Combination antagonists against IL-5 / IL-5R and IL-4 / IL-4R or IL-13 / IL-13R

Abstract

The present invention relates to a combination therapy and its use in the treatment of chronic airway diseases, particularly asthma. The combination therapy comprises (i) an IL-5:IL-5R antagonist and (ii) an IL-4:IL-4R antagonist and / or an IL-13:IL-13R antagonist. The present invention also provides a bispecific antibody comprising an antigen-binding domain that binds to IL-4Rα and an antigen-binding domain that binds to IL-5. This bispecific antibody can be used to treat chronic airway diseases, particularly asthma. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to combination therapies and their use in the treatment of chronic airway diseases, particularly asthma. The combination therapies comprise (i) an IL-5:IL-5R antagonist and (ii) an IL-4:IL-4R antagonist and / or an IL-13:IL-13R antagonist. The combination antagonists may be antibody molecules, and preferably the combination therapies comprise an antibody molecule that binds to IL-4Rα and an antibody molecule that binds IL-5. The combination therapies typically inhibit signaling mediated by the type 2 cytokines IL-4, IL-13, and IL-5. The present invention also provides bispecific antibodies comprising an antigen-binding domain that binds to IL-4Rα and an antigen-binding domain that binds IL-5. The bispecific antibodies can be used to treat chronic airway diseases, particularly asthma. [Background technology]

[0002] BACKGROUND OF THE INVENTION Chronic airway disease (or chronic respiratory disease) is a chronic disease of the airways and other lung structures. Some of the most common forms of chronic airway disease are asthma and chronic obstructive pulmonary disease, which includes chronic bronchitis and emphysema.

[0003] Asthma is a chronic inflammatory disease of the conducting airways that causes symptoms of coughing, wheezing, and chest tightness. It affects up to 300 million people worldwide. The airway obstruction that occurs in asthmatics varies, with symptom-free periods punctuated by exacerbations often triggered by environmental allergens and viral infections. A common symptom is bronchial hyperresponsiveness (BHR), a tendency for airways to constrict in response to stimuli such as cold air or exercise.

[0004] Asthma patients also exhibit symptoms of airway remodeling, which results in thickening of the airway walls and an increase in the number of mucus-producing goblet cells within the glandular epithelium or mucosal glands, a phenomenon known as goblet cell metaplasia (GCM). Goblet cells produce mucins that control the viscoelasticity and hydration of the mucus that coats the ciliary escalator. In asthma patients, sputum is often very dry, potentially leading to mucus impaction and severe airway obstruction. Currently, few treatment options exist to reduce GCM and improve mucus clearance.

[0005] Historically, asthma treatment approaches included the use of nonspecific medications such as inhaled corticosteroids and β2-agonists, with varying degrees of success. However, "asthma" is now understood to be a heterogeneous disorder representing multiple phenotypes, each with distinct clinical, physiological, and molecular characteristics (Wenzel SE (2015) Nature Medicine; 18(5): 716-725; Ray et al. (2015) Am J Physiol - Lung Cellular and Molecular Physiology; 308: 130-140).

[0006] Recognizing that asthma is caused by distinct pathogenic molecular mechanisms and therefore has a distinct etiology and may respond differently to treatment, attempts have been made to group clinical subsets of asthma (Gauthier et al., (2015) American Journal of Respiratory and Critical Care Medicine; 192(6): 660-668). Examples of identified subsets include early-onset or late-onset asthma, depending on the age at which symptoms first appear; eosinophilic, neutrophilic, or non-inflammatory asthma, depending on the presence and type of inflammation observed; exercise-induced asthma; obesity-related asthma; atopic asthma, characterized by allergic sensitization to inhaled allergens such as house dust mites (HDM) and elevated serum allergen-specific IgE; mild or moderate asthma, described as corticosteroid-responsive; severe asthma; and type 2 cytokine-related asthma, representing individuals who share a type 2 inflammatory pattern. Importantly, the lack of clear demarcation between these groups means that many phenotypes overlap and patients may exhibit clinical or pathological features of more than one group, making it difficult to predict individual responses to treatment.

[0007] As our understanding of these mechanistically distinct groups, also referred to as "endotypes," improves, associated cellular and molecular biomarkers are beginning to be identified. In bronchial biopsy or lung resection samples, asthma is often characterized by the accumulation of eosinophils, mast cells, and CD4+ T lymphocytes, which produce the type 2 cytokines IL-4 and / or IL-5 within the epithelium and lamina propria. However, this type 2 inflammatory manifestation is detectable in only 50% of patients with asthma, particularly those with early-onset asthma, atopic predisposition, and high blood eosinophil counts. Conversely, in some patients with asthma, particularly those with poor steroid response, the airway infiltrate is primarily composed of neutrophils. These neutrophils may be recruited to the airways by IL-17-producing cells, such as type 17 T helper lymphocytes or γδ T cells (Lambrecht and Hammad, (2015) Nat. Immunol. 16(1): 45-56).

[0008] Drugs specifically targeting molecular pathways involved in chronic airway disease are being developed. For example, many antibody therapies have been developed for allergic diseases (Sheridan C, (2018) Nature Biotechnology; 36: 3-5; Godar et al., (2017) Monoclonal Antibodies: Taylor & Francis; 1-12). These include dupilumab, which binds to IL-4Rα; omalizumab, which targets IgE; mepolizumab and reslizumab, which target IL-5; and tralokinumab, which binds to IL-13. However, there is still a need for improved therapies in this field. Summary of the Invention

[0009] (Summary of the Invention) The present invention is based on targeting multiple type 2 cytokine signaling pathways. Type 2 cytokines are those that activate T helper type 2 cells (T H Type 2 cytokines were originally so named because they are released by T cells. In chronic airway diseases such as asthma, type 2 cytokines are H Type II cytokines are released not only from type II cells but also from cells such as basophils, mast cells, and eosinophils. Type II cytokines play an important role in the pathogenesis of chronic airway diseases, particularly asthma. Surprisingly, it has been discovered that targeting multiple type II cytokines, particularly IL-5, IL-4, and IL-13, produces unexpected synergistic effects that alleviate the underlying conditions and symptoms of chronic airway diseases. Therefore, the combination therapy of the present invention is particularly suitable for treating chronic airway diseases.

[0010] In a first aspect, the present invention provides a combination comprising (i) an IL-5:IL-5R antagonist; and (ii) an IL-4:IL-4R antagonist and / or an IL-13:IL-13R antagonist. Because the IL-4 receptor complex and the IL-13 receptor complex share a common subunit, IL-4Rα, an IL-4Rα antagonist can function as an antagonist of both the IL-4:IL-4R and IL-13:IL-13R signaling pathways. In a subsequent preferred embodiment, the combination of the present invention comprises an IL-5:IL-5R antagonist and an IL-4Rα antagonist. The IL-5:IL-5R antagonist is preferably an IL-5 antagonist. The combination may also inhibit signaling via IL-5, IL-4, and IL-13.

[0011] The antagonists of the combination may be antibody molecules. Thus, in one embodiment, the antagonist of IL-5:IL-5R is an antibody molecule, and / or the antagonist of IL-4:IL-4R is an antibody molecule, and / or the antagonist of IL-13:IL-13R is an antibody molecule. In a preferred embodiment, the combination comprises an antagonist of IL-5:IL-5R that is an antibody molecule that binds to IL-5, preferably human IL-5, and an antagonist of IL-4Rα, preferably human IL-4Rα, that is an antibody molecule that binds to IL-4Rα.

[0012] In some embodiments, the antibody molecules of the combination are independently selected from the group consisting of an antibody light chain variable domain (VL); an antibody heavy chain variable domain (VH); a single-chain antibody (scFv); a F(ab')2 fragment; a Fab fragment; a Fd fragment; a Fv fragment; a single-arm (monovalent) antibody; a diabody, a triabody, a tetrabody, or any antigen-binding molecule formed by combining, assembling, or conjugating antigen-binding fragments thereof. In a preferred embodiment, the combination comprises an antibody molecule that binds to IL-4Rα and an antibody molecule that binds to IL-5, wherein the antibody molecules are independently selected from the group consisting of an antibody light chain variable domain (VL); an antibody heavy chain variable domain (VH); a single-chain antibody (scFv); a F(ab')2 fragment; a Fab fragment; a Fd fragment; a Fv fragment; a single-arm (monovalent) antibody; a diabody, a triabody, a tetrabody, or any antigen-binding molecule formed by combining, assembling, or conjugating antigen-binding fragments thereof. The antibody molecules of the combination may be VHH antibodies. In a preferred embodiment, the antibody molecules of the combination are IgG antibodies.

[0013] In one embodiment, the antibody molecules of the combination, e.g., the antibody molecule that binds IL-4Rα and / or the antibody molecule that binds IL-5, are humanized or germline variants of non-human antibodies, or antigen-binding fragments thereof, e.g., camelid-derived antibodies or antigen-binding fragments thereof.

[0014] In one embodiment, the antibody molecules of the combination, e.g., the antibody molecule that binds to IL-4Rα and / or the antibody molecule that binds IL-5, comprise the CH1 domain, hinge region, CH2 domain, and / or CH3 domain of human IgG. Alternatively, the antibody molecules may exhibit high homology to human IgG, preferably IgG1.

[0015] In one embodiment, the antibody molecules of the combination, e.g., the antibody molecules that bind to IL-4Rα and / or the antibody molecules that bind to IL-5, comprise an Fc domain derived from human IgG, preferably IgG1. This Fc domain may be unmodified or may be modified by one or more amino acid substitutions, e.g., to increase binding affinity to FcRn (fetal Fc receptor). In a preferred embodiment, the antibody molecule comprises an Fc domain, preferably an Fc domain derived from human IgG, containing the amino acid substitutions: H433K and N434F; or M252Y, S254T, T256E, H433K, and N434F. The numbering of the Fc domain is based on the EU numbering scheme.

[0016] In one embodiment, the antibody molecules of the combination, e.g., the antibody molecules that bind to IL-4Rα and / or the antibody molecules that bind to IL-5, exhibit pH-dependent antigen-binding activity, and in particular, exhibit lower antigen-binding activity at acidic pH than at neutral pH. The ratio of the antigen-binding activity at acidic pH to that at neutral pH may be at least 2, as assessed by the dissociation constant ratio: KD(at acidic pH) / KD(at neutral pH).

[0017] With respect to the formulation of the combinations, the IL-5:IL-5R and IL-4:IL-4R and / or IL-13:IL-13R antagonists may be co-formulated or provided separately. In embodiments in which the antagonists are co-formulated, the antagonists may be formulated in a 1:1 ratio or in a non-equimolar ratio. For example, for a combination including an IL-5:IL-5R antagonist, preferably an IL-5 antagonist, and an IL-4Rα antagonist, the antagonists may be formulated in a ratio of, for example, 1:2 or 2:1.

[0018] In embodiments where the combination comprises an antagonist of IL-4Rα and an antagonist of IL-5, and the antagonists are antibody molecules, the combination may comprise antibody molecules combined in a multispecific antibody, e.g., a bispecific antibody.

[0019] In some embodiments, the combination comprises one or more additional therapeutic agents.

[0020] In a second aspect, the present invention provides a bispecific antibody comprising an antigen-binding region that binds to IL-4Rα and an antigen-binding region that binds to IL-5. In preferred embodiments, the antigen-binding region that binds to IL-4Rα and / or the antigen-binding region that binds to IL-5 are humanized or germline variants of non-human antibodies, or antigen-binding fragments thereof, preferably camelid antibodies or antigen-binding fragments thereof. In one embodiment, the antigen-binding region that binds to IL-4Rα comprises a first variable heavy domain (VH) and variable light domain (VL) pair, and the antigen-binding region that binds to IL-5 comprises a second variable heavy domain (VH) and variable light domain (VL) pair. The bispecific antibody may be an IgG antibody having a first VH-VL pair that binds to IL-4Rα and a second VH-VL pair that binds to IL-5. In one embodiment, the bispecific antibody is an IgG antibody having at least one scFv fragment bound thereto.

[0021] The bispecific antibodies of the present invention may exhibit pH-dependent antigen binding. For example, the antigen-binding region that binds to IL-4Rα and / or the antigen-binding region that binds to IL-5 may be The antigen-binding activity may be lower at acidic pH than at neutral pH. In one embodiment, the ratio of the antigen-binding activity at acidic pH to that at neutral pH is at least 2, as determined by KD(acidic pH) / KD(neutral pH).

[0022] It has been discovered that the present combinations and bispecific antibodies, which target multiple type 2 cytokine signaling pathways, are particularly useful in the treatment of chronic airways diseases, in particular asthma. Accordingly, in a further aspect of the invention, there is provided a combination according to the first aspect of the invention, or a bispecific antibody according to the second aspect of the invention, for use in treating chronic airways diseases in a human subject. Furthermore, there is provided a method of treating chronic airways diseases in a human subject, which method comprises administering to the subject an effective amount of a combination according to the first aspect of the invention, or a bispecific antibody according to the second aspect of the invention.

[0023] In certain embodiments, the chronic airway disease is selected from: asthma; chronic rhinosinusitis (CRS); immunoglobulin G4-related disease (IgG4-RD); chronic obstructive pulmonary disease (COPD); chronic bronchitis; emphysema; chronic angioedema; diseases characterized by goblet cell metaplasia, including Barrett's esophagus; active eosinophilic esophagitis; nasal polyposis; chronic rhinosinusitis; Churg-Strauss syndrome; allergic bronchopulmonary aspergillosis (ABPA); hypereosinophilic syndrome; bullous pemphigoid, and cystic fibrosis.

[0024] The chronic airway disease treated by the methods of the invention may be characterized by increased mucus production or exacerbated bronchial hyperresponsiveness. In preferred embodiments, the chronic airway disease treated is asthma, optionally severe asthma, severe refractory asthma, Type II High asthma, atopic or allergic asthma.

[0025] The methods described herein may be useful for treating chronic airway diseases, preferably asthma, by reducing goblet cell metaplasia (or GCM). Alternatively or additionally, the methods may be useful for treating chronic airway diseases, preferably asthma, by reducing bronchial hyperresponsiveness (BHR). The methods may further comprise the additional step of, for example, administering to the patient one or more additional therapeutic agents for treating the chronic airway disease. [Brief explanation of the drawings]

[0026] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figure 1 shows the neutralizing activity of both IL-4Rα and IL-5 monospecific antibodies, as assessed in in vitro cell assays of IL-4-induced HT-2 cell proliferation and IL-5-induced TF-1 cell proliferation. IL-4Rα monospecific antibodies (squares) and IL-5 monospecific antibodies (triangles) potently inhibited murine IL-4- and IL-5-induced HT-2 and TF-1 cell proliferation, respectively. Results are shown as the mean of triplicates ± SEM from two independent experiments.

[0027] [Figure 2] Figure 2 shows surface plasmon resonance (SPR) sensorgrams displaying the interaction between monoclonal antibodies (IL-4Rα antibody, IL-5 antibody, or irrelevant IgG2a antibody) at various concentrations (0-20 μg / mL) and immobilized targets (IL-4Rα or IL-5).

[0028] [Figure 3] FIG. 3 shows SPR sensorgrams displaying the interaction between a mixture composed of a monoclonal antibody (IL-4Rα antibody, IL-5 antibody, or irrelevant IgG2a antibody) and its target (IL-4Rα or IL-5) and an immobilized protein (IL-4, IL-13Rα, or IL-5Rα).

[0029] [Figure 4] Figure 4 shows MHC class II antigen expression in purified B cells analyzed by FACS before and after treatment with IL-4Rα monoclonal antibody. IL-4Rα monoclonal antibody potently inhibited IL-4-induced MHC class II antigen expression in purified B cells.

[0030] [Figure 5]Figure 5 shows the results of an experiment to test the effects of IL-4Rα and IL-5 monoclonal antibodies in an in vivo mouse model of asthma. (A) Diagrammatic representation of the experimental setup using the house dust mite (HDM) mouse model. IL-4Rα and IL-5 antibody treatment was administered by injection into HDM-treated C57BL / 6J mice during both the sensitization and challenge phases. (B) Differential cell counts in bronchoalveolar lavage fluid (BAL) analyzed by FACS from mice administered IL-4Rα monospecific antibody, IL-5 monospecific antibody, a combination of IL-4α / IL-5 monospecific antibodies, or an irrelevant IgG2a antibody. In IgG2a-treated HDM-sensitized mice, eosinophil cell counts increased upon allergen challenge. A significant decrease in eosinophil cell counts was observed after treatment with IL-4Rα monospecific antibody, IL-5 monospecific antibody, and the IL-4α / IL-5 monospecific antibody combination compared to control IgG2a antibody treatment. P values ​​reflect one-way ANOVA tests; ns: not significant, *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001 vs. control IgG2 antibody.

[0031] [Figure 6] Figure 6 is a diagrammatic representation of the experimental setup using the house dust mite (HDM) mouse model as an in vivo mouse model of asthma. IL-4Rα and IL-5 antibody treatment was administered by injection into HDM-treated C57BL / 6J mice only during the challenge phase.

[0032] [Figure 7]Figure 7 shows differential cell counts in BAL analyzed by FACS from mice administered IL-4Rα monospecific antibody, IL-5 monospecific antibody, IL-4Rα / IL-5 monospecific antibody combination, or irrelevant IgG2a antibody. In IgG2a-treated HDM-sensitized mice, eosinophil cell counts increased upon allergen challenge. A significant decrease in eosinophil cell count was observed after treatment with IL-4Rα monospecific antibody or IL-5 monospecific antibody compared to control IgG2a antibody treatment. A further decrease in eosinophil cell count was observed after treatment with the IL-4α / IL-5 monospecific antibody combination. P values ​​reflect one-way ANOVA tests; ns: not significant, **P≦0.01, ***P≦0.001, ****P≦0.0001 relative to control IgG2 antibody.

[0033] [Figure 8] Figure 8 shows the production of IL-5 and IL-13 cytokines by mesenteric lymph node (MLN) cells restimulated with HDM ex vivo for 3 days, as determined by ELISA. In vitro production of the effector cytokines IL-5 and IL-13 in allergen-restimulated MLN cell cultures was boosted by allergen challenge in IgG2a-treated HDM-sensitized mice. However, this response was significantly reduced after treatment with the IL-4Rα monospecific antibody and the combination of both monotherapies. P values ​​reflect one-way ANOVA tests; ns: not significant, **P ≤ 0.01, ***P ≤ 0.001 versus the control IgG2 antibody.

[0034] [Figure 9]Figure 9 shows serum levels of HDM-specific IgE and IgG1, as determined by ELISA, after treatment with IL-4Rα monospecific antibody, IL-5 monospecific antibody, the IL-4Rα / IL-5 monospecific antibody combination, or an irrelevant IgG2a antibody. Serum concentrations of HDM-specific IgG1 and IgE were boosted by allergen challenge in IgG2a-treated mice. The IL-4Rα monoclonal antibody and the IL-4Rα / IL-5 monoclonal antibody combination were able to significantly reduce this allergen-induced increase in IgG1 and IgE. P values ​​reflect one-way ANOVA tests; *P≦0.05, ns: not significant, **P≦0.01, ****P≦0.0001 versus the irrelevant IgG2 antibody.

[0035] [Figure 10] Figure 10 shows the expression of mucins, Muc5AC, Agr2, and Spdef in the lungs of mice treated with IL-4Rα monospecific antibody, IL-5 monospecific antibody, and the IL-4Rα / IL-5 monospecific antibody combination. (A) Confocal staining of Muc5AC in the lungs of mice treated with IL-4Rα monospecific antibody, IL-5 monospecific antibody, the IL-4Rα / IL-5 monospecific antibody combination, or an irrelevant IgG2a antibody. (B) Pulmonary mRNA expression levels of Muc5ac and Agr2, as determined by qRT-PCR. The mRNA expression levels of these two genes were induced by HDM challenge compared to PBS challenge in mice. IL-4Rα and IL-5 antibodies alone did not significantly reverse this increase in Muc5ac or Agr2 mRNA levels. However, the combination of both monospecific IL-4Rα and IL-5 monoclonal antibodies significantly reduced the HDM-mediated increase in Muc5ac or Agr2 mRNA levels. P values ​​reflect one-way ANOVA tests; ns: not significant, **P ≤ 0.01, ****P ≤ 0.0001 vs. an irrelevant IgG2 antibody.

[0036] [Figure 11]Figure 11 shows bronchial hyperresponsiveness (BHR) measured using flexiVent (SCIREQ, Inc., ®) after exposure to increasing doses of methacholine. Data represent three independent experiments with at least n = 6 mice per group. Bronchial hyperresponsiveness was significantly reduced after treatment with the IL-4Rα / IL-5 monospecific antibody combination compared to control IgG2a antibody treatment, with resistance levels returning to those observed in unchallenged mice receiving PBS alone. Results are shown as mean ± SEM. P values ​​reflect one-way ANOVA tests; ns: not significant, *P < 0.05 vs. irrelevant IgG2 antibody.

[0037] [Figure 12] Figure 12 is a schematic diagram of the dual anti-idiotype purification process for isolating the desired bispecific IL-4Rα / IL-5 antibody with the correct pairing. (a) From a mixture of four possible combinations formed from different heavy and light chain pairs, antibodies containing the correct HC / LC pairing of the IL-4Rα monospecific antibody were isolated using an anti-idiotype VHH that recognizes only the correct HC / LC pairing of the IL-4Rα monospecific antibody. (b) A second anti-idiotype column containing a VHH antibody that recognizes only the correct HC / LC pairing of the IL-5 monospecific antibody was used to collect the bispecific antibody with the correct HC / LC pairing of the αIL-5 monospecific antibody. (c) In this way, the desired IL-4Rα / IL-5 bispecific antibody with the correct HC / LC pairing was isolated.

[0038] [Figure 13]Figure 13 demonstrates the dual-targeting properties of the IL-4Rα / IL-5 bispecific antibody. A. SPR signals were measured after sequential injection of IL-4Rα monospecific antibody or IL-4Rα / IL-5 bispecific antibody onto coated IL-4Rα-Fc, followed by a second injection of IL-4Rα-Fc or IL-5. One arm of the bispecific antibody bound to the coated IL-4Rα, and the other arm bound to the injected IL-5. B. SPR signals were measured after sequential injection of IL-5 monospecific antibody or IL-4Rα / IL-5 bispecific antibody onto coated IL-5, followed by a second injection of IL-4Rα-Fc or IL-5. One arm of the bispecific antibody bound to the coated IL-5, and the other arm bound to the injected IL-4Rα.

[0039] [Figure 14] Figure 14 shows a diagrammatic representation of the experimental setup in which antibody treatments were infused into HDM-treated C57BL / 6J mice only during the challenge phase. To compare equimolar inhibition of the target and eliminate differences in total antibody amounts, the following doses of antibody were administered to each mouse: 75 μg of each monospecific antibody combined with 75 μg of an irrelevant IgG2a antibody; 75 μg of each monospecific antibody injected in combination; or 150 μg of the IL-4Rα / IL-5 bispecific antibody.

