Anti-il-25 single-domain antibody and derivative thereof

Abstract

Provided in the present invention are an anti-IL-25 single-domain antibody and a derivative thereof. The anti-IL-25 single-domain antibody of the present invention can specifically bind to human, murine, and simian IL-25 antigens, and can block the binding of IL-25 to a receptor thereof, thereby blocking the downstream signaling pathway.

Description

Anti-il-25 single-domain antibodies and derivatives thereof

[0001] Incorporation by Reference of Related Applications

[0002] This application claims priority to the application with the application date of September 30, 2024, the application number of PCT / CN2024 / 122844, and the invention name of "Anti-il-25 single-domain antibodies and derivatives thereof", the complete content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] Generally speaking, the present application relates to the field of antibodies. Specifically, the present application relates to anti-human IL-25 single-domain antibodies and derivatives thereof. BACKGROUND

[0004] IL-25, also known as IL-17E, is a member of the IL-17 family, mainly expressed by epithelial cells, innate immune cells, Th2 cells and tissue stromal cells, etc. IL-25 acts on innate immune cells and adaptive immune cells such as ILC2, mast cells, basophils and Th2 through the action of IL-17RA and IL-17RB heterodimer: IL-25 binds to its receptor to activate the NF-κB pathway in a TNFR6-dependent manner, or directly activate extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), p38 signaling pathway to participate in the release of Th2-type inflammatory factors; IL-25 can also activate STAT5 independently of NF-κB to further enhance Th2-type cytokine production. Under the invasion of pathogens such as parasites, viruses and allergens, IL-25 combined with IL-2 can induce ILC2 to proliferate rapidly and secrete Th2-type cytokines to participate in Th2-type immune response. IL-25 can also induce intestinal ILC2 to enter the circulation and transform into inflammatory ILC2. IL-25 can significantly increase the expression of IL-4, IL-5 and IL-10 in liver, lung and kidney, and can also significantly induce the expression of neutrophil-specific chemokine GRO-α in liver, kidney, lung and heart, and can increase the serum levels of IL-5, IL-13 and IgE. The liver, heart, lung, lymph node, kidney, spleen and bladder tissues of IL-25 overexpression transgenic mice all show chronic inflammation, and cause changes in lung and gastrointestinal tissues. Currently, there is only one anti-IL-25 antibody in the pipeline of a pharmaceutical company for asthma indications in clinical phase I, no other drugs in clinical trial stage, and no anti-IL-25 nanobody in clinical trial stage or preclinical research. We are the first anti-IL-25 nanobody drug. SUMMARY

[0005] The present application provides a group of anti-IL-25 single-domain antibodies and derivatives thereof.

[0006] In one embodiment, the present application provides an antibody that specifically binds to IL-25.

[0007] 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.

[0008] The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.

[0009] "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG class antibodies are

[0010] The heavy chains of antibodies can be assigned to one of five types, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), and mu (IgM), some of which can be further divided into subclasses or isotypes, e.g., gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), gamma 4 (IgG4), alpha 1 (IgA1) and alpha 2 (IgA2).

[0011] The light chains of antibodies can be assigned to one of two types, called kappa (K) and lambda (l), based on their amino acid sequences.

[0012] "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.

[0013] Single-domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single-domain antibody was derived from the variable domain of an antibody heavy chain from camelids (nanobodies or VHHs). H H fragments).

[0014] "Specifically binds" means that the binding is selective for the 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) technology (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 the antibody to an unrelated protein is less than about 10% of the binding of the antibody to the antigen, as measured, e.g., by SPR.

[0015] "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., an antibody and an 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). As such, equivalent affinities can include different rate constants so 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).

[0016] In one embodiment, the antibody has 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.

[0017] In one embodiment, the antibody of the application is a monoclonal antibody.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the 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 the particular antigen can be isolated using a VH or VL domain from an antibody that binds the antigen by screening libraries 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)).

