acid sequence information of IL-17RA (UniProtKB: Q96F46, SEQ ID NO: 17) and IL-17RC (UniProtKB: Q8NAC3, SEQ ID NO: 18), construct a lentiviral expression vector and package lentivirus. Co-infect 293 cells, screen for recombinant 293 cells that overexpress both receptors simultaneously, and further stably transfect the NFKB-Luciferase (amino acid sequence as shown in SEQ ID NO: 19, nucleotide sequence encoding it as shown in SEQ ID NO: 20) and ACT1 genes (nucleotide sequence as

[0226] As shown in SEQ ID NO: 21, the IL-17A reporter gene cell line 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc was constructed. IL-17A protein was added for activation, and at the same time, the positive control antibody Ixekizumab was added for the detection of the blocking experiment, and its EC50 value was calculated to establish a candidate antibody targeting IL-17A and an in vitro pharmacodynamic evaluation cell line.

[0227] SEQ ID NO:17:

[0228] MGAARSPPSAVPGPLLGLLLLLLGVLAPGGASLRLLDHRALVCSQPGLNCTVKNSTCLDDSWIHPRNLTPSSPKDLQIQLHFAHTQQGDLFPVAHIEWTLQTDASILYLEGAELSVLQLNTNERLCVRFEFLSKLRHHHRRWRFTFSHFVVDPDQEYEVTVHHLPKPIPDGDPNHQSKNFLVPDCEHARMKVTTPCMSSGSLWDPNITVETLEAHQLRVSFTLWNESTHYQILLTSFPHMENHSCFEHMHHIPAPRPEEFHQRSNVTLTLRNLKGCCRHQVQIQPFFSSCLNDCLRHSATVSCPEMPDTPEPIPDYMPLWVYWFITGISILLVGSVILLIVCMTWRLAGPGSEKYSDDTKYTDGLPAADLIPPPLKPRKVWIIYSADHPLYVDVVLKFAQFLLTACGTEVALDLLEEQAISEAGVMTWVGRQKQEMVESNSKIIVLCSRGTRAKWQALLGRGAPVRLRCDHGKPVGDLFTAAMNMILPDFKRPACFGTYVVCYFSEVSCDGDVPDLFGAAPRYPLMDRFEEVYFRIQDLEMFQPGRMHRVGELSGDNYLRSPGGRQLRAALDRFRDWQVRCPDWFECENLYSADDQDAPSLDEEVFEEPLLPPGTGIVKRAPLVREPGSQACLAIDPLVGEEGGAAVAKLEPHLQPRGQPAPQPLHTLVLAAEEGALVAAVEPGPLADGAAVRLALAGEGEACPLLGSPGAGRNSVLFLPVDPEDSPLGSSTPMASPDLLPEDVREHLEGLMLSLFEQSLSCQAQGGCSRPAMVLTDPHTPYEEEQRQSVQSDQGYISRSSPQPPEGLTEMEEEEEEEQDPGKPALPLSPEDLESLRSLQRQLLFRQLQKNSGWDTMGSESEGPSA。

[0229] SEQ ID NO:18:

[0230] MPVPWFLLSLALGRSPVVLSLERLVGPQDATHCSPVSLEPWGDEERLRVQFLAQQSLSLAPVTAATARTALSGLSGADGRREERGRGKSWVCLSLGGSGNTEPQKKGLSCRLWDSDILCLPGDIVPAPGPVLAPTHLQTELVLRCQKETDCDLCLRVAVHLAVHGHWEEPEDEEKFGGAADSGVEEPRNASLQAQVVLSFQAYPTARCVLLEVQVPAALVQFGQSVGSVVYDCFEAALGSEVRIWSYTQPRYEKELNHTQQLPDCRGLEVWNSIPSCWALPWLNVSADGDNVHLVLNVSEEQHFGLSLYWNQVQGPPKPRWHKNLTGPQIITLNHTDLVPCLCIQVWPLEPDSVRTNICPFREDPRAHQNLWQAARLQLLTLQSWLLDAPCSLPAEAALCWRAPGGDPCQPLVPPLSWENVTVDKVLEFPLLKGHPNLCVQVNSSEKLQLQECLWADSLGPLKDDVLLLETRGPQDNRSLCALEPSGCTSLPSKASTRAARLGEYLLQDLQSGQCLQLWDDDLGALWACPMDKYIHKRWALVWLACLLFAAALSLILLLKKDHAKGWLRLLKQDVRSGAAARGRAALLLYSADDSGFERLVGALASALCQLPLRVAVDLWSRRELSAQGPVAWFHAQRRQTLQEGGVVVLLFSPGAVALCSEWLQDGVSGPGAHGPHDAFRASLSCVLPDFLQGRAPGSYVGACFDRLLHPDAVPALFRTVPVFTLPSQLPDFLGALQQPRAPRSGRLQERAEQVSRALQPALDSYFHPPGTPAPGRGVGPGAGPGAGDGT。

[0231] SEQ ID NO:19:

[0232] MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV。

[0233] SEQ ID NO:20:

[0234]

[0235] SEQ ID NO:21:

[0236]

[0237] Experimental results:

[0238] Construct the IL-17A reporter gene cell line 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc, and add recombinant IL-17A protein for binding. The FACS results show that: the constructed IL-17A receptor overexpressing cell line can bind to IL-17A, and the positive rate is greater than 90% (the results are as Figure 4 shown).

