Anti-IL-25 antibodies and methods of use thereof

Abstract

The present disclosure provides antibodies and antibody fragments thereof that bind to IL-25. The antibodies and antibody fragments thereof of the present disclosure can modulate the biological activity of the IL-25 / IL-25 receptor signaling axis, and thus are useful in the treatment of type 2 inflammatory diseases, autoimmune diseases, allergic diseases, or cancers.

Description

Technical Field

[0001] The present disclosure generally relates to antibodies and antibody fragments thereof that bind to interleukin-25. The antibodies can be used to prevent and / or treat type 2 inflammatory diseases, autoimmune diseases or cancer. Background Art

[0002] The interleukin-17 (IL-17) family belongs to a group of cytokines that play a key role in inflammatory responses during autoimmune pathogenesis 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) and even some cancer cells (e.g., melanoma, liver cancer, breast cancer, and cervical cancer) (Gowhari Shabgah, A. et al., Cancer Med. (2021) 10: 5191–5202). The diversity of cell sources indicates that IL-25 is involved in many immune responses and cellular processes.

[0003] IL-25 is significantly different from other IL-17 family members in both its molecular structure and biological function. As an epithelial cell cytokine, IL-25, together with TSLP and IL-33, acts as an "alarmin" or barrier surface cytokine, alerting the immune system to the presence of external environmental threats (such as allergens, bacteria, viruses and worms), and mobilizing the host's immune defense mechanism (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 shown that epithelial cell factors also have a wide range of effector functions under many pathological conditions, including type 2 inflammatory diseases, allergic diseases, viral infections, chronic inflammatory diseases, autoimmune conditions and cancer.

[0004] IL-25 plays a dual role in regulating immune responses and the pathogenesis of autoimmune diseases. As a proinflammatory cytokine, IL-25 aggravates allergic inflammation by promoting Th2 cells to produce Th2 cytokines (including IL-4, IL-5, and IL-13). In addition, IL-25 can induce the proliferation and activation of innate immune cells, the production of other proinflammatory cytokines, and the 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 as an anti-inflammatory cytokine and inhibitor of innate and adaptive immunity in autoimmune diseases (Saadoun, D. et al., Current pharmaceutical design (2011) 17: 3781–3785). For example, the anti-inflammatory and immunosuppressive activity of IL-25 in rheumatoid arthritis is attributed to the downregulation of IL-13-dependent Th17 cell responses (Liu, D. et al., Sci Rep (2016) 6: 36002).

[0006] In addition, the expression of IL-25 and its receptors is dysregulated in various cancers compared with normal tissues. Therefore, it is also pointed out that IL-25 may also play a dual role in cancer progression or regression. The tumor suppressor effect of IL-25 is mainly attributed to the infiltration of eosinophils and B cells into the tumor microenvironment and induction of apoptosis. In contrast, its tumor supportive effect depends on the deviation of the immune response and the stimulation of EMT and cell growth (Gowhari Shabgah, A. et al., Cancer Med. (2021) 10: 5191–5202).

[0007] Although the IL-25 pathway is a recognized therapeutic target, only one Phase I clinical trial evaluating the safety, tolerability and pharmacokinetic characteristics of IL-25 has been determined to date. Therefore, there is an unmet need in the art for novel IL-25 antagonists (e.g., anti-IL-25 antibodies disclosed herein) for treating diseases or conditions associated with IL-25 expression and / or signaling. Summary of the invention

[0008] The present disclosure addresses the above needs by providing antibodies and antibody fragments thereof that bind to IL-25 (including human IL-25). The antibodies and antibody fragments thereof are used to treat immune-mediated inflammatory diseases (IMIDs) (e.g., autoimmune diseases and inflammatory conditions) and cancer. Anti-IL-25 antibodies or antibody fragments thereof can be used alone (e.g., as a monotherapy) or in combination with other immunotherapeutic agents. In an alternative embodiment, the antibody fragments of the IL-25 antibodies disclosed herein can be used as components of bispecific or multispecific antibodies or fusion proteins.

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

[0010] In some embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 11, CDR2: SEQ ID NO: 12, and CDR3: SEQ ID NO: 13; and / or a light chain variable region comprising 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 antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 17, CDR2: SEQ ID NO: 18, and CDR3: SEQ ID NO: 19; and / or a light chain variable region comprising CDR1: SEQ ID NO: 20, CDR2: SEQ ID NO: 21, and CDR3: SEQ ID NO: 22.

[0012] In some embodiments, the anti-IL-25 antibody or 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 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 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 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 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 SEQ ID NOs: 1, 3, 5, 7, and 9, and a variable light chain sequence selected from 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 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:

[0024] (a) a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2;

[0025] (b) a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4;

[0026] (c) a variable heavy chain sequence comprising SEQ ID NO: 5 and a variable light chain sequence comprising SEQ ID NO: 6;

[0027] (d) a variable heavy chain sequence comprising SEQ ID NO: 7 and a variable light chain sequence comprising SEQ ID NO: 8;

[0028] (e) a variable heavy chain sequence comprising SEQ ID NO: 9 and a variable light chain sequence comprising SEQ ID NO: 10;

[0029] (f) a variable heavy chain sequence comprising SEQ ID NO: 44 and a variable light chain sequence comprising SEQ ID NO: 45;

[0030] (g) a variable heavy chain sequence comprising SEQ ID NO: 44 and a variable light chain sequence comprising SEQ ID NO: 46 or

[0031] (h) a variable heavy chain sequence comprising SEQ ID NO: 47 and a variable light chain sequence comprising SEQ ID NO: 48.

[0032] In some embodiments, the antibody is a human anti-IL-25 antibody.

[0033] In some embodiments, the antibody is a full-length antibody.

[0034] In some embodiments, the antibody is a full length antibody comprising a human IgG1 constant region selected from SEQ ID NO:55 or SEQ ID NO:56.

[0035] 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.

[0036] In some embodiments, the antibody is a monoclonal antibody.

[0037] In some embodiments, the antibody is a human antibody (eg, a fully human antibody).

[0038] In some embodiments, the antibody is a mouse antibody.

[0039] In some embodiments, the antibody is a chimeric antibody.

[0040] In some embodiments, the antibody is a bispecific antibody.

[0041] In some embodiments, the antibody is a humanized antibody.

[0042] The present disclosure also provides a pharmaceutical composition comprising the antibody or antibody fragment thereof disclosed herein and a pharmaceutically acceptable carrier.

[0043] The present disclosure further provides a method of treating and / or preventing a type 2 inflammatory disease, an autoimmune disease, an allergic disorder, or cancer in a subject in need thereof, the method comprising: administering to the subject an antibody or antibody binding fragment as disclosed herein.

[0044] The present disclosure also provides a polynucleotide composition comprising a first polynucleotide encoding a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 44 or 47; and a second polynucleotide encoding a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48.

[0045] In addition, the present disclosure provides a vector composition comprising a first vector containing a first polynucleotide disclosed herein (e.g., a polynucleotide encoding SEQ ID NO: 1, 3, 5, 7, 9, 44 or 47) and a second vector containing a second polynucleotide disclosed herein (e.g., a polynucleotide encoding SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48).

[0046] The present disclosure also provides cells (eg, CHO cells) comprising a polynucleotide composition disclosed herein or a vector composition disclosed herein.

[0047] The present disclosure also provides a method for producing the anti-IL-25 antibody or antibody fragment thereof disclosed herein, the method comprising culturing a cell expressing the IL-25 antibody or antibody fragment thereof disclosed herein in a culture medium; and recovering the anti-IL-25 antibody or antibody fragment thereof from the culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] When read in conjunction with the accompanying drawings, the foregoing summary of the invention and the specific embodiments of the present disclosure below will be better understood. For the purpose of illustrating the present disclosure, shown in the drawings are currently preferred embodiments. However, it should be understood that the present disclosure is not limited to the precise arrangements, embodiments and means shown.

[0049] Figures 1A to 1D The amino acid sequences of the VH and VL domains of the anti-IL-25 antibodies and their respective CDR sequences are provided. Sequence identifiers are provided, and the CDRs are determined by Kabat, and the CDRs are underlined in the variable domain sequences.

[0050] Figure 2A-2B The amino acid sequences of human, mouse and cynomolgus monkey IL-25, as well as the amino acid sequences of human or mouse antibody constant regions are provided.

[0051] Figure 3A-3C The binding of anti-IL-25 antibody to recombinant human, mouse and cynomolgus monkey IL-25 proteins was shown. ELISA using goat anti-human IgG-HRP detection showed that anti-IL-25 antibody bound to recombinant human ( Figure 3A ) and mice ( Figure 3B )IL-25 protein. ELISA using goat anti-human κ light chain antibody HRP detection showed that anti-IL-25 antibody bound to cynomolgus monkey ( Figure 3C ) Binding of IL-25 protein.

[0052] Figure 4 It was demonstrated that anti-IL-25 antibody inhibited human IL-25-induced NFkB signaling in HEK-Blue IL-17 reporter cells.

[0053] Figure 5 Anti-IL-25 antibody was shown to inhibit IL-25-induced CXCL-1 production in the human colon cancer cell line HT29.

[0054] Figure 6 Anti-IL-25 antibodies were shown to inhibit IL-25-induced IL-5 production in a human PBMC assay.

[0055] Figure 7A-7B Anti-IL-25 antibodies were shown to reduce airway resistance in an OVA-induced asthma model. Airway hyperresponsiveness (AHR) to methacholine stimulation in OVA-sensitized / challenged BALB / c mice. The AHR of each mouse was measured on day 31 using the Buxo whole body plethysmography (WBP) system. The AHR was expressed as a percentage change from the baseline level of pulmonary resistance (Penh value). Dose-response data are presented as group mean ± SEM ( Fig. 7A ) and the AUC of the baseline Penh percentage ( Figure 7B ). **p<0.01, ***p<0.001 compared with OVA+vehicle using repeated measures / Bonferroni ( Fig. 7A ), and using one-way Anova / Dunnett, **p<0.01, ***p<0.001 compared with OVA+vehicle ( Figure 7B ).

[0056] Figure 8 It is shown that anti-IL-25 antibody inhibits IL-5 production in the lung in the OVA-induced asthma model. **p<0.01, ***p<0.001 compared to OVA+vehicle using one-way Anova / Dunnett.

[0057] Figure 9A-9C The comparison of humanized anti-IL-25 antibody with recombinant human IL-25 ( Fig. 9A )、Cynomolgus monkey IL-25( Fig. 9B ) and mouse IL-25 ( Fig. 9C ) combination.

[0058] Fig.10 It was demonstrated that humanized anti-IL-25 antibody inhibited human IL-25-induced NFkB signaling in HEK-Blue IL-17 reporter cells.

[0059] Fig.11 Humanized anti-IL-25 antibody was shown to inhibit IL-25-induced CXCL-1 production in the human colon cancer cell line HT29.

[0060] Fig.12 The humanized anti-IL-25 antibody was shown to inhibit IL-25-induced IL-5 production in a human PBMC assay. DETAILED DESCRIPTION

[0061] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meanings commonly understood by ordinary technicians in the field to which the present disclosure belongs.

[0062] Throughout this disclosure, the following abbreviations will be used:

[0063] mAb or Mab or MAb—monoclonal antibody.

[0064] CDR—complementarity determining region in an immunoglobulin variable region.

[0065] HCDR - heavy chain complementarity determining region.

[0066] LCDR - light chain complementarity determining region.

[0067] VH—Immunoglobulin heavy chain variable region.

[0068] VL—Immunoglobulin light chain variable region.

[0069] FR—antibody framework region, the immunoglobulin variable region excluding the CDR region.

[0070] As used herein, the term "interleukin-25" (interchangeably used with "IL-25") refers to the native sequence polypeptide, isomers, 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 sequence: 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). The orthologs of IL-25 have about 92% and about 80% homology with the human protein in cynomolgus monkey and mouse, respectively.

[0071] As used herein, the term "IL-25 complex" refers to the IL-17RA / IL-17RB complex, also known as the IL-25 receptor.

[0072] As used herein, the term "IL-17RA" refers to 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 interleukin 17A with low affinity.

[0073] As used herein, the term "IL-17RB" refers to interleukin 17 receptor B. This receptor specifically binds IL-17B and IL-17E (IL-25), but not IL-17A or IL-17C.

[0074] As used herein, the term "antibody" is used in the broadest sense and includes various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies).

[0075] Exemplary antibodies such as IgG comprise two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions (referred to as complementary determining regions (CDRs)), interspersed with more conservative regions, referred to as framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0076] The hypervariable region generally comprises about amino acid residues 24-34 (LCDR1; "L" for light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region and about 31-35B (HCDR1; "H" for 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 of Health, Bethesda, Md. (1991) and / or those residues that form the hypervariable loops (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region and residues 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.

[0077] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, for example, each antibody constituting a population is identical and / or in conjunction with the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or produced in the production process of monoclonal antibody preparations (these variants are usually present in small amounts). Contrary to the polyclonal antibody preparations that usually contain different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents the characteristic of the antibody obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibody production by any method. For example, the monoclonal antibody used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma method, recombinant DNA method, phage display method and a method utilizing a transgenic animal containing all or part of a human immunoglobulin locus, and these methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0078] The term "chimeric" antibody refers to a recombinant antibody in which part of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0079] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human, and / or an antibody that has been prepared using any technique known to those skilled in the art for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including those 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 can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled, e.g., immunized HuMab mice (see, e.g., Nils Lonberg et al., 1994, Nature 368:856-859, WO 98 / 24884, WO 94 / 25585, WO 93 / 1227, WO 92 / 22645, WO 92 / 03918, and WO 01 / 09187 for HuMab mice), xenomice (see, e.g., XENOMOUSE TM Nos. 6,075,181 and 6,150,584 of the technology) or Trianni mice (see, e.g., WO 2013 / 063391, WO 2017 / 035252, and WO 2017 / 136734).

[0080] The term "humanized antibody" refers to an antibody that has been engineered to contain one or more human framework regions in the variable region and non-human (e.g., mouse, rat or hamster) complementarity determining regions (CDRs) of the heavy and / or light chains. In certain embodiments, except for the CDR regions, the humanized antibody comprises completely human sequences. Compared with non-humanized antibodies, humanized antibodies are generally less immunogenic to humans and therefore may provide therapeutic benefits in certain cases. Those skilled in the art will understand humanized antibodies and will also understand suitable techniques for their production. See, 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, 1989; U.S. Patents 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 which is incorporated herein by reference in its entirety.