[0022] "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, particularly one or two, amino acids removed 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.

[0023] In one embodiment, an antibody of the application is a humanized antibody.

[0024] 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, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0025] The term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain which are highly divergent in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain the antigen-contacting residues ("antigen contacting"). Generally, antibodies comprise six HVRs: three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). Exemplary HVRs herein include:

[0026] (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));

[0027] (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));

[0028] (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

[0029] (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).

[0030] 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.

[0031] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. Generally, the FRs of a variable domain are conserved among antibodies mediating the interaction of antibody combining sites with antigens. Thus, HVR and FR sequences generally appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4 in a VHand (or VL).

[0032] "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. Generally, 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.

[0033] 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 any one of VHH domains I01 to I30 (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 any one of VHH domains I01 to I30 (see Table 3). In one embodiment, the VHH domain comprises the amino acid sequence of any one of VHH domains I01 to I30 (see Table 3).

[0034] 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.

[0035] In one embodiment, the antibody of the application is a single domain antibody, i.e. consisting only of a VHH domain.

[0036] In one embodiment, the antibody of the application is a monospecific antibody, having only binding specificity for IL-25.

[0037] 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.

[0038] 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.

[0039] 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, including wild type and variants, in particular variants that prolong half-life. In one embodiment, the Fc region of IgG4 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: 226, 243 or 244.

[0040] 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.

[0041] 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: 225.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The application also provides a nucleic acid encoding the antibody of the application.

[0048] 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.

[0049] 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).

[0050] The application also provides a method of producing an antibody, comprising culturing the host cell of the application so that the antibody is expressed.

[0051] The application also provides a composition comprising the antibody, the nucleic acid, the vector, or the host cell of the application.

[0052] The anti-IL-25 antibody (e.g., a single-domain antibody) of the application has one or more of the following properties:

[0053] (1) is capable of binding to human IL-25 antigen with high specificity;

[0054] (2) is capable of binding to murine IL-25 antigen with high specificity;

[0055] (3) capable of binding to monkey IL-25 antigen with high specificity;

[0056] (4) capable of blocking the binding of IL-25 to its receptor; and / or

[0057] (5) capable of blocking the downstream signaling pathway of IL-25 binding to its receptor. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 shows the binding affinity K D values of the I16 antibody to human IL-25 antigen as described in Example 4.

[0059] Figure 2 shows the blocking effect of the I16, I24-I28 antibodies on human IL-25 and its receptor-mediated signaling pathway in vitro using a human IL-25 reporter cell line as described in Example 4.

[0060] Figure 3 shows the determination of the inhibitory effect of the I24 antibody on the release of the cytokine IL-5 from human PBMC in vitro as described in Example 6.

[0061] Figure 4 shows the determination of the inhibitory effect of the I24 antibody on the release of the cytokine IL-13 from human PBMC in vitro as described in Example 7. DETAILED DESCRIPTION

[0062] The present application provides the following sequences.

[0063] Table 1: CDR sequences of exemplary antibodies (VHH domains) of the present application

[0064] Table 2: FR sequences of exemplary antibodies (VHH domains) of the present application

[0065] Table 3: Sequences of exemplary antibodies (VHH domains) of the present application

[0066] Sequence of VHH specific for serum albumin (SEQ ID NO: 225)

[0067] Sequence of Fc (IgG4) (SEQ ID NO: 226)

[0068] Sequence of Fc (IgG4) variant 1 (SEQ ID NO: 243)

[0069] The sequence of Fc(IgG4) variant 2 (SEQ ID NO: 244)

[0070] Example 1: Anti-IL-25 single-domain antibodies were obtained by screening alpaca immune libraries.

