Use of Anti-il-25 antibody in treatment or prevention of asthma

Abstract

The present invention relates to the use of an anti-IL-25 antibody in the treatment or prevention of asthma.

Description

Use of anti-il-25 antibodies in the treatment or prevention of asthma

[0001] Cross-reference to Related Applications

[0002] This application claims priority to the invention patent application with the application date of October 31, 2024, the application number of CN202411547150.4, and the invention name of "Use of anti-il-25 antibodies in the treatment or prevention of asthma", the complete content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present invention relates to the use of antibodies that specifically bind interleukin-25 (IL-25) in the treatment or prevention of asthma. BACKGROUND

[0004] IL-25 is a pleiotropic cytokine, also known as IL-17E, which is one of the IL-17 family members, and is mainly expressed and secreted by epithelial cells, Th2 cells, mast cells, eosinophils, basophils, macrophages and other cells of various tissues in pathological conditions. IL-25 binds to the receptor heterodimer IL-17RA / IL-17RB to activate and up-regulate transcription factors NF-κB, STAT6, GATA3 and NFATC1, thereby activating memory Th2 cells. On the other hand, IL-25 inhibits Th1 / Th17-related transcription factors such as T-bet and Stat4, reduces the secretion of tumor necrosis factor-α, interferon-γ and IL-17A, and breaks the balance of Th1 / Th2 immune response, further exacerbating the trend of Th2 immune response. In addition to regulating Th2 cells, IL-25 also directly acts on ILC2, Th9 cells; directly regulates eosinophils, basophils, macrophages, NKT cells and epithelial cells, etc., so it is not limited to type 2 inflammatory response.

[0005] Asthma patients with high IL-25 levels have severe eosinophilic airway inflammation, more obvious epithelial fibrosis, greater basement membrane thickness, more mucus secretion, and higher IgE levels in local tissues and peripheral blood, as found by blood, bronchoscopy, and biopsy of the lower respiratory tract. It shows that the plasma IL-25 level can reflect the level of IL-25 produced by epithelial cells and the condition of eosinophilic infiltration in the airway. Asthma is a highly heterogeneous disease, and patients differ in their response to different immunotherapy. Some patients do not respond well to all existing biological targeted therapies, and new mechanism drugs are needed to meet this clinical need. IL-25 is highly expressed in allergic asthma and is upstream of the inflammatory cascade, so it may play a more prominent role in allergic asthma. IL-25 monoclonal antibodies will provide new options for asthma patients. Currently, there is only one pharmaceutical company pipeline in the clinical phase I study of asthma indications for anti-IL-25 antibodies. No other drugs in the clinical trial stage, and no anti-IL-25 nanobodies have entered the clinical trial stage or preclinical research. The present application is the first anti-IL-25 nanobody drug, which will provide new options for asthma patients.

[0006] House dust mite (HDM) continuous intranasal administration causes significant airway eosinophilic inflammation and airway hyperresponsiveness. The clinical asthma patient has a high allergy rate of up to 85% to HDM, and the use of HDM to induce mouse asthma can better simulate the occurrence process of human allergic asthma. The present application applies house dust mite to induce a mouse model to test the therapeutic effect of anti-IL-25 antibodies on asthma. SUMMARY

[0007] The present application provides the use of anti-IL-25 single-domain antibodies and derivatives thereof in the treatment or prevention of asthma.

[0008] The present application provides an antibody that specifically binds to IL-25.

[0009] The term "antibody" is used in the broadest sense, and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0010] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure.

[0011] "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG class antibodies are heterogeneous, heterotetrameric glycoproteins of about 150,000 Daltons, composed of two light chains and two heavy chains that are linked by disulfide bonds. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region.

[0012] The heavy chain of an antibody can be assigned to one of five types, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), with some of these types further divided into subclasses, e.g., gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), gamma 4 (IgG4), alpha 1 (IgA1), and alpha 2 (IgA2).

[0013] The light chain of an antibody can be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequences of their constant domains.

[0014] "Antibody fragments" refer to molecules other than intact antibodies that comprise a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g. scFv); and single-domain antibodies.

[0015] Single-domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single-domain antibodies were derived from the variable domain of heavy chain antibodies from Camelidae (nanobodies or VHHs). H H fragments).

