Single-domain antibody against il-17a and uses thereof

Abstract

The application belongs to the field of immunology and relates to a single-domain antibody against IL-17A and use thereof.The single-domain antibody is composed of a heavy chain, and the heavy chain comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3.The amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are one of (2)-(4) or (6)-(8).Compared with the prior art, the application has the beneficial effect that the single-domain antibody specific to IL-17A is screened by using biological genetic engineering technology, the antibody has good affinity, can block the release of cytokines by specific cells, has good binding activity through prokaryotic expression and eukaryotic expression, and has certain drugability.

Description

Technical Field

[0001] The present invention relates to a single-domain antibody capable of specifically binding to IL-17A (hereinafter, its abbreviation is "IL-17A single-domain antibody"), a pharmaceutical composition containing the single-domain antibody as an active ingredient, and its pharmaceutical therapeutic use. Background Art

[0002] Psoriasis is a relatively common chronic inflammatory skin disease, commonly known as psoriasis vulgaris. The disease is prone to recurrence or exacerbation in winter and mostly remits in spring and autumn. The global prevalence rate is about 2% - 3%. Among them, 1 / 3 of psoriasis patients have psoriatic arthritis (PsA), often accompanied by joint swelling, pain, stiffness and movement disorders. Some cases can involve the spine, and severe cases can cause disability, which has a serious impact on the physical and mental health of patients. Psoriasis is classified according to its clinical characteristics, mainly including the following types: vulgaris, arthropathic, pustular and erythrodermic. More than 90% of psoriasis belongs to the vulgaris type. Other types of the disease are mostly caused by the use of topical irritating drugs, excessive use of glucocorticoids and sudden discontinuation during the use of immunosuppressants during treatment.

[0003] The pathogenesis of psoriasis is still unclear. Currently, the main treatment regimens for PsA are non-steroidal anti-inflammatory drugs, glucocorticoids, anti-rheumatic drugs, azathioprine, retinoids, etc., as well as physical therapy, traditional Chinese medicine therapy and other methods to relieve symptoms and control the condition. Due to the poor efficacy and adverse reactions of traditional therapies, such as long-term use of hormones can cause various side effects (increased blood pressure, increased blood sugar, osteoporosis, peptic ulcer, skin atrophy, etc.), clinically, there is a growing tendency to study biological agents. Tumor necrosis factor inhibitors are often selected as the first choice biological therapy for PsA patients. In addition, there are also anti-IL-17 biological agents.

[0004] Psoriasis takes excessive proliferation of keratinocytes, infiltration of inflammatory cells, and formation of new blood vessels as the three elements of its histopathological changes. Patients have abnormal functions of various immune cells, immune molecules, intracellular signal transduction systems, etc. The disease has a certain incubation period, and taking antimalarial drugs, antipsychotic lithium preparations, β-blockers for hypertension and angiotensin-converting enzyme inhibitors during this period will induce the occurrence of the disease, seriously affecting the quality of life and even the physical and mental health of patients.

[0005] The pathogenesis of psoriasis is still unclear. Currently, it is considered to be an autoimmune disorder disease under a polygenic genetic background. Its onset is related to the immune response mediated by T lymphocytes, mainly CD4+Th1 lymphocytes. The pathogenic process includes the activation of naive T lymphocytes into memory-effector T lymphocytes, and the memory-effector T lymphocytes enter the circulation and migrate to the skin, aggregate at the lesion site, secrete various cytokines and exert various biological functions to cause the disease.

[0006] IL-17A is the earliest discovered member of the IL-17 cytokine family, primarily secreted by Th17 cells. In 1986, Mosmann, Coffman, and their research group discovered two subsets of helper T cells—Th1 and Th2 (Mosmann et al., 1986). Besides Th17 cells, other T cells (including CD8+ T cells, γδ T cells, and NKT cells) and innate immune cells (including NK cells and ILC3 cells) can also secrete IL-17A. In recent years, numerous studies on IL-17A and Th17 cells have shown that IL-17A participates in the pathological processes of many autoimmune diseases (such as rheumatoid arthritis and encephalomyelitis), and also plays an important protective role in antibacterial and antifungal immunity.

[0007] Psoriasis is an autoimmune skin disease characterized by excessive epidermal cell proliferation. Studies have shown that IL-17A-deficient mice exhibit reduced epidermal hyperplasia (Rizzoe et al., 2011); clinical data also demonstrate the important pathological roles of Th17 cells and IL-17A in psoriasis (Hueber et al., 2010; Wilsone et al., 2007). Based on these research findings regarding the role of IL-17A in autoimmune diseases, treating these diseases by inhibiting Th17 cell differentiation and blocking the IL-17A signaling pathway has become a current research focus.

[0008] Psoriasis, due to its stubborn and difficult-to-treat nature, is considered a major research topic in the field of dermatology worldwide and is one of the key skin diseases targeted for prevention and treatment globally. With the launch of a series of new biological drugs, psoriasis patients have more and better medication options. In the field of autoimmune diseases, TNFα antagonists and interleukin (IL) drugs are currently hot research topics, with the IL-12 family and IL-17 receiving the most attention.

[0009] Currently, there is still a lack of single-domain antibody products against IL-17A with strong affinity and medicinal value in existing technologies. Summary of the Invention

[0010] The purpose of this patent is to provide a single-domain antibody that can specifically bind to IL-17A and its uses.

[0011] A first aspect of the present invention provides a single-domain antibody against IL-17A, said single-domain antibody being composed of heavy chains, the heavy chains including heavy chain CDR1 shown in any one of SEQ ID NO:19-SEQ ID NO:25, heavy chain CDR2 shown in any one of SEQ ID NO:26-SEQ ID NO:32, and heavy chain CDR3 shown in any one of SEQ ID NO:33-SEQ ID NO:41. The single-domain antibody against IL-17A is a single-domain antibody targeting IL-17A.

[0012] Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(9): (1) CDR1 shown in SEQ ID NO:19, CDR2 shown in SEQ ID NO:28, and CDR3 shown in SEQ ID NO:36; (2) CDR1 shown in SEQ ID NO:21, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:35; (3) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:26, and CDR3 shown in SEQ ID NO:41; (4) CDR1 shown in SEQ ID NO:23, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:37; (5) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:26, and CDR3 shown in SEQ ID NO:40; (6) CDR1 shown in SEQ ID NO:20, CDR2 shown in SEQ ID NO:30, and CDR3 shown in SEQ ID NO:33; (7) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:38; (8) CDR1 shown in SEQ ID NO:24, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:34; (9) CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:32, and CDR3 shown in SEQ ID NO:39.