[0071] Alpacas were immunized five times with a mixture of recombinant human IL-25 protein (purchased from Shenzhou Yiqiao Biotechnology Co., Ltd., catalog number: 10096-H01H) and Freund's adjuvant. Peripheral blood was collected after each immunization to determine the titer. After the fifth immunization, peripheral blood was collected, and PBMCs (peripheral blood mononuclear cells) were isolated. RNA was isolated and cDNA was reverse transcribed. The VHH coding sequence was amplified by PCR, and the PCR product was recovered to construct a phage display library with a volume of 8.3 × 10⁻⁶. 8 PFU (Positive Cloning Rate) was 90%. Anti-IL-25 nanobodies were screened using phage display technology. After three rounds of "adsorption-washing-enrichment" screening, single-clone ELISA verification yielded 60 differentially expressed sequences. Fifteen sequences with high OD450 were selected and cloned into pcDNA3.4 (Invitrogen). The plasmid was transfected into CHO-S cells via electroporation, causing the transformed CHO-S cells to express the VHH+Fc(IgG4) fusion protein. After one week of incubation at 37°C, the supernatant was collected, and the antibodies were purified. Purification was performed using a protein A affinity chromatography column. The purity of the purified antibodies was detected by SDS-PAGE and SEC-HPLC, with all antibodies achieving a purity of over 95%. Seven candidate antibodies (I01 to I07) with high IL-25 binding activity were screened using surface plasmon resonance (SPR) technology.

[0072] Example 2: Analysis of the affinity of anti-IL-25 single-domain antibody for human IL-25 using BIAcore

[0073] The affinity of the VHH+Fc(IgG4) fusion protein for human IL-25 was determined using a BIAcore 8000 Biosensor instrument (BIAcore AB) via surface plasmon resonance (SPR) technology. In short, antibodies carrying the Fc band were captured using a protein A chip with a surface density of approximately 500 RU. The recombinant IL-25 protein was serially diluted with HBS-EP+ buffer, resulting in five dilutions (50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM). The instrument detected binding curves between the antibody and different concentrations of IL-25. The kinetic data obtained from the sensor recordings were evaluated using BIAcore evaluation software.

[0074] Table 4: Binding affinity of the single-domain antibody of the present invention to human IL-25 antigen

[0075] Example 3: Blocking activity of anti-IL-25 single-domain antibodies on the signal pathway triggered by human IL-25 binding to its receptor on the cell line using human IL-25 reporter cell line

[0076] The human IL-25 reporter cell line (purchased from GenScript) is a luciferase reporter cell line based on the 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 thus can be used for in vitro effect evaluation of IL-17E related drugs. The specific steps are as follows: inoculate the 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 antibody or blank control at the corresponding concentration to each well of cells, and continue to incubate in a 37°C CO2 incubator for 23 hours, then collect the sample for luminescence signal detection, and calculate the EC50 value.

[0077] Table 5: Blocking activity of single-domain antibodies of the present application on human IL-25 and its receptor pathway

[0078] Example 4: Affinity and cell level blocking activity of anti-IL-25 single-domain antibodies after humanization

[0079] The I07 antibody was humanized and drugability modified. Humanization was performed using the CDR grafting method. The basic approach is to replace the camelid framework region with a selected human germline framework region, and only retain the camelid CDR; then perform back mutation on the key amino acid sites in the FR. I08 to I28 antibodies were obtained, a total of 21 sequences. The affinity of I08 to I23 antibodies was determined using the method described in Example 2 above (Table 6). The cell level blocking activity of I08-I28 antibodies was determined using the method described in Example 3 above (Table 7), and the results showed that their activities were better than those of the control antibodies.

[0080] Table 6: Affinity of humanized single-domain antibodies for human IL-25 antigen

[0081] Table 7: Cell level blocking activity of humanized single-domain antibodies

[0082] Example 5: Affinity of humanized anti-IL-25 single-domain antibody to human, murine, and monkey IL-25

[0083] The affinity of I16 antibody to human, murine, and monkey antigens was determined using the method described in Example 2 above.