[0016] "Specifically binds" means binding that is selective for an antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind a particular antigen can be measured via enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one skilled in the art, such as surface plasmon resonance (SPR) techniques (analysis on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antibody to an unrelated protein is less than about 10% of the binding of the antibody to the antigen, as measured by, e.g., SPR.

[0017] "Affinity" or "binding affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, "binding affinity" refers to the intrinsic binding affinity reflecting 1 : 1 interactions between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ), which is the ratio of the dissociation and association rate constants (k off and k on , respectively). Thus, equivalent affinities can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. One particular method for measuring affinity is surface plasmon resonance (SPR).

[0018] In one embodiment, the antibody has a binding affinity with a K D value of 5.0 x 10 -8 M or less, 1.0 x 10 -8 M or less, 5.0 x 10 -9 M or less, 1.0 x 10 -9 M or less, 5.0 x 10 -10 M or less, 1.0 x 10 -10 M or less, 5.0 x 10 -11 M or less, 1.0 x 10 -11 M or less, 5.0 x 10 -8 M to 1.0 x 10 -13 M.

[0019] In one embodiment, the antibody of the application is a monoclonal antibody.

[0020] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope except for possible variants that can arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0021] In one embodiment, the antibody of the application is a chimeric antibody. In one embodiment, the variable region of the chimeric antibody is from a llama and the constant region of the chimeric antibody is from a human.

[0022] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portion of the heavy and / or light chain is derived from a different source or species.

[0023] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T.J. et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using a VH or VL domain from that antibody to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano, S. et al., J. Immunol. 150:880-887 (1993); Clarkson, T. et al., Nature 352:624-628 (1991)).

[0024] "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue Cys226, or Pro230, to the carboxy-terminus of the heavy chain. However, antibodies generated by host cells can undergo post-translational cleavage, with one or more, and particularly one or two, amino acids being excised from the C-terminus of the heavy chain. Thus, an antibody generated by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain can include a full-length heavy chain, or it can include a cleaved variant of the full-length heavy chain (also referred to as a "cleaved variant heavy chain"). This can be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447) of an Fc region can or can not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. A "subunit" of an Fc region refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable association with itself. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0025] In one embodiment, an antibody of the application is a humanized antibody.

[0026] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues both from a non-human HVR and from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, a humanized antibody can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0027] The term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts"). Generally, antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). Exemplary HVRs herein include:

[0028] (a) the hypervariable loops which occur at amino acid residues 26-32 (LI), 50- 52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. MoI. Biol. 196:901-917 (1987));

[0029] (b) the CDRs which occur at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (HI), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0030] (c) the antigen contacts which occur at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (HI), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. MoI. Biol. 262:732-745 (1996)); and

[0031] (d) a combination of (a), (b), and / or (c), including HVR amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35 (HI), 50-63 (H2), and 95-102 (H3).

[0032] Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in a variable domain are numbered in accordance with Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) numbering.

[0033] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. Generally, the FRs of a variable domain consist of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0034] "Human consensus framework" refers to a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup is a subgroup as in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

[0035] In one embodiment, an antibody of the application comprises a VHH domain that specifically binds to IL-25. In one embodiment, the VHH domain comprises the CDR1, CDR2, and CDR3 of VHH domain I001 (see Table 1). In one embodiment, the VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to VHH domain I001 (see Table 3). In one embodiment, the VHH domain comprises the amino acid sequence of VHH domain I001 (see Table 3).

[0036] In one embodiment, the antibody of the application is a heavy chain-only antibody, i.e. consisting only of heavy chains, devoid of light chains, e.g. a heavy chain dimer. In one embodiment, the heavy chain dimer is a homodimer. In one embodiment, the heavy chain dimer is a heterodimer. In one embodiment, the heavy chain heterodimer binds the same antigen. In one embodiment, the heavy chain heterodimer binds the same epitope of the same antigen. In one embodiment, the heavy chain heterodimer binds different epitopes of the same antigen. In one embodiment, the heavy chain heterodimer binds different antigens.

[0037] In one embodiment, the antibody of the application is a single domain antibody, i.e. consisting only of a VHH domain.

[0038] In one embodiment, the antibody of the application is a monospecific antibody, having only binding specificity for IL-25.

