Anti-il-17a nanobodies, pharmaceutical compositions, methods, and uses

Abstract

The application provides an anti-IL-17A nanobody, a pharmaceutical composition, a method and a use, and belongs to the technical field of cellular immunology. The antibody provided by the application comprises a complementarity determining region HCDR, and the complementarity determining region HCDR comprises: (1) HCDR1 with an amino acid sequence as shown in SEQ ID NO:1, HCDR2 with an amino acid sequence as shown in SEQ ID NO:2, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:3; and / or (2) an amino acid sequence with a sequence similarity of at least 80% with SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively. The antibody has high affinity, strong blocking ability and stability, and the single-domain antibody has broad application prospects in the treatment of autoimmune diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cellular immunology, and specifically relates to anti-IL-17A nanobodies, pharmaceutical compositions, methods and uses. BACKGROUND

[0002] IL-17 is a family of structurally related cytokines that share a highly conserved C-terminal region but differ from each other in the N-terminal region and different biological effects. There are six members in the IL-17 family, namely IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, which are secreted in the form of homodimers. IL-17A is a 150-amino-acid-containing, 15-kDa-molecular-weight molecule that is mainly secreted by a disulfide homodimer of 30-35-kDa glycoprotein, which is different from other known cytokines. It is mainly produced by activated CD4+ T cells, CD8+ T cells, NKT cells and neutrophils, and acts on stromal cells to induce the production of pro-inflammatory and hematopoietic activity molecules. IL-17A can synergize with inflammatory factors such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) and interleukin 22 (IL-22) and other inflammatory mediators to enhance the transcriptional expression of related inflammatory genes. In the IL-17 cytokine family, IL-17A has been extensively studied, and it has been found that IL-17A plays an important role in autoimmune diseases, such as inflammatory bowel disease, rheumatoid arthritis, psoriasis, ankylosing spondylitis, etc.

[0003] In view of the important role of IL-17A in autoimmune diseases, biological agents targeting IL-17A have been extensively studied. Chinese patent CN107325179A discloses a stable anti-human IL-17A monoclonal antibody liquid preparation. The liquid preparation of the anti-human IL-17A monoclonal antibody provided by the present application comprises an anti-human IL-17A monoclonal antibody, a surfactant, a cyclodextrin and a preservative, which is stable during manufacturing, storage and administration, has a low aggregate, and is suitable for administration at a high concentration. The stable anti-human IL-17A monoclonal antibody liquid preparation of the present application can be effectively used in the preparation of drugs for treating immune-mediated inflammatory reactions. Although the monoclonal antibody in this patent has strong stability, its affinity is poor.

[0004] Single-domain antibody, also known as nanobody, VHH antibody, variable domain of heavy chain of heavy-chain antibody, etc., refers to a genetically engineered antibody composed of a variable region of a heavy chain antibody, which is a naturally light chain-deleted antibody. Compared with traditional monoclonal antibodies, single-domain antibodies have stronger affinity, and since single-domain antibodies only contain heavy chains, their production cost is lower and their yield is higher, and their production efficiency is strong.

[0005] A single-domain antibody against IL-17A and its use are disclosed in Chinese patent CN114380906A. The single-domain antibody is composed of a heavy chain, which includes a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and in the heavy chain CDR1, 1 to 5 amino acid residues can be replaced by conservative amino acids; in the heavy chain CDR2, 1 to 5 amino acid residues can be replaced by conservative amino acids; in the heavy chain CDR3, 1 to 5 amino acid residues can be replaced by conservative amino acids. Compared with the prior art, the patent uses biological genetic engineering technology to screen a single-domain antibody specific to IL-17A, which has good antibody affinity and can block the release of cytokines by specific cells. Both prokaryotic expression and eukaryotic expression have good binding activity and have certain drug properties.

[0006] However, there is still a need in the art to develop a new anti-IL-17A single-domain antibody that can bind to IL-17A with high affinity, has stronger stability and better blocking effect.

[0007] The present application is a patent application proposed by the inventors for the anti-IL-17A antibody independently developed by the inventors.

[0008] In addition to the antibody claimed in the present application, the inventors have also developed eight other antibodies. Based on the relevant provisions of the single nature of the Patent Law, nine different antibodies are claimed respectively.

[0009] The inventors have also developed 12 tandem antibodies based on the nine antibodies. Based on the relevant provisions of the single nature of the Patent Law, 12 different tandem antibodies are claimed respectively.

[0010] The inventors have also developed gene-modified stem cell technology based on the 12 tandem antibodies. Based on the relevant provisions of the single nature of the Patent Law, three different gene-modified stem cells and three different applications are claimed respectively.

[0011] For the convenience of understanding the technical solutions of the present application, the other series of application texts of the present project can be optionally referred to. SUMMARY

[0012] Terms and statements of the present application:

[0013] 1. As used herein, the term "amino acid": includes natural amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that function in a manner similar to the natural amino acids. Natural amino acids are those encoded by the genetic code. Amino acid analogs are those having the same basic chemical structure as a naturally occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0014] 2. As used herein, the term "antibody": refers to a polypeptide or fragment thereof comprising a framework region of an immunoglobulin gene that specifically binds and recognizes an antigen. The use of the term antibody is meant to encompass whole antibodies and antigen-binding fragments thereof. The term antibody includes monospecific antibodies, bispecific antibodies, and multispecific antibodies, so long as they exhibit the desired biological activity or function. In the present invention, antibody refers to a polypeptide having IL-17A binding activity comprising a CDR1 region as set forth in SEQ ID NO: 1, a CDR2 region as set forth in SEQ ID NO: 2, and a CDR3 region as set forth in SEQ ID NO: 3, and also includes variants of polypeptides having the same function as the antibodies of the present invention comprising the above-mentioned CDR regions. The variants include deletion, insertion, and / or substitution of one or more amino acids, and addition of one or more amino acids at the C-terminus and / or the N-terminus.

[0015] 3. As used herein, the term "monoclonal antibody": refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes) of the antigen, each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0016] 4. As used herein, the terms "single-domain antibody (VHH)" and "single domain antibody" (sdAb, or nanobody) have the same meaning and refer to a single domain antibody (SDA) constructed by cloning the variable region of an antibody heavy chain. This SDA is the smallest fully functional antigen-binding fragment consisting solely of a single heavy chain variable region. Typically, antibodies naturally lacking the light chain and heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a SDA consisting solely of a single heavy chain variable region. "Single domain antibody of the present invention" and "anti-IL-17A single domain antibody of the present invention" are used interchangeably to refer to SDAs that specifically recognize and bind to IL-17A.

[0017] 5. As used herein, the terms "homology" or "identity" refer to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions within each sequence, which may be aligned for the purpose of comparison. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position.

[0018] 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 typically comprise chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation and may be a "linear" epitope or a "conformational" epitope.

[0019] 7. As used herein, the term "amino acid sequence" refers to the order in which amino acids are linked to form a peptide chain (or polypeptide). The amino acid sequence can only be read in one direction.

[0020] 8. As used herein, the term "nucleic acid molecule" may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The full-length sequence of the nucleotide molecules of the present invention or fragments thereof can generally be obtained by PCR amplification or synthetic methods.

[0021] 9. As used herein, the term "nucleotide sequence" refers to the order of bases in DNA or RNA, i.e., the order of A, T, G, C in DNA, or the order of A, U, G, C in mRNA, and also includes the order of bases in rRNA, tRNA, and mRNA.

[0022] 10. As used herein, the term "framework region" refers to the backbone region. The approximately 110 amino acids near the N-termini of immunoglobulin H and L chains vary significantly in sequence, while the remaining amino acid sequences are relatively constant. This is how the light and heavy chains are divided into variable (V) and constant (C) regions. The variable region comprises the hypervariable regions (HVRs) or complementarity-determining regions (CDRs) and the framework regions (FRs).

[0023] 11. As used herein, the term "humanized" antibody refers to an antibody in which the variable region (VH or VHH), the Fr region, and the constant region (i.e., CH and CL regions), or all of the antibody, are encoded by human antibody genes. Humanized antibodies include chimeric antibodies, remodeled antibodies, and fully humanized antibodies.

[0024] 12. As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin, which is a functional structural unit consisting solely of the CH2 and CH3 regions of the heavy chain constant domain. The Fc region lacks antigen binding capacity, but has the property of extending half-life and possesses a constant amino acid sequence.

[0025] 13. As used herein, the term "variable" refers to the sequence differences in certain portions of the variable region of an antibody, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, this variability is not evenly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the light and heavy chain variable regions known as the complementarity determining regions (CDRs) or hypervariable regions. "Variable region" and "CDR" are used interchangeably herein.