[0081] The "class" of an antibody refers to the type of constant domain or region possessed by 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 the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0082] The term "antigen binding domain" of an antibody or of an antibody (simply "binding domain") or similar terms refers to one or more fragments of an antibody that retain 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) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) isolated complementarity determining regions (CDRs) and (vii) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker.

[0083] As used herein, the term "complementarity determining region" or "CDR" refers to a short polypeptide sequence within the variable region of a heavy chain and a light chain polypeptide that is primarily responsible for mediating specific antigen recognition. There are three CDRs (called CDR1, CDR2, and CDR3) in each VL and each VH.

[0084] As will be appreciated by those skilled in the art, the exact numbering and placement of CDRs may be different in different numbering systems. However, it should be understood that disclosure of variable heavy chain sequences and / or variable light chain sequences includes disclosure of the associated CDRs. Thus, disclosure of each variable heavy chain region is disclosure of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and disclosure of each variable light chain region is disclosure of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0085] In certain embodiments, the CDRs of an antibody may be identified 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 herein by reference in its entirety. Unless otherwise indicated herein, references to residue numbers in an antibody variable domain refer to residue numbering by the Kabat numbering system.

[0086] In other embodiments, the CDRs of an antibody may be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745, which is incorporated herein by reference in its entirety. See, e.g., Martin A "Protein Sequence and Structure Analysis of Antibody Variable Domains," Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), which is incorporated herein by reference in its entirety. In other embodiments, the CDRs of an antibody may be determined according to the AbM numbering scheme, which refers to the AbM hypervariable region, which represents 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, Inc.), which is incorporated herein by reference in its entirety.

[0087] "Framework" or "framework region" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4.

[0088] "Human consensus framework" is a framework representing the most common amino acid residues in a collection of human immunoglobulin VL or VH framework sequences. Typically, a collection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, a sequence subgroup is a subgroup such as Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as described above by Kabat et al. In one embodiment, for VH, the subgroup is subgroup III as described above by Kabat et al.

[0089] The "hinge region" is generally defined as extending from 216-238 (EU numbering) or 226-251 (Kabat numbering) of human IgGl. The hinge can be further divided into three distinct regions, the upper hinge, the middle hinge (eg, the core), and the lower hinge.

[0090] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0091] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0092] An "antibody that binds to the same epitope as a reference antibody" is one that contacts an overlapping set of antigenic amino acid residues as compared to the reference antibody, or blocks 50% or more of the binding of the reference antibody to its antigen in a competition 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 in complex with the antigen or by performing hydrogen / deuterium exchange. In some embodiments, the amino acid residues located at the antigen In some embodiments, an antibody that binds to the same epitope as the reference antibody blocks 50% or more of the binding of the reference antibody to its antigen in a competition assay, and conversely, the reference antibody blocks 50% or more of the binding of the antibody to its antigen in a competition assay.

[0093] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an 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; single-chain antibody molecules (e.g., scFv). Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments and a residual "Fc" fragment, a name that reflects the ability to crystallize readily. The Fab fragment consists of an intact light (L) chain as well as the variable region domain (VH) of the heavy (H) chain and the first constant domain (CH1) of one heavy chain. Pepsin treatment of an antibody produces a single large F(ab)2 fragment that roughly corresponds to two disulfide-linked Fab fragments that have divalent antigen-binding activity and are still able to cross-link antigen. Fab fragments differ from Fab' fragments in that there are several additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH refers herein to Fab' in which the cysteine ​​residues of the constant domains carry a free thiol group. F(ab')2 antibody fragments originally were produced as a pair of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0094] "Fv" consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight non-covalent association. Six hypervariable loops (3 loops each in the H chain and L chain) are generated from the folding of these two domains, which provide amino acid residues for antigen binding and give the antibody antigen binding specificity.

[0095] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the structure required for antigen binding. For a review of sFv, see Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0096] When used to describe the various antibodies disclosed herein, the term "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from a cell or cell culture in which it is expressed. Contaminant components of its natural environment are substances that would normally interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). In one embodiment, the antibody is purified: (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.

[0097] With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or an epitope on a particular polypeptide target refers to binding that is measurably different from non-specific interactions. For example, specific binding can be measured by measuring the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be measured by competition with a control molecule similar to the target (e.g., an excess of unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. As used herein, the term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or an epitope on a particular polypeptide target can be expressed, for example, as a molecule having a Kd of 10 for the target. -4 M or lower, or 10 -5 M or lower, or 10 -6 M or lower, or 10 -7 M or lower, or 10 -8 M or lower, or 10 -9 M or lower, or 10 -10 M or lower, or 10 -11 M or lower or 10 -12 M or lower, or Kd in 10 -4 M to 10 -6 M, or 10 -6 M to 10 -10M or 10 - 7 M to 10 -9 M. Those skilled in the art will appreciate that affinity and KD values ​​are negatively correlated. 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 specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope. As used herein, the terms "specific binding", "specifically bind", and "selectively bind" refer to the binding of an antibody to an epitope of interleukin-25.

[0098] The term "affinity" as used herein refers to the strength of binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant Ka is defined as 1 / Kd. Methods for determining mAb affinity can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., ed., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993) and Muller, Meth. Enzymol. 92: 589-601 (1983), the references of which are incorporated herein by reference in their entirety. One standard method known in the art for determining mAb affinity is to use surface plasmon resonance (SPR) screening (e.g., by using BIAcore TM SPR analysis equipment).

[0099] "Epitope" is a term of art, which refers to one or more sites of interaction between an antibody and its antigen. Description is found in (Janeway, C, Jr., P. Travers et al. (2001). Immunobiology: the immune system in health and disease. Part II, Sections 3-8. Part II, Sections 3-8. New York, Garland Publishing, Inc.): Antibodies usually only recognize a small area on the surface of a macromolecule (such as a protein) ... [Some epitopes] may be composed of amino acids from different parts of the [antigen] polypeptide chain that are gathered together by protein folding. This type of antigenic determinant is called a conformational epitope or a discontinuous epitope because the structure identified is composed of segments of proteins that are discontinuous in the antigen amino acid sequence but gathered together in a three-dimensional structure. In contrast, an epitope consisting of a single segment of a polypeptide chain is referred to as a continuous epitope or linear epitope" (Janeway, C. Jr., P. Travers et al. (2001). Immunobiology: the immune system in health and disease. Part II, Sections 3-8. Part II, Sections 3-8. New York, Garland Publishing, Inc.).

[0100] As used herein, the term "KD" is intended to refer to the dissociation constant of a specific antibody-antigen interaction. It is calculated by the following formula: Koff / Kon=KD.

[0101] As used herein, the term “IC 50 " is intended to refer to the effective concentration of an antibody of the invention required to inhibit 50% of a particular biological or biochemical function.

[0102] “EC” for agents and specific activities (e.g., binding to cells, inhibiting enzyme activity, activating or inhibiting immune cells) 50 "Effective concentration of an agent that produces 50% of its maximum response or effect with respect to that activity. The "EC 100 ” refers to the effective concentration of an agent that produces a substantially maximal response for that activity.

[0103] As used herein, the terms "antibody-based immunotherapy" and "immunotherapy" are used to refer generally to any form of therapy that relies on the targeting specificity of an anti-IL-25 antibody, bispecific molecule, multispecific molecule, binder, or fusion protein comprising an IL-25-specific binder to mediate direct or indirect effects on cells characterized by abnormal expression of IL-25. The term is intended to include treatment methods using naked antibodies, bispecific antibodies (including T cell engagement, NK cell engagement, and other immune cell / effector cell engagement forms), antibody drug conjugates, cell therapy using T cells (CAR-T) or NK cells (CAR-NK) engineered to contain IL-25-specific chimeric antigen receptors, and oncolytic viruses comprising IL-25-specific binders, as well as gene therapy by delivering antigen binding sequences of anti-IL-25 antibodies and expressing corresponding antibody fragments in vivo.

[0104] As used herein, "Group 2innate lymphoid cell" (ILC2) refers to an immune cell type that plays an important role in the early immune response to parasitic worms, allergens, and other environmental insults. ILC2 is part of the innate immune system, which means that they are able to respond quickly to a variety of stimuli without previous exposure to pathogens or specific recognition of pathogens. ILC2 is characterized by the ability to produce a variety of cytokines, including interleukin (IL) -5 and IL-13, which recruit and activate other immune cells, such as eosinophils, mast cells, and T helper 2 (Th2) cells, to promote type 2 immune responses. This type of response plays an important role in responding to parasitic infections, allergic reactions, and tissue repair.

[0105] The term "type 2 inflammation," also known as type 2 immune response, as used herein, is a specific type of immune response that is activated in response to a variety of stimuli, such as parasitic infections, allergens, and tissue damage. Type 2 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.

[0106] IL-25 / IL-25R shaft

[0107] I. IL-17 cytokine family

[0108] The IL-17 family is a group of cytokines consisting of 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 lymphocytes, and they play key roles in both innate and adaptive immune responses (McGeachy, MJ Immunity. 2019 Apr 16;50(4):892–906).

[0109] IL-17A and IL-17F are the most intensively studied members of the IL-17 family, which are often co-expressed and have similar functions. They promote inflammation by inducing the expression of proinflammatory cytokines, chemokines, and adhesion molecules, and stimulate the recruitment and activation of neutrophils and other immune cells.

[0110] IL-17B, IL-17C, and IL-17E have more specific roles. IL-17B and IL-17C are involved in host defense against bacterial and fungal infections, while IL-17E plays an important role in allergic responses and protection against parasitic infections.

[0111] The IL-17 cytokine interacts with various cell types expressing different heterodimeric receptor complexes, which contain five homologous subunits IL-17-RA to IL-17RE, of which IL-17RA is common to all receptors. Each subunit of the IL-17 receptor is a protein containing a transmembrane domain and several conserved motifs, such as the extracellular fibronectin III-like motif, the transmembrane region, and the cytoplasmic SEF / IL-17R (SEFIR) domain (Zhang Q, J Biol Chem. 2013; 288(51): 36956-36965).

[0112] IL-17 family cytokines have been implicated in the pathogenesis of various inflammatory and autoimmune diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease, and are targets for therapeutic intervention in these pathologies.

[0113] II.IL-25 (IL-17E)

[0114] IL-25 has approximately 23% to 33% homology with other IL-17 family members and binds to specific homologous IL-17 receptors to transmit signals. The low sequence similarity between IL-25 and other family members suggests that anti-IL25 specific antibodies are unlikely to bind to other IL-17 family members.

[0115] IL-25 is synthesized as a precursor molecule that is processed and cleaved to produce a mature, biologically active form of the cytokine. The cleavage of the IL-25 precursor is mediated by several proteases, including furin and other proprotein convertases (PCs), which cleave the precursor protein at specific sites to produce mature cytokines. The exact cleavage site and mechanism depends on the cell type or tissue that produces IL-25. Specifically, 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).

[0116] IL-25 is secreted as a disulfide-linked homodimeric glycoprotein and is produced by a variety of cells, including epithelial cells, T cells, and innate lymphoid cells.

[0117] IL-25 interacts with the IL-25 receptor, which contains the heterodimeric IL-17RB / IL-17RA receptor subunits, and signals through the IL-25 receptor (Iwakura et al., (2010), Immunity, 34:149). IL-25 promotes and maintains Th2-type immune responses through its receptor and triggers the expansion of type 2 innate lymphocytes (ILC2) and M2 macrophages.

[0118] Although IL-25 is widely produced by various types of cells, the expression of its receptor is relatively limited. IL-25 receptors are present in innate immune cells, such as invariant natural killer T (iNKT) cells, ILC2, eosinophils, basophils, mast cells and antigen presenting cells (APC). Cells express IL-17RB, and changes in IL-17RB expression in different diseases, especially in airway inflammatory diseases and skin inflammation (Rickel EA, J Immunol. 2008; 181 (6): 4299-4310).

[0119] IL-25 interacts exclusively with IL-17RB, allosterically promoting the formation of the IL-17RB-IL-17RA "tip-to-tip" interface, a key receptor-receptor interaction required to initiate signal transduction.

[0120] After IL-25 binds to the receptor complex, it triggers the recruitment and phosphorylation of ACT1, which is a key adapter protein for downstream signal transduction. Studies have shown that IL-25 can activate multiple downstream signal cascades in a cell context-dependent manner, including nuclear factor κB (NF-kB), 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), leading to the expression of various proinflammatory cytokines, chemokines and other immune-related genes (Amini P et al. (2018). American Journal of Rhinology & Allergy, 32 (2), 115-124.).

[0121] III. Mechanism of action of IL-25 in pathogenesis

[0122] IL-25 can enhance Th2-type immune responses. Th2 cells are a subset of T cells that produce cytokines such as IL-4, IL-5, and IL-13 that 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.).

[0123] IL-25 can also stimulate innate lymphoid cells (ILC2). ILC is a group of immune cells that play a role in defense against pathogens and tissue repair. ILC2 is a subpopulation of ILC that produces Th2 cytokines and participates in allergic reactions. IL-25 can stimulate the activation and proliferation of ILC2, thereby producing Th2 cytokines and initiating immune responses (Wu. J. Front Immunol. 2022; 13: 986118).

[0124] IL-25 can also promote the accumulation of induced co-stimulatory factors (ICOS) and T1 / ST2 on new innate lymphocytes (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, which are expressed on the surface of nuocytes and participate in their activation and function (C. Wang et al., PLoS One, Vol. 11, No. 9).

[0125] IL-25 can also stimulate naive T cells to differentiate into Th2 cells, thereby producing Th2 cytokines such as IL-4, IL-5, and IL-13.

[0126] In addition, IL-25 increases the production of chemokines 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 and promote the recruitment of eosinophils, which participate in defense against parasites and allergic reactions. This recruitment can lead to inflammation and tissue damage (J. Beale et al., Sci Trans Med, Vol. 6, No. 256, (2014).

[0127] IL-25 can also promote airway remodeling, which refers to changes in the structure of the airways that occur due to chronic inflammation. This can include increased mucus secretion, extracellular collagen deposition, smooth muscle cell proliferation, and angiogenesis (formation of new blood vessels). These changes can lead to airway obstruction and dyspnea (M. Suzukawa et al., J Immunol. 2012 Oct 1; 189(7)).