[0039] In one embodiment, the antibody of the application is a monovalent antibody, i.e. comprising one binding site, e.g. consisting of one VHH domain.

[0040] In one embodiment, the antibody of the application is a multivalent antibody (e.g. bivalent antibody, trivalent antibody, tetravalent antibody, etc.), i.e. comprising multiple binding sites, e.g. each binding site consisting of one VHH domain, which can be fused to each other via a peptide linker. In one embodiment, the multivalent antibody of the application is a monospecific antibody, i.e. having only binding specificity for IL-25. In one embodiment, the multivalent antibody of the application is a monoeptopic antibody. In one embodiment, the multivalent antibody of the application is a polyepitopic antibody (e.g. bi-epitopic antibody, tri-epitopic antibody, tetra-epitopic antibody, etc.). In the embodiment of a polyepitopic antibody (e.g. bi-epitopic antibody, tri-epitopic antibody, tetra-epitopic antibody, etc.), the binding valency of the antibody of the application for each epitope is independent from each other, or the same or different.

[0041] In one embodiment, the antibody of the application comprises an Fc region. In one embodiment, the Fc region is an Fc region of IgG. In one embodiment, the Fc region is an Fc region of IgGl, IgG2 or IgG4. In one embodiment, the Fc region of IgG4 comprises an amino acid sequence which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10.

[0042] In one embodiment, the antibody of the application is a multispecific antibody (e.g., bispecific antibody, trispecific antibody, tetraspecific antibody, etc.). In one embodiment, the multispecific antibody has binding specificities for IL-25 and a different antigen. In one embodiment, the binding to the different antigen prolongs the half-life of the antibody. In one embodiment, the different antigen is serum albumin.

[0043] In one embodiment, the binding specificity for serum albumin is a VHH that specifically binds to serum albumin. In one embodiment, the VHH that specifically binds to serum albumin comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9.

[0044] In one embodiment, the VHH that specifically binds to serum albumin is located N-terminal to the VHH that specifically binds to IL-25. In one embodiment, the VHH that specifically binds to serum albumin is located C-terminal to the VHH that specifically binds to IL-25.

[0045] In one embodiment, the VHH that specifically binds to serum albumin is connected to the VHH that specifically binds to IL-25 via a linker.

[0046] In one embodiment, the antibody of the application comprises a half-life extending moiety. In one embodiment, the half-life extending moiety is an Fc region or a VHH that specifically binds to serum albumin, e.g., as described hereinabove.

[0047] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids, often glycine (G) and / or serine (S). Suitable linker peptides are, for example, (G4S) n , (SG4) n , (SG4) n S or G4(SG4) n peptide linker, wherein "n" is generally an integer from 1 to 10, typically an integer from 1 to 6, in particular 4.

[0048] In one embodiment, the antibody of the application is a membrane-integrated antibody (e.g., a chimeric antigen receptor, CAR). In one embodiment, the membrane-integrated antibody comprises a hinge region. In one embodiment, the hinge region is a hinge region of IgGl, IgG4, CD8a, CD28, Siglecs, NGFR, or CD34. In one embodiment, the membrane-integrated antibody comprises a transmembrane region. In one embodiment, the transmembrane region is a transmembrane region of CD3zeta, CD4, CD8a, CD28, ICOS, 4-1BB, or KIR2DS2. In one embodiment, the membrane-integrated antibody comprises an intracellular signaling domain. In one embodiment, the intracellular signaling domain is a signaling domain of CD3zeta or FcyR. In one embodiment, the membrane-integrated antibody comprises a costimulatory domain. In one embodiment, the costimulatory domain is a costimulatory domain of CD28, ICOS, 4-1BB, OX40, CD27, CD40, HVEM, GITR, MYD88-CD40, TLR2, or Dectin-1. In one embodiment, the membrane-integrated antibody comprises a signal peptide. In one embodiment, the signal peptide is a signal peptide of CD8a.

[0049] The application also provides a nucleic acid encoding the antibody of the application.

[0050] The application also provides a vector comprising the nucleic acid of the application. In one embodiment, the vector is a cloning vector or an expression vector. In one embodiment, the vector is a plasmid, a virus, or a cosmid.

