Novel anti-il-17a single domain antibodies, preparation and use thereof

Abstract

The application provides a novel anti-IL-17A single-domain antibody, preparation and application thereof, and belongs to the technical field of cellular immunology. The amino acid sequence of the antibody provided by the application comprises: (1) HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; and / or; (2) an amino acid sequence 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. The antibody has the activities or performances of strong binding capacity and stability, good blocking effect and the like. The application also provides application of the antibody in preparation of a drug for treating autoimmune diseases.

Description

TECHNICAL FIELD

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

[0002] IL-17A is secreted by TH17 cells, and plays a pro-inflammatory function by interacting with GM-CSF, IFN-gamma, IL-22, IL-1beta and TNF-alpha and other mediators, and the downstream signaling pathway mediated by IL-17A further induces the production of chemokines, cytokines, AMPs and restructured proteins, thereby playing an important role in cell transport, immune regulation, host defense and tissue repair. IL-17A induces the production of various cytokines and chemokines, thereby playing an important role in autoimmune suppression diseases and various types of tumors.

[0003] Research has found that specific blocking of the binding of IL-17A to its receptor can block the signal transmission of inflammatory factors, thereby achieving the purpose of treating diseases such as psoriasis. At present, there are many antibody drugs blocking IL-17 on the market worldwide, including monoclonal antibody drugs neutralizing IL-17A (Secukinumab and Ixekizumab) or IL-17RA (Brodalumab), and there are more than 50 antibody drugs or small molecules under research in China for the treatment of autoimmune diseases. Although antibodies generally have good efficacy and tolerability 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, cardiotoxicity and severe immune response; in addition, the complex structure of monoclonal antibody drugs and the relatively poor stability also lead to the need for repeated large-dose injection to achieve the best effect.

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

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

[0006] The present application is a patent application proposed by the inventor for the anti-IL-17A antibody independently developed by the inventor.

[0007] In addition to the antibody claimed in the present application, the inventor has also developed another 8 antibodies, and based on the relevant provisions of the single nature of the Patent Law, 9 different antibodies are respectively claimed.

[0008] The inventor has also developed 12 tandem antibodies based on the 9 antibodies, and based on the relevant provisions of the single nature of the Patent Law, 12 different tandem antibodies are respectively claimed.

[0009] The inventor has also developed gene-modified stem cell technology based on the 12 tandem antibodies, and based on the relevant provisions of the single nature of the Patent Law, 3 different gene-modified stem cells and 3 different applications are respectively claimed.

[0010] In order to facilitate the understanding of the technical scheme of the present application, the other series of application texts of the project can be optionally referred to. SUMMARY

[0011] Terms and statements of the present application:

[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 function in a manner similar to the naturally occurring amino acids. Natural amino acids are those encoded by the genetic code. Amino acid analogs refer to compounds that have the same basic chemical structure as the naturally occurring amino acids. 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.

[0013] 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 means that both whole antibodies and antigen-binding fragments thereof are included. 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 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), 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.

[0015] 4、As used herein, the terms "single domain antibody (VHH)", "single domain antibody" (sdAb, or nanobody) have the same meaning, referring to the variable region of the heavy chain of an antibody, which is constructed to consist of only one heavy chain variable region, which is the smallest antigen-binding fragment with full function. Usually, after obtaining an antibody naturally lacking a light chain and a heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0016] 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 in each sequence, which can be aligned for purposes of comparison. When a position in the comparison sequence is occupied by the same base or amino acid, then 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. An antigenic determinant typically comprises a chemically active surface group of a molecule, such as an amino acid or sugar side chain, and often has specific three-dimensional structural features as well as specific charge features. For example, an epitope often includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which 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 to each other to form a peptide chain (or polypeptide), which can only be read in one direction.

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

[0020] 9. As used herein, the term "framework region" or "FR" refers to the variable region of an immunoglobulin's H and L chains that is highly conserved among different species of antibodies and is not involved in antigen binding. The framework region is flanked on one side by the hypervariable region and on the other side by the constant region.

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

[0022] 11、The terms "hypervariable region," "hypervariable region," "complementarity determining region," "HVR," or "CDR," as used herein refer to the regions of an antibody variable domain that are highly variable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs or CDRs: three in the VHand three in the VL. Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L26-L32 (LI), L50-L52 (L2), L91-L96 (L3), H26-H32 (HI), H52-H56 (H2), and H96-H101 (H3) based on the Chothia definition rules (Chothia et al., J. Mol. Biol. 196:901-917 (1987)). Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L24-L34 (LI), L50-L56 (L2), L89-L97 (L3), H31-H35 (HI), H50-H65 (H2), and H95-H102 (H3) based on the Kabat definition rules (Kabat et al., Sequences of Proteins of Immunological Interest, the fifth edtion, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L27-L32 (LI), L50-L51 (L2), L89-L97 (L3), H26-H33 (HI), H51-H56 (H2), and H93-H102 (H3) based on the IMGT definition rules (Honjo, T. and Alt, F.W. (1995) Immunoglobulin genes. Academic Press pp. 3-443). It is well known to those of skill in the art that CDRs of an antibody can be defined in various ways in the art, such as the Kabat definition rules based on sequence variability, the Chothia definition rules based on the location of structurally looped regions, and the reference tool based on CDR grafting for antibody humanization design (see J Mol Biol. 273:927-48, 1997).It will be understood by those skilled in the art that the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., a variable region) are intended to encompass complementarity determining regions defined by any of the above known schemes as described by the present disclosure, unless otherwise specified. While the scope of the present disclosure is based on the sequences shown according to the IMGT definition rules, the amino acid sequences corresponding to other CDR definition rules should also fall within the scope of the present disclosure.