[0128] IV. Targeted Therapy for IL-25-Associated Diseases and Conditions

[0129] a. Type 2 inflammatory disorders

[0130] Respiratory diseases such as asthma and allergies are characterized by type 2 inflammation. IL-25 is a type 2 cytokine produced by Th2 cells that induces the production of IL-4, IL-5, and IL-13, leading to airway inflammation.

[0131] Asthma is a heterogeneous inflammatory disease characterized by airflow obstruction, wheezing, and airway infiltration with eosinophils and neutrophils. Asthma can be broadly divided into two categories: eosinophilic (T helper 2 [Th2]) and noneosinophilic (non-Th2), although it is recognized that asthma encompasses multiple phenotypes with distinct underlying biological mechanisms.

[0132] Elevated levels of IL-25 in plasma are associated with an allergic asthma phenotype (Tang W. et al., Int Arch Allergy Immunol; 163: 5–10 (2014)), and IL-25 concentrations in sputum are associated with disease severity. Patients with atopic asthma also have higher IL-25 levels in their sputum than those with non-atopic asthma (Paplinska-Goryca M. et al., Postepy Dermatol Alergol; 35: 462-469 (2018)). The relationship between IL-25 and neutrophilic airway inflammation suggests that IL-25 has pleiotropic effects in the immune response in asthma. In addition, rare alleles of the IL-25 receptor component IL-17RB are associated with a reduced incidence of asthma (Jung JS et al., Association of IL-17RB gene polymorphism with asthma. Chest 135: 1173: -1180 (2009)).

[0133] In animal models of asthma, overexpression or administration of recombinant IL-25 triggers an allergic response characterized by Th2 cytokine expression, eosinophilia, and mucus hypersecretion (U.S. Pat. No. 6,159,193). The effect of blocking IL-25 may be due to reducing the expression of Th2 cytokines in allergic reactions and inhibiting 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 hyperresponsiveness, 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)). In addition, IL-25 knockout mice showed reduced lung lesions 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 shown in preclinical studies with the anti-IL-25 monoclonal antibody ABM125 (Lee, J. et al., Biochemical and biophysical research communications, 495(1), 1391-1397)).

[0134] Psoriasis is an autoimmune disease that causes skin inflammation and is characterized by epidermal hyperplasia, increased angiogenesis, and dermal inflammation. Although the exact cause of psoriasis remains unclear, studies have suggested that an imbalance of Th1 / Th2 cells and the involvement of Th17 cells may contribute to the disease. IL-25 is a cytokine produced by keratinocytes that has been found to promote skin inflammation by recruiting neutrophils and activating macrophages. IL-25 is highly expressed in skin lesions of patients with psoriasis and promotes the proliferation of IL-17RB+ keratinocytes, thereby exacerbating the disease. IL-25 can activate keratinocytes by activating the STAT3 transcription factor, leading to the expression of IL-17RB (Xu M et al., Inflammation. Immunity (2018) 48(4):787–98). Blocking IL-17RA (a common 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.

[0135] b. Autoimmune diseases

[0136] Studies have shown that IL-25 has both pro-inflammatory and anti-inflammatory effects depending on the specific disease context and the role of the relevant cell types. In some autoimmune diseases, such as rheumatoid arthritis (RA) and multiple sclerosis (MS), IL-25 was found to have a protective role as an anti-inflammatory cytokine and an innate and adaptive immunosuppressant. In some diseases, such as inflammatory bowel disease (IBD) and systemic lupus erythematosus (SLE), IL-25 has both pro-inflammatory and anti-inflammatory effects in regulating immune responses during disease development (Deng, D. et al., Front Immunol. 2021; 12: 691559).

[0137] However, in other autoimmune diseases, such as Sjögren's syndrome (SS), studies have shown that IL-25 plays a pathogenic role in the development of SS. Studies have shown that IL-17A plays a key role in the pathogenesis of Sjögren's syndrome by promoting the activation of Th17 cells. Recently, it was observed that IL-25 was significantly increased in the SG and peripheral blood of SS patients and promoted the activation of inflammatory ILC2s. Blocking IL-25 using neutralizing antibodies improved salivary flow rate and alleviated SG tissue damage in experimental SS mice, accompanied by a decrease in ILC2 infiltration. In the SG of pSS patients, upregulation of TRAF6 in CD3+ T cells and ILC2s indicated that IL-25 signaling plays a role by coordinating the activation of ERK1 / 2 and related transcription factors (Guggino G, Arthritis Rheumatol (2018) 70(8):1265–75).

[0138] Overall, the effects of IL-25 on autoimmune diseases are complex and context-dependent, and further studies are 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.

[0139] c. Cancer

[0140] IL-25 has been shown to have potential anti-cancer effects by regulating immune responses and inhibiting tumor growth (Shabgah. A, et al., Cancer Med. 2021 Aug;10(15):5191-5202).

[0141] IL-25 can activate 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 the activity of immune cells, such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) (Sfanos, KS et al. (2014). Clinical cancer research, 20 (12), 3254-3265).

[0142] It was found that in addition to its role in enhancing immunity, IL-25 also has direct anti-tumor effects on various cancer types. 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 cell cycle progression (Zhang, J., et al. (2019). Oncology letters, 18 (5), 5179-5186).

[0143] In addition, IL-25 has been found to enhance the efficacy of chemotherapy and radiotherapy in preclinical models of cancer. IL-25 treatment has been shown to sensitize cancer cells to chemotherapy by increasing their sensitivity to cell death, and to enhance the effects of radiotherapy by stimulating immune responses and reducing tumor vascular density (Bao, H et al., (2017). Scientific reports, 7(1), 1-11).

[0144] Taken together, these findings suggest that IL-25 has good potential as an anticancer agent, either alone or in combination with other therapeutic approaches. However, further studies are needed to fully understand the underlying mechanisms of its antitumor effects and explore its potential as a clinical therapeutic agent.

[0145] Anti-IL-25 antibody

[0146] The anti-IL-25 antibodies (e.g., IL25Ab1, IL25Ab2, IL25Ab3, IL25Ab4, IL25Ab5, IL25Ab6, IL25Ab7, IL25Ab8, IL25Ab9, IL25Ab10, and IL25Ab11) of the present disclosure bind (e.g., specifically bind) to human IL-25. Such antibodies and fragments thereof can be used to disrupt IL-25 / IL-25R binding interactions. These antibodies and fragments thereof are characterized by a unique set of CDR sequences, specificity for IL-25, and can be used as a monotherapy or in combination with other agents to treat type 2 inflammatory diseases, autoimmune diseases, or cancer. More specifically, the present disclosure relates to antibodies and fragments thereof that bind to human IL-25, and their use in regulating the IL-25-mediated activity of cells located in an inflammatory disease microenvironment or a tumor microenvironment.

[0147] In one embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises a heavy chain variable region (VH) having a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 1. For example, the anti-IL-25 antibody or antibody fragment thereof may comprise a set of CDRs corresponding to those in one or more anti-IL-25 antibodies disclosed in Table 1 (e.g., the HCDRs of the IL25Ab1 antibody).

[0148] In another embodiment, the anti-IL-25 antibody or fragment thereof 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 antibody fragment thereof may comprise a set of CDRs corresponding to those in one or more anti-IL-25 antibodies disclosed in Table 2 (e.g., LCDRs of the IL25Ab1 antibody).

[0149] In an alternative embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises a VH having a set of CDRs disclosed in Table 1 (HCDR1, HCDR2, and HCDR3) and a VL having a set of CDRs disclosed in Table 2 (LCDR1, LCDR2, and LCDR3).

[0150] Table 1: CDR sequences of the murine heavy chain variable (VH) domain

[0151] Anti-IL-25 antibody CDR1 CDR2 CDR3 IL25Ab1 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 IL25Ab2 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 19 IL25Ab3 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 25 IL25Ab4 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31 IL25Ab5 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37

[0152] Table 2: CDR sequences of the murine light chain variable (VL) domain

[0153] Anti-IL-25 antibody CDR1 CDR2 CDR3 IL25Ab1 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 IL25Ab2 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 IL25Ab3 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28 IL25Ab4 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 IL25Ab5 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 40

[0154] In one embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises a humanized heavy chain variable region (VH) having a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 3. For example, the anti-IL-25 antibody or antibody fragment thereof may comprise a set of CDRs corresponding to those in one or more anti-IL-25 antibodies disclosed in Table 1 (e.g., the HCDRs of the IL25Ab6 antibody).

[0155] In another embodiment, the anti-IL-25 antibody or fragment thereof 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 antibody fragment thereof may comprise a set of CDRs corresponding to those in one or more anti-IL-25 antibodies disclosed in Table 2 (e.g., LCDRs of the IL25Ab6 antibody).

[0156] In an alternative embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises a VH having a set of CDRs disclosed in Table 3 (HCDR1, HCDR2, and HCDR3) and a VL having a set of CDRs disclosed in Table 4 (LCDR1, LCDR2, and LCDR3).

[0157] Table 3: CDR sequences of humanized heavy chain variable (VH) domain

[0158]

[0159] Table 4: CDR sequences of humanized light chain variable (VL) domain

[0160]

[0161] In one embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises a VH having a complementarity determining region group (HCDR1, HCDR2 and HCDR3) selected from:

[0162] (i) HCDR1: SEQ ID NO: 11, HCDR2: SEQ ID NO: 12, HCDR3: SEQ ID NO: 13;

[0163] (ii) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, HCDR3: SEQ ID NO: 19;

[0164] (iii) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, HCDR3: SEQ ID NO: 25;

[0165] (iv) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 30, HCDR3: SEQ ID NO: 31;

[0166] (v) HCDR1: SEQ ID NO: 35, HCDR2: SEQ ID NO: 36, HCDR3: SEQ ID NO: 37;

[0167] (vi) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 49, HCDR3: SEQ ID NO: 25, and

[0168] (vii) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 52, HCDR3: SEQ ID NO: 25.

[0169] In one embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises a VL having a complementarity determining region group (LCDR1, LCDR2, and LCDR3) selected from:

[0170] (i) LCDR1: SEQ ID NO: 14, LCDR2: SEQ ID NO: 15, LCDR3: SEQ ID NO: 16;

[0171] (ii) LCDR1: SEQ ID NO: 20, LCDR2: SEQ ID NO: 21, LCDR3: SEQ ID NO: 22;

[0172] (iii) LCDR1: SEQ ID NO: 26, LCDR2: SEQ ID NO: 27, LCDR3: SEQ ID NO: 28;

[0173] (iv) LCDR1: SEQ ID NO: 32, LCDR2: SEQ ID NO: 33, LCDR3: SEQ ID NO: 34;

[0174] (v) LCDR1: SEQ ID NO: 38, LCDR2: SEQ ID NO: 39, LCDR3: SEQ ID NO: 40; and

[0175] (vi) LCDR1: SEQ ID NO: 50, LCDR2: SEQ ID NO: 51, LCDR3: SEQ ID NO: 28.

[0176] In another embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises:

[0177] (a) a VH having a complementarity determining region group (HCDR1, HCDR2 and HCDR3) selected from:

[0178] (i) HCDR1: SEQ ID NO: 11, HCDR2: SEQ ID NO: 12, HCDR3: SEQ ID NO: 13;

[0179] (ii) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, HCDR3: SEQ ID NO: 19;

[0180] (iii) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, HCDR3: SEQ ID NO: 25;

[0181] (iv) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 30, HCDR3: SEQ ID NO: 31;

[0182] (v) HCDR1: SEQ ID NO: 35, HCDR2: SEQ ID NO: 36, HCDR3: SEQ ID NO: 37;

[0183] (vi) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 49, HCDR3: SEQ ID NO: 25, and

[0184] (vii) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 52, HCDR3: SEQ ID NO: 25.

[0185] (b) a VL having a complementarity determining region group (LCDR1, LCDR2 and LCDR3) selected from:

[0186] (i) LCDR1: SEQ ID NO: 14, LCDR2: SEQ ID NO: 15, LCDR3: SEQ ID NO: 16;

[0187] (ii) LCDR1: SEQ ID NO: 20, LCDR2: SEQ ID NO: 21, LCDR3: SEQ ID NO: 22;

[0188] (iii) LCDR1: SEQ ID NO: 26, LCDR2: SEQ ID NO: 27, LCDR3: SEQ ID NO: 28;

[0189] (iv) LCDR1: SEQ ID NO: 32, LCDR2: SEQ ID NO: 33, LCDR3: SEQ ID NO: 34;

[0190] (v) LCDR1: SEQ ID NO: 38, LCDR2: SEQ ID NO: 39, LCDR3: SEQ ID NO: 40;

[0191] (vi) LCDR1: SEQ ID NO: 50, LCDR2: SEQ ID NO: 51, LCDR3: SEQ ID NO: 28.

[0192] In one embodiment, the antibody or antibody fragment thereof comprises a combination of VH and VL having a complementarity determining region set (CDR1, CDR2 and CDR3) selected from the group consisting of:

[0193] (i) VH: CDR1: SEQ ID NO: 11, CDR2: SEQ ID NO: 12, CDR3: SEQ ID NO: 13, VL: CDR1: SEQ ID NO: 14, CDR2: SEQ ID NO: 15, CDR3: SEQ ID NO: 16;

[0194] (ii) VH: CDR1: SEQ ID NO: 17, CDR2: SEQ ID NO: 18, CDR3: SEQ ID NO: 19, VL: CDR1: SEQ ID NO: 20, CDR2: SEQ ID NO: 21, CDR3: SEQ ID NO: 22;

[0195] (iii) VH: CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 24, CDR3: SEQ ID NO: 25, VL: CDR1: SEQ ID NO: 26, CDR2: SEQ ID NO: 27, CDR3: SEQ ID NO: 28;

[0196] (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;

[0197] (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;

[0198] (vi) VH: CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 49, CDR3: SEQ ID NO: 25, VL: CDR1: SEQ ID NO: 50, CDR2: SEQ ID NO: 51, CDR3: SEQ ID NO: 28; and

[0199] (vii) VH: CDR1: SEQ ID NO: 23, CDR2: SEQ ID NO: 52, CDR3: SEQ ID NO: 25, VL: CDR1: SEQ ID NO: 50, CDR2: SEQ ID NO: 51, CDR3: SEQ ID NO: 28.

[0200] In some embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises a variable heavy chain sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 44, and 47; and / or a variable light chain sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, and 48.

[0201] In one embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises a pair of variable heavy chain 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 appreciate that variable light chains and variable heavy chains can be independently selected or mixed and matched to prepare anti-IL-25 antibodies or fragments thereof that contain combinations of variable heavy and variable light chains other than the pairs identified above.