[0051] The application also provides a host cell comprising the nucleic acid of the application or the vector of the application. In one embodiment, the host cell is a prokaryotic cell or a eukaryotic cell. In one embodiment, the host cell is an immune cell, such as a T cell or an NK cell. In one embodiment, the host cell displays the antibody of the application (particularly a membrane-integrated antibody, CAR) on the cell surface (e.g., a CAR-T cell or a CAR-NK cell).

[0052] The application also provides a method of producing an antibody, comprising culturing the host cell of the application so that the antibody is expressed.

[0053] The application also provides a composition comprising the antibody, the nucleic acid, the vector, or the host cell of the application. In one embodiment, the composition is a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.

[0054] The anti-IL-25 antibody (e.g., a single-domain antibody) of the application has one or more of the following properties:

[0055] (1) is capable of binding to human IL-25 antigen with high specificity;

[0056] (2) is capable of binding to murine IL-25 antigen with high specificity;

[0057] (3) is capable of binding to monkey IL-25 antigen with high specificity;

[0058] (4) is capable of blocking the binding of IL-25 to its receptor; and / or

[0059] (5) is capable of blocking the downstream signaling pathway of IL-25 binding to its receptor.

[0060] The present application provides a method of treating or preventing asthma comprising administering to a subject in need thereof an effective amount (e.g., a therapeutically effective amount or a prophylactically effective amount) of an anti-IL-25 antibody of the present application.

[0061] The present application provides use of an anti-IL-25 antibody of the present application for treating or preventing asthma comprising administering to a subject in need thereof an effective amount (e.g., a therapeutically effective amount or a prophylactically effective amount) of an anti-IL-25 antibody of the present application.

[0062] The present application provides use of an anti-IL-25 antibody of the present application for the manufacture of a medicament for treating or preventing asthma, wherein the treatment or prevention comprises administering to a subject in need thereof an effective amount (e.g., a therapeutically effective amount or a prophylactically effective amount) of an anti-IL-25 antibody of the present application.

[0063] The present application provides a composition comprising an effective amount (e.g., a therapeutically effective amount or a prophylactically effective amount) of an anti-IL-25 antibody of the present application for use in treating or preventing asthma in a subject in need thereof.

[0064] In one embodiment, the asthma is mild asthma, mild-to-moderate asthma, moderate asthma, moderate-to-severe asthma, severe asthma, asthma type 2, non-asthma type 2, eosinophilic asthma, non-eosinophilic asthma, neutrophilic asthma, mixed granulocytic asthma, paucigranulocytic asthma, hormone-sensitive asthma, hormone- non-sensitive asthma, allergic asthma, non-allergic asthma, atopic asthma, non-atopic asthma, acute asthma, or brittle asthma.

[0065] In one embodiment, treating or preventing asthma means one or more of:

[0066] (1) preventing the onset of asthma (e.g., as compared to standard treatment);

[0067] (2) reducing the onset of asthma (e.g., as compared to standard treatment);

[0068] (3) reducing the frequency of onset of asthma (e.g., as compared to standard treatment);

[0069] (4) reducing the severity of onset of asthma (e.g., as compared to standard treatment);

[0070] (5) inhibiting leukocytosis (e.g., compared to standard treatment);

[0071] (6) inhibiting eosinophilocytosis (e.g., compared to standard treatment);

[0072] (7) inhibiting neutrophilocytosis (e.g., compared to standard treatment);

[0073] (8) inhibiting the increase of eosinophil proportion (e.g., compared to standard treatment);

[0074] (9) inhibiting the increase of neutrophil proportion (e.g., compared to standard treatment);

[0075] (10) inhibiting the increase of serum total IgE level (e.g., compared to standard treatment);

[0076] (11) inhibiting the increase of inflammatory cell infiltration proportion in lung tissue (e.g., compared to standard treatment);

[0077] (12) inhibiting the increase of mucus secretion in lung tissue (e.g., compared to standard treatment); and / or

[0078] (13) inhibiting the increase of Th2 cytokine (e.g., IL-5 and / or IL-13) expression in lung tissue (e.g., compared to standard treatment);

[0079] (14) inhibiting airway remodeling (e.g., compared to standard treatment);

[0080] (15) inhibiting airway hyperresponsiveness (e.g., compared to standard treatment); and / or

[0081] (16) improving lung function (e.g., compared to standard treatment). BRIEF DESCRIPTION OF DRAWINGS

[0082] Figure 1 shows the body weight of mice at different time points during the experiment.

