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

Abstract

The present invention relates to combination therapy and its use in treating chronic airway diseases, in particular, asthma. The combination therapy comprises (i) IL-5: an antagonist of IL-5R and (ii) IL-4: an antagonist of IL-4R and / or IL-13: an antagonist of IL-13R. 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. The bispecific antibody can be used to treat chronic airway diseases, in particular asthma.

Description

Technical Field

[0001] The present invention relates to combination therapy and its use in treating chronic airway diseases, in particular, asthma. The combination therapy comprises (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. The antagonist of the combination can be an antibody molecule, and preferably, the combination therapy comprises an antibody molecule that binds to IL-4Rα and an antibody molecule that binds to IL-5. The combination therapy generally inhibits signal transduction via type 2 cytokines IL-4, IL-13 and IL-5. 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. The bispecific antibody can be used to treat chronic airway diseases, in particular asthma. Background Art

[0002] Chronic airway disease (or chronic respiratory disease) is a chronic disease of the airways and other structures of the lungs. 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. Asthma is a disease that affects up to 300 million people worldwide. The airway obstruction that occurs in asthmatics develops over a variable course, with asymptomatic periods interspersed with periods of exacerbations, which are often caused by environmental allergens and viral infections. The typical feature is bronchial hyperresponsiveness (BHR), which is the tendency of the airways to narrow in response to stimuli such as cold air and exercise.

[0004] Asthmatics also exhibit signs of airway remodeling, whereby the airway walls thicken and the number of mucus-producing goblet cells in the epithelium or submucosal glands increases, a phenomenon known as goblet cell metaplasia (GCM). Goblet cells produce mucins that control the viscoelasticity and hydration of the mucus that covers the ciliary escalator. In asthmatics, saliva is often too dry, which can lead to mucus impaction and severe airway obstruction. There are currently few treatment options to reduce GCM and improve mucus clearance.

[0005] Historically, treatment approaches for asthma have involved the use of nonspecific agents, such as inhaled corticosteroids and β2-agonists, with varying degrees of success. However, it is now understood that 'asthma' is a heterogeneous condition describing multiple phenotypes, each presenting distinct clinical, physiological, and molecular features (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] Attempts have been made to group clinical subgroups of asthma based on the recognition that each subgroup may be driven by distinct pathogenic molecular mechanisms and therefore may have different etiologies and respond differently to therapy (Gauthier et al. (2015) American Journal of Respiratory and Critical Care Medicine; 192(6):660-668). Examples of identified subgroups include: early-onset and late-onset asthma, depending on the age at which symptoms occur; eosinophilic, neutrophilic, or non-inflammatory asthma, depending on the presence and type of inflammation observed; exercise-induced asthma; obesity-related asthma; atopic asthma, which is characterized by allergic sensitization to inhaled allergens such as house dust mites (HDM) and increased allergen-specific IgE in serum; mild or moderate asthma, which can also be described as corticosteroid-responsive; severe asthma; and type 2 cytokine-related asthma, which represents individuals who share a type 2 inflammatory pattern. Importantly, many phenotypes overlap due to the lack of clear demarcation between these groups, and patients may exhibit clinical or pathological features of multiple groups, making it difficult to predict individual responsiveness to treatment.

[0007] There is a growing understanding of these mechanistically different groups, sometimes referred to as 'endotypes', and the relevant cellular or molecular biomarkers are beginning to be identified. In bronchial biopsies or lung resection samples, asthma is often characterized by the accumulation of eosinophils, mast cells, and CD4+T lymphocytes, which produce type 2 cytokines IL-4 and / or IL-5 in the epithelium and lamina propria. However, this type 2 inflammatory marker is only detectable in 50% of individuals with asthma, especially those with early-onset disease, atopic tendencies, and high blood eosinophil counts. In contrast, in some individuals with asthma, especially those who respond poorly to steroids, airway infiltrates are mainly composed of neutrophils. These neutrophils may be recruited to the airways by IL-17 producing cells, such as T helper type 17 lymphocytes or γδT cells (Lambrecht and Hammad (2015) Nat. Immunol. 16 (1): 45-56).

[0008] Drugs that specifically target molecular pathways involved in chronic airway diseases are currently being developed. For example, many antibody therapies for allergic diseases have been developed (Sheridan C (2018) Nature Biotechnology; 36: 3-5; Godar et al. (2017) mAbs: 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 IL-13. However, there is a need for improved therapies in the art. Summary of the invention

[0009] The present invention is based on the targeting of multiple type 2 cytokine signaling pathways. Initially called type 2 cytokines because they are produced by T helper type 2 cells (T H In chronic airway diseases such as asthma, type 2 cytokines are not only released by T cells, but also by H The present invention relates to the release of IL-2 cells and IL-4 cells, and also by cells such as basophils, mast cells and eosinophils. Type II cytokines play a key role in the pathogenesis of chronic airway diseases, particularly asthma. It has been surprisingly found that targeting multiple type II cytokines, particularly IL-5, IL-4 and IL-13, produces unexpected synergistic effects in alleviating the potential symptoms and symptoms of chronic airway diseases. Therefore, the combination therapy of the present invention is particularly suitable for treating chronic airway diseases.

[0010] In the first aspect, the present invention provides a combination comprising: (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. Because the IL-4 receptor complex and the IL-13 receptor complex share the common subunit IL-4Rα, an antagonist of IL-4Rα can act as an antagonist of both the IL-4:IL-4R and IL-13:IL-13R signal transduction pathways. Therefore, in a preferred embodiment, the combination of the present invention comprises an antagonist of IL-5:IL-5R and an antagonist of IL-4Rα. The antagonist of IL-5:IL-5R is preferably an antagonist of IL-5. The combination can inhibit signal transduction via IL-5, IL-4 and IL-13.

[0011] The antagonist of the combination can be an antibody molecule. Thus, in certain embodiments, 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, which is an antibody molecule that binds to IL-5, preferably human IL-5; and an antagonist of IL-4Rα, which is an antibody molecule that binds to IL-4Rα, preferably human IL-4Rα.

[0012] In certain embodiments, the antibody molecules of the combination are independently selected from the group consisting of: antibody light chain variable domain (VL); antibody heavy chain variable domain (VH); single chain antibody (scFv); F(ab')2 fragment; Fab fragment; Fd fragment; Fv fragment; one-arm (monovalent) antibody; two-chain antibodies, three-chain antibodies, four-chain antibodies or any antigen binding molecules formed by the combination, assembly or conjugation of such antigen binding fragments. 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: antibody light chain variable domain (VL); antibody heavy chain variable domain (VH); single chain antibody (scFv); F(ab')2 fragment; Fab fragment; Fd fragment; Fv fragment; one-arm (monovalent) antibody; two-chain antibodies, three-chain antibodies, four-chain antibodies or any antigen binding molecules formed by the combination, assembly or conjugation of such antigen binding fragments. The antibody molecule of the combination can be a VHH antibody. In a preferred embodiment, the antibody molecules of the combination are IgG antibodies.

[0013] In certain embodiments, the antibody molecules of the combination, for example, antibody molecules that bind to IL-4Rα and / or antibody molecules that bind to IL-5, are humanized or germline variants of non-human antibodies or antigen-binding fragments thereof, for example, antibodies or antigen-binding fragments thereof derived from Camelidae. In certain embodiments, the antibody molecules of the combination, for example, antibody molecules that bind to IL-4Rα and / or antibody molecules that bind to IL-5, comprise the CH1 domain, hinge region, CH2 domain and / or CH3 domain of human IgG. Alternatively, the antibody molecule may exhibit a high degree of homology with human IgG, preferably IgG1.

[0014] In certain embodiments, the antibody molecule of the combination, for example, an antibody molecule that binds to IL-4Rα and / or an antibody molecule that binds to IL-5, comprises an Fc domain derived from human IgG, preferably IgG1. The Fc domain may be unmodified or may be modified by one or more amino acid substitutions, for example, to increase binding affinity to FcRn (neonatal Fc receptor). In a preferred embodiment, the antibody molecule may comprise an Fc domain, preferably derived from human IgG and comprising the following amino acid substitutions: H433K and N434F; or M252Y, S254T, T256E, H433K and N434F. The numbering of the Fc domain is according to the EU numbering scheme.

[0015] In certain embodiments, the antibody molecules of the combination, for example, antibody molecules that bind to IL-4Rα and / or antibody molecules that bind to IL-5, exhibit pH-dependent antigen binding activity, particularly exhibiting lower antigen binding activity at acidic pH values ​​than at neutral pH values. As measured by the dissociation constant ratio: KD (at acidic pH values) / KD (at neutral pH values), the ratio of antigen binding activity at acidic pH values ​​to that at neutral pH values ​​may be at least 2.

[0016] Regarding the formulation of the combination, IL-5:IL-5R and IL-4:IL-4R and / or IL-13:IL-13R antagonists may be co-formulated or provided separately. For embodiments in which the antagonists are co-formulated, the antagonists may be formulated according to a 1:1 ratio or may be formulated according to a non-equimolar ratio. For example, for a combination comprising an antagonist of IL-5:IL-5R (preferably an antagonist of IL-5) and an antagonist of IL-4Rα, the antagonists may be formulated according to a ratio of, for example, 1:2 or 2:1.

[0017] For 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 into a multispecific antibody, such as a bispecific antibody.

[0018] In certain embodiments, the combination may include one or more additional therapeutic agents.

[0019] 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 a preferred embodiment, the antigen binding region that binds to IL-4Rα and / or the antigen binding region that binds to IL-5 is a humanized or germline variant of a non-human antibody or an antigen binding fragment thereof, preferably a camelid antibody or an antigen binding fragment thereof. In certain embodiments, the antigen binding region that binds to IL-4Rα comprises a first variable heavy chain domain (VH) and a variable light chain domain (VL) pairing 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) pairing. The bispecific antibody may be an IgG antibody having a first VH-VL pairing that binds to IL-4Rα and a second VH-VL pairing that binds to IL-5. In certain embodiments, the bispecific antibody is an IgG antibody having at least one scFv fragment connected thereto.

[0020] The bispecific antibodies of the present invention may exhibit pH-dependent antigen binding. For example, the antigen binding region bound to IL-4Rα and / or the antigen binding region bound to IL-5 may exhibit lower antigen binding activity at acidic pH values ​​than at neutral pH values. In certain embodiments, as measured by KD (at acidic pH values) / KD (at neutral pH values), the ratio of antigen binding activity at acidic pH values ​​to that at neutral pH values ​​is at least 2.

[0021] It has been found that combinations and bispecific antibodies targeting multiple type 2 cytokine signaling pathways are particularly suitable for treating chronic airway diseases, in particular asthma. Therefore, another aspect of the invention provides 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 a chronic airway disease in a human subject. A method for treating a chronic airway disease in a human subject is further provided, 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.

[0022] In certain embodiments, the chronic airway disease is selected from the group consisting of: 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 oesophagus; active eosinophilic esophagitis; nasal polyposis; chronic sinusitis; Churg-Strauss Syndrome; allergic bronchopulmonary aspergillosis (ABPA); hypereosinophilic syndrome, bullous pemphigoid, and cystic fibrosis.

[0023] The chronic airway disease to be treated according to the method of the invention may be characterized by increased mucus production or increased bronchial hyperresponsiveness. In a preferred embodiment, the chronic airway disease to be treated is asthma, optionally severe asthma, severe refractory asthma, Type II High asthma, atopic or allergic asthma.

[0024] The methods described herein can be used to treat chronic airway diseases, preferably asthma, by reducing goblet cell metaplasia (or GCM). Alternatively or in addition, the methods can be used to treat chronic airway diseases, preferably asthma, by reducing bronchial hyperresponsiveness (BHR). The methods may include additional steps, for example, further administering one or more additional therapeutic agents to the patient to treat chronic airway diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Neutralizing activity of both IL-4Rα and IL-5 monospecific Abs as assessed in in vitro cell assays of IL-4-induced HT-2 cell proliferation and IL-5-induced TF-1 cell proliferation is shown. IL-4Rα (squares) and IL-5 (triangles) monospecific Abs potently inhibited mouse IL-4- and IL-5-induced HT-2 and TF-1 cell proliferation, respectively. Results are presented as mean ± SEM of triplicates of two independent experiments.

[0026] Figure 2 Representative surface plasmon resonance (SPR) sensorgrams of the interaction between mAb (IL-4Rα Ab, IL-5 Ab, or irrelevant IgG2a Ab) and immobilized target (IL-4Rα or IL-5) at varying concentrations (0-20 μg / mL).

[0027] Figure 3Representative SPR sensorgrams of the interaction between a mixture consisting of mAb (IL-4RαAb, IL-5Ab, or irrelevant IgG2a Ab) and its target (IL-4Rα or IL-5) and an immobilized protein (IL-4, IL-13Rα, or IL-5Rα).