[0023] 12. As used herein, the terms "comprises" or "comprising" means "including, but not limited to." The terms are intended to be open-ended, to designate any stated features, elements, integers, steps, or components, but does not preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of and "consisting essentially of." In one embodiment, the term "comprising" as used throughout the application, and in the claims, can be replaced with the term "consisting of." The three letter and one letter codes for amino acid residues known to those of skill in the art or as described in J Biol. Chem, 243, p 3558 (1968) are used herein.

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

[0025] 14. As used herein, the term "about" means ±20% of the numerical value of which it is used in relation to. In some embodiments, the term "about" means ±10% of the numerical value of which it is used in relation to. In some embodiments, the term "about" means ±5% of the numerical value of which it is used in relation to.

[0026] 15. As used herein, the term "and / or" is to be taken as a specific term meaning either one of the items or a combination of any two or more of the items.

[0027] 16、As used herein, the term "IL-17A" or "interleukin-17A" refers to a cytokine belonging to the interleukin 17 family, 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, other cells including CD8+ T cells, gd T cells, NK cells, and neutrophils, mast cells and macrophages also express IL-17A. It mainly acts on immune cells such as macrophages, neutrophils and endothelial cells, inducing 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 cases. 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 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 attenuates IL-17A activity, and / or inactivates IL-17A, prevents IL-17A response or downstream pathways or other IL-17A-mediated functions mediated by IL-17A.

[0029] 18、As used herein, the term "antibody" refers to a glycoprotein comprising heavy (H) and light (L) chains interconnected by disulfide bonds (SS). Each heavy chain is composed 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 is composed of three domains, CH1, CH2 and CH3. Each light chain is composed 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 is composed of one domain, CL. The light chain is divided into two types, kappa type light chain and lambda type light chain (for example, the light chain constant region Ck / in the present invention indicates that the light chain constant region is a kappa type light chain or a lambda type light chain). The VH and VL regions can be further subdivided into hypervariable regions (also known as complementarity determining regions (CDRs)), which are interspersed with more conserved regions or framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino-terminal to the carboxy-terminal: 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 "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, 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 with non-human homologs of the targeted peptide, or with other proteins or tissues), and the ability to maintain high expression levels of the protein in mammalian cells. The aforementioned properties or characteristics are observed, measured, or evaluated using techniques known in the art, including, but not limited to, ELISA, FACS or BIACORE® plasmon resonance analysis, un-limited 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] 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 disassembly of the Fc fragment is variable, the Fc fragment of the heavy chain of human IgG generally refers to the polypeptide from A231 to the carboxy terminus thereof.

[0032] 21. As used herein, the term "affinity" or "binding affinity" 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). 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] 22. As used herein, the terms "high affinity" or "high affinity" with respect to IgG antibodies refer 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] 23. 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 via a linker. The linker can be cleavable in a particular environment (e.g., intracellular low pH environment) or by a particular action (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 biologically active compound fragment and the targeting moiety or antibody or antigen binding fragment thereof are directly linked via a covalent bond that is cleavable in a particular environment or by a particular action, thereby separating the biologically active compound fragment from the antibody or antigen binding fragment thereof moiety.

[0035] 24. The term "nucleic acid" or "polynucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing analogues of natural nucleotides, as well as analogues that have similar binding properties as the reference nucleic acid and are metabolized in a similar manner as naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridines are replaced with pseudouridines, methods of synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of

[0036] 25. The term "isolated nucleic acid" as used herein is a nucleic acid that has been identified and separated from a constituent of its natural environment. Isolated nucleic acid includes nucleic acid contained in cells that are typically found in nature that are not present in a cell, or present in a different cell than that of its natural environment.

[0037] 26、The term "vector," as used herein, refers to any recombinant polynucleotide construct that can be used for the purposes of transformation (i.e., introduction of heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double stranded DNA loop, which can be used to link additional DNA segments. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes operably linked thereto. Such vectors are referred to herein as "expression vectors."

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

[0039] 28、The terms "cell" and "cell line," as used herein, are used interchangeably and all such designations include the progeny thereof. The term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as E. 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、The term "pharmaceutical composition," as used herein, generally refers to a preparation which is in a form suitable for its intended administration to a subject in need of treatment, and which allows the biological activity of the active ingredients to be effective without additional components that are unacceptable toxic to the subject to which the composition will be administered. The composition is sterile.

[0041] 30、The term "pharmaceutically acceptable" means, within the scope of sound medical judgment, those compounds, materials, compositions, and / or dosage forms which are suitable for use with human and animal tissues without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio as commonly employed in the art.

