Anti-il-4rα single-domain antibody, fusion protein thereof, pharmaceutical composition thereof and use thereof

Abstract

Provided are an anti-IL-4Rα single-domain antibody, a fusion protein thereof, a pharmaceutical composition thereof and the use thereof. An anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof comprises at least one heavy chain variable region. The heavy chain variable region comprises three complementary determining regions CDR1-CDR3, wherein an amino acid sequence of CDR1 is set forth in SEQ ID NO: 2, an amino acid sequence of CDR2 is set forth in SEQ ID NO: 3, and an amino acid sequence of CDR3 is set forth in SEQ ID NO: 4. The single-domain antibody or fusion protein has a high affinity for IL-4 Rα, can effectively block the binding of IL-4 and IL-13 to IL-4 Rα, and can block IL-4 / IL-13-induced phosphorylation of downstream signal STAT6, and thus has good application prospects.

Description

Anti-il-4ra single-domain antibody, fusion protein thereof, pharmaceutical composition and use TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to an anti-IL-4Rα single-domain antibody, a fusion protein thereof, a pharmaceutical composition and use. BACKGROUND

[0002] Type II inflammation is an inflammation mainly mediated by Th2 cells, type 2 innate lymphoid cells and related cytokines. Type II inflammation drives the onset of various diseases. It is known that atopic dermatitis (AD), type II inflammation asthma, allergic rhinitis, some chronic rhinosinusitis with nasal polyps (CRSwNP), chronic obstructive pulmonary disease (COPD), chronic spontaneous urticaria (CSU), and prurigo nodularis (PN) are all related to type II inflammation (Gandhi NA, et al. Nat Rev Drug Discov. 2016 Jan; 15(1): 35-50.). In the process of occurrence of these type II inflammation diseases, type II cytokines IL-4 and IL-13 are the core driving factors. IL-4 can induce the differentiation of initial CD4+ T cells into Th2 cells, induce them to produce a large number of type II immune-related cytokines and chemokines, and start type II inflammatory response. Th2 cells secrete IL-4 and IL-13 to promote B cell proliferation and promote them to undergo serotype transformation to produce immunoglobulin E (IgE). After allergen-specific IgE is cross-linked with FcεRI, it promotes the degranulation of mast cells and basophils, and releases inflammatory mediators (such as histamine, leukotrienes, etc.). IL-4 and IL-13 jointly induce eosinophils to promote their migration to inflammatory tissues. In addition, IL-13 can directly act on epithelial cells and has an important role in mucus secretion, goblet cell proliferation, smooth muscle contraction, collagen production, etc. (Wynn TA. Nat Rev Immunol. 2015 May; 15(5): 271-82.).

[0003] Both IL-4 and IL-13 require binding to the specific receptor IL-4R to exert corresponding biological activity. There are two types of IL-4R on the surface of cell membranes in the human body: type I receptor is composed of IL-4R alpha subunit and gamma c subunit, and type II receptor is composed of IL-4R alpha subunit and IL-13R alpha 1 subunit (Andrews AL, et al. J Immunol. 2006 Jun 15; 176(12):7456-61.). Therefore, the IL-4R alpha subunit is a common component of IL-4 type I and type II receptors and IL-13 receptors, and can mediate signal transduction of IL-4 (type I and type II receptors) and IL-13 (type II receptor). IL-4 binding to type I IL-4R can activate JAK1 and JAK3, and type II receptor can activate JAK1 and TYK2 at the same time. JAK / TYK signal activation phosphorylates STAT6 and forms a dimer that translocates to the nucleus, initiating the transcriptional expression of target genes, thereby regulating immune response reactions (Jiang H, et al. J Allergy Clin Immunol. 2000 Jun; 105(6 Pt 1):1063-70.). IL-4R alpha, as an important component of the type II inflammatory pathway, is expressed on various inflammatory cells such as T cells, mast cells, and basophils. Blocking IL-4R alpha can simultaneously inhibit two core cytokines of type II immune response—IL-4 and IL-13, block the central pathway of Th2 inflammation, and achieve a "double target" mechanism of action (Sheridan C. Nat Biotechnol. 2018 Jan 10; 36(1): 3-5.).

[0004] Currently, only one IL-4R alpha targeting drug has been approved globally, namely Dupixent (dupilumab) of Sanofi / Regeneron. This drug is a fully humanized monoclonal antibody that blocks IL-4 and IL-13 signaling by binding to the common receptor IL-4R alpha, and has good control effect on various type 2 immune related inflammatory diseases. At present, Dupixent has been approved for multiple indications, such as atopic dermatitis (AD), asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), eosinophilic esophagitis (EoE), and nodular prurigo.

[0005] Nanobodies are currently the smallest antibody molecules, with a molecular weight of 1 / 10 of ordinary antibodies. In addition to the antigen reactivity of monoclonal antibodies, nanobodies also have some unique functional properties, such as small molecular weight, strong stability, good solubility, easy expression, weak immunogenicity, strong penetration, strong targeting, simple humanization, low preparation cost, etc., which almost perfectly overcome the defects of traditional antibodies such as long development cycle, low stability, and harsh storage conditions.

[0006] However, there is currently a lack of satisfactory nanobodies against IL-4Rα in the art. Therefore, there is an urgent need in the art to develop new specific nanobodies against IL-4Rα. SUMMARY

[0007] The present inventors, through in-depth research and creative labor, obtained an anti-IL-4Rα single domain antibody and a fusion protein comprising the same. The present inventors surprisingly found that the anti-IL-4Rα single domain antibody or antigen-binding fragment thereof of the present application or the fusion protein of the present application has a high affinity for IL-4Rα, can effectively block the binding of IL-4 and IL-13 to IL-4Rα, block IL-4 / IL-13-induced downstream signal STAT6 phosphorylation, and has a good application prospect. Thus, the following invention is provided:

[0008] One aspect of the present application relates to an anti-IL-4Rα single domain antibody or antigen-binding fragment thereof, comprising at least one heavy chain variable region, said heavy chain variable region comprising 3 complementarity determining regions CDR1-CDR3, wherein:

[0009] the amino acid sequence of CDR1 is as shown in SEQ ID NO: 2,

[0010] the amino acid sequence of CDR2 is as shown in SEQ ID NO: 3, and

[0011] the amino acid sequence of CDR3 is as shown in SEQ ID NO: 4.

[0012] In some embodiments of the present application, the anti-IL-4Rα single domain antibody or antigen-binding fragment thereof, wherein,

[0013] any one, two, three or all four of the four framework regions of the anti-IL-4Rα single domain antibody are humanized;

[0014] Preferably, the amino acid sequence of the anti-IL-4Rα single domain antibody is as shown in any one of SEQ ID NO: 1 and SEQ ID NOs: 5-11.

[0015] In some embodiments of the present application, the anti-IL-4Rα single domain antibody or antigen-binding fragment thereof, wherein,

[0016] the anti-IL-4Rα single domain antibody or antigen-binding fragment thereof binds to 293F cells stably overexpressing human IL-4Rα and 293F cells overexpressing rhesus monkey IL-4Rα with an EC 50 less than 5 nM, preferably less than 4 nM; preferably, the EC 50determined by flow cytometry and calculated; and / or

[0017] The anti-IL-4Ra single domain antibody or antigen binding fragment thereof has an IC 50 less than 3.2 nM, preferably less than 2.5 nM or less than 2 nM; preferably, the IC 50 The mean fluorescence intensity is determined by flow cytometry and calculated.

[0018] In some embodiments of the present application, the anti-IL-4Ra single domain antibody or antigen binding fragment thereof, wherein the anti-IL-4Ra single domain antibody is the anti-IL-4Ra single domain antibody numbered VHH-005, VHH-005-H1, VHH-005-H2, VHH-005-H3, VHH-005-H4, VHH-005-H5, VHH-005-H6 or VHH-005-H8 of the present application.

[0019] Another aspect of the present application relates to a fusion protein comprising one or more copies (e.g. 2, 3, 4, 5 or 6 copies) of the anti-IL-4Ra single domain antibody or antigen binding fragment thereof of any one of the present application;

[0020] Preferably, the fusion protein further comprises a Fc fragment of human IgG, a heavy chain constant region of human IgG, a human Kappa light chain constant region or a human Lambda light chain constant region.

[0021] Preferably, the fusion protein further comprises a Fc fragment of human IgG, a heavy chain constant region of human IgG, a human Kappa light chain constant region or a human Lambda light chain constant region.

[0022] Preferably, the Fc fragment of human IgG is a Fc fragment of human IgG1, human IgG2, human IgG3 or human IgG4.

[0023] Preferably, the Fc fragment of human IgG is a Fc fragment of human IgG1, and contains L234A and L235A mutations according to EU numbering system.

[0024] Preferably, the Fc fragment of human IgG has an amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 23.

[0025] Preferably, the fusion protein has an amino acid sequence as set forth in any one of SEQ ID NOs: 25-32.