[0013] The above 9 CDR combinations (1)-(9) correspond to SEQ ID NO.1-9 in sequence.

[0014] All of the above sequences can be replaced with sequences that have "at least 80% homology" or sequences that replace only one or a few amino acids; preferably "at least 85% homology", more preferably "at least 90% homology", even more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0015] In one embodiment, in any one or more CDRs of the heavy chain CDR1, CDR2, and CDR3, one to five arbitrary amino acid residues may be substituted with their conserved amino acids. Specifically, in the heavy chain CDR1, one to five amino acid residues may be substituted with their conserved amino acids; in the heavy chain CDR2, one to five amino acid residues may be substituted with their conserved amino acids; and in the heavy chain CDR3, one to five amino acid residues may be substituted with their conserved amino acids.

[0016] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% homology.

[0017] In some embodiments, the inventive objective can also be achieved by substituting only one or a few amino acids compared to the aforementioned sequence, for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. These variations include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, most preferably up to 5) at the C-terminus and / or N-terminus. In practice, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art can consider so-called “conserved” amino acid substitutions, in which case the substitution would preferably be a conserved amino acid substitution. A conserved amino acid can generally be described as an amino acid residue substituted by another amino acid residue having a similar chemical structure, and this substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. The particularly preferred conserved amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. Furthermore, those skilled in the art will understand that the inventiveness of the single-domain antibody lies in the CDR1-3 regions, while the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0018] The term "single-domain antibody against IL-17A" in this invention includes not only complete single-domain antibodies but also fragments, derivatives, and analogs of such single-domain antibodies against IL-17A. As used herein, the terms "fragment," "derivative," and "analyte" have the same meaning and refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with an Fc tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0019] In a preferred embodiment, the antibody sequence further includes a framework region FR; the framework region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the framework region FR are as follows: The FR1 or a variant of FR1 shown in any one of SEQ ID NO:42-44, wherein the variant of FR1 contains a substitution of up to 5 amino acids in the FR1; The FR2 or a variant thereof shown in any one of SEQ ID NO:45-52, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR2; FR3 or a variant thereof shown in any one of SEQ ID NO:53-59, wherein the variant thereof contains substitutions of up to 5 amino acids in the FR3; FR4 or a variant thereof shown in any one of SEQ ID NO:60-62, wherein the variant thereof contains substitutions of up to 5 amino acids.

[0020] A second aspect of the present invention is to provide the amino acid sequence of a single-domain antibody against IL-17A, wherein the amino acid sequence of the single-domain antibody is as shown in SEQ ID NO. 1-9, or the single-domain antibody has at least 80% sequence homology with the amino acid sequence of SEQ ID NO. 1-9 and is capable of specifically binding to the IL-17A protein.

[0021] In one embodiment, the anti-IL-17A single-domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with an amino acid sequence selected from SEQ ID NO: 1-9, and is capable of specifically binding to the IL-17A protein.

[0022] A third aspect of the present invention is to provide a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion. The first antigen-binding portion is a single-domain antibody, wherein the amino acid sequence of the single-domain antibody is as shown in SEQ ID NO. 1-9 or has at least 80% homology with SEQ ID NO. 1-9. The second antigen-binding portion is another antibody, for example, an antibody against TNFα, IL-6R, IL-6, IL-23, or IL-23R (which can be a monoclonal antibody, polyclonal antibody, single-domain antibody, or any other form of antibody); the second antigen-binding portion may also be an antibody against other antigens, for application in the diagnosis, prevention, or treatment of diseases or the detection of antigens.

[0023] A fourth aspect of the present invention is the use of any of the aforementioned anti-IL-17A single-domain antibodies in the preparation of bispecific antibodies.

[0024] A fifth aspect of the invention is to provide an Fc fusion antibody or humanized antibody that provides any of the aforementioned single-domain antibodies against IL-17A.

[0025] A sixth aspect of the present invention is to provide nucleotide molecules encoding the aforementioned single-domain antibody against IL-17A, or the aforementioned Fc fusion antibody, or the aforementioned humanized antibody, wherein the nucleotide sequences are as shown in SEQ ID NO: 10-18, or have at least 95% sequence homology with any one of SEQ ID NO: 10-18.

[0026] In one embodiment, the nucleic acid molecule encoding the single-domain antibody against IL-17A has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with nucleotide sequences selected from SEQ ID NO: 10-18, and the single-domain antibody against IL-17A encoded therein is capable of specifically binding to the IL-17A protein.

[0027] A seventh aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding a single-domain antibody, an Fc fusion antibody, or a humanized antibody against IL-17A, the nucleotide sequences of which are shown in SEQ ID NO: 10-18, respectively.

[0028] In a preferred embodiment, the expression vector used is RJK-V4-3 (a nucleotide molecule encoding a single-domain antibody against IL-17A or its Fc fusion antibody or humanized antibody is integrated into RJK-V4-3 by genetic engineering). Other universal expression vectors may also be selected as needed.

[0029] An eighth aspect of the invention is to provide a host cell capable of expressing the aforementioned anti-IL-17A single-domain antibody, Fc fusion antibody, or humanized antibody, or an expression vector comprising the aforementioned. Preferably, the host cell is a bacterial cell, fungal cell, or mammalian cell.

[0030] In another preferred embodiment, the host cell includes prokaryotic or eukaryotic cells, including bacteria and fungi.

[0031] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0032] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0033] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Trichoderma, or combinations thereof.

[0034] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of insect cells such as armyworms, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

[0035] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0036] In another preferred embodiment, the host cell is a suspension 293F cell.