[0202] In one embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises a pair of variable heavy chain and variable light chain sequences selected from the following combinations: a variable heavy chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 1 and a variable light chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 2; a variable heavy chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 3 and a variable light chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 4; a variable heavy chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 5 and a variable light chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 6; a variable heavy chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 7 and a variable light chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 8; a variable heavy chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 9 and a variable light chain sequence having 90%, 95% or 99% identity to SEQ ID NO: 10; NO:9 and a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:10; 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:45; 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; and a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:47 and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:48. Those skilled in the art will further appreciate that variable light chains and variable heavy chains can be independently selected or mixed and matched to prepare anti-IL-25 antibodies or antibody fragments thereof that contain combinations of variable heavy chains and variable light chains other than the pairs identified above.

[0203] In some embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises one or more conservative amino acid substitutions. One skilled in the art will recognize that a conservative amino acid substitution is one amino acid substituted with another amino acid having similar structural or chemical properties (e.g., similar side chains). Exemplary conservative substitutions are described in the art, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Publication Company, 4th Edition (1987).

[0204] "Conservative modification" refers to amino acid modifications that do not significantly affect or change the binding properties of the antibody containing the amino acid sequence. Conservative modifications include amino acid substitutions, additions and deletions. Conservative substitutions refer to amino acids being replaced by amino acid residues with similar side chains. Families of amino acid residues having similar side chains are well defined and include amino acids having the following side chains: 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), uncharged polar 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). In addition, any native residue in the polypeptide may be substituted with alanine, such as by alanine scanning mutagenesis as described previously (MacLennan et al., (1998) ActaPhysiol Scand Suppl 643:55-67; ​​Sasaki et al. (1998) Adv Biophys 35:1-24). Amino acid substitutions of the antibodies of the invention may be performed by known methods, such as by PCR mutagenesis (U.S. Pat. No. 4,683,195).

[0205] In some embodiments, the antibody or its antibody fragment comprises 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 to the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 44 or 47. In other embodiments, the antibody or its fragment retains the binding and / or functional activity of an antibody or its fragment comprising the variable heavy chain sequence of SEQ ID NO: 1, 3, 5, 7, 9, 44 or 47. In other further embodiments, the antibody or its antibody fragment comprises the variable heavy chain sequence of SEQ ID NO: 1, 3, 5, 7, 9, 44 or 47 and has one or more conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions in the heavy chain variable sequence. In yet further embodiments, one or more conservative amino acid substitutions fall in one or more framework regions in SEQ ID NO: 1, 3, 5, 7 or 9, 44 or 47 (based on the numbering system of Kabat).

[0206] In a specific embodiment, 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 to the heavy chain variable region sequence of the antibody or fragment thereof shown in SEQ ID NO: 1, 3, 5, 7, 9, 44 or 47, wherein the antibody or fragment thereof comprises one or more conservative amino acid substitutions in the framework region and retains the binding and / or functional activity of an antibody or fragment thereof comprising a variable heavy chain sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 44 or 47 and a variable light chain sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48.

[0207] In some embodiments, the antibody or antibody fragment thereof comprises a variable light chain sequence having an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48.

[0208] In other embodiments, the antibody or fragment thereof retains the binding and / or functional activity of an antibody or fragment thereof comprising the variable light chain sequence of SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48.

[0209] In other further embodiments, the antibody or antibody fragment thereof comprises a variable light chain sequence of SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48 and has one or more conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions in the light chain variable sequence. In yet further embodiments, one or more conservative amino acid substitutions fall into one or more framework regions (based on the numbering system of Kabat) in SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48.

[0210] In a specific embodiment, the antibody or fragment thereof comprises a variable light chain sequence having at least about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the antibody or fragment thereof light chain variable region sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48.

[0211] In some embodiments, the anti-IL25 antibody comprises one or more conservative amino acid substitutions in the framework regions and retains the binding and / or functional activity of an antibody or fragment thereof comprising the variable heavy chain sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 44, or 47 and the variable light chain sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 45, 46, or 48.

[0212] 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 an antigen-binding portion thereof. 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 comprising an antigen-binding portion of one or more anti-IL-25 antibodies of the present disclosure can be inserted into a bispecific antibody or a multispecific antibody or into a fusion protein.

[0213] In one embodiment, the anti-IL-25 antibody or antibody fragment thereof comprises all six murine CDR regions of IL25Ab1, IL25Ab2, IL25Ab3, IL25Ab4 or IL25Ab5 antibodies constructed as chimeric or humanized antibodies. In other embodiments, the anti-IL-25 antibody or antibody fragment thereof comprises all six CDR regions of one of the murine antibodies of the present disclosure.

[0214] In some embodiments, the anti-IL-25 antibody is a humanized antibody (eg, IL25Ab6, IL25Ab7, IL25Ab8, IL25Ab9, IL25Ab10, or IL25Ab11) comprising the VH / VL CDR regions listed in Tables 3 and 4.

[0215] In some embodiments, the antibody is a full-length antibody. In other embodiments, the antibody is an antibody fragment, including, for example, an antibody fragment selected from the group consisting of Fab, Fab', F(ab)2, Fv, domain antibodies (dAbs) and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to specifically bind IL-25 to the polypeptide.

[0216] In some embodiments, the variable region domain of the anti-IL-25 antibody disclosed herein can be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, the VH domain present in the variable region domain can be linked to an immunoglobulin CH1 domain or a fragment thereof. Similarly, the VL domain can be linked to a CK domain or a fragment thereof. In this way, for example, the antibody can be a Fab fragment, in which the antigen binding domain comprises the relevant VH and VL domains, the C-termini of which are covalently linked to the CH1 and CK domains, respectively. The CH1 domain can be extended with other amino acids, for example, to provide a hinge region or a portion of a hinge region domain as present in a Fab fragment, or to provide other domains, such as antibody CH2 and CH3 domains.

[0217] In some embodiments, the variable region domain of the anti-IL-25 antibody disclosed herein can be covalently linked to the antibody constant region at the C-terminal amino acid. For example, the VL domain can be linked to a mouse κ constant region (SEQ ID NO: 54) or a human κ constant region (SEQ ID NO: 57). Similarly, the VH domain can be linked to a mouse IgG1 constant region (SEQ ID NO: 53), a human IgG1 constant region (SEQ ID NO: 55), or another antibody constant region, such as human IgG4 or IgG2. The constant region may contain certain mutations to adjust the properties of the derived antibody. For example, the triple mutation YTE can be introduced into the human IgG1 constant region (SEQ ID NO: 56) to extend the antibody half-life. Fc mutations that regulate antibody effector functions can also be introduced.

[0218] Thus, in one embodiment, the antibody fragment comprises at least one CDR as described herein. As described herein, the antibody fragment may comprise at least two, three, four, five or six CDRs. The antibody fragment may further comprise at least one variable region domain of an antibody as described herein. The variable region domain may be of any size or amino acid composition, and will generally comprise at least one CDR sequence responsible for binding to human IL-25, such as CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and / or CDR-L3 described herein, and it is adjacent to or in frame with one or more framework sequences.

[0219] In another aspect, the anti-IL-25 antibody or antibody fragment thereof exhibits one or more of the following properties:

[0220] (a) Specific for human IL-25 and capable of blocking the binding of IL-25 to its receptor;

[0221] (b) inhibiting, interfering with or modulating the interaction between IL-25 and IL-25 receptor signal transduction;

[0222] (c) inhibiting IL-25-induced intracellular NF-kB activation;

[0223] (d) inhibiting the production of IL-5 induced by human IL-25 in human PBMCs;

[0224] (e) inhibiting the production of CXCL-1 induced by human IL-25 in human colon cancer cell lines;

[0225] (f) binds to mouse and cynomolgus monkey IL-25;

[0226] (g) reducing IL-5 production in the lungs of an OVA-induced asthma model; and

[0227] (h) Improved airway resistance in OVA-induced asthma model.

[0228] In one embodiment, the anti-IL-25 antibody or its antibody fragment can reduce, inhibit, interfere with and / or regulate at least one biological response associated with IL-25, and thus can be used to alleviate the effects of IL-25-related diseases or disorders. Such antibodies and their antibody fragments can be used, for example, to reduce, inhibit, interfere with and / or regulate IL-25 signaling, IL-25 activation of epithelial cells and type 2 lymphocytes, IL-25 activation of tumor cells, or induction of proinflammatory cytokines.

[0229] The antibodies disclosed herein (e.g., IL25Ab1, IL25Ab2, IL25Ab3, IL25Ab4, IL25Ab5, IL25Ab6, IL25Ab7, IL25Ab8, IL25Ab9, IL25Ab10, and IL25Ab11) bind to (e.g., specifically bind to) human IL25. 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)) are from UniProt. Human IL-25 has low homology with its phylogenetically close relatives. The sequence similarity of IL-25 with other family members is very low, ranging from 23.2% to 32.7%. Based on the low homology, anti-IL25 antibodies are unlikely to bind to IL17 family members.

[0230] Antibodies usually bind to their cognate antigens with high affinity and specificity, as shown by 10 -7 Up to 10 -11 M or less. Any dissociation constant (KD) greater than about 10 -6 A KD of 10 is generally considered to indicate nonspecific binding. As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen with high affinity and substantially the same antigen, meaning that the antibody has a 10 -7 KD of M or less, preferably 10 -8 M or less, even more preferably 5×10 -9 M or less, and most preferably within 10 -8 M to 10 -10 The antibodies of the present disclosure bind to human IL-25 with high affinity, with a KD of <1.0E-12 as determined by BLI.

[0231] The term "cross-reactivity" as used herein 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 IL25). As used herein, cross-reactivity can be measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR, ELISA), or binding or other functional interactions with cells that physiologically express IL-25. Methods for determining cross-reactivity include standard binding assays as described herein, for example, by using 2000SPR instrument (Biacore AB, Uppsala, Sweden) Surface plasmon resonance (SPR) analysis, bio-layer interferometry (BLI) or flow cytometry techniques.

[0232] The human IL2-5 antibodies IL25Ab1 to IL25Ab11 of the present disclosure all bind to IL-25 from cynomolgus monkeys with significant affinity. IL25Ab1 to IL25Ab11 bind to cynomolgus monkey IL-25 and mouse IL-25 in ELISA assays with an affinity comparable to that for human IL-25.

[0233] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibody descriptions are found in, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In a further example, a chimeric antibody is a "class switching" antibody, in which the class or subclass has been changed from the class or subclass of the parent antibody. Chimeric antibodies include antibody fragments thereof.

[0234] The antibody can be prepared as a chimeric antibody or antibody fragment thereof having a murine variable region and a human constant region. The human antibody constant region can be a reported allogeneic, see Jefferis et al. for a review, Human immunoglobulinallotypes: possible implications for immunogenicity. MAbs 1, 332-338 (2009). In one embodiment, the human heavy chain constant region uses a common human IgG1 constant region sequence (SEQ ID NO: 55), while the light chain constant region uses a common human κ constant region sequence (SEQ ID NO: 57). Human IgG1 can be selected because it is one of the most common subtypes for chimeric antibody production and can provide effector functions. The human κ constant region can be used because most parent murine antibodies are mouse κ light chains.

[0235] In certain embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunogenicity to people, while retaining the specificity and affinity of parent non-human antibodies. Generally, humanized antibodies include one or more variable domains, wherein HVR, for example, CDR (or part thereof)) is derived from non-human antibodies and FR (or part thereof) is derived from human antibody sequences. Humanized antibodies will optionally also include at least a portion of human constant regions. In some embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (for example, antibodies from which HVR residues are derived), for example, to restore and / or improve antibody binding specificity or affinity. In some embodiments, some CDR residues (for example, 1, 2, 3, 4 or 5 residues) in humanized antibodies can be mutated to improve the developability of antibodies.

[0236] Humanized antibodies and methods for making them have been reviewed in, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and further described in, 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. Pat. Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" method of FR shuffling).

[0237] 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 the consensus sequence of human antibodies of a particular subgroup of light 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)); 993)); human mature (somatically mutated) 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 FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0238] For human or humanized antibodies (such as IgG1 antibodies), amino acid substitutions are introduced into the Fc region of the antibody to regulate the interaction of the antibody with Fcγ receptors, FcRn or complement, thereby changing the effector function of the antibody and / or the half-life of the antibody (see, e.g., 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 the half-life of antibodies, YTE (EU numbering is M252Y, S254T and T256E) and LS (M428L and N434S) are two examples used in the literature.

[0239] Method for producing IL-25 antibody

[0240] Anti-IL-25 antibodies or antibody fragments thereof can be prepared by any method known in the art. For example, a soluble recombinant human IL-25 protein, or a fragment or peptide coupled to a carrier protein thereof can be used to immunize a recipient. Any suitable immunization method can be used. Such methods may include adjuvants, other immunostimulants, repeated booster immunizations, and the use of one or more immunization routes.

[0241] Any suitable source of human IL-25 can be used as an immunogen for producing anti-IL-25 antibodies of the compositions and methods disclosed herein. Different forms of IL-25 antigens can be used to produce antibodies sufficient to produce biological activity. Therefore, the eliciting IL-25 antigen can be a single epitope, multiple epitopes, or the entire protein alone or in combination with one or more immunogenicity enhancers. In some aspects, the eliciting antigen is a separated soluble full-length protein, or a soluble protein containing less than the full-length sequence (for example, using a peptide containing a specific part or epitope of IL-25 for immunization). As used herein, the term "part" refers to the minimum number of amino acids or nucleic acids suitable for constituting the immunogenic epitope of the target antigen. Any genetic vector suitable for transforming the target cell can be used, including but not limited to adenovirus vectors, plasmids and non-viral vectors, such as cationic liposomes.

[0242] It is desirable to prepare monoclonal antibodies (mAbs) from a variety of mammalian hosts, such as mice, rodents, primates, humans, and the like. Techniques for preparing such monoclonal antibodies are described in, for example, Sties et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lance Medical Publication, Los Altos, CA, and references cited therein: Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.) Academic Press, New York, NY. Typically, spleen cells from an animal immunized with the desired antigen are immortalized, usually 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. (1994 ed. and periodic supplements) CELL AND TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, NY. Colonies produced by single immortalized cells are screened to produce antibodies with the desired specificity and affinity for the antigen, and the production of monoclonal antibodies produced by such cells can be increased by various techniques (including injection into the peritoneal cavity of a vertebrate host). Alternatively, for example, DNA sequences encoding monoclonal antibodies or antigen-binding fragments thereof can be isolated by screening DNA libraries from human B cells according to the general protocol outlined in Huse et al. (1989) Science 246: 1275-1281. Therefore, researchers can obtain antibodies by various techniques with which they are familiar.