[0239] IL17A activation of cells experiment: Use recombinant IL-17A protein to activate 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc, and the results are as Figure 5 shown: Recombinant IL17A protein can effectively activate the luciferase expression in the 293F-IL17Ra / IL17Rc-NFκB-Luc reporter gene cell line.

[0240] Ixekizumab blocking IL17A function experiment: Add the positive control antibody Ixekizumab and recombinant IL-17A protein to 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cells. The positive control antibody Ixekizumab can inhibit the binding of IL17A protein to its membrane receptor and inhibit the intracellular NFκB signal, showing a dose effect.

[0241] 6. Animal immunization process

[0242] 6.1 Alpaca immunization

[0243] Use the above-prepared recombinant IL-17A antigen protein to immunize 2 alpacas (No. 2 and No. 3) for a total of 6 times. The immune adjuvant is GERBU, and the immune interval is 14 days. The last two immunizations are Freund's complete adjuvant.

[0244] Arrange blood collection as follows:

[0245] Collect 100 ml of blood from alpaca No. 2 after the fifth immunization, prepare a cDNA library, and construct a yeast display library (yeast display library 1); collect 100 ml of blood after the sixth immunization and prepare a cDNA library.

[0246] For alpaca No. 3, collect 100 ml of blood after the sixth immunization, prepare a cDNA library, and mix it with the cDNA library prepared from the blood collected from alpaca No. 2 after the sixth immunization to construct a yeast display library (yeast display library 2).

[0247] 6.2 Detection of immune titer

[0248] Experimental steps:

[0249] Separate sera from alpacas immunized different times, perform serial dilution, and conduct ELISA detection with the antigen-precoated 96-well plates respectively. The specific steps are as follows:

[0250] (1) Collect 5 mL of peripheral blood, place the centrifuge tube containing the blood sample in an incubator at 37 °C for 1 hour; then transfer the blood sample to 4 °C overnight;

[0251] (2) Place the centrifuge tube containing the blood sample in a centrifuge and centrifuge at 5000 rpm for 20 min; separate the upper serum, transfer the serum to a new sterile centrifuge tube, and collect the immune serum.

[0252] (3) Dilute the recombinant IL-17A protein with sterile CBS (carbonate buffer solution) to a final concentration of 1 μg / mL. Take a new 96-well ELISA plate, add 100 μL / well and coat overnight at 4 °C.

[0253] (4) Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20).

[0254] (5) Add 200 μL / well of 3% MPBS and block at 37 °C for 2 hours;

[0255] (6) After removing the blocking buffer, wash the plate 5 times with PBST;

[0256] (7) Add 100 μl of serially diluted serum (100 μL / well), incubate at room temperature for 1 hour, and the control wells are PBS;

[0257] (8) Remove the liquid in the wells and wash 5 times with PBST;

[0258] (9) Add 100 μl of HRP anti-Llama IgG (H+L) antibody (diluted 1:50000), incubate at room temperature for 1 hour;

[0259] (10) After removing the liquid in the wells, wash the plate 5 times with PBST;

[0260] (11) Add 100 μL / well of TMB chromogenic solution;

[0261] (12) Incubate at room temperature in the dark for 10 - 15 minutes;

[0262] (13) Add 50 μL / well of stop solution;

[0263] (14) Read the OD450 value in the wells using an ELISA reader.

[0264] Experimental results: The detection results are shown in Table 2 - Table 3 below. According to the ELISA detection results, the immune serum binds well with the IL17A recombinant protein, and as the immune serum is serially diluted, the OD value shows a gradient change.

[0265] Table 2. Detection results of immune titer

[0266]

[0267] Table 3. Detection results of immune titer

[0268]

[0269] 7. Construction process of antibody yeast library

[0270] 7.1 Isolation of PBMC and cloning of VHH antibody fragment

[0271] Experimental steps:

[0272] (1) Collect 100 mL of anticoagulated peripheral blood samples and isolate PBMC cells using lymphocyte separation medium.

[0273] (2) Extract RNA and perform reverse transcription using PrimeScript TM II 1st Strand cDNA Synthesis Kit to prepare cDNA.

[0274] 1) Prepare the following reaction mixture Mix in a 200 μL PCR tube:

[0275] Table 4.

[0276] Reagent Dosage Oligo dT Primer(50μM) 8μL dNTP Mixture(10mM each) 8μL Total RNA sample 20μg RNase - free water Add up to 80μL

[0277] 2) Incubate at 65 °C for 5 min and then quickly cool on ice.

[0278] 3) Prepare the reaction solution in the above PCR tube as shown in the following table:

[0279] Table 5.