[0040] [Figure 15]Figure 15 shows differential cell counts in the BAL of HDM-treated mice administered IL-4Rα monospecific antibody, IL-5 monospecific antibody, IL-4α / IL-5 monospecific antibody combination, IL-4Rα / IL-5 bispecific antibody, or an irrelevant IgG2a antibody, as analyzed by FACS. HDM challenge in sensitized mice increased the number of eosinophils in the BAL fluid. This increase in eosinophil count was significantly reduced after injection into mice administered a combination of both monospecific IL-4Rα and IL-5 antibodies (75 μg + 75 μg) and mice administered the IL-4Rα / IL-5 bispecific antibody. Both monospecific antibody combinations and the bispecific antibody resulted in a significant reduction in eosinophil counts compared to HDM-treated mice administered the control IgG2a antibody. P values ​​reflect one-way ANOVA tests; ns: not significant, ***P≦0.001, ****P≦0.0001 versus an irrelevant IgG2 antibody.

[0041] [Figure 16] Figure 16 shows IL-5 and IL-13 cytokine production by mesenteric lymph node (MLN) cells restimulated ex vivo with HDM for 3 days, as determined by ELISA. P values ​​reflect one-way ANOVA tests; ns: not significant, ***P≦0.001, ****P≦0.0001, versus an irrelevant IgG2 antibody.

[0042] [Figure 17] 17 shows serum levels of HDM-specific IgE and IgG1 after treatment with IL-4Rα monospecific antibody, IL-5 monospecific antibody, IL-4Rα / IL-5 monospecific antibody combination, IL-4Rα / IL-5 bispecific antibody, or irrelevant IgG2a antibody, as determined by ELISA. P values ​​reflect one-way ANOVA tests; ns: not significant, *P≦0.05, **P≦0.01, ***P≦0.001 vs. irrelevant IgG2 antibody.

[0043] [Figure 18]Figure 18 shows the lung mRNA expression levels of Muc5ac, Agr2, and Spdef, as determined by qRT-PCR. The mRNA expression levels of these two genes were induced by HDM challenge compared with PBS challenge in mice. IL-4Rα and IL-5 antibodies alone did not significantly reverse this increase in Muc5ac or Agr2 mRNA levels. However, the combination of both monospecific IL-4Rα and IL-5 monoclonal antibodies, and the IL-4Rα / IL-5 bispecific antibody, significantly reduced the HDM-mediated increase in Muc5ac or Agr2 mRNA levels. P values ​​reflect one-way ANOVA tests; ns: not significant; *P≦0.05; **P≦0.01; ****P≦0.0001 relative to an irrelevant IgG2 antibody.

[0044] [Figure 19] Figure 19 shows BHR measured using flexiVent (SCIREQ, Inc., ®) after exposure to increasing methacholine doses. Data represent two independent experiments with n = 6 mice per group. Bronchial hyperresponsiveness was significantly reduced after treatment with the IL-4Rα / IL-5 monospecific antibody combination and after treatment with the IL-4Rα / IL-5 bispecific antibody compared to treatment with a control IgG2a antibody. Resistance levels after treatment with the monospecific antibody combination or the bispecific antibody returned to those observed in unchallenged mice that received PBS alone. Results are shown as mean ± SEM. P values ​​reflect one-way ANOVA tests; ns: not significant, *P < 0.05 vs. an irrelevant IgG2 antibody.

[0045] [Figure 20]Figure 20 shows the structure of an IL-4Rα / IL-5 bispecific antibody having an IL-4Rα IgG linked to two IL-5scFv fragments. The Fab arm of the IL-4Rα IgG has the VH and VL domain sequences of antibody 36B7 (see SEQ ID NOs: 45 and 46, respectively). The VH and VL domains of the IL-5scFv fragment are derived from antibody 95G7 and have the sequences set forth in SEQ ID NOs: 76 and 79, respectively.

[0046] [Figure 21] Figure 21 shows the neutralizing activity of IL-4Rα / IL-5 bispecific antibodies, assessed in an in vitro cell assay of IL-5-induced proliferation of TF-1 cells. The bispecific antibodies in Figure 20 were tested together with one IL-4Rα monoclonal antibody (36B7) and two IL-5 monoclonal antibodies (95G7hIgG1 and 95A7mIgG2a).

[0047] [Figure 22] Figure 22 is a diagrammatic representation of the experimental setup using the house dust mite (HDM) mouse model as an in vivo mouse model of asthma. IL-4Rα and IL-5 antibody treatment was administered by injection into HDM-treated C57BL / 6J mice only during the challenge phase.

[0048] [Figure 23] Figure 23 shows differential cell counts in the BAL of HDM-treated mice administered the IL-4Rα / IL-5 monospecific antibody combination, the IL-4Rα / IL-5 bispecific antibody (Bs 4Rsc5), or an irrelevant IgG2a antibody, as analyzed by FACS. HDM challenge in sensitized mice increased the number of eosinophils and lymphocytes in the BAL fluid. This increase in cell counts was significantly reduced after infusion in mice administered the combination of both monospecific IL-4Rα and IL-5 antibodies and in mice administered the IL-4Rα / IL-5 bispecific antibody. P values ​​reflect one-way ANOVA tests; ns: not significant, ***P≦0.001, ****P≦0.0001 relative to the irrelevant IgG2a antibody.

[0049] [Figure 24] Figure 24 shows the lung mRNA expression levels of Muc5ac, Agr2, and Spdef as determined by qRT-PCR. The mRNA expression levels of these two genes were induced by HDM challenge compared to PBS challenge in mice. DETAILED DESCRIPTION OF THE INVENTION

[0050] (Detailed description) (A.Definition) Unless otherwise defined herein, technical and scientific terms used herein shall have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0051] "Combination Therapy"—As used herein, the term "combination therapy" refers to a treatment in which a subject, e.g., a human subject, is given two or more therapeutic agents. The "combination" of the present invention is intended to be used as a "combination therapy." Two or more therapeutic agents are typically administered to treat a single disease, herein a chronic airway disease. The combination or combination therapy of the present invention combines antagonists that target multiple type 2 cytokine signaling pathways. In particular, the combination therapy described herein targets cytokines: cytokine receptors: IL-5:IL-5R; IL-4:IL-4R; and IL-13:IL-13R. These cytokine-cytokine receptors are described in further detail herein. In a preferred embodiment, the antagonist is an antibody molecule that specifically binds to the respective cytokine or cytokine receptor target. As described herein, the antagonists included in the combination therapy may be co-formulated or provided separately, e.g., as separate compositions, for administration to a subject or patient in need thereof. In embodiments of the combination comprising an antibody molecule that binds IL-4Rα and an antibody molecule that binds IL-5, the antibody molecules of the combination may be comprised within a single antibody, e.g., in a multispecific antibody format such as a bispecific antibody.

[0052] "Antagonist" - As used herein, the term "antagonist" refers to any agent or molecule capable of inhibiting the function of its target cytokine or cytokine receptor. As used herein, an IL-5:IL-5R antagonist refers to any agent or molecule capable of inhibiting signal transduction initiated by the binding of IL-5 to its cognate receptor complex, "IL-5R." As used herein, an IL-4:IL-4R antagonist refers to any agent or molecule capable of inhibiting signal transduction initiated by the binding of IL-4 to its cognate type I receptor complex, "IL-4R." As used herein, an IL-13:IL-13R antagonist refers to any agent or molecule capable of inhibiting signal transduction initiated by the binding of IL-13 to its cognate receptor complex, "IL-13R." As explained herein, the receptor complexes to which the type 2 cytokines IL-5; IL-4, and IL-13 bind are typically composed of two receptor subunits. For example, an IL-5:IL-5R antagonist would inhibit the association of IL-5 with its receptor complex or inhibit the association between the two subunits of the IL-5R complex (IL-5Rα and βc), thereby inhibiting or blocking IL-5-mediated signaling. Similarly, an IL-4:IL-4R antagonist or an IL-13:IL-13R antagonist would inhibit the association of a cytokine (IL-4 or IL-13) with its receptor complex or inhibit the association between the two subunits of the IL-4R or IL-13R complex, thereby inhibiting or blocking IL-4- or IL-13-mediated signaling. As explained herein, the IL-4R and IL-13R complexes share a common receptor subunit, IL-4Rα. Furthermore, the cytokine IL-4 can signal not only through its own type I receptor complex, "IL-4R," but also through the IL-13R complex. Thus, an antagonist of IL-4Rα can interfere with both the IL-4R and IL-13R complexes and inhibit signaling through both IL-4 and IL-13. Furthermore, in some cases, an antagonist of the IL-13R complex may also inhibit IL-4-mediated signaling.

[0053] The IL-5:IL-5R, IL-4:IL-4R, and IL-13:IL-13R antagonists used in the combinations of the present invention may take the form of any suitable drug or molecule. In certain embodiments, the antagonist may downregulate the expression of the target, e.g., IL-4Rα or IL-5 expression, thereby inhibiting the function of that target. In other embodiments, the antagonist may directly bind to the cytokine or receptor subunit to inhibit the function of that target. As described herein, an IL-5:IL-5R antagonist may bind to the cytokine IL-5 (referred to as an IL-5 antagonist) or one of the IL-5R subunits (referred to as an IL-5Rα antagonist or βc antagonist). Similarly, an antagonist of IL-4:IL-4R may bind to the cytokine IL-4 (referred to as an IL-4 antagonist) or to one of the type I IL-4R subunits (referred to as an IL-4Rα antagonist or γc antagonist).

[0054] In preferred embodiments, the antagonist is specific for its target. For example, an antagonist of IL-4Rα (or IL-4Rα antagonist) would preferentially inhibit the function of IL-4Rα compared to other molecular targets. An IL-5 antagonist would preferentially inhibit the function of IL-5 compared to other molecular targets. Antagonists will usually achieve the desired level of specificity by directly interacting with their targets, e.g., by selectively binding to IL-4Rα or IL-5 mRNA or protein. Suitable agents or molecules that can act as antagonists include, but are not limited to, inhibitory RNA species, e.g., siRNA or shRNA, small molecule inhibitors, and biological antagonists. In a preferred embodiment, the antagonist of the combination is an antibody molecule.

[0055] "Antibody Molecule"—As used herein, the term "antibody molecule" encompasses full-length antibodies and antigen-binding fragments thereof, including variants such as engineered antibodies, humanized antibodies, germline antibodies, and antigen-binding fragments thereof. The term "antibody" generally refers to a heterotetrameric immunoglobulin polypeptide having a combination of two heavy chains and two light chains, which polypeptide has significant specific immunoreactivity to an antigen of interest (e.g., IL-4Rα or IL-5). As an IgG class antibody, an antibody contains two identical light polypeptide chains with a molecular weight of approximately 23,000 daltons and two identical heavy chains with a molecular weight of 53,000 to 70,000. These four chains are connected by disulfide bonds to form a "Y" configuration, with the light chains bracketing the heavy chains, which begin at the mouth of the "Y" and continue through the variable region. Antibody light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can associate with either kappa or lambda light chains. Generally, light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or noncovalently when immunoglobulins are produced in hybridomas, B cells, or genetically engineered host cells. Within the heavy chain, the amino acid sequence runs from the N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0056] Those skilled in the art will recognize that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within each (e.g., γ1-γ4). It is the properties of these chains that determine the "class" of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and are known to confer functional properties. As used herein, the term "antibody molecule" includes full-length antibodies or antigen-binding fragments thereof from any class or subclass of antibody.

[0057] The term "antibody molecule" as used herein is also intended to encompass "heavy chain-only antibodies" or "VHH antibodies." The term "heavy chain-only antibodies" or "VHH antibodies" refers to a type of antibody produced exclusively by species of the Camelidae family, which includes camels, llamas, and alpacas. Heavy chain-only antibodies are composed of two heavy chains and lack light chains. Each heavy chain has a variable domain at its N-terminus, and these variable domains are referred to as "VHH" domains to distinguish them from the variable domains of the heavy chains of conventional heterotetrameric antibodies, i.e., the VH domains.

[0058] With respect to antigen-binding fragments encompassed by the general term "antibody molecule," these fragments are full-length antibodies or portions or portions of antibody chains that retain antigen-binding activity while containing fewer amino acid residues than an intact or complete antibody. As used herein, the term "antibody molecule" is intended to encompass antigen-binding fragments selected from an antibody light chain variable domain (VL); an antibody heavy chain variable domain (VH); a single-chain antibody (scFv); an F(ab')2 fragment; an Fab fragment; an Fd fragment; an Fv fragment; a single-arm (monovalent) antibody; a diabody, a triabody, a tetrabody, or any antigen-binding molecule formed by the combination, assembly, or conjugation of antigen-binding fragments thereof. As used herein, the term "antibody molecule" is further intended to encompass antibody fragments selected from the group consisting of unibodies; domain antibodies; and nanobodies. Fragments can be obtained, for example, via chemical or enzymatic treatment of intact or complete antibodies or antibody chains, or by recombinant techniques.

[0059] "Variable region" or "variable domain" - The terms "variable region" and "variable domain" are used interchangeably herein and are intended to have equivalent meanings. The term "variable" refers to the fact that certain portions of the variable domains, VH and VL, differ extensively in sequence among antibodies, which is responsible for the particular binding and specificity of each antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of an antibody. It is concentrated in three segments called "hypervariable loops" in each of the VL and VH domains, which form part of the antigen-binding site. The first, second, and third hypervariable loops of the V lambda light chain domain are referred to herein as L1(λ), ​​L2(λ), and L3(λ), and may be defined within the VL domain as comprising residues 24-33 (L1(λ) consisting of 9, 10, or 11 amino acid residues), 49-53 (L2(λ) consisting of 3 residues), and 90-96 (L3(λ) consisting of 5 residues) (Morea et al., Methods 20:267-279 (2000)). The first, second, and third hypervariable loops of the V kappa light chain domain are referred to herein as L1(κ), L2(κ), and L3(κ), and may be defined within the VL domain as comprising residues 25-33 (6, 7, 8, 11, 12, or 13 residues of L1(κ)), 49-53 (3 residues of L2(κ)), and 90-97 (6 residues of L3(κ)) (Morea et al., Methods 20:267-279 (2000)). The first, second, and third hypervariable loops of the VH domain are referred to herein as H1, H2, and H3, and may be defined within the VH domain as comprising residues 25-33 (H1 consisting of 7, 8, or 9 residues), 52-56 (H2 consisting of 3 or 4 residues), and 91-105 (H3 of highly variable length) (Morea et al., Methods 20:267-279 (2000)).

[0060] Unless otherwise specified, the terms L1, L2, and L3 refer to the first, second, and third hypervariable loops, respectively, of the VL domain and include hypervariable loops from both Vkappa and Vlambda isotypes. The terms H1, H2, and H3 refer to the first, second, and third hypervariable loops, respectively, of the VH domain and include hypervariable loops from any known heavy chain isotype, including gamma, epsilon, delta, alpha, or mu.

[0061] The hypervariable loops L1, L2, L3, H1, H2, and H3 may each comprise a portion of a "complementarity-determining region" or "CDR," as defined below. The terms "hypervariable loop" and "complementarity-determining region" are not strictly synonymous, because hypervariable loops (HV) are defined based on structure, whereas complementarity-determining regions (CDRs) are defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1983), and the limits of HV and CDR may differ in some VH and VL domains.

[0062] The CDRs of the VL and VH domains can generally be defined as comprising the following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or 31-35b (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable domain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Thus, HVs may be contained within the corresponding CDRs, and references herein to the "hypervariable loops" of the VH and VL domains should be interpreted as encompassing the corresponding CDRs, and vice versa, unless otherwise specified.

[0063] The more highly conserved portions of variable domains are called framework regions (FRs), defined below. Native heavy and light chain variable domains each contain four FRs (FR1, FR2, FR3, and FR4, respectively), which primarily adopt a β-sheet configuration connected by three hypervariable loops. The hypervariable loops of each chain are held together in close proximity by the FRs and, together with the hypervariable loops of the remaining chains, contribute to the formation of the antigen-binding site of antibodies. Structural analysis of antibodies has revealed relationships between the sequence and shape of the binding site formed by the complementarity-determining regions (Chothia et al., J. Mol. Biol. 227:799-817 (1992)); Tramontano et al., J. Mol. Biol. 215:175-182 (1990)). Despite their high sequence variability, five of the six loops adopt only a small repertoire of main-chain conformations, called "canonical structures." These conformations are determined firstly by the loop length and secondly by the presence of key residues at specific positions within the loops and framework regions that determine the conformation by their packing, hydrogen bonding or ability to assume unusual main-chain conformations.

[0064] "CDR" - As used herein, the term "CDR" or "complementarity-determining region" refers to the discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specialized regions are described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), as well as Chothia et al., J. Mol. Biol. 196:901-917 (1987) and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and these definitions, when compared with each other, contain overlapping or subsets of amino acid residues. The amino acid residues encompassing the CDRs defined by each of the above references are provided for comparison. Preferably, the term "CDR" refers to the CDR defined by Kabat based on sequence comparisons.

[0065] Table 1: CDR definition [Table 1] 1: Residue numbers follow the nomenclature of Kabat et al. 2: Residue numbers follow the nomenclature of Chothia et al. 3: Residue numbers follow the nomenclature of MacCallum et al.

[0066] "Framework Region"—As used herein, the term "framework region" or "FR region" includes amino acid residues that are part of the variable region but not part of the CDRs (e.g., using the CDR definition of Kabat). Thus, the variable region framework is between about 100-120 amino acids in length and includes only amino acids outside the CDRs. For the specific example of a heavy chain variable domain, and the CDRs defined by Kabat et al., framework region 1 corresponds to the variable region domain encompassing amino acids 1-30; framework region 2 corresponds to the variable region domain encompassing amino acids 36-49; framework region 3 corresponds to the variable region domain encompassing amino acids 66-94; and framework region 4 corresponds to the variable region domain from amino acid 103 to the end of the variable region. Light chain framework regions are similarly separated by their respective light chain variable region CDRs. Similarly, using the CDR definitions of Chothia et al. or McCallum et al., framework region boundaries are separated by their respective CDR ends, as described above. In a preferred embodiment, the CDRs are as defined by Kabat.

[0067] In natural antibodies, the six CDRs present on each monomeric antibody are short, discontinuous sequences of amino acids that are specifically arranged to form an antigen-binding site when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining portions of the heavy and light chain variable domains, which exhibit less intermolecular variability in amino acid sequence, are called framework regions. The framework regions primarily adopt a β-sheet conformation, and the CDRs form loops that connect and, in some cases, form part of the β-sheet structure. Thus, these framework regions act as a scaffold for orienting the six CDRs through interchain noncovalent interactions. The antigen-binding site formed by the arranged CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes noncovalent binding of the antibody to the immunoreactive antigen epitope. The arrangement of the CDRs can be easily identified by those skilled in the art.

[0068] "Constant Region" - As used herein, the term "constant region" refers to the portion of an antibody molecule outside the variable domain or regions. Immunoglobulin light chains typically have a single domain, the "constant region," designated the "CL or CL1 domain." This domain is C-terminal to the VL domain. Immunoglobulin heavy chains differ in their constant regions depending on the immunoglobulin class (gamma, mu, alpha, delta, epsilon). Heavy chains gamma, alpha, and delta have a constant region composed of three immunoglobulin domains (designated CH1, CH2, CH3) with a flexible hinge region separating the CH1 and CH2 domains. Heavy chains mu and epsilon have a constant region composed of four domains (CH1-CH4). The heavy chain constant domain is located C-terminal to the VH domain.

[0069] The numbering of amino acids in heavy and light immunoglobulin chains runs from the N-terminus at the branched ends of the Y configuration to the C-terminus at the bottom of each chain. Different numbering schemes are used to define the constant domains of immunoglobulin heavy and light chains. According to the EU numbering scheme, the heavy chain constant domain of an IgG molecule is identified as follows: CH1 - amino acid residues 118-215; CH2 - amino acid residues 231-340; CH3 - amino acid residues 341-446. According to the Kabat numbering scheme, the heavy chain constant domain of an IgG molecule is identified as follows: CH1 - amino acid residues 114-223; CH2 - amino acid residues 244-360; CH3 - amino acid residues 361-477. The term "Fc domain" or "Fc region" generally identifies the constant region portion of the heavy chain, including the CH2 and CH3 domains. The Fc region may also include some residues from the hinge region. The term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. The term "hinge region" refers to the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux KH et al., J. Immunol. 161:4083-90 1998). Antibodies of the present invention that contain a "fully human" hinge region may contain one of the hinge region sequences shown in Table 2 below.

[0070] Table 2: Human hinge sequences [Table 2]

[0071] "Specificity" and "Multispecific Antibodies"—Antibody molecules for use in the combination therapies described herein bind to specific target antigens. Preferably, antibody molecules "specifically bind" to their target antigens, where the term "specifically binds" refers to the ability of any antibody molecule to preferentially immunoreact with a given target, e.g., IL-4Rα and IL-5. The antibody molecules of the present combinations and methods may be monospecific and contain one or more binding sites that specifically bind to a particular target. The antibody molecules of the present combinations and methods may also be incorporated into a "multispecific antibody" format, e.g., a bispecific antibody, which binds to two or more target antigens. For example, in one embodiment, a combination of the present invention comprises a bispecific antibody comprising a first antibody molecule that specifically binds to IL-4Rα and a second antibody molecule that specifically binds to IL-5. To achieve multiple specificities, "multispecific antibodies" are typically engineered to contain different combinations or pairs of heavy and light chain polypeptides with different VH-VL pairs. Multispecific, and particularly bispecific, antibodies may be engineered to adopt the overall conformation of a native antibody, e.g., a Y-shaped antibody with Fab arms of different specificities conjugated to the Fc region. Other multispecific, e.g., bispecific, antibodies may be engineered to adopt non-native conformations, e.g., in which variable domains or variable domain pairs with different specificities are positioned at opposite ends of the Fc region.

[0072] "Engineered Antibodies"—As used herein, the term "engineered antibodies" refers to synthetic forms of antibodies that are altered so as not to occur in nature, such as antibodies that contain at least two heavy chain portions but not two complete heavy chains (such as domain-deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that have been engineered to bind to two or more different antigens or different epitopes on a single antigen; and heavy chain molecules connected to scFv molecules. ScFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. Furthermore, the term "engineered antibodies" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three or more copies of the same antigen). In another embodiment, an engineered antibody of the invention is a fusion protein that contains at least one heavy chain portion lacking a CH2 domain and includes a binding domain of a polypeptide that includes the binding portion of a member of a receptor-ligand pair.

[0073] "Humanizing substitution" - As used herein, the term "humanizing substitution" refers to an amino acid substitution in which an amino acid residue present at a particular position in a VH or VL domain of an antibody is replaced with an amino acid residue occurring at the equivalent position in a reference human VH or VL domain. The reference human VH or VL domain may be a VH or VL domain encoded by human germline. Humanizing substitutions may be made in the framework regions and / or CDRs of an antibody, as defined herein.