[0026] Preferably, the heavy chain constant region of the human IgG is a heavy chain constant region of human IgG1, human IgG2, human IgG3 or human IgG4;

[0027] Preferably, the heavy chain constant region of the human IgG is a heavy chain constant region of human IgG1, and contains L234A and L235A mutations according to the EU numbering system;

[0028] Preferably, the amino acid sequence of the heavy chain constant region of the human IgG is shown in SEQ ID NO: 18 or SEQ ID NO: 24;

[0029] Preferably, the amino acid sequence of the human Kappa light chain constant region is shown in SEQ ID NO: 19;

[0030] Preferably, the anti-IL-4R alpha single-domain antibody or antigen-binding fragment thereof is directly connected with the Fc segment of human IgG, the heavy chain constant region of human IgG, the human Kappa light chain constant region or the human Lambda light chain constant region, or is connected through a first linker;

[0031] Preferably, when the anti-IL-4R alpha single-domain antibody or antigen-binding fragment thereof is in multiple copies, each copy is directly connected or connected through a second linker;

[0032] Preferably, the amino acid sequence of the first linker or the second linker is independently shown in SEQ ID NO: 20.

[0033] In the present application, the point mutation of leucine at position 234 to alanine (L234A) and the point mutation of leucine at position 235 to alanine (L235A) contained in the heavy chain constant region according to the EU numbering system are also called LALA mutations.

[0034] In some embodiments of the present application, the fusion protein comprises a peptide chain, the peptide chain comprises one or two copies of the anti-IL-4R alpha single-domain antibody or antigen-binding fragment thereof, and the Fc segment of human IgG1.

[0035] In some embodiments of the present application, the fusion protein, wherein the peptide chain comprises the following elements in order from N-terminal to C-terminal:

[0036] the first anti-IL-4R alpha single-domain antibody or antigen-binding fragment thereof, the second anti-IL-4R alpha single-domain antibody or antigen-binding fragment thereof, and the Fc segment of human IgG1;

[0037] the first anti-IL-4R alpha single-domain antibody or antigen-binding fragment thereof, the Fc segment of human IgG1, and the second anti-IL-4R alpha single-domain antibody or antigen-binding fragment thereof; or

[0038] the Fc fragment of human IgG1, the first anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof and the second anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof;

[0039] wherein,

[0040] the first anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof and the second anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof are the same or different;

[0041] Preferably, the first anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof and the second anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof are independently selected from any one of the anti-IL-4Rα single-domain antibodies or an antigen-binding fragment thereof described in the present application; preferably, independently selected from the anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof having an amino acid sequence as set forth in any one of SEQ ID NOs: 1 and 5-11; preferably, the amino acid sequences of the first anti-IL-4Rα single-domain antibody and the second anti-IL-4Rα single-domain antibody are both as set forth in SEQ ID NO: 11;

[0042] Preferably, each element is directly connected or connected through a linker;

[0043] Preferably, the amino acid sequence of the linker is as set forth in SEQ ID NO: 20.

[0044] In some embodiments of the present application, the fusion protein, wherein the peptide chain consists of the following elements in order from N-terminus to C-terminus:

[0045] the first anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof, the second anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof and the Fc fragment of human IgG1;

[0046] the first anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof, the Fc fragment of human IgG1 and the second anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof; or

[0047] the Fc fragment of human IgG1, the first anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof and the second anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof;

[0048] wherein,

[0049] the first anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof and the second anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof are the same or different;

[0050] the first anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof and the second anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof are independently selected from an anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof having an amino acid sequence as set forth in any one of SEQ ID NOs: 1 and 5-11; preferably, the amino acid sequence of the first anti-IL-4Rα single-domain antibody and the second anti-IL-4Rα single-domain antibody are both as set forth in SEQ ID NO: 11;

[0051] Preferably, the Fc segment of human IgG1 contains L234A and L235A mutations according to the EU numbering system;

[0052] Preferably, the Fc segment of human IgG contains an amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 23;

[0053] Preferably, each element is directly connected or connected through a linker;

[0054] Preferably, the amino acid sequence of the linker is as set forth in SEQ ID NO: 20.

[0055] In some embodiments of the present application, the fusion protein, wherein the peptide chain consists of the following elements in order from N-terminus to C-terminus:

[0056] the first anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof, the linker, the second anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof, and the Fc segment of human IgG1;

[0057] the first anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof, the Fc segment of human IgG1, the linker, and the second anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof; or

[0058] the Fc segment of human IgG1, the linker, the first anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof, the linker, and the second anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof;

[0059] wherein,

[0060] the first anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof and the second anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof are the same or different;

[0061] The first anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof and the second anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof are independently selected from an anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof having an amino acid sequence as set forth in any one of SEQ ID NOs: 1 and 5-11; preferably, the amino acid sequence of the first anti-IL-4Ra single-domain antibody and the second anti-IL-4Ra single-domain antibody are both as set forth in SEQ ID NO: 11;

[0062] Preferably, the Fc segment of the human IgG1 contains L234A and L235A mutations according to the EU numbering system;

[0063] Preferably, the Fc segment of the human IgG has an amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 23;

[0064] Preferably, the linker has an amino acid sequence as set forth in SEQ ID NO: 20.

[0065] In some embodiments of the present application, the fusion protein is a peptide chain having an amino acid sequence as set forth in any one of SEQ ID NO: 12 to SEQ ID NO: 16.

[0066] In some embodiments of the present application, the fusion protein comprises a peptide chain having an amino acid sequence as set forth in any one of SEQ ID NO: 12 to SEQ ID NO: 16.

[0067] Preferably, the fusion protein is a dimer of the peptide chain having an amino acid sequence as set forth in any one of SEQ ID NO: 12 to SEQ ID NO: 14; preferably, a homodimer; preferably, the two peptide chains of the dimer are connected by one or more pairs (e.g., 2 pairs or 3 pairs) of disulfide bonds.

[0068] Preferably, the fusion protein is a fusion protein in the form of an immunoglobulin formed by two peptide chains having an amino acid sequence as set forth in SEQ ID NO: 15 and two peptide chains having an amino acid sequence as set forth in SEQ ID NO: 16.

[0069] In some embodiments of the present application, the fusion protein comprises a peptide chain having an amino acid sequence as set forth in any one of SEQ ID NO: 12 to SEQ ID NO: 16.

[0070] VHH-CL Formula (I),

[0071] VHH-CH Formula (II),

[0072] VHH-CH Formula (III); and

[0073] VHH-CL Formula (IV);

[0074] wherein,

[0075] Formula (I), (II), (III), (IV) are respectively from left to right is the order from N-terminal to C-terminal;

[0076] VHH represents the anti-IL-4Ra single domain antibody or antigen binding fragment thereof described in any one of the present application; VHH in formula (I) to formula (IV) is the same or different; preferably, the amino acid sequence of the VHH is as shown in SEQ ID NO: 11;

[0077] CH represents the heavy chain constant region of human IgG;

[0078] CL represents the human kappa light chain constant region;

[0079] VHH and CH are directly connected or connected through a linker, and VHH and CL are directly connected or connected through a linker; preferably, the amino acid sequence of the linker is as shown in SEQ ID NO: 20;

[0080] Preferably, the amino acid sequences of formula (I) and formula (IV) are the same, and / or the amino acid sequences of formula (II) and formula (III) are the same;

[0081] Preferably, the heavy chain constant region of human IgG is the heavy chain constant region of human IgG1, human IgG2, human IgG3 or human IgG4;

[0082] Preferably, the heavy chain constant region of human IgG is the heavy chain constant region of human IgG1, and contains L234A and L235A mutations according to the EU numbering system;

[0083] Preferably, the amino acid sequence of the heavy chain constant region of human IgG is as shown in SEQ ID NO: 18 or SEQ ID NO: 24;

[0084] Preferably, the amino acid sequence of the human kappa light chain constant region is as shown in SEQ ID NO: 19.

[0085] In some embodiments of the present application, the fusion protein, wherein,

[0086] Formula (I) and formula (II), formula (II) and formula (III), and formula (III) and formula (IV) are connected by one or more pairs (for example, 2 pairs or 3 pairs) of disulfide bonds.

[0087] In some embodiments of the present application, the fusion protein, wherein, formula (I), formula (II), formula (III) and formula (IV) form a fusion protein in the form of immunoglobulin.

[0088] In some embodiments of the present application, the fusion protein, wherein,

[0089] The amino acid sequence of the peptide chain represented by formula (I) and formula (IV) is as shown in SEQ ID NO: 16,

[0090] The amino acid sequence of the peptide chain represented by formula (II) and formula (III) is as shown in SEQ ID NO: 15.

[0091] In some embodiments of the present application, the fusion protein, wherein,

[0092] The fusion protein blocks the binding of human IL-4 protein to 293F cells stably overexpressing human IL-4Ra with an IC 50 less than 1 nM; preferably, the IC 50 determined by flow cytometry and calculated; and / or

[0093] The fusion protein blocks the binding of human IL-4 protein to 293F cells overexpressing rhesus IL-4Ra with an IC 50 less than 2 nM, preferably less than 1 nM, less than 0.5 nM or less than 0.4 nM; preferably, the IC 50 determined by flow cytometry and calculated.

[0094] In some embodiments of the present application, the fusion protein is an antibody fusion protein.