[0280] Reagent Dosage The above - mentioned denatured reaction solution 80μL 5×PrimeScript II Buffer 32μL RNase Inhibitor(40Μ / μL) 4μL PrimeScript Ⅱ RTase(200Μ / μL) 8μL RNase - free water 36μL

[0281] 4) Pipette and mix well, then dispense 80 μL per tube, place in a PCR instrument at 42 °C for 1 hour, inactivate at 70 °C for 15 minutes, and finally store the cDNA sample on ice or at -20 °C for long term.

[0282] (3) Amplification of VHH fragment

[0283] 1) Prepare the first round of PCR reaction system (50 μL per tube):

[0284] Table 6.

[0285] Component Dosage Forward primer(5μM) 2μL Reverse primer(10μM) 1μL NuHi Power mix(2×) 25μL cDNA template 2μL Sterile water 20μL

[0286] 2) After preparing the PCR reaction system, set the PCR instrument according to the following program:

[0287] Table 7.

[0288]

[0289] 3) Agarose gel electrophoresis of PCR products

[0290] Perform electrophoresis analysis of PCR products using 1% agarose to separate fragments with a molecular weight of about 750 bp. Recover the PCR products using a gel extraction kit and measure the concentration with NanoDrop.

[0291] 4) Prepare the second-round PCR reaction system (50 μL / tube)

[0292] Table 8.

[0293] Component Dosage <![CDATA[2 nd F primer]]> 2μL <![CDATA[2 nd R primer]]> 2μL NuHi Power mix(2×) 25μL First - round PCR recovery product 200ng Sterile water Make up to 50μL

[0294] 5) After preparing the PCR reaction system, set the PCR instrument according to the following program:

[0295] Table 9.

[0296]

[0297] 6) Agarose gel electrophoresis analysis of second-round PCR products

[0298] Perform electrophoresis analysis of PCR products using 1% agarose to separate VHH fragments with a molecular weight of about 400 bp. Recover the VHH PCR products using a gel extraction kit and measure the concentration with NanoDrop.

[0299] Experimental results:

[0300] Collect peripheral blood, extract total RNA, reverse transcribe it into cDNA, and then perform three rounds of PCR using single-domain antibody amplification primers. Detect the PCR products by agarose gel electrophoresis (the results are shown in Figures 6 - 7 ): In the first round of PCR, PCR bands of about 1000 bp and 750 bp were obtained respectively. The 750-bp fragment was recovered by gel extraction and used as the template for the second round of PCR. In the second round of PCR, a band of about 400 bp was obtained, which was the VHH fragment. It was recovered by column and used as the template for the third round of PCR. In the third round of PCR, a band of about 500 bp was obtained, with homologous arms added, and then homologously recombined into the yeast display vector pDisplay subsequently.

[0301] 7.2 Construction of single-domain antibody yeast display library

[0302] (1) Linearize the yeast display vector pDisplay. The enzyme digestion system is as follows:

[0303] Table 10.

[0304]

[0305] 1) Digest the pDisplay vector with SfiI, aliquot 100 μL per tube, and digest overnight at 50 °C.

[0306] 2) Separate the pDisplay vector fragments using 1% agarose gel, excise the 5000 bp vector fragment for gel extraction, and measure the concentration with NanoDrop.

[0307] 3) Aliquot 200 μL of the recovered pDisplay digested product into each 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3 M sodium acetate, 1 μg / μL glycogen, pipette and mix well, add 880 μL of absolute ethanol, invert and mix well, and place at -80 °C.

[0308] (2) Construct a yeast display library by electroporation

[0309] 1) Streak the yeast competent strain stored at -80 °C onto a YPD solid medium plate and activate at 30 °C for 3 - 5 days;

[0310] 2) Inoculate a single colony of yeast competent cells into 50 mL of YPD medium and shake culture at 250 rpm and 30 °C for 1 - 2 days;

[0311] 3) Prepare the yeast competent strain. After mixing the linearized vector fragment and the PCR product, add them to the electroporation cuvette and perform electroporation; transfer the electroporated yeast competent cells into a culture flask and shake culture at 220 rpm and 30 °C for 1 h;

[0312] 4) Take 20 μL of the resuspension, dilute it 5000 - fold with SDCAA, pipette 100 μL, spread it on an SDCAA plate, culture for 2 - 3 days, calculate the library capacity, and continue to culture the remaining bacterial solution for 24 h;

[0313] 5) Preserve the bacteria: Collect the remaining bacterial solution into a 50 mL centrifuge tube, centrifuge at 3000 g for 5 min, discard the supernatant, add 10 mL of SDCAA to resuspend, mix with 50% glycerol:resuspension = 1:1, and store at -80 °C.

[0314] 8. Panning of the yeast display library

[0315] Experimental procedures:

[0316] 1) Add the yeast cultured in SDCAA to a 250 mL shake flask containing 50 mL of SGCAA medium, and culture it on a shaker at 30 °C and 240 rpm for 16 h.

[0317] 2) After centrifugation, discard the supernatant, resuspend with 1 mL of 0.5% PBSA, transfer to a 1.5 mL centrifuge tube, centrifuge at 3000 g for 5 min, and discard the supernatant. Wash once more with 0.5% PBSA.