[0026] 14. As used herein, the terms "hypervariable region," "hypervariable region," "complementarity determining region," "HVR," or "CDR" refer to regions within the variable domain of an antibody that are highly variable in sequence and / or form structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs or CDRs: three present in VH (H1, H2, H3) and three present in VL (L1, L2, L3). It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., a variable region) should be understood to encompass complementarity determining regions defined by any of the known schemes described herein. Although the scope of protection claimed in this disclosure is based on the sequences shown in the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0027] 15. The term "and / or" used herein should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0028] 16、As used herein, the term "IL-17A" or "interleukin-17A" refers to a cytokine belonging to the interleukin 17 family that is produced by T cells and other types of immune cells and plays an important role in the immune system. IL-17A is primarily produced by Thl 7 cells, but other cells including CD8+ T cells, γδ T cells, NK cells, neutrophils, mast cells, and macrophages also express IL-17A. It acts primarily on immune cells such as macrophages, neutrophils, and endothelial cells to induce inflammatory responses. In some examples, 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 some instances. In other embodiments, an antibody specific for human IL-17A protein can be completely specific for human IL-17A protein and not cross-react with other species or other types of proteins, or can cross-react with IL-17A protein of some, but not all, other species.

[0029] 17、As used herein, the term "anti-IL-17A (single domain or nanobody)" 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 attenuates IL-17A activity, and / or inactivates IL-17A, prevents IL-17A responses, or downstream pathways or other IL-17A-mediated functions.

[0030] 18、As used herein, the terms "activity," "functional activity," or "biological activity," or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, ability to neutralize or antagonize IL-17A activity in vivo or in vitro, IC50, antibody stability in vivo, and 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 with non-human homologs of the targeted peptide, or with other proteins or tissues), and ability to maintain high expression levels of the protein in mammalian cells. The aforementioned properties or characteristics are observed, measured, or assessed using techniques known in the art, including, but not limited to, ELISA, FACS or BIACORE® plasmon resonance analysis, unconfined in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections of different origins, including human, primate, or any other origin.

[0031] 19. The term "Fc" or "Fc region" or "Fc fragment" as used herein 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 disassembly of the Fc fragment is variable, the heavy chain Fc fragment of human IgG generally refers to the polypeptide from A231 to the carboxy terminus thereof.

[0032] 20. The term "affinity" or "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (Koffand Kon, respectively). The affinity can be measured by common methods known in the art. One particular method for measuring affinity is the ForteBio kinetic binding assay herein.

[0033] 21. The term "high affinity" or "high affinity" as used herein, for IgG antibodies, refers to a KDof 1.0 x 10 -6 M or lower, preferably 5.0 x 10 -8 M or lower, preferably 5.0 x 10 -9 M or lower, preferably 5.0 x 10 -9 M or lower, preferably 5.0 x 10 -6 M or lower, preferably 5.0 x 10 -7 M or lower, preferably 5.0 x 10 -8 M or lower.

[0034] 22. The term "antibody drug conjugate" refers to a material resulting from the linkage of a biologically active compound fragment to an antibody or antigen binding fragment thereof moiety. The biologically active compound fragment and the targeting moiety can be linked by a linker. The linker can be cleavable under certain conditions (e.g., intracellular low pH environment) or by certain actions (e.g., action of lysosomal proteases), thereby separating the biologically active compound fragment from the targeting moiety or antibody or antigen binding fragment thereof. The linker can comprise cleavable or non-cleavable units, such as a peptide or disulfide bond. The biologically active compound fragment and the targeting moiety or antibody or antigen binding fragment thereof are directly linked by a covalent bond that is cleavable under certain conditions or actions, thereby separating the biologically active compound fragment from the antibody or antigen binding fragment thereof moiety.

[0035] 23. 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 into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Following introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) integrate into the host cell's genome and are thereby replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of operatively linked genes. Such vectors are referred to herein as "expression vectors."

[0036] 24. The term "pharmaceutical composition" as used herein generally refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is to be administered. The composition is sterile.

[0037] 25. The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0038] 26. As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0039] 27. As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of an 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 a cell, tissue, or subject, is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the progression of such diseases or conditions. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in amelioration of symptoms, such as an amount to treat, cure, prevent, or ameliorate a related medical condition or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter of at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%.

[0040] 28、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.

[0041] 29、As used herein, the term "cancer" or "tumor" refers to a physiological condition describing a cell growth that is usually characterized by a lack of regulation in a mammal. This definition includes benign and malignant cancers as well as dormant tumors or micrometastases.

[0042] 30、As used herein, the term "autoimmune disease" or "autoimmune disorder" refers to a disease caused by a decrease, loss and / or breakdown of immune tolerance to self components for some reason, resulting in self-antibodies and / or sensitized lymphocytes damaging self-organ tissues, which is manifested as dysfunction of the corresponding tissues and organs.

[0043] The technical solutions of the present application are as follows:

[0044] In a first aspect, the present application provides an antibody, wherein the antibody comprises a complementarity determining region HCDR, and the complementarity determining region HCDR comprises:

[0045] (1) HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 3; and / or

[0046] (2) an amino acid sequence having at least 80% sequence similarity with SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0047] Preferably, the amino acid sequence of the antibody comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences compared with the amino acid sequence shown in SEQ ID NO: 1-3.

[0048] Preferably, the amino acid sequence of the antibody comprises an amino acid sequence obtained by at least one of modification, deletion, addition and / or substitution on the amino acid sequence shown in SEQ ID NO: 1-3.

[0049] SEQ ID NO: 1 is GFDLDLYT.

[0050] SEQ ID NO: 2 is IDLTSGAT.

[0051] SEQ ID NO: 3 is NPVAVGGHLY.

[0052] According to some embodiments of the present application, 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 sequence set forth in SEQ ID NO: 1-3.

[0053] In particular, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions preferably are substitutions of one amino acid residue for another within the same group of: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0054] Further, conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; He to Leu or to Val; Leu to He or to Val; Lys to Arg, to Gin, or to Glu; Met to Leu, to Tyr, or to He; 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 He, or to Leu.

[0055] According to some embodiments of the present application, the functionally 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 of (1), for example, a variant (mutant) that can specifically bind to IL-17A and block the binding of IL-17A to its receptor.

[0056] In a second aspect, the present application provides an antibody, wherein the antibody comprises a framework region FR;

[0057] The framework region FR comprises: FR1 set forth in SEQ ID NO: 4, FR2 set forth in SEQ ID NO: 5, FR3 set forth in SEQ ID NO: 6, and FR4 set forth in SEQ ID NO: 7; and / or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4-7.

[0058] Preferably, 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, 34, 35, 36, 37, or 38 amino acid differences compared to the amino acid sequence set forth in SEQ ID NO: 4-7.

[0059] Preferably, the amino acid sequence of the antibody comprises an amino acid sequence obtained by at least one of addition, deletion, modification, and / or substitution to the amino acid sequence set forth in SEQ ID NO: 4-7.

[0060] SEQ ID NO: 4 is: AVQLVESGGGLVQPGGSLRLSCAAS.

[0061] SEQ ID NO: 5 is: IAWFRQAPGKEREFVSL.

[0062] SEQ ID NO: 6 is: SDAGSMKGRVAISRDKDSNTVSLQLNSLKPEDTAVYYC.

[0063] SEQ ID NO: 7 is: WGQGTQVTVSS.

[0064] In a third aspect, the present application provides an antibody, wherein the amino acid sequence of the antibody comprises:

[0065] (1) the amino acid sequence of the antibody according to the first aspect described above; and

[0066] (2) the amino acid sequence of the antibody according to the second aspect described above.

[0067] In a fourth aspect, the present application provides an antibody, wherein the amino acid sequence of the antibody comprises:

[0068] FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4;

[0069] wherein HCDR1, HCDR2, and HCDR3 are selected from the following:

[0070] (1) the amino acid sequence set forth in SEQ ID NO: 1-3; or

[0071] (2) a functionally active variant of the amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences compared to (1);

[0072] FR1, FR2, FR3, FR4 are selected from the following:

[0073] 1) an amino acid sequence as shown in SEQ ID NO: 4-7; or

[0074] 2) a functionally active variant of the amino acid sequence of 1) having at least 80% sequence identity.

[0075] Preferably, the antibody comprises an amino acid sequence of HCDR1 as shown in SEQ ID NO: 1, an amino acid sequence of HCDR2 as shown in SEQ ID NO: 2, and an amino acid sequence of HCDR3 as shown in SEQ ID NO: 3.

[0076] Preferably, the amino acid sequence of FR1 is as shown in SEQ ID NO: 4, the amino acid sequence of FR2 is as shown in SEQ ID NO: 5, the amino acid sequence of FR3 is as shown in SEQ ID NO: 6, and the amino acid sequence of FR4 is as shown in SEQ ID NO: 7.

[0077] Preferably, the amino acid sequence of the antibody is as shown in SEQ ID NO: 8.

[0078] SEQ ID NO: 8 is:

[0079] AVQLVESGGGLVQPGGSLRLSCAASGFDLDLYTIAWFRQAPGKEREFVSLIDLTSGATSDAGSMKGRVAISRDKDSNTVSLQLNSLKPEDTAVYYCNPVAVGGHLYWGQGTQVTVSS.

[0080] Further, the antibody is a single domain antibody.