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

[0043] 32、As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to the amount of an anti-IL-17A antibody or antigen-binding fragment thereof of the present application, when administered alone or in combination with other therapeutic agents, to a cell, tissue, or subject, that is effective to prevent or ameliorate the symptoms of, or the progression of, one or more diseases or conditions. A therapeutically effective dose also refers to the amount of an antibody or antigen-binding fragment thereof that is sufficient to result in amelioration of symptoms, e.g., an amount that treats, cures, prevents or ameliorates a relevant medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition. When administered to an individual, a therapeutically effective dose refers only to the active ingredients administered alone. When combination therapy is administered, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement of at least 10% in a diagnostic test or parameter; usually 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 term "cancer" or "tumor" refers to a physiological condition that describes a malignant process in a mammal that is 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 term "autoimmune disease" or "autoimmune disorder" refers to a disease that results from a decrease, reduction and / or destruction of immunologic tolerance to self-antigens, resulting in self-antibodies and / or sensitized lymphocytes that damage the body's own tissues, manifested as dysfunction of the corresponding tissues and organs.

[0047] The technical solutions of the present application are as follows:

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

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

[0050] (2) an amino acid sequence having at least 80% sequence identity to 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 application, 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 shown in SEQ ID NOs: 1-3.

[0055] 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 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 substitution is preferably a substitution of one amino acid residue in one of the following groups (a) to (e) 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 Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0057] 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.

[0058] 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.

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

[0060] (1) FR1, FR2, FR3, FR4 having an amino acid sequence as shown in SEQ ID NO: 4-7; and / or;

[0061] (2) an amino acid sequence having at least 80% sequence identity to the amino acid sequence as shown in SEQ ID NO: 4-7.

[0062] Further, 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 as shown in SEQ ID NO: 4-7.

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

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

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

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

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

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

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

[0077] (1) the amino acid sequence as shown in SEQ ID NO: 1-3; or

[0078] (2) a functionally active variant of the amino acid sequence having 1, 2, 3, 4, 5 amino acid differences compared to (1);

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

[0080] 1) the amino acid sequence as shown in SEQ ID NO: 4-7; or

[0081] 2) a functionally active variant of the amino acid sequence 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] Further, the antibody of the above first to fourth aspects is a single domain antibody.

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

[0088] Preferably, the antibody comprises part or all of antibody heavy chain framework regions selected from human, murine, primate or camelid origin or variants thereof;

[0089] Further, the antibody comprises part or all of antibody heavy chain framework regions selected from camelid origin or variants thereof;

[0090] Still further, the antibody comprises part or all of antibody heavy chain framework regions selected from llama origin or variants thereof.

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

[0092] 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;

[0093] Further, the biologically active protein or a functional fragment thereof is selected from at least one of 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] Still further, the biologically active protein or a functional fragment thereof is human immunoglobulin Fc domain, preferably Fc domain of human IgG, such as Fc domain of human IgG1, IgG2, IgG3, IgG4, more preferably Fc domain of human IgG1.

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

[0096] SEQ ID NO: 10:

[0097] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0098] In particular, the nucleotide sequence encoding SEQ ID NO: 10 is set forth in SEQ ID NO: 11.

[0099] SEQ ID NO: 11:

[0100] GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA.

[0101] 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. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution, deletion, and / or insertion) at one or more amino acid positions.

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

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

[0104] 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.

[0105] In a seventh aspect, the present application provides a polyclonal antibody comprising the antibody of any one of the first to fourth aspects described above.

[0106] In an eighth aspect, the present application provides a kit comprising:

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

[0108] (2) a container loaded with the antibody preparation.

[0109] Preferably, the kit can further comprise a container, a buffer, an antibody recognizing IL-17A protein, a detection substrate, and the like.

[0110] In a ninth aspect, the present application provides an antibody drug conjugate comprising:

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

[0112] (2) a conjugating moiety binding to (1).

[0113] 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.

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

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

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

[0117] In particular, the nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 8 has a sequence as set forth in SEQ ID NO: 9.

[0118] SEQ ID NO: 9:

[0119] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCGGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTTAGTATCCACATCTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGCTGGTCGCAACTATTACTAGAGGTGGTGTAACAAATAATGCAGACTCCGTGAAGGGGCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGCGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCAGGTGGGACGAACGGGGGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

[0120] In a eleventh aspect, the present application provides a biological expression vector, wherein the biological expression vector comprises the nucleic acid molecule.

[0121] 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.

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

[0123] 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.

[0124] In a twelfth aspect, the present application provides a host cell, wherein the genome of the host cell is integrated with the nucleic acid molecule; or; comprises 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 comprises cells of Gram-negative bacterial strains and Gram-positive bacterial strains.

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

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

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

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

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

[0132] In a thirteenth aspect, the present application provides a pharmaceutical composition comprising: 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 to fourth aspects; and;

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

[0134] 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.

[0135] In a fourteenth aspect, the present application provides a 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 to fourth aspects, wherein the use is selected from at least one of:

[0136] (1) preparing a medicament for preventing and / or treating an autoimmune disease;

[0137] (2) producing a medicament for preventing and / or treating cancer;

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

[0139] Specifically, the autoimmune disease includes Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic scleroderma, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, Takayasu's syndrome, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondylarthritis, mixed connective tissue disease, Castleman's disease, Sjogren's syndrome, adult Still's disease, vasculitis, hypersensitivity granulomatous angiitis, hypersensitivity angiitis, rheumatoid angiitis, large vessel vasculitis, ANCA-associated vasculitis, Cogan's syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, anti-phospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disease, Guillain-Barre syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, Good-pasture syndrome, rapidly progressive glomerulonephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Bassen-Kornzweig disease, Hashimoto's disease, autoimmune adrenocortical hypofunction, primary hypothyroidism, Addison's disease, idiopathic Addison's disease, type I diabetes, slow progression type I diabetes, localized scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Okuda disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease, celiac disease, ankylosing spondylitis, severe asthma, chronic urticarial transplantation immunity, familial Mediterranean fever, eosinophilic chronic rhinosinusitis, dilated cardiomyopathy, systemic mastocytosis, or inclusion body myositis.