[0318] 3) Wash the streptavidin magnetic beads incubated with the antigen twice with 0.5% PBSA (rotate and incubate at 4 °C for 5 minutes each time), place on a magnetic stand for 5 min, and discard the supernatant.

[0319] 4) Add the yeast cell suspension to the magnetic beads conjugated with the antigen, rotate and incubate at 4 °C for 60 minutes, place on a magnetic stand for 15 min.

[0320] 5) Discard the yeast cell suspension and retain the magnetic beads, wash three times with 0.5% PBSA (rotate and incubate at 4 °C for 5 minutes each time).

[0321] 6) Resuspend the magnetic beads with 1 mL of SDCAA medium, pipette 0.5 - 5 μL of the resuspended solution into 100 μL of SDCAA medium for plating. Divide the resuspended solution into two equal parts. Add 500 μL of 50% glycerol to one part (store at -80 °C); add the other part to a shake flask, supplement with 2 mL of SDCAA medium, and culture at 30 °C and 240 rpm for 16 h.

[0322] 7) Transfer the bacterial liquid in the shake flask to 50 mL of SDCAA medium (250 mL shake flask), and culture overnight at 30 °C and 240 rpm.

[0323] 8) Measure the OD600 value of the bacterial liquid. Take a portion of the bacterial liquid for centrifugation according to the OD600 value, resuspend with SGCAA, transfer to 50 mL of SGCAA medium to make the final OD600 value 1, and culture overnight at 30 °C and 240 rpm. Resuspend the remaining bacterial liquid with SDCAA:50% glycerol = 1:1 and store at -80 °C.

[0324] 9. Flow cytometry detection of yeast monoclonal

[0325] Spread the sorted yeast cell suspension on an SDCAA plate, pick monoclonal colonies for culture. After inducing expression for 48 h, incubate with Biotin-antigen, use PE-Streptavidin as the secondary antibody, and perform flow cytometry detection after incubation. Lyse the yeast clones bound to the target antigen with 0.2% SDS (incubate at 95 °C for 10 min), centrifuge, and take 0.5 μL of the bacterial liquid supernatant as a template for PCR amplification and submission for testing (store the remaining bacterial liquid at -20 °C). The flow cytometry detection results are as Figure 8As shown, according to the flow cytometry results, after the second magnetic sorting, the yeast positive rate was 37.9%, and the positive clones were significantly enriched. The sorted product was directly plated on the SDCAA plate, and monoclonal colonies were selected for flow cytometry detection.

[0326] After the 2#+3# alpaca yeast library was fully bound to the Biotin-IL-17A-His protein, 2 rounds of magnetic sorting were performed using streptavidin magnetic beads; the sorted yeast cells were cultured, induced for expression, and then subjected to flow cytometry analysis. Incubate with Biotin-IL-17A-His for 1 h, use PE Streptavidin as the secondary antibody, and perform flow cytometry detection after incubation. The results showed (as Figure 9 ) that after two rounds of magnetic sorting, the proportion of yeast cells bound to Biotin-IL-17A-His was 20.59%, and the positive clones were significantly enriched. The sorted product was directly plated on the SDCAA plate, and monoclonal colonies were selected for flow cytometry detection.

[0327] 10. Antibody sequence identification

[0328] Enrich positive clones; select the enriched monoclonal clones for Phage ELISA identification and sequence analysis of the clones to obtain the nucleic acid and amino acid sequence information of the candidate single-domain antibodies. As Figures 10 - 11 shown, 20 monoclonal colonies were randomly selected for sequence analysis, and the sequence differences were large, indicating good library diversity. For the amino acid sequence information of the CDR region of the candidate single-domain antibodies, the In silico method was used to analyze the possible post-translational modification sites.

[0329] According to the flow cytometry results of yeast monoclonal colonies, positive clones that bound to IL-17A-His were selected to extract genomic DNA, and the antibody sequences were obtained by PCR. According to the sequencing results of the PCR products, differential clones were selected for overlap PCR amplification. The specific steps are as follows:

[0330] (1) First-round PCR: Amplify CMV, VHH, and FC

[0331] 1) Prepare the PCR reaction system (50 μL system / reaction)

[0332] Table 11.

[0333]

[0334]

[0335] 2) The PCR reaction program is as follows:

[0336] 95 °C, 10 min; (95 °C, 15 s; 56 °C, 30 s; 68 °C, 60 s; 25 cycles); 68 °C, 10 min.

[0337] 3) Take 50 μL of the PCR product, add 1 / 10 volume of 10× loading buffer, and perform electrophoresis analysis using 1% agarose. The band size of CMV is about 750 bp, the band size of Fc is about 1400 bp, and the band size of VHH is about 500 bp.

[0338] 4) Excise the target band from the gel, purify the PCR product, and measure the concentration using NanoDrop (if the concentration is too high, it can be diluted for subsequent reactions).

[0339] (2) Second-round PCR: Overlap Extension PCR to ligate CMV, VHH, and FC

[0340] 1) Prepare the PCR reaction system

[0341] Table 12.