[0028] Figure 4 Shown are MHC class II antigens of purified B cells before and after IL-4Rα mAb treatment as analyzed by FACS. IL-4Rα mAb potently inhibited IL-4-induced MHC class II antigens of purified B cells.

[0029] Figure 5 The experimental results for testing the effect of IL-4Rα and IL-5mAb in the in vivo murine asthma model are shown. A is a graphical representation of the experimental setting using the house dust mite (HDM) mouse model. IL-4Rα and IL-5 antibody treatments were delivered by injection to C57BL / 6J mice treated with HDM during both the sensitization and challenge phases. B shows the bronchoalveolar lavage (BAL) differential cell counts of mice receiving IL-4Rα monospecific Ab, IL-5 monospecific Ab, IL-4α / IL-5 monospecific Ab combination or irrelevant IgG2a Ab as analyzed by FACS. Increase eosinophil counts by allergen challenge in HDM sensitized mice treated with IgG2a. Compared with treatment with control IgG2a Ab, a significant reduction in eosinophil counts was observed after treatment with IL-4Rα monospecific Ab, IL-5 monospecific Ab, and after treatment with a combination of IL-4α / IL-5 monospecific Ab. 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 Ab.

[0030] Figure 6 is a graphic representation of the experimental setup using the house dust mite (HDM) mouse model as an in vivo murine asthma model. IL-4Rα and IL-5 antibody treatments were delivered by injection to HDM-treated C57BL / 6J mice only during the challenge period.

[0031] Figure 7BAL differential cell counts of mice receiving IL-4Rα monospecific Ab, IL-5 monospecific Ab, a combination of IL-4Rα / IL-5 monospecific Ab, or an unrelated IgG2a Ab as analyzed by FACS are shown. Eosinophil counts were increased by allergen challenge in HDM-sensitized mice treated with IgG2a. A significant reduction in eosinophil counts was observed after treatment with IL-4Rα monospecific Ab or after treatment with IL-5 monospecific Ab compared to treatment with control IgG2a Ab. A further reduction in eosinophil counts was observed after treatment with a combination of IL-4α / IL-5 monospecific Ab. P values ​​reflect one-way ANOVA tests, ns: not significant, **P≤0.01, ***P≤0.001, ****P≤0.0001, vs. control IgG2Ab.

[0032] Figure 8 IL-5 and IL-13 cytokines produced by mesenteric lymph node (MLN) cells restimulated with HDM for three days ex vivo as determined by ELISA are shown. In vitro production of effector cytokines IL-5 and IL-13 in allergen restimulated MLN cell cultures was enhanced by allergen challenge in HDM-sensitized mice treated with IgG2a. However, this response was significantly reduced after treatment with IL-4Rα monospecific Ab and the combination of the two monotherapies. P values ​​reflect one-way ANOVA tests, ns: not significant, **P≤0.01, ***P≤0.001, vs. control IgG2 Ab.

[0033] Fig. 9 Serum levels of HDM-specific IgE and IgG1 after treatment with IL-4Rα monospecific Ab, IL-5 monospecific Ab, a combination of IL-4Rα / IL-5 monospecific Ab, or an irrelevant IgG2a Ab are shown as determined by ELISA. Serum concentrations of HDM-specific IgG1 and IgE were increased by allergen challenge in IgG2aAb-treated mice. IL-4RαmAb and IL-4Rα / IL-5mAb combination were able to significantly reduce this increase in allergen-induced IgG1 and IgE. P values ​​reflect one-way ANOVA tests, *P≤0.05, ns: not significant, **P≤0.01, ****P≤0.0001, vs. irrelevant IgG2 Ab.

[0034] Fig.10The expression of mucin, Muc5AC, Agr2 and Spdef in the lungs of mice treated with IL-4Rα monospecific Ab, IL-5 monospecific Ab and a combination of IL-4Rα / IL-5 monospecific Ab is shown. A shows confocal staining of Muc5AC in the lungs of mice after treatment with IL-4Rα monospecific Ab, IL-5 monospecific Ab, combined IL-4Rα / IL-5 monospecific Ab or unrelated IgG2a Ab. B shows the lung mRNA expression levels of Muc5ac and Agr2 determined by qRT-PCR. Compared with PBS stimulation, the mRNA expression levels of these two genes were induced in mice by HDM stimulation. IL-4Rα and IL-5 antibodies alone did not significantly reverse this increase in Muc5ac or Agr2 mRNA levels. However, the combination of monospecific IL-4RαmAb and IL-5mAb 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. irrelevant IgG2 Ab.

[0035] Fig.11 The use of flexiVent after exposure to increasing doses of methacholine is shown Bronchial hyperresponsiveness (BHR) measured. Data represent three independent experiments with at least n=6 mice per group. Bronchial hyperresponsiveness was significantly reduced after treatment with the combination of IL-4Rα / IL-5 monospecific Ab compared to treatment with control IgG2a Ab, and resistance levels were restored to levels observed in unchallenged mice receiving only PBS. Results are expressed as mean ± SEM. P values ​​reflect one-way ANOVA test, ns: not significant, *P≤0.05, vs. irrelevant IgG2Ab.

[0036] Fig.12 Schematic representation of the dual anti-idiotypic purification process used to isolate the desired and properly paired bispecific IL-4Rα / IL-5 Ab. (a) From a mixture of four possible combinations formed by different heavy and light chain pairings, anti-idiotypic VHHs that only recognize the correct HC / LC pairing of the IL-4Rα monospecific Ab are used to extract Abs containing this pairing. (b) A second anti-idiotypic column containing VHH antibodies that only recognize the correct HC / LC pairing of the IL-5 monospecific Ab is used to collect bispecific Abs containing the properly paired HC / LC of the αIL-5 monospecific Ab. (c) This allows the isolation of the desired IL-4Rα / IL-5 bispecific Ab with the correct HC / LC pairing.

[0037] Fig.13The dual targeting properties of the IL-4Rα / IL-5 bispecific antibody were verified. A. The SPR signal was measured on the coated IL-4Rα-Fc after sequential injection of IL-4Rα monospecific Ab or IL-4Rα / IL-5 bispecific Ab, followed by a second injection of IL-4Rα-Fc or IL-5. One arm of the bispecific antibody binds to the coated IL-4Rα, and the other arm binds to the injected IL-5. B. The SPR signal was measured on the coated IL-5 after sequential injection of IL-5 monospecific Ab or IL-4Rα / IL-5 bispecific Ab, followed by a second injection of IL-4Rα-Fc or IL-5. One arm of the bispecific antibody binds to the coated IL-5, and the other arm binds to the injected IL-4Rα.

[0038] Fig.14 is a graphical representation of the experimental setup, in which antibody treatments were injected only during the challenge period in HDM-treated C57BL / 6J mice. To compare equimolar inhibition of the target and differences in the total amount of Ab eliminated, the following doses of antibody were administered to each mouse: 75 μg of each monospecific Ab combined with 75 μg of irrelevant IgG2a Ab; 75 μg of each monospecific Ab injected in combination; or 150 μg of IL-4Rα / IL-5 bispecific Ab.

[0039] Fig.15 Shown as by FACS analysis, receiving IL-4Rα monospecific Ab, IL-5 monospecific Ab, combined IL-4Rα / IL-5 monospecific Ab, IL-4Rα / IL-5 bispecific Ab or unrelated IgG2a Ab HDM treated mice BAL differential cell count.HDM stimulation in sensitized mice increased the number of eosinophils in BAL fluid.This increase in the number of eosinophils was significantly reduced after injection in mice receiving a combination of monospecific IL-4Rα and IL-5Ab (75μg+75μg) and mice receiving IL-4Rα / IL-5 bispecific Ab.The combination of two monospecific Abs and bispecific antibodies significantly reduced the number of eosinophils compared to mice receiving HDM treatment of control IgG2a Ab.P value reflects single factor ANOVA test, ns: not significant, ***P≤0.001, ****P≤0.0001, compared to unrelated IgG2 Ab.

[0040] Fig.16Shown are IL-5 and IL-13 cytokine production by mesenteric lymph node (MLN) cells restimulated ex vivo with HDM for three days as determined by ELISA. P values ​​reflect one-way ANOVA tests, ns: not significant, ***P≤0.001, ****P≤0.0001, vs. irrelevant IgG2 Ab.

[0041] Fig.17 Shown are serum levels of HDM-specific IgE and IgG1 after treatment with IL-4Rα monospecific Ab, IL-5 monospecific Ab, combined IL-4Rα / IL-5 monospecific Ab, IL-4Rα / IL-5 bispecific Ab, or irrelevant IgG2a Ab, as determined by ELISA. P values ​​reflect one-way ANOVA test, ns: not significant, *P≤0.05, **P≤0.01, ***P≤0.001, vs. irrelevant IgG2 Ab.

[0042] Fig.18 Lung mRNA expression levels of Muc5ac, Agr2 and Spdef determined by qRT-PCR are shown. Compared with PBS stimulation, the mRNA expression levels of these two genes were induced by HDM stimulation 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-4RαmAb and IL-5mAb and IL-4Rα / IL-5 bispecific Ab significantly reduced HDM-mediated increases 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, compared to irrelevant IgG2 Ab.

[0043] Fig.19 The use of flexiVent after exposure to increasing doses of methacholine is shown BHR measured. Data represent two independent experiments with n=6 mice per group. Bronchial hyperresponsiveness was significantly reduced after treatment with a combination of IL-4Rα / IL-5 monospecific Abs and after treatment with IL-4Rα / IL-5 bispecific Abs compared to treatment with control IgG2a Abs. After treatment with a combination of monospecific Abs or after treatment with bispecific Abs, resistance levels returned to levels observed in unchallenged mice receiving only PBS. Results are expressed as mean ± SEM. P values ​​reflect one-way ANOVA tests, ns: not significant, *P≤0.05, vs. irrelevant IgG2a Abs.

[0044] Fig. 20The structure of an IL-4Rα / IL-5 bispecific antibody with an IL-4RαIgG connected to two IL-5scFv fragments is shown. The Fab arm of the IL-4RαIgG 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 sequences represented by SEQ ID NOs: 76 and 79, respectively.

[0045] Fig.21 Neutralizing activity of the IL-4Rα / IL-5 bispecific antibody as assessed in an in vitro cellular assay of IL-5-induced TF-1 cell proliferation is shown. Tested with IL-4Rα mAb (36B7) and two IL-5 mAbs (95G7hIgG1 and 95A7mIgG2a) Fig. 20 bispecific antibodies.

[0046] Fig. 22 is a graphic representation of the experimental setup using the house dust mite (HDM) mouse model as an in vivo murine asthma model. IL-4Rα and IL-5 antibody treatments were delivered by injection to HDM-treated C57BL / 6J mice only during the challenge period.

[0047] Fig.23 Shown are BAL differential cell counts of HDM-treated mice receiving a combined IL-4Rα / IL-5 monospecific Ab, IL-4Rα / IL-5 bispecific Ab (Bs 4Rsc5) or an unrelated IgG2a Ab as analyzed by FACS. HDM stimulation in sensitized mice increased the number of eosinophils and lymphocytes in BAL fluid. This increase in cell number was significantly reduced after injection in mice receiving a combination of both monospecific IL-4Rα and IL-5Ab and in mice receiving IL-4Rα / IL-5 bispecific Ab. P values ​​reflect one-way ANOVA tests, ns: not significant, ***P≤0.001, ****P≤0.0001, vs. unrelated IgG2 Ab.

[0048] Fig.24 Shown are lung mRNA expression levels of Muc5ac, Agr2 and Spdef determined by qRT-PCR. The mRNA expression levels of these two genes in mice were induced by HDM challenge compared to PBS challenge. DETAILED DESCRIPTION

[0049] A. Definition

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as 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 two or more therapeutic agents are administered to a subject, such as a human subject. The "combination" of the present invention is intended for use 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 more detail herein. In a preferred embodiment, the antagonist is an antibody molecule that specifically binds to its corresponding cytokine or cytokine receptor target. As described elsewhere herein, the antagonists included in the combination therapy can be co-formulated or can be provided independently, for example, as a separate composition, for administration to a subject or patient in need. For embodiments in which the combination comprises an antibody molecule that binds to IL-4Rα and an antibody molecule that binds to IL-5, the combined antibody molecules may be contained within a single antibody, for example in the form of a multispecific antibody, such as a bispecific antibody.