[0095] In some embodiments of the present application, the fusion protein is an anti-IL-4Ra antibody fusion protein.

[0096] In some embodiments of the present application, the fusion protein is an Fc fusion protein.

[0097] In some embodiments of the present application, the fusion protein is an anti-IL-4Ra Fc fusion protein.

[0098] In some embodiments of the present application, the fusion protein is an Fc fusion protein of bivalent humanized anti-IL-4Ra single domain antibody.

[0099] In some embodiments of the present application, the fusion protein is an Fc fusion protein of tetravalent humanized anti-IL-4Ra single domain antibody.

[0100] In some embodiments of the present application, the fusion protein is selected from the fusion protein H8-H8-Fc, the fusion protein H8-Fc-H8, the fusion protein Fc-H8-H8 and the fusion protein H8-IgG of the present application.

[0101] The anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof according to any one of the present application or the fusion protein according to any one of the present application is used for treating or preventing a disease caused by type II inflammation.

[0102] Preferably, the disease caused by type II inflammation is one or more selected from the group consisting of atopic dermatitis (AD), type II inflammation asthma, allergic rhinitis, chronic rhinosinusitis with nasal polyps (CRSwNP), eosinophilic esophagitis (EoE), chronic obstructive pulmonary disease (COPD), chronic spontaneous urticaria (CSU), and prurigo nodularis (PN).

[0103] The present application relates to an isolated nucleic acid molecule encoding the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof according to any one of the present application or the fusion protein according to any one of the present application.

[0104] The present application relates to a recombinant vector comprising the isolated nucleic acid molecule of the present application.

[0105] The present application relates to a recombinant host cell comprising the isolated nucleic acid molecule of the present application or comprising the recombinant vector of the present application.

[0106] The present application relates to an antibody drug conjugate comprising an antibody moiety and a small molecule drug, wherein the antibody moiety is the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof according to any one of the present application or the fusion protein according to any one of the present application.

[0107] Preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug.

[0108] In some embodiments of the present application, the antibody drug conjugate, wherein the antibody moiety is connected to the small molecule drug through a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond.

[0109] Preferably, the molar ratio of the antibody moiety to the small molecule drug is 1:(2-4), 1:2, 1:3 or 1:4.

[0110] The present application relates to a pharmaceutical composition comprising an effective amount of the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof according to any one of the present application or the fusion protein according to any one of the present application, and one or more pharmaceutically acceptable excipients.

[0111] In yet another aspect, the present application relates to the use of the anti-IL-4Ra single domain antibody or antigen binding fragment thereof according to any one of the present application or the fusion protein according to any one of the present application in the manufacture of a medicament for treating or preventing a disease caused by type II inflammation.

[0112] Preferably, the disease caused by type II inflammation is one or more selected from the group consisting of atopic dermatitis (AD), type II inflammation asthma, allergic rhinitis, chronic rhinosinusitis with nasal polyps (CRSwNP), eosinophilic esophagitis (EoE), chronic obstructive pulmonary disease (COPD), chronic spontaneous urticaria (CSU) and prurigo nodularis (PN).

[0113] In yet another aspect, the present application relates to a method for treating or preventing a disease caused by type II inflammation, comprising the step of administering to a subject in need thereof an effective amount of the anti-IL-4Ra single domain antibody or antigen binding fragment thereof according to any one of the present application or the fusion protein according to any one of the present application.

[0114] Preferably, the disease caused by type II inflammation is one or more selected from the group consisting of atopic dermatitis (AD), type II inflammation asthma, allergic rhinitis, chronic rhinosinusitis with nasal polyps (CRSwNP), eosinophilic esophagitis (EoE), chronic obstructive pulmonary disease (COPD), chronic spontaneous urticaria (CSU) and prurigo nodularis (PN).

[0115] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art. Also, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory operations steps used herein are all conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0116] The term "single-domain antibody" (sdAb) has the meaning generally understood by those skilled in the art, and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or shark antibody). Typically, a single-domain antibody consists of four framework regions (FR1, FR2, FR3, and FR4) and three complementarity determining regions (CDR1, CDR2, and CDR3), with the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single-domain antibody can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or lack one or both of those framework regions, so long as antigen binding and specificity are substantially maintained. Single-domain antibodies are also known as nanobodies or VHH (Variable domain of heavy chain antibody), used interchangeably.

[0117] The term "heavy-chain antibody" (HCAb) refers to an antibody consisting of two heavy chains, each comprising a heavy chain variable region (VHH), a hinge region, a CH2 region, and a CH3 region, with the CH1 region and light chain being absent. Heavy-chain antibodies are naturally occurring in camelids and cartilaginous fish.

[0118] The term "antigen-binding fragment" of a single-domain antibody refers to a polypeptide comprising a fragment of a single-domain antibody that retains the ability to specifically bind the same antigen to which the single-domain antibody binds, and / or competes with the single-domain antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the antibodies of the application can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of whole single-domain antibodies of the application. In some embodiments, the "antigen-binding fragment" of the single-domain antibody can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or lack one or both of those framework regions, compared to the full-length single-domain antibody, so long as antigen binding and specificity are substantially maintained.

[0119] The term "Fc region" as used herein is an antibody fragment formed by disulfide bond linkage of the second, third constant regions of the first heavy chain with the second, third constant regions of the second heavy chain of an antibody.

[0120] In the present application, if not otherwise specified, the term Fc or Fc fragment refers to the Fc peptide chain in the state of a single peptide chain. In a dimer, two Fc peptide chains are connected by an inter-chain disulfide bond.

[0121] As used herein, the term EC 50 refers to the concentration for 50% of maximal effect, i.e. the concentration that causes 50% of maximal effect.

[0122] As used herein, the term IC 50 refers to the half maximal inhibitory concentration, i.e. the drug concentration that inhibits the activity of a biological process or a component of that process (e.g. an enzyme, a receptor, a cell, etc.) by 50% under given conditions.

[0123] As used herein, the term "isolated" or "isolated" refers to a substance or component obtained by artificial means from its natural state. If a certain "isolated" substance or component appears in nature, it is possible that the natural environment of the substance has changed, or the substance has been separated from the natural environment, or both. For example, a certain polynucleotide or polypeptide naturally exists in a living animal body, and a high-purity polynucleotide or polypeptide isolated from such a natural state is called isolated. The term "isolated" or "isolated" does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0124] As used herein, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector can be expressed in the host cell. The vector is well known to those skilled in the art, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), pox viruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site.

[0125] As used herein, 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 Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0126] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0127] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have an equilibrium dissociation constant of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D K binds to antigens (e.g., IL-4Rα). K can be measured using methods known to those skilled in the art. D For example, measurements can be taken using a Fortebio molecular interaction analyzer.

[0128] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19). thed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0129] As used herein, the term "effective amount" means an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) means an amount that is sufficient to prevent, arrest, or delay the onset of the disease (e.g., a tumor); an effective amount for treating a disease means an amount that is sufficient to cure or at least partially arrest the disease and its complications in a patient already having the disease. Determining such effective amounts is well within the capability of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight, and gender, the mode of administration of the drug, and other therapies that the patient may be undergoing, etc.

[0130] In the present application, if not otherwise specified, the "first" (e.g., first linker, etc.) or "second" (e.g., second linker, etc.) is merely for the purpose of distinction in terms of reference, and does not have a particular meaning in terms of order, nor does it mean that the two objects referred to must be different.

[0131] Advantages of the Invention

[0132] The anti-IL-4Rα single-domain antibody or antigen-binding fragment thereof of the present application or the fusion protein of the present application achieves one or more of the following technical effects (1) to (4):

[0133] (1) has a high affinity for IL-4Rα,

[0134] (2) can effectively block the binding of IL-4 and IL-13 to IL-4Rα,

[0135] (3) blocks IL-4 / IL-13-induced downstream signal STAT6 phosphorylation,

[0136] (4) can effectively treat or prevent diseases caused by type II inflammation, such as one or more selected from the group consisting of atopic dermatitis, type II inflammatory asthma, allergic rhinitis, chronic rhinosinusitis with nasal polyps, eosinophilic esophagitis, chronic obstructive pulmonary disease, chronic spontaneous urticaria, and nodular prurigo. BRIEF DESCRIPTION OF DRAWINGS

[0137] Figure 1A: Binding activity curve of Fc fusion proteins of humanized anti-IL-4Ra single domain antibodies to 293F-huIL-4Ra cells.

[0138] Figure 1B: Binding activity curve of Fc fusion proteins of humanized anti-IL-4Ra single domain antibodies to 293F-rhesus IL-4Ra cells.

[0139] Figure 2: Blocking activity curve of Fc fusion proteins of humanized anti-IL-4Ra single domain antibodies on human IL-4 protein binding to 293F-huIL-4Ra cells.

[0140] Figure 3A: Blocking activity curve of Fc fusion proteins of humanized anti-IL-4Ra single domain antibodies on human IL-4 induced phosphorylation signal of STAT6 in HEK-blue IL-4 / IL-13 cells.