[0342] Component Dosage CMV 1st product 50ng VHH 1st product 50ng Fc 1st product 50ng NuHi Power mix(2×) 25μL Sterile water Make up to 46μL

[0343] 2) The PCR reaction program is as follows:

[0344] 95°C, 10 min; (95°C, 15 s; 60°C, 30 s; 68°C, 120 s; 15 cycles).

[0345] 3) Add 2 μL each of the upstream primer and the downstream primer;

[0346] The PCR reaction program is as follows:

[0347] 95°C, 10 min; (95°C, 15 s; 60°C, 30 s; 68°C, 120 s; 20 cycles); 68°C, 10 min.

[0348] 4) Purify the overlap PCR product using the DNA fragment recovery kit from TakaRa, and measure the concentration using NanoDrop. At least 10 μg of the PCR product is required. It is used for subsequent cell transfection verification.

[0349] 5) The transfection procedure is the same as that for the transfection of eukaryotic expression vectors.

[0350] Add a signal peptide to the N-terminus of VHH and IgG1-FC to the C-terminus, transiently transfect the PCR product into HEK293 cells; take the expressed antibody supernatant for ELISA detection: Add 100 μL of the transfection supernatant to a 96-well plate pre-coated with the recombinant antibody of IL-17A and incubate. Use HRP-Protein A as the secondary antibody for ELISA detection. The results are shown in Table 13: 1-F12 binds to the IL-17A-His antigen, and a eukaryotic expression vector is constructed.

[0351] Table 13. ELISA Binding Experiment Results of Supernatant of Transfected Candidate Clones

[0352]

[0353] 11. Expression and Purification of Candidate Single-Domain Antibodies

[0354] Experimental Procedures:

[0355] 1) According to the ELISA test results of the candidate antibodies, select the positive clones. Synthesize the obtained VHH antibody sequences respectively, and subclone them in series with human IgG1Fc (SEQ ID NO: 10) into the expression vector pcDNA3.4-hIgG1-Fc. After the vector is verified by sequencing, use the Qiagen plasmid large-scale extraction kit to prepare endotoxin-free plasmids for standby.

[0356] 2) Take out the LVTransm transfection reagent and the single-chain antibody expression vector from the refrigerator, thaw them at room temperature, and mix them thoroughly by pipetting up and down. Take out the PBS buffer and warm it to room temperature. Take 2 mL of PBS into one well of a 6-well plate, add 130 μg of the antibody expression vector respectively, mix well by pipetting up and down, then add 400 μL of LVTransm, immediately mix well by pipetting, and let it stand at room temperature for 10 minutes.

[0357] 3) Add the above DNA / LVTransm complex to 30 mL of 293F cells, and gently shake to mix well. Place the cells in an incubator at 37°C and 5% CO2, and culture them at 130 rpm for 6 - 8 hours. Then add 50 mL of fresh 293 cell medium, and put the cells back into the incubator for continued culture.

[0358] 4) After continuous culture for 7 days, centrifuge to collect the culture supernatant, filter it through a 0.45 μm filter membrane, transfer the filtrate to a sterile centrifuge tube, and purify the antibody using a Protein A column.

[0359] The steps for purifying the antibody using a Protein A column are as follows:

[0360] 1) Add the sample containing the target antibody to an EP tube and gently invert the tube to mix.

[0361] 2) Mix the EP tube at room temperature or incubate it on a rotator (for 1 - 4 hours or overnight), and add 100 mM PMSF to prevent protein degradation.

[0362] 3) Use a magnetic separation rack to collect the magnetic beads and discard the supernatant.

[0363] 4) Add 1 mL of binding / washing buffer to the EP tube and mix well. Use a magnetic stand to collect the magnetic beads and discard the supernatant. Repeat the washing step three times.

[0364] 5) Add 500 μL of elution buffer to the EP tube, resuspend quickly by pipetting or vortexing, and then incubate at room temperature (about 25 °C) on a shaker or gently invert the EP tube by hand for 5 minutes.

[0365] 6) Use a magnetic separation stand to collect the magnetic beads and transfer the supernatant containing the eluted antibody to a clean EP tube.

[0366] 7) Repeat steps 1) and 2) twice.

[0367] 8) Add 1 / 10 of the neutralization buffer to every 500 μL of eluate to neutralize the pH, which helps to maintain the biological activity of the antibody and avoid antibody inactivation.

[0368] 9) Binding / washing buffer: 1×PBS, pH 7.0.

[0369] Elution buffer: (1) 0.1 M glycine, pH 2 - 3 (2) 0.1 M NaAc-HAc, pH 3.6.

[0370] Neutralization buffer: 1 M Tris, pH 8.5.

[0371] Magnetic bead regeneration buffer: 0.1 M NaOH.

[0372] The experimental results are as Figure 12 shown.

[0373] Example 2 ELISA for Detecting the Binding of Recombinant Antibody to Target Protein

[0374] Experimental procedures:

[0375] 1) Dilute the recombinant protein with sterile CBS to a final concentration of 2 μg / mL. Take a new 96-well ELISA plate and add 100 μL / well, then coat at 4 °C overnight.

[0376] 2) Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20).

