Anti-IL-25 antibody and method of use thereof

Abstract

This disclosure provides antibodies that bind to IL-25 and their antibody fragments. The disclosed antibodies and their antibody fragments can modulate the biological activity of the IL-25 / IL-25 receptor signaling axis and are therefore useful in the treatment of type 2 inflammatory diseases, autoimmune diseases, allergic disorders, or cancer.

Description

[Technical Field]

[0001] This disclosure generally relates to antibodies that bind to interleukin-25 and their antibody fragments. The antibodies may be useful in the prevention and / or treatment of type 2 inflammatory diseases, autoimmune diseases, or cancer. [Background technology]

[0002] The interleukin-17 (IL-17) family belongs to a group of cytokines that play a crucial role in inflammatory responses during autoimmune etiologies and host defense against extracellular pathogens. IL-25 (also known as IL-17E) is produced not only by immune cells (e.g., T cells, dendritic cells, and macrophages) but also by other non-immune cells (e.g., fibroblasts, epithelial cells, and keratinocytes), as well as by some cancer cells (melanoma, liver cancer, breast cancer, and cervical cancer, etc.) (Gowhari Shabgah, A., et al., Cancer Med. (2021) 10:5191-5202). This diversity of cellular sources suggests that IL-25 is involved in many immune responses and cellular processes.

[0003] IL-25 is distinctly different from other IL-17 family members in both its molecular structure and its biological function. As an epithelial cytokine, IL-25, along with TSLP and IL-33, is responsible for the role of an "aluminine" or barrier surface cytokine, alerting the immune system to exogenous environmental threats (such as allergens, bacteria, viruses, and helminths) and mobilizing host immune defense mechanisms (Ham, J. et al., Immune Netw. (2022)(1):e11, Borowczyk J. et al, Journal of Allergy and Clinical Immunology (2021)148(1)40-52). Recent studies have also suggested that epithelial cytokines have broad effector functions in numerous pathological conditions, including type 2 inflammatory diseases, allergic disorders, viral infections, chronic inflammatory disorders, autoimmune conditions, and cancer.

[0004] IL-25 plays a dual role in regulating immune responses and the pathogenesis of autoimmune diseases. As a pro-inflammatory cytokine, IL-25 exacerbates allergic inflammation by promoting the production of Th2 cytokines, including IL-4, IL-5, and IL-13, by Th2 cells. Furthermore, IL-25 can induce proliferation and activation of innate immune cells, production of other pro-inflammatory cytokines, and recruitment of immune cells (Deng, C. et al., Front. Immunol (2021) 12:691559).

[0005] On the other hand, IL-25 has been reported to play a role in autoimmune diseases as both an anti-inflammatory cytokine and an inhibitor of both innate and adaptive immunity (Saadoun, D. et al., Current pharmaceutical design (2011) 17:3781-3785). For example, the anti-inflammatory and immunosuppressive activities of IL-25 in rheumatoid arthritis are due to Th17 IL-13-dependent downregulation (Liu, D. et al., Sci Rep (2016) 6:36002).

[0006] Furthermore, the expression of IL-25 and its receptor is dysregulated in various cancers compared to normal tissues. Therefore, IL-25 has been shown to potentially play opposing roles in cancer progression or regression. The tumor-suppressive role of IL-25 is mainly due to the infiltration of eosinophils and B cells into the tumor microenvironment and the induction of apoptosis. In contrast, its tumor-supporting role depends on deviations from the immune response, as well as stimulation of EMT and cell proliferation (Gowhari Shabgah, A. et al., Cancer Med. (2021) 10: 5191-5202).

[0007] While the IL-25 pathway is a recognized therapeutic target, only a single Phase I clinical trial evaluating the safety, tolerability, and pharmacokinetic properties of IL-25 has been identified to date. Therefore, there is an unmet need in the art for novel IL-25 antagonists, such as the anti-IL-25 antibodies disclosed herein, for the treatment of diseases or disorders related to IL-25 expression and / or signaling. [Overview of the project]

[0008] This disclosure addresses the above need by providing antibodies that bind to IL-25, including human IL-25, and their antibody fragments. The antibodies and their antibody fragments are useful in the treatment of immune-mediated inflammatory diseases (IMIDs) (e.g., autoimmune diseases and inflammatory disorders) and cancer. Anti-IL-25 antibodies or their antibody fragments can be used either alone (e.g., as monotherapy) or in combination with other immunotherapeutic agents. In alternative embodiments, the antibody fragments of the IL-25 antibodies disclosed herein may be used as components of bispecific or multispecific antibodies or fusion proteins.

[0009] In some embodiments, an anti-IL-25 antibody or an antibody fragment thereof binds to the IL-25 cytokine (IL-17E) with high affinity but does not bind to any other member of the IL-17 cytokine family (e.g., does not bind specifically).

[0010] In some embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises CDR1:SEQ ID NO: 11, CDR2:SEQ ID NO: 12, and CDR3:SEQ ID NO: 13; a heavy chain variable region including CDR1; and / or a light chain variable region including CDR1:SEQ ID NO: 14, CDR2:SEQ ID NO: 15, and CDR3:SEQ ID NO: 16.

[0011] In some embodiments, the anti-IL-25 antibody or an antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 17, CDR2: SEQ ID NO: 18, and CDR1: SEQ ID NO: 19; and / or a light chain variable region comprising CDRI: SEQ DM NO: 20, CDR2: SEQ ID NO: 21, and CDR3: SEQ ID NO: 22.

[0012] In some embodiments, the anti-IL-25 antibody or an antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 24, and CDR3: SEQ ID NO: 25; and / or a light chain variable region comprising CDR1: SEQ ID NO: 26, CDR2: SEQ ID NO: 27, and CDR3: SEQ ID NO: 28.

[0013] In some embodiments, the anti-IL-25 antibody or an antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 29, CDR2: SEQ ID NO: 30, and CDR3: SEQ ID NO: 31; and / or a light chain variable region comprising CDR1: SEQ ID NO: 32, CDR2: SEQ ID NO: 33, and CDR3: SEQ ID NO: 34.

[0014] [ In some embodiments, the anti-IL-25 antibody or an antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 35, CDR2: SEQ ID NO: 36, and CDR3: SEQ ID NO: 37; and / or a light chain variable region comprising CDR1: SEQ ID NO: 38, CDR2: SEQ ID NO: 39, and CDR3: SEQ ID NO: 40.

[0015] In some embodiments, the anti-IL-25 antibody or an antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 49, and CDR3: SEQ ID NO: 25; and / or a light chain variable region comprising CDR1: SEQ ID NO: 50, CDR2: SEQ ID NO: 51, and CDR3: SEQ ID NO: 28.

[0016] In some embodiments, the anti-IL-25 antibody or an antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 52, and CDR3: SEQ ID NO: 25; and / or a light chain variable region comprising CDR1: SEQ ID NO: 50, CDR2: SEQ ID NO: 51, and CDR3: SEQ ID NO: 28.

[0017] In some embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, and 9.

[0018] In other embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, and 10.

[0019] In other embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, and 9, and a variable light chain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, and 10.

[0020] In some embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises a humanized heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 and 47.

[0021] In other embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises a humanized light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 46, and 48.

[0022] In other embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 and 47, and a variable light chain sequence selected from the group consisting of SEQ ID NOs: 45, 46, and 48.

[0023] In some embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises a heavy chain variable region sequence and a light chain variable region sequence selected from the following combinations: (a) a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2; or (b) a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4; (c) a variable heavy chain sequence comprising SEQ ID NO: 5 and a variable light chain sequence comprising SEQ ID NO: 6; (d) Variable heavy chain sequence containing Sequence ID 7 and variable light chain sequence containing Sequence ID 8; (e) Variable heavy chain sequence containing Sequence ID 9 and variable light chain sequence containing Sequence ID 10; (f) Variable heavy chain sequence containing Sequence ID 44 and variable light chain sequence containing Sequence ID 45; (g) A variable heavy chain sequence containing SEQ ID NO: 44 and a variable light chain sequence containing SEQ ID NO: 46, or (h) Variable heavy chain sequence containing sequence number 47 and variable light chain sequence containing sequence number 48.

[0024] In some embodiments, the antibody is a human anti-IL-25 antibody.

[0025] In some embodiments, the antibody is a full-length antibody.

[0026] In some embodiments, the antibody is a full-length antibody containing a human IgG1 constant region selected from SEQ ID NO: 55 or SEQ ID NO: 56.

[0027] In some embodiments, the antibody fragment is selected from the group consisting of: Fab, Fab, F(ab)2, Fd, Fv, scFv, and scFv-Fc fragments, single-chain antibodies, minibodies, and diabodies.

[0028] In some embodiments, the antibody is a monoclonal antibody.

[0029] In some embodiments, the antibody is a human antibody (e.g., a fully human antibody).

[0030] In some embodiments, the antibody is a mouse antibody.

[0031] In some embodiments, the antibody is a chimeric antibody.

[0032] In some embodiments, the antibody is a bispecific antibody.

[0033] In some embodiments, the antibody is a humanized antibody.

[0034] This disclosure also provides a pharmaceutical composition comprising an antibody or antibody fragment disclosed herein and a pharmaceutically acceptable carrier.

[0035] Furthermore, the Disclosure provides a method for treating and / or preventing a type 2 inflammatory disease, autoimmune disease, allergic disorder, or cancer in a subject requiring such treatment and / or prevention, the method comprising administering an antibody or antibody-conjugated fragment disclosed herein to the subject.

[0036] The disclosure also provides a polynucleotide composition comprising a first polynucleotide encoding a heavy chain variable region containing an amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47; and a second polynucleotide encoding a light chain variable region containing an amino acid sequence described in SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, or 48.

[0037] Furthermore, the Disclosure provides a vector composition comprising a first vector comprising a first polynucleotide disclosed herein (e.g., a polynucleotide encoding SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47); and a second vector comprising a second polynucleotide disclosed herein (e.g., a polynucleotide encoding SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, or 48).

[0038] This disclosure also provides cells (e.g., CHO cells) comprising the polynucleotide composition disclosed herein or the vector composition disclosed herein.

[0039] The Disclosure also provides a method for producing an anti-IL-25 antibody or an antibody fragment as disclosed herein, which includes culturing cells expressing an IL-25 antibody or an antibody fragment as disclosed herein in a culture medium, and recovering the anti-IL-25 antibody or an antibody fragment as disclosed herein from the culture medium.

[0040] The above summary, as well as the following detailed description of this disclosure, will be better understood when read in conjunction with the accompanying drawings. For the purposes of illustrating this disclosure, the figures show currently preferred embodiments. However, it should be understood that this disclosure is not limited to the exact arrangements, examples, and means shown. [Brief explanation of the drawing]

[0041] [Figure 1A] Figures 1A–1D provide the amino acid sequences of the VH and VL domains of the anti-IL-25 antibody, as well as their respective CDR sequences. Sequence identifiers are provided, and the CDRs determined by Kabat are underlined in relation to the variable domain sequences. [Figure 1B] Figures 1A–1D provide the amino acid sequences of the VH and VL domains of the anti-IL-25 antibody, as well as their respective CDR sequences. Sequence identifiers are provided, and the CDRs determined by Kabat are underlined in relation to the variable domain sequences. [Figure 1C] Figures 1A–1D provide the amino acid sequences of the VH and VL domains of the anti-IL-25 antibody, as well as their respective CDR sequences. Sequence identifiers are provided, and the CDRs determined by Kabat are underlined in relation to the variable domain sequences. [Figure 1D] Figures 1A–1D provide the amino acid sequences of the VH and VL domains of the anti-IL-25 antibody, as well as their respective CDR sequences. Sequence identifiers are provided, and the CDRs determined by Kabat are underlined in relation to the variable domain sequences. [Figure 2A] Figures 2A and 2B provide the amino acid sequences of human, mouse, and cynomolgus monkey IL-25, as well as the amino acid sequences of the constant region of human or mouse antibodies. [Figure 2B] Figures 2A and 2B provide the amino acid sequences of human, mouse, and cynomolgus monkey IL-25, as well as the amino acid sequences of the constant region of human or mouse antibodies. [Figure 3A]Figures 3A-3C show the binding of anti-IL-25 antibodies to recombinant human, mouse, and cynomolgus monkey IL-25 proteins. ELISA using goat anti-human IgG-HRP as detection shows the binding of anti-IL-25 antibodies to recombinant human (3A) and mouse (3B) IL-25 proteins. [Figure 3B] Figures 3A-3C show the binding of anti-IL-25 antibodies to recombinant human, mouse, and cynomolgus monkey IL-25 proteins. ELISA using goat anti-human IgG-HRP as detection shows the binding of anti-IL-25 antibodies to recombinant human (3A) and mouse (3B) IL-25 proteins. [Figure 3C] Figures 3A-3C show the binding of anti-IL-25 antibodies to recombinant human, mouse, and cynomolgus monkey IL-25 proteins. ELISA using goat anti-human kappa light chain antibody HRP as detection shows the binding of anti-IL-25 antibodies to cynomolgus monkey (3C) IL-25 protein. [Figure 4] Figure 4 shows that an anti-IL-25 antibody inhibits NFκB signaling induced by human IL-25 in HEK-Blue IL-17 reporter cells. [Figure 5] Figure 5 shows that an anti-IL-25 antibody inhibits IL-25-induced CXCL-1 production in the human colon cancer cell line HT29. [Figure 6] Figure 6 shows that the anti-IL-25 antibody inhibits IL-25-induced IL-5 production in a human PBMC assay. [Figure 7A]Figures 7A–7B show that anti-IL-25 antibodies reduce airway resistance in an OVA-induced asthma model. Airway hyperresponsiveness (AHR) in OVA-sensitive / challenged BALB / c mice to methacholine challenge. AHR for each mouse was measured on day 31 using Buxo's whole-body plethysmography (WBP) system. AHR is expressed as the percentage change from baseline levels of lung resistance (Penh value). Dose-response data are presented as AUC of % of group mean ± SEM (7A) and baseline Penh (7B) compared to OVA+ vehicle using repeated measures / bonferroni (7A), with **p<0.01, ***p<0.001. [Figure 7B] Figures 7A–7B show that anti-IL-25 antibodies reduce airway resistance in an OVA-induced asthma model. Airway hyperresponsiveness (AHR) in OVA-sensitive / challenged BALB / c mice to metacholine challenge. AHR for each mouse was measured on day 31 using Buxo's whole-body plethysmography (WBP) system. AHR is expressed as the percentage change from baseline levels of lung resistance (Penh value). Dose-response data are presented as AUC of % of group mean ± SEM (7A) and baseline Penh (7B). Compared to OVA+ vehicle using one-way Anova / Dunnett (7B), **p<0.01, ***p<0.001. [Figure 8] Figure 8 shows that an anti-IL-25 antibody inhibits IL-5 production in the lungs of an OVA-induced asthma model. Using a single-component Anova / Dunnett system, compared to an OVA+ vehicle, **p<0.01, ***p<0.001. [Figure 9A] Figures 9A-9C show the binding of humanized anti-IL-25 antibodies to recombinant human IL-25 (9A), cynomolgus monkey IL-25 (9B), and mouse IL-25 (9C) as measured by ELISA. [Figure 9B] Figures 9A-9C show the binding of humanized anti-IL-25 antibodies to recombinant human IL-25 (9A), cynomolgus monkey IL-25 (9B), and mouse IL-25 (9C) as measured by ELISA. [Figure 9C] Figures 9A-9C show the binding of humanized anti-IL-25 antibodies to recombinant human IL-25 (9A), cynomolgus monkey IL-25 (9B), and mouse IL-25 (9C) as measured by ELISA. [Figure 10] Figure 10 shows that a humanized anti-IL-25 antibody inhibits NFκB signaling induced by human IL-25 in HEK-Blue IL-17 reporter cells. [Figure 11] Figure 11 shows that a humanized anti-IL-25 antibody inhibits IL-25-induced CXCL-1 production in the human colon cancer cell line HT29. [Figure 12] Figure 12 shows that a humanized anti-IL-25 antibody inhibits IL-25-induced IL-5 production in a human PBMC assay. [Modes for carrying out the invention]

[0042] To make this disclosure more easily understandable, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which this disclosure belongs.

[0043] Throughout this disclosure, the following abbreviations will be used: mAb, Mab, or MAb monoclonal antibody. Complementarity-determining regions within the CDR-immunoglobulin variable region. HCDR - Heavy Chain Complementarity Determination Region. LCDR - Light chain complementarity determination region. VH-immunoglobulin heavy chain variable region. VL - Variable region of the immunoglobulin light chain. Immunoglobulin variable regions excluding the FR-antibody framework region and the CDR region.