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

[0053] IL-5:IL-5R, IL-4:IL-4R and IL-13:IL-13R antagonists used in the combination of the present invention can take the form of any suitable agent or molecule. In certain embodiments, the antagonist can inhibit the function of its target by downregulating the expression of the target, such as the expression of IL-4Rα or IL-5. In alternative embodiments, the antagonist can inhibit the function of its target by directly binding to a cytokine or receptor subunit. As explained herein, IL-5:IL-5R antagonists can bind to the cytokine IL-5 (referred to as IL-5 antagonists) or to one of the IL-5R subunits (referred to as IL-5Rα antagonists or βc antagonists). Similarly, IL-4:IL-4R antagonists can bind to the cytokine IL-4 (referred to as IL-4 antagonists) or to one of the type I IL-4R subunits (referred to as IL-4Rα antagonists or γc antagonists).

[0054] In a preferred embodiment, the antagonist is specific to its target. For example, compared with other molecular targets, an antagonist of IL-4Rα (or IL-4Rα antagonist) will preferentially inhibit the function of IL-4Rα. Compared with other molecular targets, an IL-5 antagonist will preferentially inhibit the function of IL-5. Antagonists will generally achieve the desired level of specificity by directly interacting with their targets, such as 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 combined antagonist is an antibody molecule.

[0055] "Antibody molecule" - As used herein, the term "antibody molecule" is intended to encompass full-length antibodies and antigen-binding fragments thereof, including variants, such as modified 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, wherein the polypeptide has significant specific immunoreactivity activity to an antigen of interest (e.g., IL-4Rα or IL-5). For antibodies of the IgG class, the antibody comprises two identical polypeptide light chains with a molecular weight of approximately 23,000 Daltons and two identical heavy chains with a molecular weight of 53,000-70,000. The four chains are connected by disulfide bonds in a "Y" configuration, wherein the light chain holds the heavy chain starting at the opening of the "Y" and continuing through the variable region. The light chains of antibodies are classified as κ or λ (κ, λ). Each heavy chain class can be combined with a κ or λ light chain. In general, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, the light chain and the heavy chain are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0056] Those skilled in the art will appreciate that heavy chains are classified as gamma, μ, alpha, delta, or epsilon (γ, μ, alpha, delta, epsilon), with some subclasses (e.g., γ1-γ4). The properties of this chain determine the "class" of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and known to confer functional specialization. As used herein, the term "antibody molecule" encompasses full-length antibodies or antigen-binding fragments thereof from any antibody class or subclass.

[0057] As used herein, the term "antibody molecule" 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 only by species of the Camelidae family, including camels, llamas, and alpacas. Heavy chain only antibodies consist of two heavy chains and do not contain light chains. Each heavy chain has a variable domain at the 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 described above, i.e., VH domains.

[0058] Regarding the antigen-binding fragments covered in the general term "antibody molecule", these fragments are full-length antibodies or antibody chains containing less amino acid residues than a complete or complete antibody while retaining antigen-binding activity. As used herein, the term "antibody molecule" is intended to cover antigen-binding fragments selected from the following: antibody light chain variable domain (VL); antibody heavy chain variable domain (VH); single-chain antibody (scFv); F(ab')2 fragment; Fab fragment; Fd fragment; Fv fragment; one-arm (monovalent) antibody; double-chain antibody, three-chain antibody, four-chain antibody or any antigen-binding molecule formed by the combination, assembly or conjugation of such antigen-binding fragments. As used herein, the term "antibody molecule" is further intended to cover antibody fragments selected from the group consisting of: single antibody (unibody); domain antibody; and nanobody. Fragments can be obtained, for example, via chemical or enzymatic treatment of a complete or complete antibody or antibody chain or by recombinant means.

[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 between antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of the antibody. The variability 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 Vλ light chain domain are referred to herein as L1(λ), ​​L2(λ) and L3(λ) and can be defined 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) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second and third hypervariable loops of the Vκ light chain domain are referred to herein as L1 (κ), L2 (κ) and L3 (κ) and can be defined as comprising residues 25-33 (L1 (κ), consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2 (κ), consisting of 3 residues) and 90-97 (L3 (κ), consisting of 6 residues) in the VL domain (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 can be defined 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, length is highly variable) in the VH domain (Morea et al., Methods 20:267-279 (2000)).

[0060] Unless otherwise indicated, the terms L1, L2, and L3 refer to the first, second, and third hypervariable loops of the VL domain, respectively, and encompass hypervariable loops obtained from both Vκ and Vλ isotypes. The terms H1, H2, and H3 refer to the first, second, and third hypervariable loops of the VH domain, respectively, and encompass hypervariable loops obtained from any of the known heavy chain isotypes, including γ, ε, δ, α, or μ.

[0061] 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 strictly synonymous, because hypervariable loops (HV) are defined on the basis of structure, while complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD., 1983), and the limits of HV and CDR may be different in some VH and VL domains.

[0062] The CDRs of 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)). Therefore, unless otherwise indicated, HVs may be contained within the corresponding CDRs and references to the "hypervariable loops" of VH and VL domains herein should be interpreted as also covering the corresponding CDRs, and vice versa.

[0063] The more highly conserved portions of the variable domain are called framework regions (FRs) as defined below. The variable domains of native heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4, respectively), which primarily adopt a β-folded configuration connected by three hypervariable loops. The hypervariable loops in each chain are held tightly together by the FRs and, together with the hypervariable loops from the other chain, contribute to the formation of the antigen binding site of the antibody. Structural analysis of antibodies reveals the relationship between the sequence and shape of the binding site formed by the complementary determining region (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol. Biol, 215: 175-182 (1990)). Despite the high degree of sequence variability, five of the six loops adopt only a small group of main chain conformations, referred to as "typical structures". These conformations are determined firstly by the length of the loops and secondly by the presence of key residues at certain positions in the loops and in the framework regions that dictate the conformation by their ability to pack, hydrogen bond or assume unusual main-chain conformations.

[0064] "CDR" - As used herein, the term "CDR" or "complementarity determining region" means the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest ("Protein sequences with immunological significance"), (1991) and by Chothia et al., J. Mol. Biol. 196: 901-917 (1987) and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), wherein definitions include overlaps or subgroups of amino acid residues when compared to each other. The amino acid residues covering the CDRs as defined by each of the references cited above are stated for comparison. Preferably, the term "CDR" is a CDR as defined by Kabat based on sequence comparison.

[0065] Table 1: CDR Definition

[0066]

[0067] 1 The residue numbering follows the nomenclature of Kabat et al. (supra).

[0068] 2 The residue numbering follows the nomenclature of Chothia et al. (supra).

[0069] 3The residue numbering follows the nomenclature of MacCallum et al. (supra).

[0070] "Framework region" - As used herein, the term "framework region" or "FR region" includes amino acid residues that are part of the variable region, but are not part of the CDR (e.g., using the Kabat definition of CDR). Thus, the variable region framework is about 100-120 amino acids in length, but includes only those amino acids outside the CDR. For the specific example of a heavy chain variable domain and for the CDRs as defined by Kabat et al., framework region 1 corresponds to the domain of the variable region covering amino acids 1-30; framework region 2 corresponds to the domain of the variable region covering amino acids 36-49; framework region 3 corresponds to the domain of the variable region covering amino acids 66-94; and framework region 4 corresponds to the domain of the variable region from amino acid 103 to the end of the variable region. The framework region of the light chain is separated in a similar manner by each of the light chain variable region CDRs. Similarly, using the CDR definitions of Chothia et al. or McCallum et al., the framework region boundaries are separated by the corresponding CDR ends as described above. In a preferred embodiment, the CDRs are as defined by Kabat.

[0071] In naturally occurring antibodies, the six CDRs present on each monomer antibody are short non-contiguous sequences of amino acids that are specifically positioned to form an antigen binding site when the antibody presents its three-dimensional configuration in an aqueous environment. The remainder of the heavy and light variable domains show less intermolecular variability in the amino acid sequence and are referred to as framework regions. The framework regions primarily adopt a β-folded conformation and the CDRs form loops that are linked together and form a part of the β-folded structure in some cases. Therefore, these framework regions are used to form a scaffold that positions the six CDRs in the correct orientation through interchain non-covalent interactions. The antigen binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. The position of the CDR can be easily identified by a person of ordinary skill in the art.

[0072] "Constant region" - As used herein, the term "constant region" refers to the portion of an antibody molecule outside of a variable domain or variable region. An immunoglobulin light chain has a single domain "constant region", commonly referred to as a "CL or CL1 domain". This domain is at the C-terminus of the VL domain. An immunoglobulin heavy chain differs in its constant region, depending on the class of immunoglobulin (γ, μ, α, δ, ε). Heavy chains γ, α and δ have a constant region consisting of three immunoglobulin domains (called CH1, CH2 and CH3), with a flexible hinge region separating the CH1 and CH2 domains. Heavy chains μ and ε have a constant region consisting of four domains (CH1-CH4). The constant domain of the heavy chain is located at the C-terminus of the VH domain.

[0073] The amino acid numbering in the immunoglobulin heavy and light chains extends from the N-terminus of the forked end 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 the immunoglobulin heavy and light chains. According to the EU numbering scheme, the heavy chain constant domains of the IgG molecule are 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 domains of the IgG molecule are identified as follows: CH1-amino acid residues 114-223; CH2-amino acid residues 244-360; CH3-amino acid residues 361-477. "Fc domain" or "Fc region" generally defines 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 "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains about 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to act independently. The hinge region can be subdivided into three different domains: upper, middle and lower hinge domains (Roux KH et al. J. Immunol. 161: 4083-90 1998). The antibody of the present invention comprising a "fully human" hinge region may contain one of the hinge region sequences shown in Table 2 below.

[0074] Table 2: Human hinge sequences

[0075]

[0076]

[0077] "Specific" and "multispecific antibodies" - antibody molecules used in the combination therapies described herein bind to specific target antigens. Preferably, the antibody molecule "specifically binds" to its target antigen, wherein the term "specifically binds" refers to the ability of any antibody molecule to preferentially immunoreact with a given target, such as IL-4Rα and IL-5. The antibody molecules of the combinations and methods of the present invention can be monospecific and contain one or more binding sites that specifically bind to a specific target. The antibody molecules of the combinations and methods of the present invention can be combined into a "multispecific antibody" format, such as a bispecific antibody, in which the multispecific antibody binds to two or more target antigens. For example, in one embodiment, the 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. In order to achieve multiple specificities, "multispecific antibodies" are typically engineered to include different combinations or pairings of heavy chain and light chain polypeptides with different VH-VL pairs. Multispecific, in particular bispecific antibodies can be engineered to adopt the overall conformation of a native antibody, for example, a Y-shaped antibody with Fab arms of different specificities conjugated to the Fc region. Alternatively, multispecific antibodies, such as bispecific antibodies, can be engineered to adopt a non-native conformation, for example, in which variable domains or pairs of variable domains with different specificities are located at opposite ends of the Fc region.

[0078] "Modified antibody" - As used herein, the term "modified antibody" includes antibodies that have been altered so that they are not naturally occurring synthetic forms, for example, antibodies that contain at least two heavy chain portions, but do not contain two complete heavy chains (such as domain-deleted antibodies or miniantibodies); multispecific forms of antibodies that have been altered to bind to two or more different antigens or to different epitopes on a single antigen (e.g., bispecific, trispecific, etc.); heavy chain molecules linked to scFv molecules, etc. ScFv molecules are known in the art and are described in, for example, U.S. Patent No. 5,892,019. In addition, the term "modified antibody" includes antibodies in multivalent forms (e.g., trivalent, tetravalent, etc. antibodies that bind to three or more copies of the same antigen). In another embodiment, the modified antibody of the present invention is a fusion protein that comprises at least one heavy chain portion that lacks a CH2 domain and comprises a polypeptide binding domain that contains a binding portion of one member of a receptor ligand pair.

[0079] "Humanizing substitutions" - As used herein, the term "humanizing substitutions" refers to amino acid substitutions 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 a human germline. Humanizing substitutions may be made in the framework regions and / or CDRs of an antibody as defined herein.

[0080] “Humanized variant” - As used herein, the term “humanized variant” or “humanized antibody” refers to a variant antibody that contains one or more “humanizing substitutions” compared to a reference antibody, wherein a portion of the reference antibody (e.g., the VH domain and / or VL domain or the portion thereof containing at least one CDR) has amino acids derived from a non-human species, and the “humanizing substitutions” occur within the amino acid sequence derived from the non-human species.

[0081] "Germlined variant" - The term "germlined variant" or "germlined antibody" is used herein to specifically refer to a "humanized variant" in which "humanizing substitutions" are made such that one or more amino acid residues present at one or more specific positions in the VH or VL domain of the antibody are replaced by amino acid residues occurring at equivalent positions in a reference human VH or VL domain encoded by a human germline. Typically, for any given "germlined variant", the replacement amino acid residues substituted into the germlined variant are obtained entirely or predominantly from a VH or VL domain encoded by a single human germline. The terms "humanized variant" and "germlined variant" are often used interchangeably. One or more "humanizing substitutions" are introduced into a VH or VL domain derived from Camelidae (e.g., from llama) to produce a "humanized variant" of a VH or VL domain derived from Camelidae (llama). If the substituted amino acid residues are derived primarily or entirely from a single human germline-encoded VH or VL domain sequence, the result can be a "humanized germline variant" of a VH or VL domain derived from Camelidae (llama).