[0377] 3) Add 200 μL / well of 3% MPBS and block at 37 °C for 2 hours;

[0378] 4) After removing the blocking buffer, wash the plate 5 times with PBST;

[0379] 5) Add the purified single-domain antibody at an initial concentration of 10 μg / mL and dilute it 7-fold in a 5-fold gradient. The results show that the EC50 value of single-domain antibody 1-F8 is 1.614, and the EC50 of the positive antibody (Ixekizumab)

[0380] = 10.06. Single-domain antibody 1-F8 has good binding activity with Human IL-17A protein ( Figure 13 ). The EC50 values of each candidate antibody are shown in Table 14.

[0381] Table 14. EC50 values of candidate antibodies

[0382] Antibody number 1-F8 Ixekizumab EC50 1.614 10.06

[0383] Example 3 Detection of the binding of IL17A to the reporter gene cell line by FACS

[0384] Experimental procedure:

[0385] 1) Resuscitate the 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cell line from liquid nitrogen and adjust the cell state to the logarithmic growth phase;

[0386] 2) Divide the cells into several portions, with the number of cells in each portion being 2×10 5 cells;

[0387] 3) Incubate the IL17A-His protein with the target cells. After thorough mixing, incubate at room temperature for 1 hour;

[0388] 4) Centrifuge at 800 x g at room temperature for 3 minutes, discard the supernatant containing the antibody, and wash the cells 3 times with PBS;

[0389] 5) Add the secondary antibody APC-His (diluted 1:500), mix well, and incubate in the dark at room temperature for 30 minutes;

[0390] 6) Centrifuge at 800 x g at room temperature for 3 minutes, discard the supernatant containing the secondary antibody, and wash the cells 3 times with PBS;

[0391] 7) Resuspend the cells with 500 μL of PBS and perform flow cytometry analysis.

[0392] Example 4 Single-domain antibody blocking function experiment

[0393] Experimental steps:

[0394] Add the detection antibodies (positive antibody: Ixekizumab; antibody to be detected) with gradient dilution to a 96-well plate. Dilute the antibodies in a 10-fold gradient, and perform 10 consecutive dilutions. The final concentrations are 100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, 0.001 μg / mL, 0.0001 μg / mL, 0.00001 μg / mL, 0.000001 μg / mL, 0.0000001 μg / mL, and 0 μg / mL respectively. Take 50 μL of the diluted gradient concentration antibodies and add them to the 96-well plate, with 2 replicates for each gradient. Then, add 50 μL of 0.4 μg / mL IL-17A protein (final concentration is 0.1 μg / mL) to each corresponding well. After mixing, place it in an incubator at 37°C and incubate for 1 hour. Pipette 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cells cultured to the logarithmic growth phase into the 96-well plate, and inoculate 2×10 4 cells per well. After co-culturing for 18 h, add 20 μL of Bright-GloTM detection reagent to each well, and use a Tecan M1000pro microplate reader to detect the luciferase activity value in the well.

[0395] Experimental results:

[0396] The results are as Figure 14 shown: The Ixekizumab positive control can block the activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc by Human IL-17A protein. Among the antibodies to be detected, the IL-17A single-domain antibody 1-F8 can block the activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc by Human IL-17A protein, but the blocking effect is weaker than that of the positive antibody.

[0397] Example 5 Stability experiment

[0398] Detect the fluorescence change by nano differential scanning fluorimetry (nanoDSF) technology. It can detect protein thermal denaturation and chemical denaturation under natural conditions, and accurately determine the temperature (Tm) when 50% of the protein is in the unfolded state and the temperature (Tagg) when aggregation begins to occur; the higher the Tm value and Tagg of thermal denaturation, the more stable the antibody protein.

[0399] Experimental procedure:

[0400] Take 100 μL of the candidate antibody prepared in the previous project and Lxekizumab (sample concentration greater than 200 μg / ml). After centrifuging at 12,000×g for 10 min at 4°C, use a capillary to aspirate the sample. Prepare two capillaries for each sample as parallel controls, and place them in the corresponding slots in sequence, ensuring that the capillaries are filled with the sample without air bubbles, and then perform detection and analysis.

[0401] The experimental results are as Figures 15 - 16 shown. It can be seen from the figure that the stability of the single-domain antibody 1-F8 is relatively strong, Tm = 62.57, Tagg = 80.09, and the Tm of the positive control is 56.1, Tagg = 61.86.

[0402] Application Example 1 An antibody preparation

[0403] The antibody preparation contains: an anti-IL-17A antibody, the amino acid sequence of which is as shown in SEQ ID NO:8; buffer, surfactant, amino acid, tonicity agent, etc.

[0404] In one embodiment of the present invention, the preparation of the antibody preparation includes: weighing each substance, dissolving and mixing with water, and adjusting the components to the following concentrations: (100 - 200) mg / ml of anti-IL-17A antibody (the amino acid sequence is as shown in SEQ ID NO:8), (1 - 10) mM citrate buffer, (0.1 - 1% w / v) Tween 80, (100 - 200) mM arginine, and (1 - 10)% sucrose, and the pH of the preparation is 5.0 - 8.0.