[0044] As used in this disclosure, the term “interleukin-25” (used interchangeably with “IL-25”) refers to the native sequence polypeptides, isoforms, chimeric polypeptides, all homologs, fragments, and precursors of human IL-25. The amino acid sequences of human, cynomolgus monkey, and mouse IL-25 are provided in the NCBI reference sequences: NP_073626.1 (human) (SEQ ID NO: 41), XP_005560919.1 (cynomolgus monkey) (SEQ ID NO: 43), and NP_542767.1 (mouse) (SEQ ID NO: 42). Orthologs of IL-25 share approximately 92% and approximately 80% homology with the human protein in cynomolgus monkeys and mice, respectively.

[0045] As used in this disclosure, the term “IL-25 complex” refers to the IL-17RA / IL-17RB complex, also known as the IL-25 receptor.

[0046] As used in this disclosure, the term "IL-17RA" refers to the interleukin-17 receptor A, also known as CDw217 (cluster of differentiation w217). The protein encoded by this gene (interleukin-17A receptor; IL-17RA) is a ubiquitous type I membrane glycoprotein that binds to interleukin-17A with low affinity.

[0047] As used in this disclosure, the term "IL-17RB" refers to interleukin-17 receptor B. This receptor specifically binds to IL-17B and IL-17E (IL-25), but does not bind to either IL-17A or IL-17C.

[0048] The term "antibody" as used in this disclosure is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies).

[0049] Exemplary antibodies such as IgG include two heavy chains and two light chains. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this disclosure) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL in this disclosure) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0050] The hypervariable regions generally consist of approximately 24-34 amino acid residues (LCDR1; "L" indicates light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region, and approximately 31-35B (HCDR1; "H" indicates heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), and / or residues that form hypervariable loops (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region, and 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region, including amino acid residues from Chothia and Lesk (1987) J. Mol Biol. 196:901-917).

[0051] As used in this disclosure, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. For example, the individual antibodies comprising this population are identical and / or bind to the same epitope, except for any variant antibodies that may exist, such as naturally occurring mutations or those that arise during the production of the monoclonal antibody preparation and are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed to different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Thus, the modifier “monoclonal” characterizes the antibody as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any arbitrary method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of methods, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus. Such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0052] The term "chimeric antibody" refers to a recombinant antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining portion of the heavy chain and / or light chain originates from a different source or species.

[0053] A “human antibody” is an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or is produced using any technique for producing human antibodies known to those skilled in the art. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including the methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al, J. Immunol, 147(I):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodies are produced by administering antigens to transgenic humanized animals that have been modified to produce the antibody in response to antigen exposure, but whose endogenous gene loci have been rendered inoperable, for example, immunized HuMab mice (see, e.g., Nils Lonberg et al., 1994, Nature 368:856-859, WO98 / 24884, WO94 / 25585, WO93 / 1227, WO92 / 22645, WO92 / 03918, and WO01 / 09187 on HuMab mice), xeno mice (see, e.g., U.S. Patents 6,075,181 and 6,150,584 on XENOMOUSE® technology). Alternatively, it can be prepared using Trianni mice (e.g., WO2013 / 063391, WO2017 / 035252, and WO2017 / 136734).

[0054] The term "humanized antibody" refers to an antibody that has been engineered to include one or more human framework regions in its variable region, along with non-human (e.g., mouse, rat, or hamster) complementarity-determining regions (CDRs) in its heavy and / or light chains. In certain embodiments, a humanized antibody contains a sequence that is entirely human except for the CDR regions. Humanized antibodies typically exhibit lower immunogenicity to humans compared to non-humanized antibodies and therefore offer therapeutic benefits in certain situations. Those skilled in the art will recognize humanized antibodies and the appropriate techniques for their production. For example, Hwang, WYK, et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature, 321:522-25, 1986; Riechmann et al., Nature, 332:323-27, 1988;Verhoeyen et al., Science, 239:1534-36, 1988;Orlandi et al., Proc. Natl. Acad. Sci. USA,86:3833-37, See U.S. Patent Nos. 5,225,539, 5,530,101, 5,585,089, 5,693,761, 5,693,762, 6,180,370; and Selick et al., WO 90 / 07861. Each of these is incorporated in whole within this disclosure by reference.

[0055] The "class" of an antibody refers to the type of constant domain or constant region present in its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0056] The term “antigen-binding domain” (or simply “binding domain”) or similar terms refer to one or more fragments of an antibody that possess the ability to specifically bind to an antigen complex. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include: (i) the Fab fragment, a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH domain; (ii) the F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide crosslink at a hinge region; (iii) the Fd fragment, consisting of a VH domain and a CH domain; (iv) the Fv fragment, consisting of the VL domain and VH domain of a single arm of the antibody; (v) the dAb fragment, consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a combination of two or more isolated CDRs that can be arbitrarily joined by a synthetic linker.

[0057] When these terms are used in this disclosure, “complementarity-determining regions” or “CDRs” refer to short polypeptide sequences located within the variable regions of both heavy-chain and light-chain polypeptides, which are primarily involved in mediating the recognition of a particular antigen. Within each VL and each VH, there are three CDRs (referred to as CDR1, CDR2, and CDR3).

[0058] As will be understood by those skilled in the art, the exact numbering and arrangement of CDRs may differ between different numbering systems. However, it should be understood that the disclosure of a variable heavy chain sequence and / or a variable light chain sequence includes the disclosure of the corresponding CDR. Thus, the disclosure of each variable heavy chain region is a disclosure of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light chain region is a disclosure of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0059] In certain embodiments, the CDR of an antibody can be determined according to the IMGT numbering system described in Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al, (1999) Nucleic Acids Res 27: 209-212, each of which is incorporated in whole by reference in this disclosure. Unless otherwise stated in this disclosure, references to residue numbers in the variable domain of an antibody mean residue numbering according to the Kabat numbering system.

[0060] In other embodiments, the antibody CDR can be determined according to MacCallum RM et al, (1996) J Mol Biol 262: 732-745, which is incorporated in its entirety by reference. See also, for example, Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), which is incorporated in its entirety by reference. In other embodiments, the antibody CDR can be determined according to the AbM numbering rule, which refers to the AbM hypervariable region, representing a compromise between the Kabat CDR and the Chothia structural loop, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group), which is incorporated in its entirety by reference.

[0061] "Framework," "framework region," or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4.

[0062] The "Human Consensus Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), Vols. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I, as described in Kabat et al. (above). In one embodiment, for VH, the subgroup is subgroup III, as described in Kabat et al. above.

[0063] The "hinge region" is generally defined as the extent of human IgG1 between 216 and 238 (EU numbering) or 226 and 251 (Kabat numbering). The hinge can be further divided into three distinct regions: the upper hinge, the middle (e.g., core) hinge, and the lower hinge.

[0064] The term “Fc region” as used in this disclosure is used to define the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of the constant region. This term includes the Fc region of the natural sequence and the Fc region of variants. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the lysine (Lys447) at the C-terminus of the Fc region may or may not be present. Unless otherwise specified in this disclosure, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0065] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of an antigen.

[0066] An antibody that "binds to the same epitope as the reference antibody" refers to an antibody that contacts a set of overlapping amino acid residues of the antigen compared to the reference antibody, or that blocks the binding of the reference antibody to the antigen by 50% or more in a competitive assay. The amino acid residues of the antibody that contact the antigen can be determined, for example, by determining the crystal structure of the antibody complexed with the antigen, or by performing hydrogen / deuterium exchange. In some embodiments, antibody residues within 5 Å of the antigen are considered to be in contact with the antigen. In some embodiments, an antibody that binds to the same epitope as the reference antibody blocks the binding of the reference antibody to that antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of its antibody to that antigen by 50% or more in a competitive assay.

[0067] The term "antibody fragment" refers to molecules other than the intact antibody, including a portion of the intact antibody that binds to 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, linear antibodies, and single-chain antibody molecules (e.g., scFv). Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments and the remaining "Fc" fragment, whose name reflects its ability to crystallize easily. A Fab fragment consists of the entire light (L) chain, the variable region domain (VH) of the heavy (H) chain, and the first constant domain (CH1) of a single heavy chain. Pepsin treatment of an antibody yields a single large F(ab)2 fragment, which roughly corresponds to two disulfide-linked Fab fragments that retain bivalent antigen-binding activity and are still capable of crosslinking antigens. Fab fragments differ from Fab' fragments in that they have a small number of additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines derived from the antibody hinge region. Fab'-SH is the name used in this disclosure for Fab' having a free thiol group in the cysteine ​​residue of the constant domain. The F(ab')2 antibody fragment was originally produced as a pair with Fab' fragments having a hinged cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0068] "Fv" consists of a dimer of one heavy chain variable domain and one light chain variable domain in a tight, non-covalent association. From the folding of these two domains, six hypervariable loops (three from the H chain and three from the L chain) are generated, which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0069] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing a VH antibody domain and a VL antibody domain linked to a single polypeptide chain. Preferably, the sFv polypeptide further includes a polypeptide linker between the VH domain and the VL domain, thereby enabling the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0070] When used to describe the various antibodies disclosed herein, the term “isolated antibody” means an antibody identified, isolated, and / or recovered from the cells or cell cultures on which it is expressed. Its natural environment contaminant components are typically materials that interfere with the diagnostic or therapeutic use of polypeptides and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, antibodies are purified to a purity greater than 95% or 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For an overview of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007). In one embodiment, the antibody is purified (1) by using a spinning cup sequencer to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (2) by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue staining or preferably silver staining until homogeneous.

[0071] With respect to the binding of an antibody to a target molecule, the terms "specific binding" or "specifically binds thereto" or "is specific for" with respect to a particular polypeptide or an epitope on a particular polypeptide target mean a binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule as compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target, etc. In this case, specific binding is indicated when the binding of the labeled target and the probe is competitively inhibited by the excess unlabeled target. As used in the present disclosure, the terms "specific binding to" or "specifically binds to" or "is specific for" an epitope on a particular polypeptide or a particular polypeptide target mean, for example, 10 -4 M or less, or 10 -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or less, or 10 -11 M or less, or 10 -12 M or less for the Kd for the target, or 10 -4 M to 10 -6 M, or 10 -6 M to 10 -10 M, or 10 -7 M to 10 -9 M and can be shown by a molecule having a Kd in the range. As will be understood by those skilled in the art, affinity and KD value are inversely proportional. High affinity for an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to a binding in which a molecule binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. As used in the present disclosure, the terms "specific binding", "specifically binds", and "selectively binds" refer to antibody binding to an epitope of interleukin-25.

[0072] As used in this disclosure, the term "affinity" means the strength of binding of an antibody to an epitope. The affinity of an antibody is given by a dissociation constant Kd, defined as [Ab] × [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody, and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1 / Kd. Methods for determining the affinity of mAbs are described in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), and these references are fully incorporated herein by reference. One standard method well known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (e.g., by analysis using a BIAcore® SPR analyzer).

[0073] An "epitope" refers to one or more sites of interaction between an antibody and its antigen. According to Janeway, C. Jr., P. Travers, et al. (2001). Immunobiology: the immune system in health and disease. Part II, Section 3-8. New York, Garland Publishing, Inc., "Antibodies generally recognize only small regions on the surface of macromolecules such as proteins... [Specific epitopes] are often composed of amino acids derived from different parts of the [antigen] polypeptide chain joined by protein folding. This type of antigenic determinant is known as a three-dimensional or discontinuous epitope because the recognized structure is discontinuous in the amino acid sequence of the antigen but consists of segments of the protein joined in the three-dimensional structure. In contrast, epitopes composed of a single segment of the polypeptide chain are called continuous or linear epitopes" (Janeway, C. Jr., P. Travers, et al. (2001). Immunobiology: the immune system in health and disease. Part II, Section 3-8. New York, Garland Publishing, Inc.).

[0074] When used in this disclosure, the term "KD" is intended to refer to the dissociation constant of a particular antibody-antigen interaction. This is calculated by the following formula: Koff / Kon = KD.

[0075] When used in this disclosure, "IC" 50 The term "effective concentration" is intended to refer to the effective concentration of the antibody of the present invention required to inhibit a specific biological or biochemical function by 50%.

[0076] "EC" regarding active substances and their specific activities (e.g., binding to cells, inhibition of enzyme activity, activation or inhibition of immune cells) 50"Efficiency of the active substance" refers to the efficient concentration of the active substance that produces 50% of the maximum response or effect related to such activity. 100 "The efficient concentration of the active substance that produces substantially the maximum response with respect to such activity."

[0077] As used in this disclosure, the terms “antibody-based immunotherapy” and “immunotherapy” are used to broadly refer to any form of therapy that relies on the targeting specificity of an anti-IL-25 antibody, bispecific molecule, multispecific molecule, conjugate, or fusion protein containing an IL-25-specific conjugate to mediate a direct or indirect effect on cells characterized by the abnormal expression of anti-IL-25. The terms are intended to encompass therapeutic methods using naked antibodies, and include, but are not limited to, bispecific antibodies (including T cell engagement, NK cell engagement, and other immune cell / effector cell engagement forms), antibody-drug conjugates, cell therapies using T cells (CAR-T) or NK cells (CAR-NK) engineered to contain an IL-25-specific chimeric antigen receptor, oncolytic viruses containing an IL-25-specific conjugate, and gene therapies by delivering an antigen-binding sequence of an anti-IL-25 antibody and expressing the corresponding antibody fragment in vivo.

[0078] As used in this disclosure, the term “Group II Innate Lymphocytes” (ILC2) refers to a type of immune cell that plays a crucial role in the early stages of the immune response to parasites, allergens, and other environmental damage. ILC2s are part of the innate immune system, meaning they can respond rapidly to a variety of stimuli without prior exposure to or specific recognition of the pathogen. ILC2s are characterized by their ability to produce a variety of cytokines, including interleukin (IL)-5 and IL-13, which mobilize and activate other immune cells such as eosinophils, mast cells, and T helper 2 (Th2) cells, in order to promote a type II immune response. This type of response is important in dealing with parasitic infections, as well as in allergic reactions and tissue repair.

[0079] As used in this disclosure, the term “Type II inflammation” is also known as Type II immune response and is a specific type of immune response that is activated in response to a variety of stimuli, including parasitic infections, allergens, and tissue damage. Type II inflammation is characterized by the activation of immune cells such as Group II innate lymphoid cells (ILC2), eosinophils, mast cells, and T helper 2 (Th2) cells, which produce cytokines such as interleukin (IL)-4, IL-5, and IL-13.

[0080] IL-25 / IL-25R shaft I. IL-17 Cytokine Family The IL-17 family is a group of cytokines consisting of the following six members: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (also known as IL-25), and IL-17F. These cytokines are produced by various types of immune cells, including T cells, mast cells, and innate lymphoid cells, and play important roles in both innate and adaptive immune responses (McGeachy, MJ Immunity. 2019 Apr 16; 50(4): 892-906).

[0081] IL-17A and IL-17F are the most well-studied members of the IL-17 family, often co-expressed, and possess similar functions. They promote inflammation by inducing the expression of pro-inflammatory cytokines, chemokines, and adhesion molecules, as well as by stimulating the recruitment and activation of neutrophils and other immune cells.

[0082] IL-17B, IL-17C, and IL-17E have more specialized roles. IL-17B and IL-17C are involved in host defense against bacterial and fungal infections, while IL-17E is important for protection against allergic reactions and parasitic infections.

[0083] The IL-17 cytokine interacts with various cell types expressing different heterodimeric receptor complexes consisting of five homologous subunits from IL-17-RA to IL-17RE, with IL-17RA being common to all receptors. Each subunit of the IL-17 receptor is a protein containing a single transmembrane domain with several conserved motifs, including an extracellular fibronectin III-like motif, a transmembrane region, and a cytoplasmic SEF / IL-17R (SEFIR) domain (Zhang Q, J Biol Chem. 2013;288(51):36956-36965).

[0084] IL-17 family cytokines are involved in the pathogenesis of various inflammatory and autoimmune diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease, and are targets for therapeutic interventions in these conditions.

[0085] II. IL-25 (IL-17E) IL-25 shares approximately 23%–33% homology with other IL-17 family members and transmits signals by binding to specific homologous IL-17 receptors. The low sequence similarity between IL-25 and other family members suggests that anti-IL25 specific antibodies are less likely to bind to other IL-17 family members.

[0086] IL-25 is synthesized as a precursor molecule that undergoes processing and cleavage to produce the mature, biologically active form of cytokines. Cleavage of the IL-25 precursor is mediated by several proteases, including furin and other proprotein converters (PCs) that cleave the precursor protein at specific sites to produce mature cytokines. The exact cleavage site and mechanism vary depending on the cell type or tissue in which it is produced. In particular, proteolytic cleavage of IL-25 at specific sites can enhance or inhibit its ability to activate downstream signaling pathways (Matsushita, K. et al. JCI Insight. 2020 Feb 27;5(4):e131480).