[0037] A ninth aspect of the present invention is to provide a recombinant protein comprising the aforementioned single-domain antibody against IL-17A. The recombinant protein may be a single-domain antibody as shown in SEQ ID NO. 1-9, or a single-domain antibody having at least 80% homology with SEQ ID NO. 1-9, or a multi-epitope antibody, a multi-specific antibody, or a multivalent antibody; for example, the multi-epitope antibody may consist of more than one sequence from SEQ ID NO. 1-9; the multivalent antibody may consist of one sequence from SEQ ID NO. 1-9 repeated a certain number of times; the multi-specific antibody includes, but is not limited to, the aforementioned bispecific antibody and trispecific antibody; furthermore, the recombinant protein may be a fragment, derivative, or analog of the aforementioned antibody.

[0038] A tenth aspect of the invention is to provide a pharmaceutical composition comprising the aforementioned anti-IL-17A single-domain antibody and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally determined based on the isoelectric point of the antibody (the pH of the aqueous carrier medium must deviate from the isoelectric point of the antibody by approximately 2). The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous, transdermal (direct application or patching to the affected area).

[0039] The pharmaceutical composition of the present invention can be directly used to bind to the IL-17A protein molecule, and therefore can be used to treat psoriasis. Furthermore, it can be used in combination with other psoriasis treatment agents.

[0040] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the aforementioned single-domain antibody and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0041] The eleventh aspect of the present invention is to provide a medicament for treating psoriasis, comprising the aforementioned anti-IL-17A single-domain antibody as an active ingredient.

[0042] A twelfth aspect of the present invention is to provide a kit for detecting IL-17A levels, comprising the aforementioned anti-IL-17A single-domain antibody. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc.

[0043] In a preferred embodiment, the kit includes an antibody that recognizes the IL-17A protein, a lysis medium for dissolving the sample, and universal reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0044] In a preferred embodiment, the kit further contains a second antibody and an enzyme or fluorescent or radiolabeled marker for detection, as well as a buffer solution.

[0045] In a preferred embodiment, the second antibody in the kit may be an antibody (as an anti-antibody) of the aforementioned single-domain antibody against IL-17A, and may be a single-domain antibody, monoclonal antibody, polyclonal antibody, or any other form of antibody.

[0046] The thirteenth aspect of the present invention provides a method for generating a single-domain antibody against IL-17A, comprising the steps of: (a) culturing a host cell as described in the eighth aspect of the present invention under conditions suitable for generating a single-domain antibody, thereby obtaining a culture containing the single-domain antibody against IL-17A; and (b) Isolating or recovering the single-domain antibody against IL-17A from the culture; and (c) Optionally, purify and / or modify the single-domain antibody against IL-17A obtained in step (b).

[0047] The fourteenth aspect of the present invention is to provide the use of the aforementioned anti-IL-17A single-domain antibody or pharmaceutical composition in the preparation of a medicament for inhibiting IL-17A gene expression or an anti-psoriasis medicament.

[0048] The fifteenth aspect of the invention is to provide the use of the aforementioned anti-IL-17A single-domain antibody or the aforementioned pharmaceutical composition in the preparation of a medicament for treating a disease.

[0049] In a preferred embodiment, the disease is an autoimmune disease, or the disease is a condition associated with abnormal expression of IL-17A.

[0050] In a preferred embodiment, the disease is psoriasis.

[0051] In a preferred embodiment, the disease is arthritis.

[0052] The sixteenth aspect of the present invention is to provide the use of the aforementioned anti-IL-17A single-domain antibody in the preparation of a medicament that blocks the binding of IL-17A to IL-17R.

[0053] Beneficial effects Compared with the prior art, the beneficial effects of the present invention are: (1) The single-domain antibody of the present invention is specifically targeted at the IL-17A protein with the correct spatial structure.

[0054] (2) The single-domain antibody of the present invention has good specificity, can block the interaction between the IL-17A and IL-17R binding sites, and has higher blocking activity than existing drugs, and has great application prospects in the preparation of drugs for treating autoimmune diseases.

[0055] (3) The single-domain antibody obtained by the present invention has a flexible expression system. It can be expressed in a prokaryotic system or in a eukaryotic system of yeast cells or mammalian cells. Moreover, its expression cost in the prokaryotic expression system is low, which can reduce the production cost in the later stage.

[0056] (4) The single-domain antibody obtained by the present invention has simple multi-combination form modification. It can be obtained by simple tandem through genetic engineering to obtain multivalent and multispecific antibodies. Moreover, its immune heterogeneity is very low and it will not produce a strong immune response without humanization modification.

[0057] (5) The present invention provides a single-domain antibody with a wider affinity range. Before affinity maturation, its affinity range can be from nM to pM, providing multiple options for antibodies for different purposes in the later stage. Attached Figure Description

[0058] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0059] Figure 1 The enrichment status of the library for screening antibodies targeting IL-17A in Example 3; Figure 2 This is a partial clone of the antibody-antigen binding dose-response curve determination diagram from Example 13. Figure 3 This is a dose-response curve of antibody-antigen binding in Example 13 (another clone, Tab1, hIgG); Figure 4 This is a chromatogram (partial clone) of the antibody blocking the binding of IL-17A and IL-17R in Example 14; Figure 5 The image shows the assay results of antibody blocking the binding of IL-17A and IL-17R in Example 14 (another clone, hIgG); Figure 6The image shows the experimental results of antibody neutralization of human IL-17A (eukaryotic sample) inducing the release of IL-6 in HeLa cells in Example 15 (partial clone, Tab, hIgG). Figure 7 The image shows the experimental results of antibody neutralization of human IL-17A (eukaryotic sample) inducing the release of IL-6 in HeLa cells in Example 15 (another clone); Figure 8 The image shows the experimental results (Tab, hIgG, 2G3) of HeLa cells induced by antibody neutralization of human IL-17A (humanized sample) in Example 16. Figure 9 The image shows the experimental results (2G3, 1A10) of HeLa cells induced by antibody neutralization of human IL-17A (humanized sample) in Example 16. Figure 10 The image shows the experimental results (1A10) of the antibody neutralization of human IL-17A (humanized sample) in Example 16, which induced the release of IL-6 from HeLa cells. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0061] As used herein, “sdAb” (also referred to as nanobodies or VHH by the developer Ablynx) is well known to those skilled in the art. A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Therefore, a single-domain antibody contains a single complementarity-determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies include antibodies containing only heavy chains (which naturally do not contain light chains), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0062] Single-domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species such as camels, dromedaries, llamas, and guanacos. Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. Single-domain antibodies may contain only variable domains of the immunoglobulin chain, which have CDR1, CDR2, and CDR3, as well as a frame region.