[0081] Still further, the antibody is an anti-IL-17A antibody.

[0082] Preferably, the antibody comprises part or all of antibody heavy chain framework regions selected from human, murine, primate or camelid origin or variants thereof;

[0083] Further preferably, part or all of antibody heavy chain framework regions selected from camelid origin or variants thereof are comprised;

[0084] More preferably, part or all of antibody heavy chain framework regions selected from alpaca origin or variants thereof are comprised.

[0085] In a fifth aspect, the present application provides an antibody preparation comprising the antibody of any one of the above first to fourth aspects and a pharmaceutically acceptable carrier.

[0086] In particular, the "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical administration.

[0087] In a sixth aspect, the present application provides a polyclonal antibody comprising the antibody of any one of the first aspect to the fourth aspect.

[0088] In a seventh aspect, the present application provides a recombinant protein comprising the antibody of any one of the first aspect to the fourth aspect.

[0089] Preferably, the recombinant protein further comprises a biologically active protein or a functional fragment thereof that assists in its expression and / or secretion, or prolongs its half-life in vivo;

[0090] Further preferably, the biologically active protein or the functional fragment thereof is selected from at least one of an immunoglobulin Fc domain, serum albumin, an albumin-binding polypeptide, prealbumin, a carboxy-terminal peptide, an elastin-like polypeptide, a His tag, a GST tag, an MBP tag, a FLAG tag, and a SUMO tag.

[0091] Preferably, the biologically active protein or the functional fragment thereof is a human immunoglobulin Fc domain, preferably an Fc domain of human IgG, for example, an Fc domain of human IgG1, IgG2, IgG3, IgG4, more preferably an Fc domain of human IgG1.

[0092] According to some embodiments of the present application, the amino acid sequence of the human IgG1 Fc is shown in SEQ ID NO: 10.

[0093] SEQ ID NO: 10:

[0094] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0095] In particular, the nucleotide sequence encoding SEQ ID NO: 10 is set forth in SEQ ID NO: 11.

[0096] SEQ ID NO: 11:

[0097] GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA.

[0098] According to some embodiments of the application, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to thereby generate an Fc region variant. Fc region variants can comprise human Fc region sequences (e.g., of an IgGl, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions, deletions, and insertions) at one or more amino acid positions.

[0099] According to some embodiments of the application, the recombinant protein can be a monomer, a dimer, or a multimer.

[0100] In an eighth aspect, the present application provides a kit, which comprises:

[0101] (1) the antibody, the recombinant protein, the antibody preparation or the polyclonal antibody according to any one of the first to fourth aspects; and

[0102] (2) a container loaded with the antibody preparation.

[0103] Preferably, the kit can further comprise a container, a buffer, an antibody recognizing IL-17A protein, a detection substrate, etc.

[0104] In a ninth aspect, the present application provides an antibody drug conjugate, which comprises:

[0105] (1) the antibody, the antibody preparation, the polyclonal antibody, the recombinant protein according to any one of the first to fourth aspects; and

[0106] (2) a conjugating moiety binding to (1).

[0107] According to some embodiments of the present application, the conjugating moiety comprises a detectable label, a drug, a toxin, a cytokine, a radionuclide and / or an enzyme.

[0108] In a tenth aspect, the present application provides a nucleic acid molecule encoding the antibody, the polyclonal antibody, the recombinant protein according to any one of the first to fourth aspects.

[0109] According to some embodiments of the present application, the nucleic acid can be RNA, DNA or cDNA.

[0110] According to some embodiments of the present application, the nucleic acid of the present application can also be in the form of, can exist in 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 generally comprises at least one nucleic acid of the present application operably linked to one or more suitable expression control elements (e.g. a promoter, an enhancer, a terminator, etc.).

[0111] In particular, the nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO: 8 has a sequence as set forth in SEQ ID NO: 9.

[0112] SEQ ID NO: 9 is:

[0113] GCGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCGATTTGGATTTGTATACCATAGCCTGGTTCCGCCAGGCTCCGGGGAAGGAGCGCGAGTTTGTGTCACTCATAGATTTGACTAGTGGTGCCACATCCGACGCGGGCTCCATGAAGGGCCGAGTCGCCATCTCCAGAGACAAAGACAGCAACACGGTGTCGCTGCAATTGAATAGCCTGAAACCTGAAGATACGGCCGTCTACTACTGTAATCCAGTCGCGGTGGGAGGACACTTGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

[0114] In a eleventh aspect, the present application provides a biological expression vector, characterized in that the biological expression vector comprises the nucleic acid molecule.

[0115] According to some embodiments of the present application, the biological expression vector can be a eukaryotic expression vector or a prokaryotic expression vector, preferably a eukaryotic expression vector.

[0116] Preferably, the eukaryotic expression vector is selected from a yeast expression vector, an insect expression vector or a mammalian expression vector.

[0117] More preferably, the mammalian expression vector is selected from a retroviral expression vector, a lentiviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector.

[0118] In a twelfth aspect, the present application provides a host cell, characterized in that the genome of the host cell is integrated with the nucleic acid molecule; or; the host cell comprises the biological expression vector.

[0119] Preferably, the host cell is a bacterial cell, a fungal cell or a mammalian cell.

[0120] Further, the bacterial cell comprises a cell of a gram-negative bacterial strain and a gram-positive bacterial strain.

[0121] Still further, the gram-negative bacterial strain comprises an Escherichia coli strain, a Pseudomonas strain and a Proteus strain.

[0122] Still further, the gram-positive bacterial strain comprises a Streptomyces strain, a Bacillus strain and a Staphylococcus strain.

[0123] Further, the fungal cell comprises a cell of Trichoderma, Neurospora, and Aspergillus.

[0124] Further, the mammalian cell comprises a HEK293 cell, a HeLa cell, a CHO cell, and a COS cell.

[0125] Still further, the mammalian cell is a HEK293 cell.

[0126] In a thirteenth aspect, the present application provides a pharmaceutical composition comprising: the antibody, the antibody preparation, the polyclonal antibody, the recombinant protein, the antibody-drug conjugate, the nucleic acid molecule, the biological expression vector, or the host cell according to any one of the first to fourth aspects; and;

[0127] Optionally, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0128] Specifically, the pharmaceutically acceptable excipient is selected from at least one of a solvent, a diluent, a disintegrant, a precipitation inhibitor, a surfactant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a thickening agent, an emulsifying agent, a preservative, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a coloring agent, a flavoring agent, a sweetening agent, an ion exchanger, a release agent, a coating agent, a flavoring agent, an antioxidant.

[0129] In a fourteenth aspect, the present application provides a use of the antibody, the antibody preparation, the polyclonal antibody, the recombinant protein, 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 to fourth aspects, wherein the use is selected from at least one of:

[0130] (1) preparing a detection reagent or a kit;

[0131] (2) preparing a drug for preventing and / or treating an autoimmune disease;

[0132] (3) preparing a drug for preventing and / or treating a cancer.

[0133] In particular, the autoimmune disease includes psoriasis, systemic lupus erythematosus, malignant rheumatoid arthritis, rheumatoid arthritis, adult Still's disease, Behcet's disease, scleroderma, Hashimoto's disease, allergic angiitis, juvenile idiopathic arthritis, temporal arteritis, myasthenia gravis, rheumatoid vasculitis, autoimmune optic neuropathy, polymyalgia rheumatica, polymyositis, multiple sclerosis, fibromyalgia, systemic scleroderma, dermatomyositis, rapidly progressive glomerulonephritis, Behcet's disease, progressive systemic sclerosis, Sjogren's syndrome, psoriasis vulgaris, pernicious anemia, vasculitis, large vessel vasculitis, bullous pemphigoid, Addison's disease, Castleman's disease, giant cell arteritis, idiopathic Addison's disease, psoriatic arthritis, multifocal motor neuropathy, chronic inflammatory demyelinating polyneuropathy, periarteritis nodosa, amyotrophic lateral sclerosis, idiopathic azoospermia, gestational herpes, aortitis syndrome, autoimmune hemolytic anemia, eosinophilic fasciitis, pemphigus, spondylarthritis, vitiligo, primary biliary cirrhosis, mixed connective tissue disease, chronic atrophic gastritis, antiphospholipid antibody syndrome, allergic granulomatous angiitis, Good-pasture syndrome, Cogan syndrome, ANCA-associated vasculitis, linear IgA bullous dermatosis, RS3PE syndrome, chronic discoid lupus erythematosus, IgG4-related disease, Guillain-Barre syndrome, autoimmune hepatitis, epidermolysis bullosa acquisita, non-alcoholic steatohepatitis, lupus nephritis, type I diabetes, megaloblastic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, autoimmune adrenal hypofunction, primary hypothyroidism, slow progression type I diabetes, localized scleroderma, pemphigus, alopecia areata, neuromyelitis optica, sarcoidosis, Harada disease, habitual abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplantation immunity, familial Mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis, or inclusion body myositis.