[0243] Other suitable techniques include selecting antibody libraries in phage, yeast, viruses or similar vectors. See, for example, Huse et al., supra; and Ward et al. (1989) Nature 341:544-546. The polypeptides and antibodies disclosed herein can be used with or without modification, including chimeric or humanized antibodies. Typically, the polypeptides and antibodies will be labeled by covalently or non-covalently linking a substance that provides a detectable signal. A variety of labeling and coupling techniques are known and widely reported in the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents that teach the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. In addition, recombinant immunoglobulins can be produced, see Cabilly U.S. Pat. No. 4,816,567; and Queen et al., (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10023; or made 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, Harbor and other technologies.

[0244] In some embodiments, the ability of the produced antibodies to bind to IL-25 can be evaluated using standard binding assays, such as surface plasmon resonance (SPR), Octet (BLI), ELISA, Western blot, immunofluorescence, flow cytometry analysis, chemotaxis assays, and cell migration assays. In some aspects, the ability of the produced antibodies to inhibit IL-25 blocking IL-25 receptor signal transduction and inhibiting IL-25-mediated inflammatory microenvironment secondary effects can also be evaluated, including inhibition of IL-25-induced NFkB signaling, IL-5 production, and / or CXCL1 production.

[0245] Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a common purification technique. The suitability of protein A as an affinity ligand depends on the type 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 human γ3 (see, e.g., Guss et al., 1986 EMBO J. 5: 1567-1575). The matrix to which the affinity ligand is attached is typically agarose, but other matrices may also be used. Mechanically stable matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, can achieve faster flow rates and shorter processing times than agarose. When the antibody comprises a CH3 domain, Bakerbond ABX TM Resin (JT Baker, Phillipsburg, NJ) was used for purification. Other protein purification techniques such as ion exchange column fractionation, ethanol precipitation, reversed phase HPLC, silica gel chromatography, heparin SEPHAROSE can also be used depending on the antibody to be recovered. TM Chromatography, chromatography on anion or cation exchange resins (eg, polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0246] Following any preliminary purification steps, the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, typically at low salt concentrations (e.g., about 0-0.25 M salt).

[0247] Also included are nucleic acids that hybridize under low, medium, and high stringency conditions as defined herein to all or part of a nucleotide sequence represented by an isolated polynucleotide sequence encoding an antibody or antibody fragment of the present disclosure (e.g., a portion encoding a variable region). The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid has at least 80%, such as at least 90%, at least 95%, at least 98%, or at least 99% identity to a portion or all of a nucleic acid encoding an anti-IL-25 polypeptide (e.g., a heavy or light chain variable region), or its complement. Hybridizing nucleic acids of the type described herein can be used, for example, as cloning probes, primers (e.g., PCR primers), or diagnostic probes.

[0248] Polynucleotides, vectors and cells

[0249] Other embodiments include isolated polynucleotides comprising sequences encoding anti-IL-25 antibodies or antibody fragments thereof, vectors and cells comprising the polynucleotides, and recombinant techniques for producing antibodies. The isolated polynucleotides may encode any desired form of 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, miniantibodies, and multispecific antibodies formed from antibody fragments.

[0250] Some embodiments include isolated polynucleotides comprising a sequence encoding a heavy chain variable region of an antibody or antibody fragment having an amino acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 44, and 47. Some embodiments include isolated polynucleotides comprising a sequence encoding a light chain variable region of an antibody or antibody fragment having an amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 45, 46, and 48.

[0251] In one embodiment, the isolated polynucleotide sequence encodes an antibody or antibody fragment having heavy and light chain variable regions comprising the amino acid sequence:

[0252] (a) a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2;

[0253] (b) a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4;

[0254] (c) a variable heavy chain sequence comprising SEQ ID NO: 5 and a variable light chain sequence comprising SEQ ID NO: 6;

[0255] (d) a variable heavy chain sequence comprising SEQ ID NO: 7 and a variable light chain sequence comprising SEQ ID NO: 8;

[0256] (e) a variable heavy chain sequence comprising SEQ ID NO: 9 and a variable light chain sequence comprising SEQ ID NO: 10;

[0257] (f) a variable heavy chain sequence comprising SEQ ID NO: 44 and a variable light chain sequence comprising SEQ ID NO: 45;

[0258] (g) a variable heavy chain sequence comprising SEQ ID NO: 44 and a variable light chain sequence comprising SEQ ID NO: 46; or

[0259] (h) a variable heavy chain sequence comprising SEQ ID NO: 47 and a variable light chain sequence comprising SEQ ID NO: 48.

[0260] In one embodiment, the isolated polynucleotide sequence encodes an antibody or antibody fragment having light and heavy chain variable regions comprising the amino acid sequence:

[0261] (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;

[0262] (b) 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;

[0263] (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;

[0264] (d) 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;

[0265] (e) a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:9 and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:10;

[0266] (f) 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:45;

[0267] (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

[0268] (h) and a variable heavy chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:47 and a variable light chain sequence that is 90%, 95% or 99% identical to SEQ ID NO:48.

[0269] The polynucleotides comprising sequences encoding anti-IL-25 antibodies or antibody fragments thereof may be fused to one or more regulatory or control sequences known in the art and may be contained in suitable expression vectors or cells known in the art. Each polynucleotide molecule encoding a heavy or light chain variable domain may be independently fused to a polynucleotide sequence encoding a constant domain (e.g., a human constant domain) to produce a complete antibody. Alternatively, polynucleotides or portions thereof may be fused together to provide a template for producing a single-chain antibody.

[0270] For recombinant production, the polynucleotide encoding the antibody or its antibody fragment is inserted into a reproducible vector for cloning (amplification of the DNA) or for expression. Many suitable vectors for expressing recombinant antibodies are available. The 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, an enhancer element, a promoter, and a transcription termination sequence.

[0271] Anti-IL-25 antibodies or antibody fragments thereof can also be produced as fusion polypeptides, wherein the antibody or fragment thereof is fused to a heterologous polypeptide (e.g., a signal sequence, or other polypeptides having a specific cleavage site at the amino terminus of a mature protein or polypeptide). The selected heterologous signal sequence is typically a sequence that is recognized and processed by the cell (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 by a prokaryotic signal sequence. The signal sequence can be, for example, an alkaline phosphatase, penicillinase, lipoprotein, heat-stable enterotoxin II leader sequence, and the like. When used for yeast secretion, the natural signal sequence can be replaced by, for example, a leader sequence obtained from yeast invertase α-factor (including Saccharomyces and Kluyveromyces α-factor leader sequences), acid phosphatase, Candida albicans (C.albicans) glucoamylase, or a signal described in WO90 / 13646. In mammalian cells, mammalian signal sequences and viral secretion leaders, such as herpes simplex gD signals, can be used. The DNA for this precursor region is linked in reading frame to the DNA encoding the anti-IL-25 antibody.

[0272] Expression and cloning vectors contain nucleic acid sequences that make the vector replicate in one or more selected cells. Usually, in cloning vectors, the sequence is a sequence that makes the vector replicate independently of the host chromosome DNA, including a replication origin or an autonomous replication sequence. Such sequences of various bacteria, yeasts and viruses are well known. The replication origin from plasmid pBR322 is applicable to most gram-negative bacteria, the 2-υ plasmid origin is applicable to yeast, and various viral origins (SV40, polyoma virus, adenovirus, VSV and BPV) can be used for cloning vectors in mammalian cells. Generally speaking, mammalian expression vectors do not need a replication origin assembly (usually the SV40 origin can be used, just because it contains an early promoter).

[0273] Expression and cloning vectors may contain genes encoding selectable markers to facilitate identification of expression. Typical selectable marker genes encode proteins that confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), or alternatively, encode proteins that complement auxotrophic deficiencies, or alternatively, encode proteins that provide specific nutrients not present in complex media, such as the gene encoding D-alanine racemase for Bacilli.

[0274] Non-therapeutic use

[0275] The anti-IL-25 antibodies or antibody fragments thereof described herein can be used as affinity purification agents. In this process, the antibody is fixed to a solid phase, such as a protein A resin, using methods well known in the art. The fixed antibody is contacted with a sample containing the IL-25 protein (or fragment thereof) to be purified, and then the support is washed with a suitable solvent, which will substantially remove all substances in the sample except the IL-25 protein bound to the fixed antibody. Finally, the support is washed with another suitable solvent, which will release the IL-25 protein from the antibody.

[0276] Anti-IL-25 antibodies or antibody fragments thereof can also be used in diagnostic assays to detect and / or quantify IL-25 protein, for example, to detect the expression of IL-25 in specific cells, tissues or serum. Anti-IL-25 antibodies can be used diagnostically, for example, to monitor the development or progression of a disease as part of a clinical test method, for example, to determine the efficacy of a given treatment and / or prevention regimen. Detection can be facilitated by coupling 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 using various positron emission tomography, and non-radioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents according to the present disclosure, see, for example, U.S. Patent No. 4,741,900.

[0277] Anti-IL-25 antibodies or antibody fragments thereof can be used to diagnose IL-25-related disorders (e.g., disorders characterized by abnormal expression of IL-25) or to determine whether a subject has an 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 an antibody 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 cell source that may express IL-25. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0278] In some embodiments, the method can further include comparing the IL-25 level in the patient sample to a control sample (e.g., a subject not suffering from an IL-25-associated disorder) to determine whether the patient suffers from, or is at risk of developing, an IL-25-associated disorder.

[0279] In some embodiments, for example, it would be advantageous to use a detectable moiety labeled antibody for diagnostic purposes. Many detectable labels are available, including radioisotopes, fluorescent labels, enzyme substrate labels, and the like. Various known techniques can be used to indirectly couple the label to the antibody. For example, the antibody can be coupled to biotin, and any of the three major types of labels can be coupled to avidin, and vice versa. Biotin selectively binds to avidin, so the label can be bound to the antibody in this indirect manner. Alternatively, in order to achieve indirect coupling of the label to the antibody, the antibody can be coupled to a small hapten (such as digoxin), and one of the different types of labels described above can be coupled to an anti-hapten antibody (e.g., anti-digoxin antibody). Therefore, indirect coupling of the label to the antibody can be achieved.

[0280] Exemplary radioisotope labels include 35S, 14C, 125I, 3H and 131I. Antibodies can be labeled with radioisotopes, and the techniques that can be used are described in, for example, Current Protocols in Immunology, Vols. 1 and 2, 1991, Coligen et al., ed., Wiley-Interscience, New York, NY. For example, radioactivity can be measured by scintillation counting.

[0281] Exemplary fluorescent labels that can be used include labels derived from rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin and Texas Red. Fluorescent labels can be coupled to antibodies by known techniques, for example, as disclosed in Current Protocols in Immunology. Fluorescence can be quantified using a fluorometer.

[0282] There are various well-characterized enzyme-substrate labels known in the art (see, e.g., U.S. Patent No. 4,275,149). The enzyme typically catalyzes a chemical change in a chromogenic substrate, which can be measured using various techniques. For example, the change can be a change in the color of the substrate, which can be measured by spectrophotometry. Alternatively, the enzyme can change the fluorescence or chemiluminescence of the substrate. Techniques for quantitative fluorescence changes are described above. Chemiluminescent substrates are electronically excited by a chemical reaction and can then emit light that can be measured (e.g., using a chemiluminometer), or provide energy to a fluorescent receptor.

[0283] Examples of enzyme labels include luciferases, such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase (such as horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. Techniques for conjugating enzymes to 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.

[0284] Examples of enzyme substrate combinations include, for example: horseradish peroxidase (HRPO) with catalase as a substrate, where the catalase oxidizes a dye precursor such as o-phenylenediamine (OPD) or 3,3,5,5-tetramethylbenzidine hydrochloride (TMB); alkaline phosphatase (AP) with p-nitrophenyl phosphate as a chromogenic substrate; and β-D-galactosidase (β-D-Gal) with a chromogenic substrate such as p-nitrophenyl-β-D-galactosidase or a fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

[0285] In another embodiment, the anti-IL-25 antibody or antibody fragment thereof used is unlabeled and is detected using a labeled antibody that binds to the anti-IL-25 antibody or antibody fragment thereof.

[0286] The antibodies and antibody fragments thereof described herein can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. See, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0287] Anti-IL-25 antibodies or antibody fragments thereof can be used to inhibit the binding of ligands to IL-25 receptors. Such methods include administering anti-IL-25 antibodies to cells (e.g., mammalian cells) or cell environments, thereby inhibiting signal transduction mediated by IL-25 receptors. These methods can be performed in vitro or in vivo. "Cell environment" refers to the tissue, medium or extracellular matrix surrounding the cell.

[0288] Compositions and methods of treatment

[0289] The present disclosure also provides compositions, including, for example, pharmaceutical compositions comprising anti-IL-25 antibodies or antibody fragments thereof. Such compositions have a variety of therapeutic uses for treating, preventing, or ameliorating diseases or conditions (e.g., diseases or conditions involving biological activities mediated by the IL-25 / IL-25 receptor signaling axis), such as immune-mediated inflammatory conditions or autoimmune diseases.

[0290] The anti-IL-25 antibodies or antibody fragments thereof disclosed herein can be used to treat various diseases or conditions, including type 2 inflammatory diseases, autoimmune diseases or cancer. Specifically, the antibodies disclosed herein are used to treat immune-mediated airway inflammatory conditions.

[0291] The method for treating IL-25-related disorders comprises administering a therapeutically effective amount of an anti-IL-25 antibody or an antibody fragment thereof to a subject in need thereof. The present disclosure also provides a method for treating or preventing type 2 inflammatory diseases, comprising administering a composition or formulation comprising an anti-IL-25 antibody or an antibody fragment thereof to a subject in need thereof, and optionally another immunotherapy-based therapy. The anti-IL-25 antibody of the present disclosure can be administered as an additional treatment or in combination with other cytokine inhibitors.

[0292] The antibodies disclosed herein can also be used in methods for 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 conditions. In addition, the term includes virus-induced asthma exacerbations.

[0293] The antibodies of the present disclosure may also be used in methods of preventing or treating chronic obstructive pulmonary disease (COPD) in a patient in need thereof by administering an anti-IL-25 antibody or antigen-binding fragment thereof described herein.