[0063] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% homology.

[0064] As used in this article, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc fragment of a target antibody with a biologically active functional protein molecule using genetic engineering techniques.

[0065] The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or by transplanting the complementarity-determining region (CDR1-3 sequence) of a target antibody into the variable region of a human antibody, or by mutating the target antibody according to the characteristics of the human antibody backbone region (FR1-4). Humanized antibodies can be produced using synthetic methods or site-directed mutagenesis methods.

[0066] In this invention, sequences with high homology to the CDR1-3 sequences disclosed herein can also yield single-domain antibodies against IL-17A. In some embodiments, sequences having "at least 80% homology," or "at least 85% homology," "at least 90% homology," "at least 95% homology," or "at least 98% homology" with the sequences in SEQ ID NO. 1-9 can achieve the purpose of the invention.

[0067] In some embodiments, the inventive objective can also be achieved by replacing only one or a few amino acids compared to the sequences in SEQ ID NO: 1-9, for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. In practice, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art can consider so-called “conserved” amino acid substitutions. In the case of substitution, the substitution will preferably be a conserved amino acid substitution, which can generally be described as an amino acid residue replaced by another amino acid residue having a similar chemical structure, and the substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. The particularly preferred conserved amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. Furthermore, those skilled in the art will understand that the inventiveness of the single-domain antibody lies in the CDR1-3 regions, while the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0068] The preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0069] This patent involves preparing a target protein and a truncated form of the target protein using genetic engineering technology. The obtained antigen protein is then used to immunize Bactrian camels in Alashan, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. Through genetic engineering, the variable region coding sequence of the camel-derived antibody is recombined into a phage display vector. Specific antibodies against the antigen protein are screened using phage display technology, and their ability to bind to the antigen and their application in the treatment of autoimmune diseases are further tested.

[0070] The above technical solution will now be broken down and explained in detail, and described with specific embodiments: Example 1: Preparation of recombinant human IL-17A extracellular domain protein: The human recombinant extracellular domain protein used in this patent was expressed and purified by the company itself. The specific design scheme of the human IL-17A recombinant extracellular domain protein expression vector is as follows: (1) The coding sequence of IL-17A was obtained by searching in NCBI. Its accession number is NM_002190.2, the accession number of the amino acid sequence encoded by this sequence is NP_002181.1, and the Uniprot ID is Q16552.

[0071] (2) The amino acid sequence corresponding to NP_002181.1 was analyzed for transmembrane region and extracellular terminus using TMHMM and SMART websites, respectively.

[0072] (3) The analysis results show that IL-17A is a secreted protein, and positions 1-23 are the signal peptides of this protein.

[0073] (4) The nucleotide sequence encoding amino acids 24-155 of the IL-17A protein was cloned into the vector pcDNA3.4 using gene synthesis.

[0074] (5) The constructed vector was subjected to Sanger sequencing. After comparing with the original sequence and confirming that there were no errors, the recombinant plasmid was extracted in batches to remove endotoxins. It was then transfected with suspension 293F for expression and purification of the target protein. The purified protein had a purity of up to 90%, which met the needs of animal immunity.

[0075] Example 2: Construction of a single-domain antibody library targeting the IL-17A protein: One mg of the purified human recombinant IL-17A protein obtained in Example 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alashan, Inner Mongolia. The camel was immunized once a week for a total of 7 weeks. Except for the first immunization, the remaining six immunizations were performed by mixing one mg of IL-17A protein with an equal volume of Freund's incomplete adjuvant. This immunization process was intended to concentrate the stimulation of the camel to produce antibodies against IL-17A protein.

[0076] After animal immunization, 150 mL of peripheral blood lymphocytes were collected from camels, and RNA was extracted from the cells. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified using nested PCR with the cDNA as a template.

[0077] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments were ligated to the vector. The ligated fragments were electroporated into competent TG1 cells to construct a phage display library of IL-17A protein, and the library size was measured to be approximately 1 × 10⁻⁶. 9 Meanwhile, the correct insertion rate of the target fragment in the library was detected by colony PCR.

[0078] The results showed that after PCR amplification of 40 colonies randomly selected from the library, 38 clones were able to amplify bands of the predicted size, while 2 clones amplified bands incorrectly. Therefore, the correct insertion rate was 38 ÷ 40 × 100% ≈ 95%.

[0079] Example 3: Screening for single-domain antibodies against IL-17A protein: Take 200 μL of the recombinant TG1 cells from Example 2 and culture them in 2×TY medium. During the culture, add 40 μL of helper phage VCSM13 to infect the TG1 cells and culture them overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and collect the amplified phage by centrifugation.

[0080] 500 μg of IL-17A protein diluted in 100 mM pH 8.3 NaHCO3 was coupled onto an ELISA plate and incubated overnight at 4°C. A negative control well (culture medium control) was also included. The next day, 200 μL of 3% skim milk was added, and the plate was blocked at room temperature for 2 hours. After blocking, 100 μL of the amplified phage library (approximately 2 × 10⁻⁶) was added. 11 (1 phage particle), incubate at room temperature for 1 hour; after 1 hour, wash 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0081] Phages that specifically bind to IL-17A protein were dissociated using trypsin at a final concentration of 25 mg / mL and then used to infect E. coli TG1 cells in the logarithmic growth phase. The cells were cultured at 37°C for 1 h to produce and collect phages for the next round of screening. The same screening process was repeated once to gradually enrich the cells.

[0082] When the enrichment factor reaches 10 times or more, the enrichment effect is as follows: Figure 1 As shown.

[0083] Figure 1In this context, P / N = the number of monoclonal bacteria grown from phages eluted from positive wells in the biopanning process after infecting TG1 bacteria / the number of monoclonal bacteria grown from phages eluted from negative wells after infecting TG1 bacteria. This parameter gradually increases after enrichment occurs. I / E = the total number of phages added to positive wells in each round of the biopanning process / the total number of phages eluted from positive wells in each round of the biopanning process. This parameter gradually approaches 1 after enrichment occurs.