[0134] In particular, the cancer includes oral cancer, lymphoma, respiratory system cancer, prostate cancer, leukemia, retinoblastoma, choriocarcinoma, peritoneal cancer, eye cancer, urinary system cancer, bone cancer, endometrial cancer, uterine cancer, bladder cancer, thyroid cancer, esophageal cancer, laryngeal cancer, head and neck cancer, acute lymphoblastic leukemia, melanoma, hairy cell leukemia, neuroblastoma, salivary gland cancer, digestive system cancer, myeloma, gastric cancer, liver cancer, skin cancer, lung cancer, rectal cancer, ovarian cancer, kidney cancer, pancreatic cancer, liver cancer, sarcoma, glioblastoma, basal cell carcinoma, cholangiocarcinoma, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, connective tissue cancer, rhabdomyosarcoma, squamous cell carcinoma, testicular cancer, B-cell lymphoma, chronic lymphocytic leukemia, chronic myeloblastic leukemia.

[0135] In a fifteenth aspect, the present application provides a method for detecting IL-17A in a sample in vitro for non-diagnostic purposes, the method comprising the steps of:

[0136] (1) combining the antibody, the antibody preparation, the polyclonal antibody, the recombinant protein, the antibody drug conjugate of any one of the first to fourth aspects above with the sample to be detected;

[0137] (2) detecting the antigen-antibody complex and interpreting the results.

[0138] In a sixteenth aspect, the present application provides a method for preventing and / or treating autoimmune diseases, the method comprising: administering to a subject a therapeutically effective amount of the antibody, the antibody preparation, the polyclonal antibody, the recombinant protein, the antibody drug conjugate, the nucleic acid molecule, the biological expression vector, the host cell or the pharmaceutical composition of any one of the first to fourth aspects above.

[0139] In a seventeenth aspect, the present application provides a method for preventing and / or treating cancer, the method comprising:

[0140] administering to a subject a therapeutically effective amount of the antibody, the antibody preparation, the polyclonal antibody, the recombinant protein, the antibody drug conjugate, the nucleic acid molecule, the biological expression vector, the host cell or the pharmaceutical composition of any one of the first to fourth aspects above.

[0141] The beneficial effects of the present application include:

[0142] The single-domain antibody 1-H9 of the present application has a higher affinity than traditional monoclonal antibodies, and the EC50 value of the single-domain antibody 1-H9 is 21.08, while the EC50 value of the positive antibody (Ixekizumab) is 10.06. The single-domain antibody 1-H9 has better binding activity with Human IL-17A protein.

[0143] The single-domain antibody 1-H9 of IL-17A has good blocking effect and can block the activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc by Human IL-17A protein.

[0144] The single-domain antibody 1-H9 has strong stability, with Tm=58 and Tagg=80.15. BRIEF DESCRIPTION OF DRAWINGS

[0145] Figure 1 Figure 4 is a graph showing the results of SDS-PAGE detection of IL-17A recombinant protein.

[0146] Figure 2 Figure for the results of the experiment of activating NIH-3T3 cells by the purchased IL-17A (Acro) protein.

[0147] Figure 3 Figure for the results of the experiment of activating NIH-3T3 cells by the recombinant IL-17A (TEST) protein.

[0148] Figure 4 Figure for the results of the experiment of binding of IL-17A to the reporter cell strain.

[0149] Figure 5 Figure for the results of the experiment of activating cells by IL-17A

[0150] Figure 6 Figure for the results of the agarose gel electrophoresis of 2# alpaca.

[0151] Figure 7 Figure for the results of the agarose gel electrophoresis of 2#+3# alpaca.

[0152] Figure 8 Figure for the results of the flow detection of the yeast library of 2# alpaca, top left: NC group: primary antibody: none, secondary antibody: PE-streptavidin; top right: original library experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, Alexa Fluor 647-V5; bottom left: 1MACS experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, Alexa Fluor 647-V5; bottom right: 2MACS experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, Alexa Fluor 647-V5.

[0153] Figure 9 Figure for the results of the flow detection of the yeast library of 2#+3# alpaca, A: NC group: primary antibody: none, secondary antibody: PE-streptavidin; B: original library experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, Alexa Fluor 647-V5; C: 1MACS experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, Alexa Fluor 647-V5; D: 2MACS experimental group: primary antibody: IL-17A-His-Biotin, secondary antibody: PE-streptavidin, Alexa Fluor 647-V5.

[0154] Figure 10FACS detection of 2# alpaca IL-17A target monoclonal binding to target.

[0155] Figure 11 FACS detection of 2#+3# alpaca IL-17A target monoclonal binding to target.

[0156] Figure 12 SDS-PAGE results of antibody purification, left lane marker, right lane 1-H9.

[0157] Figure 13 Candidate antibody ELISA binding experiment results.

[0158] Figure 14 Candidate antibody blocking function experiment results.

[0159] Figure 15 Thermal stability detection results of single domain antibody 1-H9.

[0160] Figure 16 Thermal stability detection results of positive control antibody. DETAILED DESCRIPTION

[0161] In order to make the technical means, creative features, purposes and effects realized by the present application easy to understand, the following specific embodiments are combined to further illustrate the present application. However, the following embodiments are only preferred embodiments of the present application, not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application. In the following examples, if not otherwise specified, the operation method used is the conventional operation method, the equipment used is the conventional equipment, and the equipment and materials used in each example are the same.

[0162] Experimental reagents:

[0163] Agar (Sigma, CAT# A1296); Proteose peptone (Sigma, CAT# 93926); Yeast extract (OXOID, CAT#: LP0021); Sodium chloride (Aladdin, CAT#: C111533); Potassium chloride (Aladdin, CAT#: P112133); Magnesium sulfate (National Pharmaceutical, CAT#: 10013018); Magnesium chloride (National Pharmaceutical, CAT#: 10012818); Glucose (Shenguo, CAT#: GT1991); Sfil (NEB, CAT#: R0123L); T4 DNA ligase (TaKaRa, CAT#: 2011A); PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#: 6210B); NuHipower mix (Xin Hai Biological, CAT#: NH9303); 3M Sodium acetate (pH 5.2-6) (Sigma, CAT#: 126-96-5); DNA fragment recovery kit (TakaRa, CAT#: 9761); Gel recovery kit (Qiagen, CAT#: 28706); 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).

[0164] Experimental supplies:

[0165] 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 flask (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).

[0166] Experimental equipment:

[0167] Eppendorf Multiporator; centrifuge (Thermo FRESCO-17); incubator (Shanghai Jinghong DNP-9052); constant temperature shaking incubator (Jingqi CO-O6U); clean bench (Su Nengantai 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.

[0168] The primers used in the following examples for screening, cloning VHH fragments, and constructing nanobodies are designed according to the following references:

[0169] Maass DR, Sepulveda J, Pernthaner A, Shoemaker CB. Alpaca (Lama paco) as a convenient source of recombinant camelid heavy chain antibodies (VHHs). J Immunol Methods. 2007; 324(1-2): 13-25.

[0170] 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.

[0171] Studies on design of single domain antibodies by Alpaca VHH phage library and high throughput sequencing to construct Fab antibody purification system (http: / / hdl.handle.net / 10232 / 00030916).

[0172] Example 1

[0173] 1. Preparation of IL-17A (Human) recombinant protein

[0174] Retrieved the sequence information of Human IL-17A (Q16552-1) from UniProt database (SEQ ID NO: 12), added 6xHis tag at C terminal, optimized according to prokaryotic codon, then synthesized gene and subcloned into pET28a vector; after Sanger sequencing verification, extracted plasmid.

[0175] Transformed BL21 competent with recombinant plasmid, induced overnight with 0.5mM IPTG, collected bacterial lysate; purified recombinant protein using nickel column.

[0176] Detected the purity of target protein by SDS-PAGE, the results are shown in Figure 1 After purification, the purity of IL-17A antigen protein was greater than 90%.

[0177] SEQ ID NO: 12:

[0178] MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA.

[0179] 2. Preparation of positive control antibody Ixekizumab

[0180] (1) Gene synthesis of Ixekizumab heavy chain and light chain variable regions (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14), the heavy chain variable region is subcloned into the pcDNA3.4-hIgG4 (the amino acid sequence of IgG4 is shown in SEQ ID NO: 15) vector, and the light chain variable region is subcloned into the pcDNA3.4-hIgKc vector (the amino acid sequence of IgG KC is shown in SEQ ID NO: 16); after Sanger sequencing verification, the endotoxin-free plasmid is prepared using the plasmid large extraction kit for standby.

[0181] (2) Take the LVTransm transfection reagent and the heavy chain expression vector and the light chain expression vector from the refrigerator, thaw at room temperature, and mix thoroughly with a pipette gun. Take PBS buffer and warm it to room temperature. Take 2 mL of PBS into one well of a 6-well plate, add 50 μg of heavy chain expression vector and 50 μg of light chain expression vector, mix thoroughly with a pipette gun, then add 300 μL of LVTransm, immediately mix with a pipette, and stand at room temperature for 10 minutes.