[0140] Specifically, the cancer includes basal cell carcinoma, bile duct cancer, 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 application provides a method for detecting IL-17A in a sample in vitro for non-diagnostic purposes, the method comprising the following steps:

[0142] (1) combining the antibody, the recombinant protein, the antibody preparation, the polyclonal antibody, the antibody drug conjugate according to any one of the first to fourth aspects with the detection sample;

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

[0144] 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 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 according to any one of the first to fourth aspects.

[0145] In a seventeenth aspect, the present application 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 according to any one of the first to fourth aspects.

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

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

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

[0150] The single domain antibody 1-F8 has strong stability, Tm = 62.57, Tagg = 80.09. BRIEF DESCRIPTION OF DRAWINGS

[0151] Figure 1 Figure is the SDS-PAGE detection result of IL-17A recombinant protein.

[0152] Figure 2 Figure is the experimental result of IL-17A (Acro) protein activation of NIH-3T3 cells.

[0153] Figure 3 Figure is the experimental result of recombinant IL-17A (TEST) protein activation of NIH-3T3 cells.

[0154] Figure 4 Figure is the experimental result of IL17A binding to reporter gene cell lines.

[0155] Figure 5 Figure is the experimental result of IL17A activation of cells.

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

[0157] Figure 7 Figure is the agarose gel electrophoresis map of 2#+3# alpaca.

[0158] Figure 8 Figure is the flow detection result of 2# alpaca yeast library, A: NC group: primary antibody: no addition, 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 9Figure 2 is a flow cytometry detection result of a yeast library of 2#+3# alpacas, 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.

[0160] Figure 10 Figure 2 is a flow cytometry detection result of a yeast library of 2#+3# alpacas, 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.

[0161] Figure 11 Figure 2 is a flow cytometry detection result of a yeast library of 2#+3# alpacas, 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.

[0162] Figure 12 Figure 3 is a result of SDS-PAGE of antibody purification, the left lane is marker, and the right lane is 1-F8.

[0163] Figure 13 Figure 4 is a result of ELISA binding experiment of candidate antibodies.

[0164] Figure 14 Figure 5 is a result of blocking function experiment of candidate antibodies.

[0165] Figure 15 Figure 6 is a result of thermal stability detection of single-domain antibody 1-F8.

[0166] Figure 16 Figure 7 is a result of thermal stability detection of positive control antibody. DETAILED DESCRIPTION

[0167] 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 further described to illustrate the present application. However, the following embodiments are only preferred embodiments of the present application, and are 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 embodiments, if not otherwise specified, the operation method used is a conventional operation method, the equipment used is a conventional equipment, and the equipment and materials used in each embodiment are the same.

[0168] Experimental reagents:

[0169] 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 TM II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#: 6210B); NuHi power mix (Xin Hai Biological, CAT#: NH9303); 3M Sodium acetate (pH5.2-6) (Sigma, CAT#: 126-96-5); DNA fragment recovery kit (TakaRa, CAT#: 9761); Gel recovery kit (Qiagen, CAT#: 28706); Tian Gen Plasmid Large Extraction 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 (iCarTab); BL21 Competent (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 tube (Corning, CAT# 352070); Shock cup (Bio-Rad 0.2 cm); RNase free 1.5 mL EP tube (QSP, CAT#: 509-GRD-Q); 200 μL RNase free PCR tube (Axygen, PCR-02D-C); T125 shake flask (Corning, CAT# 431143); 15 mL Falcon centrifuge tube (Corning, CAT# 430052); 6-well plate (Corning, CAT# 3516); 96-well plate (Corning, CAT# 3365); 96-well black plate (F-BOTTOM (CHIMNEY WELL) BLACK).

[0172] Experimental equipment:

[0173] Electrotransformation instrument (Eppendorf Multiporator); centrifuge (Thermo FRESCO-17); constant temperature incubator (Shanghai Jinghong DNP-9052); constant temperature shaking incubator (Jingqi CO-O6U); clean bench (Su Nian Tai, 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 in the following examples to screen, clone VHH fragments, construct Nanobodies are designed according to the following references:

[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 singledomain antibodies by Alpaca VHH 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 IL-17A (Human) recombinant protein

[0180] The sequence information of Human IL-17A (Q16552-1) was retrieved from UniProt database (SEQ ID NO: 12), a 6xHis tag was added at the C-terminus, and after optimization according to prokaryotic codon, the gene was synthesized and subcloned into pET28a vector; after Sanger sequencing verification, the plasmid was extracted.

[0181] The recombinant plasmid was transformed into BL21 competent cells, induced overnight with 0.5 mM IPTG, and the bacterial solution was collected and lysed; the recombinant protein was purified using a nickel column.

[0182] SDS-PAGE was used to detect the purity of the target protein, and the results showed that the purity of the purified IL-17A antigen protein was greater than 90%. Figure 1

[0183] SEQ ID NO: 12:

[0184] MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNI HNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINAD GNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA.