[0084] Table 8: Affinity of humanized single-domain antibody I16 to human, murine, and monkey IL-25 antigens

[0085] Example 6: In vitro inhibition of cytokine IL-5 release from human PBMC by humanized anti-IL-25 single-domain antibody

[0086] Human PBMC were resuspended in RPMI1640 medium containing 10% FBS, and the cell density was adjusted to 3.4 x 10 6 cells / mL. A 96-well plate was seeded with a volume of 150 μL per well, and the number of cells per well was about 5 x 10 5 Three groups of experiments were set up: 1. No IL-2 and IL-25 were added as a negative control group; 2. IL-2 protein 10 ng / mL and IL-25 protein 10 ng / mL were added to stimulate PBMC; 3. IL-2 protein 10 ng / mL and IL-25 protein 10 ng / mL were added to stimulate PBMC, and I24 antibody 1 μg / mL was added for inhibition. Two wells were set up in each group for repetition. After 5 days of cell culture, the supernatant was collected by centrifugation at 400 g for 5 minutes. The level of IL-5 was detected using a human IL-5 ELISA detection kit (purchased from Doctor De, item number EK0407). The results showed that the anti-IL-25 single-domain antibody inhibited the release of IL-5 from human PBMC.

[0087] Example 7: In vitro inhibition of cytokine IL-13 release from human PBMC by humanized anti-IL-25 single-domain antibody

[0088] Human PBMC were resuspended in RPMI1640 medium containing 10% FBS, and the cell density was adjusted to 3.4 x 10 6 cells / mL. A 96-well plate was seeded with a volume of 150 μL per well, and the number of cells per well was about 5 x 10 5Three groups of experiments were set: 1. No IL-2 and IL-25 were added as a negative control group; 2. IL-2 protein 10 ng / mL and IL-25 protein 30 ng / mL were added to stimulate PBMC; 3. IL-2 protein 10 ng / mL and IL-25 protein 30 ng / mL were added to stimulate PBMC, and I24 antibody 100 nM was added for inhibition; 4. IL-2 protein 10 ng / mL and IL-25 protein 30 ng / mL were added to stimulate PBMC, and a phase I anti-IL-25 IgG antibody (reference antibody) 100 nM was added for inhibition. Two wells were set for each group. After 5 days of cell culture, centrifugation at 400g for 5 minutes, the supernatant was collected. The human IL-13 ELISA detection kit (purchased from Cobio, item number ELK2266) was used to detect the IL-13 level. The results showed that the anti-IL-25 single-domain antibody inhibited the release of IL-13 from human PBMC, and the inhibition activity was better than that of the reference antibody.

[0089] Example 8: Anti-IL-25 single-domain antibody obtained by screening using a llama natural library (unimmunized)

[0090] PBMC (peripheral blood mononuclear cells) were isolated from the peripheral blood of 40 llamas, RNA was extracted and reverse transcribed into cDNA, VHH coding sequences were amplified by PCR, and the PCR product was recovered to construct a phage display library with a capacity of 3.3 x 1011pfu. 10 Using phage display technology to screen anti-IL-25 nanobodies, 15 sequences were picked and cloned into pcDNA3.4 (Invitrogen), and the plasmid was transfected into CHO-S cells by electroporation. The transformed CHO-S cells expressed VHH+Fc (IgG4) fusion proteins. After incubation at 37°C for one week, the supernatant was collected, and two candidate antibodies with high IL-25 binding activity were screened by bio-layer interference technology. Further mutation of CDR amino acids was performed using internal AI software, and then a solid mutation library was established for screening to determine the two optimal sequences I29 and I30.