[0182] (3) Add the above DNA / LVTransm complex to 100 mL of 293F cells, mix thoroughly, and place the cells in a 37°C, 5% CO2 incubator at 130 RPM for continuous culture.

[0183] (4) After continuous culture for 5-7 days, centrifuge to collect the culture supernatant, filter with a 0.45 μm filter membrane, transfer the filtrate to a sterile centrifuge tube, and purify the antibody using a Protein A column.

[0184] SDS-PAGE is used to detect the purity of the target antibody protein, and the purity is >95%.

[0185] SEQ ID NO: 13:

[0186] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMY GTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQ GTLVTVSS.

[0187] SEQ ID NO: 14:

[0188] DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKVSNR FIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIK.

[0189] SEQ ID NO: 15:

[0190] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0191] SEQ ID NO: 16:

[0192] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0193] 3. ELISA assay for binding activity of Human IL-17A recombinant protein to control antibodies

[0194] (1) Use sterile PBS to dilute IL-17A recombinant protein to a final concentration of 2 μg / mL. Take a new 96-well plate and add 100 μL / well of 4°C overnight coating.

[0195] (2) Remove the antigen coating solution and wash 3 times with PBST (containing 0.5% Tween).

[0196] (3) Add 200 μL / well of 3% MPBS 37°C blocking for 2 hours.

[0197] (4) After removing the blocking buffer, wash the plate 3 times with PBST.

[0198] (5) Positive control antibody Ixekizumab is diluted with PBS to 10 μg / ml, 5-fold dilution of 7 points, 100 μL / well is added to the enzyme-labeled plate, and incubated at room temperature for 1 hour. The control wells are PBS.

[0199] (6) Remove the liquid in the wells and wash 3 times with PBST.

[0200] (7) Add secondary antibody HRP-ProteinA (Boster, BA1080) diluted 1:10000, 100 μL / well is added to the enzyme-labeled plate, and incubated at room temperature for 1 hour.

[0201] (8) After removing the liquid in the wells, wash the plate 3 times with PBST.

[0202] (9) Add 100 μL / well TMB color developing solution.

[0203] (10) Incubate at room temperature for 15 minutes in the dark.

[0204] (11) Add 50 μL / well of stop solution (2M HCL).

[0205] (12) Use a microplate reader to read the OD450 value in the wells.

[0206] The results of the binding ability of IL-17A recombinant protein to positive antibody are shown in the following table. As can be seen from the table, the positive antibody binds well to IL-17A antigen protein and can be used for immunization.

[0207] Table 1.

[0208]

[0209] 4、Human IL-17A recombinant protein activity detection

[0210] Experimental procedure:

[0211] (1) Resuscitate the NIH-3T3 cells from liquid nitrogen, continuously subculture to make the cells in logarithmic growth phase, after cell counting, inoculate 2x10 5 cells / well into 96-well plates.

[0212] (2) Add 100 μl of IL-17A (ACRO, Cat#ILA-H5118) of different concentrations and prepared IL-17A recombinant protein (ACRO protein as control) into 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, 10 μg / ml, respectively.

[0213] (3) Incubate at 37℃, 5% CO2 for 48 hours, after the incubation, gently take out the 96-well plate, centrifuge to collect the culture supernatant, and use mouse IL-6 ELISA kit to detect the secretion of IL-6.

[0214] (4) Use PRISM GraphPad to process the data, draw the curve graph, and calculate the EC50 value.

[0215] Experimental results:

[0216] The results are shown in Figures 2-3 According to the detection results, the IL-17A antigen protein has the activity of activating the expression of mIL-6 of NIH-3T3 cells, and the activity of IL-17A (TEST, hereinafter referred to as IL-17A recombinant protein) is positively correlated with its concentration, which can be used for immunization.

[0217] 5, IL-17A reporter gene cell strain construction

[0218] Experimental steps:

[0219] 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), a lentivirus expression vector is constructed and lentivirus is packaged, 293 cells are co-infected, and recombinant 293 cells overexpressing the two receptors are screened, and NFKB-Luciferase (the amino acid sequence is shown as SEQ ID NO: 19, and the nucleotide sequence encoding the same is shown as SEQ ID NO: 20) and ACT1 gene (the nucleotide sequence is shown as SEQ ID NO: 21) are further stably transfected to construct IL-17A reporter cell line 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc. IL-17A protein is added for activation, and positive control antibody Ixekizumab is added for blocking experiment detection, and the EC50 value is calculated to establish a candidate antibody targeting IL-17A for in vitro pharmacodynamic evaluation of the cell line.

[0220] SEQ ID NO: 17:

[0221] MGAARSPPSAVPGPLLGLLLLLLGVLAPGGASLRLLDHRALVCSQPGLNCTVKNSTCLDDSWIHPRNLTPSSPKDLQIQLHFAHTQQGDLFPVAHIEWTLQTDASILYLEGAELSVLQLNTNERLCVRFEFLSKLRHHHRRWRFTFSHFVVDPDQEYEVTVHHLPKPIPDGDPNHQSKNFLVPDCEHARMKVTTPCMSSGSLWDPNITVETLEAHQLRVSFTLWNESTHYQILLTSFPHMENHSCFEHMHHIPAPRPEEFHQRSNVTLTLRNLKGCCRHQVQIQPFFSSCLNDCLRHSATVSCPEMPDTPEPIPDYMPLWVYWFITGISILLVGSVILLIVCMTWRLAGPGSEKYSDDTKYTDGLPAADLIPPPLKPRKVWIIYSADHPLYVDVVLKFAQFLLTACGTEVALDLLEEQAISEAGVMTWVGRQKQEMVESNSKIIVLCSRGTRAKWQALLGRGAPVRLRCDHGKPVGDLFTAAMNMILPDFKRPACFGTYVVCYFSEVSCDGDVPDLFGAAPRYPLMDRFEEVYFRIQDLEMFQPGRMHRVGELSGDNYLRSPGGRQLRAALDRFRDWQVRCPDWFECENLYSADDQDAPSLDEEVFEEPLLPPGTGIVKRAPLVREPGSQACLAIDPLVGEEGGAAVAKLEPHLQPRGQPAPQPLHTLVLAAEEGALVAAVEPGPLADGAAVRLALAGEGEACPLLGSPGAGRNSVLFLPVDPEDSPLGSSTPMASPDLLPEDVREHLEGLMLSLFEQSLSCQAQGGCSRPAMVLTDPHTPYEEEQRQSVQSDQGYISRSSPQPPEGLTEMEEEEEEEQDPGKPALPLSPEDLESLRSLQRQLLFRQLQKNSGWDTMGSESEGPSA.

[0222] SEQ ID NO: 18:

[0223] MPVPWFLLSLALGRSPVVLSLERLVGPQDATHCSPVSLEPWGDEERLRVQFLAQQSLSLAPVTAATARTALSGLSGADGRREERGRGKSWVCLSLGGSGNTEPQKKGLSCRLWDSDILCLPGDIVPAPGPVLAPTHLQTELVLRCQKETDCDLCLRVAVHLAVHGHWEEPEDEEKFGGAADSGVEEPRNASLQAQVVLSFQAYPTARCVLLEVQVPAALVQFGQSVGSVVYDCFEAALGSEVRIWSYTQPRYEKELNHTQQLPDCRGLEVWNSIPSCWALPWLNVSADGDNVHLVLNVSEEQHFGLSLYWNQVQGPPKPRWHKNLTGPQIITLNHTDLVPCLCIQVWPLEPDSVRTNICPFREDPRAHQNLWQAARLQLLTLQSWLLDAPCSLPAEAALCWRAPGGDPCQPLVPPLSWENVTVDKVLEFPLLKGHPNLCVQVNSSEKLQLQECLWADSLGPLKDDVLLLETRGPQDNRSLCALEPSGCTSLPSKASTRAARLGEYLLQDLQSGQCLQLWDDDLGALWACPMDKYIHKRWALVWLACLLFAAALSLILLLKKDHAKGWLRLLKQDVRSGAAARGRAALLLYSADDSGFERLVGALASALCQLPLRVAVDLWSRRELSAQGPVAWFHAQRRQTLQEGGVVVLLFSPGAVALCSEWLQDGVSGPGAHGPHDAFRASLSCVLPDFLQGRAPGSYVGACFDRLLHPDAVPALFRTVPVFTLPSQLPDFLGALQQPRAPRSGRLQERAEQVSRALQPALDSYFHPPGTPAPGRGVGPGAGPGAGDGT.

[0224] SEQ ID NO: 19:

[0225] MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV.

[0226] SEQ ID NO:20:

[0227]

[0228] SEQ ID NO:21:

[0229]

[0230] Experimental results:

[0231] The IL-17A reporter gene cell line 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc was constructed, and IL-17A recombinant protein was added for binding. FACS results showed that the constructed IL-17A receptor overexpressing cell line could bind to IL-17A with a positive rate of more than 90% (results as shown in Figure 4 shown).