[0074] "Humanized variant" - As used herein, the term "humanized variant" or "humanized antibody" refers to a variant antibody that comprises one or more "humanizing substitutions" compared to a reference antibody, wherein portions of the reference antibody (e.g., the VH domain and / or VL domain or a portion thereof comprising at least one CDR) have amino acids derived from a non-human species, and the "humanizing substitutions" occur within the amino acid sequence derived from the non-human species.

[0075] "Germline variant"—The term "germline variant" or "germline antibody" is used herein specifically to refer to a "humanized variant" in which "humanizing substitutions" result in one or more amino acid residues present at a particular position(s) in an antibody VH or VL domain being replaced with amino acid residues occurring at the equivalent position(s) in a reference human VH or VL domain encoded by the human germline. For all "germline variants," the substituted amino acid residues in the germline variant are typically taken exclusively or primarily from a single human germline-encoded VH or VL domain. The terms "humanized variant" and "germline variant" are often used interchangeably. Introducing one or more "humanizing substitutions" into a camelid-derived (e.g., llama-derived) VH or VL domain results in a "humanized variant" of the camelid (llama)-derived VH or VL domain. The result may be a "human germline variant" of a camelid (llama)-derived VH or VL domain if the substituted amino acid residues therein are derived primarily or exclusively from a single human germline-encoded VH or VL domain sequence.

[0076] "Affinity variant" - As used herein, the term "affinity variant" refers to a variant antibody that exhibits one or more changes in its amino acid sequence compared to a reference antibody, such that the affinity variant exhibits altered affinity for a target antigen compared to the reference antibody. For example, the affinity variant will exhibit altered affinity for a target, e.g., IL-4Rα or IL-5, compared to a reference IL-4Rα or IL-5 antibody. Preferably, the affinity variant will exhibit improved affinity for the target antigen compared to the reference antibody. Affinity variants typically exhibit one or more changes in the amino acid sequence of a CDR compared to the reference antibody. Such substitutions may be natural or non-natural amino acid residues, replacing the original amino acid present at a given position in the CDR with a different amino acid residue. The amino acid substitutions may be conservative or non-conservative.

[0077] "IL-5:IL-5R" - As used herein, the term "IL-5" refers to the interleukin-5 cytokine, and the term "IL-5R" refers to the receptor complex to which the interleukin-5 cytokine binds. The term "IL-5:IL-5R" is used herein to refer to the IL-5 cytokine / cytokine receptor signaling complex. The cytokine IL-5 is also known as B-cell differentiation factor I, eosinophil differentiation factor, and T-cell replacement factor (TRF). The human homolog of IL-5 is 134 amino acids long (http: / / www.uniprot.org / uniprot / P05113). As used herein, the term "IL-5" is intended to encompass all splice variants of the protein. Monomeric IL-5 is inactive and requires a homodimer for function. One IL-5 homodimer binds to one IL-5 receptor (IL-5R). The IL-5 receptor is composed of two subunits. The first subunit is the "IL-5 receptor subunit alpha" or "IL-5Rα," also known as IL-5R-alpha, IL-5RA, CDw125, and CD antigen, CD125; this subunit forms the ligand-binding portion of the receptor complex. The human homolog of IL-5Rα is 420 amino acids long (http: / / www.uniprot.org / uniprot / Q01344). The second subunit of the IL-5R complex is the nonligand-binding common signal-transducing beta subunit or beta chain (βc). IL-5 is secreted by a limited number of mesenchymal cell types. Cells known to express IL-5 include eosinophils, NK cells, TC2CD8+ T cells, mast cells, CD45+CD4+ T cells, gamma delta T cells, and IL-1beta-activated endothelial cells. IL-5 is known to regulate the expression of genes involved in proliferation, cell survival, and maturation of B cells and eosinophils, as well as effector functions.

[0078] "IL-4:IL-4R" - As used herein, the term "IL-4" refers to the interleukin-4 cytokine, and the term "IL-4R" refers to the type I receptor complex to which the interleukin-4 cytokine binds. The term "IL-4:IL-4R" is used to refer to the IL-4 cytokine / type I cytokine receptor signaling complex. The cytokine IL-4 is also known as B cell stimulatory factor 1 (BSF-1), binetrakin, and lymphocyte stimulatory factor 1. The human homolog of IL-4 is 153 amino acids long (http: / / www.uniprot.org / uniprot / P05112). As used herein, the term "IL-4" is intended to encompass all splice variants of the protein. The type I receptor for IL-4 is composed of two subunits. The first subunit is also known as "IL-4Rα," interleukin-4 receptor subunit alpha, interleukin-4 binding subunit, and CD124. IL-4Rα is a 140-kDa transmembrane glycoprotein widely expressed within the class I cytokine receptor family. The human homolog of IL-4Rα is 825 amino acids long (http: / / www.uniprot.org / uniprot / P24394). As used herein, the term "IL-4Rα" is intended to encompass all splice variants of the protein. Within the type I IL-4 receptor complex, IL-4Rα associates with a second subunit, the common gamma chain (γc), which enhances the affinity of IL-4Rα for IL-4 and influences downstream IL-4 signaling. The IL-4 receptor complex is present, for example, in B cells, T cells, monocytes, eosinophils, and fibroblasts, and IL-4Rα couples to the JAK1 / 2 / 3-STAT6 pathway. IL-4 responses are involved in promoting Th2 differentiation and regulating IgE production at sites of allergic inflammation, as well as chemokine and mucus production.

[0079] "IL-13:IL-13R" - As used herein, the term "IL-13" refers to the interleukin-13 cytokine, and the term "IL-13R" refers to the receptor complex to which the interleukin-13 cytokine binds. The IL-13 receptor also acts as a type II receptor for IL-4. The term "IL-13:IL-13R" is used to refer to the IL-13 cytokine / cytokine receptor signaling complex. The human homolog of IL-13 is 146 amino acids long (http: / / www.uniprot.org / uniprot / P35225). As used herein, the term "IL-13" is intended to encompass all splice variants of the protein. The receptor for IL-13 is composed of two subunits. The first subunit is "IL-4Rα," also known as interleukin-4 receptor subunit alpha, interleukin-4 binding subunit, and CD124. As previously described, this subunit is common to both the IL-4 receptor and the IL-13 receptor. Within the IL-13 receptor complex, IL-4Rα associates with a second subunit, interleukin-13 receptor subunit alpha 1, or "IL-13Ralpha1," or "IL-13Rα1," or "IL-13RA1." This second subunit acts as a ligand-binding subunit for IL-13. The IL-13R complex, composed of the IL-4Rα and IL-13Rα1 subunits, responds to IL-13 (through binding to IL-13Rα1) and IL-4 (through binding to IL-4Rα), and cytokines that bind to this receptor complex initiate signaling via the JAK1 / 2 / 3-STAT6 pathway. Similar to the IL-4:IL-4R complex, signaling through the IL-13R complex, which is composed of the IL-4Rα and IL-13Rα1 subunits, is involved in regulating IgE production at sites of allergic inflammation, as well as chemokine and mucus production. Additional information regarding the IL-4 and IL-13 cytokine-receptor signaling pathways can be found, for example, in McCormick and Heller (2015) Cytokine 75(1):38-50, the contents of which are incorporated herein in their entirety.

[0080] "Chronic Airway Disease" - As used herein, the term "chronic airway disease," sometimes used interchangeably with "chronic respiratory disease (CRD)," is intended to mean any disease of the airways and other structures of the lungs. Some of the most common forms of chronic airway disease are chronic obstructive pulmonary disease (COPD), asthma, occupational lung disease, and pulmonary hypertension. In addition to tobacco smoke, other risk factors include air pollution, occupational chemicals and dust, and frequent lower respiratory tract infections during childhood. Other chronic airway diseases include, but are not limited to, chronic rhinosinusitis (CRS); immunoglobulin G4-related disease (IgG4-RD); chronic bronchitis; emphysema; chronic angioedema; diseases characterized by goblet cell metaplasia such as Barrett's esophagus; active eosinophilic esophagitis; nasal polyposis; chronic sinusitis; Churg-Strauss syndrome; allergic bronchopulmonary aspergillosis (ABPA); hypereosinophilic syndrome; bullous pemphigoid; and cystic fibrosis.

[0081] "Asthma"—As used herein, "asthma" is intended to mean any disease or condition associated with inflammation of the airways in the lungs. Normally, inflammation affects the sensitivity of nerve endings in the airways, making them easily irritated. During an asthma attack, the lining of the airways swells, narrowing the airways and reducing airflow in and out of the lungs. Asthma is a heterogeneous disease with multiple phenotypes, each exhibiting distinct clinical, physiological, and molecular characteristics. Exemplary asthma subtypes include severe asthma, severe refractory asthma, mild or moderate asthma, obesity-related asthma, exercise-induced asthma, aspirin-induced asthma, atopic or allergic asthma, eosinophilic asthma, neutrophilic asthma, paucigranulocytic or non-inflammatory asthma, early-onset asthma, late-onset asthma, type II high asthma, type II low asthma, and type I / Th17 asthma.

[0082] B. Combination Therapy for Inhibiting Type 2 Cytokine Signaling The present invention relates to combinations or combination therapies and their use in the treatment of chronic airway diseases, particularly in the treatment of asthma. The combinations of the present invention relate to antagonists that target type 2 cytokines and / or their respective receptors. The cytokines and cytokine receptors targeted by the combinations of the present invention include IL-5 and its receptor IL-5R; IL-4 and its type 1 receptor IL-4R; and IL-13 and its receptor IL-13R.

[0083] In a first aspect, the present invention provides a combination comprising (i) an IL-5:IL-5R antagonist and (ii) an IL-4:IL-4R antagonist and / or an IL-13:IL-13R antagonist. In one embodiment, the combination comprises an IL-5:IL-5R antagonist and an IL-4:IL-4R antagonist. In a further embodiment, the combination comprises an IL-5:IL-5R antagonist and an IL-13:IL-13R antagonist. In an even further embodiment, the combination comprises an IL-5:IL-5R antagonist, an IL-4:IL-4R antagonist, and an IL-13:IL-13R antagonist. In one embodiment, the combination inhibits signaling via IL-5 and IL-4. In one embodiment, the combination inhibits signaling through IL-5 and IL-13. In a preferred embodiment, the combination inhibits signaling through IL-5, IL-4 and IL-13.

[0084] Without being bound by theory, it is believed that the combination of the present invention is particularly effective in treating chronic airway diseases, particularly asthma, due to the combined effect of blocking type 2 cytokine activity. IL-4, IL-13, and IL-5 are type 2 cytokines, and the combination of the present invention can inhibit signaling through all three of these cytokines. A "type 2 immune response" is one that is initiated by T helper (T H 2) Refers to an immune response regulated primarily by a subpopulation of CD4+ T cells known as CD4+ T cells. However, airway type 2 immune responses also include eosinophils, mast cells, basophils, and T HIt is also mediated by group 2 cells, group 2 innate lymphoid cells (ILC2s), and IgE-producing B cells.

[0085] IL-4, IL-13, and IL-5 induce IgE production by B cells, eosinophil activation and recruitment, and mucus production. Type 2 cytokines drive a cascade of downstream events, including airway epithelial cell activation, chemoattraction of effector cells (mast cells, eosinophils, and basophils), and remodeling of the epithelial and subepithelial matrix. In chronic airway diseases, such as asthma, aberrant signaling through IL-4, IL-13, and IL-5 can lead to airway eosinophilia, airway remodeling, and bronchial hyperresponsiveness (see Lambrecht and Hammad (2015) ibid.). Associated remodeling changes may include smooth muscle cell changes (hyperplasia and hypertrophy), mucus cell changes (goblet cell metaplasia), and ductal remodeling. Together, these inflammatory and pathological changes in the airways increase the likelihood that subjects will exhibit an exacerbated response to inhaled exacerbating agents.

[0086] The present inventors have demonstrated that a surprising synergistic effect is observed by blocking signaling through IL-4, IL-13, and IL-5. This effect can be confirmed when compared with treatment with IL-4Rα monotherapy, which blocks only IL-4 and IL-13, or IL-5 monotherapy, which blocks only IL-5. The data provided herein demonstrate that the combination therapy of the present invention, which includes an antagonist targeting the IL-5:IL-5R signaling axis and an antagonist targeting the IL-4:IL-4R and / or IL-13:IL-13R signaling axis, produces a synergistic effect. This effect can be confirmed by reduced levels of mucin production (an indicator of goblet cell metaplasia) and reduced levels of bronchial hyperresponsiveness in an in vivo model of chronic airway disease. Importantly, the combination therapy of the present invention, which includes an antagonist targeting type 2 cytokines, has been shown to completely prevent bronchial hyperresponsiveness in an in vivo model.

[0087] The combinations of the present invention comprise antagonists that target (i) IL-5:IL-5R; and (ii) IL-4:IL-4R and / or IL-13:IL-13R. As used herein, the term "antagonist" means: The term "antibody" is used broadly to refer to any agent or molecule capable of inhibiting the function of its target. For example, an IL-5:IL-5R antagonist would inhibit IL-5-mediated signaling, thereby inhibiting the function of the IL-5 cytokine-IL-5 receptor complex. Similarly, an IL-4:IL-4R antagonist would inhibit IL-4-mediated signaling, thereby inhibiting the function of the IL-4 cytokine-IL-4 receptor complex. Similarly, an IL-13:IL-13R antagonist would inhibit IL-13-mediated signaling, thereby inhibiting the function of the IL-13 cytokine-IL-13 receptor complex.

[0088] Antagonists of type 2 cytokine-receptor pairs described herein, i.e., antagonists of IL-5:IL-5R, IL-4:IL-4R, and IL-13:IL-13R, typically target or interact with a component of the cytokine-receptor complex. For example, an antagonist of IL-5:IL-5R may interact with IL-5 and inhibit signaling through the IL-5:IL-5R signaling pathway. Alternatively, an antagonist of IL-5:IL-5R may interact with the receptor subunit IL-5Rα and inhibit signaling through the IL-5:IL-5R signaling pathway. In a preferred embodiment, the antagonist of IL-5:IL-5R is an antagonist of IL-5. Similarly, IL-4:IL-4R antagonists and IL-13:IL-13R antagonists may interact with the cytokines IL-4 and IL-13, respectively, or with the receptor subunits to inhibit IL-4 and / or IL-13 signaling, respectively.

[0089] Particularly preferred is a combination of the present invention that includes an antagonist of the receptor subunit IL-4Rα. Even more preferably, the combination includes an antagonist of IL-5 and an antagonist of IL-4Rα. Targeting IL-4Rα is preferred because this receptor subunit forms part of the type I receptor complex for IL-4 (together with γc) and, similarly, part of the type I receptor complex for IL-13 (together with IL-13Rα1). Thus, an antagonist of IL-4Rα can simultaneously act as an antagonist of IL-4:IL-4R and IL-13:IL-13R, thereby inhibiting signaling mediated by both IL-4 and IL-13. In particular, a combination comprising an antagonist of IL-5:IL-5R, preferably an antagonist of IL-5, and an antagonist of IL-4Rα can inhibit signaling mediated by IL-5, IL-4, and IL-13. The antagonists of the combinations described herein preferably bind to their respective human targets.

[0090] In a preferred embodiment, the antagonists of IL-5:IL-5R, IL-4:IL-4R and / or IL-13:IL-13R are antibody molecules. More preferably, the combination comprises an antibody molecule that binds to IL-5 and an antibody molecule that binds to IL-4Rα.

[0091] In one embodiment, the combination comprises an antibody molecule that binds to IL-5 as the IL-5:IL-5R antagonist. Alternatively, the combination may comprise an antibody molecule that binds to IL-5Rα as the IL-5:IL-5R antagonist. IL-5 and IL-5R antibody molecules that can be incorporated into the combinations described herein include any suitable IL-5 and IL-5Rα antibodies known in the art. Exemplary antibodies include mepolizumab (Nucala, ®) and reslizumab (Cinquair, ®), which bind to IL-5, and benralizumab (Fasenra, ®), which binds to IL-5Rα.

[0092] In one embodiment, the combination comprises an antibody molecule that binds to IL-4 as an IL-4:IL-4R antagonist. In one embodiment, the combination comprises an antibody molecule that binds to IL-13 as an IL-13:IL-13R antagonist. In a preferred embodiment, the combination comprises an antibody molecule that binds to IL-4Rα. As previously described, this antagonist acts as an antagonist of both IL-4:IL-4R and IL-13:IL-13R because the IL-4Rα subunit is common to both IL-4 and IL-13 receptor complexes. Antibody molecules targeting the IL-4 and IL-13 signaling pathways that can be incorporated into the combinations described herein include any suitable IL-4, IL-13, IL-4Rα, and IL-13Rα1 antibodies known in the art. These antibodies include dupilumab (Dupixent, Inc.®), a fully humanized monoclonal antibody directed against the IL-4Rα receptor. See also Sheridan C. (2018) Nat. Biotechnol. 36(1): 3-5.

[0093] The antibody molecules of the combination, e.g., the antibody molecule that binds IL-4Rα and the antibody molecule that binds IL-5, may be selected from any suitable antibody molecules that are immunoreactive with their respective targets. As noted above, the term "antibody molecule" is used herein to refer to full-length antibodies as well as antigen-binding fragments thereof.

[0094] Because the antibodies in the combinations described herein are intended for human therapeutic use, they are usually of the IgA, IgD, IgE, IgG, or IgM type, and often of the IgG type, which can belong to any of the four subclasses: IgG1, IgG2a and IgG2b, IgG3, or IgG4. In a preferred embodiment, the antibody molecules in the combination are IgG antibodies, optionally IgG1 antibodies. The antibodies may be monoclonal, polyclonal, or multispecific (e.g., bispecific) antibodies, provided they exhibit appropriate immunospecificity for their targets. Monoclonal antibodies are preferred because they are highly specific and directed against a single antigenic site.

[0095] The antigen-binding fragments of the combinations described herein can typically comprise a portion of a full-length antibody, typically the antigen-binding or variable domain thereof. Examples of such antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab' fragments, and Fv fragments, linear antibodies, single-chain antibody molecules, single-chain variable fragments (scFv), and multispecific antibodies formed from antibody fragments (see Holliger and Hudson (2005) Nature Biotechnol. 23:1126-36, incorporated herein by reference).

[0096] The antibody molecules of the combinations described herein may exhibit high human homology. These antibody molecules exhibiting high human homology may include antibodies comprising VH and VL domains of native non-human antibodies that exhibit a sufficiently high percent sequence identity to human germline sequences. In some embodiments, the antibody molecules are humanized or germline variants of the non-human antibody.

[0097] In some embodiments, the antibody molecules of the combinations described herein may be derived from camelids. Camelid-derived antibodies may be heavy chain-only antibodies, i.e., VHH antibodies, or may be conventional heterotetrameric antibodies. In a preferred embodiment, the antibody molecules of the combinations are derived from camelid heterotetrameric antibodies.

[0098] For example, the antibody molecules may be selected from immune libraries obtained by a method comprising immunizing a camelid with a target of interest. The camelid may be immunized with the target protein or a polypeptide fragment thereof, or with an mRNA or cDNA molecule expressing the protein or a polypeptide fragment thereof. Methods for generating antibodies within camelid species and selecting antibodies against preferred targets from camelid immune libraries are described, for example, in International Patent Application No. WO 2010 / 001251, which is incorporated herein by reference.

[0099] In one embodiment, the antibody molecule may be derived from a Camelidae species in that it comprises at least one hypervariable (HV) loop or complementarity determining region derived from a VH or VL domain of a species in the Camelidae family. In particular, the antibody molecule may comprise a VH and / or VL domain, or CDRs thereof, derived from active immunization of an outbred Camelid, such as a llama, with, for example, IL-4Rα and IL-5.

[0100] In this context, the term "derived from" refers to a structural relationship in the sense that the HV or CDR of the antibody molecule embody an amino acid sequence originally encoded by an immunoglobulin gene of a Camelidae (or a minor variant thereof), but this does not necessarily imply a particular relationship with respect to the engineering process used to prepare the antibody molecule.

[0101] The camelid-derived antibody molecule may be derived from any camelid species, including, inter alia, llama, dromedary, alpaca, vicuña, guanaco or camel.

[0102] Antibody molecules comprising VH and VL domains, or their CDRs, from camelids are usually recombinantly expressed polypeptides and may be chimeric polypeptides. The term "chimeric polypeptide" refers to an artificial (non-natural) polypeptide created by the juxtaposition of two or more peptide fragments that are not otherwise contiguous. Included in this definition are "species" chimeric polypeptides created by the juxtaposition of peptide fragments encoded by two or more species, e.g., camelids and humans.

[0103] In one embodiment, the complete VH domain and / or the complete VL domain can be obtained from a species in the family Camelidae. This camelid-derived VH domain and / or camelid-derived VL domain may then be subjected to protein engineering, in which one or more amino acid substitutions, insertions, or deletions are introduced into the camelid amino acid sequence. These engineered changes preferably include amino acid substitutions relative to the camelid sequence. Such changes include "humanization" or "germlining," in which one or more amino acid residues in a camelid-encoded VH or VL domain are replaced with the same residue from a homologous, human-encoded VH or VL domain.

[0104] Isolated camelid VH and VL domains, obtained by active immunization of camelids (e.g., llamas) with, for example, IL-4Rα or IL-5, can be used as the basis for designing antibody molecules for use in the combinations described herein. Starting with intact camelid VH and VL domains, one or more amino acid substitutions, insertions, or deletions can also be designed that deviate from the starting camelid sequence. In one embodiment, these substitutions, insertions, or deletions may be within the framework regions of the VH and / or VL domains.

[0105] In another embodiment, "chimeric" antibody molecules are provided, comprising VH and VL domains (or engineered variants thereof) derived from a camelid and one or more constant domains derived from a non-camelid antibody, e.g., human-encoded constant domains (or engineered variants thereof). In such embodiments, it is preferred that both VH and VL domains are derived from the same species of camelid, e.g., (prior to the introduction of engineered amino acid sequence variations) both VH and VL are derived from a llama, or both VH and VL are derived from an alpaca. In such embodiments, both VH and VL domains may be derived from a single animal, particularly a single animal that has been actively immunized with an antigen of interest.

[0106] Where changes within the primary amino acid sequence of the Camelid VH and / or VL domains are otherwise designed, hypervariable loops or CDRs from individual Camelid species, or combinations thereof, can also be isolated from Camelid VH / VL domains and grafted into another (i.e. non-Camelid) framework, for example a human VH / VL framework, by CDR grafting techniques.