[0084] Example 4: Screening for specific positive clones against IL-17A using phage enzyme-linked immunosorbent assay (ELISA): Following the screening method described in Example 3 above, three rounds of screening were performed on single-domain antibodies against IL-17A protein. The phage enrichment factor against IL-17A protein reached 10 or higher. After screening, 384 single colonies were selected from the positive clones and inoculated into 96-well plates containing 100 μg / mL ampicillin in 2×TY medium. A blank control was also set up. After incubation at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and incubated overnight at 28°C.

[0085] Crude antibody was obtained using the osmotic burst method. IL-17A recombinant protein was released into 100 mM NaHCO3 (pH 8.3), and 100 μg of protein was coated overnight at 4°C in an ELISA plate. 100 μL of the obtained crude antibody extract was transferred to an ELISA plate containing the antigen and incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and 100 μL of Mouse Anti-HA tag Antibody (HRP) (mouse anti-HA horseradish peroxidase labeled antibody, ThermoFisher) diluted 1:2000 was added. The plate was incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and horseradish peroxidase chromogenic solution was added. The reaction was carried out at 37°C for 15 min, and then stop solution was added. The absorbance was read at 450 nm using an ELISA reader.

[0086] When the OD value of the sample well is more than 5 times that of the control well, it is determined to be a positive clone well. The bacteria in the positive clone well are transferred to LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0087] Gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while clones with different sequences were considered different clones. Ultimately, single-domain antibodies specifically targeting the IL-17A protein (SEQ ID NO. 1-9 and other single-domain antibodies with sequences not shown, including 1C9, 1D1, etc. in the attached figure) were obtained.

[0088] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, constituting the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system to ultimately obtain the single-domain antibody protein.

[0089] The CDR and FR sequences of the nine single-domain antibodies are shown in Tables 1-6. The amino acid sequences of the nine single-domain antibodies are shown in SEQ ID NO. 1-9, and the DNA sequences encoding the nine single-domain antibodies are shown in SEQ ID NO. 10-18.

[0090] Table 1. CDR1 sequences of 9 single-domain antibodies Table 2. CDR2 sequences of 9 single-domain antibodies Table 3. CDR3 sequences of 9 single-domain antibodies Table 4. FR1 sequences of 9 single-domain antibodies Table 5. FR2 sequences of 9 single-domain antibodies Table 6. FR3 sequences of 9 single-domain antibodies Table 7. FR4 sequences of 9 single-domain antibodies The amino acid sequences SEQ ID NO. 1-9 of the single-domain antibodies correspond one-to-one with the single-domain antibody sequences 1A10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2, and 4A12, respectively. The nucleotide sequences SEQ ID NO. 10-18 of the single-domain antibodies also correspond one-to-one with the single-domain antibody sequences 1A10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2, and 4A12, respectively.

[0091] Example 5: Purification and expression of IL-17A protein-specific single-domain antibody in host bacterium Escherichia coli. The plasmid (pMECS-VHH) of different clones obtained from the sequencing analysis in Example 4 was electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated overnight at 37°C. Single colonies were selected and inoculated into 5 mL of LB medium containing ampicillin and incubated overnight on a shaker at 37°C.

[0092] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium and incubate at 37°C in a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and incubate overnight at 28°C in a shaker. Centrifuge to collect E. coli and obtain crude antibody extract using the osmotic rupture method. The antibody was purified by nickel column affinity chromatography, and the purified single-domain antibody included VHH1-9.

[0093] VHH1-9 correspond to the single-domain antibodies of the amino acid sequences SEQ ID NO.1-9, that is, VHH1-9 correspond to: 1A10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2, 4A12 respectively.

[0094] Example 6: Humanization of a single-domain antibody against IL-17A The humanization method employs high-throughput screening of antibody framework region mutation libraries constructed based on big data analysis results. The detailed steps are as follows: (1) Sequence analysis of human / camel antibody data: Amino acid preference analysis was performed on 13,873 Nb (Human) sequences downloaded in batches from the NCBI website, and amino acid preference analysis was also performed on 2,000 nanobody sequences from our company to obtain the amino terminus ratio data of each site in the framework region; (2) Human-Camel-Derived Weighted Analysis: The above-mentioned human / camel-derived antibody sequences were uniformly numbered according to the IMGT numbering rules and matched one by one. Combined with the amino acid ratio analysis results of the two species, a weighted analysis was performed with a weight of 90% human and 10% camel. The weighted proportion of amino acids at each site was calculated and sorted from high to low. Based on the final weighted results, only amino acid species with a proportion >10% were retained at each site in the framework region. The final weight of amino acids with a proportion >10% was calculated based on the standard that the proportion after retention is combined to 1, which serves as the basis for the design of subsequent custom amino acid libraries. (3) Design of the custom amino acid library: For each individual site to be mutated, the number of amino acids in the >10% range is defined as n, and the ratio of the highest to the lowest proportion in the >10% range is defined as V. The site to be mutated is then judged by its properties: if V≥3 and n≤2, the site is considered to be a "high concentration site", otherwise it is considered to be a "medium-low concentration site". Based on this method, the custom amino acid library is divided into two "high / medium-low concentration libraries" and the custom amino acid libraries are constructed separately. The final weight in (2) above is the reference basis for the types and proportions of amino acids in the site in the library.

[0095] (4) High-throughput screening of custom amino acid libraries: Humanized antibody libraries were constructed for antibody strains 1A10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2, and 4A12. The constructed libraries were then panned with the corresponding antigens to obtain antibody sequences with high affinity and high degree of humanization.

[0096] For example, the following humanized antibodies can be obtained: eight humanized antibodies (2G3-V17, 2G3-V18, 2G3-V24, 2G3-V25, 2G3-V46, 2G3-V50, 2G3-V31, and 2G3-V34) obtained by humanizing 2G3 single-domain antibodies (the sequences of these eight humanized 2G3 antibodies are not completely identical), and eight humanized antibodies (1A10-V1, 1A10-V4, 1A10-V9, 1A10-V10, 1A10-V5, 1A10-V6, 1A10-V15, or 1A10-V14) obtained by humanizing 1A10 single-domain antibodies (the sequences of these eight humanized 1A10 antibodies are not completely identical).