[0087] IL-25 is secreted as a disulfide-linked homodimeric glycoprotein and is produced by various cells, including epithelial cells, T cells, and innate lymphoid cells.

[0088] IL-25 interacts with the IL-25 receptor, which includes the heterodimeric IL-17RB / IL-17RA receptor subunit, and signals through it (Iwakura, et.al., (2010), Immunity, 34:149). Through its receptor, IL-25 promotes and maintains the Th2 immune response and induces the proliferation of type 2 innate lymphoid cells (ILC2) and M2 macrophages.

[0089] IL-25 is widely produced by various cell types, but the expression of its receptor is more limited. IL-25 receptors are present on innate immune cells such as immutable natural killer T (iNKT) cells, ILC2s, eosinophils, basophils, mast cells, and antigen-presenting cells (APCs). How cellular expression of IL-17RB and IL-17RB expression are altered in different disorders, particularly airway inflammatory disorders and skin inflammation (Rickel EA, J Immunol. 2008;181(6):4299-4310).

[0090] IL-25 interacts exclusively with IL-17RB, allosterically facilitating the formation of the IL-17RB-IL-17RA "tip-to-tip" interface, a crucial receptor-receptor interaction necessary to initiate signal transduction.

[0091] Upon binding to its receptor complex, IL-25 induces the recruitment and phosphorylation of ACT1, which plays a crucial role as an adapter protein for downstream signaling. IL-25 has been shown to activate several downstream signaling cascades in a cellular context-dependent manner, including nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and Janus kinase / signal transducer and activator of transcription (JAK / STAT) (Borowczyk J et. Al., J Allergy Clin Immunol. Jul;148(1):40-52(2021), resulting in the expression of various pro-inflammatory cytokines, chemokines, and other immune-related genes (Amini P., et al. (2018). American Journal of Rhinology & Allergy, 32(2), 115-124.).

[0092] III. Mechanism of IL-25 action in pathogenesis IL-25 may enhance the Th2 immune response. Th2 cells are a subset of T cells that produce cytokines such as IL-4, IL-5, and IL-13, which are involved in allergic reactions and defense against parasites. IL-25 can stimulate the differentiation of naive T cells into Th2 cells and enhance the production of Th2 cytokines. This effect is important in regulating allergic reactions and host defense against certain infections (Liu et al, J Immunol Res 2018).

[0093] IL-25 can also stimulate innate lymphoid cells (ILC2s). ILCs are a group of immune cells that play a role in defense against pathogens and tissue repair. ILC2s are a subset of ILCs that produce Th2 cytokines and are involved in allergic reactions. IL-25 stimulates the activation and proliferation of ILC2s, leading to the production of Th2 cytokines and the initiation of an immune response (Wu. J. Front Immunol. 2022; 13: 986118).

[0094] IL-25 may also promote the accumulation of inductive costimulators (ICOS) and T1 / ST2 on nuocytes. Nuocytes are a recently discovered subset of innate immune cells that produce Th2 cytokines and play a role in allergy and host defense. IL-25 can promote the accumulation of ICOS and T1 / ST2, molecules expressed on the surface of nuocytes that are involved in their activation and function (C. Wang, et al. PLoS One, vol. 11, no. 9).

[0095] IL-25 can also stimulate the differentiation of naive T cells into Th2 cells, leading to the production of Th2 cytokines such as IL-4, IL-5, and IL-13.

[0096] Furthermore, IL-25 increases chemokine production and promotes the recruitment of eosinophils and inflammation. Chemokines regulate the migration and activation of immune cells. IL-25 can increase the production of chemokines that promote the recruitment of eosinophils involved in defense against parasites and allergic reactions. This recruitment may lead to inflammation and tissue damage (J. Beale, et al Sci Trans Med, v6, no.256, (2014)).

[0097] IL-25 can also promote airway remodeling, which refers to structural changes in the airways that occur in response to chronic inflammation. This may include increased mucus secretion, deposition of extracellular collagen, proliferation of smooth muscle cells, and angiogenesis (formation of new blood vessels). These changes can lead to airway obstruction and respiratory problems (M. SuzuLNG, et al., J Immunol. 2012 Oct 1;189(7)).

[0098] IV. Therapeutic targeting of IL-25-related diseases and disorders a. Type 2 inflammatory disorder Respiratory diseases such as asthma and allergies are characterized by type 2 inflammation. IL-25, a type 2 cytokine produced by Th2 cells, induces the production of IL-4, IL-5, and IL-13, leading to respiratory tract inflammation.

[0099] Asthma is a heterogeneous inflammatory disease characterized by airway obstruction, wheals, eosinophilia, and neutrophilia. While asthma can be broadly categorized into two types—eosinophilic (T-helper 2 [Th2]) and non-eosinophilic (non-Th2)—multiple phenotypes with diverse underlying biological makeups are recognized.

[0100] Elevated plasma levels of IL-25 are associated with the allergic asthma phenotype (Tang W., et al. Int Arch Allergy Immunol; 163: 5-10 (2014)), and salivary IL-25 concentration correlates with disease severity. Salivary IL-25 is also increased in atopic asthma patients compared to non-atopic asthma patients (Paplinska-Goryca M. et al. Postepy Dermatol Alergol; 35: 462-469 (2018)). The relationship between IL-25 and neutrophilic airway inflammation suggests a multifaceted role of IL-25 in the immune response in asthma. Furthermore, rare alleles of the IL-25 receptor, IL-17RB, have been associated with a reduced incidence of asthma (Jung JS, et al. Association of IL-17RB gene polymorphism with asthma. Chest 135: 1173-1180 (2009)).

[0101] In animal models of asthma, overexpression or administration of recombinant IL-25 induces allergic reactions characterized by Th2 cytokine expression, eosinophilia, and mucus secretion (U.S. Patent No. 6,159,193). The effect of blocking IL-25 may be due to both a reduction in Th2 cytokines in allergic reactions and inhibition of the expression of key chemokines that promote exacerbated inflammatory responses (Petersen BC et al., Future Med Chem;4:833-6(2012)). Administration of anti-IL-25 antibodies has been shown to significantly reduce airway hyperreactivity, Th2-related cytokine levels, IgE levels, and goblet cell hyperplasia (Fort MM, et al., Immunity;15:985-95(2001), Rickel EA, et al., J Immunol;181:4299-310(2008), Ballantyne, SJ et al., Journal of Allergy and Clinical Immunology:120(6):1324-1331(2007)). Furthermore, IL-25 knockout mice showed reduced pulmonary pathology in an asthma model (Ballantyne, SJ, et al., Borowczyk J et al., J Allergy Clin Immunol. Jul;148(1):40-52(2021)). These data suggest that targeting IL-25 or IL-17RB+ immune cells may be a promising strategy for treating allergic inflammation, as demonstrated in preclinical studies using the anti-IL-25 monoclonal antibody ABM125 (Lee, J., et al. anti-IL-25 Biochemical and biophysical research communications, 495(1), 1391-1397).

[0102] Psoriasis is an autoimmune disease characterized by epidermal hyperplasia, increased angiogenesis, and skin inflammation. While the exact cause of psoriasis is unknown, research suggests that Th1 / Th2 cell imbalance and Th17 cell involvement may contribute to the disease. IL-25, a cytokine produced by keratinocytes, has been found to promote skin inflammation by recruiting neutrophils and activating macrophages. IL-25 is highly expressed in skin lesions of psoriasis patients, promoting the proliferation of IL-17RB+ keratinocytes and exacerbating the disease. Keratinocytes can be activated by IL-25 via the activation of the STAT3 transcription factor, which leads to IL-17RB expression (Xu M, et al., Inflammation. Immunity (2018) 48(4):787-98). Blocking IL-17RA, a co-receptor for IL-17A, IL-17F, and IL-25, has shown high efficacy in the treatment of psoriasis, suggesting that blocking IL-25 may be a promising strategy for targeting skin inflammation.

[0103] b.Autoimmune disease Studies suggest that IL-25 may possess both pro-inflammatory and anti-inflammatory effects, depending on the specific disease state and the cells involved. In some autoimmune diseases, such as rheumatoid arthritis (RA) and multiple sclerosis (MS), IL-25 has been found to have protective effects as both an anti-inflammatory cytokine and an inhibitor of both innate and adaptive immunity. In several diseases, such as inflammatory bowel disease (IBD) and systemic lupus erythropathy (SLE), IL-25 plays a dual role in regulating the immune response during disease development, exhibiting both pro-inflammatory and anti-inflammatory effects. (Deng, D., et.al. Front Immunol. 2021;12:691559).

[0104] However, in other autoimmune diseases such as Sjögren's syndrome (SS), studies have shown that IL-25 plays a pathogenic role during the development of SS. Studies have shown that IL-17A plays a crucial role in the pathogenesis of Sjögren's syndrome by promoting Th17 cell activity. Recently, IL-25 has been observed to be significantly increased in SG and peripheral blood of SS patients and to promote inflammatory ILC2 activation. Blocking IL-25 using neutralizing antibodies improved salivary flow and SG tissue damage in mice with experimental SS, accompanied by a decrease in ILC2 infiltration. Upregulation of TRAF6 in CD3+ T cells and ILC2 in SG in pSS patients suggests that IL-25 signaling is functional via coordinated activation of ERK1 / 2 and relative transcription factors (Guggino G, Arthritis Rheumatol (2018) 70(8):1265-75).

[0105] Overall, the impact of IL-25 on autoimmune diseases is complex and context-dependent, and further research is needed to fully understand its role in these diseases and explore its potential as a therapeutic target. Blocking IL-25 may be a promising strategy for targeting autoimmune diseases such as Sjögren's syndrome.

[0106] c. Cancer IL-25 has been shown to have potential anti-cancer effects by modulating the immune response and inhibiting tumor growth (Shabgah.A, et al Cancer Med. 2021 Aug; 10(15): 5191-5202).

[0107] IL-25 can stimulate the activity of immune cells such as natural killer (NK) cells, dendritic cells, and CD8+ T cells, which can directly kill cancer cells. It can also promote the production of other cytokines that enhance immune cell activity, such as interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) (Sfanos, KS, et.al. (2014). Clinical cancer research, 20(12), 3254-3265).

[0108] In addition to its immune-boosting effects, IL-25 has been found to have direct antitumor effects in various types of cancer. For example, IL-25 has been shown to induce apoptosis (programmed cell death) in prostate cancer cells and inhibit the growth of colon cancer cells by blocking the progression of the cell cycle (Zhang, J., et.al (2019). Oncology letters, 18(5), 5179-5186).

[0109] Furthermore, IL-25 has been found to enhance the effectiveness of chemotherapy and radiotherapy in preclinical models of cancer. IL-25 therapy has been shown to enhance the effects of radiotherapy by sensitizing cancer cells to chemotherapy, stimulating immune responses, and reducing tumor vascular density by increasing their sensitivity to cell death (Bao, H et al. (2017). Scientific Reports, 7(1), 1-11).

[0110] In summary, these findings suggest that IL-25 has promising potential as an anticancer agent, either alone or in combination with other therapies. However, further research is needed to fully understand the mechanism underlying its antitumor effect and to explore its potential as a therapeutic agent in clinical practice.

[0111] Anti-IL-25 antibody The anti-IL-25 antibodies of this disclosure (e.g., IL25Ab1, IL25Ab2, IL25Ab3, IL25Ab4, IL25Ab5, IL25Ab6, IL25Ab7, IL25Ab8, IL25Ab9, IL25Ab10, and IL25Ab11) bind to human IL-25 (e.g., specifically). Such antibodies and their fragments may be useful for disrupting the IL-25 / IL-25R binding interaction. These antibodies and their fragments are characterized by a unique set of CDR sequences, specificity for IL-25, and are useful as monotherapy or in combination with other anticancer agents in type 2 inflammatory diseases, autoimmune diseases, or cancer. More specifically, this disclosure relates to antibodies and their fragments that bind to human IL-25, and their use for modulating IL-25-mediated activity of cells localized in the inflammatory disease microenvironment or tumor microenvironment.

[0112] In one embodiment, an anti-IL-25 antibody or an antibody fragment thereof includes a heavy chain variable region (VH) having a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 1. For example, an anti-IL-25 antibody or an antibody fragment thereof may be configured to include a set of CDRs corresponding to those CDRs in one or more of the anti-IL-25 antibodies disclosed in Table 1 (e.g., the HCDRs of the IL25Ab1 antibody).

[0113] In another embodiment, the anti-IL-25 antibody or its fragment comprises a light chain variable region (VL) having a set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 2. For example, the anti-IL-25 antibody or its antibody fragment may be configured to include a set of CDRs corresponding to those CDRs in one or more of the anti-IL-25 antibodies disclosed in Table 2 (e.g., LCDRs of IL25Ab1 antibody).

[0114] In yet another embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises a VH having a set of CDRs (HCDR1, HCDR2, and HCDR3) as disclosed in Table 1, and a VL having a set of CDRs (LCDR1, LCDR2, and LCDR3) as disclosed in Table 2.

[0115] [Table 1]

[0116] [Table 2]

[0117] In one embodiment, an anti-IL-25 antibody or an antibody fragment thereof includes a humanized heavy chain variable region (VH) having a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 3. For example, an anti-IL-25 antibody or an antibody fragment thereof may be configured to include a set of CDRs corresponding to those CDRs in one or more anti-IL-25 antibodies disclosed in Table 1 (e.g., the HCDRs of the IL25Ab6 antibody).

[0118] In another embodiment, the anti-IL-25 antibody or its fragment comprises a humanized light chain variable region (VL) having a set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 4. For example, the anti-IL-25 antibody or its antibody fragment may be configured to include a set of CDRs corresponding to those CDRs in one or more of the anti-IL-25 antibodies disclosed in Table 2 (e.g., LCDRs of IL25Ab6 antibodies).

[0119] In yet another embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises a VH having a set of CDRs (HCDR1, HCDR2, and HCDR3) as disclosed in Table 3, and a VL having a set of CDRs (LCDR1, LCDR2, and LCDR3) as disclosed in Table 4.

[0120] [Table 3]

[0121] [Table 4]

[0122] In one embodiment, the anti-IL-25 antibody or the antibody fragment thereof is (i) HCDR1: Sequence ID 11, HCDR2: Sequence ID 12, HCDR3: Sequence ID 13; (ii) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, HCDR3: SEQ ID NO: 19; (iii) HCDR1: Sequence ID 23, HCDR2: Sequence ID 24, HCDR3: Sequence ID 25; (iv) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 30, HCDR3: SEQ ID NO: 31; (v)HCDR1:Sequence ID 35, HCDR2:Sequence ID 36, HCDR3:Sequence ID 37; (vi) HCDR1: Sequence ID 23, HCDR2: Sequence ID 49, HCDR3: Sequence ID 25, and (vii) A VH having a set of complementarity determination regions (HCDR1, HCDR2, and HCDR3) selected from the group consisting of HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 52, and HCDR3: SEQ ID NO: 25.

[0123] In another embodiment, an anti-IL-25 antibody or an antibody fragment thereof is (i)LCDR1: Sequence ID 14, LCDR2: Sequence ID 15, LCDR3: Sequence ID 16; (ii) LCDR1: Sequence ID 20, LCDR2: Sequence ID 21, LCDR3: Sequence ID 22; (iii) LCDR1: Sequence ID 26, LCDR2: Sequence ID 27, LCDR3: Sequence ID 28; (iv)LCDR1:Sequence ID 32, LCDR2:Sequence ID 33, LCDR3:Sequence ID 34; (v)LCDR1:Sequence ID 38, LCDR2:Sequence ID 39, LCDR3:Sequence ID 40; and (vi) A VL having a set of complementarity determination regions (LCDR1, LCDR2, and LCDR3) selected from the group consisting of LCDR1: SEQ ID NO: 50, LCDR2: SEQ ID NO: 51, and LCDR3: SEQ ID NO: 28.

[0124] In another embodiment, an anti-IL-25 antibody or an antibody fragment thereof is (a) (i) HCDR1: Sequence ID 11, HCDR2: Sequence ID 12, HCDR3: Sequence ID 13; (ii) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, HCDR3: SEQ ID NO: 19; (iii) HCDR1: Sequence ID 23, HCDR2: Sequence ID 24, HCDR3: Sequence ID 25; (iv) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 30, HCDR3: SEQ ID NO: 31; (v)HCDR1:Sequence ID 35, HCDR2:Sequence ID 36, HCDR3:Sequence ID 37; (vi) HCDR1: Sequence ID 23, HCDR2: Sequence ID 49, HCDR3: Sequence ID 25, and (vii) A VH having a set of complementarity determination regions (HCDR1, HCDR2, and HCDR3) selected from the group consisting of HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 52, and HCDR3: SEQ ID NO: 25, (b) (i)LCDR1: Sequence ID 14, LCDR2: Sequence ID 15, LCDR3: Sequence ID 16; (ii) LCDR1: Sequence ID 20, LCDR2: Sequence ID 21, LCDR3: Sequence ID 22; (iii) LCDR1: Sequence ID 26, LCDR2: Sequence ID 27, LCDR3: Sequence ID 28; (iv)LCDR1:Sequence ID 32, LCDR2:Sequence ID 33, LCDR3:Sequence ID 34; (v)LCDR1:Sequence ID 38, LCDR2:Sequence ID 39, LCDR3:Sequence ID 40; (vi) A VL having a set of complementarity determination regions (LCDR1, LCDR2, and LCDR3) selected from the group consisting of LCDR1: SEQ ID NO: 50, LCDR2: SEQ ID NO: 51, and LCDR3: SEQ ID NO: 28.