[0232] IL-17A activated cell experiment: IL-17A recombinant protein was used to activate 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc. The results are as follows Figure 5 As shown: IL-17A recombinant protein can effectively activate luciferase expression in the 293F-IL17Ra / IL17Rc-NFκB-Luc reporter gene cell line.

[0233] Ixekizumab blocked IL-17A function: The positive control antibody Ixekizumab was added to 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cells together with IL-17A recombinant protein. The positive control antibody Ixekizumab inhibited the binding of IL-17A protein to its membrane receptor and inhibited the intracellular NFκB signal, showing a dose effect.

[0234] 6. Animal immunization process

[0235] 6.1 Alpaca Immunity

[0236] Two alpacas (2# and 3#) were immunized with the IL-17A recombinant antigen protein prepared above for a total of 6 immunizations. The immune adjuvant was GERBU, the immunization interval was 14 days, and the last two immunizations were with Freund's complete adjuvant.

[0237] Arrange blood collection as follows:

[0238] 100 ml of blood was collected from 2# alpaca after five vaccinations to prepare a cDNA library and construct a yeast display library (yeast display library 1); 100 ml of blood was collected after six vaccinations to prepare a cDNA library.

[0239] 100 ml of blood was collected from alpaca 3# after six vaccinations to prepare a cDNA library, which was mixed with the cDNA library prepared from blood collected from alpaca 2# after six vaccinations to construct a yeast display library (yeast display library 2).

[0240] 6.2 Detection of immune titer

[0241] Experimental steps:

[0242] Sera were isolated from alpacas immunized at different times, subjected to limiting dilution, and tested by ELISA on 96-well plates pre-coated with antigens. The specific steps are as follows:

[0243] (1) Collect 5 mL of peripheral blood and place the centrifuge tube containing the blood sample in a 37°C incubator for 1 hour; then transfer the blood sample to 4°C overnight;

[0244] (2) Place the centrifuge tube containing the blood sample in a centrifuge and centrifuge at 5000 rpm for 20 minutes; separate the upper serum and transfer the serum to a new sterile centrifuge tube to collect the immune serum.

[0245] (3) Dilute the IL-17A recombinant protein in sterile CBS (carbonate buffer) to a final concentration of 1 μg / mL. Add 100 μL / well to a new 96-well microtiter plate and coat overnight at 4°C.

[0246] (4) Remove the antigen coating solution and wash five times with PBST (containing 0.05% Tween 20).

[0247] (5) Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours;

[0248] (6) After removing the blocking buffer, wash the plate 5 times with PBST;

[0249] (7) Add 100 μl of serially diluted serum (100 μL / well) and incubate at room temperature for 1 hour. The control wells were filled with PBS.

[0250] (8) Remove the liquid in the wells and wash with PBST 5 times;

[0251] (9) Add 100 μl of HRP anti-Llama IgG (H+L) antibody (1:50,000 dilution) and incubate at room temperature for 1 hour;

[0252] (10) After removing the liquid in the wells, wash the plate 5 times with PBST;

[0253] (11) Add 100 μL / well TMB colorimetric solution;

[0254] (12) Incubate at room temperature in the dark for 10-15 minutes;

[0255] (13) Add 50 μL / well stop solution;

[0256] (14) Use a microplate reader to read the OD450 value in the well.

[0257] The experimental results are shown in Tables 2-3 below. According to the ELISA detection results, the immune serum is well combined with the IL-17A recombinant protein, and the OD value changes in gradient with the gradient dilution of the immune serum.

[0258] Table 2. Immune titer detection results

[0259]

[0260] Table 3. Immune titer detection results

[0261]

[0262] 7. Construction of antibody yeast library

[0263] 7.1 PBMC separation and VHH antibody fragment cloning

[0264] Experimental steps:

[0265] (1) Collect 100 mL of peripheral blood anticoagulant sample, and separate PBMC cells using lymphocyte separation medium.

[0266] (2) Extract RNA, and perform reverse transcription using PrimeScript™ II 1st Strand cDNA Synthesis Kit to prepare cDNA.

[0267] 1) Prepare the following reaction mixture Mix in a 200 μL PCR tube:

[0268] Table 4.

[0269]

[0270] 2) After 5 min of 65°C incubation, quickly cool on ice.

[0271] 3) Prepare the following reaction solution in the above PCR tube:

[0272] Table 5.

[0273]

[0274] 4) After mixing, aliquot 80 μL / tube, and place in a PCR instrument at 42°C for 1 hour, and then heat inactivate at 70°C for 15 minutes. Finally, place the cDNA sample on ice or at -20°C for long-term storage.

[0275] (3) Amplification of VHH fragments

[0276] 1) Prepare the first round of PCR reaction system (50 μL / tube):

[0277] Table 6.

[0278]

[0279] 2) After the PCR reaction system was configured, the PCR instrument was set according to the following program:

[0280] Table 7.

[0281]

[0282] 3) Agarose electrophoresis of PCR products

[0283] 1% agarose was used for electrophoretic analysis of PCR products, and fragments with a molecular weight of about 750 bp were separated. The PCR product was recovered using a gel recovery kit, and the concentration was determined by NanoDrop.

[0284] 4) Configuration of the second round of PCR reaction system (50 μL / tube)

[0285] Table 8.

[0286]

[0287] 5) After the PCR reaction system was configured, the PCR instrument was set according to the following program:

[0288] Table 9.

[0289]

[0290] 6) Agarose electrophoresis analysis of the second round of PCR products

[0291] 1% agarose was used for electrophoretic analysis of PCR products, and VHH fragments with a molecular weight of about 400 bp were separated. The VHH PCR product was recovered using a gel recovery kit, and the concentration was determined by NanoDrop.

[0292] Experimental results:

[0293] Peripheral blood was collected, total RNA was extracted, and after reverse transcription to cDNA, single-domain antibody amplification primers were used for three rounds of PCR, and the PCR products were detected by agarose gel electrophoresis (results are shown in Figures 6-7 ): The first round of PCR obtained PCR bands of about 1000 bp and 750 bp, and the 750 bp fragments were recovered as templates for the second round of PCR. The second round of PCR obtained a band of about 400 bp, which was a VHH fragment, and was column recovered as a template for the third round of PCR. The third round of PCR obtained a band of about 500 bp, which added a homologous arm and was subsequently homologously recombined into the yeast display vector pDisplay.

[0294] 7.2 Construction of single-domain antibody yeast display library

[0295] ​(1) Linearization of the yeast display vector pDisplay, and the enzyme digestion system is as follows:

[0296] Table 10.

[0297]

[0298] 1) Digest the pDisplay vector with SfiI, aliquot 100 μL / tube, and incubate at 50°C overnight.

[0299] 2) Separate the pDisplay vector fragment using a 1% agarose gel, excise a 5000 bp fragment, recover it from the gel, and determine its concentration using a NanoDrop.

[0300] 3) Aliquot 200 μL of the recovered pDisplay digestion product into each 1.5 mL centrifuge tube. Add 1 / 10 volume (20 μL) of 3M sodium acetate and 1 μg / μL glycogen. Mix by pipetting. Add 880 μL of anhydrous ethanol, mix by inversion, and store at -80°C.

[0301] (2) Construction of yeast display library by electroporation

[0302] 1) Streak the competent yeast strain frozen at -80°C onto a YPD solid medium plate and activate at 30°C for 3-5 days.

[0303] 2) Inoculate a single competent yeast colony into 50 mL of YPD medium and shake at 250 rpm and 30°C for 1-2 days.

[0304] 3) Prepare a competent yeast strain. Mix the linearized vector fragment and PCR product, add them to an electroporation cuvette, and electroporate. After electroporation, shake the competent yeast in a transfection culture flask at 220 rpm and 30°C for 1 hour.

[0305] 4) Take 20 μL of the resuspension, dilute it 5000-fold with SDCAA, aspirate 100 μL, apply it to SDCAA plates, incubate for 2-3 days, calculate the reservoir volume, and continue to incubate the remaining bacterial solution for 24 hours;

[0306] 5) Preservation: Collect the remaining bacterial suspension into a 50 mL centrifuge tube and centrifuge at 3000 g for 5 min. Discard the supernatant and resuspend in 10 mL of SDCAA. Mix the suspension with 50% glycerol and resuspension solution in a ratio of 1:1 and freeze at -80°C.

[0307] 8. Yeast display library selection

[0308] Experimental steps:

[0309] 1) Add yeast cultured in SDCAA to a 250 mL shake flask containing 50 mL of SGCAA medium and incubate in a shaker at 30°C, 240 rpm, for 16 h.

[0310] 2) After centrifugation, discard the supernatant, resuspend the tube in 1 mL of 0.5% PBSA, transfer to a 1.5 mL centrifuge tube, centrifuge at 3000 g for 5 minutes, discard the supernatant, and wash again with 0.5% PBSA.

[0311] 3) Wash the streptavidin magnetic beads incubated with antigen twice with 0.5% PBSA (incubating at 4°C with rotation for 5 minutes each time), place on a magnetic stand for 5 minutes, and discard the supernatant.