[0405] Application Example 2 A kit

[0406] The kit contains: anti-IL-17A antibody, recombinant protein, antibody preparation and / or polyclonal antibody, a container loaded with the antibody preparation, buffer, etc.

[0407] In one embodiment of the present invention, the kit includes: (100 - 200) mg / ml of anti-IL-17A antibody (the amino acid sequence is as shown in SEQ ID NO:8), and a buffer with a pH of 5.0 - 8.0.

[0408] Application Example 3 Antibody-drug conjugate

[0409] The antibody-drug conjugate contains: anti-IL-17A antibody, recombinant protein, antibody preparation and / or polyclonal antibody, the drug is a physiologically active substance (such as nucleic acid, etc.) and a linker connecting the antibody and the drug (the linker includes maleimide linker, Val-Cit linker, SS linker, and DMSS linker).

[0410] In one embodiment of the present invention, the IL-17A antibody is linked to a drug via an SS linker, and an aqueous solution of (100-200) mM is added and mixed at room temperature to terminate the linker reaction, thereby obtaining the antibody-drug conjugate.

[0411] Application Example 4 Drug Composition

[0412] The drug composition comprises an anti-IL-17A antibody, a recombinant protein, an antibody preparation, a polyclonal antibody, a nucleic acid molecule, a biological expression vector, and / or a host cell, and further comprises a pharmaceutically acceptable excipient.

[0413] In one embodiment of the present invention, the preparation of the drug composition comprises: preparing an anti-IL-17A antibody or its antigen-binding fragment with a concentration of (100-200) mg / ml, and adding sucrose at (1-20 w / v), histidine at (10-300) mM, and Tween 80 at (0.1-10)%, thereby obtaining the drug composition.

[0414] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An antibody, characterized in that: The amino acid sequence of the described antibody comprises: (1) HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively; and / or; (2) Amino acid sequences having at least 80% sequence identity with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:

3.

2. The antibody according to claim 1, wherein: The amino acid sequence of the described antibody comprises: an amino acid sequence obtained by at least one of addition, deletion, modification, and / or substitution on the amino acid sequences shown in SEQ ID NO:1 - 3.

3. The antibody according to claim 2, wherein: The amino acid sequence of the described antibody comprises: an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid differences compared to the amino acid sequences shown in SEQ ID NO:1 - 3.

4. An antibody, characterized in that: The amino acid sequence of the described antibody comprises: (1) FR1, FR2, FR3, and FR4 with amino acid sequences as shown in SEQ ID NO:4 - 7; and / or; (2) Amino acid sequences having at least 80% sequence identity with the amino acid sequences shown in SEQ ID NO:4 - 7.

5. The antibody according to claim 4, wherein: The amino acid sequence of the described antibody comprises: an amino acid sequence obtained by at least one of addition, deletion, modification, and / or substitution on the amino acid sequences shown in SEQ ID NO:4 - 7.

6. The antibody according to claim 5, wherein: The amino acid sequence of the described antibody comprises: an amino acid sequence having 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, 38, or 39 amino acid differences compared to the amino acid sequences shown in SEQ ID NO:4 - 7.

7. An antibody, characterized in that: The amino acid sequence of the described antibody comprises: (1) The amino acid sequence of the antibody according to any one of claims 1 - 3; and (2) The amino acid sequence of the antibody according to any one of claims 4 - 6.

8. An antibody, characterized in that: The amino acid sequence of the described antibody comprises: FR1 - HCDR1 - FR2 - HCDR2 - FR3 - HCDR3 - FR4; Wherein HCDR1, HCDR2, and HCDR3 are selected from the following: (1) Amino acid sequences as shown in SEQ ID NO:1 - 3; or (2) Functional active variants of amino acid sequences having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid differences compared to (1). FR1, FR2, FR3, and FR4 are selected from the following: 1) Amino acid sequences as shown in SEQ ID NO:4 - 7; or 2) Functional active variants of amino acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with 1).

9. The antibody according to claim 8, wherein: The amino acid sequence of the HCDR1 is as shown in SEQ ID NO:1, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:2, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

3.

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

7.

11. The antibody according to claim 10, wherein: The amino acid sequence of the antibody is as shown in SEQ ID NO:

8.

12. The antibody according to any one of claims 1-11, characterized in that: The antibody is a single-domain antibody.

13. The antibody according to any one of claims 1-12, characterized in that: The antibody is an anti-IL-17A antibody.

14. The antibody according to any one of claims 1-13, characterized in that: Comprising a partial or complete antibody heavy chain framework region or a variant thereof selected from human, murine, primate or camelid sources; Preferably, comprising a partial or complete antibody heavy chain framework region or a variant thereof selected from camelid sources; More preferably, comprising a partial or complete antibody heavy chain framework region or a variant thereof selected from alpaca sources.

15. A recombinant protein, characterized in that: Comprising the antibody according to any one of claims 1-14.

16. The recombinant protein according to claim 15, wherein: Also comprising a bioactive protein or a functional fragment thereof that assists in its expression and / or secretion, or prolongs its in vivo half-life; Preferably, the bioactive protein or the functional fragment thereof is selected from at least one of an immunoglobulin Fc domain, serum albumin, albumin-binding polypeptide, prealbumin, carboxyl-terminal peptide, elastin-like polypeptide, His tag, GST tag, MBP tag, FLAG tag and SUMO tag.