[0125] In the embodiment, the antibody or antibody fragment comprises a VH and VL combination having a set of complementarity-determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of: (i) VH:CDR1:Sequence ID 11, CDR2:Sequence ID 12, CDR3:Sequence ID 13, VL:CDR1:Sequence ID 14, CDR2:Sequence ID 15, CDR3:Sequence ID 16; (ii) VH:CDR1:Sequence ID 17, CDR2:Sequence ID 18, CDR3:Sequence ID 19, VL:CDR1:Sequence ID 20, CDR2:Sequence ID 21, CDR3:Sequence ID 22; (iii) VH:CDR1:Sequence ID 23, CDR2:Sequence ID 24, CDR3:Sequence ID 25, VL:CDR1:Sequence ID 26, CDR2:Sequence ID 27, CDR3:Sequence ID 28; (iv) VH:CDR1:SEQ ID NO: 29, CDR2:SEQ ID NO: 30, CDR3:SEQ ID NO: 31, VL:CDR1:SEQ ID NO: 32, CDR2:SEQ ID NO: 33, CDR3:SEQ ID NO: 34; (v)VH:CDR1:SEQ ID NO: 35, CDR2:SEQ ID NO: 36, CDR3:SEQ ID NO: 37, VL:CDR1:SEQ ID NO: 38, CDR2:SEQ ID NO: 39, CDR3:SEQ ID NO: 40; (vi) VH:CDR1:Sequence ID 23, CDR2:Sequence ID 49, CDR3:Sequence ID 25, VL:CDR1:Sequence ID 50, CDR2:Sequence ID 51, CDR3:Sequence ID 28; and (vii)VH:CDR1:Sequence ID 23, CDR2:Sequence ID 52, CDR3:Sequence ID VL:CDR1:Sequence ID 50, CDR2:Sequence ID 51, CDR3:Sequence ID 28.

[0126] In one embodiment, the anti-IL-25 antibody or the antibody fragment thereof comprises a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 44, and 47; and / or a variable light chain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, and 48.

[0127] In one embodiment, an anti-IL-25 antibody or an antibody fragment thereof comprises a pair of variable heavy chain sequences and variable light chain sequences selected from the following combinations: a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2; a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4; a variable heavy chain sequence comprising SEQ ID NO: 5 and a variable light chain sequence comprising SEQ ID NO: 6; a variable heavy chain sequence comprising SEQ ID NO: 7 and a variable light chain sequence comprising SEQ ID NO: 8; a variable heavy chain sequence comprising SEQ ID NO: 9 and a variable light chain sequence comprising SEQ ID NO: 10; a variable heavy chain sequence comprising SEQ ID NO: 44 and a variable light chain sequence comprising SEQ ID NO: 45; a variable heavy chain sequence comprising SEQ ID NO: 44 and a variable light chain sequence comprising SEQ ID NO: 46; and a variable heavy chain sequence comprising SEQ ID NO: 47 and a variable light chain sequence comprising SEQ ID NO: 48. Those skilled in the art will further understand that the variable light chains and variable heavy chains may be independently selected or mixed and fitted together to prepare an anti-IL-25 antibody or an antibody fragment thereof comprising combinations of variable heavy chains and variable light chains different from the pairs specified above.

[0128] In one embodiment, an anti-IL-25 antibody or an antibody fragment thereof comprises a pair of variable heavy chain sequences and variable light chain sequences selected from the following combinations: a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 1 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 2; a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 3 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 4; a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 5 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 6; a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 7 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 8 Variable light chain sequences that are 90%, 95%, or 99% identical; variable heavy chain sequences that are 90%, 95%, or 99% identical to sequence number 9 and variable light chain sequences that are 90%, 95%, or 99% identical to sequence number 10; variable heavy chain sequences that are 90%, 95%, or 99% identical to sequence number 44 and variable light chain sequences that are 90%, 95%, or 99% identical to sequence number 45; variable heavy chain sequences that are 90%, 95%, or 99% identical to sequence number 44 and variable light chain sequences that are 90%, 95%, or 99% identical to sequence number 46; and variable heavy chain sequences that are 90%, 95%, or 99% identical to sequence number 47 and variable light chain sequences that are 90%, 95%, or 99% identical to sequence number 48. Those skilled in the art will further understand that variable light chains and variable heavy chains may be independently selected or mixed and fitted together to prepare anti-IL-25 antibodies or antibody fragments thereof containing combinations of variable heavy chains and variable light chains different from the pairings specified above.

[0129] In some embodiments, an anti-IL-25 antibody or an antibody fragment thereof contains one or more conservative amino acid substitutions. Those skilled in the art will recognize that a conservative amino acid substitution is a substitution of one amino acid using another amino acid having similar structural or chemical properties, such as similar side chains. Exemplary conservative substitutions are described in the Art, for example, in Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Publication Company, 4th Ed. (1987).

[0130] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody, including its amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative substitution is when an amino acid is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains are clearly defined and include acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), non-charged side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), and beta-branched side chains (e.g., threonine, valine, The polypeptide contains an amino acid having isoleucine and a sulfur-containing side chain (cysteine, methionine). Furthermore, any native residue in the polypeptide may be substituted with alanine as previously described for alanine scanning mutagenesis (MacLennan et al. (1998) Acta Physiol Sc and Suppl 643: 55-67; ​​Sasaki et al. (1998) Adv Biophys 35: 1-24). Amino acid substitutions in the antibody of the present invention can be produced by known methods, for example, PCR mutagenesis (U.S. Patent No. 4,683,195).

[0131] In some embodiments, the antibody or fragment thereof includes a variable heavy chain sequence comprising an amino acid sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47. In other embodiments, the antibody or fragment thereof retains the binding and / or functional activity of the antibody or fragment thereof comprising the variable heavy chain sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47. In further embodiments, the antibody or fragment thereof comprises the variable heavy chain sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47, and the variable heavy chain sequence has one or more conserved amino acid substitutions, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5. In further embodiments, one or more conserved amino acid substitutions fall within one or more framework regions of SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47 (based on the Kabat numbering system).

[0132] In certain embodiments, the antibody or fragment thereof comprises a variable heavy chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the heavy chain variable region sequence of the antibody or fragment thereof described in SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47, comprising one or more conserved amino acid substitutions in the framework region, and retaining the binding and / or functional activity of the antibody or fragment thereof comprising the variable heavy chain sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47 and the variable light chain sequence described in SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, or 48.

[0133] In some embodiments, the antibody or fragment thereof includes a variable light chain sequence comprising an amino acid sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the amino acid sequence described in SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, or 48.

[0134] In other embodiments, the antibody or fragment thereof retains the binding and / or functional activity of the antibody or fragment thereof containing the variable light chain sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, or 48.

[0135] In further embodiments, the antibody or fragment thereof comprises the variable light chain sequence of SEQ ID NOs. 2, 4, 6, 8, 10, 45, 46, or 48, and the variable light chain sequence has one or more conserved amino acid substitutions, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5. In further embodiments, one or more conserved amino acid substitutions fall within one or more framework regions of SEQ ID NOs. 2, 4, 6, 8, 10, 45, 46, or 48 (based on the Kabat numbering system).

[0136] In certain embodiments, the antibody or fragment thereof includes a variable light chain sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the antibody or fragment light chain variable region sequence described in SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, or 48.

[0137] In some embodiments, the anti-IL25 antibody contains one or more conserved amino acid substitutions in its framework region and retains the binding and / or functional activity of an antibody or fragment thereof containing a variable heavy chain sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47 and a variable light chain sequence described in SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, or 48.

[0138] In some embodiments, the anti-IL-25 antibody is a monoclonal antibody. In some embodiments, the anti-IL-25 antibody is a human antibody or its antigen-binding portion. In alternative embodiments, the anti-IL-25 antibody is a mouse antibody or a chimeric antibody. In some embodiments, the anti-IL-25 antibody is a humanized antibody. In some embodiments, the anti-IL-25 antibody is a fully human antibody. In alternative embodiments, an antibody fragment containing one or more antigen-binding portions of the disclosed anti-IL-25 antibodies may be incorporated into a bispecific antibody, a multispecific antibody, or a fusion protein.

[0139] In one embodiment, the anti-IL-25 antibody or its antibody fragment comprises all six mouse CDR regions of IL25Ab1, IL25Ab2, IL25Ab3, IL25Ab4, or IL25Ab5 antibodies formatted as a chimeric or humanized antibody. In another embodiment, the anti-IL-25 antibody or its antibody fragment comprises all six CDR regions of one of the disclosed mouse antibodies.

[0140] In some embodiments, the anti-IL-25 antibody is a humanized antibody containing the VH / VL CDR region as described in Tables 3 and 4 (e.g., IL25Ab6, IL25Ab7, IL25Ab8, IL25Ab9, IL25Ab10, or IL25Ab11).

[0141] In some embodiments, the antibody is a full-length antibody. In other embodiments, the antibody is an antibody fragment selected from the group consisting of, for example, Fab, Fab', F(ab)2, Fv, domain antibodies (dAb), and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, miniantibodies, and polypeptides containing at least a portion of immunoglobulin sufficient to confer IL-25 specific binding to the polypeptide.

[0142] In some embodiments, the variable region domain of the anti-IL-25 antibody disclosed herein may be covalently bound to at least one other antibody domain or fragment thereof at its C-terminal amino acid. For example, a VH domain present in the variable region domain may be linked to an immunoglobulin CH1 domain or fragment thereof. Similarly, a VL domain may be linked to a CK domain or fragment thereof. Thus, for example, the antibody may be a Fab fragment, and the antigen-binding domain contains the relevant VH and VL domains covalently bound to the CH1 domain and CK domain, respectively, at its C-terminus. The CH1 domain may be extended with further amino acids, for example, to provide a hinge region or a portion of a hinge region domain found in the Fab fragment, or to provide further domains such as antibody CH2 and CH3 domains.

[0143] In some embodiments, the variable region domain of the anti-IL-25 antibody disclosed herein may be covalently bound to the antibody constant region at a C-terminal amino acid. For example, the VL domain may be linked to the mouse kappa constant region (SEQ ID NO: 54) or the human kappa constant region (SEQ ID NO: 57). Similarly, the VH domain may be linked to the mouse IgG1 constant region (SEQ ID NO: 53), the human IgG1 constant region (SEQ ID NO: 55), or another antibody constant region such as human IgG4 or IgG2. The constant region may be configured to contain specific mutations to modulate the properties of the induced antibody. For example, a triple mutant YTE may be introduced into the human IgG1 constant region (SEQ ID NO: 56) to extend the antibody half-life. Fc mutations that modulate the effector function of the antibody may also be introduced.

[0144] Accordingly, in one embodiment, the antibody fragment comprises at least one CDR as described in this disclosure. The antibody fragment may also be configured to comprise at least two, three, four, five, or six CDRs as described in this disclosure. The antibody fragment may further comprise at least one variable region domain of the antibody as described in this disclosure. The variable region domain may be of any size or amino acid composition and generally comprises at least one CDR sequence involved in binding to human IL-25, for example, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 as described in this disclosure, which are adjacent to or within one or more framework sequences.

[0145] In a further embodiment, the anti-IL-25 antibody or the antibody fragment thereof exhibits one or more of the following characteristics: (a) It is specific to human IL-25 and has the ability to block IL-25 binding to its receptor. (b) Inhibiting, interfering with, or modulating IL-25 interaction with IL-25 receptor signaling, (c) Inhibiting intracellular NF-κB activation induced by IL-25, (d) Inhibiting IL-5 production induced by human IL-25 in human PBMCs, (e) Inhibiting human IL-25-induced CXCL-1 production in human colon cancer cell lines, (f) To bind to mouse and cynomolgus monkey IL-25, (g) To reduce IL-5 production in the lungs of an OVA-induced asthma model, and (h) To improve airway resistance in an OVA-induced asthma model.

[0146] In one embodiment, an anti-IL-25 antibody or an antibody fragment thereof can reduce, inhibit, interfere with, and / or modulate at least one of the biological responses related to IL-25, and is therefore useful in improving the effects of IL-25-related diseases or disorders. Such antibodies and their antibody fragments may be used, for example, to reduce, inhibit, interfere with, and / or modulate IL-25 signaling, IL-25 activation in epithelial cells and type 2 lymphocytes, IL-25 activation in tumor cells, or to induce the production of pro-inflammatory cytokines.

[0147] The disclosed antibodies (e.g., IL25Ab1, IL25Ab2, IL25Ab3, IL25Ab4, IL25Ab5, IL25Ab6, IL25Ab7, IL25Ab8, IL25Ab9, IL25Ab10, and IL25Ab11) bind to human IL-25 (e.g., specifically). The amino acid sequences of closely related human IL-17 family members (IL-17A (Q16552), IL-17B (Q9UHF5), IL-17C (Q9P0M4), IL-17D (Q8TAD2), and IL-17F (Q96PD4)) were obtained from UniProt. Human IL-25 shows low homology to its phylogenetic relatives. IL-25 shows very low sequence similarity to other family members in the range of 23.2% to 32.7%. Based on low homology, anti-IL25 antibodies are unlikely to bind to IL17 family members.

[0148] Antibodies are typically 10 -7~10 -11 It binds specifically to its congener antigen with high affinity, as reflected by the dissociation constant (KD) of M or less. -6 Any KD greater than M is generally considered to exhibit nonspecific binding. As used in this disclosure, an antibody that specifically binds to an antigen refers to an antibody that binds with high affinity to the antigen and substantially the same antigen, which is 10 -7 M or less, preferably 10 -8 M or less, more preferably 5 × 10 -9 M or less, most preferably 10 -8 M~10 -10 This means that the antibody has a KD of M or less, but does not bind to unrelated antigens with high affinity. The disclosed antibody has a KD determined by BLI < 1.0E-12 and binds to human IL25 with high affinity.

[0149] As used in this disclosure, the term “cross-reactivity” refers to the ability of the anti-human IL-25 specific antibodies described herein to bind to IL-25 from different species. For example, the antibodies described herein may also bind to IL-25 from another species (e.g., cynomolgus monkey or mouse IL-25). As used in this disclosure, cross-reactivity may be measured by binding assays (e.g., SPR, ELISA) or by detecting specific reactivity to purified antigens that bind to or otherwise functionally interact with cells that physiologically express IL-25. Methods for determining cross-reactivity include, for example, the standard binding assays described herein by BIACORE® 2000 SPR instrument (Biacore AB, Uppsala, Sweden), biolayer interferometry (BLI), or BIACORE® surface plasmon resonance (SPR) analysis using flow cytometry techniques.

[0150] All of the disclosed human IL-2-5 antibodies, IL25Ab1 to IL25Ab11, bind to cynomolgus monkey-derived IL-25 with remarkable affinity. IL25Ab1 to IL25Ab11 bind to cynomolgus monkey IL-25 and mouse IL-25 in ELISA assays with affinities comparable to their binding affinity to human IL-25.

[0151] In certain embodiments, the antibodies provided in this disclosure are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one embodiment, the chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, the chimeric antibody is a “class-switching” antibody in which the class or subclass is changed from that of the parent antibody. The chimeric antibody includes its antibody fragment.

[0152] The antibody may be prepared as a chimeric antibody or antibody fragment having a mouse variable region and a human constant region. The human antibody constant region may be any of the reported allotypes outlined in Jefferis et al., Human immunoglobulin allotypes: possible implications for immunogenicity. MAbs 1, 332-338 (2009). In one embodiment, the human heavy chain constant region uses the consensus human IgG1 constant region sequence (SEQ ID NO: 55), while the light chain constant region uses the consensus human kappa constant region sequence (SEQ ID NO: 57). Human IgG1 may be selected because it is one of the most common subtypes for chimeric antibody production and can provide effector function. The human kappa constant region may be used because the majority of parental mouse antibodies have a mouse kappa light chain.

[0153] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains in which HVR, e.g., CDR (or a portion thereof), is derived from the non-human antibody and FR (or a portion thereof), is derived from the human antibody sequence. The humanized antibody also optionally contains at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived) to restore and / or improve antibody binding specificity or affinity, for example. In some embodiments, some CDR residues (e.g., residues 1, 2, 3, 4, or 5) in the humanized antibody may be mutated to improve antibody development potential.