[0082] "Affinity variant" - As used herein, the term "affinity variant" refers to a variant antibody that exhibits one or more changes in the amino acid sequence compared to a reference antibody, wherein the affinity variant exhibits an altered affinity for the target antigen compared to the reference antibody. For example, the affinity variant will exhibit a changed affinity for the target, such as IL-4Rα or IL-5, compared to the reference IL-4Rα or IL-5 antibody. Preferably, the affinity variant will exhibit an 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 the CDR compared to the reference antibody. Such substitutions can cause the original amino acid present at a given position in the CDR to be replaced by a different amino acid residue, which can be a naturally occurring amino acid residue or a non-naturally occurring amino acid residue. Amino acid substitutions can be conservative or non-conservative.

[0083] "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 indicate the IL-5 cytokine / cytokine receptor signaling complex. 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 in length (http: / / www.uniprot.org / uniprot / P05113). As used herein, the term "IL-5" is intended to encompass any splice variant of the protein. Monomeric IL-5 is inactive and requires homodimers for function. One IL-5 homodimer engages one IL-5 receptor (IL-5R). The receptor for IL-5 consists of two subunits. The first subunit is the "IL-5 receptor subunit alpha" or "IL-5Rα", also known as IL-5R-α, 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 in length (http: / / www.uniprot.org / uniprot / Q01344). The second subunit of the IL-5R complex is the non-ligand-binding universal signaling β subunit or β chain (βc). IL-5 is secreted by a limited number of interstitial cell types. Cells known to express IL-5 include eosinophils, NK cells, TC2 CD8+T cells, mast cells, CD45+CD4+T cells, γδT cells, and IL-1β-activated endothelial cells. IL-5 is known to regulate the expression of genes involved in the proliferation, cell survival and maturation of B cells and eosinophils, and effector functions.

[0084] "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 indicate the IL-4 cytokine / type I cytokine receptor signaling complex. Cytokine IL-4 is also known as B-cell stimulating factor 1 (BSF-1), human interleukin 4 (Binetrakin), and lymphocyte stimulating factor 1. The human homolog of IL-4 is 153 amino acids in length (http: / / www.uniprot.org / uniprot / P05112). As used herein, the term "IL-4" is intended to cover any splice variant of the protein. The type I receptor of IL-4 consists of two subunits. The first subunit is "IL-4Rα", also known as interleukin-4 receptor subunit α, interleukin-4 binding subunit, and CD124. IL-4Rα is a 140kDa transmembrane glycoprotein widely expressed in the class I cytokine receptor family. The length of the human homolog of IL-4Rα is 825 amino acids (http: / / www.uniprot.org / uniprot / P24394). As used herein, the term "IL-4Rα" is intended to cover any splice variant of the protein. In the type I IL-4 receptor complex, IL-4Rα associates with the second subunit universal γ chain (γc), and the γc subunit increases the affinity of IL-4Rα to IL-4 to achieve downstream signaling via IL-4. The IL-4 receptor complex is present in, for example, B-cells, T-cells, monocytes, eosinophils, and fibroblasts, and IL-4Rα is coupled to the JAK1 / 2 / 3-STAT6 path. The IL-4 response is involved in promoting Th2 differentiation, and regulating IgE production at sites of allergic inflammation as well as chemokine and mucus production.

[0085] "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 indicate the IL-13 cytokine / cytokine receptor signaling complex. The human homolog of IL-13 is 146 amino acids in length (http: / / www.uniprot.org / uniprot / P35225). As used herein, the term "IL-13" is intended to encompass any splice variant of the protein. The receptor for IL-13 consists of two subunits. The first subunit is "IL-4Rα", also known as interleukin-4 receptor subunit α, interleukin-4 binding subunit and CD124. As mentioned above, this subunit is common to both IL-4 and IL-13 receptors. In the IL-13 receptor complex, IL-4Rα associates with a second subunit, interleukin-13 receptor subunit α1, or "IL-13Rα1," 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 (via binding to IL-13Rα1) and IL-4 (via binding to IL-4Rα), and cytokine binding to this receptor complex initiates signaling via the JAK1 / 2 / 3-STAT6 pathway. Similar to the IL-4:IL-4R complex, signaling via the IL-13R complex 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 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.

[0086] "Chronic airway disease" - As used herein, the term "chronic airway disease" can be used interchangeably with 'chronic respiratory disease (CRD)' and is intended to refer to 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 smoking, other risk factors include air pollution, occupational chemicals and dust, and frequent lower respiratory tract infections in childhood. Other chronic airway diseases may include, but are not limited to: chronic rhinosinusitis (CRS), immunoglobulin G4-related disease (IgG4-RD), chronic bronchitis, emphysema, chronic angioedema, goblet cell metaplasia-specific diseases (such as Barrett's esophagus), active eosinophilic esophagitis, nasal polyposis, chronic sinusitis, Chag-Strauss syndrome, allergic bronchopulmonary aspergillosis (ABPA), high eosinophilic syndrome, bullous pemphigoid and cystic fibrosis.

[0087] "Asthma" - As used herein, "asthma" is intended to mean any disease or condition associated with inflammation of the air passages in the lungs. Typically, inflammation affects the sensitivity of the nerve endings in the airways to make them susceptible to irritation. During an asthma attack, the lining of the passage swells, causing the airways to narrow and reducing air flow into and out of the lungs. Asthma is a heterogeneous disorder describing a variety of phenotypes, each of which presents different clinical, physiological and molecular characteristics. Exemplary asthma subtypes may 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, oligogranulocytic or non-inflammatory asthma, early-onset asthma, late-onset asthma, high type II asthma, low type II asthma (type II low asthma) and type I / Th17 asthma.

[0088] B. Combination Therapies to Inhibit Type 2 Cytokine Signaling

[0089] The present invention relates to combinations or combination therapies and their use in the treatment of chronic airway diseases, particularly asthma. The combinations of the present invention comprise antagonists targeting type 2 cytokines and / or their corresponding 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 I receptor IL-4R; and IL-13 and its receptor IL-13R.

[0090] In the first aspect, the present invention provides a combination comprising: (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. In one embodiment, the combination comprises an antagonist of IL-5:IL-5R and an antagonist of IL-4:IL-4R. In another embodiment, the combination comprises an antagonist of IL:5:IL-5R and an antagonist of IL-13:IL-13R. In yet another embodiment, the combination comprises an antagonist of IL-5:IL-5R, an antagonist of IL-4:IL-4R, and an antagonist of IL-13:IL-13R. In certain embodiments, the combination inhibits signal transduction via IL-5 and IL-4. In certain embodiments, the combination inhibits signal transduction via IL-5 and IL-13. In preferred embodiments, the combination inhibits signal transduction via IL-5, IL-4, and IL-13.

[0091] Without wishing to be 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 its combined action in blocking the activity of type 2 cytokines. IL-4, IL-13 and IL-5 are type 2 cytokines and the combination of the present invention can inhibit signaling via all three of these cytokines. "Type 2 immune response" refers to a response that is primarily mediated by a cell called T helper (T H However, the airway type 2 immune response can also be regulated by eosinophils, mast cells, basophils, T H 2 cells, group 2 innate lymphoid cells (ILC2), and IgE-producing B cells.

[0092] IL-4, IL-13 and IL-5 are responsible for the production of IgE, eosinophil activation and recruitment, and mucus production by B cells. Type 2 cytokines drive a series of downstream events, including the activation of airway epithelial cells, the chemoattraction of effector cells (mast cells, eosinophils and basophils), and the remodeling of epithelial and subepithelial matrix. In chronic airway diseases, such as asthma, abnormal signaling via IL-4, IL-13 and IL-5 can lead to airway eosinophilia, airway remodeling and bronchial hyperresponsiveness (see Lambrecht and Hammad (2015) supra). Related remodeling changes can include smooth muscle cell changes (hyperplasia and hypertrophy), mucous cell changes (goblet cell metaplasia) and vascular remodeling. In short, these inflammatory and pathological changes in the airway predispose individuals to an increased response to inhaled exacerbants.

[0093] The inventors have shown that by blocking signaling via IL-4, IL-13 and IL-5, a surprising synergistic effect is observed. This effect is observed relative to treatment with IL-4Rα monotherapy that blocks only IL-4 and IL-13 or IL-5 monotherapy that blocks only IL-5. The data provided herein show that the combination therapy of the present invention comprising 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. In an in vivo model of chronic airway disease, this effect is observed at the level of reduced mucin production (indicating goblet cell metaplasia) and reduced bronchial hyperresponsiveness. Importantly, it has been shown that the combination therapy according to the present invention comprising an antagonist targeting type 2 cytokines completely prevents bronchial hyperresponsiveness in an in vivo model.

[0094] The combination of the present invention comprises an antagonist that targets (i) IL-5:IL-5R; and (ii) IL-4:IL-4R and / or IL-13:IL-13R. As described herein, the term "antagonist" is used in a broad sense to mean any agent or molecule that is capable of inhibiting the function of its target. For example, an IL-5:IL-5R antagonist will inhibit the function of the IL-5 cytokine-IL-5 receptor complex, thereby inhibiting signal transduction via IL-5. Similarly, an IL-4:IL-4R antagonist will inhibit the function of the IL-4 cytokine-IL-4 receptor complex, thereby inhibiting signal transduction via IL-4. Similarly, an IL-13:IL-13R antagonist will inhibit the function of the IL-13 cytokine-IL-13 receptor complex, thereby inhibiting signal transduction via IL-13.

[0095] 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 will generally target a component of the cytokine-receptor complex or interact with such a component. For example, an antagonist of IL-5:IL-5R can interact with IL-5 to inhibit signal transduction via the IL-5:IL-5R signal transduction pathway. Alternatively, an antagonist of IL-5:IL-5R can interact with the receptor subunit IL-5Rα to inhibit signal transduction via the IL-5:IL-5R signal transduction pathway. In a preferred embodiment, the antagonist of IL-5:IL-5R is an antagonist of IL-5. Similarly, antagonists of IL-4:IL-4R and antagonists of IL-13:IL-13R can interact with cytokines IL-4 and IL-13, respectively, or can interact with receptor subunits to inhibit IL-4 and / or IL-13 signal transduction, respectively.

[0096] It is particularly preferred that the combination of the present invention comprises an antagonist of the receptor subunit IL-4Rα. More preferably, the combination comprises an antagonist of IL-5 and an antagonist of IL-4Rα. The reason for preferably targeting IL-4Rα is that this receptor subunit forms a part of the type I receptor complex of IL-4 (together with γc) and a part of the IL-13 receptor complex (together with IL-13Rα1). Therefore, the antagonist of IL-4Rα can simultaneously act as an antagonist of IL-4:IL-4R and IL-13:IL-13R, thereby inhibiting signal transduction via both IL-4 and IL-13. In particular, a combination of an antagonist of IL-5:IL-5R (preferably an antagonist of IL-5) and an antagonist of IL-4Rα can inhibit signal transduction via IL-5, IL-4 and IL-13. The antagonist of the combination described herein is preferably bound to its corresponding human target.

[0097] In a preferred embodiment, the antagonist of IL-5:IL-5R, IL-4:IL-4R and / or IL-13:IL-13R is an antibody molecule. More preferably, the combination comprises an antibody molecule that binds to IL-5 and an antibody molecule that binds to IL-4Rα.

[0098] In certain embodiments, the combination comprises an antibody molecule that binds to IL-5 as an IL-5:IL-5R antagonist. Alternatively, the combination may comprise an antibody molecule that binds to IL-5Rα as an IL-5:IL-5R antagonist. The IL-5 and IL-5R antibody molecules that may be incorporated into the combinations described herein include any suitable IL-5 and IL-5Rα antibodies known to those skilled in the art. Exemplary antibodies include mepolizumab that binds to IL-5. and reslizumab and benralizumab, which binds to IL-5Rα

[0099] In certain embodiments, the combination comprises an antibody molecule that binds to IL-4 as an IL-4:IL-4R antagonist. In certain embodiments, 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 mentioned above, this antagonist acts as an antagonist for both IL-4:IL-4R and IL-13:IL-13R, because the IL-4Rα subunit is shared by both IL-4 and IL-13 receptor complexes. Antibody molecules targeting 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 to those skilled in the art. Such antibodies include dupilumab It is a fully humanized mAb against the IL-4Rα receptor. See also: Sheridan C. (2018) Nat. Biotechnol. 36(1): 3-5.