[0185] 2. Preparation of positive control antibody Ixekizumab

[0186] (1) The heavy chain and light chain variable regions of Ixekizumab were synthesized by gene synthesis (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 was subcloned into the pcDNA3.4-hIgG4 vector (the amino acid sequence of IgG4 is shown in SEQ ID NO: 15), and the light chain variable region was 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 was prepared using the plasmid large extraction kit for standby use.

[0187] (2) The LVTransm transfection reagent and the heavy chain expression vector and the light chain expression vector were taken out from the refrigerator, thawed at room temperature, and completely mixed by blowing up and down with a pipette gun. PBS buffer was taken out and warmed to room temperature. 2 mL of PBS was taken to one well of a 6-well plate, 50 μg of the heavy chain expression vector and the light chain expression vector were added respectively, and after mixing well by blowing up and down with a pipette gun, 300 μL of LVTransm was added, immediately mixed by blowing up and down with a pipette, and incubated at room temperature for 10 minutes.

[0188] (3) The above DNA / LVTransm complex was added to 100 mL of 293F cells, mixed well by gently shaking, and the cells were incubated at 37°C, 5% CO2 incubator, 130 RPM.

[0189] (4) After continuous culture for 5-7 days, the culture medium supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube. The antibody was purified using a Protein A column.

[0190] ​SDS-PAGE was used to detect the purity of the target antibody protein, and the purity was >95%.

[0191] SEQ ID NO: 13:

[0192] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMY GTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWG QGTLVTVSS.

[0193] SEQ ID NO: 14:

[0194] DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKV SNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIK.

[0195] SEQ ID NO: 15:

[0196] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK.

[0197] SEQ ID NO: 16:

[0198] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC.

[0199] 3. ELISA detection of Human IL-17A recombinant protein binding activity with control antibody

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

[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 37°C blocking for 2 hours.

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

[0204] (5) Dilute the positive control antibody Ixekizumab with PBS to 10 μg / ml, 5-fold dilution 7 points, add 100 μL / well to the enzyme-labeled plate, incubate at room temperature for 1 hour, and the control wells are PBS.

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

[0206] (7) Add secondary antibody HRP-Protein A (Boster, BA1080) diluted 1:10,000, add 100 μL / well to the enzyme-labeled plate, 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 color developing solution.

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

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

[0211] (12) Read the OD450 value in the wells using an enzyme-labeled instrument.

[0212] 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.

[0213] Table 1.

[0214]

[0215] 4. Detection of Human IL-17A recombinant protein activity

[0216] Experimental procedure:

[0217] (1) Resuscitate the NIH-3T3 cells from liquid nitrogen, continuously subculture to make the cells in logarithmic growth phase, after cell counting, inoculate into 96-well plates at a cell amount of 2x10 5 cells / well.

[0218] (2) Add 100 μl of IL-17A (ACRO, Cat#ILA-H5118) of different concentrations and the prepared IL-17A recombinant protein (ACRO protein as control) into each well, with a final concentration 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.

[0219] (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 the mouse IL-6 ELISA kit to detect the secretion of IL-6.

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

[0221] Experimental results:

[0222] 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 the 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.

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

[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 recombinant 293 cells that overexpress the two receptors, and further stably transfer NFKB-Luciferase (the amino acid sequence is shown in SEQ ID NO: 19, and the nucleotide sequence encoding it is shown in SEQ ID NO: 20) and ACT1 gene (the nucleotide sequence is shown in SEQ ID NO: 21, and the amino acid sequence encoded by it is shown in SEQ ID NO: 22).

[0226] SEQ ID NO: 21), and the IL-17A reporter gene cell line 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc was constructed. The IL-17A protein was added for activation, and the positive control antibody Ixekizumab was added for blocking experiment detection, and the EC50 value was calculated, and the candidate antibody targeting IL-17A was established, and the in vitro pharmacodynamic evaluation cell line was established.

[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] The IL-17A reporter gene cell line 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc was constructed and combined with IL-17A recombinant protein. The FACS results showed that the constructed IL-17A receptor overexpression cell line could bind with IL-17A, and the positive rate was greater than 90% (results shown in Figure 4

[0239] IL17A activation cell experiment: IL-17A recombinant protein was used to activate 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc, and the results are shown in Figure 5

[0240] Ixekizumab blocking IL17A function experiment: positive control antibody Ixekizumab and IL-17A recombinant protein were added 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] Two alpacas (2# and 3#) were immunized with the prepared IL-17A recombinant antigen protein, a total of 6 times, with GERBU as the immunization adjuvant, an interval of 14 days, and the last two immunizations with Freund's complete adjuvant.

[0244] Blood sampling was arranged as follows:

[0245] 2# alpaca, 100ml of blood was collected after five immunizations, a cDNA library was prepared, and a yeast display library (yeast display library 1) was constructed; 100ml of blood was collected after six immunizations, and a cDNA library was prepared.

[0246] 3# alpaca, 100ml of blood was collected after six immunizations, a cDNA library was prepared, and mixed with the cDNA library prepared from the blood collected after six immunizations from 2# alpaca to construct a yeast display library (yeast display library 2).