[0107] In a non-limiting embodiment, the antibody molecules of the combination may comprise a CH1 domain and / or a CL domain (from the heavy and light chain, respectively), the amino acid sequences of which may be fully or substantially human. For antibody molecules intended for human therapeutic use, typically the complete antibody constant region, or at least a portion thereof, will have a fully or substantially human amino acid sequence. Thus, one or more, or any combination of, the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and CH4 domain, if present) may be fully or substantially human with respect to their amino acid sequence. The CH1 domain, hinge region, CH2 domain, CH3 domain, and / or CL domain (and / or CH4 domain, if present) may be derived from a human antibody, preferably a human IgG antibody, more preferably a human IgG1 antibody of the subtype IgG1, IgG2, IgG3, or IgG4.

[0108] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and CH4 domain, if present) may all have fully or substantially human amino acid sequences. In the context of the constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially human" refers to at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% amino acid sequence identity with a human constant region. The term "human amino acid sequence" in this context refers to an amino acid sequence encoded by a human immunoglobulin gene, including germline, rearranged, and somatically mutated genes. The present invention also contemplates polypeptides comprising constant domains of "human" sequence, altered by one or more amino acid additions, deletions, or substitutions with respect to the human sequence, except in embodiments where the presence of a "fully human" hinge region is expressly required.

[0109] (Fc region modification) The antibody molecules of the combination may also have one or more amino acid substitutions, insertions, or deletions within the heavy and / or light chain constant regions, particularly within the Fc region. Amino acid substitutions may result in replacement of the substituted amino acid with a different natural amino acid or with a non-natural or modified amino acid. Other structural modifications, such as changes in glycosylation patterns (e.g., by addition or deletion of N- or O-linked glycosylation sites), are also permissible.

[0110] The antibody molecules of the combination may be modified within the Fc region to increase their binding affinity to the fetal receptor FcRn. The increased binding affinity may be measurable at an acidic pH (e.g., approximately pH 5.5 to approximately pH 6.0). The increased binding affinity may also be measurable at a neutral pH (e.g., approximately pH 6.9 to approximately pH 7.4). "Increased binding affinity" refers to increased binding affinity to FcRn relative to an unmodified Fc region. Typically, the unmodified Fc region retains the wild-type amino acid sequence of human IgG1, IgG2, IgG3, or IgG4. In such embodiments, the increased FcRn binding affinity of an antibody molecule having an altered Fc region will be measured relative to the binding affinity to FcRn of wild-type IgG1, IgG2, IgG3, or IgG4.

[0111] In a preferred embodiment, one or more amino acid residues in the Fc region may be substituted with different amino acids to increase binding to FcRn. Several Fc substitutions have been reported that enhance FcRn binding, thereby improving the pharmacokinetics of antibodies. Such substitutions are reported, for example, in Zalevsky et al. (2010) Nat. Biotechnol. 28(2):157-9; Hinton et al. (2006) J. Immunol. 176:346-356; Yeung et al. (2009) J. Immunol. 182:7663-7671; Presta LG. (2008) Curr. Op. Immunol. 20:460-470; and Vaccaro et al. (2005) Nat. Biotechnol. 23(10):1283-88, the contents of which are incorporated herein in their entireties.

[0112] In a preferred embodiment, one or more antibody molecules of a combination described herein comprise a modified human IgG Fc domain comprising or consisting of the amino acid substitutions H433K and N434F, wherein said Fc domain numbering is according to EU numbering. In a further preferred embodiment, one or more antibody molecules of a combination described herein comprise a modified human IgG Fc domain comprising or consisting of the amino acid substitutions M252Y, S254T, T256E, H433K and N434F, wherein said Fc domain numbering is according to EU numbering.

[0113] In one embodiment, the antibody molecules of the combination, e.g., the IL-4Rα and / or IL-5 antibody molecules, comprise an altered human IgG Fc domain consisting of no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 12, no more than 15, or no more than 20 substitutions relative to the corresponding wild-type IgG sequence.

[0114] These antibody molecules may also be modified to form immunoconjugates comprising antibodies conjugated to cytotoxic agents such as chemotherapeutic drugs, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes (i.e., radioconjugates). The Fc region may also be engineered for half-life extension, as described in Chan and Carter (2010) Nature Reviews: Immunology 10:301-316, incorporated herein by reference.

[0115] In particular embodiments, the Fc region can be engineered to have no effector function. In some embodiments, the antibody molecules of the present invention can have an Fc region derived from a native IgG isotype, such as IgG4, with reduced effector function. The IgG4-derived Fc region may be further modified to increase therapeutic utility, for example, by introducing modifications that minimize arm exchange between IgG4 molecules in vivo. The IgG4-derived Fc region may also be engineered to include an S228P substitution.

[0116] In certain embodiments, the antibody molecules of the combination may be modified with respect to glycosylation. For example, aglycoslated antibodies can be generated (i.e., glycosylation-deficient antibodies). Glycosylation can be altered to, for example, increase the affinity of the antibody for a target antigen. Such glycomodifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation will increase the affinity of the antibody for the antigen.

[0117] (pH dependent antibody) The antibody molecules of the combination may exhibit pH-dependent antigen binding.

[0118] Antibody binding to an antigen is internalized by cells and transported to the endosomal-lysosomal degradation pathway. Antibodies that can be separated from their antigens in early endosomes can be recycled back to the cell surface. Antibodies that bind to their antigens with high affinity in the endosomal compartment are typically transported to lysosomes for degradation. It has previously been shown that if an antibody molecule has pH-dependent antigen-binding activity, it will be recycled back to the cell surface more efficiently if it has a lower binding affinity for its antigen at early endosomal pH compared to plasma pH. This extends the plasma half-life of the antibody and enables the same antibody to bind to multiple antigens. Therefore, it is advantageous for the antibody molecules of the combinations described herein, such as IL-4Rα and / or IL-5 antibody molecules, to exhibit pH-dependent antigen binding.

[0119] Methods for engineering pH-dependent antigen binding activity in antibody molecules are described, for example, in European Patent Application No. EP2275443, which is incorporated herein by reference. Methods for engineering pH-dependent antigen binding in antibody molecules are also described in International Patent Application No. WO2018 / 206748, which is incorporated herein by reference. The antibody molecules described herein may be modified to exhibit pH-dependent antigen binding according to the methods described in European Patent Application No. EP2275443 or International Patent Application No. WO2018 / 206748.

[0120] The antigen-binding activity of the pH-dependent antibody molecules in the combination described herein is lower at endosomal pH compared to plasma pH. Endosomal pH is typically acidic, while plasma pH is typically neutral. Thus, the antibody molecules in the combination, such as the IL-4Rα and / or IL-5 antibody molecules described herein, may exhibit pH-dependent antigen binding such that their antigen-binding activity is lower at acidic pH compared to neutral pH. Endosomal pH or "acidic pH" may be about pH 4.0 to about pH 6.5, preferably about pH 5.5 to about pH 6.5, preferably about pH 5.5 to about pH 6.0, and preferably pH 5.5, pH 5.6, pH 5.7, or pH 5.8. Plasma pH or "neutral pH" may be a pH of about pH 6.9 to about pH 8.0, preferably about pH 7.0 to about pH 8.0, preferably about pH 7.0 to about pH 7.4, preferably pH 7.0 or pH 7.4.

[0121] In certain embodiments, the antibody molecules, e.g., IL-4Rα antibody molecules and / or IL-5 antibody molecules, exhibit pH-dependent binding, such that the antigen-binding activity at pH 5.8 is lower than that at pH 7.4. These pH-dependent antibody molecules, e.g., IL-4Rα antibody molecules and / or IL-5 antibody molecules, may be characterized in that the dissociation constant (KD) of the antibody-antigen interaction at acidic pH or pH 5.8 is higher than the dissociation constant (KD) of the antibody-antigen interaction at neutral pH or pH 7.4. In certain embodiments, the antibody molecules, e.g., IL-4Rα antibody molecules and / or IL-5 antibody molecules, exhibit pH-dependent binding, such that the ratio of the KD for the antigen at pH 5.8 to the KD for the antigen at pH 7.4 (KD(pH5.8) / KD(pH7.4)) is 2 or greater, 4 or greater, 6 or greater, 8 or greater, 10 or greater, or 12 or greater.

[0122] The pH-dependent antigen-binding activity of the antibody molecule may be engineered by modifying the antibody molecule to impair its antigen-binding ability at acidic pH and / or increase its antigen-binding ability at neutral pH. For example, the antibody molecule may be modified by substituting at least one amino acid of the antibody molecule with histidine or by inserting at least one histidine into the antibody molecule. The site of such histidine mutation (substitution or insertion) is not particularly limited, and any site is acceptable as long as the antigen-binding activity at endosomal pH (e.g., pH 5.8) is lower than that at plasma pH (e.g., pH 7.4) compared to before the mutation or insertion.

[0123] In some embodiments, the antibody molecules, e.g., IL-4Rα and / or IL-5 antibody molecules, may be engineered to exhibit pH-dependent antigen binding by introducing one or more substitutions into the variable domains. In a preferred embodiment, the antibody molecules, e.g., IL-4Rα and / or IL-5 antibody molecules, may be engineered to exhibit pH-dependent antigen binding by introducing one or more substitutions into the CDRs of the antibody molecule. The substitutions may confer pH-dependent antigen binding by introducing one or more His residues into one or more sites in the variable domains, preferably the heavy and / or light chain CDRs. Non-histidine substitutions may also be incorporated into the variable domains, particularly the CDRs, of the pH-dependent antibodies described herein. The antibody molecules, e.g., IL-4Rα and / or IL-5 antibody molecules, may be engineered according to the methods described in International Patent Application No. WO2018 / 206748.

[0124] In a preferred embodiment, the exemplary IL-4Rα and IL-5 antibodies described herein having the above CDR, VH, and / or VL domain sequences are engineered such that they exhibit pH-dependent antigen binding. For example, the CDR sequences of the exemplary IL-4Rα and / or IL-5 antibody molecules described herein may be modified by the introduction of one or more histidine substitutions to generate antibody molecules that exhibit pH-dependent antigen binding.

[0125] In particularly preferred embodiments, the pH-dependent antibodies, e.g., IL-4Rα and / or IL-5 antibody molecules of the combinations described herein, comprise an Fc domain with increased binding affinity to the fetal receptor FcRn. Potential substitutions that increase the binding affinity of the Fc domain to FcRn are described elsewhere herein, and such substitutions can be incorporated into the pH-dependent antibody molecules of the combinations.

[0126] In a particularly preferred embodiment, the combination comprises a pH-dependent IL-4Rα antibody molecule and a pH-dependent IL-5 antibody molecule, wherein one or both antibody molecules comprise a modified human IgG Fc domain comprising or consisting of the amino acid substitutions H433K and N434F, and the Fc domain numbering is according to EU numbering. In a further preferred embodiment, the combination comprises a pH-dependent IL-4Rα antibody molecule and a pH-dependent IL-5 antibody molecule, wherein one or both antibody molecules comprise a modified human IgG Fc domain comprising or consisting of the amino acid substitutions M252Y, S254T, T256E, H433K, and N434F, and the Fc domain numbering is according to EU numbering.

[0127] Exemplary IL-4Rα and IL-5 Antibodies As noted above, in a preferred embodiment, the combination comprises an antibody molecule that binds IL-5 and an antibody molecule that binds IL-4Rα. In such an embodiment, the combination is capable of inhibiting signaling through all three type 2 cytokines, IL-5, IL-4, and IL-13.

[0128] Antibody molecules that bind to human IL-4Rα and that can be incorporated into the combinations described herein include antibody molecules that comprise a combination of a variable heavy chain CDR3 (HCDR3), a variable heavy chain CDR2 (HCDR2) and a variable heavy chain CDR1 (HCDR1), a variable light chain CDR3 (LCDR3), a variable light chain CDR2 (LCDR2) and a variable light chain CDR1 (LCDR1), which combinations are as follows: (i) an HCDR3 comprising or consisting of SEQ ID NO:3; an HCDR2 comprising or consisting of SEQ ID NO:2; an HCDR1 comprising or consisting of SEQ ID NO:1; an LCDR3 comprising or consisting of SEQ ID NO:12; an LCDR2 comprising or consisting of SEQ ID NO:11; an LCDR1 comprising or consisting of SEQ ID NO:10; (ii) an HCDR3 comprising or consisting of SEQ ID NO:6; an HCDR2 comprising or consisting of SEQ ID NO:5; an HCDR1 comprising or consisting of SEQ ID NO:4; an LCDR3 comprising or consisting of SEQ ID NO:15; an LCDR2 comprising or consisting of SEQ ID NO:14; an LCDR1 comprising or consisting of SEQ ID NO:13; (iii) an HCDR3 comprising or consisting of SEQ ID NO:9; an HCDR2 comprising or consisting of SEQ ID NO:8; an HCDR1 comprising or consisting of SEQ ID NO:7; an LCDR3 comprising or consisting of SEQ ID NO:18; an LCDR2 comprising or consisting of SEQ ID NO:17; an LCDR1 comprising or consisting of SEQ ID NO:16; (iv) an HCDR3 comprising or consisting of SEQ ID NO:91; an HCDR2 comprising or consisting of SEQ ID NO:8; an HCDR1 comprising or consisting of SEQ ID NO:7; an LCDR3 comprising or consisting of SEQ ID NO:18; an LCDR2 comprising or consisting of SEQ ID NO:17; an LCDR1 comprising or consisting of SEQ ID NO:16; (v) an HCDR3 comprising or consisting of SEQ ID NO:92; an HCDR2 comprising or consisting of SEQ ID NO:8; an HCDR1 comprising or consisting of SEQ ID NO:7; an LCDR3 comprising or consisting of SEQ ID NO:97; an LCDR2 comprising or consisting of SEQ ID NO:17; an LCDR1 comprising or consisting of SEQ ID NO:16; (vi) an HCDR3 comprising or consisting of SEQ ID NO: 93; an HCDR2 comprising or consisting of SEQ ID NO: 8; an HCDR1 comprising or consisting of SEQ ID NO: 7; an LCDR3 comprising or consisting of SEQ ID NO: 98; an LCDR2 comprising or consisting of SEQ ID NO: 96; an LCDR1 comprising or consisting of SEQ ID NO: 16; (vii) an HCDR3 comprising or consisting of SEQ ID NO: 94; an HCDR2 comprising or consisting of SEQ ID NO: 8; an HCDR1 comprising or consisting of SEQ ID NO: 7; an LCDR3 comprising or consisting of SEQ ID NO: 98; an LCDR2 comprising or consisting of SEQ ID NO: 17; an LCDR1 comprising or consisting of SEQ ID NO: 16; and (viii) an HCDR3 comprising or consisting of SEQ ID NO: 95; an HCDR2 comprising or consisting of SEQ ID NO: 8; an HCDR1 comprising or consisting of SEQ ID NO: 7; an LCDR3 comprising or consisting of SEQ ID NO: 98; an LCDR2 comprising or consisting of SEQ ID NO: 17; or an LCDR1 comprising or consisting of SEQ ID NO: 16.

[0129] Antibody molecules that bind to IL-4Rα and that can be incorporated into the combinations described herein also include antibody molecules that comprise a combination of a variable heavy chain CDR3 (HCDR3), a variable heavy chain CDR2 (HCDR2) and a variable heavy chain CDR1 (HCDR1), a variable light chain CDR3 (LCDR3), a variable light chain CDR2 (LCDR2) and a variable light chain CDR1 (LCDR1), which combinations are as follows: (i) an HCDR3 comprising or consisting of SEQ ID NO: 27; an HCDR2 comprising or consisting of SEQ ID NO: 26; an HCDR1 comprising or consisting of SEQ ID NO: 25; an LCDR3 comprising or consisting of SEQ ID NO: 36; an LCDR2 comprising or consisting of SEQ ID NO: 35; an LCDR1 comprising or consisting of SEQ ID NO: 34; (ii) an HCDR3 comprising or consisting of SEQ ID NO: 30; an HCDR2 comprising or consisting of SEQ ID NO: 29; an HCDR1 comprising or consisting of SEQ ID NO: 28; an LCDR3 comprising or consisting of SEQ ID NO: 39; an LCDR2 comprising or consisting of SEQ ID NO: 38; an LCDR1 comprising or consisting of SEQ ID NO: 37; and (iii) an HCDR3 comprising or consisting of SEQ ID NO: 33; an HCDR2 comprising or consisting of SEQ ID NO: 32; an HCDR1 comprising or consisting of SEQ ID NO: 31; an LCDR3 comprising or consisting of SEQ ID NO: 42; an LCDR2 comprising or consisting of SEQ ID NO: 41; or an LCDR1 comprising or consisting of SEQ ID NO: 40.

[0130] In one embodiment, the antibody molecule that binds to human IL-4Rα is selected from antibody molecules comprising or consisting of a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains are as follows: (i) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 19, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 20, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (ii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 21, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 22, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (iii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 23, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 24, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (iv) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 99, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 100, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (v) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 101, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 102, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (vi) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 103, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 104, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (vii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 105, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 106, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; and (viii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 107, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 108, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0131] In one embodiment, the antibody molecule that binds to IL-4Rα is selected from antibody molecules comprising or consisting of a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains are as follows: (i) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 43, or an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 44, or an amino acid sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (ii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 45, or an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% identical thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 46, or an amino acid sequence that has at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% identity thereto; and (iii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 47, or an amino acid sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 48, or an amino acid sequence which has at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0132] Antibody molecules that bind to human IL-5 and that can be incorporated into the combinations described herein include antibody molecules that comprise a combination of a variable heavy chain CDR3 (HCDR3), a variable heavy chain CDR2 (HCDR2) and a variable heavy chain CDR1 (HCDR1), a variable light chain CDR3 (LCDR3), a variable light chain CDR2 (LCDR2) and a variable light chain CDR1 (LCDR1), which combinations are as follows: (i) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 54; an LCDR2 comprising or consisting of SEQ ID NO: 53; an LCDR1 comprising or consisting of SEQ ID NO: 52; (ii) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 57; an LCDR2 comprising or consisting of SEQ ID NO: 56; an LCDR1 comprising or consisting of SEQ ID NO: 55; (iii) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 59; an LCDR2 comprising or consisting of SEQ ID NO: 56; an LCDR1 comprising or consisting of SEQ ID NO: 58; (iv) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 61; an LCDR2 comprising or consisting of SEQ ID NO: 60; an LCDR1 comprising or consisting of SEQ ID NO: 58; and (v) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 62; an LCDR2 comprising or consisting of SEQ ID NO: 56; or an LCDR1 comprising or consisting of SEQ ID NO: 58.

[0133] Antibody molecules that bind to IL-5 and that can be incorporated into the combinations described herein include antibody molecules that comprise a combination of a variable heavy chain CDR3 (HCDR3), a variable heavy chain CDR2 (HCDR2) and a variable heavy chain CDR1 (HCDR1), a variable light chain CDR3 (LCDR3), a variable light chain CDR2 (LCDR2) and a variable light chain CDR1 (LCDR1), which combinations are as follows: (i) an HCDR3 comprising or consisting of SEQ ID NO: 72; an HCDR2 comprising or consisting of SEQ ID NO: 71; an HCDR1 comprising or consisting of SEQ ID NO: 70; an LCDR3 comprising or consisting of SEQ ID NO: 74; an LCDR2 comprising or consisting of SEQ ID NO: 38; an LCDR1 comprising or consisting of SEQ ID NO: 73; and (ii) an HCDR3 comprising or consisting of SEQ ID NO: 72; an HCDR2 comprising or consisting of SEQ ID NO: 71; an HCDR1 comprising or consisting of SEQ ID NO: 70; an LCDR3 comprising or consisting of SEQ ID NO: 75; an LCDR2 comprising or consisting of SEQ ID NO: 38; or an LCDR1 comprising or consisting of SEQ ID NO: 73.

[0134] In one embodiment, the antibody molecule that binds human IL-5 is selected from antibody molecules comprising or consisting of a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains are as follows: (i) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 63, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 64, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (ii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 63, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 65, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (iii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 63, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 66, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (iv) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 63, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 67, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (v) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 63, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 68, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; and (vi) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 63, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 69, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0135] In one embodiment, the antibody molecule that binds IL-5 is selected from antibody molecules comprising or consisting of a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains are as follows: (i) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 76, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 77, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (ii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 76, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 78, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; and (iii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 76, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 79, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0136] In a preferred embodiment, the combination comprises an antibody molecule having a CDR, VH and / or VL sequence that binds to human IL-4Rα, and an antibody molecule having a CDR, VH and / or VL sequence that binds to human IL-5.

[0137] Where a particular antibody molecule is identified as comprising a combination of a VH domain or heavy chain, defined relative to a particular amino acid sequence, and a VL domain or light chain, also defined relative to a particular amino acid sequence, it will be recognized that this definition for each particular VH / VL or heavy chain / light chain combination recited (unless otherwise stated) is intended to include antibody molecules formed by combining a VH domain / heavy chain that has at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the recited VH / heavy chain amino acid sequence, and a VL domain / light chain that has at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the recited VL / light chain amino acid sequence.

[0138] Each domain / chain, defined by % sequence identity with a given domain / chain amino acid sequence, may retain the same CDR sequence as present in the given VH / VL domain or heavy / light chain amino acid sequence, while exhibiting amino acid sequence variation within the framework regions or other regions outside of the CDR regions.

[0139] In one embodiment, the IL-4Rα and / or IL-5 antibody molecules defined as having the above CDR sequences or having a particular percentage of identity with the above particular VH / VL domain amino acid sequences are humanized, germlined, or affinity variants of the antibodies or antigen-binding fragments thereof from which the CDR, VH, and / or VL sequences are derived.

[0140] In preferred embodiments, exemplary IL-4Rα and IL-5 antibody molecules having the above CDR sequences are humanized or germline variants of the antibodies or antigen-binding fragments thereof from which the CDR sequences are derived, e.g., exhibiting high human homology.

[0141] In non-limiting embodiments, exemplary IL-4Rα and IL-5 antibody molecules having the CDR, VH, and / or VL sequences described herein comprise a CH1 domain and / or a CL domain (from the heavy and light chains, respectively), the amino acid sequences of which may be fully or substantially human. For antibody molecules intended for human therapeutic use, the complete antibody constant region, or at least a portion thereof, typically has a fully or substantially human amino acid sequence. Thus, one or more, or any combination, of the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and, if present, the CH4 domain) may be fully or substantially human in terms of their amino acid sequences.

[0142] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and, if present, the CH4 domain) may all have fully or substantially human amino acid sequences. In the context of the constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially human" refers to at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% amino acid sequence identity with a human constant region. In this context, the term "human amino acid sequence" refers to an amino acid sequence encoded by a human immunoglobulin gene, including germline, rearranged, and somatically mutated genes. The present invention also contemplates polypeptides comprising constant domains of "human" sequence, altered by one or more amino acid additions, deletions, or substitutions relative to the human sequence, except in embodiments where the presence of a "fully human" hinge region is expressly required. Any of the exemplary Fc region modifications described herein may be incorporated into IL-4Rα and / or IL-5 antibodies having the CDR and / or VH / VL domain sequences described above. In a preferred embodiment, the IL-4Rα and / or IL-5 antibodies having the above CDR and / or VH / VL domain sequences comprise a modified human IgG Fc domain comprising or consisting of the amino acid substitutions H433K and N434F, wherein the Fc domain numbering is according to EU numbering. In a preferred embodiment, the IL-4Rα and / or IL-5 antibodies having the above CDR and / or VH / VL domain sequences comprise a modified human IgG Fc domain comprising or consisting of the amino acid substitutions M252Y, S254T, T256E, H433K, and N434F.