[0141] Figure 3B: Blocking activity curve of Fc fusion proteins of humanized anti-IL-4Ra single domain antibodies on human IL-13 induced phosphorylation signal of STAT6 in HEK-blue IL-4 / IL-13 cells.

[0142] Figure 4A: Blocking activity curve of Fc fusion proteins of humanized anti-IL-4Ra single domain antibodies on human IL-4 induced proliferation of TF-1 cells.

[0143] Figure 4B: Blocking activity curve of Fc fusion proteins of humanized anti-IL-4Ra single domain antibodies on human IL-13 induced proliferation of TF-1 cells.

[0144] Figures 5A to 5D: Antibody structure schematic of Fc fusion proteins of tetravalent humanized anti-IL-4Ra single domain antibodies H8-H8-Fc, H8-Fc-H8, Fc-H8-H8 and H8-IgG, respectively.

[0145] Figure 6A: Blocking activity curve of Fc fusion proteins of tetravalent humanized anti-IL-4Ra single domain antibodies on human IL-4 protein binding to 293F-huIL-4Ra cells.

[0146] Figure 6B: Blocking activity curve of Fc fusion proteins of tetravalent humanized anti-IL-4Ra single domain antibodies on rhesus IL-4 protein binding to 293F-rhesus IL-4Ra cells.

[0147] Figure 7A: Blocking activity curve of Fc fusion proteins of tetravalent humanized anti-IL-4Ra single domain antibodies on human IL-4 induced phosphorylation signal of STAT6 in HEK-blue IL-4 / IL-13 cells.

[0148] Figure 7B: The blocking activity curve of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody on the phosphorylation signal of human IL-13 induced STAT6 in HEK-blue IL-4 / IL-13 cells.

[0149] Figure 8A: The blocking activity curve of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody on the proliferation of human IL-4 induced TF-1 cells.

[0150] Figure 8B: The blocking activity curve of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody on the proliferation of human IL-13 induced TF-1 cells.

[0151] Figure 9A: The blocking activity curve of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody on the expression of CD23 of B cells induced by human IL-4.

[0152] Figure 9B: The blocking activity curve of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody on the expression of CD23 of B cells induced by human IL-13.

[0153] Figure 10A: The bar chart of the effect of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody on the number of CD45+ cells in the lung tissue of mice in a mouse asthma model.

[0154] Figure 10B: The bar chart of the effect of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody on the number of eosinophils in the lung tissue of mice in a mouse asthma model.

[0155] Figure 10C: The bar chart of the effect of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody on the proportion of eosinophils in CD45+ cells in the lung tissue of mice in a mouse asthma model.

[0156] Figure 10D: The bar chart of the effect of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody on the content of serum IgE of mice in a mouse asthma model.

[0157] Figure 10E: The bar chart of the effect of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody on the content of IgE in the lung alveolar lavage fluid of mice in a mouse asthma model.

[0158] Figure 10F: The results of the pathological changes of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody after administration in a mouse asthma model. Six or seven pictures in each group represent the results of 6 or 7 mouse samples in the same group, respectively.

[0159] The information of part of the sequences involved in the present application is provided in Table 1 below.

[0160] Table 1: Information of partial sequences involved in the present application DETAILED DESCRIPTION

[0161] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not noted in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not noted by the manufacturer are all conventional products that can be obtained by purchase.

[0162] Example 1: Construction of nanobody library

[0163] 1 mg of human IL-4Rα antigen (purchased from AcroBiosystems) was mixed with an equal volume of Freund's adjuvant, and two llamas were immunized, once a week, for a total of 4 times, to stimulate B cells to express single domain antibodies specific to the antigen. After the 4 times of immunization, 50 mL of peripheral blood of the llamas was extracted, and lymphocytes were obtained by using lymphocyte separation medium. Total RNA was extracted by using Trizol (purchased from Invitrogen). The total cDNA of the llamas was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen). IgG2 and IgG3 sequences were amplified from the cDNA by first-round PCR. The first-round PCR product was subjected to agarose gel electrophoresis, and the fragment at 750 bp was recovered by gel cutting and used for second-round VHH sequence amplification. The second-round PCR product was used as a template for third-round PCR to add homologous arms to the VHH gene. Finally, the target fragment was recovered by using a PCR purification kit (purchased from QIAGEN).

[0164] The linearized yeast display vector after enzyme digestion and the third-round PCR product were mixed and then electroporated into Saccharomyces cerevisiae (20828) to construct the anti-IL-4Rα single domain antibody library from two llamas and determine the library capacity. 20828) to construct the anti-IL-4Rα single domain antibody library from two llamas and determine the library capacity.

[0165] Example 2: Screening of anti-IL-4Rα single domain antibodies

[0166] Dissolve the human IL-4Rα protein (purchased from AcroBiosystems) in double distilled water, and mix with the biotin solution according to the product manual of the biotin labeling kit (purchased from Thermo). Incubate at 4°C for 2 hours. Remove the excess biotin with a desalting column (purchased from Thermo). The pretreatment of the desalting column and the collection of the sample are performed according to the product manual.

[0167] The VHH library constructed in Example 1 was inoculated into SD-CAA expansion medium (6.7 g YNB, 5 g casein amino acid, 13.62 g Na2HPO4·12H2O, 7.44 g NaH2PO4, and 2% glucose were added to 1 L of SD-CAA expansion medium), and the inoculated yeast cells were cultured overnight at 30°C and 225 rpm, with a cell number >10 times the library capacity (initial expansion concentration = 0.5 OD600 / mL). 10 times the library capacity of yeast cells were removed, centrifuged to remove the culture medium, and resuspended in SD-CAA induction medium. The concentration of the induced library was determined, and 10 times the library capacity of yeast cells were removed, centrifuged to remove the culture medium. The yeast cells were resuspended in 50 mL of washing buffer (PBS + 0.5% BSA + 2 mM EDTA), and centrifuged to remove the supernatant. The yeast cells were resuspended in 10 mL of washing buffer.

[0168] Biotin-labeled IL-4Rα protein (final concentration 100 nM) was added, and the mixture was incubated at room temperature for 30 min. The yeast cells were collected by centrifugation, and the yeast was washed 3 times with 50 mL of washing buffer. The yeast cells were resuspended in 5 mL of washing buffer, and 200 μL of SA magnetic beads (purchased from Miltenyi) were added. The mixture was incubated by inversion for 10 min. The yeast and magnetic bead mixture was washed 3 times with washing buffer, and then added to an LS column (purchased from Miltenyi). The LS column was placed on a magnetic stand, and the non-specifically bound yeast cells were removed by washing with washing buffer. The column was removed from the magnetic stand, and the yeast was eluted with washing buffer. The eluted yeast was centrifuged and transferred to 200 mL of SD-CAA expansion medium for expansion.

[0169] The yeast cells enriched by MACS (Magnetic Activated Cell Sorting) were inoculated in the SD-CAA expansion medium, and the initial expansion concentration was 0.5 OD600 / mL. The flask was cultured at 30°C, 225 rpm overnight. The yeast cells were resuspended with the induction medium and induced overnight. The anti-c-Myc mouse antibody (purchased from Thermo) diluted 1:200 and 100 nM biotin-labeled IL-4Rα antigen were added, and the mixture was incubated at room temperature for 10 min. The yeast cells were washed with PBS for 3 times, and then 1:500 diluted goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 (purchased from Invitrogen) and streptavidin APC conjugated fluorescent antibody (purchased from Invitrogen) were added, and the mixture was incubated at 4°C in the dark for 15 min. The cells were resuspended with PBS, and the BD FACSAria II instrument was used for sorting to obtain the yeast cells with high binding capacity to the IL-4Rα antigen.

[0170] The yeast cells with high binding capacity to the IL-4Rα antigen obtained by MACS and FACS enrichment were cultured in the SD-CAA expansion medium at 30°C, 225 rpm overnight. The yeast plasmid was extracted according to the yeast plasmid extraction kit (purchased from Tiangen). The plasmid was transformed into Top10 competent cells (purchased from Tiangen) by electroporation, and then coated on ampicillin-resistant plates and cultured at 37°C overnight. The single colonies were picked and sequenced to obtain the VHH gene sequence.

[0171] Thus, the anti-IL-4Rα single domain antibody VHH-005 was obtained, the amino acid sequence of which is shown as SEQ ID NO: 1, and according to the kabat numbering system, the amino acid sequences of the 3 CDRs contained therein are shown as SEQ ID NO: 2 to SEQ ID NO: 4, respectively.

[0172] Example 3: Humanization of the anti-IL-4Rα single domain antibody

[0173] Based on the anti-IL-4Rα single domain antibody VHH-005 (SEQ ID NO: 1) obtained in Example 2, humanization was performed to obtain the humanized anti-IL-4Rα single domain antibodies VHH-005-H1, VHH-005-H2, VHH-005-H3, VHH-005-H4, VHH-005-H5, VHH-005-H6 and VHH-005-H8, the amino acid sequences of which are shown as SEQ ID NO: 5 to SEQ ID NO: 11 in Table 1, respectively, and the amino acid sequences of the 3 CDRs are the same as VHH-005, which are shown as SEQ ID NO: 2 to SEQ ID NO: 4, respectively.