[0100] The antibody molecules of the combination of the present invention, for example, antibody molecules that bind to IL-4Rα and antibody molecules that bind to IL-5, can be selected from any suitable antibody molecules that exhibit immunoreactivity to their respective targets. As mentioned above, the term "antibody molecule" is used herein to refer to full-length antibodies as well as antigen-binding fragments thereof.

[0101] The antibodies of the combinations described herein are intended for human therapeutic use and therefore will generally be of IgA, IgD, IgE, IgG, IgM type, often of IgG type, in which case the antibody may belong to any of the four subclasses IgG1, IgG2a and b, IgG3 or IgG4. In a preferred embodiment, the antibody molecule of the combination is an IgG antibody, optionally an IgG1 antibody. The antibody may be a monoclonal, polyclonal, multispecific (e.g., bispecific antibody) antibody, provided that the antibody exhibits appropriate immunospecificity to its target. Monoclonal antibodies are preferred because they are highly specific for a single antigenic site.

[0102] The combined antigen-binding fragments described herein will generally comprise a portion of a full-length antibody, generally its antigen-binding or variable domain. Examples of antibody fragments include Fab, Fab', F(ab')2, bispecific Fab' and Fv fragments, linear antibodies formed from antibody fragments, single-chain antibody molecules, single-chain variable fragments (scFv) and multispecific antibodies (see Holliger and Hudson (2005) Nature Biotechnol. 23: 1126-36, incorporated herein by reference).

[0103] The antibody molecules of the combinations described herein can exhibit high human homology. Such antibody molecules with high human homology can include antibodies comprising the VH and VL domains of native non-human antibodies, which exhibit sufficiently high sequence identity % with human germline sequences. In certain embodiments, the antibody molecules are humanized or germline variants of non-human antibodies.

[0104] In certain embodiments, the antibody molecules of the combinations described herein may be derived from Camelidae. The antibodies derived from Camelidae 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 Camelidae heterotetrameric antibodies.

[0105] For example, the antibody molecule may be selected from an immune library obtained by a method comprising the step of immunizing Camelidae with a target of interest. Camelidae may be immunized with a target protein or polypeptide fragment thereof, or with an mRNA molecule or cDNA molecule expressing the protein or polypeptide fragment thereof. Methods for generating antibodies in Camelidae species and selecting antibodies against preferred targets from Camelidae immune libraries are described, for example, in International Patent Application No. WO2010 / 001251, which is incorporated herein by reference.

[0106] In certain embodiments, the antibody molecule may be derived from Camelidae in that it comprises at least one hypervariable (HV) loop or complementarity determining region of a VH domain or VL domain obtained from a species of Camelidae. In particular, the antibody molecule may comprise a VH and / or VL domain or its CDR obtained by active immunization of an outbred Camelidae, such as a llama, with, for example, IL-4Rα and IL-5.

[0107] The term "derived from" in this context means a structural relationship in the sense that the HV or CDR of the antibody molecule embodies an amino acid sequence originally encoded by a camelid immunoglobulin gene (or a minor variant thereof). However, this does not necessarily mean a specific relationship with respect to the production process used to prepare the antibody molecule.

[0108] The camelid-derived antibody molecule may be derived from any species of Camelidae, including, inter alia, llama, dromedary, alpaca, llama, guanaco or camel.

[0109] Antibody molecules comprising VH and VL domains or their CDRs derived from Camelidae are typically recombinantly expressed polypeptides, and may be chimeric polypeptides. The term "chimeric polypeptide" refers to an artificial (non-naturally occurring) polypeptide created by the concatenation of two or more peptide fragments that would not otherwise appear contiguous. Included within this definition are "species" chimeric polypeptides created by the concatenation of peptide fragments encoded by two or more species (e.g., Camelidae and humans).

[0110] In certain embodiments, the entire VH domain and / or the entire VL domain may be obtained from a Camelidae species. The Camelidae-derived VH domain and / or the Camelidae-derived VL domain may then be subjected to protein engineering, wherein one or more amino acid substitutions, insertions or deletions are introduced into the Camelidae amino acid sequence. These engineered changes preferably include amino acid substitutions relative to the Camelidae sequence. Such changes include "humanization" or "germlining", wherein one or more amino acid residues in a Camelidae-encoded VH or VL domain are replaced by equivalent residues from a homologous human-encoded VH or VL domain.

[0111] Isolated Camelidae VH and VL domains obtained by active immunization of Camelidae (e.g., llamas) with, for example, IL-4Rα or IL-5 can be used as the basis for engineering antibody molecules for use in the combinations described herein. Starting with intact Camelidae VH and VL domains, it is possible to engineer one or more amino acid substitutions, insertions or deletions away from the starting Camelidae sequence. In certain embodiments, such substitutions, insertions or deletions may be present in the framework regions of the VH domain and / or the VL domain.

[0112] In other embodiments, "chimeric" antibody molecules are provided, comprising VH and VL domains derived from Camelidae (or engineered variants thereof) and one or more constant domains from a non-Camelidae antibody, e.g., a human-encoded constant domain (or an engineered variant thereof). In such embodiments, it is preferred that both the VH domain and the VL domain are obtained from the same Camelidae species, e.g., both the VH and VL may be from Lama glama or both the VH and VL may be from Lama pacos (before the introduction of engineered amino acid sequence variations). In such embodiments, both the 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.

[0113] As an alternative to engineering changes in the primary amino acid sequence of the Camelid VH and / or VL domains, individual Camelid-derived hypervariable loops or CDRs or combinations thereof may be isolated from Camelid VH / VL domains and transferred by CDR grafting into alternative (i.e., non-Camelid) frameworks, such as human VH / VL frameworks.

[0114] In non-limiting embodiments, the antibody molecule of the combination may comprise a CH1 domain and / or a CL domain (from a heavy chain and a light chain, respectively), the amino acid sequence of which is completely or substantially human. For antibody molecules intended for human therapeutic use, typically, the entire constant region of the antibody or at least a portion thereof has a completely 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 completely or substantially human with respect to its 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 subtype IgG1, IgG2, IgG3 or IgG4.

[0115] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain, if present) may all have completely or substantially human amino acid sequences. In the case of the constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially human" refers to an amino acid sequence identity of at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% 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 encompasses polypeptides comprising a constant domain of a "human" sequence, which has been altered by one or more amino acid additions, deletions, or substitutions with respect to a human sequence, except for those embodiments in which a "fully human" hinge region is explicitly required.

[0116] Modification of the Fc region

[0117] The antibody molecules of the combination may have one or more amino acid substitutions, insertions or deletions in the constant region of the heavy chain and / or light chain, particularly in the Fc region. Amino acid substitutions may be such that the substituted amino acid is replaced by a different naturally occurring amino acid or by a non-natural or modified amino acid. Other structural modifications are also permitted, for example, changes in glycosylation patterns (e.g., by the addition or deletion of N-linked or O-linked glycosylation sites).

[0118] The antibody molecule of combination can be modified in Fc district to increase the binding affinity to neonatal receptor FcRn. The increased binding affinity can be measured at acidic pH value (e.g., about pH 5.5 to about pH 6.0). The increased binding affinity can also be measured at neutral pH value (e.g., about pH 6.9 to about pH 7.4). "Increased binding affinity" means that the binding affinity to FcRn increases relative to unmodified Fc district. Typically, unmodified Fc district will have the wild-type amino acid sequence of human IgG1, IgG2, IgG3 or IgG4. In such embodiments, the increased FcRn binding affinity of the antibody molecule with modified Fc district will be measured relative to the binding affinity of wild-type IgG1, IgG2, IgG3 or IgG4 to FcRn.

[0119] In a preferred embodiment, one or more amino acid residues in the Fc region can be substituted with different amino acids to increase binding to FcRn. Several Fc substitutions that increase FcRn binding, thereby improving antibody pharmacokinetics, have been reported. 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 entirety.

[0120] In a preferred embodiment, one or more of the antibody molecules of the combinations described herein 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 another preferred embodiment, one or more of the antibody molecules of the combinations described herein comprise a modified human IgG Fc domain comprising or consisting of the amino acid substitutions M252Y, S254T, T256E, H433K and N434F, wherein the Fc domain numbering is according to EU numbering.

[0121] In certain embodiments, the combined antibody molecules, e.g., IL-4Rα and / or IL-5 antibody molecules, comprise a modified human IgG Fc domain consisting of at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 12, at most 15, at most 20 substitutions relative to the corresponding wild-type IgG sequence.

[0122] Antibody molecules can also be modified to form immunoconjugates comprising antibodies conjugated to cytotoxic agents, such as chemotherapeutic agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof) or radioactive isotopes (i.e., radioconjugates). Fc regions can also be engineered to extend half-life, as described by Chan and Carter (2010) Nature Reviews: Immunology 10: 301-316, which is incorporated herein by reference.

[0123] In certain embodiments, the Fc region can be engineered so that effector functions are absent. In certain embodiments, the antibody molecules of the present invention can have an Fc region derived from a naturally occurring IgG isotype having reduced effector functions, such as IgG4. The Fc region derived from IgG4 can be further modified to increase therapeutic utility, for example, by introducing modifications that minimize arm exchange between IgG4 molecules in vivo. The Fc region derived from IgG4 can be modified to include an S228P substitution.

[0124] In certain embodiments, the antibody molecules of the combination are modified with respect to glycosylation. For example, a deglycosylated antibody (i.e., the antibody lacks glycosylation) can be made. Glycosylation can be altered, for example, to increase the affinity of the antibody for the target antigen. Such carbohydrate modifications 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 deglycosylation can increase the affinity of the antibody for the antigen.

[0125] pH-dependent antibodies

[0126] The combined antibody molecules can exhibit pH-dependent antigen binding.

[0127] The antibody bound to the antigen is absorbed into the cell and transported to the endosomal-lysosomal degradation path. The antibody that can be dissociated from its antigen in the early endosome can be recycled back to the cell surface. The antibody that is bound to its antigen with high affinity in the endosomal compartment is usually transported to the lysosome for degradation. It has been previously shown that if the antibody molecule has pH-dependent antigen binding activity, so that it has a lower binding affinity to its antigen compared with the plasma pH value under the early endosomal pH value, then the antibody will be more effectively recycled to the cell surface. This can extend the antibody plasma half-life and allow the same antibody to be bound to multiple antigens. For this reason, it is advantageous that the antibody molecules of the combination described herein, such as IL-4Rα and / or IL-5 antibody molecules, exhibit pH-dependent antigen binding.

[0128] Methods for engineering pH-dependent antigen binding activity in antibody molecules are described, for example, in EP2275443, which is incorporated herein by reference. Methods for engineering pH-dependent antigen binding in antibody molecules are also described in WO2018 / 206748, which is incorporated herein by reference. The antibody molecules described herein can be modified according to the methods described in EP2275443 or WO2018 / 206748 to exhibit pH-dependent antigen binding.

[0129] For the pH-dependent antibody molecules of the combinations described herein, the antigen binding activity at endosomal pH is lower than the antigen binding activity at plasma pH. Endosomal pH is generally an acidic pH, while plasma pH is generally a neutral pH. Therefore, the combined antibody molecules, for example, the IL-4Rα and / or IL-5 antibody molecules described herein, can exhibit pH-dependent antigen binding, so that their antigen binding activity at acidic pH is lower than that at neutral pH. Endosomal pH or "acidic pH" can be the following pH: 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, preferably pH 5.5, pH 5.6, pH 5.7 or pH 5.8. The 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.

[0130] In certain embodiments, the antibody molecule, e.g., IL-4Rα antibody molecule and / or IL-5 antibody molecule, exhibits pH-dependent binding, such that the antigen binding activity at pH 5.8 is lower than the antigen binding activity at pH 7.4. The pH-dependent antibody molecule, e.g., IL-4Rα antibody molecule and / or IL-5 antibody molecule, is 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 molecule, e.g., IL-4Rα antibody molecule and / or IL-5 antibody molecule, exhibits 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 more, 4 or more, 6 or more, 8 or more, 10 or more, 12 or more.

[0131] The pH-dependent antigen-binding activity of the antibody molecule can be engineered by modifying the antibody molecule to confer antigen-binding ability at an acidic pH and / or to increase the antigen-binding ability at a neutral pH. For example, the antibody molecule can be modified by replacing at least one amino acid of the antibody molecule with histidine, or by inserting at least one histidine into the antibody molecule. Such histidine mutation (replacement or insertion) sites are not particularly limited, and any site is acceptable, as long as the antigen-binding activity at an endosomal pH (e.g., pH 5.8) is lower than the antigen-binding activity at a plasma pH (e.g., pH 7.4) compared to before the mutation or insertion.