[0083] Figure 2 shows the proportion of eosinophils in the bronchoalveolar lavage fluid (BALF).

[0084] Figure 3 shows the total IgE concentration in serum.

[0085] Figure 4A shows the results of lung tissue HE staining, and Figure 4B shows the results of quantitative statistical analysis of inflammatory cell infiltration.

[0086] Figure 5A shows the results of lung tissue PAS immunohistochemical staining, and Figure 5B shows the results of quantitative statistical analysis of PAS staining.

[0087] Figure 6 shows the expression amount of IL-5 in lung tissue.

[0088] Figure 7 shows the amount of lung tissue IL-13 expression.

[0089] In the figures, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. DETAILED DESCRIPTION

[0090] The present application provides the following sequences.

[0091] Table 1: CDR sequences of exemplary antibodies (VHH domains) of the present application

[0092] Table 2: FR sequences of exemplary antibodies (VHH domains) of the present application

[0093] Table 3: Sequences of exemplary antibodies (VHH domains) of the present application

[0094] Sequence of VHH that specifically binds to serum albumin (SEQ ID NO: 9)

[0095] Sequence of Fc (IgG4) (SEQ ID NO: 10)

[0096] Sequence of Fc (IgG4) variant 1 (SEQ ID NO: 11)

[0097] Sequence of Fc (IgG4) variant 2 (SEQ ID NO: 12)

[0098] The present application is further illustrated, but not limited, by the following examples.

[0099] Example 1: Screening of anti-IL-25 single-domain antibodies using llama immunization library

[0100] The llama was immunized with a mixture of human IL-25 recombinant protein (purchased from Shengzhou Yiqiao Biotechnology Co., Ltd., item number: 10096-H01H) and Freund's adjuvant for a total of five times. The titer was determined after each immunization by taking the peripheral blood of the llama. After the fifth immunization, the peripheral blood was collected and PBMC (peripheral blood mononuclear cells) were isolated, RNA was isolated and cDNA was reverse transcribed, VHH coding sequences were amplified by PCR and the PCR product was recovered to construct a phage display library with a library capacity of 8.3 x 10 8pfu, the positive clone rate was 90%. After three rounds of "adsorption-washing-enrichment" screening of the anti-IL-25 nanobodies by phage display technology, single clone ELISA verification was performed, and 60 different sequences were obtained. The 15 sequences with higher OD450 were selected and cloned into pcDNA3.4 (Invitrogen). The plasmid was transfected into CHO-S cells by electroporation to make the transformed CHO-S cells express VHH+Fc (IgG4) fusion protein. After incubation at 37°C for one week, the supernatant was collected and the antibody was purified. The antibody was purified using a protein A affinity chromatography column, and the purity of the antibody was detected by SDS-PAGE and SEC-HPLC detection methods after purification. The purity of all antibodies reached more than 95%. Seven candidate antibodies with high binding activity to IL-25 were screened by surface plasmon resonance technology.

[0101] Example 2: Affinity of humanized anti-IL-25 single-domain antibody to human IL-25 and cell level blocking activity

[0102] One of the candidate antibodies was humanized and drugable. The humanization used the CDR grafting method. The basic method is to replace the camelid framework region with the selected human germline framework region, and only retain the camelid CDR; then perform back mutation on the key amino acid sites in FR. The I001 antibody was obtained.

[0103] Example 3: Analysis of the affinity of anti-IL-25 single-domain antibody to human IL-25 by BIAcore

[0104] The BIAcore 8000 Biosensor instrument (BIAcore AB) was used to determine the affinity of VHH+Fc (IgG4) fusion protein to human IL-25 using surface plasmon resonance technology. Briefly, the protein A chip was used to capture the Fc-containing antibody with a surface density of about 500 RU, and the IL-25 recombinant protein was diluted with the HBS-EP+ buffer series, so that the obtained series of dilutions (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, a total of 5 gradients) passed through the surface of the sensor chip. The instrument detected the binding curve of the antibody with different concentrations of IL-25. The sensor record obtained above was evaluated by BIAcore evaluation software to obtain kinetic data.