[0174] Example 4: Construction and expression of Fc fusion proteins of humanized anti-IL-4Rα single domain antibodies

[0175] The nucleotide sequences encoding VHH-005, VHH-005-H1, VHH-005-H2, VHH-005-H3, VHH-005-H4, VHH-005-H5, VHH-005-H6 and VHH-005-H8 were respectively connected with the nucleotide sequence encoding human hlgG1 heavy chain Fc (the sequence contains part of the hinge region, CH2 and CH3 regions of the heavy chain, and contains L234A / L235A mutations to reduce Fc effector function, SEQ ID NO: 17) using homologous recombination enzyme (purchased from Vazyme) to construct into EcoR I / Not I double enzyme linearized pcDNA3.1 vector, and the process was operated according to the product instruction. The homologous recombination product was transformed into Top10 competent cells, coated on ampicillin resistant plates, incubated at 37°C overnight, and single colonies were selected for sequencing.

[0176] ExpiCHO TM Expression system kit (purchased from Thermo), the plasmid was transformed into Expi-CHO cells, and the transfection method was according to the product instruction. The supernatant was collected after 5 days of cell culture, and the target protein was purified by protein A magnetic bead (purchased from Jin Sui) sorting method. The magnetic beads were resuspended (1-4 times the volume of the magnetic beads) with an appropriate volume of binding buffer (PBS+0.1% Tween 20, pH 7.4) and added to the sample to be purified, and incubated at room temperature for 1 hour with gentle shaking. The sample was placed on a magnetic stand (purchased from Suzhou Beaver Biology), and the supernatant was discarded. The magnetic beads were washed with binding buffer for 3 times. According to 3-5 times the volume of the magnetic beads, elution buffer (0.1M sodium citrate, pH 3.2) was added, and the mixture was shaken at room temperature for 5-10 min, and then placed on the magnetic stand. The elution buffer was collected and transferred to a collection tube containing neutralization buffer (1M Tris, pH 8.54) and mixed well to obtain VHH-Fc fusion protein, which was named VHH-005-Fc, VHH-005-H1-Fc, VHH-005-H2-Fc, VHH-005-H3-Fc, VHH-005-H4-Fc, VHH-005-H5-Fc, VHH-005-H6-Fc, and VHH-005-H8-Fc, respectively.

[0177] Example 5: Affinity determination of Fc fusion proteins of humanized anti-IL-4Rα single domain antibodies

[0178] ForteBio affinity assay was performed according to the existing method (Estep, P, et al. Solution-based high-throughput antibody-antigen affinity and epitope binning measurements. MAbs, 2013. 5(2): p. 270-8). Briefly, AHC sensors were equilibrated for 30 min under the line, then detected for 60 s under the line to establish a baseline, and then loaded with purified VHH-Fc fusion proteins obtained as described above onto the AHC sensor. The sensor was then placed in 100 nM IL-4Ra antigen for 150 s, after which the sensor was transferred to PBS for dissociation for 150 s. Kinetics were analyzed using a 1:1 binding model.

[0179] Table 2: Affinity of Fc fusion proteins of humanized anti-IL-4Ra single domain antibodies

[0180] The results are shown in Table 2.

[0181] The results show that the Fc fusion proteins VHH-005-H1-Fc, VHH-005-H2-Fc, VHH-005-H3-Fc, VHH-005-H4-Fc, VHH-005-H5-Fc and VHH-005-H6-Fc of the single domain antibodies of the present application all maintain monovalent affinity equivalent to VHH-005-Fc for binding to human IL-4Ra protein. The Fc fusion protein VHH-005-H8-Fc of the humanized single domain antibody has better monovalent affinity for binding to human IL-4Ra than VHH-005-Fc.

[0182] Example 6: Binding of Fc fusion proteins of humanized anti-IL-4Ra single domain antibodies to IL-4Ra overexpressing cells

[0183] The nucleotide sequence encoding the full-length human IL-4Rα amino acid sequence (SEQ ID NO: 21), the nucleotide sequence encoding the full-length rhesus IL-4Rα amino acid sequence (SEQ ID NO: 22) were inserted into the multiple cloning site of the PLVX-puro vector (purchased from Addgene) by EcoR I and Hind III double enzyme digestion, respectively. The 293T cells were used to package the lentivirus and infect the 293F cells, and the stable overexpression of human IL-4Rα 293F cells (293F-huIL-4Rα cells), overexpression of rhesus IL-4Rα 293F cells (293F-rhesusIL-4Rα cells) were obtained by adding puromycin (purchased from Invitrogen) to screen. The overexpressed cells expanded in culture were adjusted to the appropriate cell density in a 96-well flow cytometry plate, centrifuged, and then added with the test sample diluted by 4-fold gradient from 200 nM, incubated at 4°C for 30 min. Washed twice with 1×PBS, added with Goat F(ab')2 Anti-Human IgG-Fc (PE) (purchased from abcam) diluted 200 times with 1×PBS, incubated at 4°C for 30 min. Washed twice with 1×PBS, and then added with 1×PBS to resuspend the cells, and then detected by CytoFlex flow cytometry and calculated the median fluorescence intensity.

[0184] Table 3: Fc fusion protein of humanized anti-IL-4Rα single-domain antibody binding or blocking IL-4Rα overexpression cell activity

[0185] The results are shown in FIG. 1A and Table 3. The results show that the binding activity of humanized molecules VHH-005-H1-Fc, VHH-005-H2-Fc, VHH-005-H3-Fc, VHH-005-H4-Fc, VHH-005-H5-Fc, VHH-005-H6-Fc and VHH-005-H8-Fc to 293F-huIL-4Rα cells is significantly better than that of the molecule VHH-005-Fc before humanization, and VHH-005-H8-Fc performs best.

[0186] The results are shown in FIG. 1B and Table 3. The results show that the binding activity of humanized molecules VHH-005-H1-Fc, VHH-005-H2-Fc, VHH-005-H3-Fc, VHH-005-H4-Fc, VHH-005-H5-Fc, VHH-005-H6-Fc and VHH-005-H8-Fc to 293F-rhesusIL-4Rα cells is significantly better than that of the molecule VHH-005-Fc before humanization, and VHH-005-H8-Fc performs best.

[0187] Example 7: Fc fusion proteins of humanized anti-IL-4Rα single-domain antibody block IL-4 binding to IL-4Rα overexpressing cells

[0188] The expanded 293F-huIL-4Rα cells were adjusted to a cell density of 2 x 10 6 The cells were centrifuged and resuspended in 1 x PBS at a cell density of 2 x 10

[0189] The results are shown in Figure 2 and Table 3.

[0190] The results show that the humanized molecules VHH-005-H1-Fc, VHH-005-H2-Fc, VHH-005-H3-Fc, VHH-005-H4-Fc, VHH-005-H5-Fc, VHH-005-H6-Fc and VHH-005-H8-Fc have significantly better blocking activity on the binding of human IL-4 protein to 293F-huIL-4Rα cells than the molecule VHH-005-Fc before humanization, and VHH-005-H8-Fc performs best.

[0191] Example 8: Fc fusion proteins of humanized anti-IL-4Rα single-domain antibody block IL-4 / IL-13 induced downstream signal STAT6 phosphorylation

[0192] The trypsin-digested HEK-blue IL-4 / IL-13 reporter cells (purchased from Invivogen) were washed once with 50 mL of PBS. The cells were resuspended in DMEM medium containing 10% inactivated FBS, and the cell density was adjusted to 5 x 10 5The HEK-blue IL-4 / IL-13 cells were diluted to 2 x 105cells / mL, 100 μL / well added into a 96-well flat-bottom plate. The test samples were diluted with DMEM medium containing 10% inactivated FBS, 4-fold gradient dilution starting from 40 nM, then 50 μL / well added into the above 96-well flat-bottom plate. The human IL-13 protein (purchased from Acro) and human IL-4 protein (purchased from Acro) were diluted with DMEM medium containing 10% inactivated FBS, respectively, 50 μL / well added into the above 96-well flat-bottom plate. Incubated overnight at 37°C in a 5% CO2incubator. The next day, a new 96-well flat-bottom plate was prepared, 180 μL / well QUANTI-Blue Solution (purchased from Invivogen) was added, and 20 μL / well of the supernatant of the above overnight incubation was added. Incubated for 4 h at 37°C in the dark, and the absorbance value at 620-655 nm was read on a microplate reader.

[0193] Table 4: Fc fusion proteins of humanized anti-IL-4Rα single-domain antibodies block IL-4 / IL-13-induced functional activity

[0194] The results are shown in FIGS. 3A-3B and Table 4.

[0195] The results show that the humanized molecules VHH-005-H1-Fc, VHH-005-H2-Fc, VHH-005-H3-Fc, VHH-005-H4-Fc, VHH-005-H5-Fc, and VHH-005-H6-Fc have a blocking activity on the phosphorylation signal of human IL-4 / human IL-13-induced intracellular STAT6 of HEK-blue IL-4 / IL-13 cells that is equivalent to that of VHH-005-Fc. VHH-005-H8-Fc has a significantly better blocking activity on the phosphorylation activity of human IL-4 / human IL-13-induced intracellular STAT6 of HEK-blue IL-4 / IL-13 cells than VHH-005-Fc.