[0154] Humanized antibodies and methods for producing them are outlined, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (describes specificity-determining region (SDR) transplantation), Padlan, Mol. Immunol. 28:489-498 (1991). (Describes "re-surfaced"); Dall'Acqua et al., Methods 36:43-60 (2005) (Describes "(FR shuffling)"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (Describes the "guide selection" approach to FR shuffling).

[0155] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "Best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008) and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. See Chem.272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996).

[0156] For human or humanized antibodies such as IgG1 antibodies, amino acid substitutions are introduced into the Fc region to modulate antibody interactions with the Fcγ receptor, FcRn, or complement, resulting in regulated antibody effector function and / or antibody half-life (see, for example, Damelang, T., et al. "Impact of structural modifications of IgG antibodies on effector functions." Front Immunol 14: 1304365 (2023)). Among the Fc mutations that extend antibody half-life, YTE (M252Y, S254T, and T256E in EU numbering) and LS (M428L and N434S) are two examples used in the literature.

[0157] Method for generating IL-25 antibodies Anti-IL-25 antibodies or their antibody fragments can be prepared by any method known in the art. For example, a recipient may be immunized with soluble recombinant human IL-25 protein, or a fragment or peptide conjugated with its carrier protein. Any suitable immunization method can be used. Such methods include the use of adjuvants, other immunostimulants, repeated booster immunization, and one or more immune pathways.

[0158] Any suitable source of human IL-25 can be used as an immunogen to produce anti-IL-25 antibodies of the compositions and methods disclosed herein. Different forms of the IL-25 antigen may be used to produce antibodies sufficient to generate biological activity. Thus, the induced IL-25 antigen may be a single epitope, multiple epitopes, or the entire protein, or in combination with one or more immunogenic retrieval agents. In some embodiments, the induced antigen is an isolated soluble full-length protein, or a soluble protein containing less than the full-length sequence (e.g., immunized with a peptide containing a specific portion or epitope of IL-25). As used in this disclosure, the term “portion” means, as necessary, the minimum number of amino acids or nucleic acids that constitute the immunogenic epitope of the antigen of interest. Any gene vector suitable for the transformation of the cells of interest may be employed, such as, but are not limited to, adenovirus vectors, plasmids, and nonviral vectors such as cationic lipids.

[0159] It is desirable to prepare monoclonal antibodies (mAbs) from various mammalian hosts, such as mice, rodents, primates, and humans. Descriptions of techniques for preparing such monoclonal antibodies can be found, for example, in Sties et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lance Medical Publication, Los Altos, CA, and the references cited herein; Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; and Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.) Academic Press, New York, NY. Typically, spleen cells from animals immunized with the desired antigen are generally immortalized by fusion with myeloma cells. See Kohler and Milstein (196) Eur. J. Immunol. 6:511-519. Alternative methods of immortalization include transformation with Epstein-Barr virus, oncogenes, or retroviruses, or other methods known in the art. See, for example, Doyle et al. (eds. 1994 and periodic supple supples) CELL and TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, NY. Colonies arising from a single immortalized cell are screened for the production of antibodies of desired specificity and affinity for an antigen, and the yield of monoclonal antibodies produced by such cells can be enhanced by various techniques, including intraperitoneal injection of a vertebrate host. Alternatively, DNA sequences encoding monoclonal antibodies or their antigen-binding fragments may be isolated by screening a DNA library from human B cells according to a general protocol outlined, for example, Huse et al. (1989) Science 246: 1275-1281.Thus, antibodies can be obtained through various techniques that are well-versed in by experienced researchers in this field.

[0160] Other suitable techniques include the selection of a library of antibodies in phages, yeasts, viruses, or similar vectors. See, for example, Huse et al. (above); Ward et al., (1989) Nature 341:544-546. The polypeptides and antibodies disclosed herein may be used with or without modifications, including chimeric antibodies or humanized antibodies. Frequently, polypeptides and antibodies are labeled by covalently or noncovalently linking substances that provide a detectable signal. A wide variety of labeling and conjugation techniques are known and have been extensively reported in both scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patents No. 3,817,837; No. 3,850,752; No. 3,996,345; No. 4,277,437; No. 4,275,149; and No. 4,366,241. Recombinant immunoglobulins may also be produced; see U.S. Patent No. 4,816,567 of Cabilly; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10023; or they may be produced in transgenic mice. See Nils Lonberg et al., (1994), Nature 368:856-859; and Mendez et al. (1997) Nature Genetics 15: 146-156; TRANSGENIC ANIMALS AND METHODS OF USE (WO 2012 / 62118), Medarex, Trianni, Abgenix, Ablexis, OminiAb, Harbour, and other techniques.

[0161] In some embodiments, the ability of the produced antibody to bind to IL-25 can be evaluated using standard binding assays, such as surface plasmon resonance (SPR), Octet (BLI), ELISA, Western blotting, immunofluorescence, flow cytometry, chemotaxis assays, and cell migration assays. In some embodiments, the produced antibody may also be evaluated for its ability to inhibit the IL-25-mediated inflammatory microenvironment chain of effects, including IL-25 blocking IL-25 receptor signaling and inhibiting IL-25-induced NFκB signaling, IL-5 production, and / or CXCL1 production.

[0162] Antibody compositions prepared from cells can be purified using, for example, hydroxyl apatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a typical purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (see, e.g., Lindmark et al., 1983 J.Immunol. Meth. 62:1-13). Protein G is recommended for all mouse isotypes and for human γ3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575). The matrix to which affinity ligands bind is most often agarose, but other matrices are available. Physically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene enable faster flow rates and shorter processing times than those achievable with agarose. When the antibody contains a CH3 domain, Bakerbond ABX® resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation with ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography with silica, chromatography with heparin SEPHAROSE® chromatography on anion or cation exchange resins (e.g., polyaspartate columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody being recovered.

[0163] Following any preliminary purification steps, the mixture containing the antibody of interest and any contaminants can be subjected to low-pH hydrophobic interaction chromatography using elution buffers at a pH between approximately 2.5 and 4.5, typically performed at low salt concentrations (e.g., approximately 0–0.25 M salt).

[0164] Also included are nucleic acids that hybridize, as defined in this disclosure, under low, medium, and high stringency conditions to all or part of a nucleotide sequence (e.g., a portion encoding a variable region) represented by an isolated polynucleotide sequence encoding an antibody or antibody fragment of this disclosure. The hybridized portion of the hybridized nucleic acid is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides in length. The hybridized portion of the hybridized nucleic acid is at least 80%, for example, at least 90%, at least 95%, at least 98%, or at least 99%, identical to part or all of the sequence of the nucleic acid encoding the anti-IL-25 polypeptide (e.g., heavy chain or light chain variable region) or its complement. Hybridized nucleic acids of the types described in this disclosure can be used, for example, as cloning probes, primers, such as PCR primers, or diagnostic probes.

[0165] Polynucleotides, vectors, and cells Other embodiments include isolated polynucleotides comprising sequences encoding an anti-IL-25 antibody or an antibody fragment thereof, vectors and cells comprising the polynucleotide, and recombinant techniques for the production of the antibody. The isolated polynucleotide may encode any desired form of the anti-IL-25 antibody, including, for example, full-length monoclonal antibodies, Fab, Fab, F(ab)2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, mini-antibodies, and multispecific antibodies formed from antibody fragments.

[0166] Some embodiments include isolated polynucleotides comprising sequences encoding the heavy chain variable region of an antibody or antibody fragment having the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 44, and 47. Some embodiments include isolated polynucleotides comprising sequences encoding the light chain variable region of an antibody or antibody fragment having the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, and 48.

[0167] In one embodiment, the isolated polynucleotide sequence encodes an antibody or antibody fragment having a heavy chain variable region and a light chain variable region containing the following amino acid sequence: (a) A variable heavy chain sequence containing SEQ ID NO: 1 and a variable light chain sequence containing SEQ ID NO: 2; (b) Variable heavy chain sequence containing Sequence ID 3 and variable light chain sequence containing Sequence ID 4; (c) Variable heavy chain sequence containing Sequence ID No. 5 and variable light chain sequence containing Sequence ID No. 6; (d) Variable heavy chain sequence containing Sequence ID 7 and variable light chain sequence containing Sequence ID 8; (e) Variable heavy chain sequence containing Sequence ID 9 and variable light chain sequence containing Sequence ID 10; (f) Variable heavy chain sequence containing Sequence ID 44 and variable light chain sequence containing Sequence ID 45; (g) A variable heavy chain sequence containing SEQ ID NO: 44 and a variable light chain sequence containing SEQ ID NO: 46; or (h) Variable heavy chain sequence containing sequence number 47 and variable light chain sequence containing sequence number 48.

[0168] In another embodiment, the isolated polynucleotide sequence encodes an antibody or antibody fragment having a light chain variable region and a heavy chain variable region comprising the following amino acid sequence: a) A variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 1, and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 2; b) Variable heavy chain sequences that are 90%, 95%, or 99% identical to SEQ ID NO: 3, and variable light chain sequences that are 90%, 95%, or 99% identical to SEQ ID NO: 4; c) A variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 5, and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 6; d) Variable heavy chain sequences that are 90%, 95%, or 99% identical to sequence number 7, and variable light chain sequences that are 90%, 95%, or 99% identical to sequence number 8; e) Variable heavy chain sequences that are 90%, 95%, or 99% identical to sequence number 9, and variable light chain sequences that are 90%, 95%, or 99% identical to sequence number 10; f) Variable heavy chain sequences that are 90%, 95%, or 99% identical to sequence number 44, and variable light chain sequences that are 90%, 95%, or 99% identical to sequence number 45; g) A variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 44, and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 46; or h) A variable heavy chain sequence that is 90%, 95%, or 99% identical to sequence number 47, and a variable light chain sequence that is 90%, 95%, or 99% identical to sequence number 48.

[0169] A polynucleotide containing a sequence encoding an anti-IL-25 antibody or an antibody fragment thereof can be fused to one or more regulatory sequences known in the art and can be incorporated into a suitable expression vector or cell known in the art. Each polynucleotide molecule encoding a heavy chain or light chain variable domain can be independently fused to a polynucleotide sequence encoding a constant domain, such as a human constant domain, enabling the production of intact antibodies. Alternatively, a polynucleotide, or a portion thereof, can be fused together to provide a template for the production of single-chain antibodies.

[0170] For recombinant production, a polynucleotide encoding an antibody or an antibody fragment is inserted into a replicable vector for cloning (DNA amplification) or expression. Many suitable vectors for expressing recombinant antibodies are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence.

[0171] Anti-IL-25 antibodies or their antibody fragments can also be produced as fusion polypeptides, in which the antibody or its fragment is fused with a heterologous polypeptide, such as a signal sequence having a specific cleavage site at the amino terminus of a mature protein or polypeptide, or other polypeptides. The selected heterologous signal sequence is typically recognized and processed by cells (i.e., cleaved by a signal peptidase). For prokaryotic cells that do not recognize and process the anti-IL-25 antibody signal sequence, the signal sequence can be replaced with a prokaryotic signal sequence. The signal sequence may be, for example, an alkaline phosphatase, penicillinase, lipoprotein, or a heat-stable enterotoxin II leader. For yeast secretion, the native signal sequence can be replaced with, for example, a yeast invertase alpha factor (including the alpha factor leaders of Saccharomyces and Kluyveromyces), an acid phosphatase, a leader sequence obtained from C. albicans glucoamylase, or the signals described in WO90 / 13646. In mammalian cells, mammalian signal sequences and viral secretion readers, such as the herpes simplex gD signal, can be used. The DNA of these precursor regions is ligated to the DNA encoding the anti-IL-25 antibody in the reading frame.

[0172] Expression vectors and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected cells. Generally, in cloning vectors, this sequence enables the vector to replicate independently of host chromosomal DNA and includes an origin of replication or an autonomous replication sequence. Such sequences are well known for various bacteria, yeasts, and viruses. The origin of replication derived from plasmid pBR322 is suitable for most Gram-negative bacteria, the 2-υ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, and BPV) are useful for cloning vectors in mammalian cells. Generally, an origin of replication is not required for mammalian expression vectors (the SV40 origin can typically be used because it contains an early promoter).

[0173] Expression vectors and cloning vectors may contain genes encoding selection markers to facilitate the identification of expression. Typical selection marker genes encode proteins that supply specific nutrients absent in the complex medium, such as conferring resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, or alternatively, complement nutrient deficiency, or other alternatives, for example, a gene encoding D-alanine racemase for bacilli.

[0174] Non-therapeutic use The anti-IL-25 antibodies or antibody fragments described herein are useful as affinity purifiers. In this process, the antibody is immobilized on a solid phase, such as a protein A resin, using methods well known in the art. The immobilized antibody is brought into contact with a sample containing IL-25 (or a fragment thereof) to be purified, and the carrier is then washed with a suitable solvent to remove substantially all substances in the sample other than the IL-25 protein bound to the immobilized antibody. Finally, the carrier is washed with another suitable solvent to release the IL-25 protein from the antibody.

[0175] Anti-IL-25 antibodies or their antibody fragments are also useful in diagnostic assays for detecting and / or quantifying IL-25 proteins, such as detecting IL-25 expression in specific cells, tissues, or serum. Anti-IL-25 antibodies can be used diagnostically, for example, to monitor the onset or progression of a disease as part of a clinical laboratory procedure, to determine the effectiveness of a given therapeutic and / or prophylactic regimen. Detection can be facilitated by conjugating the anti-IL-25 antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals used in various positron emission tomography (PTMF) scans, and non-radioactive paramagnetic metal ions. For example, see U.S. Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents according to this disclosure.

[0176] Anti-IL-25 antibodies or fragments thereof may be used in methods for diagnosing IL-25-related disorders (disorders characterized by abnormal expression of IL-25) or for determining whether a subject is at increased risk of developing an IL-25-related disorder. Such methods include contacting a biological sample from a subject with an anti-IL-25 antibody or fragment thereof and detecting the binding of the antibody to IL-25. "Biological sample" refers to any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing IL-25. Methods for obtaining tissue biopsies and bodily fluids from mammals are well known in the art.

[0177] In some embodiments, the method may further include comparing the level of IL-25 in a patient sample with that of a control sample (e.g., a subject without IL-25-related disorders) to determine whether the patient has an IL-25-related disorder or is at risk of developing one.

[0178] In some embodiments, for example, for diagnostic purposes, it may be advantageous to label an antibody with a detectable portion. Numerous detectable labels are available, including radioisotopes, fluorescent labels, enzyme substrate labels, and the like. The label may be indirectly conjugated to the antibody using various known techniques. For example, an antibody can be conjugated with biotin, and any of the three broad categories of labels mentioned above may be configured to be conjugated with avidin, or vice versa. Biotin selectively binds to avidin, and thus the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with an antibody, the antibody can be conjugated with a small hapten (e.g., digoxin), and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., an anti-digoxin antibody). In this way, indirect conjugation of the label with an antibody can be achieved.

[0179] Examples of radioisotope labeling include 35S, 14C, 125I, 3H, and 131I. Antibodies can be labeled with radioisotopes using techniques described, for example, in Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs. Radioactivity can be measured, for example, by scintillation counting.

[0180] Examples of readily available fluorescent labels include those derived from rare earth chelates (europium chelate) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lysamine, phycoerythrin, and Texas Red. Fluorescent labels can be conjugated to antibodies via known techniques, such as those disclosed in Current Protocols in Immunology. Fluorescence can be quantified using a fluorometer.

[0181] There are various well-characterized enzyme-substrate labels known in the art (see, for example, U.S. Patent No. 4,275,149). Enzymes generally catalyze chemical changes in chromogenic substrates that can be measured using various techniques. For example, the change may be a color change of the substrate that can be measured by spectrophotometry. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying fluorescence changes are described above. The chemiluminescent substrate is gradually electronically excited by a chemical reaction and can then emit measurable light or provide energy to a fluorescence acceptor, for example, using a chemiluminometer.

[0182] Examples of enzyme labeling include luciferases such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozymes, sugar oxidases (glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, etc.), heterocyclic oxidases (uricase and xanthine oxidase, etc.), lactoperoxidase, and microperoxidase. Techniques for conjugating enzymes with antibodies are described, for example, in O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J. Langone & H. Van Vunakis, eds.), Academic press, NY, 73: 147-166.

[0183] Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO), which has a hydrogen peroxidase as a substrate, and the hydrogen peroxidase oxidizes a dye precursor such as orthophenylenediamine (OPD) or 3,3,5,5-tetramethylbenzidine hydrochloride (TMB); alkaline phosphatase (AP), which has p-nitrophenyl phosphate as a chromogenic substrate; β-D-galactosidase (β-D-Gal), which has a fluorescent substrate such as p-nitrophenyl-β-D-galactosidase, or β-D-Gal, which has a chromogenic substrate such as 4-methylumbelliferyl-β-D-galactosidase.