[0312] 4) Add the yeast solution to the antigen-bound magnetic beads, incubate with rotation at 4°C for 60 minutes, and place on a magnetic stand for 15 minutes.

[0313] 5) Discard the yeast solution and retain the magnetic beads. Wash three times with 0.5% PBSA (rotating and incubating at 4°C for 5 minutes each time).

[0314] 6) Resuspend the magnetic beads in 1 mL of SDCAA medium. Pipette 0.5-5 μL of the resuspension into 100 μL of SDCAA medium and spread on a plate. Divide the resuspension into two equal portions. Add 500 μL of 50% glycerol (stored at -80°C) to one portion; add the other portion to a shaker tube and add 2 mL of SDCAA medium. Incubate at 30°C, 240 rpm, and incubate for 16 h.

[0315] 7) Transfer the bacterial solution from the shake tube to 50 mL of SDCAA medium (in a 250 mL shake flask) and culture overnight at 30°C and 240 rpm.

[0316] 8) Measure the OD600 value of the bacterial solution. Centrifuge a portion of the culture according to the OD600 value, resuspend in SGCAA, and transfer to 50 mL of SGCAA medium to a final OD600 value of 1. Incubate overnight at 30°C, 240 rpm. Resuspend the remaining culture in a 1:1 ratio of SDCAA:50% glycerol and freeze at -80°C.

[0317] 9. Yeast monoclonal flow cytometry detection

[0318] After sorting, the yeast liquid was spread on SDCAA plates, and single clones were picked and cultured. After induction of expression for 48 hours, it was incubated with Biotin-antigen and PE-Streptavidin as the secondary antibody. After incubation, flow cytometry was performed. The yeast clones bound to the target antigen were lysed with 0.2% SDS (incubated at 95℃ for 10 minutes), centrifuged, and 0.5μL of the supernatant was taken as a template for PCR amplification and sent for testing (the remaining bacterial liquid was stored at -20℃). The flow cytometry results are as follows: Figure 8As shown, according to the flow detection results, after the second magnetic separation, the positive rate of the yeast was 37.9%, and the positive clones were significantly enriched. The separation product was directly coated on the SDCAA plate, and single clones were selected for flow detection.

[0319] 2#+3#Alpaca yeast library was combined with Biotin-IL-17A-His protein, and 2 rounds of magnetic separation were performed using streptavidin magnetic beads; the separated yeast cells were cultured, induced for expression, and then subjected to flow analysis. Incubation with Biotin-IL-17A-His for 1 h, secondary antibody using PE Streptavidin, and flow detection after completion of incubation, the results show (as shown in Figure 9 ): After two rounds of magnetic separation, the yeast combined with Biotin-IL-17A-His accounted for 20.59%, and the positive clones were significantly enriched. The separation product was directly coated on the SDCAA plate, and single clones were selected for flow detection.

[0320] 10. Antibody sequence identification

[0321] Positive clones were enriched; single clones after enrichment were selected for Phage ELISA identification, and the clones were sequenced for analysis to obtain nucleic acid and amino acid sequence information of the candidate single domain antibody. As shown in Figures 10-11 , 20 single clones were randomly selected for sequencing analysis, and the sequences were significantly different, indicating good library diversity. According to the amino acid sequence information of the CDR region of the candidate single domain antibody, In silico method was used to analyze the possible post-translational modification sites.

[0322] According to the flow detection results of the yeast single clone, the positive clones combined with IL-17A-His were selected for extraction of genomic DNA, and the antibody sequence was obtained by PCR. According to the sequencing results of the PCR product, the difference clones were selected for overlap PCR amplification, and the specific steps were as follows:

[0323] (1) First round of PCR: amplification of CMV, VHH and FC

[0324] 1) Configuration of PCR reaction system (50 μL system / reaction)

[0325] Table 11.

[0326]

[0327] 2) The PCR reaction program is as follows:

[0328] 95℃, 10min; (95℃, 15s; 56℃, 30s; 68℃, 60s; 25 cycles); 68℃, 10min.

[0329] 3) Take 50 μL of PCR product, add 1 / 10 volume of 10x loading buffer, use 1% agarose for electrophoresis analysis, 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.

[0330] 4) Cut the target band from the gel, purify the PCR product, and measure the concentration with NanoDrop (if the concentration is too high, dilute it for subsequent reactions).

[0331] (2) Second round of PCR: Overlap Extension PCR connecting CMV, VHH and FC

[0332] 1) Configure the PCR reaction system

[0333] Table 12.

[0334]

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

[0336] 95℃, 10min; (95℃, 15s; 60℃, 30s; 68℃, 120s; 15 cycles).

[0337] 3) Add primers, 2 μL of upstream primer and 2 μL of downstream primer;

[0338] The PCR reaction program is as follows:

[0339] 95℃, 10min; (95℃, 15s; 60℃, 30s; 68℃, 120s; 20 cycles); 68℃, 10min.

[0340] 4) Purify the overlap PCR product using TakaRa DNA fragment recovery kit, and measure the concentration with NanoDrop, at least 10 μg of PCR product is required. For subsequent cell transfection verification.

[0341] 5) The transfection steps are the same as those of the eukaryotic expression vector.

[0342] Add a signal peptide to the N terminus of VHH and IgG1-FC to the C terminus, and transiently transfect HEK293 cells with the PCR product; take the expressed antibody supernatant for ELISA detection: 100 uL of transfection supernatant is added to IL-17A recombinant antibody pre-coated 96-well plates for incubation, HRP-Protein A is used as the secondary antibody for ELISA detection, and the results are shown in Table 13: 1-H9 binds to IL-17A-His antigen, and a eukaryotic expression vector is constructed.

[0343] Table 13. Results of candidate clone transfection supernatant ELISA binding experiments

[0344]

[0345] 11. Expression and purification of candidate single domain antibodies

[0346] Experimental procedure:

[0347] 1) According to the ELISA detection results of the candidate antibodies, positive clones were selected, and the obtained VHH antibody sequences were respectively genetically synthesized and subcloned into the expression vector pcDNA3.4-hIgG1-Fc in series with human IgG1 Fc (SEQ ID NO: 10). After the vector was sequenced and verified to be correct, a Qiagen plasmid maxi kit was used to prepare a endotoxin-free plasmid for standby.

[0348] 2) Take LVTransm transfection reagent and single-chain antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly with a pipette gun. Take 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 antibody expression vector, mix thoroughly with a pipette gun, then add 400 μL of LVTransm, immediately mix with a pipette, and stand at room temperature for 10 minutes.

[0349] 3) Add the above DNA / LVTransm complex to 30 mL of 293F cells and mix thoroughly. Place the cells in a 37°C, 5% CO2 incubator at 130 rpm for 6-8 hours, then add 50 mL of fresh 293 cell culture medium and continue to culture in the incubator.

[0350] 4) After 7 days of continuous culture, centrifuge to collect the culture supernatant, filter with a 0.45 μm filter membrane, and transfer the filtrate to a sterile centrifuge tube. Use a Protein A column to purify the antibody.

[0351] The steps for purifying the antibody with a Protein A column are as follows:

[0352] 1) Add the sample containing the target antibody to an EP tube and mix by gently inverting the tube.

[0353] 2) Mix the EP tube at room temperature or incubate it on a rotator (1-4 hours or overnight), and add 100 mM PMSF to prevent protein degradation.

[0354] 3) Collect the magnetic beads using a magnetic separation rack and discard the supernatant.

[0355] 4) Add 1 mL binding / washing buffer to the EP tube and mix well, collect the magnetic beads using a magnetic stand and discard the supernatant, repeat the washing step three times.

[0356] 5) Add 500 μL elution buffer to the EP tube, resuspend quickly under pipetting or vortexing, then incubate at room temperature (about 25°C) for 5 minutes in a rotating mixer or gently flip the EP tube by hand.

[0357] 6) Collect the magnetic beads using a magnetic separation stand, and transfer the supernatant containing the eluted antibodies to a clean EP tube.

[0358] 7) Repeat steps 1) and 2) twice.

[0359] 8) Add 1 / 10 of neutralization buffer to each 500 μl eluate to neutralize the pH, so as to maintain the biological activity of the antibodies and avoid inactivation of the antibodies.

[0360] 9) Binding / washing buffer: 1 × PBS, pH 7.0.

[0361] Elution buffer: (1) 0.1 M glycine, pH 2-3 (2) 0.1 M NaAc-HAc, pH 3.6.

[0362] Neutralization buffer: 1 M Tris, pH 8.5.

[0363] Magnetic bead regeneration buffer: 0.1 M NaOH.

[0364] The experimental results are shown in Figure 12 .

[0365] Example 2 ELISA detection of the binding of recombinant antibodies to target proteins

[0366] Experimental steps:

[0367] 1) Dilute the recombinant protein to a final concentration of 2 μg / mL using sterile CBS. Take a new 96-well enzyme-labeled plate, and add 100 μL / well of 4°C overnight coating.

[0368] 2) Remove the antigen coating solution, and wash 5 times using PBST (containing 0.05% Tween 20).