[0097] Example 7: Construction of a eukaryotic expression vector for an Fc fusion antibody against a single domain antibody of anti-IL-17A (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened in Example 4 was obtained by Sanger sequencing to obtain its nucleotide sequence; (2) The above nucleotide sequence (SEQ ID NO:10-18) was synthesized into the vector RJK-V4-3 designed and modified by our company by sequence synthesis to obtain a recombinant eukaryotic expression vector. The modification method of the vector is as described in Example 11. (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culture it for plasmid extraction, and remove endotoxins; (4) The extracted plasmids were then sequenced and identified. (5) After confirming the recombinant vector, prepare it for subsequent eukaryotic cell transfection and expression. After expressing the Fc protein of VHH using the method in Example 8 or 9, purify the antibody using the method in Example 10.

[0098] Example 8: Expression of a single-domain antibody against IL-17A protein in suspension ExpiCHO-S cells (1) Three days before transfection, use 2.5×10 5 / mL cell passage and expansion culture ExpiCHO-S TM Cells, the calculated desired cell volume, were transferred to 120 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium, the cell concentration was increased to approximately 4 × 10⁻⁶ cells / mL.6 -6×10 6 live cells / mL; (2) One day before transfection, ExpiCHO-S TM Cells were diluted to a concentration of 3.5 × 10⁻⁶. 6 Live cells / mL, incubate cells overnight; (3) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 7 × 10⁻⁶ cells / day before transfection. 6 -10×10 6 live cells / mL; (4) Use fresh ExpiCHO preheated to 37°C TM The expression medium was used to dilute the cells to 6 × 10⁶. 6 viable cells / mL. The calculated desired cell volume was transferred to 100 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium; (5) Gently invert to mix ExpiFectamine TM CHO reagent, using 3.7 mL OptiPRO TM Culture medium for diluting ExpiFectamine TM CHO reagent, vortex or mix well; (6) Use 4 mL of refrigerated OptiPRO TM Dilute the plasmid DNA in the culture medium and vortex to mix. (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-IL-17A single domain antibody prepared in Example 7) complex at room temperature for 1-5 minutes, and then gently add it to the prepared cell suspension while gently shaking the flask during the addition process. (8) The cells were cultured with shaking in humidified air at 37°C and 8% CO2. (9) Add 600ul of ExpiFectamine on the first day after transfection (18-22 hours later). TM CHO Enhancer and 24mLExpiCHO feed.

[0099] (10) Collect the supernatant about 8 days after transfection (when cell viability is less than 70%).

[0100] Example 9: Expression of single-domain antibody against IL-17A protein in suspension 293F cells Recombinant single-domain antibody expression experimental procedure (taking a 500mL shake flask as an example): (1) Three days before transfection, use 2.5×10 5After passage and expansion of 293F cells at / mL, the calculated desired cell volume was transferred to a 500mL shake flask containing 120mL (final volume) of fresh, preheated OPM-293CD05 Medium to achieve a cell concentration of approximately 2×10⁻⁶. 6 -3×10 6 Live cells / mL.

[0101] (2) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 2 × 10⁻⁶ cells / day before transfection. 6 -3×10 6 Live cells / mL.

[0102] (3) Dilute the cells to 1×10⁻⁵ using preheated OPM-293CD05 Medium. 6 1 live cells / mL. Calculate the required cell volume and transfer it to a 500 mL shake flask containing 100 mL (final volume) of fresh, preheated culture medium.

[0103] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, and mix by swirling or pipetting; dilute plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the anti-IL-17A single-domain antibody prepared in Example 7) with 4 mL Opt-MEM medium, mix by swirling, and filter with a 0.22 μm filter. Incubate at room temperature for 5 min.

[0104] (5) Add the diluted PEI reagent to the diluted DNA and mix by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15-20 minutes, then gently add it to the prepared cell suspension while gently shaking the flask during the addition process.

[0105] (6) Culture the cells at 37°C, 5% CO2, and 120 rpm with shaking.

[0106] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0107] (8) Collect the supernatant about 7 days after transfection (when cell viability is less than 70%).

[0108] Example 10: Purification of single-domain antibody against IL-17A protein (1) The protein expression supernatant obtained in Example 8 or 9 was filtered with a 0.45 μm disposable filter to remove insoluble impurities; (2) The above filtrate was purified by affinity chromatography using a protein purifier. The agarose packing material coupled with Protein A was used to purify the filtrate by utilizing the ability of human Fc to bind to Protein A. (3) Pass the filtrate through a pre-packed Protein A column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the packing material. (4) Wash the impurity proteins bound to the column with low-salt and high-salt buffers; (5) Use a low pH buffer to systemically bind the target protein on the column; (6) Quickly add the eluent to a Tris-HCl solution with a pH of 9.0 to neutralize it; (7) After dialysis of the above-neutralized protein solution, perform SDS-PAGE analysis to confirm that the protein purity is above 95% and the concentration is above 0.5 mg / mL, and then store it at low temperature for later use.

[0109] Example 11: Construction of the single-domain antibody eukaryotic expression vector RJK-V4-3 The aforementioned universal target vector for nanobodies, RJK-V4-3, is a modified version of Invitrogen's commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) by incorporating the Fc region of the human IgG4 heavy chain coding sequence. Specifically, this vector contains the CH2 and CH3 hinge regions of the IgG4 heavy chain. The specific modification scheme is as follows: (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4; (2) Multiple cloning sites (MCS) and 6×His tags were introduced at the 5' and 3' ends of the Fc fragment coding sequence, respectively, by overlapping PCR. (3) The above fragment was amplified by PCR using a pair of primers with XbaI and AgeI restriction sites respectively; (4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction endonucleases XbaI and AgeI, respectively; (5) The digested vector and the insert fragment were ligated with T4 ligase, and then the ligation product was transformed into E. coli, amplified, sequenced and verified to obtain the recombinant plasmid.

[0110] Example 12: Expression and purification of a tool antibody (Tab1) targeting human IL-17A. In this article, Tab 1 is referred to as Tab. Tab1 (Secukinumab) is a secukinumab whose sequence is derived from IMGT.