[0091] Example 9: Analysis of the affinity of anti-IL-25 single-domain antibody to human IL-25 by BIAcore

[0092] The affinity of the VHH+Fc (IgG4) fusion proteins of I29 and I30 to human IL-25 was determined using surface plasmon resonance technology with a BIAcore 8000 Biosensor instrument (BIAcore AB). Briefly, the Fc-bearing antibodies were captured with a Protein A chip at a surface density of about 500 RU, and the IL-25 recombinant protein was diluted in a HBS-EP+ buffer series, and the resulting series of dilutions (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, for a total of 5 gradients) were passed over the sensor chip surface. The instrument detected the binding curves of the antibodies to IL-25 at different concentrations. The sensorgrams obtained were evaluated with BIAcore evaluation software to obtain kinetic data.

[0093] Table 9: Binding affinity of the single-domain antibodies of the application to human IL-25 antigen

Claims

1. An antibody that specifically binds to IL-25 comprising a VHH domain that specifically binds to IL-25, the VHH domain comprising: (1) a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3; (2) a CDR1 as set forth in SEQ ID NO: 4, a CDR2 as set forth in SEQ ID NO: 5, and a CDR3 as set forth in SEQ ID NO: 6; (3) a CDR1 as set forth in SEQ ID NO: 7, a CDR2 as set forth in SEQ ID NO: 8, and a CDR3 as set forth in SEQ ID NO: 9; (4) a CDR1 as set forth in SEQ ID NO: 10, a CDR2 as set forth in SEQ ID NO: 11, and a CDR3 as set forth in SEQ ID NO: 12; (5) a CDR1 as set forth in SEQ ID NO: 13, a CDR2 as set forth in SEQ ID NO: 14, and a CDR3 as set forth in SEQ ID NO: 15; (6) a CDR1 as set forth in SEQ ID NO: 16, a CDR2 as set forth in SEQ ID NO: 17, and a CDR3 as set forth in SEQ ID NO: 18; (7) a CDR1 as set forth in SEQ ID NO: 19, a CDR2 as set forth in SEQ ID NO: 20, and a CDR3 as set forth in SEQ ID NO: 21; (8) a CDR1 as set forth in SEQ ID NO: 22, a CDR2 as set forth in SEQ ID NO: 23, and a CDR3 as set forth in SEQ ID NO: 24; (9) a CDR1 as set forth in SEQ ID NO: 25, a CDR2 as set forth in SEQ ID NO: 26, and a CDR3 as set forth in SEQ ID NO: 27; (10) a CDR1 as set forth in SEQ ID NO: 28, a CDR2 as set forth in SEQ ID NO: 29, and a CDR3 as set forth in SEQ ID NO: 30; (11) a CDR1 as set forth in SEQ ID NO: 31, a CDR2 as set forth in SEQ ID NO: 32, and a CDR3 as set forth in SEQ ID NO: 33; (12) a CDR1 as set forth in SEQ ID NO: 34, a CDR2 as set forth in SEQ ID NO: 35, and a CDR3 as set forth in SEQ ID NO: 36; (13) a CDR1 as set forth in SEQ ID NO: 37, a CDR2 as set forth in SEQ ID NO: 38, and a CDR3 as set forth in SEQ ID NO: 39; (14) a CDR1 as set forth in SEQ ID NO: 40, a CDR2 as set forth in SEQ ID NO: 41, and a CDR3 as set forth in SEQ ID NO: 42; (15) a CDR1 as depicted in SEQ ID NO: 43, a CDR2 as depicted in SEQ ID NO: 44 and a CDR3 as depicted in SEQ ID NO: 45; (16) a CDR1 as depicted in SEQ ID NO: 46, a CDR2 as depicted in SEQ ID NO: 47 and a CDR3 as depicted in SEQ ID NO: 48; (17) a CDR1 as depicted in SEQ ID NO: 49, a CDR2 as depicted in SEQ ID NO: 50 and a CDR3 as depicted in SEQ ID NO: 51 ; (18) a CDR1 as depicted in SEQ ID NO: 52, a CDR2 as depicted in SEQ ID NO: 53 and a CDR3 as depicted in SEQ ID NO: 54; (19) a CDR1 as depicted in SEQ ID NO: 55, a CDR2 as depicted in SEQ ID NO: 56 and a CDR3 as depicted in SEQ ID NO: 57; (20) a CDR1 as depicted in SEQ ID NO: 58, a CDR2 as depicted in SEQ ID NO: 59 and a CDR3 as depicted in SEQ ID NO: 60; (21) a CDR1 as depicted in SEQ ID NO: 61, a CDR2 as depicted in SEQ ID NO: 62 and a CDR3 as depicted in SEQ ID NO: 63; (22) a CDR1 as depicted in SEQ ID NO: 64, a CDR2 as depicted in SEQ ID NO: 65 and a CDR3 as depicted in SEQ ID NO: 66; (23) a CDR1 as depicted in SEQ ID NO: 67, a CDR2 as depicted in SEQ ID NO: 68 and a CDR3 as depicted in SEQ ID NO: 69; (24) a CDR1 as depicted in SEQ ID NO: 70, a CDR2 as depicted in SEQ ID NO: 71 and a CDR3 as depicted in SEQ ID NO: 72; (25) a CDR1 as depicted in SEQ ID NO: 73, a CDR2 as depicted in SEQ ID NO: 74 and a CDR3 as depicted in SEQ ID NO: 75; (26) a CDR1 as depicted in SEQ ID NO: 76, a CDR2 as depicted in SEQ ID NO: 77 and a CDR3 as depicted in SEQ ID NO: 78; (27) a CDR1 as depicted in SEQ ID NO: 79, a CDR2 as depicted in SEQ ID NO: 80 and a CDR3 as depicted in SEQ ID NO: 81 ; (28) a CDR1 as depicted in SEQ ID NO: 82, a CDR2 as depicted in SEQ ID NO: 83 and a CDR3 as depicted in SEQ ID NO: 84; (29) a CDR1 as depicted in SEQ ID NO: 227, a CDR2 as depicted in SEQ ID NO: 228 and a CDR3 as depicted in SEQ ID NO: 229; or (30) a CDR1 as depicted in SEQ ID NO: 230, a CDR2 as depicted in SEQ ID NO: 231 and a CDR3 as depicted in SEQ ID NO:

232.

2. The antibody of 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 any one of SEQ ID NOs: 197-224, 241, and 242.

3. The antibody of claim 1 or 2, comprising an Fc region, optionally, of an IgG, further optionally, of an IgGl, IgG2, or IgG4, still further optionally, an Fc region comprising 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: 226, 243, or 244.

4. The antibody of claim 1 or 2, comprising a half-life prolonging moiety, optionally, a moiety that binds to serum albumin, further optionally, a VHH domain that specifically binds to serum albumin, still further optionally, a VHH domain comprising 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: 225, optionally, which is N- or C-terminal to a VHH domain that specifically binds to IL-25.

5. The antibody of any one of claims 1 or 2, comprising a hinge region (optionally, of an IgGl, IgG4, CD8a, CD28, Siglecs, NGFR, or CD34), a transmembrane region (optionally, of CD3zeta, CD4, CD8a, CD28, ICOS, 4-1BB, or KIR2DS2), an intracellular signaling domain (optionally, of CD3zeta or FcyR), and optionally a costimulatory domain (optionally, of CD28, ICOS, 4-1BB, OX40, CD27, CD40, HVEM, GITR, MYD88-CD40, TLR2, or Dectin-1), and optionally a signal peptide (optionally, of CD8a).

6. A nucleic acid encoding the antibody of any one of claims 1-5.

7. A vector comprising the nucleic acid of claim 6.

8. A host cell comprising the nucleic acid of claim 6 or the vector of claim 7.

9. A method of producing an antibody, comprising culturing the host cell of claim 8 such that the antibody is expressed.

10. A composition comprising the antibody of any one of claims 1-5, the nucleic acid of claim 6, the vector of claim 7, or the host cell of claim 8.

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