[0184] In another embodiment, the anti-IL-25 antibody or its antibody fragment is used unlabeled and detected with a labeled antibody that binds to the anti-IL-25 antibody or its antibody fragment.

[0185] The antibodies and antibody fragments described herein may be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. See, for example, Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0186] The binding of ligands to the IL-25 receptor can be inhibited using anti-IL-25 antibodies or fragments thereof. Such methods involve administering the anti-IL-25 antibody to cells (e.g., mammalian cells) or the cellular environment, thereby inhibiting signaling mediated by the IL-25 receptor. These methods can be carried out in vitro or in vivo. The "cellular environment" refers to the tissue, culture medium, or extracellular matrix surrounding the cells.

[0187] Therapeutic composition and therapeutic method This disclosure also provides compositions comprising, for example, an anti-IL-25 antibody or a fragment thereof. Such compositions have many therapeutic applications for the treatment, prevention, or improvement of diseases or disorders (e.g., diseases or disorders involving biological activity mediated by the IL-25 / IL-25 receptor signaling axis), such as immune-mediated inflammatory disorders or autoimmune diseases.

[0188] The anti-IL-25 antibodies or antibody fragments disclosed herein are useful in the treatment of a variety of diseases or disorders, including type 2 inflammatory diseases, autoimmune diseases, or cancer. In particular, the disclosed antibodies are useful in the treatment of immune-mediated airway inflammatory disorders.

[0189] A method for treating IL-25-related disorders comprises administering a therapeutically effective dose of an anti-IL-25 antibody or a fragment thereof to a subject in need. The disclosure also provides a composition or formulation comprising an anti-IL-25 antibody or a fragment thereof, and optionally, a method for treating or prophylacticizing type 2 inflammatory disease, comprising administering another immune-based therapy to a subject in need. The disclosed anti-IL-25 antibody may be administered as an add-on therapy or in combination with other cytokine inhibitors.

[0190] The disclosed antibodies are also useful in methods of preventing or treating airway hyperresponsiveness (AHR) or airway inflammation, including but not limited to allergic asthma, non-allergic asthma, severe refractory asthma, asthma exacerbations, virus-induced asthma, virus-induced asthma exacerbations, steroid-resistant asthma, steroid-sensitive asthma, eosinophilic asthma, non-eosinophilic asthma, and related disorders. Furthermore, this term includes virus-induced asthma exacerbations.

[0191] The disclosed antibodies are also useful in methods for preventing or treating chronic obstructive pulmonary disease (COPD) in patients who require such treatment, by administering the anti-IL-25 antibody or its antigen-binding fragment described herein.

[0192] The disclosed antibodies are also useful in methods for preventing or treating inflammatory bowel disease (IBD). In this context, “IBD” refers to a group of disorders affecting the mucosal layer of the colon or colon, including ulcerative colitis, Crohn’s disease, collagenous colitis, lymphocytic colitis, ischemic colitis, diffractional colitis, Behçet’s syndrome, infectious colitis, indeterminate colitis, and other related conditions characterized by inflammation.

[0193] The antibodies disclosed herein are also useful in methods for preventing or treating atopic dermatitis (AD), an inflammatory skin condition characterized by scaly, dry, severe itching and eczematous lesions. AD can result from epidermal barrier dysfunction, exposure to certain foods, pollen, mold, dust mites, animals, and / or asthma. The methods disclosed herein can be used to treat various degrees of AD, including mild, moderate, moderate-severe, and severe forms.

[0194] The disclosed antibodies are also useful in preventing or treating various diseases and disorders such as eosinophilic granulomatosis with polyangiitis or EGPA (also known as Churg-Strauss syndrome), allergies, allergic rhinitis, allergic airway inflammation, food hypersensitivity, urticaria (including chronic idiopathic urticaria), eosinophilic pneumonia, eosinophilic esophagitis, eosinophilic syndrome, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, rheumatoid arthritis, vasculitis, uveitis, cancer, and graft-versus-host disease.

[0195] The disclosed antibodies are also useful in methods of treating cancer, either alone (e.g., as monotherapy) or in combination with other immunotherapeutic agents and / or chemotherapy.

[0196] Antibodies can be administered alone or in combination with other compositions useful for treating immune-mediated inflammatory disorders or autoimmune diseases. In some embodiments, a composition comprising, for example, a pharmaceutical composition containing an anti-IL-25 antibody may further comprise a therapeutic agent conjugated to or unconjugated to a binder.

[0197] In some embodiments, pharmaceutical compositions are provided, for example, pharmaceutical compositions comprising one or more antibodies disclosed herein. The pharmaceutical compositions may be formulated in combination with a pharmaceutically acceptable carrier or diluent, as well as any other known adjuvants and excipients, according to conventional methods, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

[0198] Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. If necessary, the drug may also contain solubilizers and local anesthetics such as lignocaines to reduce pain at the injection site. Generally, the components are supplied separately or mixed together in unit dosage forms, for example, as dry lyophilized powders or anhydrous concentrates in sealed containers such as ampoules or sachets that indicate the amount of the active drug. When the drug is administered by injection, it can be dispensed in injection bottles containing sterile pharmaceutical-grade water or saline. When the drug is administered by injection, ampoules of sterile water for injection or saline may be provided, so the components can be mixed before administration.

[0199] When used in this disclosure, “pharmaceutically acceptable carriers” include any and all physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders. Preferably, the carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial administration (e.g., by injection or infusion). Depending on the route of administration, the active compounds, i.e., antibodies, bispecific and multispecific molecules, may be coated in materials to protect the compounds from the action of acids and other natural conditions that may inactivate them.

[0200] The composition can be administered by various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome. The active compound can be prepared using a carrier that protects the compound for controlled-release formulations, such as rapid-release formulations including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations are generally known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0201] The dosage level of the active ingredient in a pharmaceutical composition may be varied to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular subject, composition, and mode of administration, without causing toxicity to the subject. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical technology field.

[0202] The pharmaceutical compositions described herein may be administered in effective doses. “Effective dose” means an amount that achieves the desired response or effect alone or in combination with additional doses. In the case of treating a particular disease or condition, the desired response preferably relates to inhibiting the course of the disease. This includes slowing the progression of the disease, in particular interrupting or reversing its progression.

[0203] In some embodiments, the compositions described herein are administered to a patient, for example, in vivo, to treat or prevent a variety of disorders, including those described herein. Preferred patients include human patients with disorders that can be corrected or improved by the administration of the drug, which modulate the biological activity of the IL-25 / IL-25 receptor signaling axis.

[0204] In some embodiments, nucleic acids encoding antibodies or derivatives thereof described herein can be introduced into mammalian cells or target tissues using conventional viral and nonviral-based gene transfer methods. Such methods can be used to administer antibody-encoding nucleic acids to cells in vitro. In some embodiments, nucleic acids encoding antibodies or derivatives thereof are administered for use in vivo or ex vivo gene therapy. In other embodiments, gene delivery technologies are used to test the activity of antibodies in cell-based or animal models. Nonviral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles, such as liposomes. Viral vector delivery systems include DNA viruses and RNA viruses, which have either an episomal genome or an integrated genome after delivery to cells. Such methods are well known in the art.

[0205] The nonviral delivery methods of nucleic acids encoding operational polypeptides described herein include lipofection, microinjection, bioristic methods, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection methods and lipofection reagents are well known in the art (e.g., Transfectam® and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO91 / 17424, and WO91 / 16024. Delivery may be to cells (ex vivo administration) or target tissues (in vivo administration). Preparation of lipid:nucleic acid conjugates, including targeted liposomes such as immunolipid complexes, is well known to those skilled in the art.

[0206] The use of RNA or DNA virus-based systems for the delivery of antibody-encoding nucleic acids described herein utilizes highly evolved processes for targeting viruses to specific cells throughout the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to a patient (in vivo), or they can be used to treat cells in vitro, with the modified cells being administered to the patient (ex vivo). Conventional virus-based systems for polypeptide delivery in this disclosure may include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated vectors, and herpes simplex virus vectors for gene transfer. Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration into the host genome is possible by retroviral, lentiviral, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiencies have been observed in many different cell types and target tissues.

[0207] All identified patents and publications are expressly incorporated by reference in this disclosure for the purpose of describing and disclosing methodologies described in such publications, which may be used, for example, in connection with this disclosure. These publications are provided only for their disclosure prior to the filing date of this application. In this regard, nothing should be construed as an admission that the inventors do not have prior rights to such disclosure by prior invention or for any other reason. All statements regarding dates or representations relating to the contents of these documents are based on information available to the applicant and do not constitute any endorsement of the accuracy of the dates or contents of these documents.

[0208] Beyond what has not yet been shown, those skilled in the art will understand that any one of the various embodiments described and illustrated in this disclosure may be further modified to incorporate features shown in any of the other embodiments disclosed in this disclosure.

[0209] The broad scope of this disclosure is best understood by referring to the following embodiments, which are not intended to limit this disclosure to any particular embodiment. The specific embodiments described herein are provided for illustrative purposes only, and this disclosure is limited by the language of the appended claims and by the entire scope of equivalents to which such claims are entitled.

[0210] Examples of subject matter as clauses Various examples of the embodiments are provided for convenience as numbered clauses (1, 2, 3, etc.). These are provided as examples and do not limit the technology of the subject matter. Reference number identifications are provided below solely as examples and for illustrative purposes, and the clauses are not limited by their identifications.

[0211] Clause 1: An anti-IL-25 antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 (HCDR1), the amino acid sequence of SEQ ID NO: 49 (HCDR2), and the amino acid sequence of SEQ ID NO: 25 (HCDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50 (LCDR1), the amino acid sequence of SEQ ID NO: 51 (LCDR2), and the amino acid sequence of SEQ ID NO: 28 (LCDR3); and (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 (HCDR1), the amino acid sequence of SEQ ID NO: 52 (HCDR2), and the amino acid sequence of SEQ ID NO: 25 (HCDR3). (c) a light chain variable region including the amino acid sequence of SEQ ID NO: 50 (LCDR1), the amino acid sequence of SEQ ID NO: 51 (LCDR2), and the amino acid sequence of SEQ ID NO: 28 (LCDR3), (d) a heavy chain variable region including the amino acid sequence of SEQ ID NO: 11 (HCDR1), the amino acid sequence of SEQ ID NO: 12 (HCDR2), and the amino acid sequence of SEQ ID NO: 13 (HCDR3), as well as a light chain variable region including the amino acid sequence of SEQ ID NO: 14 (LCDR1), the amino acid sequence of SEQ ID NO: 15 (LCDR2), and the amino acid sequence of SEQ ID NO: 16 (LCDR3), (d) the amino acid sequence of SEQ ID NO: 17 (e) A heavy chain variable region including the amino acid sequence of sequence number 18 (HCDR1), the amino acid sequence of sequence number 18 (HCDR2), and the amino acid sequence of sequence number 19 (HCDR3), and a light chain variable region including the amino acid sequence of sequence number 20 (LCDR1), the amino acid sequence of sequence number 21 (LCDR2), and the amino acid sequence of sequence number 22 (LCDR3), (e) a heavy chain variable region including the amino acid sequence of sequence number 23 (HCDR1), the amino acid sequence of sequence number 24 (HCDR2), and the amino acid sequence of sequence number 25 (HCDR3), and the amino acid sequence of sequence number 26 (LCDR1), sequence number 27 (f) Light chain variable region including amino acid sequence (LCDR2) and amino acid sequence (LCDR3) of SEQ ID NO: 29, heavy chain variable region including amino acid sequence (HCDR1) of SEQ ID NO: 29, amino acid sequence (HCDR2) of SEQ ID NO: 30, and amino acid sequence (HCDR3) of SEQ ID NO: 31, and light chain variable region including amino acid sequence (LCDR1) of SEQ ID NO: 32, amino acid sequence (LCDR2) of SEQ ID NO: 33, and amino acid sequence (LCDR3) of SEQ ID NO: 34, or (g) amino acid sequence (HCDR1) of SEQ ID NO: 35, amino acid sequence (HCDR2) of SEQ ID NO: 36,An anti-IL-25 antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 37 (HCDR3), and a light chain variable region containing the amino acid sequence of SEQ ID NO: 38 (LCDR1), the amino acid sequence of SEQ ID NO: 39 (LCDR2), and the amino acid sequence of SEQ ID NO: 40 (LCDR3).

[0212] Clause 2: An anti-IL-25 antibody or antigen-binding fragment thereof as described in Clause 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region containing one amino acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47, and a light chain variable region containing one amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, or 48.

[0213] Clause 3: An anti-IL-25 antibody as described in Clause 1, wherein the antibody comprises (a) the heavy chain variable region sequence of SEQ ID NO: 44 and the light chain variable region sequence of SEQ ID NO: 45; (b) the heavy chain variable region sequence of SEQ ID NO: 44 and the light chain variable region sequence of SEQ ID NO: 46; (c) the heavy chain variable region sequence of SEQ ID NO: 47 and the light chain variable region sequence of SEQ ID NO: 48; (d) the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 2; (e) the heavy chain variable region sequence of SEQ ID NO: 3 and the light chain variable region sequence of SEQ ID NO: 4; (f) the heavy chain variable region sequence of SEQ ID NO: 5 and the light chain variable region sequence of SEQ ID NO: 6; (g) the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8; or (h) the heavy chain variable region sequence of SEQ ID NO: 9 and the light chain variable region sequence of SEQ ID NO: 10.

[0214] Clause 4: The anti-IL-25 antibody described in Clause 1, wherein the antibody is an anti-human anti-IL-25 antibody.

[0215] Clause 5: The anti-IL-25 antibody according to Clause 1, wherein the antibody is a full-length antibody containing a human IgG1 constant region selected from SEQ ID NO: 55 or SEQ ID NO: 56.

[0216] Clause 6: The anti-IL-25 antibody described in Clause 1, wherein the antibody is an antibody fragment.

[0217] Clause 7: An anti-IL-25 antibody as described in Clause 4, wherein the antibody fragment is selected from the group consisting of Fab, Fab, F(ab)2, Fd, Fv, scFv, and scFv-Fc fragments, single-chain antibodies, minibodies, and diabodies.

[0218] Clause 8: The anti-IL-25 antibody described in Clause 1, wherein the antibody is a monoclonal antibody.

[0219] Clause 9: The anti-IL-25 antibody described in Clause 1, wherein the antibody is a human antibody.

[0220] Clause 10: The anti-IL-25 antibody described in Clause 1, wherein the antibody is a mouse antibody.

[0221] Clause 11: The anti-IL-25 antibody described in Clause 1, wherein the antibody is a chimeric antibody.

[0222] Clause 12: The anti-IL-25 antibody according to Clause 1, wherein the antibody is a bispecific antibody or a multispecific antibody.

[0223] Clause 13: The anti-IL-25 antibody described in Clause 1, wherein the antibody is a humanized antibody.

[0224] Clause 14: A pharmaceutical composition comprising the antibody described in Clause 1 and a pharmaceutically acceptable carrier.

[0225] Clause 15: A method for treating and / or preventing a subject in need of treatment and / or prevention of a type 2 inflammatory disease, autoimmune disease, allergic disorder, or cancer, comprising administering the antibody of Clause 1 to the subject.

[0226] Clause 16: a) containing the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47 A polynucleotide composition comprising a first polynucleotide encoding a heavy chain variable region, and a) a second polynucleotide encoding a light chain variable region containing an amino acid sequence described in SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, or 48.

[0227] Clause 17: A vector composition comprising: a) a first vector comprising the first polynucleotide described in Clause 16; and b) a second vector comprising the second polynucleotide described in Clause 16.

[0228] Clause 18: A cell comprising the polynucleotide composition described in Clause 16, or the vector composition described in Clause 17.

[0229] Clause 19: A method for producing the anti-IL-25 antibody described in Clause 1, comprising culturing the cells described in Clause 18 in a culture medium and recovering the anti-IL-25 antibody from the medium. [Examples]

[0230] General method Methods for protein purification, including immunoprecipitation, chromatography, and electrophoresis, are described. See, for example, Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, fusion protein production, and protein glycosylation are also described. For example, see Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391. The production, purification, and fragmentation of polyclonal and monoclonal antibodies are described. Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra.

[0231] Hybridomas or cell culture supernatants containing anti-IL-25 antibody were purified via a HiTrap Protein G column (GE, catalog no. 17040401) according to the manufacturer's protocol. Briefly, the column was equilibrated with DPBS (Gibco, catalog no. 14190-136) over 5 CVs, and the supernatant was loaded via a syringe / injection pump (Legato 200, KD Scientific) at ambient temperature with a residence time of 3 minutes. The column was washed with 5 CVs of DPBS, eluted with 4 CVs of pH 2.8 elution buffer (Fisher Scientific, catalog no. PI21004), neutralized with 1 M Tris-HCl, pH 8.5 (Fisher Scientific, catalog no. 50-843-270), and assayed with A280 (Drop sense96, Trinan). Next, the purified material was buffered with DPBS via a 30 kDa MWCO centrifugal filter (EMD Millipore, catalog number UFC803024). The final pool was analyzed using A280 and stored at either 2–8°C or -20°C.