[0143] In a non-limiting embodiment, IL-4Rα and IL-5 antibody molecules having the exemplary CDR, VH, and / or VL sequences described herein may be modified as described above to exhibit pH-dependent antigen binding. For example, IL-4Rα and IL-5 antibody molecules having the exemplary CDR, VH, and / or VL sequences described herein may be modified to have pH-dependent binding such that their antigen-binding activity at pH 5.8 is reduced compared to their antigen-binding activity at pH 7.4. The above methods for engineering antibodies to confer pH-dependent antigen binding may be applied to any of the exemplary IL-4Rα and IL-5 antibody molecules having the CDR, VH, and / or VL sequences described herein. For example, the variable domain and / or CDR regions may be modified with histidine substitutions or insertions to confer pH-dependent binding.

[0144] Unless otherwise specified in this application, the percent sequence identity between two amino acid sequences can be determined by comparing the two sequences aligned in an optimal manner, where the compared amino acid sequence may contain additions or deletions relative to the reference sequence for optimal alignment between the two sequences. The percent identity can be calculated by determining the number of identical positions where the amino acid residue is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage identity between the two sequences. For example, the BLAST program "BLAST2 sequences" (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), available on the Internet at http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html, can be used, with the parameters set to default (in particular, the matrix chosen for the parameters "open gap penalty": 5 and "extension gap penalty": 2 is, for example, the matrix "BLOSUM 62" recommended by the program), and the percentage of identity between the two sequences being compared can be calculated directly by the program.

[0145] (C. Combination Formulation) The different antagonist or antibody molecules of the combination may be combined or formulated in any manner and the combination therapy administered to a subject or patient, preferably a human subject or patient, in need thereof. The combination may be formulated for single dose administration or for multiple dose administration.

[0146] In some embodiments, the antagonist or antibody molecules of the combination are separate molecules that are co-formulated, i.e., formulated as a single pharmaceutical composition. In embodiments in which the antagonist or antibody molecules are co-formulated, the combination or composition is suitable for simultaneous administration of the two components. The composition can also be formulated for single-dose or multi-dose administration. In embodiments in which the antagonist or antibody molecules are co-formulated, the antagonist or antibody molecules can be formulated in equal amounts, for example, in a 1:1 ratio for combinations comprising first and second antagonist or antibody molecules targeting different cytokines or receptors. Alternatively, the antagonist or antibody molecules can be formulated such that the ratio of different antagonist or antibody molecules is not 1:1. For example, in embodiments in which the combination comprises or consists of first and second antagonist or antibody molecules binding to different targets, the ratio of the first and second antagonist or antibody molecules can be 2:1, optionally 3:1, or optionally 4:1. Alternatively, the antagonist or antibody molecules may be formulated according to a ratio of 1:2, optionally 1:3, optionally 1:4.

[0147] In some embodiments, the antagonists or antibody molecules of the combination are formulated separately, e.g., as individual compositions. In embodiments in which the antagonists or antibody molecules are formulated separately, the possibility exists for the different components or compositions to be administered simultaneously or separately. When the antagonists or antibody molecules or separate compositions comprising them are administered separately, the antagonists / antibody molecules or compositions may be administered sequentially in any order. For example, the antibody molecule that binds IL-4Rα may be administered first, followed by the antibody molecule that binds IL-5, or vice versa. The interval between administrations of the antagonists / antibody molecules or compositions may be any suitable time interval. The different compositions may be administered once (for single-dose administration) or repeatedly (for multiple-dose administration).

[0148] In one embodiment, the antagonists are antibody molecules, and the antibody molecules of the combination may be combined as a multispecific antibody, e.g., a bispecific antibody. For example, if the combination includes a Fab fragment that binds IL-4Rα and a Fab fragment that binds IL-5, the two Fab fragments may be combined into a single bispecific antibody molecule having the two Fab regions conjugated to an IgG Fc portion.

[0149] The bispecific or multispecific antibodies of the present invention may be constructed according to any suitable bispecific / multispecific antibody format. For example, the antibody molecules of the combination can be incorporated into a bispecific or multispecific antibody format in which the antibodies bind to different targets in a "trans" configuration, e.g., with each Fab arm of a Y-shaped antibody having a different binding specificity. In another embodiment, the antibody molecules can be incorporated into a bispecific or multispecific antibody format in which the targets are bound in a "cis" configuration. For example, the Fab regions or variable domains capable of binding to antigens can be placed at opposite ends of an IgG Fc portion.

[0150] Bispecific or multispecific antibodies may have a native IgG structure with two Y-shaped Fab arms that have binding specificity for a first target, and one or more additional antigen-binding domains located at the C-terminus of the Fc domain that have binding specificity for a second target. For example, in one embodiment, a bispecific antibody of the invention is configured such that two classical antigen-binding domains in the Fab region present on the native IgG structure bind to IL-4Rα, and one or more VHH domains located at the C-terminus of the Fc domain bind to IL-5. The reverse configuration is also possible, where two classical antigen-binding domains in the Fab region present on the native IgG structure bind to IL-5, and one or more VHH domains located at the C-terminus of the Fc domain bind to IL-4Rα.

[0151] Alternatively, a bispecific or multispecific antibody may have a native IgG structure with two Y-shaped Fab arms that have binding specificity for a first target, and one or more scFv fragments with binding specificity for a second target, located at the C-terminus of the Fc domain. For example, in one embodiment, a bispecific antibody of the invention is configured such that two classical antigen-binding domains in the Fab region present on the native IgG structure bind to IL-4Rα, and one or more scFv domains located at the C-terminus of the Fc domain bind to IL-5. The reverse configuration is also possible, in which two classical antigen-binding domains in the Fab region present on the native IgG structure bind to IL-5, and one or more scFv domains located at the C-terminus of the Fc domain bind to IL-4Rα. In such an embodiment, a bispecific antibody may be composed of two scFv domains located at the C-terminus of an IgG Fc domain.

[0152] Bispecific or multispecific antibodies may also be asymmetric IgG antibodies in which one Fab region has been replaced by a different antigen-binding domain, such as a VHH domain. For example, in another embodiment, a bispecific antibody of the invention may be configured to resemble a native IgG structure, with an Fab region on one intact arm of the antibody that binds to IL-4Rα and a VHH domain that binds to IL-5 and replaces the second Fab region. The reverse configuration is also possible, in which the Fab region on one intact arm of the antibody binds to IL-5 and a VHH domain that binds to IL-4Rα replaces the second Fab region.

[0153] For embodiments in which the antagonists are antibody molecules and the antibody molecules are co-formulated, and / or for embodiments in which multiple antibody molecules are provided as separate compositions and / or in which the antibody molecules are provided in a multispecific antibody format, the antibody molecules can be formulated using any suitable pharmaceutical carrier or excipient. Techniques for formulating antibodies for human therapeutic use are well known in the art and are reported, for example, in Wang et al. (2007) Journal of Pharmaceutical Sciences, 96:1-26, the contents of which are incorporated herein in their entirety. In embodiments in which the antibody molecules are formulated separately, the pharmaceutical carrier or excipient can be different or the same for the different compositions.

[0154] Pharmaceutically acceptable excipients that can be used to formulate the present compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0155] In certain embodiments, the compositions are formulated for administration to a subject via any suitable route of administration, including, but not limited to, intramuscular, intravenous, intradermal, intraperitoneal injection, subcutaneous, epidural, nasal, oral, rectal, topical, inhalation, buccal (e.g., sublingual), and transdermal administration. In embodiments in which the antagonist or antibody molecule are formulated separately, each composition may be formulated to be administered via a different route.

[0156] In some embodiments, the composition comprises one or more additional therapeutic agents. The additional therapeutic agents may be agents suitable for the prevention or treatment of chronic airway diseases. The one or more additional agents may be formulated for administration via the same route or via a different route than the antagonist or antibody molecule of the combination.

[0157] D. Bispecific IL-4Rα and IL-5 Antibodies As mentioned above, the antibody molecules of the combinations described herein may be provided in a multispecific antibody format, e.g., a bispecific antibody format. In a preferred embodiment, the combination comprises an antibody molecule that binds IL-5 and an antibody molecule that binds IL-4Rα, provided in a bispecific antibody format.

[0158] Accordingly, in a further aspect of the invention, provided herein is a bispecific antibody comprising an antigen-binding region that binds to IL-4Rα and an antigen-binding region that binds to IL-5. Such bispecific antibodies are referred to herein as "IL-4Rα / IL-5 bispecific" antibodies. All embodiments described herein with respect to the separate IL-4Rα and IL-5 antibody molecules equally apply to this further aspect of the invention. In particular, the "antigen-binding region" of the bispecific antibodies described herein can take the form of any antibody or antigen-binding fragment described herein, including VHH antibodies.

[0159] Bispecific antibodies of the invention can be constructed according to any suitable bispecific / multispecific antibody format as described herein. For example, bispecific antibodies may be constructed so that the targets bind in either a "trans" configuration, i.e., at the same end of the molecule, or in a "cis" configuration, i.e., at opposite ends of the molecule.

[0160] In one embodiment, a bispecific antibody has a native IgG structure in which the antigen-binding regions are contained within two Fab arms. A first Fab arm may exhibit binding specificity for IL-4Rα, and a second Fab arm may exhibit binding specificity for IL-5. In another embodiment, a bispecific antibody has a native IgG structure and can additionally comprise one or more additional antigen-binding regions disposed at the C-terminus of the Fc region. In such an embodiment, the two Fab arms of the native IgG may bind to the same target, e.g., IL-4Rα, and the one or more additional antigen-binding regions disposed at the C-terminus of the Fc region may bind to a second target, e.g., IL-5. The one or more additional antigen-binding regions may take any suitable antigen-binding form, including, but not limited to, a VHH domain or scFv.

[0161] In one embodiment, an IL-4Rα / IL-5 bispecific antibody of the invention comprises an Fab region that binds to IL-4Rα and a VHH domain that binds to IL-5. In one embodiment, an IL-4Rα / IL-5 bispecific antibody of the invention comprises an Fab region that binds to IL-5 and a VHH domain that binds to IL-4Rα.

[0162] In one embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention comprises an IL-4Rα-binding Fab region and an IL-5-binding scFv. In one embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention comprises an IL-5-binding Fab region and an IL-4Rα-binding scFv. In a preferred embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention comprises or is composed of an IL-4Rα-binding IgG antibody and one or more IL-5-binding scFvs. In such an embodiment, the IL-4Rα / IL-5 bispecific antibody may comprise or be composed of an IL-4Rα-binding IgG antibody and two IL-5-binding scFv fragments. The one or more IL-5-binding scFv fragments are preferably located at the C-terminus of the Fc region of the IgG antibody, i.e., at the end of the Fc region opposite the two IL-4Rα-binding Fab arms.

[0163] The bispecific antibodies of the present invention may be configured as asymmetric IgG antibodies in which one Fab region (or Fab arm) is replaced by a different antigen-binding region or domain, such as a VHH domain. In one embodiment, the IL-4Rα / IL-5 bispecific antibody is configured similarly to a native IgG structure, with the Fab region on one intact arm of the antibody binding to IL-4Rα and a VHH domain replacing the second Fab region binding to IL-5. The reverse configuration is also possible, in which the Fab region on one intact arm of the antibody binds to IL-5 and the VHH domain replacing the second Fab region binds to IL-4Rα.

[0164] The IL-4Rα / IL-5 bispecific antibody of the present invention, i.e., configured according to any of the formats described herein, can comprise an antigen-binding region that binds to human IL-4R, and the antigen-binding region can comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains are: (i) an HCDR3 comprising or consisting of SEQ ID NO:3; an HCDR2 comprising or consisting of SEQ ID NO:2; an HCDR1 comprising or consisting of SEQ ID NO:1; an LCDR3 comprising or consisting of SEQ ID NO:12; an LCDR2 comprising or consisting of SEQ ID NO:11; an LCDR1 comprising or consisting of SEQ ID NO:10; (ii) an HCDR3 comprising or consisting of SEQ ID NO:6; an HCDR2 comprising or consisting of SEQ ID NO:5; an HCDR1 comprising or consisting of SEQ ID NO:4; an LCDR3 comprising or consisting of SEQ ID NO:15; an LCDR2 comprising or consisting of SEQ ID NO:14; an LCDR1 comprising or consisting of SEQ ID NO:13; (iii) an HCDR3 comprising or consisting of SEQ ID NO:9; an HCDR2 comprising or consisting of SEQ ID NO:8; an HCDR1 comprising or consisting of SEQ ID NO:7; an LCDR3 comprising or consisting of SEQ ID NO:18; an LCDR2 comprising or consisting of SEQ ID NO:17; an LCDR1 comprising or consisting of SEQ ID NO:16; (iv) an HCDR3 comprising or consisting of SEQ ID NO:91; an HCDR2 comprising or consisting of SEQ ID NO:8; an HCDR1 comprising or consisting of SEQ ID NO:7; an LCDR3 comprising or consisting of SEQ ID NO:18; an LCDR2 comprising or consisting of SEQ ID NO:17; an LCDR1 comprising or consisting of SEQ ID NO:16; (v) an HCDR3 comprising or consisting of SEQ ID NO:92; an HCDR2 comprising or consisting of SEQ ID NO:8; an HCDR1 comprising or consisting of SEQ ID NO:7; an LCDR3 comprising or consisting of SEQ ID NO:97; an LCDR2 comprising or consisting of SEQ ID NO:17; an LCDR1 comprising or consisting of SEQ ID NO:16; (vi) an HCDR3 comprising or consisting of SEQ ID NO: 93; an HCDR2 comprising or consisting of SEQ ID NO: 8; an HCDR1 comprising or consisting of SEQ ID NO: 7; an LCDR3 comprising or consisting of SEQ ID NO: 98; an LCDR2 comprising or consisting of SEQ ID NO: 96; an LCDR1 comprising or consisting of SEQ ID NO: 16; (vii) an HCDR3 comprising or consisting of SEQ ID NO: 94; an HCDR2 comprising or consisting of SEQ ID NO: 8; an HCDR1 comprising or consisting of SEQ ID NO: 7; an LCDR3 comprising or consisting of SEQ ID NO: 98; an LCDR2 comprising or consisting of SEQ ID NO: 17; an LCDR1 comprising or consisting of SEQ ID NO: 16; and (viii) comprises a CDR sequence selected from the group consisting of: an HCDR3 comprising or consisting of SEQ ID NO: 95; an HCDR2 comprising or consisting of SEQ ID NO: 8; an HCDR1 comprising or consisting of SEQ ID NO: 7; an LCDR3 comprising or consisting of SEQ ID NO: 98; an LCDR2 comprising or consisting of SEQ ID NO: 17; and an LCDR1 comprising or consisting of SEQ ID NO: 16.

[0165] The IL-4Rα / IL-5 bispecific antibody of the present invention may comprise an antigen-binding region that binds to IL-4R, and the antigen-binding region may comprise a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains are: (i) an HCDR3 comprising or consisting of SEQ ID NO: 27; an HCDR2 comprising or consisting of SEQ ID NO: 26; an HCDR1 comprising or consisting of SEQ ID NO: 25; an LCDR3 comprising or consisting of SEQ ID NO: 36; an LCDR2 comprising or consisting of SEQ ID NO: 35; an LCDR1 comprising or consisting of SEQ ID NO: 34; (ii) an HCDR3 comprising or consisting of SEQ ID NO: 30; an HCDR2 comprising or consisting of SEQ ID NO: 29; an HCDR1 comprising or consisting of SEQ ID NO: 28; an LCDR3 comprising or consisting of SEQ ID NO: 39; an LCDR2 comprising or consisting of SEQ ID NO: 38; an LCDR1 comprising or consisting of SEQ ID NO: 37; and (iii) comprises a CDR sequence selected from the group consisting of: an HCDR3 comprising or consisting of SEQ ID NO: 33; an HCDR2 comprising or consisting of SEQ ID NO: 32; an HCDR1 comprising or consisting of SEQ ID NO: 31; an LCDR3 comprising or consisting of SEQ ID NO: 42; an LCDR2 comprising or consisting of SEQ ID NO: 41; and an LCDR1 comprising or consisting of SEQ ID NO: 40.

[0166] The antigen-binding region that binds to human IL-4Rα can comprise a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains are: (i) a VH domain comprising an amino acid sequence comprising SEQ ID NO: 19, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising SEQ ID NO: 20, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (ii) a VH domain comprising an amino acid sequence comprising SEQ ID NO: 21, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising SEQ ID NO: 22, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (iii) a VH domain comprising an amino acid sequence comprising SEQ ID NO: 23, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising SEQ ID NO: 24, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (iv) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 99, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 100, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (v) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 101, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 102, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (vi) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 103, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 104, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; (vii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 105, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 106, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; and (viii) a VH domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 107, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto; and a VL domain comprising an amino acid sequence comprising or consisting of SEQ ID NO: 108, or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0167] The antigen-binding region that binds to IL-4Rα can comprise a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains are: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 70% identity thereto; (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 70% identity thereto; and (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 70% identity thereto.

[0168] The IL-4Rα / IL-5 bispecific antibody of the present invention may comprise an antigen-binding region that binds to human IL-5, and the antigen-binding region may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains are: (i) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 54; an LCDR2 comprising or consisting of SEQ ID NO: 53; an LCDR1 comprising or consisting of SEQ ID NO: 52; (ii) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 57; an LCDR2 comprising or consisting of SEQ ID NO: 56; an LCDR1 comprising or consisting of SEQ ID NO: 55; (iii) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 59; an LCDR2 comprising or consisting of SEQ ID NO: 56; an LCDR1 comprising or consisting of SEQ ID NO: 58; (iv) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 61; an LCDR2 comprising or consisting of SEQ ID NO: 60; an LCDR1 comprising or consisting of SEQ ID NO: 58; and (v) comprises a CDR sequence selected from the group consisting of: an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 62; an LCDR2 comprising or consisting of SEQ ID NO: 56; and an LCDR1 comprising or consisting of SEQ ID NO: 58.

[0169] The IL-4Rα / IL-5 bispecific antibody of the present invention may comprise an antigen-binding region that binds to IL-5, and the antigen-binding region may comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains are: (i) an HCDR3 comprising or consisting of SEQ ID NO: 72; an HCDR2 comprising or consisting of SEQ ID NO: 71; an HCDR1 comprising or consisting of SEQ ID NO: 70; an LCDR3 comprising or consisting of SEQ ID NO: 74; an LCDR2 comprising or consisting of SEQ ID NO: 38; an LCDR1 comprising or consisting of SEQ ID NO: 73; and (ii) comprises a CDR sequence selected from the group consisting of: an HCDR3 comprising or consisting of SEQ ID NO: 72; an HCDR2 comprising or consisting of SEQ ID NO: 71; an HCDR1 comprising or consisting of SEQ ID NO: 70; an LCDR3 comprising or consisting of SEQ ID NO: 75; an LCDR2 comprising or consisting of SEQ ID NO: 38; and an LCDR1 comprising or consisting of SEQ ID NO: 73.

[0170] The antigen-binding region that binds to human IL-5 can comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains are: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least 70% identity thereto; (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 70% identity thereto; (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 70% identity thereto; (iv) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence having at least 70% identity thereto; (v) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 70% identity thereto; and (vi) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 70% identity thereto.

[0171] The antigen-binding region that binds to IL-5 can comprise a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains are: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 70% identity thereto; (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence having at least 70% identity thereto; and (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence having at least 70% identity thereto.

[0172] Each domain / chain, defined by % sequence identity with a given domain / chain amino acid sequence, may retain the same CDR sequence as present in the given VH / VL domain or heavy / light chain amino acid sequence, while exhibiting amino acid sequence variation within the framework regions or other regions outside of the CDR regions.

[0173] In a preferred embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention may comprise an antigen-binding region that binds to IL-4Rα and an antigen-binding region that binds to IL-5, wherein the antigen-binding region that binds to IL-4Rα comprises a first pair of variable heavy chain domain (VH) and variable light chain domain (VL), and the antigen-binding region that binds to IL-5 comprises a second pair of variable heavy chain domain (VH) and variable light chain domain (VL); The first VH-VL domain pair is: (i) an HCDR3 comprising or consisting of SEQ ID NO:3; an HCDR2 comprising or consisting of SEQ ID NO:2; an HCDR1 comprising or consisting of SEQ ID NO:1; an LCDR3 comprising or consisting of SEQ ID NO:12; an LCDR2 comprising or consisting of SEQ ID NO:11; an LCDR1 comprising or consisting of SEQ ID NO:10; (ii) an HCDR3 comprising or consisting of SEQ ID NO:6; an HCDR2 comprising or consisting of SEQ ID NO:5; an HCDR1 comprising or consisting of SEQ ID NO:4; an LCDR3 comprising or consisting of SEQ ID NO:15; an LCDR2 comprising or consisting of SEQ ID NO:14; an LCDR1 comprising or consisting of SEQ ID NO:13; and (iii) comprises a CDR sequence selected from: an HCDR3 comprising or consisting of SEQ ID NO:9; an HCDR2 comprising or consisting of SEQ ID NO:8; an HCDR1 comprising or consisting of SEQ ID NO:7; an LCDR3 comprising or consisting of SEQ ID NO:18; an LCDR2 comprising or consisting of SEQ ID NO:17; an LCDR1 comprising or consisting of SEQ ID NO:16; and the second VH-VL domain pair being: (i) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 54; an LCDR2 comprising or consisting of SEQ ID NO: 53; an LCDR1 comprising or consisting of SEQ ID NO: 52; (ii) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 57; an LCDR2 comprising or consisting of SEQ ID NO: 56; an LCDR1 comprising or consisting of SEQ ID NO: 55; (ii) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 59; an LCDR2 comprising or consisting of SEQ ID NO: 56; an LCDR1 comprising or consisting of SEQ ID NO: 58; (iv) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 61; an LCDR2 comprising or consisting of SEQ ID NO: 60; an LCDR1 comprising or consisting of SEQ ID NO: 58; and (v) comprises a CDR sequence selected from: an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 62; an LCDR2 comprising or consisting of SEQ ID NO: 56; and an LCDR1 comprising or consisting of SEQ ID NO: 58.