[0247] 6.2 Detection of immunization titer

[0248] Experimental steps:

[0249] ​​Serum was isolated from different times of immunized alpaca, and ELISA was performed with antigen pre-coated 96-well plates by limiting dilution, and the specific steps were as follows:

[0250] (1) 5 mL of peripheral blood was collected, and the centrifuge tube containing the blood sample was placed in a 37°C incubator for 1 hour; then the blood sample was transferred to 4°C overnight;

[0251] (2) The centrifuge tube containing the blood sample was placed in a centrifuge, and centrifuged at 5000 rpm for 20 min; the upper serum was separated, and the serum was transferred to a new sterile centrifuge tube to collect the immune serum.

[0252] (3) The IL-17A recombinant protein was diluted with sterile CBS (carbonate buffer) to a final concentration of 1 μg / mL. A new 96-well enzyme-labeled plate was taken, and 100 μL / well was added 4°C coated overnight.

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

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

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

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

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

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

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

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

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

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

[0263] (14) Use the enzyme-labeled instrument to read the OD450 value in the well.

[0264] The experimental results are shown in Tables 2-3 below. According to the ELISA detection results, the immune serum is well combined with the IL17A recombinant protein, and the OD value changes in gradient with the gradient dilution of the immune serum.

[0265] Table 2. Immune titer detection results

[0266]

[0267] Table 3. Immune titer detection results

[0268]

[0269] 7. Construction of antibody yeast library

[0270] 7.1 PBMC separation and VHH antibody fragment cloning

[0271] Experimental steps:

[0272] (1) Collect 100 mL of peripheral blood anticoagulant sample, and separate 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 Amount Oligo dT Primer (50 μΜ) 8 μL dNTP Mixture (10 mM each) 8 μL Total RNA sample 20 μg RNase-free water Added to 80 μL

[0277] 2) After 5 min of 65℃ incubation, quickly cool on ice.

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

[0279] Table 5.

[0280] Reagent Amount Denatured reaction above 80 μL 5x PrimeScript II Buffer 32 μL RNase Inhibitor (40 Μ / μL) 4 μL PrimeScript II RTase (200 Μ / μL) 8 μL RNase-free water 36 μL

[0281] 4) After mixing, aliquot 80 μL / tube, and place in a PCR instrument for 42℃, 1 hour, 70℃ heat inactivation for 15 minutes, and finally place the cDNA sample on ice or at -20℃ for long-term storage.

[0282] (3) Amplification of VHH fragments

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

[0284] Table 6.

[0285] Ingredient Amount Upstream primer (5 μΜ) 2 μL Downstream primer (10 μΜ) 1 μL NuHi Power mix (2x) 25 μL cDNA template 2 μL Sterile water 20 μL

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

[0287] Table 7.

[0288]

[0289] 3) Agarose electrophoresis of PCR products

[0290] The PCR products were analyzed by electrophoresis using 1% agarose, and the fragments with a molecular weight of about 750 bp were separated. The PCR products were recovered using a gel recovery kit, and the concentration was determined using NanoDrop.

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

[0292] Table 8.

[0293] Ingredient Amount 2 nd F primer 2 μL 2 nd R primer 2 μL NuHi Power mix (2x) 25 μL One round of PCR recovered product 200 ng Sterile water Bring up to 50 μL

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

[0295] Table 9.

[0296]

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

[0298] The PCR products were analyzed by electrophoresis using 1% agarose, and the VHH fragments with a molecular weight of about 400 bp were separated. The VHH PCR products were recovered using a gel recovery kit, and the concentration was determined using NanoDrop.

[0299] Experimental results:

[0300] 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.

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

[0302] (1) Linearization of yeast display vector pDisplay, enzyme digestion system as follows:

[0303] Table 10.

[0304]

[0305] 1) Use Sfil to digest pDisplay vector, 100 μL / tube, 50°C enzyme digestion overnight.

[0306] 2) Use 1% agarose gel to separate pDisplay vector fragments, cut 5000 bp of vector fragments for gel recovery, and use NanoDrop to determine the concentration.

[0307] 3) Divide the recovered pDisplay enzyme digestion product 200 μL per 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3M sodium acetate, 1 μg / μL glycogen, mix well by blowing and sucking, add 880 μL of anhydrous ethanol, mix well by inverting, and place at -80°C.

[0308] (2) Electric transformation to construct yeast display library

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

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

[0311] 3) Prepare the yeast competent strain. After mixing the linearized vector fragments and PCR products, add them to the electroporation cup, and then perform electroporation; after electroporation, transfer the yeast competent transfection culture bottle to 220 rpm, 30°C, shake culture for 1 h;

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

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

[0314] 8, Yeast display library screening

[0315] Experimental steps:

[0316] 1) Take the yeast from SDCAA culture and add to 250 mL flask with 50 mL SGCAA medium, 30°C, 240 rpm, shake culture for 16 h.

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

[0318] 3) Wash the streptavidin magnetic beads that have been incubated with the antigen twice with 0.5% PBSA (4°C rotation incubation for 5 min each time), place on the magnetic stand for 5 min, discard the supernatant.

[0319] 4) Add the yeast solution to the magnetic beads that have been combined with the antigen, 4°C rotation incubation for 60 min, place on the magnetic stand for 15 min.

[0320] 5) Discard the yeast solution and leave the magnetic beads, wash three times with 0.5% PBSA (4°C rotation incubation for 5 min each time).