[0232] Standard methods in molecular biology are described. For example, see Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods are also found in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0233] The sequences of the heavy-chain and light-chain variable regions of the hybridoma clone were determined as described below. Using the RNeasy Plus Mini Kit from Qiagen (Germantown, Maryland, USA), 1–2 × 10⁻¹⁶ sequences were determined. 6Total RNA was extracted from individual hybridoma cells. cDNA was generated by performing 5’ RACE reaction using the SMARTer RACE 5’ / 3’ Kit from Takara Bio Inc. (Mountain View, CA, USA). PCR was performed using Q5 High-Fidelity DNA Polymerase from NEB Laboratories (Ipswich, MA, USA), and the variable regions derived from the heavy and light chains were amplified using the Takara Universal Primer Mix in combination with gene-specific primers for the 3’ mouse constant region of the appropriate immunoglobulin. The amplified variable regions of the heavy and light chains were electrophoresed on a 2% agarose gel, appropriate bands were excised, and then gel-purified using the Mini Elute Gel Extraction Kit from Qiagen. The purified PCR products were cloned using the Zero Blunt PCR Cloning Kit from Invitrogen (Carlsbad, CA, USA), transformed into Stellar Competent E. Coli cells from Takara Bio Inc., and plated on LB agar + 50 μg / ml kanamycin plates. Direct colony Sanger sequencing was performed by GeneWiz (South Plainfield, NJ, USA). The resulting nucleotide sequences were analyzed using IMGT V-QUEST to identify productive recombinants and the translated protein sequences were analyzed. The determination of CDRs was based on Kabat numbering.

[0234] The selected VH or VL chain was PCR amplified and cloned into a pcDNA3.4-based expression vector, which retains the constant region from human IgG1 (Uniprot P01857 or its allotype) or human kappa light chain (UniProt P01834). A pair of heavy chain expression plasmid and light chain expression plasmid was transfected into Expi293 cells (Thermo Fisher Scientific) according to the protocol of the supplier's Expi293 expression system. Five days after transfection, the culture supernatant was collected by centrifugation. The chimeric antibody was purified using a Protein A column and buffer exchanged into PBS pH 7.2 or 20 mM histidine pH 5.5.

[0235] Methods for flow cytometry are available, including fluorescence-activated cell sorting detection systems (FACS®). See, for example, Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, N.J. Fluorescent reagents suitable for modifying nucleic acids are available, including nucleic acid primers and probes, polypeptides, and antibodies for use as diagnostic reagents. Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

[0236] Standard techniques are available for characterizing ligand / receptor interactions. See, for example, Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York. Standard methods for antibody functional characterization suitable for characterizing antibodies with specific mechanisms of action are well known to those skilled in the art.

[0237] Two in-hospital IL-25 specific antibodies, referred to in this disclosure as IL-25-PC2 and IL-25-PC4, were prepared. IL-25-PC2 was prepared based on publicly available information published in WO2016 / 049000 A2 (VH, SEQ ID NO: 114, and VL, SEQ ID NO: 122), and IL-25-PC4 was prepared based on publicly available information published in WO2020 / 102935 A1 (VH, SEQ ID NO: 12, and VL, SEQ ID NO: 13). Using both antibodies, functional assays were established for conjugation and for evaluating and characterizing the anti-IL-25 specific antibodies disclosed in this disclosure.

[0238] For example, software packages and databases are available for determining antigen fragments, leader sequences, protein folding, functional domains, CDR annotations, glycosylation sites, and sequence alignment.

[0239] Example 1: Production of anti-IL-25 antibody Anti-IL-25 antibodies were generated by immunizing wild-type mice or transgenic / humanized mice.

[0240] Immunization. Mice were immunized with recombinant human IL-25 protein intraperitoneally, subcutaneously, in the sole of the foot, or at the base of the tail. The immune response was monitored by post-orbital blood sampling. Plasma was screened by ELISA (described below), and mice with sufficient titers of anti-human IL-25 were used for fusion. Mice were boosted with recombinant human IL-25 protein intraperitoneally, intravenously, in the sole of the foot, or at the base of the tail, then sacrificed, and the spleen and lymph nodes were removed.

[0241] Selection of Balb / c mice producing anti-IL-25 antibodies. To select Balb / c mice producing antibodies that bind to IL-25, serum from immunized mice was screened for IL-25 binding by ELISA. Briefly, recombinant human IL-25 protein-coated ELISA plates were incubated with dilutions of serum from immunized mice at room temperature for 1 hour, the plates were washed, and specific antibody binding was detected with HRP-labeled anti-mouse IgG antibody. The plates were read using an ELISA reader. Hybridoma supernatants were tested for anti-IL-25 specific binding by ELISA as described above.

[0242] Generation of hybridomas that produce antibodies against IL-25. To generate the antibody-producing hybridomas of the present invention, splenocytes and lymph node cells were isolated from immunized mice and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas were screened for the production of antigen-specific antibodies. For example, single-cell suspensions of splenocytes and lymph node cells from immunized mice were fused by electrofusion to an equal number of Sp2 / 0 non-secreting mouse IgG myeloma cells (ATCC, CRL 1581). After seeding the cells in a flat-bottomed 96-well tissue culture plate, they were incubated in selective medium (HAT medium) for two weeks, and then replaced with hybridoma culture medium. Approximately 10-14 days after cell seeding, the supernatant obtained from each well was screened by ELISA as described above. Antibody-secreting hybridomas were transferred to 24-well plates and screened again. If still positive for anti-IL-25, the positive hybridomas were subcloned by sorting using a single-cell sorter. Stable subclones were then cultured in vitro to generate small amounts of antibody for purification and characterization.

[0243] Example 2: Binding of an anti-IL-25 specific antibody In this study, the ability of five disclosed anti-IL-25 antibodies (IL25Ab1-Ab5) that bind to both human and mouse IL-25 was evaluated using ELISA. Briefly, recombinant human IL-25 protein (Acro, catalog number: IL5-H4221, lot number: 401-20CNF1-UT) or recombinant mouse IL-25 protein (Sino Biological Inc, catalog number: 50138-M07H, lot number: LC15AP0607) was directly coated onto ELISA plates. Recombinant antibodies were then added to the plates and subsequently detected using goat anti-human IgG-HRP (Jackson ImmunoResearch, catalog number: 109-035-098, lot number: 157400). After adding ABTS (Moss Inc., catalog number: ABTS-1000, lot number: 03086202) substrate, the ELISA plates were read using an ELISA plate reader (Bioteck).

[0244] Figure 3A shows that five disclosed anti-IL-25 antibodies have an EC level ranging from 0.010 nM to 0.034 nM. 50 The values ​​indicate dose-dependent binding to the human recombinant IL-25 protein. The positive control antibodies IL-25-PC2 (recombinant antibody produced by NovaRock, lot number: P09282021JLF) and IL-25-PC4 (recombinant antibody produced by NovaRock, lot number: A10032022JLF) had EC values ​​of 0.026 nM and 0.028 nM, respectively. 50 The value was found. Human IgG1 (InVivoMab, catalog number BE0297, lot number 760620M1) isotype control did not show binding.

[0245] Figure 3B shows that five disclosed anti-IL-25 antibodies have an EC level ranging from 0.012 nM to 0.035 nM. 50 The values ​​indicate that the antibodies also bound to recombinant mouse IL-25 protein in a dose-dependent manner. The positive control antibodies IL-25-PC2 and IL-25-PC4 had EC50 values ​​of 0.022 nM and 0.021 nM, respectively.

[0246] In another study, the binding ability of five disclosed anti-IL-25 antibodies (IL-25Ab1-Ab5) to cynomolgus monkey IL-25 antibodies was evaluated using ELISA. Briefly, recombinant human IL-25 protein (Acro, catalog number: IL5-H4221, lot number: 401-20CNF1-UT) or recombinant cynomolgus monkey IL-25 protein (Sino Biological Inc, catalog number: customized order, lot number: MB17MA2715) was directly coated onto ELISA plates. The recombinant antibodies were then added to the plates and subsequently detected using goat anti-human kappa light chain antibody HRP (Novus, catalog number: NBP1-75064, lot number: 68-188-010920). After adding ABTS (Moss Inc., catalog number ABTS-1000, lot number 03086202) substrate, the ELISA plate was read using an ELISA plate reader (Bioteck).

[0247] Figure 3C shows that the five disclosed anti-IL-25 antibodies bound to the cynomolgus monkey recombinant IL-25 protein in a dose-dependent manner, with similar binding activity compared to the positive control antibodies IL-25-PC2 and IL-25-PC4.

[0248] [Table 5]

[0249] Example 3: Binding kinetics of anti-IL-25 specific antibody The binding kinetics of the disclosed anti-IL-25 antibody to recombinant human IL-25 were determined by the Octet® Bio-Layer Interferometry (BLI) system developed by Sartorius. The assay used the anti-IL-25 monoclonal antibody as the ligand and the recombinant human IL-25 protein as the analyte.

[0250] Briefly stated, the assay was performed by first capturing 5 μg / ml of anti-IL-25 antibody using an anti-human Fc Octet biosensor. The mAb-captured biosensor was then immersed in wells containing serially diluted human IL-25 for 4 - 6 minutes, followed by a dissociation time of 10 - 15 minutes. Binding sensorgrams were collected and analyzed by Octet Data Analysis software.

[0251] Table 6 summarizes the KD values of the anti-IL-25 antibodies disclosed in this study. The data suggest that these antibodies have strong binding affinities for human recombinant IL-25 and exhibit both fast and slow kinetic properties.

[0252]

Table 6

[0253] Example 4: Blocking of IL-25-induced NF-kB signaling IL-25 interacts and signals through the IL-25 receptor, which contains the heterodimeric IL-17RA and IL-17RB subunits. The activated heterodimeric receptor recruits the Act1 adapter, leading to the ubiquitination of TNF receptor-associated factor 6 (TRAF6). This then triggers a signaling cascade, resulting in the activation of NF-κB and AP-1.

[0254] The blocking activity of anti-IL-25 antibodies was studied using the HEK-293 reporter cell line. HEK-Blue™ IL-17 cells (InvivoGen, San Diego, CA) were generated by stably transfecting the human embryonic kidney HEK293 cell line with a human gene encoding the IL-17RA / IL-17RC heterodimeric receptor and the Act1 adapter molecule. These cells also express a secreted embryonic alkaline phosphatase (SEAP) reporter gene that is inducible by NF-κB and AP-1.

[0255] In this study, HEK-Blue® IL-17 cells were treated with 5 ng / ml recombinant human IL-25 along with serially diluted IL-25 antibody. After overnight incubation, the NF-κB response was determined using QUANTI-Blue® solution, a SEAP detection reagent, by reading the optical density (OD) at 655 nm.

[0256] As summarized in Figure 4 and Table 7, the five disclosed anti-IL-25 antibodies effectively dose-dependently block IL-25-induced NF-κB signaling and IC 50 The values ​​ranged from 0.049 nM to 0.073 nM. Positive control antibodies IL-25-PC2 and IL-25-PC4 had IC50 values ​​of 0.062 nM and 0.456 nM, respectively. Human IgG1 isotype controls showed no blocking activity.

[0257] [Table 7]

[0258] Example 5: Blockade of IL-25-induced CXCL1 production Interleukin-17 (IL-17) expression is significantly elevated in the peripheral blood of patients with inflammatory bowel disease (IBD), suggesting that IL-17 may play a crucial role in the physiological and pathological processes of the disease. The intestinal epithelial cell line HT-29 is the most common cell line used in the laboratory to study the immune mechanisms of the intestinal mucosa because it possesses normal colonic epithelial structure and function.

[0259] To determine whether the disclosed anti-IL-25 antibodies could block the production of the inflammatory cytokine CXCL1 induced by human IL-25, HT-29 cells were seeded into assay plates. Subsequently, serially diluted anti-IL-25 antibody combinations with hIL-25 were added to each well, and the cells were incubated at 37°C for 72 hours. The supernatant was then collected for CXCL1 ELISA using the Human CXCL1 ELISA Ready-SET-Go Kit (R&D System #DY275). The results, presented in Figure 5 and Table 8, demonstrate that five disclosed anti-IL-25 antibodies effectively blocked the production of IL-25-stimulated CXCL1, thereby potentially reducing the recruitment of immune cells and preventing the development of inflammation.

[0260] I C 50 The values ​​ranged from 0.067 nM to 0.089 nM. Positive control antibodies IL-25-PC2 and IL-25-PC4 had IC50 values ​​of 0.066 nM and 0.877 nM, respectively. Human IgG1 isotype controls showed no blocking activity.

[0261] [Table 8]

[0262] Example 6: Blocking IL-25-induced IL-5 production IL-25 was involved as a type 2 cytokine produced by Th2 cells, and it was able to induce the expression of IL-4, IL-5, and IL-13 genes. The induction of these cytokines resulted in a Th2-like response characterized by increased serum IgE, IgG, and IgA levels, blood eosinophilia, and pathological changes in the lungs and gastrointestinal tract, including eosinophil infiltration, increased mucus production, and epithelial cell hyperplasia / hypertrophy.

[0263] To further characterize the biological function of IL-25 antibodies and evaluate their potential in the treatment of inflammatory diseases, human PBMCs were used in this assay. When human PBMCs were treated with 30 U / ml recombinant hIL-2 and 2 ng / ml recombinant hIL-25 for 6 days, significant amounts of IL-5 were produced. However, when the cells were co-treated with an IL-25 blocking antibody, IL-5 levels were significantly reduced, suggesting that IL-25 antibodies can suppress the type 2 response.

[0264] As summarized in Figure 6 and Table 9, all five disclosed IL-25 antibodies effectively dose-dependently block IL-25-induced IL-5 production and IC 50 The values ​​ranged from 0.12 nM to 0.26 nM. The positive control antibodies IL-25-PC2 and IL-25-PC4 showed IC50 values ​​of 0.26 nM and 12.99 nM, respectively. 50 The value was found. The human IgG1 isotype control showed no blocking activity whatsoever.

[0265] [Table 9]

[0266] Example 7: In vivo efficacy of an anti-IL-25 specific antibody in an OVA-induced asthma model The in vivo efficacy of anti-IL-25 specific antibodies was studied using an OVA-induced asthma model. Six-to-seven-week-old male BALB / c mice were divided into six groups (10 mice per group): (1) control group, (2) OVA group + vehicle, and (3-6) OVA group + anti-IL-25 antibody. Mice in the OVA group were sensitized by intraperitoneal injection of an OVA solution containing aluminum hydroxide on days 1 and 14, and challenged on days 28, 29, and 31 by aerosolizing a 1% OVA solution for 30 minutes using a BUXCO aerosolization system. Anti-IL-25 antibodies were administered subcutaneously at a dose of 10 mg / kg on days 14, 17, 20, 23, 26, 29, and 31. All tested antibodies were produced in mouse IgG1 format. On day 31, lung resistance in response to the range of aerosolized methacholine was measured by whole-body plethysmography (WBP, Buxco BFE0100 WBP). On day 32, mice were anesthetized and BALF was obtained by flushing the lungs with PBS containing 1% FBS.

[0267] Airway hyperresponsiveness is a characteristic feature of asthma. In Figure 7, mice in the OVA+ vehicle group showed a significant increase in airway tolerance to metacholine compared to the control group. Treatment with IL25Ab3 significantly reduced airway resistance. IL25Ab4, IL25Ab5, and IL25-PC4 showed slight decreases in airway resistance; however, these decreases did not reach statistical significance. Airway resistance was presented as dose-response data for metacholine, shown as the percentage change from baseline levels of lung resistance (Penh value) (Figure 7A) and the AUC of % of baseline Penh (Figure 7B).

[0268] Interleukin-5 (IL-5) plays a central pathogenic role in eosinophil differentiation, recruitment, survival, and degranulation. IL-5 has been reported to have potent effects on the induction, maintenance, and amplification of eosinophilic inflammation, such as asthma. Figure 8 shows a significant increase in IL-5 in bronchoalveolar lavage fluid (BALF) in an OVA-induced asthma model compared to control mice. Treatment with IL25Ab3, IL25Ab5, and IL25-PC4 significantly reduced IL-5 production in BALF. Treatment with IL25Ab4 reduced IL-5 production (but not significantly).