[0132] In certain embodiments, antibody molecules, for example, IL-4Rα antibody molecules and / or IL-5 antibody molecules, can be engineered by introducing one or more substitutions into the variable domains to exhibit pH-dependent antigen binding. In a preferred embodiment, antibody molecules, for example, IL-4Rα antibody molecules and / or IL-5 antibody molecules, can be engineered by introducing one or more substitutions into the CDRs of antibody molecules to exhibit pH-dependent antigen binding. Replacement can introduce one or more His residues into the variable domains, preferably one or more sites of the heavy chain and / or light chain CDRs, to confer pH-dependent antigen binding. Non-histidine substitutions can also be incorporated into the variable domains of the pH-dependent antibodies described herein, particularly in the CDRs. Antibody molecules, for example, IL-4Rα antibody molecules and / or IL-5 antibody molecules can be engineered according to the methods described in WO2018 / 206748.

[0133] In a preferred embodiment, the exemplary IL-4Rα and IL-5 antibodies described herein having the CDR, VH and / or VL domain sequences listed above are engineered to exhibit pH-dependent antigen binding. For example, the CDR sequences of the exemplary IL-4Rα and / or IL-5 antibody molecules listed herein can be modified by introducing one or more histidine substitutions to generate antibody molecules that exhibit pH-dependent antigen binding.

[0134] In a particularly preferred embodiment, the pH-dependent antibodies of the combinations described herein, e.g., IL-4Rα and / or IL-5 antibody molecules, comprise an Fc domain with increased binding affinity for the neonatal receptor FcRn. Substitutions that can increase the binding affinity of the Fc domain to FcRn are described elsewhere herein and any such substitutions can be incorporated into the pH-dependent antibody molecules of the combinations of the invention.

[0135] 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 of the antibody molecules comprises a modified human IgG Fc domain comprising or consisting of amino acid substitutions H433K and N434F, wherein the Fc domain numbering is according to EU numbering. In another preferred embodiment, the combination comprises a pH-dependent IL-4Rα antibody molecule and a pH-dependent IL-5 antibody molecule, wherein one or both of the antibody molecules comprises a modified human IgG Fc domain comprising or consisting of amino acid substitutions M252Y, S254T, T256E, H433K and N434F, wherein the Fc domain numbering is according to EU numbering.

[0136] Exemplary IL-4Rα Antibodies and IL-5 Antibodies

[0137] As mentioned above, in a preferred embodiment, the combination comprises an antibody molecule that binds to IL-5 and an antibody molecule that binds to IL-4Rα. In such embodiments, the combination may inhibit signaling through all three type 2 cytokines IL-5, IL-4 and IL-13.

[0138] Antibody molecules that bind to human IL-4Rα and that can be incorporated into the combinations described herein include antibody molecules comprising 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) selected from:

[0139] (i) HCDR3 comprising or consisting of SEQ ID NO: 3; HCDR2 comprising or consisting of SEQ ID NO: 2; HCDR1 comprising or consisting of SEQ ID NO: 1; LCDR3 comprising or consisting of SEQ ID NO: 12; LCDR2 comprising or consisting of SEQ ID NO: 11; LCDR1 comprising or consisting of SEQ ID NO: 10;

[0140] (ii) HCDR3 comprising or consisting of SEQ ID NO:6; HCDR2 comprising or consisting of SEQ ID NO:5; HCDR1 comprising or consisting of SEQ ID NO:4; LCDR3 comprising or consisting of SEQ ID NO:15; LCDR2 comprising or consisting of SEQ ID NO:14; LCDR1 comprising or consisting of SEQ ID NO:13;

[0141] (iii) HCDR3 comprising or consisting of SEQ ID NO:9; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:18; LCDR2 comprising or consisting of SEQ ID NO:17; LCDR1 comprising or consisting of SEQ ID NO:16;

[0142] (iv) HCDR3 comprising or consisting of SEQ ID NO:91; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:18; LCDR2 comprising or consisting of SEQ ID NO:17; LCDR1 comprising or consisting of SEQ ID NO:16;

[0143] (v) HCDR3 comprising or consisting of SEQ ID NO:92; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:97; LCDR2 comprising or consisting of SEQ ID NO:17; LCDR1 comprising or consisting of SEQ ID NO:16;

[0144] (vi) HCDR3 comprising or consisting of SEQ ID NO:93; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:98; LCDR2 comprising or consisting of SEQ ID NO:96; LCDR1 comprising or consisting of SEQ ID NO:16;

[0145] (vii) HCDR3 comprising or consisting of SEQ ID NO:94; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:98; LCDR2 comprising or consisting of SEQ ID NO:17; LCDR1 comprising or consisting of SEQ ID NO:16; and

[0146] (viii) HCDR3 comprising or consisting of SEQ ID NO:95; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:98; LCDR2 comprising or consisting of SEQ ID NO:17; LCDR1 comprising or consisting of SEQ ID NO:16.

[0147] Antibody molecules that bind to IL-4Rα and that can be incorporated into the combinations described herein also include antibody molecules comprising 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) selected from:

[0148] (i) HCDR3 comprising or consisting of SEQ ID NO:27; HCDR2 comprising or consisting of SEQ ID NO:26; HCDR1 comprising or consisting of SEQ ID NO:25; LCDR3 comprising or consisting of SEQ ID NO:36; LCDR2 comprising or consisting of SEQ ID NO:35; LCDR1 comprising or consisting of SEQ ID NO:34;

[0149] (ii) HCDR3 comprising or consisting of SEQ ID NO:30; HCDR2 comprising or consisting of SEQ ID NO:29; HCDR1 comprising or consisting of SEQ ID NO:28; LCDR3 comprising or consisting of SEQ ID NO:39; LCDR2 comprising or consisting of SEQ ID NO:38; LCDR1 comprising or consisting of SEQ ID NO:37; and

[0150] (iii) HCDR3 comprising or consisting of SEQ ID NO:33; HCDR2 comprising or consisting of SEQ ID NO:32; HCDR1 comprising or consisting of SEQ ID NO:31; LCDR3 comprising or consisting of SEQ ID NO:42; LCDR2 comprising or consisting of SEQ ID NO:41; LCDR1 comprising or consisting of SEQ ID NO:40.

[0151] In certain embodiments, the antibody molecule that binds to human IL-4Rα is selected from an antibody molecule comprising or consisting of a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from:

[0152] (i) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 19, 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 or consisting of the amino acid sequence of SEQ ID NO: 20, 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;

[0153] (ii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21, 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 or consisting of the amino acid sequence of SEQ ID NO: 22, 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;

[0154] (iii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23, 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 or consisting of the amino acid sequence of SEQ ID NO: 24, 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;

[0155] (iv) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 99, 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 or consisting of the amino acid sequence of SEQ ID NO: 100, 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;

[0156] (v) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 101, 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 or consisting of the amino acid sequence of SEQ ID NO: 102, 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;

[0157] (vi) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 103, 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 or consisting of the amino acid sequence of SEQ ID NO: 104, 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;

[0158] (vii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence 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 or consisting of the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% identical thereto; and

[0159] (viii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 107, 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 or consisting of the amino acid sequence of SEQ ID NO: 108, 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.

[0160] In certain embodiments, the antibody molecule that binds to IL-4Rα is selected from an antibody molecule comprising or consisting of a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from:

[0161] (i) a VH domain comprising or consisting of the amino acid sequence 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%, at least 99% identical thereto, and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 44, 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;

[0162] (ii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence 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 or consisting of the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% identical thereto; and

[0163] (iii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 47, 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 or consisting of the amino acid sequence of SEQ ID NO: 48, 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.

[0164] Antibody molecules that bind to human IL-5 and that can be incorporated into the combinations described herein include antibody molecules comprising 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) selected from:

[0165] (i) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:54; LCDR2 comprising or consisting of SEQ ID NO:53; LCDR1 comprising or consisting of SEQ ID NO:52;

[0166] (ii) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:57; LCDR2 comprising or consisting of SEQ ID NO:56; LCDR1 comprising or consisting of SEQ ID NO:55;

[0167] (ii) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:59; LCDR2 comprising or consisting of SEQ ID NO:56; LCDR1 comprising or consisting of SEQ ID NO:58;

[0168] (iv) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:61; LCDR2 comprising or consisting of SEQ ID NO:60; LCDR1 comprising or consisting of SEQ ID NO:58; and

[0169] (v) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:62; LCDR2 comprising or consisting of SEQ ID NO:56; LCDR1 comprising or consisting of SEQ ID NO:58.

[0170] Antibody molecules that bind to IL-5 and that can be incorporated into the combinations described herein include antibody molecules comprising 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) selected from:

[0171] (i) HCDR3 comprising or consisting of SEQ ID NO:72; HCDR2 comprising or consisting of SEQ ID NO:71; HCDR1 comprising or consisting of SEQ ID NO:70; LCDR3 comprising or consisting of SEQ ID NO:74; LCDR2 comprising or consisting of SEQ ID NO:38; LCDR1 comprising or consisting of SEQ ID NO:73; and

[0172] (ii) HCDR3 comprising or consisting of SEQ ID NO:72; HCDR2 comprising or consisting of SEQ ID NO:71; HCDR1 comprising or consisting of SEQ ID NO:70; LCDR3 comprising or consisting of SEQ ID NO:75; LCDR2 comprising or consisting of SEQ ID NO:38; LCDR1 comprising or consisting of SEQ ID NO:73.

[0173] In certain embodiments, the antibody molecule that binds to human IL-5 is selected from an antibody molecule comprising or consisting of a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from:

[0174] (i) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 63, 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 or consisting of the amino acid sequence of SEQ ID NO: 64, 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;

[0175] (ii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 63, 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 or consisting of the amino acid sequence of SEQ ID NO: 65, 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;

[0176] (iii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 63, 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 or consisting of the amino acid sequence of SEQ ID NO: 66, 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;

[0177] (iv) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 63, 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 or consisting of the amino acid sequence of SEQ ID NO: 67, 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;

[0178] (v) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence 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 or consisting of the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% identical thereto; and

[0179] (vi) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO:63, 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 or consisting of the amino acid sequence of SEQ ID NO:69, 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.

[0180] In certain embodiments, the antibody molecule that binds to IL-5 is selected from an antibody molecule comprising or consisting of a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from:

[0181] (i) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76, 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 or consisting of the amino acid sequence of SEQ ID NO: 77, 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;

[0182] (ii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76, 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 or consisting of the amino acid sequence of SEQ ID NO: 78, 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

[0183] (iii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76, 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 or consisting of the amino acid sequence of SEQ ID NO: 79, 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.

[0184] In a preferred embodiment, the combination comprises an antibody molecule having CDR, VH and / or VL sequences that bind to human IL-4Rα and an antibody molecule having CDR, VH and / or VL sequences that bind to human IL-5.

[0185] When a particular antibody molecule is identified as comprising a combination of a VH domain or heavy chain defined by reference to a particular amino acid sequence and a VL domain or light chain also defined by reference to a particular amino acid sequence, then for each particular VH / VL or heavy chain / light chain combination listed (unless otherwise specified), this definition can be taken to include antibody molecules formed by a combination of a VH domain / heavy chain having at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the specified VH / heavy chain amino acid sequence and a VL domain / light chain having at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the specified VL / light chain amino acid sequence.

[0186] In each case, the domain / chain defined by the % sequence identity to a specified domain / chain amino acid sequence may retain CDR sequences identical to those present in a specified VH / VL domain or heavy / light chain amino acid sequence, while exhibiting amino acid sequence variation within the framework or other regions outside of the CDR regions.

[0187] In certain embodiments, the IL-4Rα and / or IL-5 antibody molecules defined as having the CDR sequences listed above, or as having a particular percent identity with a particular VH / VL domain amino acid sequence listed above, are humanized, germlined, or affinity variants of the antibody or antigen-binding fragment thereof from which the CDR, VH and / or VL sequences are derived.

[0188] In preferred embodiments, the exemplary IL-4Rα and IL-5 antibody molecules having the CDR sequences listed above exhibit a high degree of human homology, such as being humanized or germlined variants of the antibodies or antigen-binding fragments thereof from which the CDR sequences are derived.

[0189] In non-limiting embodiments, exemplary IL-4Rα and IL-5 antibody molecules having CDR, VH and / or VL sequences described herein may comprise a CH1 domain and / or a CL domain (from the heavy chain and light chain, respectively) whose amino acid sequence is completely or substantially human. For antibody molecules intended for human therapeutic use, typically the entire constant region of the antibody or at least a portion thereof has a completely 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 completely or substantially human with respect to their amino acid sequence.