[0196] Example 9: Fc fusion proteins of humanized anti-IL-4Rα single-domain antibodies block IL-4 / IL-13-induced proliferation of TF-1 cells

[0197] The expanded TF-1 cells (purchased from ATCC) were centrifuged to remove the supernatant, washed once with 50 mL of PBS, resuspended with 10% FBS-containing RPMI1640, and adjusted to a cell density of 2 x 105cells / mL. 100 μL / well was added into a 96-well flat-bottom plate. The test samples were diluted with DMEM medium containing 10% inactivated FBS, 4-fold gradient dilution starting from 40 nM, then 50 μL / well added into the above 96-well flat-bottom plate. The human IL-13 protein (purchased from Acro) and human IL-4 protein (purchased from Acro) were diluted with DMEM medium containing 10% inactivated FBS, respectively, 50 μL / well added into the above 96-well flat-bottom plate. Incubated overnight at 37°C in a 5% CO2incubator. The next day, a new 96-well flat-bottom plate was prepared, 180 μL / well QUANTI-Blue Solution (purchased from Invivogen) was added, and 20 μL / well of the supernatant of the above overnight incubation was added. Incubated for 4 h at 37°C in the dark, and the absorbance value at 620-655 nm was read on a microplate reader. 5The cells were inoculated in a 96-well cell culture flat-bottom plate at a density of 1 x 104cells / mL, 100 μL / well. The samples were diluted in RPMI1640 medium containing 10% FBS by gradient dilution, starting from 200 nM, and then 50 μL / well was added to the 96-well cell plate. Human IL-4 protein (purchased from Acro) and human IL-13 protein (purchased from Novoprotein) were diluted in RPMI1640 medium containing 10% FBS, respectively, and then 50 μL / well was added to the 96-well cell plate. Incubation was performed at 37°C in a 5% CO2 incubator for 3 days. 20 μL of CCK8 solution (purchased from Dojindo) was added to each well, and after incubation at room temperature for 8 h, the absorbance value at 450 nm was detected.

[0198] The results are shown in FIGS. 4A to 4B and Table 4.

[0199] The results show that the humanized molecules VHH-005-H1-Fc, VHH-005-H2-Fc, VHH-005-H3-Fc, VHH-005-H4-Fc, VHH-005-H5-Fc, VHH-005-H6-Fc and VHH-005-H8-Fc have equivalent or better blocking activity on the proliferation of TF-1 cells induced by human IL-4 and human IL-13 than the molecule VHH-005-Fc before humanization. Among them, VHH-005-H8-Fc performs best, and the blocking activity of IL-4 and IL-13 induced TF-1 cell proliferation is increased by 2.7 times and 3.0 times, respectively, compared with VHH-005-Fc.

[0200] Example 10: Construction and expression and purification of Fc fusion proteins of tetravalent humanized anti-IL-4Rα single-domain antibody

[0201] Using the humanized antibody VHH-005-H8, four tetravalent VHH-Fc fusion proteins with structures shown in FIGS. 5A to 5D were constructed. The steps are as follows:

[0202] The nucleotide sequences of the peptide segments H8-H8-Fc (SEQ ID NO: 12), H8-Fc-H8 (SEQ ID NO: 13), Fc-H8-H8 (SEQ ID NO: 14), H8-IgG-HC (SEQ ID NO: 15) and H8-IgG-LC (SEQ ID NO: 16) were synthesized by total gene synthesis, and the nucleotide sequences of these peptide segments were constructed into pcDNA3.1 expression frame by molecular cloning technology. The pcDNA3.1 plasmids containing the above sequences were linearized by enzyme digestion, and then transfected into 293 expression system for conventional expression. The proteins shown in FIGS. 5A-5D were obtained by the same purification protocol as in Example 4, and were named as fusion protein H8-H8-Fc, fusion protein H8-Fc-H8, fusion protein Fc-H8-H8 and fusion protein H8-IgG, respectively, and were abbreviated as H8-H8-Fc, H8-Fc-H8, Fc-H8-H8 and H8-IgG, respectively.

[0203] wherein:

[0204] The fusion protein H8-H8-Fc is assembled from two H8-H8-Fc peptide segments by two pairs of disulfide bonds located in the hinge region;

[0205] The fusion protein H8-Fc-H8 is assembled from two H8-Fc-H8 peptide segments by two pairs of disulfide bonds located in the hinge region;

[0206] The fusion protein Fc-H8-H8 is assembled from two Fc-H8-H8 peptide segments by two pairs of disulfide bonds located in the hinge region;

[0207] The fusion protein H8-IgG is assembled from two H8-IgG-HC peptide segments and two H8-IgG-LC peptide segments by two pairs of disulfide bonds located in the heavy chain hinge region and two pairs of disulfide bonds between CH1-CL (one pair of disulfide bonds between each CH1 and the corresponding CL).

[0208] Example 11: Affinity determination of Fc fusion protein of tetravalent humanized anti-IL-4Rα single-domain antibody

[0209] ForteBio affinity assay was performed according to the existing method (Estep, P, et al. Solution-based high-throughput antibody-antigen affinity and epitope ranking measurements. MAbs, 2013. 5(2): p. 270-8). Briefly, AHC sensors were equilibrated for 30 min under the line, then detected for 60 s under the line to establish the baseline, and then the purified samples obtained as described above were loaded onto the AHC sensor. The sensor was then placed in 100 nM IL-4Rα antigen for 200 s, and then the sensor was transferred to PBS for dissociation for 2000 s. The kinetics were analyzed using a 1:1 binding model.

[0210] IL-4Rα control antibody: English generic name Dupilumab, Chinese generic name Dupilimumab, trade name Dupixent, purchased from Sanofi.

[0211] Table 5: Affinity of Fc fusion protein of tetravalent humanized anti-IL-4Rα single domain antibody

[0212] The results are shown in Table 5.

[0213] The results show that the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single domain antibody of the application maintains a high affinity for monovalent binding of human IL-4Rα protein, in which H8-Fc-H8, Fc-H8-H8, H8-IgG is equivalent to the control antibody Dupilumab.

[0214] Example 12: Fc fusion protein of tetravalent humanized anti-IL-4Rα single domain antibody blocks IL-4 binding to IL-4Rα overexpressing cells

[0215] The 293F-huIL-4Rα cells were adjusted to a cell density of 2×10 6 The cells were centrifuged and stored for use. The purified protein was diluted with 1×PBS, and 3-fold gradient dilution was performed starting from 400 nM. The diluted sample was added to the 96-well flow plate with cells at 60 μL / well, and incubated at 4°C for 30 min. Then 60 μL / well of biotinylated human IL-4 protein (Acro) diluted with 1×PBS was added, and incubated at 4°C for 30 min. 1×PBS was washed twice, 100 μL / well of SAPE antibody (Thermo) diluted 100 times with 1×PBS was added, and incubated at 4°C for 30 min. 1×PBS was washed twice, 100 μL / well of 1×PBS resuspended cells was added, and detected on a CytoFlex flow cytometer and calculated the corresponding Mean Fluorescence Intensity (MFI).

[0216] The expanded 293F-rhesusIL-4Rα cells were adjusted to a cell density of 2 x 10 6 Cells / mL, 100 μL / well were added to a 96-well flow plate and centrifuged for later use. The purified protein was diluted with 1 x PBS, and 3-fold gradient dilution was performed starting from 400 nM. The diluted sample was added to the above-mentioned 96-well flow plate with cells at 60 μL / well, and incubated at 4°C for 30 minutes. Then 60 μL / well of Mouse IgG2a Fc-tagged Rhesus IL-4 protein (Biotheus synthetic) diluted with 1 x PBS was added, and incubated at 4°C for 30 minutes. Twice washing with 1 x PBS, 100 μL / well of APC anti-mouse IgG antibody (Biolegend) diluted 200 times with 1 x PBS was added, and incubated at 4°C for 30 minutes. Twice washing with 1 x PBS, 100 μL / well of cells resuspended with 1 x PBS was added, and detected on a CytoFlex flow cytometer and calculated the corresponding MFI.

[0217] Table 6: Blocking activity of Fc fusion proteins of tetravalent humanized anti-IL-4Rα single domain antibodies on the binding of IL-4 to IL-4Rα overexpressed cells

[0218] The results are shown in FIG. 6A, Table 6. The results show that the blocking activity of Fc fusion proteins of tetravalent humanized anti-IL-4Rα single domain antibodies H8-H8-Fc, H8-Fc-H8, Fc-H8-H8, H8-IgG on the binding of human IL-4 protein to 293F-huIL-4Rα cells is equivalent or superior to that of the control antibody Dupilumab.

[0219] The results are shown in FIG. 6B, Table 6. The results show that the blocking activity of Fc fusion proteins of tetravalent humanized anti-IL-4Rα single domain antibodies H8-H8-Fc, H8-Fc-H8, Fc-H8-H8, H8-IgG on the binding of monkey IL-4 protein to 293F-rhesusIL-4Rα cells is significantly superior to that of the control antibody Dupilumab.