[0294] The antibodies disclosed herein can also be used in methods for preventing or treating inflammatory bowel disease (IBD). In the context of this article, "IBD" refers to a group of disorders that affect the mucosal layer of the large intestine or colon, including ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, shunt colitis, Behcet's syndrome, infectious colitis, indeterminate colitis and other related conditions characterized by inflammation.

[0295] The antibodies disclosed herein can also be used in methods for preventing or treating atopic dermatitis (AD), including but not limited to inflammatory skin conditions characterized by severe itching and scaly, dry eczematous lesions. AD may be caused by epidermal barrier dysfunction, allergies to certain foods, pollen, mold, dust mites, animal radiation exposure, and / or asthma. The methods disclosed herein can be used to treat varying degrees of AD, including mild, moderate, moderate to severe, and severe forms.

[0296] The antibodies of the present disclosure can also be used in methods of 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 allergies, urticaria (including chronic idiopathic urticaria), eosinophilic pneumonia, eosinophilic esophagitis, hypereosinophilic syndrome, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, rheumatoid arthritis, vasculitis, uveitis, cancer, and graft-versus-host disease.

[0297] The antibodies of the present disclosure may also be used alone (eg, as monotherapy) or in combination with other immunotherapeutic agents and / or chemotherapeutic agents in methods of treating cancer.

[0298] The antibody can be administered alone or in combination with other compositions that can be used to treat immune-mediated inflammatory disorders or autoimmune diseases. In some embodiments, the composition (including, for example, a pharmaceutical composition) comprising an anti-IL-25 antibody can further comprise a therapeutic agent that is coupled or uncoupled to the binding agent.

[0299] In some aspects, compositions (e.g., pharmaceutical compositions) comprising one or more antibodies disclosed herein are provided. Pharmaceutical compositions can be formulated with pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients according to conventional techniques, for example, those disclosed in Remington: The Science and Practice of Pharmacy, 19th edition, Gennaro, ed., Mack Publishing Co., Easton, Pa., 1995.

[0300] Typically, the composition administered by injection is a solution in a sterile isotonic aqueous buffer. If necessary, the drug may also include a solubilizer and a local anesthetic (such as lidocaine) to relieve pain at the injection site. Typically, the ingredients are provided separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or pouch indicating the active dose). When the drug is administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline may be used for distribution. When the drug is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed before administration.

[0301] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (i.e., antibody, bispecific and multispecific molecule) can be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0302] The composition can be applied by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route of administration and / or mode will vary according to the desired result. The active compound can be prepared together with a carrier (such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems) that will protect the compound from rapid release. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The preparation methods of such preparations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, compiled by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0303] The dosage level of the active ingredient in the pharmaceutical composition can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the subject. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and previous medical history of the patient to be treated, and similar factors well known in the medical field.

[0304] The pharmaceutical compositions described herein can be administered in an effective amount. An "effective amount" refers to an amount that, alone or together with other doses, achieves a desired response or desired effect. In the case of treating a specific disease or a specific condition, the desired response preferably involves inhibiting the course of the disease. This includes slowing the progression of the disease, particularly interrupting or reversing the progression of the disease.

[0305] In some aspects, the compositions described herein are administered to a patient (e.g., in vivo) to treat or prevent a variety of disorders, such as those described herein. Preferred patients include human patients suffering from disorders that can be corrected or improved by administering an agent that modulates the biological activity of the IL-25 / IL-25 receptor signaling axis.

[0306] In some aspects, conventional viral and non-viral gene transfer methods can be used to import nucleic acids encoding antibodies or derivatives thereof described herein in mammalian cells or target tissues. Such methods can be used to apply nucleic acids encoding antibodies to cells in vitro. In some embodiments, nucleic acids encoding antibodies or derivatives thereof are used for in vivo or ex vivo gene therapy purposes. In other embodiments, gene delivery technology is used to study the activity of antibodies based on cells or animal models. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids compounded with delivery vehicles (such as liposomes). Viral vector delivery systems include DNA and RNA viruses, which have free or integrated genomes after delivery to cells. Such methods are well known in the art.

[0307] Non-viral delivery methods of nucleic acids encoding engineered polypeptides of the present disclosure include lipofection, microinjection, biolistic methods, virosomes, liposomes, immunoliposomes, polycationic or lipid:nucleic acid conjugates, naked DNA, artificial viral particles, and agents that enhance DNA uptake. Lipofection methods and lipofection agents are well known in the art (e.g., Transfectam TM Lipofectin TM ). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those of Felgner, WO 91 / 17424, WO 91 / 16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration). The preparation of lipid:nucleic acid complexes containing targeted liposomes (such as immunolipid complexes) is well known to those skilled in the art.

[0308] The use of RNA or DNA virus-based systems to deliver nucleic acids encoding antibodies described herein utilizes a highly evolved process for targeting specific cells in the virus body and transporting the viral payload to the nucleus. Viral vectors can be directly applied to patients (in vivo), or they can be used for in vitro treatment of cells and for applying modified cells to patients (ex vivo). Conventional viral-based systems for delivering polypeptides disclosed herein can include retroviruses, slow viruses, adenoviruses, adeno-associated viruses, and herpes simplex virus vectors for gene transfer. Viral vectors are the most effective and versatile methods for gene transfer in target cells and tissues at present. By retrovirus, slow virus, and adeno-associated virus gene transfer methods, it is possible to be integrated into the host genome, usually resulting in long-term expression of the inserted transgene. In addition, high transduction efficiency has been observed in many different cell types and target tissues.

[0309] For the purpose of description and disclosure, all patents and publications mentioned (identified) are expressly incorporated herein by reference, for example, the methods described in these publications can be used in conjunction with the present disclosure. These publications are provided only because they are disclosed before the date of filing of the present application. Any content in this regard should not be interpreted as admitting that the inventor has no right to rely on previous inventions or for any other reason and prior to these disclosures. All statements about dates, or statements of the contents of these documents are based on information at the applicant's disposal and do not constitute any recognition of the correctness of the dates or contents of these documents.

[0310] To the extent not already indicated, one of ordinary skill in the art will appreciate that any of the various embodiments described and illustrated herein may be further modified to incorporate features shown in any other embodiment disclosed herein.

[0311] The broad scope of the present disclosure may be best understood with reference to the following examples, which are not intended to limit the present disclosure to the specific examples. The specific embodiments described herein are provided by way of example only, and the present disclosure will be limited to the terms of the appended claims and the full range of equivalents to which these claims are entitled.

[0312] Describe the subject technology in terms of terms

[0313] For convenience, various aspects are described as numbered clauses (1, 2, 3, etc.). These are provided only as examples and do not limit the technology of the subject matter. The following reference numbers are provided only as examples and for illustrative purposes, and the clauses are not limited by these labels.

[0314] Item 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); (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); 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); (c) a heavy chain variable region comprising 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 a light chain variable region comprising 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) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 (HCDR1), the amino acid sequence of SEQ ID NO: 18 (HCDR2) and the amino acid sequence of SEQ ID NO: 19 (HCDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 (LCDR1), the amino acid sequence of SEQ ID NO: 21 (LCDR2) and the amino acid sequence of SEQ ID NO: 22 (LCDR3); (e) a heavy chain variable region comprising 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 a light chain variable region comprising 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 comprising 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 comprising 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) a heavy chain variable region comprising 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 a light chain variable region comprising 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). ;

[0315] Item 2: An anti-IL-25 antibody or antigen-binding fragment thereof according to Item 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising any one of the amino acid sequences of SEQ ID NO: 1, 3, 5, 7, 9, 44 or 47, and a light chain variable region comprising any one of the amino acid sequences of SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48.

[0316] Item 3: An anti-IL-25 antibody according to Item 1, wherein the antibody comprises: (a) a heavy chain variable region sequence of SEQ ID NO: 44 and a light chain variable region sequence of SEQ ID NO: 45; (b) a heavy chain variable region sequence of SEQ ID NO: 44 and a light chain variable region sequence of SEQ ID NO: 46; (c) a heavy chain variable region sequence of SEQ ID NO: 47 and a light chain variable region sequence of SEQ ID NO: 48; (d) a heavy chain variable region sequence of SEQ ID NO: 1 and a light chain variable region sequence of SEQ ID NO: 2; (e) a heavy chain variable region sequence of SEQ ID NO: 3 and a light chain variable region sequence of SEQ ID NO: 4; (f) a heavy chain variable region sequence of SEQ ID NO: 5 and a light chain variable region sequence of SEQ ID NO: 6; (g) a heavy chain variable region sequence of SEQ ID NO: 7 and a light chain variable region sequence of SEQ ID NO: 8; or (h) a heavy chain variable region sequence of SEQ ID NO: 9 and a light chain variable region sequence of SEQ ID NO: 10.

[0317] Clause 4: The anti-IL-25 antibody according to Clause 1, wherein the antibody is an anti-human anti-IL-25 antibody.

[0318] Clause 5: An anti-IL-25 antibody according to Clause 1, wherein the antibody is a full length antibody comprising a human IgG1 constant region selected from SEQ ID NO: 55 or SEQ ID NO: 56.

[0319] Clause 6: The anti-IL-25 antibody according to Clause 1, wherein the antibody is an antibody fragment.

[0320] Clause 7: An anti-IL-25 antibody according to 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.

[0321] Clause 8: The anti-IL-25 antibody according to Clause 1, wherein the antibody is a monoclonal antibody.

[0322] Clause 9: An anti-IL-25 antibody according to Clause 1, wherein the antibody is a human antibody.

[0323] Clause 10: An anti-IL-25 antibody according to Clause 1, wherein the antibody is a murine antibody.

[0324] Clause 11: The anti-IL-25 antibody according to Clause 1, wherein the antibody is a chimeric antibody.

[0325] Clause 12: The anti-IL-25 antibody according to Clause 1, wherein the antibody is a bispecific or multispecific antibody.

[0326] Clause 13: The anti-IL-25 antibody according to Clause 1, wherein the antibody is a humanized antibody.

[0327] Item 14: A pharmaceutical composition comprising the antibody of Item 1 and a pharmaceutically acceptable carrier.

[0328] Clause 15: A method of treating and / or preventing a type 2 inflammatory disease, an autoimmune disease, an allergic disorder or cancer in a subject in need thereof, the method comprising: administering the antibody of Clause 1 to the subject.

[0329] Item 16: A polynucleotide composition comprising: a) a first polynucleotide encoding a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 44 or 47; and b) a second polynucleotide encoding a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48.

[0330] Item 17: A vector composition comprising: a) a first vector comprising the first polynucleotide of Item 16; and b) a second vector comprising the second polynucleotide of Item 16.

[0331] Item 18: A cell comprising the polynucleotide composition of Item 16 or the vector composition of Item 17.

[0332] Item 19: A method for producing the anti-IL-25 antibody of Item 1, the method comprising culturing the cell of Item 18 in a culture medium; and recovering the anti-IL-25 antibody from the culture medium.

[0333] Example

[0334] General approach

[0335] Protein purification methods including immunoprecipitation, chromatography, and electrophoresis are described. See, e.g., 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, production of fusion proteins, and glycosylation of proteins are described. See, e.g., 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) Bio Directory, Piscataway, NJ, pp. 384-391. The production, purification and fragmentation of polyclonal and monoclonal antibodies are described. Coligan et al. (2001) Current Protcols in Immunology, Volume 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.

[0336] According to the manufacturer's operating protocol, the hybridoma or cell culture supernatant containing anti-binding IL-25 antibody was purified by HiTrap protein G column (GE, catalog number: 17040401). In short, the column was balanced with 5CV of DPBS (Gibco, catalog number: 14190-136), and the supernatant was loaded by a syringe / infusion pump (Legato 200, KDS) at ambient temperature and a residence time of 3 minutes. The column was washed with 5CV of DPBS, eluted with 4CV of pH 2.8 elution buffer (FisherScientific, catalog number: PI21004), neutralized with 1M Tris-HCL (pH 8.5, Fisher Scientific, catalog number: 50-843-270), and measured by A280 (DropSense96, Trinean). The purified material was then buffer exchanged into DPBS through 30 kDa MWCO centrifugal filters (EMD Millipore, Cat. No: UFC803024). The final pool was analyzed by A280 and stored at 2-8°C or -20°C.

[0337] Standard methods of molecular biology are described. See, for example, 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, California. Standard methods also appear in Ausbel et al., (2001) Current Protocols in Molecular Biology, Vols. 1 to 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).

[0338] The sequences of the heavy and light chain variable regions of the hybridoma clones were determined as follows. 1-2 × 10 6 Total RNA was extracted from each hybridoma cell. 5'RACE reaction was performed to generate cDNA by using the SMARTer RACE 5' / 3' kit from Takara (Mountainview, CA, USA). Takara Universal Primer Mix was used to combine gene-specific primers for the 3' mouse constant region of the corresponding immunoglobulin, and PCR was performed using Q5 high-fidelity DNA polymerase from NEB (Ipswitch, MA, USA) to amplify the variable regions of heavy and light chains. The amplified heavy and light chain variable regions were run on a 2% agarose gel, the corresponding bands were cut, and then the gel was purified using the Mini Elute Gel Extraction kit from Qiagen. The purified PCR product was cloned using the Zero Blunt PCR Cloning kit from Invitrogen (Carlsbad, CA, USA), and transformed into Stellar competent Escherichia coli cells from Takara, and plated on LB agar + 50ug / 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 rearrangements and analyze the translated protein sequence. CDR assignment was based on Kabat numbering.

[0339] The selected VH or VL chain was PCR amplified and cloned into an expression vector based on pcDNA3.4 with constant regions from human IgG1 (Uniprot P01857 or its allotype) or human kappa light chain (Uniprot P01834). According to the supplier's Expi293 expression system protocol, plasmids expressing paired heavy and light chains were transfected into Expi293 cells (Thermo Fisher Scientific). Five days after transfection, the culture supernatant was collected by centrifugation. The chimeric antibody was purified by 1-step affinity purification using a protein A column and buffer exchanged to PBS pH 7.2 or 20 mM histidine (pH 5.5).

[0340] Methods are available for flow cytometry, including fluorescence-activated cell sorting detection systems See, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd Edition; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Fluorescent reagents suitable for modifying nucleic acids (including nucleic acid primers and probes), polypeptides, and antibodies are available, e.g., for use as diagnostic reagents. Molecular Probes (2003) catalog (Molecular Probes, Inc., Eugene, Oreg.); Sigma-Aldrich (2003) catalog (St. Louis, Mo).