[0174] In a preferred embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention may comprise an antigen-binding region that binds to IL-4Rα and an antigen-binding region that binds to IL-5, wherein the antigen-binding region that binds to IL-4Rα comprises a first pair of variable heavy chain domain (VH) and variable light chain domain (VL), and the antigen-binding region that binds to IL-5 comprises a second pair of variable heavy chain domain (VH) and variable light chain domain (VL); The first VH-VL domain pair is: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 70% identity thereto; (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 70% identity thereto; and (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 70% identity thereto; and the second VH-VL domain pair being: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least 70% identity thereto; (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 70% identity thereto; (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 70% identity thereto; (iv) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence having at least 70% identity thereto; (v) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 70% identity thereto; and (vi) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 70% identity thereto.

[0175] In a preferred embodiment, the antigen-binding regions of the bispecific antibodies described herein exhibit a high degree of human homology, for example, the antigen-binding regions may be humanized or germline variants of the VH and / or VL domains from which their CDR sequences are derived.

[0176] Bispecific antibodies of the invention may also comprise one or more constant domains having amino acid sequences that are fully or substantially human, for example, a bispecific antibody of the invention may comprise an Fc domain derived from a human IgG antibody, such as human IgG1, IgG2, IgG3, or IgG4.

[0177] Bispecific antibodies of the present invention having an Fc domain may be modified within the Fc region as described herein to enhance binding affinity to the fetal receptor FcRn. For example, one or more amino acid residues within the Fc region may be substituted with different amino acid residues to increase binding to FcRn. Preferred amino acid substitutions within the Fc region are described herein and apply equally to the bispecific antibodies of the present invention.

[0178] The bispecific antibodies of the present invention may be modified to have pH-dependent antigen-binding activity, as described herein. In particular, the bispecific antibodies may be modified so that their IL-4Rα and / or IL-5 binding activity is reduced at acidic pH compared to their binding activity at neutral pH. The bispecific antibodies may also be modified by substituting at least one amino acid in the IL-4Rα antigen-binding region and / or the IL-5 antigen-binding region of the antibody molecule with histidine, or by inserting at least one histidine into one or both antigen-binding regions of the antibody molecule. The histidine substitution and / or insertion is preferably introduced at one or more sites within the CDRs of the heavy chain variable domain and / or the light chain variable domain, as described herein.

[0179] (E. Treatment Method) The combination therapies and bispecific antibodies described herein are used in methods of treating chronic airway diseases in human subjects.

[0180] Accordingly, the present invention provides a combination comprising (i) an IL-5:IL-5R antagonist; and (ii) an IL-4:IL-4R antagonist and / or an IL-13:IL-13R antagonist, for use in treating chronic airway disease in a human subject. The present invention also provides a bispecific antibody comprising an antigen-binding region that binds to IL-4Rα and an antigen-binding region that binds IL-5, for use in treating chronic airway disease in a human subject. The present invention provides an IL-5:IL-5R IL-5 antagonist for use in treating chronic airway disease in a human subject, which antagonist is administered in combination with an IL-4:IL-4R antagonist and / or an IL-13:IL-13R antagonist. The present invention provides an antagonist of IL-4:IL-4R and / or an antagonist of IL-13:IL-13R, which is administered in combination with an antagonist of IL-5:IL-5R, for use in treating chronic airway disease in a human subject. In a preferred embodiment, the present invention provides an antagonist of IL-5, which is administered in combination with an antagonist of IL-4Rα, for use in treating chronic airway disease in a human subject. In a further preferred embodiment, the present invention provides an antagonist of IL-4Rα, which is administered in combination with an antagonist of IL-5, for use in treating chronic airway disease in a human subject. In a further preferred embodiment, the antagonist used in the method is an antibody molecule.

[0181] In a still further aspect, the present invention provides a method of treating a chronic airway disease in a human subject, the method comprising administering to the subject an effective amount of a combination according to the first aspect of the invention, or a bispecific antibody according to the second aspect of the invention. All embodiments described above relating to the combination according to the first aspect of the invention and the bispecific antibody according to the second aspect of the invention are equally applicable to the methods described herein.

[0182] In some embodiments, the methods described herein are for treating chronic airway diseases. As described herein, chronic airway diseases include any disease of the airways and other structures of the lungs. Non-limiting examples include asthma, chronic rhinosinusitis (CRS), immunoglobulin G4-related disease (IgG4-RD), chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, chronic angioedema, diseases characterized by goblet cell metaplasia, including Barrett's esophagus, active eosinophilic esophagitis, nasal polyposis, chronic sinusitis, Churg-Strauss syndrome, allergic bronchopulmonary aspergillosis (ABPA), hypereosinophilic syndrome, bullous pemphigoid, and cystic fibrosis.

[0183] In some embodiments, the methods described herein are for treating chronic airway diseases characterized by increased mucus production. One example of a disease characterized by increased mucus production is cystic fibrosis. In further embodiments, the methods described herein are for treating chronic airway diseases characterized by bronchial hyperresponsiveness.

[0184] In a preferred embodiment, the methods described herein are for treating asthma. Exemplary asthma subtypes include, but are not limited to, severe asthma, severe refractory asthma, mild or moderate asthma, obesity-related asthma, exercise-induced asthma, aspirin-induced asthma, atopic or allergic asthma, eosinophilic asthma, neutrophilic asthma, paucigranulocytic or non-inflammatory asthma, early-onset asthma, late-onset asthma, Type II high asthma, Type II low asthma, and Type I / Th17 asthma.

[0185] In a preferred embodiment, the method described herein is for the treatment of atopic or allergic asthma. Atopic or allergic asthma is a form of asthma clinically defined as a disease occurring simultaneously with allergic sensitization, which is manifested by the presence of serum IgE antibodies and / or a positive skin prick test against inhaled or ingested common allergen (lipo)proteins, such as house dust mites (HDM), animal dander, fungal spores, plant or tree pollen, or peanuts (Lambrecht and Hammad, (2015) supra). The majority of early-onset asthma cases are of the allergic or atopic type. Therefore, the method of the present invention is also for the treatment of early-onset asthma.

[0186] In a further preferred embodiment, the methods described herein are for treating type II high (or type II Hi) asthma. Type II high asthma is characterized by the presence of type 2 cytokine molecules characteristic of type II high asthma. Patients with type II high asthma typically exhibit higher IL-13 and IL-5 levels, have greater numbers of eosinophils and mast cells, and exhibit more severe atopy and SBM (subepithelial basement membrane) thickening compared to type II low asthma patients (see Wenzel, (2012) ibid., and Gauthier et al., (2015) ibid.). The combinations and bispecific antibodies of the present invention target multiple type 2 cytokine signaling pathways. Thus, the combinations and bispecific antibodies described herein may be particularly effective in treating human subjects with type II high asthma.

[0187] In some embodiments, the methods described herein are for treating severe asthma or severe refractory asthma.

[0188] Nearly all forms of asthma are pathologically associated with goblet cell metaplasia, or GCM. GCM is caused by the transdifferentiation of ciliated cells and Clara cells into goblet cells under the influence of IL-4 and / or IL-13 and epidermal growth factor receptor ligands. Thus, in some embodiments, the methods described herein are for treating asthma by reducing goblet cell metaplasia. In such embodiments, goblet cell metaplasia is reduced compared to the level of goblet cell metaplasia before treatment.

[0189] Patients with chronic airway diseases, including the majority of asthma patients, also suffer from bronchial hyperresponsiveness (BHR). BHR is defined as increased sensitization to various airway-narrowing stimuli. Most patients with asthma and chronic obstructive pulmonary disease (COPD) exhibit such increased sensitization. In certain embodiments, the methods described herein are for treating chronic airway diseases by reducing bronchial hyperresponsiveness. In preferred embodiments, the methods described herein are for treating asthma by reducing bronchial hyperresponsiveness. In such embodiments, bronchial hyperresponsiveness is reduced compared to the level of bronchial hyperresponsiveness before treatment. Bronchial hyperresponsiveness can be assessed by a bronchial challenge test. Products such as methacholine or histamine are most commonly used for this purpose. These chemicals cause bronchospasm in normal individuals, but individuals with bronchial hyperresponsiveness have a lower threshold.

[0190] Patients with chronic airway diseases, including the majority of asthma patients, also exhibit excessive mucus production. In certain embodiments, the methods described herein are for treating chronic airway diseases by reducing mucus production. In preferred embodiments, the methods described herein are for treating asthma by reducing mucus production. In such embodiments, mucus production is reduced compared to the level of mucus production before treatment. Mucus production can be assessed by measuring mucin expression and / or activity, for example, muc5AC expression and / or activity. In certain embodiments, the methods described herein are for treating chronic airway diseases by reducing mucin expression and / or activity compared to expression and / or activity before treatment. In certain embodiments, the methods described herein are for treating asthma by reducing mucin expression and / or activity compared to expression and / or activity before treatment.

[0191] Chronic airway disease is also characterized by decreased lung function. Forced expiratory volume (FEV1) is a measure of lung function and can be assessed using a spirometer. Forced expiratory volume (FEV1) measures how much air a person can exhale during a forced breath. Forced expiratory volume (FVC) is the total volume of air exhaled during an FEV1 test. The FEV1 / FVC ratio, also referred to as the Tiffeneau-Pinelli index, is a calculated ratio that represents the percentage of the lung capacity a subject can exhale in the first second of a forced exhalation relative to the total lung capacity. Normal values ​​for the FEV1 / FVC ratio are approximately 70-80%. In certain embodiments, the methods described herein are for treating chronic airway disease by improving lung function. In preferred embodiments, the methods described herein are for treating asthma by improving lung function. In such embodiments, lung function is improved compared to pre-treatment lung function. In certain embodiments, the methods described herein are for treating chronic airway disease by increasing the FEV1 / FVC ratio compared to the FEV1 / FVC ratio before treatment. In certain embodiments, the methods described herein are for treating asthma by increasing the FEV1 / FVC ratio compared to the FEV1 / FVC ratio before treatment.

[0192] The methods described herein can also be used to treat comorbidities associated with chronic airway diseases, such as asthma. Severe asthma is associated with many comorbidities, including, but not limited to, chronic sinusitis, nasal polyposis, allergic rhinitis, respiratory dysfunction, vocal cord dysfunction, anxiety and depression, obesity, obstructive sleep apnea syndrome (OSAS), gastroesophageal reflux disease (GERD), bronchiectasis, allergic bronchopulmonary aspergillosis (ABPA), and eosinophilic granulomatosis with polyangiitis (EGPA) (see Porsbjerg and Menzies-Gow, (2017) Respirology 22:651-661). The methods described herein can be used to treat any of the above-mentioned asthma comorbidities.

[0193] The methods described herein may further comprise the administration of a therapeutic agent. In some embodiments, the methods described herein comprise the administration of one or more additional therapeutic agents for the treatment of chronic airway disease. The combination or bispecific antibodies of the invention may be administered with the additional therapeutic agents separately, simultaneously, sequentially, or simultaneously in overlapping fashion.

[0194] A patient or subject treated with the methods described herein may already be undergoing treatment, such as corticosteroid treatment. A patient or subject treated with the methods described herein may be classified as "corticosteroid responsive" or "corticosteroid non-responsive." A patient or subject may also exhibit one or more symptoms associated with a chronic airway disease. In some embodiments, a patient or subject may be an individual currently receiving medical care for the treatment of a chronic airway disease and / or actively seeking medical care for the treatment of a chronic airway disease.

[0195] In some embodiments, the patient or subject treated with the methods described herein may be a patient with a disease characterized by elevated eosinophilia. In patients with elevated eosinophil levels, certain existing drugs targeting IL-4Rα, such as dupilumab, cannot be prescribed because they cause the patient's circulating eosinophil levels to become unacceptably high. Without being bound by theory, combined treatment with an IL-4Rα antagonist, e.g., an IL-4Rα antibody, and an IL-5 antagonist, e.g., an IL-5 antibody, is believed to be appropriate for patients with elevated eosinophil levels because IL-5 promotes eosinophil proliferation. Combined inhibition of IL-5 and IL-4Rα may prevent or reduce the increased eosinophil levels observed with IL-4Rα antibody therapy.

[0196] (Incorporating References) Various publications are cited in the foregoing description and throughout the examples which follow, each of which is incorporated herein by reference in its entirety. [Example]

[0197] (Experimental Embodiment) The present invention will be further understood from the following non-limiting examples.

[0198] Example 1: Production of neutralizing IL-4Rα and IL-5 monoclonal antibodies A. Llama immunization and library construction: Two llamas, raised outdoors in accordance with French animal welfare regulations, were immunized intramuscularly with recombinant murine IL-4Rα-Fc in one shoulder and recombinant murine IL-5 (R&D Systems) in the other shoulder, with weekly booster immunizations for six weeks. Briefly, for the first two weeks, they received 100 μg of IL-4Rα-Fc and 50 μg of IL-5 buffered in phosphate-buffered saline (PBS) and mixed with incomplete Freund's adjuvant (Sigma-Aldrich). For the remaining four weeks, they received 50 μg of IL-4Rα-Fc and 25 μg of IL-5. Fab library generation was performed using the applicant's proprietary SIMPLE antibody platform, as previously described (see International Patent Application No. WO 2010 / 001251, the contents of which are incorporated herein in their entirety). Five days after the final immunization, 400 mL of blood containing peripheral blood lymphocytes was collected from the llama, purified by centrifugation on a Ficoll-Paque gradient, and used to extract total RNA. The total RNA was then converted to randomly primed cDNA using reverse transcriptase, and the gene sequences encoding the VH-CH1 region and VL-CL domains (kappa and lambda) of llama IgG1 were isolated and subcloned into the phagemid vector pCB3. The pCB3 vector allows expression of recombinant antibodies as Fab fragments fused to the phage pIII envelope protein.

[0199] Parallel studies were carried out to generate recombinant antibodies that bind to human IL-4Rα and human IL-5. Llamas were immunized intramuscularly with recombinant human IL-4Rα-Fc and recombinant human IL-5 according to the same protocol as described above.

[0200] B. Selection of Fabs that bind to IL-4Rα and IL-5 The E. coli strain TG1 (Netherlands Culture Collection of Bacteria) was transformed with the recombinant phagemid to generate Fab-expressing phage libraries (one lambda and one kappa library per immunized llama). Since llamas were immunized with IL-4Rα linked to the crystallizable fragment (Fc) portion, counterselection for phages expressing Fc-binding antigen-binding fragments (Fab) was first performed with an irrelevant human antibody. The resulting Fab-expressing phages were collected at 10 8 ~10 9 The phage expressing a wide range of Fab fragments were then adsorbed onto immobilized recombinant biotinylated IL-4Rα-Fc or IL-5 and eluted using trypsin, as previously described (De Haard et al., (1999) Journal of Biological Chemistry, 274: 18218-30). Three rounds of selection were performed to enrich for phages expressing IL-4Rα- or IL-5-specific Fabs. Finally, TG1 E. coli was infected with the selected phages, and individual colonies were isolated. Secretion of Fab fragments into the periplasm of the E. coli strain TG1 was induced using isopropyl β-D-1-thiogalactopyranoside (Sigma-Aldrich) under low glucose concentrations (0.1% w / v), and the Fab-containing periplasmic fraction of the bacteria was collected.

[0201] C. Screening, Characterization, and Generation of Fabs The binding of Fabs to mouse or human targets, respectively, and their ability to neutralize them were determined by surface plasmon resonance (SPR) using a Biacore 3000 instrument (GE Healthcare). IL-4Rα-Fc and IL-5 were immobilized on a carboxymethyl dextran sensor chip (CM-5) using amine coupling in sodium acetate buffer (GE Healthcare). Fab-containing periplasmic extracts were loaded at a flow rate of 30 μL / min. Fab off-rates were measured over 90 seconds.

[0202] D. Monospecific Antibody Generation, Purification, and Characterization cDNAs encoding the VH and VL (lambda or kappa) domains of potently neutralizing IL-4Rα- and IL-5-specific selected Fab fragments were engineered into two separate pUPE mammalian expression vectors containing cDNAs encoding the CH1, CH2, and CH3 domains of mouse IgG2a (or human IgG1) containing mutations that abolish antibody effector functions mediated by Fc receptors or CL (lambda or kappa), respectively. The best potent neutralizing anti-IL-4Rα and anti-IL-5 IgG2a (or IgG1) molecules were then generated (by transient transfection of mammalian cells) and purified (by protein A affinity chromatography) as previously described (Basilico et al., (2014) Journal of Clinical Investigation 124:3172). The CDR, VH, and VL sequences of the selected antibodies are shown in Tables 3 to 14 below.

[0203] [Table 3]

[0204] [Table 4]

[0205] [Table 5]

[0206] [Table 6]

[0207] [Table 7]

[0208] [Table 8]

[0209] [Table 9]

[0210] [Table 10]

[0211] [Table 11]

[0212] [Table 12]

[0213] [Table 13]

[0214] [Table 14]

[0215] Example 2: In vitro characterization of IL-4Rα and IL-5 monoclonal antibodies Murine IL-4Rα and IL-5 monoclonal antibodies (m4RMP36B7 and m5MP95G7) were tested for their ability to bind to their respective targets in vitro and to inhibit the cellular effects mediated by IL-4Rα and IL-5 signaling.

[0216] A. Inhibition of IL4- and IL5-induced HT-2 and TF-1 cell proliferation The neutralizing activity of both IL-4Rα and IL-5 monospecific antibodies was assessed in an in vitro cell assay in which murine IL-4 and murine IL-5, respectively, induce proliferation of HT-2 and TF-1 cells.

[0217] Human TF-1 cells (erythroblasts, ATCC® CRL-2003™) and murine HT-2 clone A5E cells (IL-2-dependent T lymphocytes, ATCC® CRL-1841™) were cultured at 37°C in 5% (v / v) CO2 in growth medium containing RPMI 1640 (Sigma), 10% (v / v) heat-inactivated fetal bovine serum (Sigma), 1X gentamycin (Sigma), and 2 ng / mL human granulocyte-macrophage colony-stimulating factor (R&D Systems) or human IL-2 (R&D Systems), respectively. The assay medium was growth medium without human GM-CSF for TF-1 cells and without human IL-2 for HT-2 cells. 0.5 ng / mL mouse IL-5 (R&D Systems) for TF-1 cells or mouse IL-4 (R&D Systems) for HT-2 cells was added to the assay medium. The antibodies were serially diluted 10-fold for TF-1 cells and 5-fold for HT-2 cells in assay medium containing 1 ng of mouse IL-5 for TF-1 cells or 0.75 ng of mouse IL-4 for HT-2 cells. Cells were incubated at 37°C for 1 hour, washed, and TF-1 cells were diluted to 1.1 x 10 6 cells / mL, HT-2 cells were 0.2 × 10 6 The cells were resuspended to a final volume of 1000 cells / mL. Cells were then added to each well, followed by the addition of CellTiter 96® AQueous One Solution Reagent (Promega). After a 3-hour incubation, absorbance was measured.

[0218] As shown in Figure 1, IL-4Rα (squares) and IL-5 (triangles) monospecific antibodies potently inhibited murine IL-4- and IL-5-induced HT-2 and TF-1 cell proliferation, respectively. In particular, IL-4Rα and IL-5 antibodies could block murine IL-4- and IL-5-induced cellular proliferation with comparable EC50 values ​​of 0.2 nM and 0.6 nM, respectively.

[0219] B. Binding of IL-4Rα and IL-5 Monoclonal Antibodies to IL-4Rα and IL-5, Respectively Figure 2 shows representative surface plasmon resonance (SPR) sensorgrams of the interaction between monoclonal antibodies (IL-4Rα antibody, IL-5 antibody, or irrelevant IgG2a antibody) at various concentrations (0-20 μg / mL) and immobilized targets (IL-4Rα or IL-5). Both monoclonal antibodies bound to their targets, whereas the irrelevant IgG2a did not bind to either target. The IL-4Rα and IL-5 monospecific antibodies bound to their targets with affinities of 8E-11 M and 2E-12 M, respectively.

[0220] Furthermore, the ability of monospecific antibodies to compete with their respective targets was tested by SPR. Figure 3 shows representative SPR sensorgrams of competitive interactions between a mixture of a monoclonal antibody (IL-4Rα, IL-5, or irrelevant IgG2a) and its target (IL-4Rα or IL-5) and an immobilized protein (IL-4, IL-13Rα, or IL-5Rα). The mixture of IL-4Rα monoclonal antibody and IL-4Rα inhibited the binding of IL-4Rα to the immobilized IL-4 ligand. The mixture of IL-4Rα monoclonal antibody and IL-13 inhibited the binding of IL-13 to immobilized IL-13Rα. Similarly, the mixture of IL-5 monoclonal antibody and IL-5 inhibited the binding of IL-5 to immobilized IL-5Rα.

[0221] C. Inhibition of IL-4-induced MHC class II antigen expression in purified B cells analyzed by FACS. FACS analysis was used to test whether the IL-4Rα monospecific antibody competes with mouse IL-4, which is known to induce MHC class II translocation to the surface of B cells.

[0222] For FACS analysis, B cells were collected using magnetic-activated cell sorting mouse anti-CD19 microbeads (Miltenyi Biotec) according to the manufacturer's protocol. Purified B cells (5 × 10 5 Cells (1000 cells / mL) were cultured in 24-well plates (Costar) for 16 hours with or without IL-4 (0.1 ng / mL), in the presence of 500 ng / mL anti-IL-4Rα or irrelevant IgG2a antibody, or in medium control with or without inhibitors. Cells were washed and preincubated on ice for 20 minutes with rat IgG2b anti-mouse FcγR monospecific Ab 2.4G2 (Bioceros) to block IgG Fc receptors. Cells were then stained with antibodies against MHC class II (M5 / 114.15.2, eBioscience) and CD19 (1D3, eBioscience), incubated for 30 minutes at 4°C, and then washed. Dead cells were excluded from analysis using a fixable viability stain (eFluor 506, eBioscience). Stained cells were analyzed on an LSR Fortessa flow cytometer (BD Biosciences). Final analysis and image output were performed using FlowJo v10.0.7 software (Tree Star).

[0223] As shown in Figure 4, MHC class II antigen expression in B cells was significantly stronger in the presence of IL-4 than in the absence of IL-4. IL-4Rα monoclonal antibody potently inhibited IL-4-induced MHC class II antigen expression in purified B cells.

[0224] Example 3: In vivo characterization of anti-mouse IL-4Rα and IL-5 monoclonal antibodies in a murine house dust mite (HDM) model House dust mites (HDM, Dermatophagoides species) are one of the most common allergens worldwide. 50–85% of asthma patients are allergic to HDM. Prolonged exposure to HDM leads to airway remodeling with increased mucus cell density and airway hyperresponsiveness, which persists even after cessation of HDM exposure. The murine HDM model is a useful in vivo model for studying asthma.