17. The recombinant protein according to claim 16, wherein: The bioactive protein or the functional fragment thereof is a human immunoglobulin Fc domain, preferably the Fc domain of human IgG, such as the Fc domains of human IgG1, IgG2, IgG3, IgG4, and more preferably the Fc domain of human IgG1.

18. An antibody preparation, characterized in that: The antibody preparation comprises the antibody according to any one of claims 1-14 and a pharmaceutically acceptable carrier.

19. A polyclonal antibody, characterized in that: Comprising the antibody according to any one of claims 1-14.

20. A kit, characterized in that: The kit includes: (1) The antibody according to any one of claims 1-14, the recombinant protein according to any one of claims 15-17, the antibody preparation according to claim 18 or the polyclonal antibody according to claim 19; and; (2) A container for loading the antibody preparation.

21. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate includes: (1) The antibody according to any one of claims 1-14, the recombinant protein according to any one of claims 15-17, the antibody preparation according to claim 18 or the polyclonal antibody according to claim 19; and; (2) A conjugate moiety conjugated to (1).

22. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the antibody according to any one of claims 1-14, the recombinant protein according to any one of claims 15-17 or the polyclonal antibody according to claim 19.

23. A biological expression vector, characterized in that: The biological expression vector contains the nucleic acid molecule according to claim 22.

24. A host cell, characterized in that: The genome of the host cell is integrated with the nucleic acid molecule according to claim 22; or; contains the biological expression vector according to claim 23.

25. A pharmaceutical composition, characterized in that: The described pharmaceutical composition comprises: the antibody according to any one of claims 1-14, the recombinant protein according to any one of claims 15-17, the antibody preparation according to claim 18, the polyclonal antibody according to claim 19, the antibody-drug conjugate according to claim 21, the nucleic acid molecule according to claim 22, the biological expression vector according to claim 23 or the host cell according to claim 24; and; Optionally, the described pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

26. Use of the antibody according to any one of claims 1-14, the recombinant protein according to any one of claims 15-17, the antibody preparation according to claim 18, the polyclonal antibody according to claim 19, the kit according to claim 20, the antibody-drug conjugate according to claim 21, the nucleic acid molecule according to claim 22, the biological expression vector according to claim 23 or the host cell according to claim 24, characterized in that: The described use is selected from at least one of the following: (1) Preparing a drug for preventing and / or treating an autoimmune disease; (2) Preparing a drug for preventing and / or treating cancer; (3) Preparing a detection reagent or kit.

27. The use according to claim 26, wherein: The autoimmune diseases include Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic scleroderma, progressive systemic scleroderma, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, aortitis syndrome, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Castleman disease, Sjogren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, allergic vasculitis, rheumatoid vasculitis, large vessel vasculitis, ANCA-associated vasculitis, Cogan syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disease, Guillain-Barre syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic fatty liver disease, primary biliary cirrhosis, Good-pasture syndrome, rapidly progressive glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Basedow's disease, Hashimoto's disease, autoimmune adrenocortical hypofunction, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type I diabetes mellitus, slowly progressive type I diabetes mellitus, localized scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, vulgaris vitiligo, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada 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 rhinosinusitis, dilated cardiomyopathy, systemic mastocytosis or inclusion body myositis.

28. The use according to claim 26 or 27, characterized in that: The cancers described above include basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, peritoneal cancer, cervical cancer, cholangiocarcinoma, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, 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 system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, B-cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic myelogenous leukemia.

29. A method for in vitro detecting IL-17A in a sample for non-diagnostic purposes, characterized in that: The method described above includes the following steps: (1) Binding the antibody described in any one of claims 1-14, the recombinant protein described in any one of claims 15-17, the antibody preparation described in claim 18, the polyclonal antibody described in claim 19, and the antibody-drug conjugate described in claim 21 to a test sample; (2) Detecting the antigen-antibody complex and interpreting the result.

30. A method for preventing and / or treating an autoimmune disease, characterized in that: The method described above includes: administering to a subject a therapeutically effective amount of the antibody described in any one of claims 1-14, the recombinant protein described in any one of claims 15-17, the antibody preparation described in claim 18, the polyclonal antibody described in claim 19, the antibody-drug conjugate described in claim 21, the nucleic acid molecule described in claim 22, the biological expression vector described in claim 23, the host cell described in claim 24, or the pharmaceutical composition described in claim 25.

31. A method for preventing and / or treating cancer, characterized in that: The method described above includes: administering to a subject a therapeutically effective amount of the antibody described in any one of claims 1-14, the recombinant protein described in any one of claims 15-17, the antibody preparation described in claim 18, the polyclonal antibody described in claim 19, the antibody-drug conjugate described in claim 21, the nucleic acid molecule described in claim 22, the biological expression vector described in claim 23, the host cell described in claim 24, or the pharmaceutical composition described in claim 25.

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