[0220] Example 13: Blocking activity of Fc fusion proteins of tetravalent humanized anti-IL-4Rα single domain antibodies on IL-4 / IL-13 induced downstream signal STAT6 phosphorylation

[0221] The experimental method is referred to Example 8, and the experimental results are shown in FIGS. 7A to 7B, Table 7.

[0222] Table 7: Blocking activity of Fc fusion proteins of tetravalent humanized anti-IL-4Rα single domain antibodies on IL-4 / IL-13 induced functional activity

[0223] The results show that the blocking activity of the Fc fusion proteins of the four tetravalent humanized anti-IL-4R alpha single domain antibodies of the application on the phosphorylation signal of intracellular STAT6 of HEK-blue IL-4 / IL-13 cells induced by human IL-4 and human IL-13 is basically equivalent to that of the control antibody Dupilumab. Among them, H8-Fc-H8, Fc-H8-H8 and H8-IgG show activities slightly better than the control antibody Dupilumab.

[0224] Example 14: Fc fusion proteins of tetravalent humanized anti-IL-4R alpha single domain antibodies block IL-4 / IL-13-induced proliferation of TF-1 cells

[0225] The experimental method is referred to Example 9, and the experimental results are shown in FIGS. 8A to 8B and Table 7.

[0226] The results show that the blocking activity of the Fc fusion proteins of the four tetravalent humanized anti-IL-4R alpha single domain antibodies of the application on the phosphorylation signal of intracellular STAT6 of HEK-blue IL-4 / IL-13 cells induced by human IL-4 and human IL-13 is basically equivalent to that of the control antibody Dupilumab. Among them, H8-Fc-H8, Fc-H8-H8 and H8-IgG show activities slightly better than the control antibody Dupilumab.

[0227] Example 15: Fc fusion proteins of tetravalent humanized anti-IL-4R alpha single domain antibodies block IL-4 / IL-13-induced B cell CD23 expression

[0228] The fresh PBMC cells stored in liquid nitrogen were thawed and centrifuged to remove the supernatant. Resuspend in X-VIVO15 medium and adjust the cell density to 4x10 6The sample was diluted with X-VIVO 15 medium in a gradient, starting from 400 nM, and then 50 μL / well was added to the above 96-well cell plate. Human IL-4 protein (ACRO) and human IL-13 protein (ACRO) were diluted with X-VIVO 15 medium, respectively, and 50 μL / well was added to the above 96-well cell plate. Incubation was carried out at 37°C in a 5% CO2 incubator for 2 days. The cells in the above culture plate were mixed by blowing and all the liquid was transferred to a new 96-well U-bottom plate, centrifuged, and the supernatant was discarded. 100 μL / well of 1% FBS-containing 1xPBS was added and mixed, and incubated at 4°C for 20 min. Each well was washed once with 100 μL of 1xPBS, centrifuged, and the supernatant was discarded. 100 μL / well of 1xPBS-diluted Anti-huCD19 (purchased from Biolegend) and Anti-huCD23 (purchased from Biolegend) fluorescent antibodies were added, and incubated at 4°C in the dark for 20 min. Washed twice with 1xPBS, and then 100 μL / well of 1xPBS was added to resuspend the cells, and detection was performed on a CytoFlex flow cytometer.

[0229] The results are shown in FIGS. 9A-9B.

[0230] The results show that the Fc fusion protein Fc-H8-H8 (IC 50 = 22.87 nM) of the tetravalent humanized anti-IL-4Rα single-domain antibody of the present application has a blocking activity on human IL-4-induced B cell expression of CD23 that is equivalent to that of the control antibody Dupilumab (IC 50 = 23.28 nM), and the Fc-H8-H8 (IC 50 = 4.259 nM) has a blocking activity on human IL-13-induced B cell expression of CD23 that is equivalent to that of the control antibody Dupilumab (IC 50 = 3.377 nM).

[0231] Example 16: Pharmacodynamics of the Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody in an ovalbumin-induced mouse asthma model

[0232] An ovalbumin (purchased from Sigma)-induced asthma model of huIL4 / huIL4RαKI C57BL / 6 mice (purchased from Biocytogen) was used to detect the pharmacodynamics of the Fc-H8-H8 Fc fusion protein of the tetravalent humanized anti-IL-4Rα single-domain antibody of the present application.

[0233] Table 8: Test scheme for pharmacodynamics in an ovalbumin-induced mouse asthma model

[0234] First, the mice were sensitized by subcutaneous injection of 0.2 mL of 500 μg / mL ovalbumin at multiple points. Each group had 6-7 mice, and the injection was performed once a week for a total of three weeks. The day before the aerosol challenge, the mice were injected intraperitoneally with an equal volume of the antibody to be tested. In the fourth week, the mice were challenged with aerosolized ovalbumin at a concentration of 2.5% m / v, and the challenge was performed for 6 consecutive days. On the seventh day, the mice were anesthetized, the trachea was exposed from the neck, and an equal volume of pre-cooled PBS was used to perform lung lavage and collect the lung lavage fluid. After centrifugation of the lung lavage fluid, the supernatant was stored at -80°C for later use. The cell pellet after centrifugation was used for flow cytometry to detect the number of CD45-positive immune cells and eosinophils. The mouse blood was collected by orbital bleeding, and the serum was collected after centrifugation and stored at -80°C for later use. At the same time, the lung tissue was collected and fixed with 4% paraformaldehyde, and H&E staining was performed after fixation. The collected mouse serum and lung lavage fluid supernatant were used to determine the IgE content in the mouse serum and lung lavage fluid using an ELISA kit (purchased from Thermo).

[0235] The results of flow cytometry are shown in FIGS. 10A-10C. The results show that, compared with the OVA model group, the number of CD45+ leukocytes in the lung lavage fluid of the antibody Fc-H8-H8 administration group was significantly reduced, and the number and proportion of eosinophils were also significantly reduced, and the number and proportion of cell reduction were comparable to the control antibody Dupilumab.

[0236] The results of ELISA detection are shown in FIGS. 10D-10E. The results show that, compared with the OVA model group, the total IgE concentration in the lung lavage fluid and serum of the antibody Fc-H8-H8 administration group was significantly reduced, and the degree of IgE content reduction was comparable to the control antibody Dupilumab, close to the blank control group.

[0237] The results of pathological staining are shown in FIG. 10F. The results show that, compared with the OVA model group, the inflammatory cell infiltration around the bronchi in the lung tissue of the antibody Fc-H8-H8 administration group was significantly reduced, and the production of mucus near the bronchi was also significantly reduced. From the pathological tissue staining, it can be seen that the antibody Fc-H8-H8 administration group has a pathological change in the mouse lung tissue comparable to the control antibody Dupilumab.

[0238] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details in accordance with all the teachings disclosed herein, and such changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. An anti-IL-4Rα single-domain antibody or its antigen-binding fragment, comprising at least one heavy chain variable region, wherein the heavy chain variable region comprises three complementarity-determining regions CDR1-CDR3, wherein: The amino acid sequence of CDR1 is shown in SEQ ID NO:

2. The amino acid sequence of CDR2 is shown in SEQ ID NO:3, and The amino acid sequence of CDR3 is shown in SEQ ID NO:

4.

2. The anti-IL-4Rα single-domain antibody or its antigen-binding fragment according to claim 1, wherein, Humanization of any one, two, three, or all four framework regions of the anti-IL-4Rα single-domain antibody; Preferably, the amino acid sequence of the anti-IL-4Rα single-domain antibody is shown in any one of SEQ ID NO:11, SEQ ID NO:1 and SEQ ID NOs:5-10.

3. The anti-IL-4Rα single-domain antibody or its antigen-binding fragment according to any one of claims 1 to 2, wherein, EC50 of the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof binding to 293F cells stably overexpressing human IL-4Ra and to 293F cells overexpressing rhesus monkey IL-4Ra 50 less than 5 nM, preferably less than 4 nM; preferably, the EC50 of the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof binding to 293F cells stably overexpressing human IL-4Ra and to 293F cells overexpressing rhesus monkey IL-4Ra is less than 5 nM, preferably less than 4 nM 50 determined by flow cytometry and calculated; and / or The anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof has an IC50 of less than 3.2 nM, preferably less than 2.5 nM or less than 2 nM for blocking the binding of human IL-4 protein to 293F cells stably overexpressing human IL-4Ra 50 less than 3.2 nM, preferably less than 2.5 nM or less than 2 nM; preferably, the IC50 of the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof for blocking the binding of human IL-4 protein to 293F cells stably overexpressing human IL-4Ra is less than 2 nM 50 The mean fluorescence intensity is determined by flow cytometry and calculated.