[0225] (Experimental setting 1) Female C57Bl / 6J wild-type mice were obtained from The Jackson Laboratory. This experiment was approved by the VIB-UGent Center Ethics Committee for Inflammation Research. All mice were between 6 and 8 weeks of age. Experiments were performed using age-matched groups. As shown in Figure 5A, the experimental design included antibody treatment given throughout the full sensitization and challenge periods. On day 0, mice were lightly anesthetized with isoflurane (2.5% in air) via the intratracheal route and administered 1 μg of HDM (Greer Laboratories). From days 6 to 10, mice were lightly anesthetized with isoflurane (2.5% in air) via the intranasal route and challenged daily with 10 μg of HDM. On days 1, 1, 6, 8, and 10, mice were treated with one of the following: (i) IL-4Rα monospecific antibody (m4RMP36B7), (ii) IL-5 monospecific antibody (m5MP95G7), (iii) a combination of IL-4Rα and IL-5 monospecific antibodies (m4RMP36B7 and m5MP95G7), or (iv) an irrelevant IgG2a antibody. Days 1 and 1 represent the sensitization phase, and days 6, 8, and 10 represent the challenge phase. At least n = 6 mice were treated per group.

[0226] A. Potent neutralizing IL-4Rα and IL-5 monoclonal antibodies injected in combination into a murine HDM model of asthma reduce eosinophilia. BAL fluid collection and analysis. On day 14, mice were euthanized. Bronchoalveolar lavage (BAL) was performed via cannulation using 3 × 1 mL of PBS containing EDTA, and the cellular composition of BAL fluid was determined by fluorescence-activated cell sorting (FACS) as previously described (Deckers et al. (2017) Journal of Allergy and Clinical Immunology 140(5), 1364-1377; Dullaers et al. (2017) Journal of Allergy and Clinical Immunology, 140:76-88; Schuijs et al. (2015) Science, 349:1106-10).

[0227] Figure 5B shows the differential cell counts in bronchoalveolar lavage fluid from mice administered IL-4Rα monoclonal antibody, IL-5 monoclonal antibody, a combination of IL-4Ra and IL-5 monoclonal antibodies, or an irrelevant IgG2a antibody. Cells were counted by FACS analysis. In IgG2a-treated HDM-sensitized mice, the number of eosinophil cells increased upon allergen challenge. A significant decrease in the number of eosinophil cells was observed after treatment with the IL-4α / IL-5 monospecific antibody combination compared to treatment with the control IgG2a antibody.

[0228] (Experimental setting 2) A second experimental protocol was designed to further test the effects of IL-4Rα and IL-5 antibodies in a mouse HDM model in a clinically relevant therapeutic setting. The experimental design shown in Figure 6 differs from that of Figure 5A in that the procedure involving antibody treatment was performed only during the challenge phase, not during the sensitization phase. Therefore, mice received their treatment (IL-4Rα monospecific antibody; IL-5 monospecific antibody; IL-4Rα / IL-5 monospecific antibodies, or an irrelevant IgG2a antibody) 4 hours before challenge only on days 6, 8, and 10. Mice were administered 150 μg of either monospecific antibody, 150 μg of each monospecific antibody in combination, or 150 μg of an irrelevant mouse IgG2a monoclonal antibody. At least n = 6 mice were treated per group.

[0229] Mice treated according to this protocol were subsequently evaluated for total inflammatory cell counts in BAL fluid (Figure 7); cytokine production in mediastinal lymph nodes (Figure 8); serum immunoglobulin production (Figure 9); signs of goblet cell metaplasia (Figure 10); and bronchial hyperresponsiveness (Figure 11).

[0230] A potently neutralizing IL-4Rα and IL-5 monoclonal antibody reduces eosinophilia when infused in combination into a murine HDM model of asthma. BAL fluid collection and analysis. On day 14, mice were euthanized. Bronchoalveolar lavage (BAL) was performed via cannulation using 3 × 1 mL of PBS containing EDTA, and the cellular composition of BAL fluid was determined by fluorescence-activated cell sorting (FACS) as previously described (Deckers et al. (2017) Journal of Allergy and Clinical Immunology 140(5), 1364-1377; Dullaers et al. (2017) Journal of Allergy and Clinical Immunology, 140:76-88; Schuijs et al. (2015) Science 349:1106-10).

[0231] Figure 7 shows differential cell counts in BAL analyzed by FACS from HDM-treated mice administered IL-4Rα monoclonal antibody, IL-5 monoclonal antibody, IL-4Rα / IL-5 combination monoclonal antibody, or irrelevant IgG2a antibody. In mice treated with irrelevant IgG2a antibody, airway HDM exposure increased the total number of inflammatory cells in BAL fluid compared with mice challenged with PBS. More specifically, it increased the numbers of eosinophils, lymphocytes, and macrophages. Interestingly, eosinophil counts were significantly reduced after monotherapy treatment compared with treatment with the control IgG2a antibody. The lowest number of eosinophils was observed in mice treated with this combination therapy.

[0232] B. IL-4Rα monoclonal antibody and IL-4Rα / IL-5 monoclonal antibody combination reduce cytokine production. The effects of IL-4Rα and IL-5 monoclonal antibodies on cytokine production in allergen-restimulated mesenteric lymph node (MLN) cells were examined in vitro. Single cell suspensions (2 × 10 6 Cells (cells / mL) were obtained from MLNs by homogenizing the organs through a 100 μm cell sieve. Cells were restimulated ex vivo in 96-well round-bottom plates with 15 μg / mL HDM for 3 days, supernatants were collected, and cytokine production was determined using Read-SET-Go!® ELISA sets (eBioscience).

[0233] As shown in Figure 8, in vitro production of the effector cytokines IL-5 and IL-13 in cultures of allergen-restimulated MLN cells was boosted by allergen challenge in IgG2a-treated HDM-sensitized mice, but this response was significantly reduced after treatment with IL-4Rα monospecific antibody and the IL-4Rα and IL-5 monoclonal antibody combination, whereas IL-5 monospecific antibody had no significant effect.

[0234] C. IL-4Rα monoclonal antibody and IL-4Rα / IL-5 monoclonal antibody combination reduce HDM-specific IgE and IgG1 production. Blood was collected from the iliac vein, serum was then prepared, and the amount of HDM-specific IgG1 and IgE was determined as previously described (Schuijs et al., (2015) Science, 349:1106-10).

[0235] As shown in Figure 9, serum concentrations of HDM-specific IgG1 and IgE were boosted by allergen challenge in IgG2a antibody-treated mice. IL-4Rα monoclonal antibody and the IL-4Rα / IL-5 monoclonal antibody combination were able to significantly reduce this allergen-induced increase in IgG. IL-4Rα monoclonal antibody, IL-5 monoclonal antibody, and the IL-4Rα / IL-5 monoclonal antibody combination were able to significantly reduce the allergen-induced increase in IgE.

[0236] D. The combination of IL-4Rα and IL-5 monoclonal antibody reduces Muc5AC and Agr2 expression. Mucin expression in the lungs was assessed by immunostaining. Lungs were infused with PBS / OCT (1:1) solution, flash-frozen in liquid nitrogen, and kept at −80°C until further processing for Muc5AC immunofluorescence staining as previously described (Deckers et al. (2017) Journal of Allergy and Clinical Immunology 140(5), 1364-1377).

[0237] Goblet cell metaplasia (GCM) is induced by IL-13 and is characterized by increased production of Muc5AC, a gel-forming mucin present in the airways of asthmatic mice and humans. In IgG2a antibody-treated, HDM-sensitized mice, HDM challenge upregulated Muc5AC expression in the airway epithelium compared with PBS challenge (Figure 10A). The increased Muc5AC staining was not affected by IL-4Rα or IL-5 antibody monotherapy. However, strikingly, lungs of mice treated with the IL-4Rα / IL-5 antibody combination showed significantly reduced Muc5AC staining intensity compared with the other HDM-sensitized and challenged groups.

[0238] To confirm and quantify the effects of IL-4Rα and IL-5 monoclonal antibodies on GCM, the mRNA expression levels of Muc5ac, as well as Agr2, another IL-13 / STAT6 downstream target gene involved in GCM, were measured by qRT-PCR in lung tissues.

[0239] Lungs were snap-frozen in liquid nitrogen and kept at -80°C until further processing for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) as previously described (Dullaers et al., (2017) Journal of Allergy and Clinical Immunology, 140:76-88). Briefly, RNA was obtained and isolated using TriPure Isolation Reagent (Roche, Mannheim, Germany) according to the manufacturer's protocol. RNA was reverse-transcribed using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche), and samples were analyzed using SYBR Green-based qRT-PCR on a LightCycler 480 system (Roche) against reference genes (Rpl13a, Hprt, and Sdha).

[0240] As shown in Figure 10B, the mRNA expression levels of these two genes were induced by HDM challenge in mice. IL-4Rα and IL-5 antibodies alone did not significantly reverse this increase in Muc5ac mRNA or Agr2 mRNA levels. However, the combination of IL-4Rα and IL-5 monoclonal antibodies significantly reduced the HDM-mediated increase in Muc5ac or Agr2 mRNA levels.

[0241] (E. The combination of IL-4Rα and IL-5 monoclonal antibodies reduces lung resistance.) Bronchial hyperresponsiveness (BHR) is the tendency of airways to constrict in response to small doses of bronchoconstrictors, such as the muscarinic receptor agonist methacholine. Pulmonary function was measured using invasive measurements of dynamic resistance (Flexivent, Scireq).

[0242] Twenty-four hours after the final HDM challenge, nonspecific airway responsiveness was measured by exposing awake mice to aerosolized PBS using an ultrasonic nebulizer to establish a baseline value, followed by increasing concentrations of aerosolized methacholine (0–400 μg / kg). For invasive measurements of dynamic resistance, mice were anesthetized with urethane, tracheotomized, and intubated with an 18-gauge catheter, followed by mechanical ventilation using a Flexivent device (SCIREQ). Respiratory frequency was set at 120 breaths / min with a tidal volume of 0.2 mL, and a positive end-expiratory pressure of 2 mL HO was applied. Dynamic resistance was recorded after standardized inhalation maneuvers every 10 seconds for 2 minutes. Baseline resistance was allowed to recover before the next dose of methacholine was administered.

[0243] As shown in Figure 11, methacholine increased airway resistance in control IgG2a antibody-treated mice and HDM-challenged sensitized mice compared with PBS-challenged HDM-sensitized mice. Treatment with IL-5 and IL-4Rα antibodies did not significantly reduce BHR. However, mice treated with a combination of IL-4Rα and IL-5 monoclonal antibodies were completely protected from developing BHR. These results demonstrate the synergistic effect of the IL-4Rα / IL-5 antibody combination in treating fundamental aspects of chronic airway disease.

[0244] Overall, these results demonstrated that the αIL-5 and αIL-4Rα monospecific antibody combination given during challenge of HDM-treated mice has synergistic effects on important aspects of asthma, including GCM and BHR.

[0245] Statistical analysis was performed using GraphPad Prism software v7.01 and Genstat software v19. For all experiments, results were expressed as mean ± standard error of the mean (SEM), and differences between groups were calculated using one-way ANOVA tests. Differences between groups were considered significant if *P≦0.05, **P≦0.01, ***P≦0.001, and ****P≦0.0001 relative to the HDM-sensitized and challenged control IgG2 antibody-treated group.

[0246] Example 4: Generation and in vitro characterization of IL-4Rα / IL-5 bispecific antibodies An IL-4Rα / IL-5 bispecific antibody was generated using the sequences of the 4RMP36B7 IL-4Rα antibody and the 5MP95G7 IL-5 antibody. The Fc portion of the antibody was modified to contain "knob-into-hole"-like mutations that facilitate correct chain pairing in the context of a bispecific antibody (Ridgway et al., (1996) Protein Engineering, Design and Selection, 9:617-21). Specifically, the IL-4Rα antibody 4RMP36B7 was engineered to introduce substitutions T366S, L368A, and Y407V in the CH3 domain of the mouse Fc region. The IL-5 antibody 5MP95G7 was engineered to introduce substitution T366W in the CH3 domain of the mouse Fc region.

[0247] As described in Godar et al. (2016, Scientific Reports, 6:31621), two mutant heavy chains and two light chains of IL-4Rα and IL-5 antibody were coexpressed, and bispecific antibodies containing the correct heavy-light chain pair were purified using anti-idiotypic VHH antibodies. The purification process is illustrated schematically in Figure 12.

[0248] The purity of the IL-4Rα / IL-5 bispecific antibody was confirmed using high-resolution spectroscopy, and the dual targeting properties of the antibody were confirmed by BIAcore.

[0249] A. Binding of IL-4Rα / IL-5 Bispecific Antibodies to IL-4Rα and IL-5 The bispecificity of the IL-4Rα / IL-5 antibody was confirmed using SPR, as shown in Figure 13. The bispecific antibody specifically bound to IL-4Rα-Fc immobilized on a chip, and then an increase in signal was observed upon addition of IL-5. This indicated that the bispecific antibody was able to bind to the coated IL-4Rα and also to IL-5 in solution (Figure 13A). The experiment was performed in the reverse order, confirming the bispecificity of the molecule, as shown in Figure 13B.

[0250] Taken together, these results demonstrate that the dual anti-idiotype approach allows the isolation of pure and functional IL-4Rα / IL-5 bispecific antibodies.

[0251] Example 5: In vivo characterization of anti-mouse IL-4Rα / IL-5 bispecific antibodies in a mouse HDM model The IL-4Rα / IL-5 bispecific antibody generated in Example 4 was tested in the mouse HDM model described above in Example 3. The experimental protocol is shown in Figure 14. Mice were subjected to an HDM sensitization and challenge protocol, with antibody treatment only during the challenge phase.

[0252] The in vivo activity of IL-4Rα / IL-5 bispecific antibodies was compared to monotherapy (IL-4Rα or IL-5 monospecific antibodies) and combinations (αIL-4Rα / αIL-5 monospecific antibodies). To compare equimolar inhibition of the target and eliminate differences in total antibody amounts, antibodies were dosed as follows: 75 μg of each monospecific antibody and 75 μg of an irrelevant IgG2a antibody combination for testing individual antibodies; 75 μg of each monospecific antibody combined at the time of injection; and 150 μg of the bispecific antibody.

[0253] Mice treated according to this protocol were subsequently evaluated for total inflammatory cell counts in BAL fluid (Figure 15), cytokine production in mediastinal lymph nodes (Figure 16), serum immunoglobulin production (Figure 17), signs of goblet cell metaplasia (Figure 18), and bronchial hyperresponsiveness (Figure 19). The experimental protocol was performed as described in Example 3 above.

[0254] A. IL-4Rα / IL-5 bispecific antibody infused into a murine HDM model of asthma reduces eosinophilia. As shown in Figure 15, HDM challenge in sensitized mice increased the number of eosinophils in the BAL fluid. This increase in eosinophil count was significantly reduced in mice administered the combination of IL-4Rα and IL-5 antibodies (75 μg + 75 μg). Notably, the bispecific IL-4Rα / IL-5 antibody (150 μg) was as effective as the combination of both IL-4Rα and IL-5 monoclonal antibodies in reducing airway eosinophilia and lymphocytosis induced by allergen challenge.

[0255] B. IL-4Rα / IL-5 bispecific antibodies reduce cytokine production. As shown in Figure 16, HDM-induced MLN2-type cytokine (IL-5 and IL-13) levels were significantly reduced after treatment with the combination of IL-4Rα and IL-5 antibodies, as well as the bispecific antibody.

[0256] C. IL-4Rα / IL-5 bispecific antibodies reduce HDM-specific IgE and IgG1 production. As shown in Figure 17, HDM-specific IgE and IgG1 were also significantly reduced after treatment with the combination of IL-4Rα and IL-5 antibodies and the bispecific antibody.

[0257] D. IL-4Rα / IL-5 bispecific antibodies reduce Muc5AC and Agr2 expression. To examine and compare the effects of bispecific antibodies on goblet cell metaplasia (GCM), we measured mRNA expression levels of Muc5ac and Agr2 in lung tissue. The increased expression of Muc5ac and Agr2 observed in HDM-sensitized and challenged mice was significantly reduced by the combination of IL-4Rα and IL-5 antibodies, as well as by the bispecific antibody (Figure 18). Individual IL-4Rα and IL-5 antibody treatments had no significant effect.

[0258] (E. IL-4Rα / IL-5 bispecific antibodies reduce lung resistance.) Treatment with the combination of IL-4Rα and IL-5 antibodies, as well as with the bispecific antibody, similarly protected the lungs from HDM-induced BHR, as assessed by methacholine-induced bronchoconstriction. As shown in Figure 19, mice treated with the combination of IL-4Rα and IL-5 monoclonal antibodies and the bispecific antibody were completely protected from the development of BHR. In contrast, the resistance observed in HDM-sensitized and challenged mice treated with individual IL-4Rα and IL-5 antibodies was not significantly reduced. This demonstrates the synergistic effect of combination therapy (either the co-administration format of individual IL-4Rα and IL-5 antibodies, or the bispecific antibody) in treating cardinal aspects of chronic airway disease, particularly asthma.

[0259] Taken together, these results demonstrate that a single bispecific antibody simultaneously targeting IL-4Rα and IL-5 is effective in alleviating all prominent asthmatic features when administered during the challenge phase of an HDM-induced asthma model.

[0260] Example 6: In vitro characterization of IL-4Rα and IL-5 monoclonal antibodies Human IL-4Rα and IL-5 monoclonal antibodies (h4RMP5D1, h4RMP3B2, 4RMP3D6 and h5MP90A9, h5MP90D9, h5MP92B4, h5MP90C8, h5MP90E7, h5MP90G7) were tested for their ability to bind to their respective targets in vitro and inhibit the cellular effects mediated by IL-4Rα and IL-5 signaling as described in Example 2. The proliferation assay was modified to use hIL-4 and hIL-5 at concentrations that resulted in suboptimal cell proliferation. The results are shown in the table below.

[0261] Table 15 [Table 15]

[0262] Table 16 [Table 16]

[0263] Example 7: Generation and in vitro characterization of a second IL-4Rα / IL-5 bispecific antibody A second IL-4Rα / IL-5 bispecific antibody was constructed using the sequences of the 4RMP36B7 IL-4Rα antibody and the 5MP95G7 IL-5 antibody. The VH-VL domains of the 5MP95G7 antibody were constructed as single-chain Fv (scFv) fragments with either a 15- or 20-amino acid linker (15GS = (GGGS)3 or 20GS = (GGGS)4), and two of these scFv fragments were connected to the C-terminus of the Fc domain of the 4RMP36B7 IgG antibody via a (GGGS)3 connector (15GS). Furthermore, two mutations were introduced into VH95G7 (G44C) and VL97G7 (G100C) to stabilize the scFv. A schematic diagram of this second IL-4Rα / IL-5 bispecific antibody is shown in Figure 20.

[0264] This second IL-4Rα / IL-5 bispecific antibody was tested for its ability to inhibit IL-5-induced proliferation of TF-1 cells. The bispecific antibody was tested together with the IL-4Rα antibody 36B7hIgG1 and the IL-5 antibodies 95G7hIgG1 and 95G7mIgG2a.

[0265] The assay was performed essentially as described in Example 2 above. Specifically, a fixed concentration of mIL-5 (R&D Systems) was preincubated with 5-fold serial dilutions of antibody (starting at 100 nM) and added to 500,000 human TF-1 cells (erythroblasts, ATCC® CRL-2003™) (final murine IL-5 concentration (R&D Systems) 0.25 ng / mL), and the cells were incubated at 37°C with 5% CO2 for 48 hours. 20 μl of CellTiter 96® AQueous One solution reagent (Promega) was added, and the cells were incubated at 37°C for 3 hours. Absorbance was measured at A490 vs. A655. The results are shown in Figure 21 and Table 17 below.

[0266] Table 17 [Table 17]

[0267] As expected, the IL-4Ra antibody had no effect on TF-1 cell proliferation, whereas the 95A7 antibody, formulated as either a human IgG1 or mouse IgG2a antibody, was able to inhibit IL-5-induced cell proliferation. Interestingly, the IL-4Rα / IL-5 bispecific antibody was able to inhibit IL-5-induced cell proliferation with much greater potency than the 95A7 IgG antibody, a 100-fold increase in potency. In other words, the VH-VL domain of the 95A7 antibody, formulated as an scFv fragment at the C-terminus of the Fc region of the IL-4Rα IgG antibody, was much more potent than when formulated as a Fab arm in the native IgG conformation.

[0268] Example 8: In vivo characterization of anti-mouse IL-4Rα / IL-5 IgG-scFv bispecific antibodies in a mouse HDM model The IL-4Rα / IL-5 bispecific antibody described in Example 7 above was tested in the murine HDM model described in Examples 3 and 5 above. The experimental protocol is shown in Figure 22. Mice underwent the HDM sensitization and challenge protocol, with antibody treatment only during the challenge phase.

[0269] The in vivo activity of the IL-4Rα / IL-5 bispecific antibody (4Rsc5) was compared with the combination of anti-mIL-4Rα (36B7) and anti-mIL5 (95G7). Each injection contained either 150 μg or 75 μg of the bispecific (4Rsc5), or the combination (75 μg of each antibody for a total of 150 μg, or 36.75 μg of each antibody for a dose of 75 μg per injection).

[0270] Mice treated according to this protocol were subsequently evaluated for total eosinophil and lymphocyte cell counts in BAL fluid (Figure 23), and expression of muc5a, spdef, and agr2 (Figure 24). The experimental protocol was performed as described in Example 3 above.

[0271] Example 9: Design of human IL-4Rα antibody The human IL-4Rα monoclonal antibody, 4RMP3D6, was designed to cross-react with Cyno / Rhesus (cynomolgus / rhesus) IL-4Rα. Variants of 4RMP3D6 were generated by random mutagenesis using an error-prone PCR approach. For this purpose, the GeneMorphII EZClone Domain Mutagenesis Kit (Agilent) was used. Mutations were introduced into the VH and VK of 4RMP3D6 according to the supplier's recommendations. The mutated VH (VHm * ) and VK(VKm * ) was further cloned into a phagemid vector (PCB13) containing the CH1 and CK constant domains to generate a mutant Fab library.

[0272] Sequencing showed that this procedure led to approximately 3–5 amino acid substitutions within each V domain (VH and VK). * / VKwt, VHwt / VKm * and VHm * / VKm * ) were generated and used for several rounds of phage display selection on cynomolgus monkey recombinant IL-4Rα (agroBioscience cat#ILR-C52H8). After three rounds of selection, clones (from rounds 2 and 3) were selected, and Fabs were generated from periplasmic extracts and tested by SPR. For SPR, a Biacore 3000 was used in combination with CM5 chips coated with either human IL-4Rα or cynomolgus monkey IL-4Rα. Binding (R0) and dissociation (kd, s-1) were recorded as shown in Table 18 below.

[0273] Table 18 [Table 18]

[0274] The CDR, VH and VL sequences of the selected antibodies are shown in Tables 19 to 21 below.