[0105] Table 4: Affinity of humanized single-domain antibody to human IL-25 antigen

[0106] Example 4: Determination of the blocking activity of anti-IL-25 single-domain antibody to the signal pathway triggered by the binding of human IL-25 to its receptor on the cell line using human IL-25 reporter cell line

[0107] Human IL-25 reporter cell line (purchased from GenScript) is a luciferase reporter cell line based on IL-17 signaling pathway. When IL-17E binds to IL-17RA-RB, Act1 is recruited and binds to TRAF6, further activating the expression of luciferase. The luminescence reading represents the activation effect of the signaling pathway, and therefore can be used for in vitro effect evaluation of IL-17E related drugs. The specific steps are as follows: inoculate cells in a 96-well plate at a density of 5 x 10 5 cells / well, with a volume of 100 μL per well; add IL-25 recombinant protein to activate the cell line, so that the final concentration of IL-25 per well is 6.7 ng / mL; after 1 hour of co-incubation, add the antibodies I01 to I07 to be tested at an initial concentration of 70 μM, with 5-fold gradient dilution, a total of 10 gradients; add 33 μL of the corresponding concentration of antibodies or blank control to each well of cells, and continue to incubate in a 37°C CO2 incubator for 23 hours, then collect samples for luminescence signal detection, and calculate the EC50 value.

[0108] Table 5: Cell level blocking activity of humanized single domain antibodies

[0109] Example 5: Evaluation of the therapeutic effect of anti-IL-25 antibodies on house dust mite (HDM) induced asthma

[0110] Methods

[0111] This drug efficacy experiment sets up 4 groups, with 6 mice in each group:

[0112] G1: blank group, healthy control;

[0113] G2: negative control group, disease model;

[0114] G3: positive control group, reference drug Dupilumab, 10 mpk BIW (mpk = mg / kg, BIW = twice a week);

[0115] G4: test group, test drug anti-IL-25 antibody (VHH + Fc (IgG4) fusion protein) of the present application, 10 mpk BIW.

[0116] The specific treatment is as follows:

[0117] G1: hIL-4 / hIL-4R humanized transgenic mice (female, 6 weeks old, body weight 20-25 g, Shanghai South Model Organism Co., Ltd.), 30 μL of normal saline was given intranasally (i.n.) every day for the first five days of each week;

[0118] G2: the same mice as G1 were given 30 μg of HDM dissolved in 30 μL of normal saline intranasally (i.n.) every day for the first five days of each week for 4 weeks;

[0119] G3: In addition to the treatment in group G2, administer 10 mpk of the reference drug Dupilumab intraperitoneally on the 2nd and 5th days of each week;

[0120] G4: In addition to the treatment in group G2, administer 10 mpk of test drug intraperitoneally (ip) on days 2 and 5 of each week.

[0121] Mice were weighed and recorded twice weekly. On day 27, mice were sacrificed, and whole blood was collected from the eyeballs to separate serum. The total IgE concentration in the serum was detected using a mouse IgE ELISA kit (abcam). The right lung tissue was washed with 10 mL of PBS to obtain bronchoalveolar lavage fluid (BALF). The number of CD45+ cells and eosinophils in the BALF was detected by flow cytometry, and the proportion of eosinophils to CD45+ cells was calculated. Half of the left lung was fixed in PFA, embedded to obtain paraffin-embedded tissue blocks, and sectioned for H&E and PAS staining. The percentage of inflammatory cells and the percentage of PAS-positive areas were calculated using HALO software (v3.6.4134.362). The other half was cryopreserved in liquid nitrogen, and RNA was extracted and reverse transcribed into cDNA using a kit (SuperScript II Reverse Transcriptase Kit, Life Technologies). The expression of IL-5 and IL-13 was detected using Q-PCR.

[0122] result

[0123] The body weight values ​​of mice in each group at each time point are shown in Figure 1 and Table 6, which fluctuated within the normal range.

[0124] Table 6: Relative body weight changes in mice (g)

[0125] The percentage of eosinophils in BALF was detected by flow cytometry. The percentage of eosinophils in the G2 group was significantly higher than that in the G1 group, while the percentage of eosinophils in the G3 and G4 groups was significantly lower than that in the G2 group (Figure 2).