[0269] Example 8: Humanization of anti-IL-25Ab3 Mouse anti-IL-25Ab3 was humanized using a CDR transplantation approach. The heavy and light chain variable regions of mouse Ab3 were derived from mouse germline IGHV1-18*01 and IGKV10-94*01, respectively. Using the heavy chain variable region, a search was performed against a human antibody germline database, and human IGHV1-2*02, the human germline sequence with the highest similarity, was selected as the template for heavy chain CDR transplantation. Similarly, human IGKV1-33*01 was selected as the template for light chain CDR transplantation. During lattice formation, a structural model of the antibody was generated, and selected reverse mutations were introduced to maintain proper antibody folding. A total of five heavy chain variants and four light chain variants were designed and cloned into expression constructs. Twenty pairs of heavy and light chain humanized variants were produced as both Fab antibodies and human IgG1 antibodies. Humanized variant Fab fragments were used to measure binding affinity on a BLI instrument, and human IgG1 antibodies were used for ELISA conjugation and functional assays. Table 10 shows that the top three selected humanized Fabs have similar binding affinity to the mouse parent Fab.

[0270] [Table 10]

[0271] Example 9: Conjugation of human anti-IL-25 antibody to human, mouse, and cynomolgus monkey IL-25 recombinant protein. In this study, three humanized anti-IL-25 antibodies produced in hIgG1 (IL25Ab6-Ab8) and hIgG1 YTE format (IL25Ab9-Ab11) that bind to both human, mouse, and cynomolgus monkey IL-25 were evaluated using ELISA. Briefly, recombinant human IL-25 protein (Acro, catalog number: IL5-H4221, lot number: 401-20CNF1-UT), recombinant mouse IL-25 protein (Sino Biological Inc, catalog number: 50138-M07H, lot number: LC15AP0607), or recombinant cynomolgus monkey IL-25 protein (Sino Biological Inc, catalog number: custom order, lot number: MB17MA2715) were directly coated onto ELISA plates. Next, recombinant antibodies were added to the plates and subsequently detected using goat anti-human kappa light chain antibody HRP (Novus, catalog number: NBP1-75064, lot number: 68-188-010920). After adding ABTS (Moss Inc., catalog number ABTS-1000, lot number 03086202) substrate, the ELISA plates were read using an ELISA plate reader (Bioteck).

[0272] Figure 9A shows that all humanized anti-IL-25 antibodies have an EC level in the range of 0.302 nM to 0.404 nM. 50 The values ​​indicate dose-dependent binding to the human IL-25 protein. The mouse-derived anti-IL-25 specific antibody (IL25Ab3), positive control antibodies IL-25-PC2 and IL-25-PC4 had EC50 values ​​of 0.506 nM, 0.334 nM, and 0.585 nM, respectively. The human IgG1 isotype control did not show binding.

[0273] Figure 9B shows that all humanized anti-IL-25 antibodies have an EC level in the range of 0.222 nM to 0.295 nM. 50The values ​​indicate dose-dependent binding to the cynomolgus monkey IL-25 protein. The mouse-derived anti-IL-25 specific antibody (IL25Ab3), positive control antibodies IL-25-PC2 and IL-25-PC4 had EC50 values ​​of 0.364 nM, 0.365 nM, and 0.408 nM, respectively. The human IgG1 isotype control did not show binding.

[0274] Figure 9C shows that all humanized anti-IL-25 antibodies have an EC level in the range of 0.232 nM to 0.332 nM. 50 The values ​​indicate dose-dependent binding to the mouse IL-25 protein. The mouse-derived anti-IL-25 specific antibody (IL25Ab3), positive control antibodies IL-25-PC2 and IL-25-PC4 had EC50 values ​​of 0.390 nM, 0.420 nM, and 0.407 nM, respectively. The human IgG1 isotype control showed no binding. The results are summarized in Table 11.

[0275] [Table 11]

[0276] Example 10: Blockade of IL-25-induced NF-κB signaling In this study, the blocking activity of a humanized anti-IL-25 antibody was investigated using HEK-Blue® IL-17 cells, as described in Example 4. Briefly, reporter cells were treated with 5 ng / ml recombinant human IL-25 along with serially diluted IL-25 antibody. After overnight incubation, the NF-κB response was determined using QUANTI-Blue® solution, a SEAP detection reagent, and the optical density (OD) was read at 655 nm.

[0277] As summarized in Figure 10 and Table 12, all humanized anti-IL-25 antibodies effectively dose-dependently block IL-25-induced NF-κB signaling and IC 50The values ​​ranged from 0.037 nM to 0.052 nM. The mouse-derived anti-IL-25 specific antibody (IL25Ab3), and the positive control antibodies IL-25-PC2 and IL-25-PC4 had IC50 values ​​of 0.037 nM, 0.030 nM, and 0.513 nM, respectively. The human IgG1 isotype control showed no blocking activity.

[0278] [Table 12]

[0279] Example 11: Blockade of IL-25-induced CXCL1 production To determine whether a humanized anti-IL-25 antibody can block the production of the inflammatory cytokine CXCL1 induced by human IL-25, we used an intracellularly expressed IL17 receptor in HT-29 cells, as described in Example 5. Briefly, HT-29 cells were stimulated with hIL-25 for 72 hours at 37°C with serially diluted combinations of anti-IL-25 antibodies. The supernatant was then collected for measurement of CXCL1 levels using the human CXCL1 ELISA Ready-SET-Go kit (R&D System #DY275). The results presented in Figure 11 and Table 13 demonstrate that the humanized anti-IL-25 antibody retains potent blocking activity against IL-25-induced CXCL1 production, thus potentially reducing the recruitment of immune cells and preventing the development of inflammation.

[0280] IC of humanized antibodies (IL25Ab6-Ab11) 50 The values ​​ranged from 0.183 nM to 0.225 nM. The mouse-derived anti-IL-25 specific antibody (IL25Ab3), positive control antibodies IL-25-PC2 and IL-25-PC4 had IC50 values ​​of 0.144 nM, 0.148 nM, and 1.84 nM, respectively. The human IgG1 isotype control showed no blocking activity.

[0281] [Table 13]

[0282] Example 12: Blocking IL-25-induced IL-5 production To further characterize the biological functions of humanized IL-25 antibodies and evaluate their potential in the treatment of inflammatory diseases, human PBMC assays were performed as described in Example 6. Briefly, IL-25 induced significant amounts of IL-5 production by human PBMCs in the presence of 30 U / ml recombinant hIL-2. The blocking activity of humanized anti-IL-25 activity was determined to indicate their inhibitory activity against type 2 inflammatory responses.

[0283] As summarized in Figure 12 and Table 14, all humanized IL-25 antibodies effectively block IL-25-induced IL-5 production in a dose-dependent manner, and IC 50 The values ​​ranged from 0.381 nM to 1.43 nM. The mouse-derived anti-IL-25 specific antibody (IL25Ab3), positive control antibodies IL-25-PC2 and IL-25-PC4 had IC50 values ​​of 0.302 nM, 0.394 nM, and 7.15 nM, respectively. The human IgG1 isotype control showed no blocking activity.

[0284] [Table 14]

[0285] Unless otherwise indicated, all numbers used in this disclosure and the claims to express properties such as quantities, molecular weights, and reaction conditions of components should be understood in all cases to be modified by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired properties to be obtained by this disclosure. At the very least, without attempting to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be interpreted by considering at least the reported number of significant figures and by applying common rounding techniques.

[0286] Although the numerical ranges and parameters describing the broad scope of this disclosure are approximations, the numerical values ​​described in specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors that inevitably arise from the standard deviation observed in each test measurement.

[0287] The terms “a,” “an,” “the,” and similar references used in the context describing this disclosure (particularly in the context of the claims below) should be construed to encompass both singular and plural forms unless otherwise indicated in this disclosure or unless the context clearly contradicts it. The enumeration of ranges of values ​​in this disclosure is intended to function merely as a concise way of referring individually to each separate value that falls within that range. Unless otherwise indicated in this disclosure, each separate value is incorporated into this disclosure as if it were described separately herein. All methods described in this disclosure may be performed in any appropriate order unless otherwise indicated in this disclosure or unless the context clearly contradicts it. Any and all examples or exemplary language (e.g., “etc.”) provided in this disclosure are intended merely to better illustrate this disclosure and do not impose any limitation on the scope of this disclosure as otherwise described in the claims. Nothing in this specification should be construed as indicating any non-claimed element essential to the practice of this disclosure.

[0288] The grouping of alternative elements or embodiments of the Disclosure disclosed herein should not be construed as limitation. Each group member may be referenced and described in the claims individually or in any combination with other members or elements of the group found herein. It is anticipated that one or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. In the event of such inclusion or omission, this Specification shall be considered to include that group as modified and therefore satisfy the written description of all Markush groups used in the appended claims.

[0289] The specific embodiments of this disclosure are described in this disclosure, including the best mode known to the inventors for carrying out the disclosure. Needless to say, variations of these described embodiments will be apparent to those skilled in the art by reading the foregoing description. The inventors expect that those skilled in the art will adopt such variations as needed, and the inventors intend that this disclosure will be practiced in ways other than those specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter described in the claims attached to this disclosure, as permitted by applicable law. Furthermore, unless otherwise indicated in this disclosure, or unless it is clearly inconsistent with the context, any combination of the elements described above in all possible variations thereof is encompassed by this disclosure.

[0290] The specific embodiments disclosed in this disclosure may be further limited in the claims using the language “consisting of” or “essentially consisting of.” When used in the claims, whether at the time of filing or added with each amendment, the transitional term “consisting of” excludes any elements, processes, or components not specified in the claims. The transitional term “essentially consisting of” limits the claims to the specified materials or processes, as well as materials or processes that do not substantially affect the basic and novel features. Embodiments of the disclosure described in the claims in this manner are essentially or expressly described and enabled in this disclosure.

[0291] It should be understood that the embodiments of the Disclosure disclosed herein are illustrative of the principles of the Disclosure. Other possible modifications are within the scope of the Disclosure. Therefore, alternative configurations of the Disclosure, for example but not limited to them, may be used in accordance with the teachings of the Disclosure. Accordingly, the Disclosure is not limited to those precisely shown and described herein.

[0292] While this disclosure is described and illustrated by reference to various specific materials, procedures, and examples, it should be understood that this disclosure is not limited to specific combinations of materials and procedures selected for its purposes. Multiple variations of such details may be implied to be understood by those skilled in the art. This specification and examples are intended to be illustrative only, and the true scope and spirit of this disclosure is intended to be shown by the following claims. All references, patents, and patent applications referenced herein are incorporated in their entirety by reference.

Claims

1. It is an anti-IL-25 antibody, (a) A heavy chain variable region including the amino acid sequence of SEQ ID NO: 23 (HCDR1), the amino acid sequence of SEQ ID NO: 49 (HCDR2), and the amino acid sequence of SEQ ID NO: 25 (HCDR3), and a light chain variable region including the amino acid sequence of SEQ ID NO: 50 (LCDR1), the amino acid sequence of SEQ ID NO: 51 (LCDR2), and the amino acid sequence of SEQ ID NO: 28 (LCDR3), (b) Heavy chain variable region including the amino acid sequence of SEQ ID NO: 23 (HCDR1), the amino acid sequence of SEQ ID NO: 52 (HCDR2), and the amino acid sequence of SEQ ID NO: 25 (HCDR3), and light chain variable region including the amino acid sequence of SEQ ID NO: 50 (LCDR1), the amino acid sequence of SEQ ID NO: 51 (LCDR2), and the amino acid sequence of SEQ ID NO: 28 (LCDR3), (c) Heavy chain variable region including the amino acid sequence of SEQ ID NO: 11 (HCDR1), the amino acid sequence of SEQ ID NO: 12 (HCDR2), and the amino acid sequence of SEQ ID NO: 13 (HCDR3), and light chain variable region including the amino acid sequence of SEQ ID NO: 14 (LCDR1), the amino acid sequence of SEQ ID NO: 15 (LCDR2), and the amino acid sequence of SEQ ID NO: 16 (LCDR3), (d) Heavy chain variable region including amino acid sequence 17 (HCDR1) of SEQ ID NO: (e) Heavy chain variable region including the amino acid sequence of SEQ ID NO: 23 (HCDR1), the amino acid sequence of SEQ ID NO: 24 (HCDR2), and the amino acid sequence of SEQ ID NO: 25 (HCDR3), and light chain variable region including the amino acid sequence of SEQ ID NO: 26 (LCDR1), the amino acid sequence of SEQ ID NO: 27 (LCDR2), and the amino acid sequence of SEQ ID NO: 28 (LCDR3), (f) A heavy chain variable region including the amino acid sequence of SEQ ID NO: 29 (HCDR1), the amino acid sequence of SEQ ID NO: 30 (HCDR2), and the amino acid sequence of SEQ ID NO: 31 (HCDR3), and a light chain variable region including the amino acid sequence of SEQ ID NO: 32 (LCDR1), the amino acid sequence of SEQ ID NO: 33 (LCDR2), and the amino acid sequence of SEQ ID NO: 34 (LCDR3), or (g) Heavy chain variable region including the amino acid sequence of SEQ ID NO: 35 (HCDR1), the amino acid sequence of SEQ ID NO: 36 (HCDR2), and the amino acid sequence of SEQ ID NO: 37 (HCDR3), and light chain variable region including the amino acid sequence of SEQ ID NO: 38 (LCDR1), the amino acid sequence of SEQ ID NO: 39 (LCDR2), and the amino acid sequence of SEQ ID NO: 40 (LCDR3) Anti-IL-25 antibodies, including [specific antibody name].

2. An anti-IL-25 antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region containing any one amino acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47, and a light chain variable region containing any one amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, or 48.

3. The anti-IL-25 antibody according to claim 1, (a) Heavy chain variable region sequence of Sequence ID No. 44 and light chain variable region sequence of Sequence ID No. 45; (b) Heavy chain variable region sequence of Sequence ID No. 44 and light chain variable region sequence of Sequence ID No. 46; (c) Heavy chain variable region sequence of Sequence ID No. 47 and light chain variable region sequence of Sequence ID No. 48; (d) Heavy chain variable region sequence of Sequence ID No. 1 and light chain variable region sequence of Sequence ID No. 2; (e) Heavy chain variable region sequence of Sequence ID No. 3 and light chain variable region sequence of Sequence ID No. 4; (f) Heavy chain variable region sequence of Sequence ID No. 5 and light chain variable region sequence of Sequence ID No. 6; (g) Heavy chain variable region sequence of Sequence ID No. 7 and light chain variable region sequence of Sequence ID No. 8; or (h) Heavy chain variable region sequence of Sequence ID No. 9 and light chain variable region sequence of Sequence ID No. 10, Anti-IL-25 antibodies, including [specific antibody name].

4. The anti-IL-25 antibody according to claim 1, wherein the antibody is an anti-human anti-IL-25 antibody.

5. The anti-IL-25 antibody according to claim 1, wherein the antibody is a full-length antibody containing a human IgG1 constant region selected from SEQ ID NO: 55 or SEQ ID NO:

56.

6. The anti-IL-25 antibody according to claim 1, wherein the antibody is an antibody fragment.

7. An anti-IL-25 antibody according to claim 4, wherein the antibody fragment is selected from the group consisting of Fab, Fab, F(ab)2, Fd, Fv, scFv, and scFv-Fc fragments, single-chain antibodies, minibodies, and diabodies.

8. The anti-IL-25 antibody according to claim 1, wherein the antibody is a monoclonal antibody.

9. The anti-IL-25 antibody according to claim 1, wherein the antibody is a human antibody.

10. The anti-IL-25 antibody according to claim 1, wherein the antibody is a mouse antibody.

11. The anti-IL-25 antibody according to claim 1, wherein the antibody is a chimeric antibody.

12. The anti-IL-25 antibody according to claim 1, wherein the antibody is a bispecific or multispecific antibody.

13. The anti-IL-25 antibody according to claim 1, wherein the antibody is a humanized antibody.

14. A pharmaceutical composition comprising the antibody described in claim 1 and a pharmaceutically acceptable carrier.

15. A method for treating and / or preventing a subject in need of treatment and / or prevention of a type 2 inflammatory disease, autoimmune disease, allergic disorder, or cancer, comprising administering the antibody described in claim 1 to the subject.

16. A polynucleotide composition, a) A first polynucleotide encoding a heavy chain variable region containing the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 44, or 47, and b) A second polynucleotide encoding a light chain variable region containing the amino acid sequence described in SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, or 48, A polynucleotide composition containing the following:

17. A vector composition, a) A first vector comprising the first polynucleotide described in claim 16, b) A second vector comprising the second polynucleotide described in claim 16, A vector composition containing the following:

18. A cell comprising the polynucleotide composition according to claim 16, or the vector composition according to claim 17.

19. A method for producing an anti-IL-25 antibody according to claim 1, wherein the method comprises culturing the cells according to claim 18 in a culture medium and recovering the anti-IL-25 antibody from the culture medium.

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