[0275] [Table 19]

[0276] [Table 20]

[0277] [Table 21] The present application provides the following aspects of the invention. (Aspect 1) (i) an antagonist of IL-5:IL-5R; and (ii) an antagonist of IL-4:IL-4R and / or an antagonist of IL-13:IL-13R; A combination including: (Aspect 2) (i) an antagonist of IL-5:IL-5R; and (ii) IL-4Rα antagonist 2. The combination of embodiment 1, comprising: (Aspect 3) 3. The combination according to embodiment 1 or 2, which inhibits signaling mediated by IL-5, IL-4 and IL-13. (Aspect 4) The combination according to any one of aspects 1 to 3, wherein the antagonist of (i) is an antibody molecule and / or at least one antagonist of (ii) is an antibody molecule. (Aspect 5) The combination according to any one of Aspects 1 to 4, wherein the antagonist (i) is an antibody molecule that binds to IL-5, preferably human IL-5. (Aspect 6) The combination according to any one of Aspects 2 to 5, wherein the antagonist (ii) is an antibody molecule that binds to IL-4Rα, preferably human IL-4Rα. (Aspect 7) The combination according to any one of Aspects 4 to 6, wherein the antibody molecule of (i) and / or the antibody molecule of (ii) is independently selected from the group consisting of an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single-chain antibody (scFv), an F(ab')2 fragment, an Fab fragment, an Fd fragment, an Fv fragment, a single-arm (monovalent) antibody, a diabody, a triabody, a tetrabody, a VHH antibody, or an antigen-binding molecule formed by a combination, assembly, or conjugation of antigen-binding fragments thereof. (Aspect 8) The combination according to any one of Aspects 4 to 6, wherein the antibody molecule (i) and / or the antibody molecule (ii) is an IgG antibody. (Aspect 9) The combination according to any one of aspects 4 to 8, wherein the antibody molecule of (i) and / or the antibody molecule of (ii) is a humanized or germline variant of a non-human antibody. (Aspect 10) 10. The combination of embodiment 9, wherein the non-human antibody is of camelid origin. (Aspect 11) The combination according to any one of Aspects 4 to 10, wherein the antibody molecule (i) and / or the antibody molecule (ii) comprises a CH1 domain, a hinge region, a CH2 domain, and / or a CH3 domain of human IgG. (Aspect 12) 12. The combination according to any one of aspects 4 to 11, wherein the antibody molecule (i) and / or the antibody molecule (ii) exhibits high homology to human IgG, preferably IgG1. (Aspect 13) The combination according to any one of Aspects 4 to 12, wherein the antibody molecule (i) and / or the antibody molecule (ii) comprises an Fc domain derived from human IgG, preferably IgG1. (Aspect 14) 14. The combination of embodiment 13, wherein the Fc domain has been modified by one or more amino acid substitutions to increase binding affinity to FcRn. (Aspect 15) 15. The combination of embodiment 14, wherein the Fc domain comprises the amino acid substitutions H433K and N434F. (Aspect 16) 15. The combination of embodiment 14, wherein the Fc domain comprises the following amino acid substitutions: M252Y, S254T, T256E, H433K, and N434F. (Aspect 17) The antagonist (ii) is an antibody molecule that binds to IL-4Rα, the antibody molecule comprising a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains are (i) an HCDR3 comprising or consisting of SEQ ID NO:3; an HCDR2 comprising or consisting of SEQ ID NO:2; an HCDR1 comprising or consisting of SEQ ID NO:1; an LCDR3 comprising or consisting of SEQ ID NO:12; an LCDR2 comprising or consisting of SEQ ID NO:11; an LCDR1 comprising or consisting of SEQ ID NO:10, (ii) an HCDR3 comprising or consisting of SEQ ID NO:6; an HCDR2 comprising or consisting of SEQ ID NO:5; an HCDR1 comprising or consisting of SEQ ID NO:4; an LCDR3 comprising or consisting of SEQ ID NO:15; an LCDR2 comprising or consisting of SEQ ID NO:14; an LCDR1 comprising or consisting of SEQ ID NO:13; and (iii) an HCDR3 comprising or consisting of SEQ ID NO:9; an HCDR2 comprising or consisting of SEQ ID NO:8; an HCDR1 comprising or consisting of SEQ ID NO:7; an LCDR3 comprising or consisting of SEQ ID NO:18; an LCDR2 comprising or consisting of SEQ ID NO:17; an LCDR1 comprising or consisting of SEQ ID NO:16, 17. The combination according to any one of embodiments 2 to 16, comprising a CDR sequence selected from the group consisting of: (Aspect 18) The antagonist (ii) is an antibody molecule that binds to IL-4Rα, the antibody molecule comprising a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains are (i) an HCDR3 comprising or consisting of SEQ ID NO: 27; an HCDR2 comprising or consisting of SEQ ID NO: 26; an HCDR1 comprising or consisting of SEQ ID NO: 25; an LCDR3 comprising or consisting of SEQ ID NO: 36; an LCDR2 comprising or consisting of SEQ ID NO: 35; an LCDR1 comprising or consisting of SEQ ID NO: 34, (ii) an HCDR3 comprising or consisting of SEQ ID NO: 30; an HCDR2 comprising or consisting of SEQ ID NO: 29; an HCDR1 comprising or consisting of SEQ ID NO: 28; an LCDR3 comprising or consisting of SEQ ID NO: 39; an LCDR2 comprising or consisting of SEQ ID NO: 38; an LCDR1 comprising or consisting of SEQ ID NO: 37; and (iii) an HCDR3 comprising or consisting of SEQ ID NO: 33; an HCDR2 comprising or consisting of SEQ ID NO: 32; an HCDR1 comprising or consisting of SEQ ID NO: 31; an LCDR3 comprising or consisting of SEQ ID NO: 42; an LCDR2 comprising or consisting of SEQ ID NO: 41; an LCDR1 comprising or consisting of SEQ ID NO: 40, 17. The combination according to any one of embodiments 2 to 16, comprising a CDR sequence selected from the group consisting of: (Aspect 19) The antagonist (ii) is an antibody molecule that binds to IL-4Rα, and the antibody molecule is: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 70% identity thereto; (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 70% identity thereto; and (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 70% identity thereto; 19. The combination according to any one of aspects 2 to 18, comprising a variable heavy domain (VH) and a variable light domain (VL) selected from the group consisting of: (Aspect 20) The antagonist (ii) is an antibody molecule that binds to IL-4Rα, and the antibody molecule is: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 70% identity thereto; (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 70% identity thereto; and (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 70% identity thereto; 19. The combination according to any one of aspects 2 to 18, comprising a variable heavy domain (VH) and a variable light domain (VL) selected from the group consisting of: (Aspect 21) The antagonist of (i) is an antibody molecule that binds to IL-5, the antibody molecule comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains are: (i) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 54; an LCDR2 comprising or consisting of SEQ ID NO: 53; an LCDR1 comprising or consisting of SEQ ID NO: 52, (ii) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 57; an LCDR2 comprising or consisting of SEQ ID NO: 56; an LCDR1 comprising or consisting of SEQ ID NO: 55, (iii) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1:X comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 59; an LCDR2 comprising or consisting of SEQ ID NO: 56; an LCDR1 comprising or consisting of SEQ ID NO: 58, (iv) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 61; an LCDR2 comprising or consisting of SEQ ID NO: 60; an LCDR1 comprising or consisting of SEQ ID NO: 58; and (v) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 62; an LCDR2 comprising or consisting of SEQ ID NO: 56; an LCDR1 comprising or consisting of SEQ ID NO: 58, 21. The combination according to any one of embodiments 2 to 20, comprising a CDR sequence selected from the group consisting of: (Aspect 22) The antagonist of (i) is an antibody molecule that binds to IL-5, the antibody molecule comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL domains are: (i) an HCDR3 comprising or consisting of SEQ ID NO: 72; an HCDR2 comprising or consisting of SEQ ID NO: 71; an HCDR1 comprising or consisting of SEQ ID NO: 70; an LCDR3 comprising or consisting of SEQ ID NO: 74; an LCDR2 comprising or consisting of SEQ ID NO: 38; an LCDR1 comprising or consisting of SEQ ID NO: 73; and (ii) an HCDR3 comprising or consisting of SEQ ID NO: 72; an HCDR2 comprising or consisting of SEQ ID NO: 71; an HCDR1 comprising or consisting of SEQ ID NO: 70; an LCDR3 comprising or consisting of SEQ ID NO: 75; an LCDR2 comprising or consisting of SEQ ID NO: 38; an LCDR1 comprising or consisting of SEQ ID NO: 73, 21. The combination according to any one of embodiments 2 to 20, comprising a CDR sequence selected from the group consisting of: (Aspect 23) The antagonist (i) is an antibody molecule that binds to IL-5, and the antibody molecule is: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least 70% identity thereto; (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 70% identity thereto; (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 70% identity thereto; (iv) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence having at least 70% identity thereto; (v) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 70% identity thereto; and (vi) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 70% identity thereto; 23. The combination according to any one of aspects 2 to 22, comprising a variable heavy domain (VH) and a variable light domain (VL) selected from the group consisting of: (Aspect 24) The antagonist (i) is an antibody molecule that binds to IL-5, and the antibody molecule is: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 70% identity thereto; (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence having at least 70% identity thereto; and (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 70% identity thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence having at least 70% identity thereto; 23. The combination according to any one of aspects 2 to 22, comprising a variable heavy domain (VH) and a variable light domain (VL) selected from the group consisting of: (Aspect 25) The combination according to any one of aspects 4 to 24, wherein the antibody molecule (i) and / or the antibody molecule (ii) exhibits lower antigen-binding activity at an acidic pH than at a neutral pH. (Aspect 26) 26. The combination according to embodiment 25, wherein the antibody molecule of (i) is an antibody molecule that binds to IL-5, and the antibody molecule of (ii) is an antibody molecule that binds to IL-4Rα. (Aspect 27) 27. The combination according to aspect 25 or 26, wherein the ratio between the antigen-binding activity at acidic pH and that at neutral pH is at least 2, as assessed by KD(acidic pH) / KD(neutral pH). (Aspect 28) A combination according to any one of aspects 1 to 27, wherein the antagonist of (i) and the antagonist of (ii) are co-formulated. (Aspect 29) 29. The combination according to embodiment 28, wherein the antagonist in (i) is an antibody molecule that binds to IL-5 and the antagonist in (ii) is an antibody molecule that binds to IL-4Rα. (Aspect 30) 30. The combination according to aspect 28 or 29, wherein said antagonist of (i) and said antagonist of (ii) are combined according to a ratio of 1:1 or 1:2 or 2:1. (Aspect 31) A combination according to any one of aspects 1 to 27, wherein the antagonist (i) and the antagonist (ii) are provided separately. (Aspect 32) 32. The combination of embodiment 31, wherein the antagonist of (i) is an antibody molecule that binds to IL-5 and the antagonist of (ii) is an antibody molecule that binds to IL-4Rα. (Aspect 33) 28. The combination according to any one of aspects 1 to 27, comprising antibody molecules that bind to IL-4Rα and antibody molecules that bind to IL-5, wherein said antibody molecules are combined in a multispecific antibody. (Aspect 34) 34. A combination according to aspect 33, wherein the antibody molecule that binds to IL-4Rα and the antibody molecule that binds IL-5 are combined in a bispecific antibody. (Aspect 35) The combination according to any one of aspects 1 to 34, wherein the combination comprises one or more additional therapeutic agents. (Aspect 36) A bispecific antibody comprising an antigen-binding region that binds to IL-4Rα and an antigen-binding region that binds to IL-5. (Aspect 37) 37. The bispecific antibody according to aspect 36, wherein the antigen-binding region that binds to IL-4Rα comprises a first variable heavy chain domain (VH) and variable light chain domain (VL) pair, and the antigen-binding region that binds to IL-5 comprises a second variable heavy chain domain (VH) and variable light chain domain (VL) pair. (Aspect 38) 38. The bispecific antibody of embodiment 37, wherein the antibody is an IgG antibody having a first VH-VL pair that binds to IL-4Rα and a second VH-VL pair that binds to IL-5. (Aspect 39) 38. The bispecific antibody of embodiment 36 or 37, wherein the antibody is an IgG antibody having at least one scFv fragment attached thereto. (Aspect 40) 40. The bispecific antibody of aspect 39, wherein the antigen-binding region that binds to IL-4Rα is contained within the IgG, and the antigen-binding region that binds to IL-5 is contained within at least one of the scFv fragments. (Aspect 41) the antigen-binding region that binds to IL-4Rα comprises a first variable heavy chain domain (VH) and a variable light chain domain (VL) pair, and the antigen-binding region that binds to IL-5 comprises a second variable heavy chain domain (VH) and a variable light chain domain (VL) pair; The first VH-VL domain pair is: (i) an HCDR3 comprising or consisting of SEQ ID NO:3; an HCDR2 comprising or consisting of SEQ ID NO:2; an HCDR1 comprising or consisting of SEQ ID NO:1; an LCDR3 comprising or consisting of SEQ ID NO:12; an LCDR2 comprising or consisting of SEQ ID NO:11; an LCDR1 comprising or consisting of SEQ ID NO:10; (ii) an HCDR3 comprising or consisting of SEQ ID NO:6; an HCDR2 comprising or consisting of SEQ ID NO:5; an HCDR1 comprising or consisting of SEQ ID NO:4; an LCDR3 comprising or consisting of SEQ ID NO:15; an LCDR2 comprising or consisting of SEQ ID NO:14; an LCDR1 comprising or consisting of SEQ ID NO:13; and (iii) an HCDR3 comprising or consisting of SEQ ID NO:9; an HCDR2 comprising or consisting of SEQ ID NO:8; an HCDR1 comprising or consisting of SEQ ID NO:7; an LCDR3 comprising or consisting of SEQ ID NO:18; an LCDR2 comprising or consisting of SEQ ID NO:17; an LCDR1 comprising or consisting of SEQ ID NO:16; and the second VH-VL domain pair being: (i) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 54; an LCDR2 comprising or consisting of SEQ ID NO: 53; an LCDR1 comprising or consisting of SEQ ID NO: 52; (ii) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 57; an LCDR2 comprising or consisting of SEQ ID NO: 56; an LCDR1 comprising or consisting of SEQ ID NO: 55; (ii) an HCDR3 comprising or consisting of SEQ ID NO:51; an HCDR2 comprising or consisting of SEQ ID NO:50; an HCDR1:X comprising or consisting of SEQ ID NO:49; an LCDR3 comprising or consisting of SEQ ID NO:59; an LCDR2 comprising or consisting of SEQ ID NO:56; an LCDR1 comprising or consisting of SEQ ID NO:58; (iv) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 61; an LCDR2 comprising or consisting of SEQ ID NO: 60; an LCDR1 comprising or consisting of SEQ ID NO: 58; and (v) an HCDR3 comprising or consisting of SEQ ID NO: 51; an HCDR2 comprising or consisting of SEQ ID NO: 50; an HCDR1 comprising or consisting of SEQ ID NO: 49; an LCDR3 comprising or consisting of SEQ ID NO: 62; an LCDR2 comprising or consisting of SEQ ID NO: 56; an LCDR1 comprising or consisting of SEQ ID NO: 58, 41. The bispecific antibody according to any one of embodiments 36 to 40, comprising a CDR sequence selected from: (Aspect 42) 42. The bispecific antibody according to any one of Aspects 36 to 41, wherein the antigen-binding region that binds to IL-4Rα and / or the second antigen-binding region that binds IL-5 is a humanized or germline variant of a non-human antibody, or an antigen-binding fragment thereof. (Aspect 43) 43. The bispecific antibody of aspect 42, wherein the non-human antibody is a camelid antibody or an antigen-binding fragment thereof. (Aspect 44) The bispecific antibody according to any one of Aspects 36 to 43, wherein the antigen-binding region that binds to IL-4Rα and / or the antigen-binding region that binds to IL-5 exhibits lower antigen-binding activity at an acidic pH than at a neutral pH. (Aspect 45) 45. The bispecific antibody of embodiment 44, wherein the ratio between the antigen-binding activity at acidic pH and that at neutral pH is at least 2, as assessed by KD(acidic pH) / KD(neutral pH). (Aspect 46) 46. ​​A combination according to any one of embodiments 1 to 35, or a bispecific antibody according to any one of embodiments 36 to 45, for use in treating a chronic airway disease in a human subject. (Aspect 47) 1. An antagonist of IL-5:IL-5R for use in treating chronic airway disease in a human subject, wherein the antagonist is administered in combination with an antagonist of IL-4:IL-4R and / or an antagonist of IL-13:IL-13R. (Aspect 48) 1. An antagonist of IL-4:IL-4R and / or an antagonist of IL-13:IL-13R for use in treating chronic airway disease in a human subject, wherein the antagonist is administered in combination with an antagonist of IL-5:IL-5R. (Aspect 49) 49. The antagonist for use according to aspect 47 or 48, wherein the antagonist of IL5:IL-5R is an antibody molecule that binds to IL-5 and the antagonist of IL-4:IL-4R or IL-13:IL-13R is an antibody molecule that binds to IL-4Rα. (Aspect 50) 46. ​​A method of treating a chronic airway disease in a human subject, the method comprising administering to the subject an effective amount of a combination according to any one of aspects 1 to 35, or a bispecific antibody according to any one of aspects 36 to 45. (Aspect 51) 51. The method of claim 50, wherein the chronic airway disease is selected from: asthma; chronic rhinosinusitis (CRS); immunoglobulin G4-related disease (IgG4-RD); chronic obstructive pulmonary disease (COPD); chronic bronchitis; emphysema; chronic angioedema; diseases characterized by goblet cell metaplasia, including Barrett's esophagus; active eosinophilic esophagitis; nasal polyposis; chronic rhinosinusitis; Churg-Strauss syndrome; allergic bronchopulmonary aspergillosis (ABPA); hypereosinophilic syndrome; bullous pemphigoid, and cystic fibrosis. (Aspect 52) 52. The method of embodiment 50 or 51, wherein said chronic airway disease is characterized by increased mucus production. (Aspect 53) 53. The method of any one of aspects 50 to 52, wherein the chronic airway disease is characterized by bronchial hyperresponsiveness. (Aspect 54) 54. The method of any one of aspects 50 to 53, wherein the chronic airway disease is asthma. (Aspect 55) 55. The method of embodiment 54, wherein said method is for the treatment of severe asthma, severe refractory asthma, or type II asthma. (Aspect 56) 55. The method of embodiment 54, wherein the method is for the treatment of atopic or allergic asthma. (Aspect 57) The method of any one of embodiments 50 to 56, wherein goblet cell metaplasia (GCM) is reduced. (Aspect 58) The method of any one of embodiments 50 to 57, wherein bronchial hyperresponsiveness (BHR) is alleviated. (Aspect 59) The method of any one of embodiments 50 to 58, further comprising administering one or more additional therapeutic agents for treating a chronic airway disease.

Claims

1. A bispecific antibody comprising an antigen-binding region that binds to IL-4Rα and an antigen-binding region that binds to IL-5, the antigen-binding region that binds to IL-4Rα comprises a first pair of variable heavy chain domain (VH) and variable light chain domain (VL), and the antigen-binding region that binds to IL-5 comprises a second pair of variable heavy chain domain (VH) and variable light chain domain (VL); the first VH-VL domain pair being: (a) an HCDR3 consisting of SEQ ID NO: 30; an HCDR2 consisting of SEQ ID NO: 29; an HCDR1 consisting of SEQ ID NO: 28; an LCDR3 consisting of SEQ ID NO: 39; an LCDR2 consisting of SEQ ID NO: 38; and an LCDR1 consisting of SEQ ID NO: 37; (b) HCDR3 consisting of SEQ ID NO: 3; HCDR2 consisting of SEQ ID NO: 2; HCDR1 consisting of SEQ ID NO: 1; LCDR3 consisting of SEQ ID NO: 12; LCDR2 consisting of SEQ ID NO: 11; LCDR1 consisting of SEQ ID NO: 10; (c) an HCDR3 consisting of SEQ ID NO: 95; an HCDR2 consisting of SEQ ID NO: 8; an HCDR1 consisting of SEQ ID NO: 7; an LCDR3 consisting of SEQ ID NO: 98; an LCDR2 consisting of SEQ ID NO: 17; and an LCDR1 consisting of SEQ ID NO: 16; and (d) HCDR3 consisting of SEQ ID NO: 6; HCDR2 consisting of SEQ ID NO: 5; HCDR1 consisting of SEQ ID NO: 4; LCDR3 consisting of SEQ ID NO: 15; LCDR2 consisting of SEQ ID NO: 14; LCDR1 consisting of SEQ ID NO: 13 and the second VH-VL domain pair being: (a) an HCDR3 consisting of SEQ ID NO: 72; an HCDR2 consisting of SEQ ID NO: 71; an HCDR1 consisting of SEQ ID NO: 70; an LCDR3 consisting of SEQ ID NO: 74; an LCDR2 consisting of SEQ ID NO: 38; and an LCDR1 consisting of SEQ ID NO: 73; (b) HCDR3 consisting of SEQ ID NO: 51; HCDR2 consisting of SEQ ID NO: 50; HCDR1 consisting of SEQ ID NO: 49; LCDR3 consisting of SEQ ID NO: 59; LCDR2 consisting of SEQ ID NO: 56; LCDR1 consisting of SEQ ID NO: 58; (c) an HCDR3 consisting of SEQ ID NO: 51; an HCDR2 consisting of SEQ ID NO: 50; an HCDR1 consisting of SEQ ID NO: 49; an LCDR3 consisting of SEQ ID NO: 57; an LCDR2 consisting of SEQ ID NO: 56; and an LCDR1 consisting of SEQ ID NO: 55; and (d) HCDR3 consisting of SEQ ID NO: 51; HCDR2 consisting of SEQ ID NO: 50; HCDR1 consisting of SEQ ID NO: 49; LCDR3 consisting of SEQ ID NO: 62; LCDR2 consisting of SEQ ID NO: 56; LCDR1 consisting of SEQ ID NO: 58 The bispecific antibody, comprising a CDR sequence selected from:

2. 10. A pharmaceutical composition for use in treating a chronic airway disease in a human subject, the pharmaceutical composition comprising the bispecific antibody of claim 1.

3. 3. The pharmaceutical composition of claim 2, wherein the chronic airway disease is selected from: asthma; chronic rhinosinusitis (CRS); immunoglobulin G4-related disease (IgG4-RD); chronic obstructive pulmonary disease (COPD); chronic bronchitis; emphysema; chronic angioedema; diseases characterized by goblet cell metaplasia, including Barrett's esophagus; active eosinophilic esophagitis; nasal polyposis; chronic rhinosinusitis; Churg-Strauss syndrome; allergic bronchopulmonary aspergillosis (ABPA); hypereosinophilic syndrome; bullous pemphigoid and cystic fibrosis.

4. 4. The pharmaceutical composition of claim 3, wherein the chronic airway disease is characterized by increased mucus production or bronchial hyperresponsiveness.

5. 5. The pharmaceutical composition according to claim 3, wherein the chronic airway disease is asthma.

6. 10. Use of the bispecific antibody of claim 1 in the manufacture of a medicament for the treatment of a chronic airway disease in a human subject.

7. 7. The use of claim 6, wherein the chronic airway disease is asthma.

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