[0190] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain, if present) may all have completely or substantially human amino acid sequences. In the case of the constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially human" refers to an amino acid sequence identity of at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% 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 encompasses polypeptides comprising a constant domain of a "human" sequence, which has been altered by one or more amino acid additions, deletions, or substitutions with respect to a human sequence, except for those embodiments in which a "fully human" hinge region is explicitly required. Any of the exemplary Fc region modifications described herein can be incorporated into an IL-4Rα and / or IL-5 antibody having the CDR and / or VH / VL domain sequences listed above. In a preferred embodiment, the IL-4Rα and / or IL-5 antibody having the CDR and / or VH / VL domain sequences listed above includes 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 antibody having the CDR and / or VH / VL domain sequences listed above includes a modified human IgG Fc domain comprising or consisting of the amino acid substitutions M252Y, S254T, T256E, H433K and N434F.

[0191] In non-limiting embodiments, exemplary IL-4Rα and IL-5 antibody molecules having CDR, VH and / or VL sequences described herein can be modified as described above to exhibit pH-dependent antigen binding. For example, exemplary IL-4Rα and IL-5 antibody molecules having CDR, VH and / or VL sequences described herein can be modified to have pH-dependent binding, so that the antigen binding activity at pH 5.8 is lower than the antigen binding activity at pH 7.4. The methods described above for engineering antibodies to confer pH-dependent antigen binding can be applied to any of the exemplary IL-4Rα and IL-5 antibody molecules having CDR, VH and / or VL sequences described herein. For example, variable domains and / or CDR regions can be modified by histidine substitution or insertion to confer pH-dependent binding.

[0192] Unless otherwise specified in the present application, the sequence identity % between two amino acid sequences can be determined by comparing the two sequences aligned in an optimal manner and wherein the compared amino acid sequence may comprise additions or deletions with respect to the reference sequence for optimal alignment between the two sequences. The identity percentage is calculated in the following manner: determine the number of identical positions where the amino acid residues are identical between the two sequences, divide this number of identical positions by the total number of positions in the comparison window, and multiply the result obtained by 100 to obtain the identity percentage between the two sequences. For example, it is possible to use the BLAST program "BLAST 2 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 website http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html, the parameters used being those given by the default values ​​(in particular for the parameters "open gap penalty": 5, and "extend gap penalty": 2; the matrix selected is, for example, the matrix "BLOSUM 62" proposed by the program), and the percentage identity between the two sequences being compared can be calculated directly by the program.

[0193] C. Combination preparation

[0194] The different antagonist or antibody molecules of the combination may be combined or formulated by any means that permits administration of the combination therapy 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.

[0195] In certain embodiments, the antagonist or antibody molecule of combination is co-formulated, that is, formulated into an independent molecule of a single pharmaceutical composition. For the embodiment of co-formulated antagonist or antibody molecule, combination or composition is suitable for simultaneous administration of two components. Composition can be formulated for single dose administration or multiple dose administration. For the embodiment of co-formulated antagonist or antibody molecule, antagonist or antibody molecule can be prepared in equivalent amount, for example, for the combination of the first and second antagonist or antibody molecule comprising targeting different cytokines or receptors according to 1:1 ratio. Alternatively, antagonist or antibody molecule can be prepared so that the ratio of different antagonists or antibody molecules is not 1:1. For example, for the embodiment of the combination comprising or consisting of the first and second antagonist or antibody molecule being bound to different targets, the ratio of the first and second antagonist or antibody molecule can be 2:1, optionally 3:1, optionally 4:1. Alternatively, antagonist or antibody molecule can be prepared according to 1:2, optionally 1:3, optionally 1:4 ratio.

[0196] In certain embodiments, the antagonist or antibody molecule of the combination is independently formulated, for example, as an individual composition. For embodiments in which antagonists or antibody molecules are independently formulated, it is possible to apply different components or compositions simultaneously or independently. If antagonists or antibody molecules or independent compositions containing them are applied independently, the antagonist / antibody molecule or composition can be applied sequentially in any order. For example, an antibody molecule that binds to IL-4Rα can be applied first, followed by an antibody molecule that binds to IL-5, and vice versa. The interval between the application of antagonists / antibody molecules or compositions can be any suitable time interval. The application of different compositions can be performed once (for single dose application) or repeatedly (for multiple dose application).

[0197] In certain embodiments where the antagonist is an antibody molecule, the combined antibody molecules can be combined into a multispecific antibody, such as a bispecific antibody. For example, if the combination comprises a Fab fragment that binds to IL-4Rα and a Fab fragment that binds to IL-5, the two Fab fragments can be incorporated into a single bispecific antibody molecule having two Fab regions conjugated to an IgG Fc portion.

[0198] Bispecific or multispecific antibodies according to the present invention can be constructed according to any suitable bispecific / multispecific antibody format.For example, the antibody molecule of combination can be incorporated into the bispecific or multispecific antibody format so that the antibody is bound to different targets in "trans", for example, each Fab arm of a Y-shaped antibody has a situation of different binding specificity.In alternative embodiments, the antibody molecule can be incorporated into the bispecific or multispecific antibody format so that the target is bound in the "cis" position.For example, the Fab region or variable domains capable of binding antigens can be positioned at the relative end of the IgGFc part.

[0199] A bispecific or multispecific antibody may have a native IgG structure having two Y-shaped Fab arms with binding specificity for a first target, and one or more additional antigen binding domains located at the C-terminus of the Fc domain with binding specificity for a second target. For example, in one embodiment, a bispecific antibody according to the present invention is constructed so that the two typical antigen binding domains of the Fab region present on the native IgG structure bind to IL-4Rα, and the one or more VHH domains located at the C-terminus of the Fc domain bind to IL-5. The opposite configuration is also possible, in which the two typical antigen binding domains of the Fab region present on the native IgG structure bind to IL-5, and the one or more VHH domains present at the C-terminus of the Fc domain bind to IL-4Rα.

[0200] Alternatively, the bispecific or multispecific antibody may have a native IgG structure having two Y-shaped Fab arms with 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, the bispecific antibody according to the present invention is constructed so that the two typical antigen binding domains of the Fab region present on the native IgG structure bind to IL-4Rα, and the one or more scFv domains located at the C-terminus of the Fc domain bind to IL-5. The opposite configuration is also possible, in which the two typical antigen binding domains of the Fab region present on the native IgG structure bind to IL-5, and the one or more scFv domains present at the C-terminus of the Fc domain bind to IL-4Rα. In such embodiments, the bispecific antibody may consist of two scFv domains present at the C-terminus of the IgG Fc domain.

[0201] The bispecific or multispecific antibody may be an asymmetric IgG antibody such that one Fab region is replaced by a different antigen binding domain, such as a VHH domain. For example, in another embodiment, the bispecific antibody according to the present invention may be constructed similarly to a native IgG structure comprising a Fab region on one complete arm of an antibody that binds to IL-4Rα and a VHH domain that replaces the second Fab region that binds to IL-5. The opposite configuration is also possible, in which the Fab region on one complete arm of the antibody binds to IL-5 and the VHH domain that binds to IL-4Rα replaces the second Fab region.

[0202] For antagonist is antibody molecule and co-preparation of antibody molecule embodiment and / or for providing antibody molecule embodiment as independent composition and / or when providing antibody molecule in multi-specific antibody form, can use any suitable drug carrier or excipient to prepare antibody molecule.Techniques for preparing antibody for human therapeutic use are well known in the art and for example in Wang et al. (2007) Journal of Pharmaceutical Sciences, 96:1-26, which content is incorporated herein in full.For independently preparing antibody molecule embodiment, drug carrier or excipient may be different or identical for different compositions.

[0203] Pharmaceutically acceptable excipients that can be used to formulate the composition 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 silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (e.g., sodium carboxymethyl cellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0204] In certain embodiments, the composition is prepared 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, inhaled, buccal (e.g., sublingual) and transdermal administration. For embodiments in which antagonists or antibody molecules are independently prepared, each composition can be prepared for administration via different routes.

[0205] In certain embodiments, the composition comprises one or more additional therapeutic agents. The additional therapeutic agent may be an agent suitable for preventing or treating chronic airway disease. One or more additional agents may be formulated for administration via the same route or via a different route compared to the antagonist or antibody molecule of the combination.

[0206] D. Bispecific IL-4Rα and IL-5 Antibodies

[0207] As discussed above, the antibody molecules of the combinations described herein may be provided in a multispecific antibody format, such as a bispecific antibody format. In a preferred embodiment, the combination comprises an antibody molecule that binds to IL-5 and an antibody molecule that binds to IL-4Rα, which are provided in a bispecific antibody format.

[0208] Therefore, according to another aspect of the present invention, a bispecific antibody is provided herein, comprising an antigen binding region that binds to IL-4Rα and an antigen binding region that binds to IL-5. Such a bispecific antibody is referred to herein as an "IL-4Rα / IL-5 bispecific" antibody. All embodiments described above with respect to independent IL-4Rα and IL-5 antibody molecules are equally applicable to this other aspect of the present invention. In particular, the "antigen binding region" of the bispecific antibody described herein may take the form of any of the antibodies or antigen binding fragments described elsewhere herein, including VHH antibodies.

[0209] As described elsewhere herein, the bispecific antibodies of the invention can be constructed according to any suitable bispecific / multispecific antibody format. For example, the bispecific antibodies can be constructed so that the target is bound in a "trans" position (i.e., at the same end of the molecule) or in a "cis" position (i.e., at the opposite end of the molecule).

[0210] In certain embodiments, the bispecific antibody has a native IgG structure, wherein the antigen binding region is contained in two Fab arms. The first Fab arm can exhibit binding specificity to IL-4Rα and the second Fab arm can exhibit binding specificity to IL-5. In alternative embodiments, the bispecific antibody has a native IgG structure and additionally comprises one or more additional antigen binding regions located at the C-terminus of the Fc region. In such embodiments, the two Fab arms of the native IgG can bind to the same target, such as IL-4Rα, and the one or more additional antigen binding regions located at the C-terminus of the Fc region can bind to a second target, such as IL-5. One or more additional antigen binding regions can adopt any suitable antigen binding form, including but not limited to VHH domains or scFv.

[0211] In one embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention comprises a Fab region that binds to IL-4Rα and a VHH domain that binds to IL-5. In one embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention comprises a Fab region that binds to IL-5 and a VHH domain that binds to IL-4Rα.

[0212] In one embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention comprises a Fab region that binds to IL-4Rα and a scFv that binds to IL-5. In one embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention comprises a Fab region that binds to IL-5 and a scFv that binds to IL-4Rα. In a preferred embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention comprises or consists of an IgG antibody that binds to IL-4Rα and one or more scFvs that bind to IL-5. In such embodiments, the IL-4Rα / IL-5 bispecific antibody may comprise or consist of an IgG antibody that binds to IL-4Rα and two scFv fragments that bind to IL-5. The one or more scFv fragments that bind to IL-5 are preferably located at the C-terminus of the Fc region of the IgG antibody, that is, at the end of the Fc region opposite to the two Fab arms that bind to IL-4Rα.

[0213] The bispecific antibodies of the present invention can be constructed 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 constructed similar to the native IgG structure, comprising a Fab region on one complete arm of the antibody that binds to IL-4Rα and a VHH domain that replaces the second Fab region that binds to IL-5. The opposite configuration is also possible, in which the Fab region on one complete arm of the antibody binds to IL-5, and the VHH domain that replaces the second Fab region binds to IL-4Rα.

[0214] The present invention, i.e., the IL-4Rα / IL-5 bispecific antibody constructed according to any of the formats described herein, may comprise an antigen binding region that binds to human IL-4R, wherein the antigen binding region comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains comprise CDR sequences selected from the group consisting of:

[0215] (i) HCDR3 comprising or consisting of SEQ ID NO: 3; HCDR2 comprising or consisting of SEQ ID NO: 2; HCDR1 comprising or consisting of SEQ ID NO: 1; LCDR3 comprising or consisting of SEQ ID NO: 12; LCDR2 comprising or consisting of SEQ ID NO: 11; LCDR1 comprising or consisting of SEQ ID NO: 10;

[0216] (ii) HCDR3 comprising or consisting of SEQ ID NO:6; HCDR2 comprising or consisting of SEQ ID NO:5; HCDR1 comprising or consisting of SEQ ID NO:4; LCDR3 comprising or consisting of SEQ ID NO:15; LCDR2 comprising or consisting of SEQ ID NO:14; LCDR1 comprising or consisting of SEQ ID NO:13;

[0217] (iii) HCDR3 comprising or consisting of SEQ ID NO:9; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:18; LCDR2 comprising or consisting of SEQ ID NO:17; LCDR1 comprising or consisting of SEQ ID NO:16;

[0218] (iv) HCDR3 comprising or consisting of SEQ ID NO:91; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:18; LCDR2 comprising or consisting of SEQ ID NO:17; LCDR1 comprising or consisting of SEQ ID NO:16;

[0219] (v) HCDR3 comprising or consisting of SEQ ID NO:92; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:97; LCDR2 comprising or consisting of SEQ ID NO:17; LCDR1 comprising or consisting of SEQ ID NO:16;

[0220] (vi) HCDR3 comprising or consisting of SEQ ID NO:93; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:98; LCDR2 comprising or consisting of SEQ ID NO:96; LCDR1 comprising or consisting of SEQ ID NO:16;

[0221] (vii) HCDR3 comprising or consisting of SEQ ID NO:94; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:98; LCDR2 comprising or consisting of SEQ ID NO:17; LCDR1 comprising or consisting of SEQ ID NO:16; and

[0222] (viii) HCDR3 comprising or consisting of SEQ ID NO:95; HCDR2 comprising or consisting of SEQ ID NO:8; HCDR1 comprising or consisting of SEQ ID NO:7; LCDR3 comprising or consisting of SEQ ID NO:98; LCDR2 comprising or consisting of SEQ ID NO:17; LCDR1 comprising or consisting of SEQ ID NO:16.