[0111] The searched sequences were commissioned to General Biosystems (Anhui) Co., Ltd. for codon optimization in mammalian cell expression systems and cloned into the pcDNA3.1 vector. After antibiotic selection, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50). 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) was added per 100 mL of cells, and PEI was used to transiently express the plasmid in 293F cells (medium: FreeStyle 293Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032). 6–24 h after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added, and the cells were cultured in 8% CO2 at 130 rpm for approximately 7–8 days. When cell viability decreased to 50%, the expression supernatant was collected and purified using a gravity column with Protein A (GE, Cat#17-5438-02). After dialysis with PBS, the concentration was determined using Nanodrop, purity was identified by SEC, and binding capacity was verified by indirect ELISA. The Tab obtained by this method has a concentration of not less than 2 mg / ml and a purity greater than 95%.

[0112] Example 13: Determination of Antibody-Antigen Binding Dose-Response Curve This embodiment uses the standard enzyme-linked immunosorbent assay (ELISA) procedure.

[0113] (1) Coat 50 μL of 1 μg / mL IL-17A&F (Acro,Cat#ILF-H52WF,Lot#G41-88ZF1-RD) overnight at 4℃.

[0114] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 2 hours.

[0115] (3) VHH-hFc was diluted to 2 μg / mL, and then the antibody was serially diluted 5-fold to a total of 8 concentration gradients. Here, VHH-hFc refers to the Fc fusion antibody of the single-domain antibody against IL-17A protein prepared in Example 9 (expressed in 293F cells) purified in Example 10. In addition, hIgG and Tab controls were set up respectively; Tab1 was prepared in Example 12. (4) Wash the plate; add 50 μL of the single-domain antibody obtained by dilution in step (3), double replicates, and incubate at 37°C for 1 h.

[0116] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 min.

[0117] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought to room temperature beforehand, and react at room temperature in the dark for 15 min.

[0118] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0119] (8) Plot the curve and calculate EC50, as follows: Figure 2-3 As shown, hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is commercially available; it can be seen that single-domain antibodies 1A10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2, and 4A12 all exhibit excellent binding efficacy and specificity to IL-17A protein.

[0120] Figure 2 In Figure 2A, the binding affinity of single-domain antibodies 1A10, 1C9, 1D1, 1D4, 1D7, and 1E2 to the IL-17A protein is shown. In Figure 2B, the binding affinity of single-domain antibodies 1E6, 1H2, 2D8, 2E2, 2F5, and 2G3 to the IL-17A protein is shown. Figure 3 In the diagram, 3A shows the binding affinity of single-domain antibodies 3A6, 3A8, 3B11, 3D1, 3E1, and 3E2 to IL-17A protein; 3B shows the binding affinity of single-domain antibodies 3F8, 3H2, 4A1, 4A12, 4F4, and 1H3 to IL-17A protein; and 3C shows the binding affinity of single-domain antibodies 3B10, 4A1, 4G4, Tab1, and hIgG to IL-17A protein.

[0121] Example 14: Determination of antibody blocking IL-17A and IL-17R binding This embodiment uses the standard enzyme-linked immunosorbent assay (ELISA) procedure.

[0122] (1) Coat 50 μL of 1 μg / mL IL-17RA & IL-17RC (Acro,Cat#ILC-H5257,Lot#G173a-2085F1-TE) at 4℃ overnight.

[0123] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 2 hours.

[0124] (3) Biotin-IL-17A (Acro, Cat#ILA-H82Q1, Lot#CBV296P1-97RF1-QJ) was mixed with serially diluted VHH-hFc to obtain a mixture (the concentration of Biotin-IL-17A in the mixture was 3.8 ng / ml). Here, VHH-hFc refers to the Fc fusion antibody of the single-domain antibody against IL-17A protein prepared in Example 9 (expressed in 293F cells) purified in Example 10. In addition, an hIgG control was also set up; hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase; (4) Wash the plate; add 50 μL of the above mixture, double-well, and incubate at 37°C for 1 h.

[0125] (5) Wash the plate; add 50 μL Streptavidin-HRP and incubate at 37°C for 30 min.

[0126] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought to room temperature beforehand, and react at room temperature in the dark for 15 min.

[0127] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0128] (8) Plot the curve and calculate EC50. The result is as follows: Figure 4-5 As shown, Figure 4-5 The horizontal axis represents the specific concentration of VHH-hFc after gradient dilution. It can be seen that single-domain antibodies 1A10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2, and 4A12 all effectively blocked the binding of IL-17A to IL-17R.

[0129] Figure 4 In the table, 4A shows the assay results for 1A10, 1C9, 1D7, 1E2, 1D1, and 1D4; 4B shows the assay results for single-domain antibodies 3A6, 3A8, 3E1, 3E2, 3B11, and 3D1; and 4C shows the assay results for 3F8, 3H2, 4F4, 1H3, 4A1, and 4A12. Figure 5 In the diagram, 5A shows the results of the assays for 3B10, 4A1, hIgG, and 4G4, while 5B shows the results of the assays for single-domain antibodies 1E6, 1H2, 2F5, 2G3, 2D8, and 2E2.

[0130] Example 15: The experiment of neutralizing human IL-17A with antibody (eukaryotic sample) to induce the release of IL-6 from HeLa cells was performed according to the methods generally known to those skilled in the art: (1) After being revived and passaged 3 or more times, HeLa cells were seeded into 96-well plates at a rate of 10,000 cells per well and 50 μl per well. (2) The serially diluted antibody was mixed with an equal volume of 4*55ng / ml human IL-17A (Novo, Cat#C774, Lot#0331872-4147), and 50μl of this mixture was added to each cell culture well. The antibody here is the Fc fusion antibody of the single-domain antibody against IL-17A protein prepared in Example 9 (expressed in 293F cells) purified in Example 10. In addition, hIgG and Tab controls were set up respectively. hIgG refers to the isotype control, which is an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase. Tab was prepared in Example 12. (3) After incubation at 37℃ for 24 h in a cell culture incubator, the concentration of IL-6 in the supernatant was detected using a human IL-6 ELISA kit; the EC50 of antibody neutralization of IL-17A-induced IL-6 release from HeLa cells was calculated based on the detection results, and the results are as follows. Figure 6-7 As shown, the horizontal axis represents the molar concentration of the aforementioned serially diluted antibodies; the vertical axis represents the IL-6 concentration in the supernatant (in pg / ml); from Figure 6-7 It is known that eukaryotic antibodies can effectively neutralize IL-17A and induce HeLa cells to release IL-6.