[0341] Standard techniques for characterizing ligand / receptor interactions are available. See, for example, Coligan et al. (2001) Current Protocols in Immunology, Volume 4, John Wiley, Inc., New York. Standard methods for characterizing antibody function suitable for characterizing antibodies with a specific mechanism of action are also well known to those skilled in the art.

[0342] Two in-house IL-25 specific antibodies were prepared, referred to herein as IL-25-PC2 and IL-25-PC4. IL-25-PC2 was prepared according to the public information published in WO 2016 / 049000 A2 (VH, SEQ ID NO: 114; and VL, SEQ ID NO: 122), and IL-25-PC4 was prepared according to the public information published in WO 2020 / 102935 A1 (VH, SEQ ID NO: 12; and VL, SEQ ID NO: 13). Both antibodies were used to establish binding and functional assays to evaluate and characterize the anti-IL-25 specific antibodies disclosed herein.

[0343] Software packages and databases are available for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, CDR annotations, glycosylation sites and sequence alignments.

[0344] Example 1: Production of anti-IL-25 antibodies

[0345] Anti-IL-25 antibodies were generated by immunizing wild-type mice or transgenic / humanized mice.

[0346] Immunization. Mice were immunized with recombinant human IL-25 protein, either intraperitoneally, subcutaneously, in the footpad, or at the base of the tail. Immune responses were monitored by retro-orbital bleeding. Plasma was screened by ELISA (as described below) and mice with adequate anti-human IL-25 titers were used for fusion. Mice were boosted with recombinant human IL-25 protein intraperitoneally, intravenously, or at the footpad or base of the tail before being sacrificed and spleen and lymph nodes removed.

[0347] Select Balb / c mice that produce anti-IL-25 antibodies. In order to select Balb / c mice that produce antibodies that bind IL-25, the serum of the mice that received immunity was screened by ELISA to detect its binding to IL-25. In short, the ELISA plates coated with recombinant human IL-25 protein were incubated at room temperature for 1 hour with dilutions of serum from immune mice, the assay plates were washed, and then HRP-labeled anti-mouse IgG antibodies were used to detect specific antibody binding. The plates were read using an ELISA microplate reader (Biotek). As described above, the anti-IL-25 specific binding of the hybridoma supernatant was tested by ELISA.

[0348] Generate hybridomas that produce antibodies against IL-25. In order to generate hybridomas that can produce antibodies of the present invention, spleen cells and lymph node cells isolated from immunized mice are fused with suitable immortalized cell lines (such as mouse myeloma cell lines). The resulting hybridomas are screened for the antigen-specific antibodies produced. For example, single cell suspensions of spleen cells and lymph node cells from immunized mice are fused with equal numbers of Sp2 / 0 non-secreting mouse IgG myeloma cells (ATCC, CRL 1581) by electrofusion. The cells are placed in a flat-bottomed 96-well tissue culture plate, then incubated in a selection medium (HAT medium) for 2 weeks, and then switched to hybridoma culture medium. After about 10-14 days of cell plating, the supernatant of each well is screened by ELISA as described above. The hybridomas that secrete antibodies are transferred to 24-well plates and screened again. If the hybridoma is still positive for anti-IL-25 activity, a single cell sorter is used to subclone the positive hybridomas by limiting dilution or sorting. The stable subclones are then cultured in vitro to generate small amounts of antibody for purification and characterization.

[0349] Example 2: Binding of anti-IL-25 specific antibodies

[0350] In this study, ELISA was used to evaluate the binding ability of five public anti-IL-25 antibodies (IL25 Ab1 to Ab5) to human and mouse IL-25. In brief, recombinant human IL-25 protein (Acro, catalog number: IL5-H4221, batch number: 401-20CNF1-UT) or recombinant mouse IL-25 protein (Sino Biological Inc, catalog number: 50138-M07H, batch number: LC15AP0607) was directly coated on an ELISA plate. Then, the recombinant antibody was added to the plate and then detected by goat anti-mouse IgG-HRP (Jackson ImmunoResearch, catalog number: 109-035-098, batch number: 157400). After adding ABTS (Moss Inc., catalog number: ABTS-1000, batch number: 03086202) substrate, the ELISA plate was read using an ELISA plate reader (Bioteck).

[0351] Figure 3A The results showed that the five anti-IL-25 antibodies disclosed bound to the human recombinant IL-25 protein in a dose-dependent manner, with EC50 values ​​ranging from 0.010 nM to 0.034 nM. The EC50 values ​​of the positive control antibodies IL-25-PC2 (recombinant antibody produced by NovaRock, batch number: P09282021JLF) and IL-25-PC4 (recombinant antibody produced by NovaRock, batch number: A10032022JLF) were 0.010 nM to 0.034 nM. 50 The values ​​were 0.026 nM and 0.028 nM, respectively. The human IgG1 (InVivoMab, Cat. No.: BE0297, Batch No.: 760620M1) isotype control did not show any binding.

[0352] Figure 3B The five anti-IL-25 antibodies disclosed also bound to mouse recombinant IL-25 protein in a dose-dependent manner, and EC 50 The values ​​ranged from 0.012 nM to 0.035 nM. The EC50 values ​​of the positive control antibodies IL-25-PC2 and IL-25-PC4 were 0.022 nM and 0.021 nM, respectively.

[0353] In another study, the binding ability of five public anti-IL-25 antibodies (IL25 Ab1 to Ab5) to cynomolgus monkey IL-25 was evaluated using ELISA. Briefly, recombinant human IL-25 protein (Acro, catalog number: IL5-H4221, batch number: 401-20CNF1-UT) or recombinant cynomolgus monkey IL-25 protein (Sino Biological Inc, catalog number (customized), batch number: MB17MA2715) was directly coated on ELISA plates. The recombinant antibodies were then added to the plate, followed by detection using goat anti-human kappa light chain antibody HRP (Novus, catalog number: NBP1-75064, batch number: 68-188-010920). After adding ABTS (Moss Inc., catalog number: ABTS-1000, batch number: 03086202) substrate, the ELISA plate was read using an ELISA plate reader (Bioteck).

[0354] Figure 3C It was shown that the five published anti-IL-25 antibodies also 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.

[0355] Table 5 :Binding EC of IL-25 antibody and IL-25 protein 50

[0356]

[0357] Example 3: Binding kinetics of anti-IL-25 specific antibodies

[0358] The binding kinetics of the anti-IL-25 antibodies of the present disclosure to recombinant human IL-25 were determined by Sartorius The assay was determined using a Bio-Layer Interferometry (BLI) system using an anti-IL-25 monoclonal antibody as the ligand and recombinant human IL-25 protein as the analyte.

[0359] Briefly, the assay was first performed by 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 10-15 minute dissociation time. The bound sensorgrams were collected and analyzed by Octet data analysis software.

[0360] Table 6 summarizes the KD values ​​of the anti-IL-25 antibodies disclosed in this study. The data show that these antibodies have strong binding affinity to human recombinant IL-25 and exhibit fast-on and slow-off kinetics.

[0361] Table 6 :Binding kinetics of anti-IL-25 antibodies

[0362] Antibody ka(1 / Ms) kd(1 / s) KD(M) IL25Ab1 1.25E+06 <1.0E-07 <1.0E-12 IL25Ab2 1.36E+06 <1.0E-07 <1.0E-12 IL25Ab3 8.75E+05 <1.0E-07 <1.0E-12 IL25Ab4 1.20E+06 <1.0E-07 <1.0E-12 IL25Ab5 2.48E+06 <1.0E-07 <1.0E-12 IL25-PC2 7.80E+05 <1.0E-07 <1.0E-12

[0363] Example 4: Blocking IL-25-induced NF-κB signaling

[0364] IL-25 interacts with and signals through the IL-25 receptor, which contains heterodimeric IL-17RA and IL-17RB subunits. The activated heterodimeric receptor recruits the Act1 adaptor, leading to ubiquitination of TNF receptor-associated factor 6 (TRAF6). This in turn triggers a signaling cascade leading to NF-κB and AP-1 activation.

[0365] HEK-293 reporter cell line was used to study the blocking activity of anti-IL-25 antibodies. HEK-Blue was generated by stably transfecting human genes encoding the IL-17RA / IL-17RC heterodimeric receptor and the Act1 linker molecule into the human embryonic kidney HEK293 cell line. TM IL-17 cells (InvivoGen, San Diego, CA) These cells also express a secreted embryonic alkaline phosphatase (SEAP) reporter gene, which is inducible by NF-κB and AP-1.

[0366] In this study, HEK-Blue TM IL-17 cells were treated with 5 ng / ml recombinant human IL-25 and serially diluted IL-25 antibodies. After overnight incubation, QUANTI-Blue TM Solution (a SEAP detection reagent) was added and the NF-κB response was determined by reading the optical density (OD) at 655 nm.

[0367] like Figure 4 As shown and summarized in Table 7, the five published anti-IL-25 antibodies effectively blocked IL-25-induced NF-kB signaling in a dose-dependent manner, with IC 50 The values ​​ranged from 0.049 nM to 0.073 nM. IC values ​​of positive control antibodies IL-25-PC2 and IL-25-PC4 50 The values ​​were 0.062 nM and 0.456 nM, respectively. The human IgG1 isotype control did not show any blocking activity.

[0368] Table 7 .Inhibitory activity of IL-25 antibody in NFkB signaling assay

[0369]

[0370]

[0371] Example 5: Blocking IL-25-induced CXCL1 production

[0372] The expression of interleukin-17 (IL-17) in the peripheral blood of patients with inflammatory bowel disease (IBD) is significantly increased, indicating that IL-17 may play a key role in the physiological and pathological processes of the disease. Since the intestinal epithelial cell line HT-29 has normal colonic epithelial structure and function, it has become the most commonly used cell line in the laboratory to study the intestinal mucosal immune mechanism.

[0373] To determine whether the anti-IL-25 antibodies disclosed herein can block the production of the inflammatory cytokine CXCL1 caused by human IL-25, HT-29 cells were seeded into the assay plate. Subsequently, a combination of serially diluted anti-IL-25 antibodies and hIL-25 was added to the respective wells, 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&Dsystem, #DY275). The results are shown in FIG. Figure 5 As shown in Table 8, it is shown that the five disclosed anti-IL-25 antibodies effectively blocked IL-25-stimulated CXCL1 production, thereby potentially reducing the recruitment of immune cells and preventing the development of inflammation.

[0374] IC 50 The values ​​ranged from 0.067 nM to 0.089 nM. IC values ​​of positive control antibodies IL-25-PC2 and IL-25-PC4 50 The values ​​were 0.066 nM and 0.877 nM, respectively. The human IgG1 isotype control did not show any blocking activity.

[0375] Table 8 Activity of IL-25 antibody in blocking CXCL1 production

[0376] <![CDATA[IC 50 ,nM]]> IL25Ab1 0.071 IL25Ab2 0.089 IL25Ab3 0.067 IL25Ab4 0.076 IL25Ab5 0.077 IL25-PC2 0.066 IL25-PC4 0.877

[0377] Example 6: Blocking IL-25-induced IL-5 production

[0378] IL-25 is considered a type 2 cytokine produced by Th2 cells and is able to induce IL-4, IL-5, and IL-13 gene expression. The induction of these cytokines results in a Th2-like response characterized by elevated serum IgE, IgG, and IgA levels, blood eosinophilia, and pathological changes in the lungs and digestive tract, including eosinophil infiltration, increased mucus production, and epithelial cell hyperplasia / hypertrophy.

[0379] To further characterize the biological function of the IL-25 antibody and evaluate its potential in treating 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, large amounts of IL-5 were produced. However, if the cells were co-treated with an IL-25 blocking antibody, IL-5 levels were greatly reduced, indicating that the IL-25 antibody can inhibit the type 2 response.

[0380] like Figure 6 As shown and summarized in Table 9, the five disclosed anti-IL-25 antibodies effectively blocked IL-25-induced IL-5 production in a dose-dependent manner, IC 50 The values ​​ranged from 0.12 nM to 0.26 nM. IC values ​​of positive control antibodies IL-25-PC2 and IL-25-PC4 50 The values ​​were 0.26 nM and 12.99 nM, respectively. The human IgG1 isotype control did not show any blocking activity.

[0381] Table 9 Activity of IL-25 antibody in blocking CXCL1 production

[0382] <![CDATA[IC 50 ,nM]]> IL25Ab1 0.12 IL25Ab2 0.14 IL25Ab3 0.16 IL25Ab4 0.15 IL25Ab5 0.26 IL25-PC2 0.26 IL25-PC4 12.99

[0383] Example 7: In vivo efficacy of anti-IL-25 specific antibodies in an OVA-induced asthma model

[0384] The in vivo efficacy of anti-IL-25 specific antibodies was investigated using an OVA-induced asthma model. Male BALB / c mice aged 6 to 7 weeks were divided into 6 groups (10 mice per group): (1) control group; (2) OVA group + vehicle; (3-6) OVA group + anti-IL-25 antibody. Mice in the OVA group were sensitized by intraperitoneal injection of OVA solution containing aluminum hydroxide on days 1 and 14, and challenged on days 28, 29, and 31 by nebulizing 1% OVA solution for 30 minutes using a BUXCO nebulizer delivery system. Anti-IL-25 antibodies were injected subcutaneously at 10 mg / kg on days 14, 17, 20, 23, 26, 29, and 31. All test antibodies were prepared in mouse IgG1 form. On day 31, changes in lung resistance in response to a series of nebulized methacholine were measured by whole body plethysmography (WBP, Buxco BFE0100 WBP). On day 32, the mice were anesthetized, and the lungs were lavaged with PBS containing 1% FBS to obtain BALF.

[0385] Airway hyperresponsiveness is a hallmark of asthma. In Figure 7, mice in the OVA+vehicle group showed significantly increased airway resistance to methacholine compared to the control group. IL25Ab3 treatment significantly reduced airway resistance. IL25Ab4, IL25Ab5, and IL25-PC4 showed a slight decrease in airway resistance. However, this decrease did not reach statistical significance. Airway resistance is expressed as dose-response data for methacholine, presented as the percentage change in lung resistance (Penh value) from baseline level ( Fig. 7A ) and the percentage of baseline Penh ( Figure 7B ).