[0369] 3) Add 200 μL / well of 3% MPBS and incubate at 37°C for 2 hours;

[0370] 4) After removing the blocking buffer, wash the plate 5 times using PBST;

[0371] 5) Add purified single-domain antibody, starting concentration is 10 μg / mL, 5-fold gradient dilution 7, the results show that the EC50 value of single-domain antibody 1-H9 is 21.08, the EC50 of positive antibody (Ixekizumab) = 10.06, single-domain antibody 1-H9 has better binding activity with HumanIL-17A protein Figure 13 The EC50 of each candidate antibody is shown in Table 14.

[0372] Table 14. EC50 values of candidate antibodies

[0373]

[0374] Example 3 FACS detection of IL-17A binding to reporter cell lines

[0375] Experimental process:

[0376] 1) Resuscitate 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cell lines from liquid nitrogen, adjust cell state to logarithmic growth phase;

[0377] 2) Divide the cells into several parts, and the number of cells in each part is 2x10 5

[0378] 3) Incubate IL-17A-His protein with target cells, mix well, and incubate at room temperature for 1 hour;

[0379] 4) Centrifuge at 800xg for 3 minutes at room temperature, remove the supernatant containing the antibody, and wash the cells with PBS 3 times;

[0380] 5) Add secondary antibody APC-His (1:500 dilution), mix well, and incubate at room temperature for 30 minutes in the dark;

[0381] 6) Centrifuge at 800xg for 3 minutes at room temperature, remove the supernatant containing the secondary antibody, and wash the cells with PBS 3 times;

[0382] 7) Resuspend the cells with 500 μL PBS and perform flow cytometry analysis.

[0383] Example 4 Single-domain antibody blocking function experiment

[0384] Experimental steps:

[0385] ​In the 96-well plate, add the gradient dilution of the detection antibody (positive antibody: Ixekizumab; antibody to be detected), dilute the antibody by 10 times gradient, dilute 10 times in sequence, and the final concentration is 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, 0 μg / mL, take 50 μL of the diluted gradient concentration antibody and add it to the 96-well plate, 2 replicates for each gradient. Then add 50 μL of 0.4 μg / mL IL-17A protein (final concentration 0.1 μg / mL) to the corresponding wells. After mixing, incubate in a 37°C incubator for 1 hour. Take the 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cells cultured to the logarithmic growth phase in the 96-well plate, inoculate 2x10 4 cells per well. After 18 hours of co-culture, 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.

[0386] Experimental results:

[0387] The results are shown in Figure 14 : Ixekizumab positive control can block Human IL-17A protein activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc. IL-17A single domain antibody 1-H9 in the antibody to be detected can block Human IL-17A protein activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc, but the blocking effect is weaker than that of the positive antibody.

[0388] Example 5 Stability experiment

[0389] The fluorescence change is detected by micro-differential scanning fluorescence technology (nanoDSF) technology, which can detect protein thermal denaturation and chemical denaturation under natural conditions, accurately determine the temperature (Tm) at which the protein is 50% unfolded and the temperature (Tagg) at which aggregation begins; the higher the thermal denaturation Tm value and Tagg, the more stable the antibody protein.

[0390] Experimental steps:

[0391] Take 100 μL of the candidate antibody prepared in the previous project and Ixekizumab (sample concentration greater than 200 μg / ml), centrifuge at 4°C, 12000xg for 10 min, then use capillary to suck the sample, prepare two capillaries for each sample as parallel controls, and place them in the corresponding card slots in order, ensuring that the capillary is full of sample without air bubbles, and then perform detection analysis.

[0392] Experimental results:

[0393] The experimental results are shown in Figures 15-16 From the figure, it can be seen that the single-domain antibody 1-H9 has strong stability, Tm=58, Tagg=80.15, and the positive control has Tm=56.1, Tagg=61.86.

[0394] Application Example 1: An antibody preparation

[0395] The antibody preparation comprises: an anti-IL-17A antibody, the amino acid sequence of which is shown in SEQ ID NO: 8; a buffer, a surfactant, an amino acid, a tonicity agent, etc.

[0396] In an embodiment of the present application, the preparation of the antibody preparation comprises: weighing each substance, dissolving in water and mixing uniformly, and adjusting each component to the following concentrations: (100-200) mg / ml of anti-IL-17A antibody (the amino acid sequence is 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.

[0397] Application Example 2: A kit

[0398] The kit comprises: an anti-IL-17A antibody, a recombinant protein, an antibody preparation and / or a polyclonal antibody, a container loaded with the antibody preparation, a buffer, etc.

[0399] In an embodiment of the present application, the kit comprises: (100-200) mg / ml of anti-IL-17A antibody (the amino acid sequence is shown in SEQ ID NO: 8), and a buffer with a pH of 5.0-8.0.

[0400] Application Example 3: An antibody drug conjugate

[0401] The antibody drug conjugate comprises: an anti-IL-17A antibody, a recombinant protein, an antibody preparation and / or a polyclonal antibody, a drug which is a physiologically active substance (such as nucleic acid, etc.) and a linker connecting the antibody and the drug (the linker includes a maleimide linker, a Val-Cit linker, an SS linker and a DMSS linker).

[0402] In one embodiment of the present application, the IL-17A antibody is connected with the drug through the SS linker, and a (100-200) mM aqueous solution is added and mixed at room temperature to terminate the linker reaction, thereby obtaining the antibody-drug conjugate.

[0403] Pharmaceutical composition

[0404] The pharmaceutical composition includes the anti-IL-17A antibody, the recombinant protein, the antibody preparation, the polyclonal antibody, the nucleic acid molecule, the biological expression vector and / or the host cell, and further includes the pharmaceutically acceptable excipient.

[0405] In one embodiment of the present application, the preparation of the pharmaceutical composition includes: preparing the anti-IL-17A antibody or the antigen binding fragment thereof with a concentration of (100-200) mg / ml, adding (1-20 w / v) sucrose, (10-300) mM histidine and (0.1-10)% Tween 80, thereby obtaining the pharmaceutical composition.

[0406] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. An anti-IL-17A single domain antibody, characterized in that: The antibody comprises a complementary determining region (HCDR), wherein the complementary determining region (HCDR) comprises: HCDR1 having an amino acid sequence as shown in SEQ ID NO: 1, HCDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and HCDR3 having an amino acid sequence as shown in SEQ ID NO:

3.

2. The antibody according to claim 1, characterized in that: The antibody includes a framework region FR; the framework region FR includes: 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.

3. The antibody according to claim 2, characterized in that: The amino acid sequence of the antibody comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 4-7.

4. The antibody according to claim 2, characterized in that: The amino acid sequence of the antibody comprises: an amino acid sequence obtained by at least one of addition, deletion, modification and / or substitution on the amino acid sequence shown in SEQ ID NO: 4-7.

5. The antibody according to claim 1, wherein: The amino acid sequence of the antibody is shown in SEQ ID NO:

8.

6. The antibody according to any one of claims 1 to 5, characterized in that: The invention also comprises an antibody heavy chain framework region partially or entirely selected from alpaca sources or a variant thereof.

7. An antibody preparation, characterized in that: The antibody preparation comprises the antibody according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. A polyclonal antibody, characterized in that: Comprising the antibody according to any one of claims 1 to 6.

9. A kit, characterized in that: The kit comprises: (1) the antibody according to any one of claims 1 to 6, the antibody preparation according to claim 7, or the polyclonal antibody according to claim 8; and (2) A container for containing the antibody preparation according to claim 7.

10. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the antibody according to any one of claims 1 to 6 or the polyclonal antibody according to claim 8.

11. A biological expression vector, characterized in that: The biological expression vector comprises the nucleic acid molecule according to claim 10.

12. A host cell, characterized in that: The genome of the host cell is integrated with the nucleic acid molecule of claim 10; or comprises the biological expression vector of claim 11.

13. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises: the antibody according to any one of claims 1 to 6, the antibody preparation according to claim 7, the polyclonal antibody according to claim 8, the nucleic acid molecule according to claim 10, the biological expression vector according to claim 11, or the host cell according to claim 12.

14. The pharmaceutical composition according to claim 13, characterized in that The pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

15. Use of the antibody according to any one of claims 1 to 6, the antibody preparation according to claim 7, the polyclonal antibody according to claim 8, the kit according to claim 9, the nucleic acid molecule according to claim 10, the biological expression vector according to claim 11, or the host cell according to claim 12, characterized in that: The use is selected from at least one of the following: (1) Preparation of reagents or kits for detecting IL-17A; (2) Preparation of drugs for treating autoimmune diseases, wherein the autoimmune diseases are plaque psoriasis, rheumatoid arthritis, and psoriatic arthritis.

16. A method for detecting IL-17A in a sample in vitro for non-diagnostic purposes, characterized in that: The method comprises the following steps: (1) combining the antibody according to any one of claims 1 to 6, the antibody preparation according to claim 7, or the polyclonal antibody according to claim 8 with a test sample; (2) Detect antigen-antibody complexes and interpret the results.

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