[0131] Figure 6 In the diagram, 6A shows the experimental results for Tab and hIgG, 6B shows the experimental results for 2G3, 3D1, 1D4, and 2E2, and 6C shows the experimental results for 1A10, 1D1, 3E1, and 3H2. Figure 7 In the diagram, 7A shows the experimental results for 4A12 and 4F4, 7B shows the experimental results for 1C9, 1D7, 1E2 and 1E6, 7C shows the experimental results for 3E2, 3F8, 2F5 and 3A6, and 7D shows the experimental results for 4A1 and 1H3.

[0132] Example 16: The experiment of neutralizing human IL-17A with antibody (humanized sample) to induce IL-6 release from HeLa cells was performed according to the methods generally known to those skilled in the art: (1) After being revived and passaged 3 or more times, HeLa cells were seeded into 96-well plates at a rate of 10,000 cells per well and 50 μl per well. (2) The serially diluted antibody was mixed with an equal volume of 4*55ng / ml human IL-17A, and 50μl of this mixture was added to each cell culture well. The antibody was: humanized antibody 2G3-V17, 2G3-V18, 2G3-V24, 2G3-V25, 2G3-V46, 2G3-V50, 2G3-V31, 2G3-V34, 1A10-V1, 1A10-V4, 1A10-V9, 1A10-V10, 1A10-V5, 1A10-V6, 1A10-V15 or 1A10-V14. The humanized single-domain antibody against IL-17A was constructed in Example 7 to form an Fc fusion antibody eukaryotic expression vector (the nucleotide sequence of the humanized antibody was constructed into the vector RJK-V4-3), and then expressed in Example 9 and purified in Example 10. In addition, hIgG and Tab controls were set up separately; hIgG refers to the isotype control, which is an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase; Tab was prepared in Example 12. (3) After incubation at 37℃ for 24 h in a cell culture incubator, the concentration of IL-6 in the supernatant was detected using a human IL-6 ELISA kit; the EC50 of antibody neutralization of IL-17A-induced IL-6 release from HeLa cells was calculated based on the detection results, and the results are as follows. Figure 8-10 As shown, the horizontal axis represents the specific concentration of the aforementioned serially diluted antibodies; the vertical axis represents the IL-6 concentration in the supernatant (unit: pg / ml); from Figure 8-10 It is known that humanized antibodies can effectively neutralize IL-17A and induce HeLa cells to release IL-6, and their technical performance is superior to that of antibodies (eukaryotic samples).

[0133] Figure 8 In the diagram, 8A shows the experimental results for Tab and hIgG, 8B shows the experimental results for 2G3-V17 and 2G3-V18, and 8C shows the experimental results for 2G3-V24 and 2G3-V25. Figure 9 In the diagram, 9A shows the experimental results for 2G3-V46 and 2G3-V50, 9B shows the experimental results for 2G3-V31 and 2G3-V34, and 9C shows the experimental results for 1A10-V1 and 1A10-V4. Figure 10 In the diagram, 10A shows the experimental results for 1A10-V9 and 1A10-V10, 10B shows the experimental results for 1A10-V5 and 1A10-V6, and 10C shows the experimental results for 1A10-V15 and 1A10-V14.

[0134] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A single-domain antibody against IL-17A, characterized in that: The single-domain antibody is composed of heavy chains, including heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are as follows (1) or (2): (1) CDR1 shown in SEQ ID NO:23, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:37; (2) CDR1 shown in SEQ ID NO:20, CDR2 shown in SEQ ID NO:30, and CDR3 shown in SEQ ID NO:

33.

2. The single-domain antibody against IL-17A according to claim 1, characterized in that: The single-domain antibody further includes a frame region FR; the frame region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the frame region FR are as follows: The FR1 or a variant thereof shown in SEQ ID NO:44 or 42, wherein the variant thereof contains up to 5 amino acid substitutions in the FR1; The FR2 or a variant thereof shown in SEQ ID NO:45 or 46, wherein the variant thereof contains substitutions of up to 5 amino acids; The FR3 or a variant thereof shown in SEQ ID NO:55 or 58, wherein the variant thereof contains substitutions of up to 5 amino acids in the FR3; The FR4 or a variant thereof shown in SEQ ID NO:61, wherein the variant thereof contains substitutions of up to 5 amino acids.

3. A single-domain antibody against IL-17A, characterized in that: The amino acid sequences of the single-domain antibodies are shown in SEQ ID NO.4 or SEQ ID NO.6, respectively.

4. A bispecific antibody, characterized in that: The bispecific antibody includes a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is a single-domain antibody against IL-17A as described in any one of claims 1 to 3.

5. Use of the single-domain antibody against IL-17A according to any one of claims 1 to 3 in the preparation of bispecific antibodies.

6. The Fc fusion antibody or humanized antibody of the single-domain antibody against IL-17A according to any one of claims 1 to 3.

7. A polynucleotide molecule encoding a single-domain antibody against IL-17A as described in any one of claims 1 to 3, characterized in that: Their nucleotide sequences are shown in SEQ ID NO: 13 or SEQ ID NO: 15, respectively.

8. An expression carrier, characterized in that: It comprises a single-domain antibody encoding anti-IL-17A as described in any one of claims 1 to 3, or a polynucleotide molecule of the Fc fusion antibody or humanized antibody as described in claim 6, or a polynucleotide molecule as described in claim 7.

9. A host cell, characterized in that: It can express the single-domain antibody against IL-17A as described in any one of claims 1 to 3, or the Fc fusion antibody or humanized antibody as described in claim 6, or the expression vector as described in claim 8.

10. The host cell according to claim 9, characterized in that: The host cell is a eukaryotic cell or a prokaryotic cell.

11. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a single-domain antibody against IL-17A selected from any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

12. A medicine for treating psoriasis, characterized in that: It comprises an anti-IL-17A single-domain antibody as the active ingredient of any one of claims 1 to 3.

13. A kit for detecting IL-17A levels, characterized in that: The kit contains a single-domain antibody against IL-17A as described in any one of claims 1 to 3.

14. The use of the single-domain antibody against IL-17A according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating a disease, characterized in that: The disease in question is psoriasis.

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