[0321] 6) Resuspend the magnetic beads with 1 mL SDCAA medium, take 0.5-5 μL of the resuspension and plate with 100 μL SDCAA medium, divide the resuspension into two parts, one part add 500 μL 50% glycerol (-80°C storage); one part add to a shake tube, supplement with 2 mL SDCAA medium, 30°C, 240 rpm, culture for 16 h.

[0322] 7) Transfer the yeast solution in the shake tube to 50 mL SDCAA medium (250 mL flask), 30°C, 240 rpm, culture overnight.

[0323] 8) Measure the OD600 value of the yeast solution, according to the OD600 value, centrifuge a part of the yeast solution, resuspend with SGCAA, transfer to 50 mL SGCAA medium, so that the final OD600 value is 1, 30°C, 240 rpm, culture overnight, the remaining yeast solution is resuspended with SDCAA: 50% glycerol = 1:1, -80°C storage.

[0324] 9, Yeast monoclonal flow detection

[0325] After sorting, the yeast solution is plated with SDCAA, single clones are picked and cultured, after 48 h of induction, incubated with Biotin-antigen, the second antibody uses PE-Streptavidin, after completion of the incubation, flow detection is performed. The yeast clones that are combined with the target antigen are lysed with 0.2% SDS (95°C incubation for 10 min), centrifuged, 0.5 μL of the supernatant is taken as a template for PCR amplification and testing (the remaining solution is stored at -20°C). The flow detection results are as follows Figure 8As shown, according to the flow detection results, after the second magnetic separation, the positive rate of 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.

[0326] 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, flow detection after incubation was completed, the results showed (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.

[0327] 10. Antibody sequence identification

[0328] Enriched positive clones; selected single clones after enrichment, Phage ELISA identification was performed, 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 sequence difference was large, and the library diversity was good. 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.

[0329] According to the flow detection results of yeast single clones, the positive clones combined with IL-17A-His were selected to extract 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:

[0330] (1) First round of PCR: amplification of CMV, VHH and FC

[0331] 1) Configuration of PCR reaction system (50 μL system / reaction)

[0332] Table 11.

[0333]

[0334]

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

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

[0337] 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.

[0338] 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).

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

[0340] 1) Configure the PCR reaction system

[0341] Table 12.

[0342] Ingredient Amount CMV 1st product 50 ng VHH 1st product 50 ng Fc 1st product 50 ng NuHi Power mix (2x) 25 μL Sterile water Bring up to 46 μL

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

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

[0345] 3) Add primers, 2 μL of upstream primer and 2 μL of downstream primer;

[0346] The PCR reaction program is as follows:

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

[0348] 4) Use TakaRa's DNA fragment recovery kit to purify the overlap PCR product, and measure the concentration with NanoDrop, at least 10 μg of PCR product is required. For subsequent cell transfection verification.

[0349] 5) The transfection steps are the same as those of the eukaryotic expression vector.

[0350] Add a signal peptide to the N terminus of VHH and IgG1-FC to the C terminus, 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-F12 binds to IL-17A-His antigen, and a eukaryotic expression vector is constructed.

[0351] Table 13. Candidate clone transfection supernatant ELISA binding experiment results

[0352]

[0353] 11. Candidate single domain antibody expression and purification

[0354] Experimental procedures:

[0355] 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.

[0356] 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.

[0357] 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 and culture at 130 rpm for 6-8 hours, then add 50 mL of fresh 293 cell culture medium and return the cells to the incubator for continued culture.

[0358] 4) After 7 days of continuous culture, 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.

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

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

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

[0362] 3) Collect the magnetic beads using a magnetic separation rack and discard the supernatant.

[0363] 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.

[0364] 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.

[0365] 6) Collect the magnetic beads using a magnetic separation stand, 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 neutralization buffer to each 500 μL eluate to neutralize the pH, so as to maintain the biological activity of the antibody and avoid inactivation of the antibody.

[0368] 9) Binding / washing buffer: 1 x 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 shown in Table 1. Figure 12

[0373] Example 2 ELISA detection of binding of recombinant antibody to target protein

[0374] Experimental procedure:

[0375] 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.

[0376] 2) Remove the antigen coating solution, and wash 5 times using 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 using PBST;

[0379] ​5) Add purified single domain antibody, starting concentration is 10 μg / mL, 5-fold dilution gradient 7, the results show that the EC50 value of single domain antibody 1-F8 is 1.614, the EC50 value of positive antibody (Lxekizumab) is 10.06, and the single domain antibody 1-F8 has better binding activity with Human IL-17A protein.

[0380] = 10.06, and the single domain antibody 1-F8 has better binding activity with Human IL-17A protein. Figure 13 The EC50 of each candidate antibody is shown in Table 14.

[0381] Table 14. EC50 value of candidate antibody

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

[0383] Example 3 FACS detection of IL17A binding to reporter cell strain

[0384] Experimental process:

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

[0386] 2) Divide the cells into several parts, and the number of cells in each part is 2x10 5 cells;

[0387] 3) Incubate IL17A-His protein with target cells, mix well, and incubate at room temperature for 1 hour;

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

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

[0390] 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;

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

[0392] Example 4 Single domain antibody blocking function experiment

[0393] Experimental steps:

[0394] 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 18h 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.

[0395] Experimental results:

[0396] 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-F8 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.