[0126] IgE is an important indicator for assessing the severity of allergic diseases. The total serum IgE level in group G2 was significantly higher than that in group G1, while the total serum IgE level in groups G3 and G4 was significantly inhibited compared to group G2 (Figure 3).

[0127] After HE staining of pathological sections, the proportion of inflammatory cell area in lung tissue was significantly higher in group G2 compared to group G1; the proportion of inflammatory cell area in groups G3 and G4 was significantly lower than that in group G2 (Figure 4). PAS staining is used to detect carbohydrates in tissues and can reflect the mucus secretion in lung tissue. The proportion of PAS-positive area was significantly higher in group G2 compared to group G1, while the proportion of PAS-positive area in groups G3 and G4 showed a decreasing trend compared to group G2 (Figure 5).

[0128] The expression level of IL-5 in lung tissue was significantly higher in group G2 than in group G1, and the expression level of IL-5 in group G4 showed a decreasing trend compared with group G2, but group G3 did not inhibit the expression of IL-5 (Figure 6); the expression level of IL-13 in lung tissue was significantly higher in group G2 than in group G1, and group G4 significantly inhibited the expression of IL-13 compared with group G2, but group G3 did not inhibit the expression of IL-13 (Figure 7); indicating that the test drug of this invention has a good inhibitory effect on the expression of Th2 cytokines.

[0129] in conclusion

[0130] The anti-IL-25 antibody of this invention significantly reduced the percentage of eosinophils in BALF, the total IgE concentration in serum, the percentage of inflammatory cells in lung tissue, and the expression level of IL-13 in an asthma model; the expression level of IL-5 showed a decreasing trend, which reduced mucus secretion in lung tissue.

[0131] The foregoing description of the embodiments and certain implementations should be considered illustrative and not limiting of the invention as defined in the claims. It will be readily understood that many variations and combinations of the above features may be used without departing from the invention as set forth in the claims. All such variations are intended to be included within the scope of the invention. All cited references are incorporated herein by reference in their entirety.

Claims

1. Use of an antibody that specifically binds to IL-25 in the preparation of a medicament for the treatment or prevention of asthma, wherein the antibody comprises a VHH domain that specifically binds to IL-25, the VHH domain comprising: CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO:

3.

2. The use according to claim 1, wherein the VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

8.

3. The use according to claim 1 or 2, wherein the antibody comprises an Fc region, optionally, an Fc region of IgG, further optionally, an Fc region of IgG1, IgG2 or IgG4, and even more optionally, an Fc region comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the same amino acid sequence as SEQ ID NO:

10.

4. The use according to claim 1 or 2, wherein the antibody comprises a module for extending half-life, optionally a module for binding to serum albumin, further optionally a VHH domain specifically binding to serum albumin, and even more optionally a VHH domain comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 9, optionally located at the N-terminus or C-terminus of the VHH domain specifically binding to IL-25.

5. The use according to claim 1 or 2, wherein the asthma is mild asthma, mild to moderate asthma, moderate asthma, moderate to severe asthma, severe asthma, type 2 asthma, non-type 2 asthma, eosinophilic asthma, non-eosinophilic asthma, neutrophilic asthma, mixed cellular asthma, oligogranulocytic asthma, hormone-sensitive asthma, hormone-insensitive asthma, allergic asthma, non-allergic asthma, atopic asthma, non-atopic asthma, acute asthma, or fragile asthma.

6. An antibody that specifically binds to IL-25, wherein the antibody comprises a VHH domain that specifically binds to IL-25, the VHH domain comprising: CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO:

3.

7. The antibody according to claim 6, wherein the VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:

8.

8. The antibody according to claim 6 or 7, wherein the antibody comprises an Fc region, optionally, an Fc region of IgG, further optionally, an Fc region of IgG1, IgG2 or IgG4, and even more optionally, an Fc region comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the same amino acid sequence as SEQ ID NO:

10.

9. The antibody according to claim 6 or 7, wherein the antibody comprises a module for extending half-life, optionally a module for binding to serum albumin, further optionally a VHH domain specifically binding to serum albumin, and even more optionally a VHH domain comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 9, optionally located at the N-terminus or C-terminus of the VHH domain specifically binding to IL-25.

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