4. A fusion protein comprising one or more copies of the anti-IL-4Rα single-domain antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 3; Preferably, the fusion protein further comprises the Fc segment of human IgG, the heavy chain constant region of human IgG, the light chain constant region of human Kappa, or the light chain constant region of human Lambda. Preferably, the Fc segment of the human IgG is the Fc segment of human IgG1, human IgG2, human IgG3, or human IgG4; Preferably, the Fc segment of the human IgG is the Fc segment of human IgG1, and according to the EU numbering system, it contains L234A and L235A mutations; Preferably, the amino acid sequence of the Fc segment of the human IgG is as shown in SEQ ID NO:17 or SEQ ID NO:23; Preferably, the amino acid sequence of the fusion protein is as shown in any one of SEQ ID NO:25-32; Preferably, the heavy chain constant region of human IgG is the heavy chain constant region of human IgG1, human IgG2, human IgG3 or human IgG4; Preferably, the heavy chain constant region of the human IgG is the heavy chain constant region of human IgG1, and contains L234A and L235A mutations according to the EU numbering system; Preferably, the amino acid sequence of the heavy chain constant region of the human IgG is as shown in SEQ ID NO:18 or SEQ ID NO:24; Preferably, the amino acid sequence of the constant region of the human Kappa light chain is shown in SEQ ID NO:19; Preferably, the anti-IL-4Rα single-domain antibody or its antigen-binding fragment is directly linked to the Fc region of human IgG, the heavy chain constant region of human IgG, the light chain constant region of human Kappa, or the light chain constant region of human Lambda, or linked through a first linker. Preferably, when the anti-IL-4Rα single-domain antibody or its antigen-binding fragment is in multiple copies, the copies are directly linked or linked through a second linker; Preferably, the amino acid sequence of the first or second linker is independently as set forth in SEQ ID NO:

20.

5. The fusion protein of claim 4, comprising a peptide chain, which is one or two copies, comprising an anti-IL-4Ra single domain antibody or antigen binding fragment thereof, and an Fc fragment of human IgG1.

6. The fusion protein of claim 5, wherein, The peptide chain comprises the following elements in order from N-terminus to C-terminus: an anti-IL-4Ra single domain antibody or antigen binding fragment thereof, an Fc fragment of human IgG1, and an anti-IL-4Ra single domain antibody or antigen binding fragment thereof; an anti-IL-4Ra single domain antibody or antigen binding fragment thereof, an Fc fragment of human IgG1, and an anti-IL-4Ra single domain antibody or antigen binding fragment thereof; an Fc fragment of human IgG1, an anti-IL-4Ra single domain antibody or antigen binding fragment thereof, and an anti-IL-4Ra single domain antibody or antigen binding fragment thereof; wherein, the first and second anti-IL-4Ra single domain antibody or antigen binding fragment thereof are the same or different; Preferably, the first and second anti-IL-4Ra single domain antibody or antigen binding fragment thereof are independently selected from the anti-IL-4Ra single domain antibody or antigen binding fragment thereof of any one of claims 1 to 3; preferably, independently selected from the anti-IL-4Ra single domain antibody or antigen binding fragment thereof having an amino acid sequence as set forth in any one of SEQ ID NOs: 1 and 5-11; preferably, the amino acid sequence of the first and second anti-IL-4Ra single domain antibody is as set forth in SEQ ID NO: 11; Preferably, each element is directly connected or connected via a linker; Preferably, the Fc fragment of human IgG1 contains L234A and L235A mutations according to the EU numbering system; Preferably, the Fc fragment of human IgG has an amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 23; Preferably, the amino acid sequence of the linker is as set forth in SEQ ID NO:

20.

7. The fusion protein of claim 4, comprising a peptide chain having an amino acid sequence as set forth in any one of SEQ ID NO: 12 to SEQ ID NO:

16. Preferably, the fusion protein is a dimer of the peptide chain as set forth in any one of SEQ ID NO: 12 to SEQ ID NO: 14; preferably, a homodimer; preferably, the two peptide chains of the dimer are connected via one or more pairs of disulfide bonds; Preferably, the fusion protein is an immunoglobulin-form fusion protein formed by two peptide chains as set forth in SEQ ID NO: 15 and two peptide chains as set forth in SEQ ID NO:

16.

8. The fusion protein of claim 5, comprising a peptide chain as set forth in formula (I), formula (II), formula (III), and formula (IV): VHH-CL formula (I), VHH-CH Formula (II), VHH-CH Formula (III); and VHH-CL Formula (IV); wherein, Formula (I), (II), (III), (IV) are in the order from left to right, i.e. from N-terminus to C-terminus, respectively; VHH represents the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 3; VHH in Formula (I) to Formula (IV) are the same or different; preferably, the amino acid sequences of the VHHs are all as set forth in SEQ ID NO: 11; CH represents the heavy chain constant region of human IgG; CL represents the kappa light chain constant region of human; VHH is directly connected to CH or connected through a linker, and VHH is directly connected to CL or connected through a linker; preferably, the amino acid sequence of the linker is as set forth in SEQ ID NO: 20; Preferably, the amino acid sequences of Formula (I) and Formula (IV) are the same, and / or the amino acid sequences of Formula (II) and Formula (III) are the same.

9. The fusion protein of claim 8, wherein, the heavy chain constant region of human IgG is the heavy chain constant region of human IgG1, human IgG2, human IgG3 or human IgG4; Preferably, the heavy chain constant region of human IgG is the heavy chain constant region of human IgG1, and contains L234A and L235A mutations according to the EU numbering system; Preferably, the amino acid sequence of the heavy chain constant region of human IgG is as set forth in SEQ ID NO: 18 or SEQ ID NO: 24; Preferably, the amino acid sequence of the kappa light chain constant region of human is as set forth in SEQ ID NO:

19.

10. The fusion protein of any one of claims 8 to 9, wherein, Formula (I) and Formula (II), Formula (II) and Formula (III), and Formula (III) and Formula (IV) are connected through one or more pairs of disulfide bonds; Preferably, the amino acid sequences of the peptide chains shown in Formula (I) and Formula (IV) are both as set forth in SEQ ID NO: 16, and the amino acid sequences of the peptide chains shown in Formula (II) and Formula (III) are both as set forth in SEQ ID NO:

15.

11. The fusion protein of any one of claims 4 to 10, wherein, The fusion protein blocks the binding of human IL-4 protein to 293F cells stably overexpressing human IL-4Rα with an IC 50 less than 1 nM; preferably, the IC 50 The mean fluorescence intensity is determined by flow cytometry and calculated; and / or The fusion protein has an IC50 of less than 2 nM, preferably less than 1 nM, less than 0.5 nM or less than 0.4 nM; preferably, the IC50 is less than 0.2 nM. 50 The fusion protein has an IC50 of less than 2 nM, preferably less than 1 nM, less than 0.5 nM or less than 0.4 nM; preferably, the IC50 is less than 0.2 nM. 50 The mean fluorescence intensity is determined by flow cytometry and calculated.

12. An isolated nucleic acid molecule encoding the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 3 or the fusion protein as claimed in any one of claims 4 to 11.

13. A recombinant vector comprising the isolated nucleic acid molecule of claim 12.

14. A recombinant host cell comprising the isolated nucleic acid molecule of claim 12 or comprising the recombinant vector of claim 13.

15. An antibody drug conjugate comprising an antibody moiety and a small molecule drug, wherein, the antibody moiety is the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 3 or the fusion protein as claimed in any one of claims 4 to 11; Preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapeutic drug.

16. The antibody drug conjugate of claim 15, wherein, the antibody moiety is linked to the small molecule drug via a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond; Preferably, the molar ratio of the antibody moiety to the small molecule drug is 1:(2-4).

17. A pharmaceutical composition comprising an effective amount of the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof of any one of claims 1 to 3 or the fusion protein of any one of claims 4 to 11, and one or more pharmaceutically acceptable excipients.

18. Use of the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof of any one of claims 1 to 3 or the fusion protein of any one of claims 4 to 11 in the manufacture of a medicament for treating or preventing a disease caused by type II inflammation. Preferably, the disease caused by type II inflammation is one or more selected from the group consisting of atopic dermatitis, type II inflammation asthma, allergic rhinitis, chronic sinusitis with nasal polyps, eosinophilic esophagitis, chronic obstructive pulmonary disease, chronic spontaneous urticaria and nodular prurigo.

19. The anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof of any one of claims 1 to 3 or the fusion protein of any one of claims 4 to 11 for use in treating or preventing a disease caused by type II inflammation. Preferably, the disease caused by type II inflammation is one or more selected from the group consisting of atopic dermatitis, type II inflammation asthma, allergic rhinitis, chronic sinusitis with nasal polyps, eosinophilic esophagitis, chronic obstructive pulmonary disease, chronic spontaneous urticaria and nodular prurigo.

20. A method of treating or preventing a disease caused by type II inflammation, comprising the step of administering to a subject in need thereof an effective amount of the anti-IL-4Ra single-domain antibody or antigen-binding fragment thereof of any one of claims 1 to 3 or the fusion protein of any one of claims 4 to 11. Preferably, the disease caused by type II inflammation is one or more selected from the group consisting of atopic dermatitis, type II inflammation asthma, allergic rhinitis, chronic sinusitis with nasal polyps, eosinophilic esophagitis, chronic obstructive pulmonary disease, chronic spontaneous urticaria and nodular prurigo.

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