[0397] Example 5 Stability experiment

[0398] 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 50% of the protein is in a unfolded state and the temperature (Tagg) at which aggregation begins; the higher the thermal denaturation Tm value and Tagg, 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), 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.

[0401] The experimental results are shown in Figures 15-16 The results show that the single-domain antibody 1-F8 has strong stability, Tm = 62.57, and Tagg = 80.09, while the positive control has Tm = 56.1 and Tagg = 61.86.

[0402] Application Example 1: An antibody preparation

[0403] The antibody preparation comprises: an anti-IL-17A antibody having an amino acid sequence as shown in SEQ ID NO: 8; a buffer, a surfactant, an amino acid, a tonicity agent, etc.

[0404] 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 (having an amino acid sequence 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 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.

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

[0408] Application Example 3: An antibody-drug conjugate

[0409] 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 a 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).

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

[0411] Pharmaceutical composition of application example 4

[0412] The pharmaceutical composition comprises 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 comprises the pharmaceutically acceptable excipient.

[0413] In one embodiment of the present application, the preparation of the pharmaceutical composition comprises: 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.

[0414] 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 amino acid sequence of the single-domain antibody comprises: HCDR1, HCDR2, HCDR3, the amino acid sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

2. The single domain antibody of claim 1, wherein: The amino acid sequence of the single-domain antibody comprises: FR1, FR2, FR3, FR4, the amino acid sequences of which are shown in SEQ ID NO: 4-7.

3. An anti-IL-17A single-domain antibody, characterized in that: The amino acid sequence of the single-domain antibody comprises: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3; The amino acid sequence of the FR1 is shown in SEQ ID NO: 4, the amino acid sequence of the FR2 is shown in SEQ ID NO: 5, the amino acid sequence of the FR3 is shown in SEQ ID NO: 6, and the amino acid sequence of the FR4 is shown in SEQ ID NO:

7.

4. The single domain antibody of claim 3, wherein: The amino acid sequence of the single-domain antibody is shown in SEQ ID NO:

8.

5. The single-domain antibody of claim 1, wherein: comprises part or all of an antibody heavy chain framework region selected from a human source, a murine source, a primate source, or a camelid source, or a variant thereof.

6. The single domain antibody of claim 5, wherein: comprises part or all of an antibody heavy chain framework region selected from a camelid source, or a variant thereof.

7. The single domain antibody of claim 6, wherein: comprises part or all of an antibody heavy chain framework region selected from a camelid source, or a variant thereof.

8. A recombinant protein, characterized in that: The single-domain antibody of any one of claims 1-7.

9. The recombinant protein according to claim 8, characterized in that: 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.

10. The recombinant protein according to claim 9, characterized in that: The biologically active protein or a 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.

11. The recombinant protein according to claim 10, characterized in that: The biologically active protein or a functional fragment thereof is a human immunoglobulin Fc domain.

12. The recombinant protein according to claim 11, characterized in that: The Fc domain is of human IgG, including Fc domains of human IgG1, IgG2, IgG3, and IgG4.

13. The recombinant protein according to claim 12, characterized in that: The Fc domain is of human IgG1.

14. An antibody formulation, characterized by: The antibody preparation comprises the single-domain antibody of any one of claims 1-7 and a pharmaceutically acceptable carrier.

15. A polyclonal antibody, characterized in that: The antibody comprises the single-domain antibody of any one of claims 1-7.

16. A kit characterized in that: The kit comprises: (1) the antibody of any one of claims 1-7, the recombinant protein of any one of claims 8-13, the antibody preparation of claim 14, or the polyclonal antibody of claim 15; and; (2) a container loaded with the antibody preparation.

17. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the antibody of any one of claims 1-7, the recombinant protein of any one of claims 8-13, or the polyclonal antibody of claim 15.

18. A biological expression vector, characterized by: The biological expression vector comprises the nucleic acid molecule of claim 17.

19. A host cell, characterized in that: The genome of the host cell is integrated with the nucleic acid molecule of claim 17; or; comprises the biological expression vector of claim 18.

20. A pharmaceutical composition, characterized by: The pharmaceutical composition comprises: the antibody of any one of claims 1-7, the recombinant protein of any one of claims 8-13, the antibody preparation of claim 14, the polyclonal antibody of claim 15, the nucleic acid molecule of claim 17, the biological expression vector of claim 18 or the host cell of claim 19.

21. The pharmaceutical composition of claim 20, wherein: The pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

22. Use of the antibody according to any one of claims 1 to 7, the recombinant protein according to any one of claims 8 to 13, the antibody preparation according to claim 14, the polyclonal antibody according to claim 15, the kit according to claim 16, the nucleic acid molecule according to claim 17, the biological expression vector according to claim 18 or the host cell according to claim 19, characterized in that: The use is selected from at least one of the following: (1) preparing a drug for preventing and / or treating an autoimmune disease, the autoimmune disease being psoriasis, psoriatic arthritis, ankylosing spondylitis; (2) preparing a reagent or kit for detecting IL-17A.

23. A method for the in vitro detection of IL-17A in a sample for non-diagnostic purposes, characterized in that: The method comprises the following steps: (1) combining the antibody of any one of claims 1-7, the recombinant protein of any one of claims 8-13, the antibody preparation of claim 14, the polyclonal antibody of claim 15 with a detection sample; (2) detecting the antigen-antibody complex and interpreting the results.

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