[0386] Interleukin 5 (IL-5) plays a central pathogenic role in the differentiation, recruitment, survival, and degranulation of eosinophils. IL-5 has been reported to have a potent effect on the induction, maintenance, and amplification of eosinophilic inflammation, such as asthma. Figure 8 In the OVA-induced asthma model, IL-5 in bronchoalveolar lavage fluid (BALF) was significantly increased in OVA+vehicle mice compared to control mice. IL-5 production in BALF was significantly reduced by IL25Ab3, IL25Ab5, and IL25-PC4 treatment. IL-5 production was reduced by IL25Ab4 treatment (not significant).

[0387] Example 8: Humanization of anti-IL-25 Ab3

[0388] Mouse anti-IL-25Ab3 was humanized using the CDR transplantation method. The variable regions of the heavy and light chains of mouse Ab3 were derived from mouse germline IGHV1-18*01 and IGKV10-94*01, respectively. The heavy chain variable region was used to search the human antibody germline database, and the human germline sequence human IGHV1-2*02 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 the transplantation process, a structural model of the antibody was generated, and selected back mutations were introduced to maintain correct antibody folding. A total of 5 heavy chain variants and 4 light chain variants were designed and cloned into expression constructs. 20 pairs of heavy and light chain humanized variants were made into Fab and human IgG1 antibodies. The Fab fragments of the humanized variants were used to measure binding affinity on the BLI instrument, and the human IgG1 antibodies were used for ELISA binding and functional assays. Table 10 shows that the top 3 humanized Fabs selected have similar binding affinity to the mouse parental Fab.

[0389] Table 10 :Binding affinity of humanized IL-25 antibody Fab to IL-25 protein

[0390] KD(M) kon(1 / Ms) kdis(1 / s) IL25Ab6 Fab 1.86E-09 1.34E+05 2.49E-04 IL25Ab7 Fab 1.80E-09 1.61E+05 2.90E-04 IL25Ab8 Fab 1.78E-09 1.41E+05 2.51E-04 IL25Ab3 Fab 1.89E-09 1.50E+05 2.85E-04

[0391] Example 9: Binding of humanized anti-IL-25 antibodies to human, mouse and cynomolgus monkey IL-25 recombinant proteins

[0392] In this study, the binding ability of three humanized anti-IL-25 antibodies produced in hIgG1 (IL25Ab6 to Ab8) and hIgG1YTE format (IL25Ab9 to Ab11) to human, mouse and cynomolgus monkey IL-25 was 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) was directly coated onto ELISA plates. The recombinant antibody was then added to the plate, followed by detection 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).

[0393] Fig. 9AAll humanized anti-IL-25 antibodies bound to human IL-25 protein in a dose-dependent manner. 50 The values ​​ranged from 0.302 nM to 0.404 nM. The EC50 values ​​of mouse-derived anti-IL-25 specific antibody (IL25Ab3), positive control antibodies IL-25-PC2 and IL-25-PC4 were 0.506 nM, 0.334 nM, and 0.585 nM, respectively. The human IgG1 isotype control did not show any binding.

[0394] Fig. 9B All humanized anti-IL-25 antibodies bound to cynomolgus monkey IL-25 protein in a dose-dependent manner. 50 The values ​​ranged from 0.222 nM to 0.295 nM. The EC50 values ​​of mouse-derived anti-IL-25 specific antibody (IL25Ab3), positive control antibodies IL-25-PC2 and IL-25-PC4 were 0.364 nM, 0.365 nM, and 0.408 nM, respectively. The human IgG1 isotype control did not show any binding.

[0395] Fig. 9C All humanized anti-IL-25 antibodies bound to mouse IL-25 protein in a dose-dependent manner. 50 The EC50 values ​​of mouse-derived anti-IL-25 specific antibody (IL25Ab3), positive control antibodies IL-25-PC2 and IL-25-PC4 were 0.390 nM, 0.420 nM and 0.407 nM, respectively. The human IgG1 isotype control did not show any binding. These results are summarized in Table 11.

[0396] Table 11 :Binding EC of IL-25 antibody and IL-25 protein 50

[0397]

[0398] Example 10: Blockade of IL-25-induced NF-kB signaling

[0399] In this study, we used HEK-Blue TM IL-17 cells (InvivoGen, San Diego, CA) were used to examine the blocking activity of humanized anti-IL-25 antibodies as described in Example 4. Briefly, reporter cells were treated with 5 ng / ml recombinant human IL-25 and serial dilutions of IL-25 antibodies. After overnight incubation, the SEAP detection reagent QUANTI-Blue was used to detect the presence of IL-25 in the reporter cells. TMThe solution was used to determine the NF-κB response and the optical density (OD) was read at 655 nm.

[0400] like Fig.10 As shown and summarized in Table 12, all humanized anti-IL-25 antibodies effectively blocked IL-25-induced NF-kB signaling in a dose-dependent manner, with IC 50 The values ​​ranged from 0.037 nM to 0.052 nM. IC values ​​of mouse-derived anti-IL-25 specific antibody (IL25Ab3), positive control antibodies IL-25-PC2 and IL-25-PC4 50 The values ​​were 0.037 nM, 0.030 nM and 0.513 nM respectively. The human IgG1 isotype control did not show any blocking activity.

[0401] Table 12 .Inhibitory activity of IL-25 antibody in NFkB signaling assay

[0402] <![CDATA[IC 50 ,nM]]> IL25Ab3 0.037 IL25Ab6 0.048 IL25Ab7 0.042 IL25Ab8 0.041 IL25Ab9 0.043 IL25Ab10 0.052 IL25Ab11 0.037 IL25-PC2 0.030 IL25-PC4 0.513

[0403] Example 11: Blocking IL-25-induced CXCL1 production

[0404] To determine whether humanized anti-IL-25 antibodies can block the production of human IL-25-induced inflammatory cytokine CXCL1, HT-29 cells endogenously expressing IL17 receptors were used as described in Example 5. Briefly, HT-29 cells were stimulated with hIL-25 in combination with serially diluted anti-IL-25 antibodies at 37°C for 72 hours. The supernatant was then collected and CXCL1 levels were measured using the Human CXCL1 ELISA Ready-SET-Go Kit (R&D system #DY275). The results are shown in Fig.11 As shown in Table 13, it was shown that the humanized anti-IL-25 antibody maintained a strong blocking activity on IL-25-induced CXCL1 production, thereby potentially reducing the recruitment of immune cells and preventing the development of inflammation.

[0405] IC of humanized antibody (IL25Ab6-Ab11) 50 The values ​​ranged from 0.183 nM to 0.225 nM. IC values ​​of mouse-derived anti-IL-25 specific antibody (IL25Ab3), positive control antibodies IL-25-PC2 and IL-25-PC4 50 The values ​​were 0.144 nM, 0.148 nM and 1.84 nM respectively. The human IgG1 isotype control did not show any blocking activity.

[0406] Table 13 Activity of IL-25 antibody in blocking CXCL1 production

[0407] IC50,nM IL25Ab3 0.144 IL25Ab6 0.225 IL25Ab7 0.183 IL25Ab8 0.199 IL25Ab9 0.225 IL25Ab10 0.198 IL25Ab11 0.187 IL25-PC2 0.148 IL25-PC4 1.84

[0408] Example 12: Blocking IL-25-induced IL-5 production

[0409] To further characterize the biological function of humanized IL-25 antibodies and evaluate their potential in treating inflammatory diseases, human PBMC assays were performed as described in Example 6. Briefly, IL-25 induced human PBMCs to produce large amounts of IL-5 in the presence of 30 U / ml recombinant hIL-2. The blocking activity of humanized anti-IL-25 activity was determined to indicate its inhibitory activity against type 2 inflammatory responses.

[0410] like Picture 12 As shown and summarized in Table 14, all humanized IL-25 antibodies effectively blocked IL-25-induced IL-5 production in a dose-dependent manner, with IC 50 The values ​​ranged from 0.381 nM to 1.43 nM. IC values ​​of mouse-derived anti-IL-25 specific antibody (IL25Ab3), positive control antibodies IL-25-PC2 and IL-25-PC4 50 The values ​​were 0.302 nM, 0.394 nM and 7.15 nM respectively. The human IgG1 isotype control did not show any blocking activity.

[0411] Table 14 Activity of IL-25 antibodies in blocking IL-5 production

[0412] IC50,nM IL25Ab3 0.302 IL25Ab6 0.381 IL25Ab7 0.850 IL25Ab8 1.04 IL25Ab9 0.435 IL25Ab10 1.43 IL25Ab11 0.953 IL25-PC2 0.394 IL25-PC4 7.15

[0413] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties (such as molecular weight), reaction conditions, and the like used in the specification and claims are to be understood as being modified in all cases by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and the appended claims are approximate values ​​that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0414] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0415] Unless otherwise indicated herein or clearly contradicted by context, the terms "a", "an", "the" and similar references used in the context of describing the present disclosure (especially in the context of the following claims) should be interpreted as covering both the singular and the plural. The numerical ranges listed herein are intended only to be used as a shorthand method of individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if the value were listed individually herein. Unless otherwise indicated herein or clearly contradicted by context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present disclosure, rather than to limit the scope of the present disclosure otherwise claimed. No language in the specification should be interpreted as indicating any unclaimed element essential to the practice of the present disclosure.

[0416] The grouping of the alternative elements or embodiments of the disclosure disclosed herein should not be construed as limiting. Each group member can be referred to and claimed individually or with other members of the group or other elements found herein in any combination. For reasons of convenience and / or patentability, it can be expected that one or more members of the group may be included in the group or deleted from the group. When any such inclusion or deletion occurs, the specification is deemed to contain the group of modification, thereby satisfying the written description of all Markush groups used in the appended claims.

[0417] Certain embodiments of the present disclosure are described herein, including the best mode known to the inventor for implementing the present disclosure. Of course, after reading the above description, variations of the embodiments described will become apparent to those of ordinary skill in the art. The inventor expects that skilled technicians will appropriately adopt such variations, and the inventor hopes to put the present disclosure into practice in a manner different from that specifically described herein. Therefore, the present disclosure includes all modifications and equivalents to the subject matter listed in the attached claims where permitted by applicable law. In addition, unless otherwise stated herein or clearly contradictory to the context, the present disclosure covers any combination of the above-mentioned elements in all possible variations thereof.

[0418] The use of "consisting of" or "consisting essentially of" language in the claims may further limit the specific embodiments disclosed herein. When used in a claim, whether as filed or added by amendment, the transition term "consisting of" excludes any elements, steps, or ingredients not specified in the claim. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics. Embodiments of the present disclosure as claimed are inherently or expressly described and enabled herein.

[0419] It will be understood that the embodiments of the present disclosure disclosed herein are illustrations of the principles of the present disclosure. Other modifications that may be adopted are within the scope of the present disclosure. Therefore, by way of example and not limitation, the optional configurations of the present disclosure may be used according to the teachings herein. Therefore, the present disclosure is not limited to exactly as shown and described.

[0420] Although the disclosure has been described and illustrated herein by reference to various specific materials, methods and embodiments, it should be understood that the disclosure is not limited to the specific combination of materials and methods selected for this purpose. As will be appreciated by those skilled in the art, many variations of such details may be implied. This is intended to treat the specification and embodiments as merely exemplary, and the true scope and spirit of the disclosure are indicated by the appended claims. All references, patents and patent applications mentioned in this application are incorporated herein by reference in their entirety.

Claims

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); (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); 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 (LCDR2); (c) a heavy chain variable region comprising 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 a light chain variable region comprising 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) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 (HCDR1), the amino acid sequence of SEQ ID NO: 18 (HCDR2), and the amino acid sequence of SEQ ID NO: 19 (HCDR3); and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 (LCDR1), the amino acid sequence of SEQ ID NO: 21 (LCDR2), and the amino acid sequence of SEQ ID NO: 22 (LCDR3); (e) a heavy chain variable region comprising 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 a light chain variable region comprising 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 comprising 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 comprising 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) a heavy chain variable region comprising 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 a light chain variable region comprising 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).

2. The anti-IL-25 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NO: 1, 3, 5, 7, 9, 44 or 47 and a light chain variable region comprising an amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48.

3. The anti-IL-25 antibody according to claim 1, wherein the antibody comprises: (a) a heavy chain variable region sequence of SEQ ID NO: 44 and a light chain variable region sequence of SEQ ID NO: 45; (b) a heavy chain variable region sequence of SEQ ID NO: 44 and a light chain variable region sequence of SEQ ID NO: 46; (c) a heavy chain variable region sequence of SEQ ID NO: 47 and a light chain variable region sequence of SEQ ID NO: 48; (d) a heavy chain variable region sequence of SEQ ID NO: 1 and a light chain variable region sequence of SEQ ID NO: 2; (e) a heavy chain variable region sequence of SEQ ID NO: 3 and a light chain variable region sequence of SEQ ID NO: 4; (f) a heavy chain variable region sequence of SEQ ID NO: 5 and a light chain variable region sequence of SEQ ID NO: 6; (g) a heavy chain variable region sequence of SEQ ID NO: 7 and a light chain variable region sequence of SEQ ID NO: 8; or (h) The heavy chain variable region sequence is SEQ ID NO: 9 and the light chain variable region sequence is SEQ ID NO:

10.

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 of claim 1 , wherein the antibody is a full-length antibody comprising a human IgG1 constant region selected from SEQ ID NO: 55 or SEQ ID NO:

56. The anti-IL-25 antibody of claim 1 , wherein the antibody is an antibody fragment.

7. The 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 of claim 1, wherein the antibody is a monoclonal antibody.

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

10. The anti-IL-25 antibody of claim 1, wherein the antibody is a murine antibody. The anti-IL-25 antibody of claim 1 , wherein the antibody is a chimeric antibody.

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

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

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

15. A method of treating and / or preventing a type 2 inflammatory disease, an autoimmune disease, an allergic disorder or cancer in a subject in need thereof, the method comprising: The antibody of claim 1 is administered to the subject.

16. A polynucleotide composition comprising: a) a first polynucleotide encoding a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 44 or 47; and b) a second polynucleotide encoding a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 45, 46 or 48.

17. A carrier composition comprising: a) a first vector comprising the first polynucleotide of claim 16; and b) a second vector comprising the second polynucleotide of claim 16.

18. A cell comprising the polynucleotide composition of claim 16 or the vector composition of claim 17.

19. A method for producing the anti-IL-25 antibody of claim 1, the method comprising culturing the cell of claim 18 in a culture medium; and recovering the anti-IL-25 antibody from the culture medium.

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