[0223] The IL-4Rα / IL-5 bispecific antibody of the present invention may comprise an antigen binding region that binds to IL-4R, wherein the antigen binding region comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains comprise a CDR sequence selected from the group consisting of:

[0224] (i) HCDR3 comprising or consisting of SEQ ID NO:27; HCDR2 comprising or consisting of SEQ ID NO:26; HCDR1 comprising or consisting of SEQ ID NO:25; LCDR3 comprising or consisting of SEQ ID NO:36; LCDR2 comprising or consisting of SEQ ID NO:35; LCDR1 comprising or consisting of SEQ ID NO:34;

[0225] (ii) HCDR3 comprising or consisting of SEQ ID NO:30; HCDR2 comprising or consisting of SEQ ID NO:29; HCDR1 comprising or consisting of SEQ ID NO:28; LCDR3 comprising or consisting of SEQ ID NO:39; LCDR2 comprising or consisting of SEQ ID NO:38; LCDR1 comprising or consisting of SEQ ID NO:37; and

[0226] (iii) HCDR3 comprising or consisting of SEQ ID NO:33; HCDR2 comprising or consisting of SEQ ID NO:32; HCDR1 comprising or consisting of SEQ ID NO:31; LCDR3 comprising or consisting of SEQ ID NO:42; LCDR2 comprising or consisting of SEQ ID NO:41; LCDR1 comprising or consisting of SEQ ID NO:40.

[0227] The antigen binding region that binds to human IL-4Rα may comprise a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from the group consisting of:

[0228] (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 19, 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 the amino acid sequence of SEQ ID NO: 20, 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;

[0229] (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 21, 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 the amino acid sequence of SEQ ID NO: 22, 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;

[0230] (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 23, 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 the amino acid sequence of SEQ ID NO: 24, 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;

[0231] (iv) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 99, 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 or consisting of the amino acid sequence of SEQ ID NO: 100, 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;

[0232] (v) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 101, 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 or consisting of the amino acid sequence of SEQ ID NO: 102, 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;

[0233] (vi) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 103, 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 or consisting of the amino acid sequence of SEQ ID NO: 104, 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;

[0234] (vii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence 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 or consisting of the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% identical thereto; and

[0235] (viii) a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 107, 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 or consisting of the amino acid sequence of SEQ ID NO: 108, 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.

[0236] The antigen binding region that binds to IL-4Rα may comprise a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from the group consisting of:

[0237] (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence that is at least 70% identical thereto;

[0238] (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence that is at least 70% identical thereto; and

[0239] (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence that is at least 70% identical thereto.

[0240] The IL-4Rα / IL-5 bispecific antibody of the present invention may comprise an antigen binding region that binds to human IL-5, wherein the antigen binding region comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains comprise a CDR sequence selected from the group consisting of:

[0241] (i) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:54; LCDR2 comprising or consisting of SEQ ID NO:53; LCDR1 comprising or consisting of SEQ ID NO:52;

[0242] (ii) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:57; LCDR2 comprising or consisting of SEQ ID NO:56; LCDR1 comprising or consisting of SEQ ID NO:55;

[0243] (ii) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:59; LCDR2 comprising or consisting of SEQ ID NO:56; LCDR1 comprising or consisting of SEQ ID NO:58;

[0244] (iv) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:61; LCDR2 comprising or consisting of SEQ ID NO:60; LCDR1 comprising or consisting of SEQ ID NO:58; and

[0245] (v) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:62; LCDR2 comprising or consisting of SEQ ID NO:56; LCDR1 comprising or consisting of SEQ ID NO:58.

[0246] The IL-4Rα / IL-5 bispecific antibody of the present invention may comprise an antigen binding region that binds to IL-5, wherein the antigen binding region comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH and VL domains comprise a CDR sequence selected from the group consisting of:

[0247] (i) HCDR3 comprising or consisting of SEQ ID NO:72; HCDR2 comprising or consisting of SEQ ID NO:71; HCDR1 comprising or consisting of SEQ ID NO:70; LCDR3 comprising or consisting of SEQ ID NO:74; LCDR2 comprising or consisting of SEQ ID NO:38; LCDR1 comprising or consisting of SEQ ID NO:73; and

[0248] (ii) HCDR3 comprising or consisting of SEQ ID NO:72; HCDR2 comprising or consisting of SEQ ID NO:71; HCDR1 comprising or consisting of SEQ ID NO:70; LCDR3 comprising or consisting of SEQ ID NO:75; LCDR2 comprising or consisting of SEQ ID NO:38; LCDR1 comprising or consisting of SEQ ID NO:73.

[0249] The antigen binding region that binds to human IL-5 may comprise a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from the group consisting of:

[0250] (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence that is at least 70% identical thereto;

[0251] (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that is at least 70% identical thereto;

[0252] (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence that is at least 70% identical thereto;

[0253] (iv) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that is at least 70% identical thereto;

[0254] (v) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence that is at least 70% identical thereto; and

[0255] (vi) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence at least 70% identical thereto.

[0256] The antigen binding region that binds to IL-5 may comprise a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from the group consisting of:

[0257] (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence that is at least 70% identical thereto;

[0258] (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence that is at least 70% identical thereto; and

[0259] (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence that is at least 70% identical thereto.

[0260] In each case, the domain / chain defined by the % sequence identity to a specified domain / chain amino acid sequence may retain CDR sequences identical to those present in a specified VH / VL domain or heavy / light chain amino acid sequence, while exhibiting amino acid sequence variation in the framework or other regions outside of the CDR regions.

[0261] 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 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,

[0262] wherein the first VH-VL domain pairing comprises a CDR sequence selected from the group consisting of:

[0263] (i) HCDR3 comprising or consisting of SEQ ID NO: 3; HCDR2 comprising or consisting of SEQ ID NO: 2; HCDR1 comprising or consisting of SEQ ID NO: 1; LCDR3 comprising or consisting of SEQ ID NO: 12; LCDR2 comprising or consisting of SEQ ID NO: 11; LCDR1 comprising or consisting of SEQ ID NO: 10;

[0264] (ii) HCDR3 comprising or consisting of SEQ ID NO:6; HCDR2 comprising or consisting of SEQ ID NO:5; HCDR1 comprising or consisting of SEQ ID NO:4; LCDR3 comprising or consisting of SEQ ID NO:15; LCDR2 comprising or consisting of SEQ ID NO:14; LCDR1 comprising or consisting of SEQ ID NO:13; and

[0265] (iii) a HCDR3 comprising or consisting of SEQ ID NO: 9; a HCDR2 comprising or consisting of SEQ ID NO: 8; a HCDR1 comprising or consisting of SEQ ID NO: 7; a LCDR3 comprising or consisting of SEQ ID NO: 18; a LCDR2 comprising or consisting of SEQ ID NO: 17; a LCDR1 comprising or consisting of SEQ ID NO: 16; and

[0266] wherein the second VH-VL domain pairing comprises a CDR sequence selected from the group consisting of:

[0267] (i) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:54; LCDR2 comprising or consisting of SEQ ID NO:53; LCDR1 comprising or consisting of SEQ ID NO:52;

[0268] (ii) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:57; LCDR2 comprising or consisting of SEQ ID NO:56; LCDR1 comprising or consisting of SEQ ID NO:55;

[0269] (ii) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:59; LCDR2 comprising or consisting of SEQ ID NO:56; LCDR1 comprising or consisting of SEQ ID NO:58;

[0270] (iv) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:61; LCDR2 comprising or consisting of SEQ ID NO:60; LCDR1 comprising or consisting of SEQ ID NO:58; and

[0271] (v) HCDR3 comprising or consisting of SEQ ID NO:51; HCDR2 comprising or consisting of SEQ ID NO:50; HCDR1 comprising or consisting of SEQ ID NO:49; LCDR3 comprising or consisting of SEQ ID NO:62; LCDR2 comprising or consisting of SEQ ID NO:56; LCDR1 comprising or consisting of SEQ ID NO:58.

[0272] 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 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,

[0273] wherein the first VH-VL domain pairing is selected from:

[0274] (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that is at least 70% identical thereto;

[0275] (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that is at least 70% identical thereto; and

[0276] (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that is at least 70% identical thereto,

[0277] and wherein the second VH-VL domain pairing is selected from:

[0278] (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence that is at least 70% identical thereto;

[0279] (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that is at least 70% identical thereto;

[0280] (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence that is at least 70% identical thereto;

[0281] (iv) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that is at least 70% identical thereto;

[0282] (v) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence that is at least 70% identical thereto; and

[0283] (vi) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence at least 70% identical thereto.

[0284] In preferred embodiments, 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 germlined variants of the VH and / or VL domains from which the CDR sequences are derived.

[0285] The bispecific antibodies of the present invention may also comprise one or more constant domains having a completely or substantially human amino acid sequence. For example, the bispecific antibodies of the present invention may comprise an Fc domain derived from a human IgG antibody, such as human IgG1, IgG2, IgG3 or IgG4.

[0286] As described elsewhere herein, the bispecific antibodies of the invention having an Fc domain can be modified within the Fc region to increase binding affinity to the neonatal receptor FcRn. For example, one or more amino acid residues within the Fc region can be substituted with a different amino acid residue to increase binding to FcRn. Preferred amino acid substitutions within the Fc region are described elsewhere herein and are equally applicable to the bispecific antibodies of the invention.

[0287] As described elsewhere herein, the bispecific antibodies of the present invention can be modified to have pH-dependent antigen binding activity. In particular, the bispecific antibodies can be modified so that the IL-4Rα and / or IL-5 binding activity at acidic pH values ​​is lower than the binding activity at neutral pH values. The bispecific antibodies can be modified by replacing 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 of the antigen binding regions of the antibody molecule. As described herein, histidine substitution and / or insertion is preferably introduced at one or more sites within the CDR of the heavy chain variable domain and / or the light chain variable domain.

[0288] E. Treatment methods

[0289] The combination therapy and bispecific antibodies as described herein are used in a method of treating a chronic airway disease in a human subject.

[0290] Therefore, the present invention provides a combination comprising (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, which is used to treat chronic airway diseases in human subjects. 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 to IL-5, which is used to treat chronic airway diseases in human subjects. The present invention provides an antagonist of IL-5:IL-5R IL-5, which is used to treat chronic airway diseases in human subjects, wherein the antagonist is administered in combination with an antagonist of IL-4:IL-4R and / or an antagonist of IL-13:IL-13R. The present invention provides an antagonist of IL-4:IL-4R and / or an antagonist of IL-13:IL-13R, which is used to treat chronic airway diseases in human subjects, wherein the antagonist is administered in combination with an antagonist of IL-5:IL-5R. In a preferred embodiment, the present invention provides an antagonist of IL-5 for use in treating a chronic airway disease in a human subject, wherein the antagonist is administered in combination with an antagonist of IL-4Rα. In other preferred embodiments, the present invention provides an antagonist of IL-4Rα for use in treating a chronic airway disease in a human subject, wherein the antagonist is administered in combination with an antagonist of IL-5. In other preferred embodiments, the antagonist used in the method is an antibody molecule.

[0291] In yet another aspect, the present invention provides a method for 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 present invention or a bispecific antibody according to the second aspect of the present invention. All embodiments described above with respect to the combination of the first aspect of the present invention or the bispecific antibody of the second aspect of the present invention are also applicable to the methods described herein.

[0292] In certain embodiments, the methods described herein are used to treat chronic airway diseases. As described elsewhere herein, chronic airway diseases encompass 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, Chag-Strauss syndrome, allergic bronchopulmonary aspergillosis (ABPA), high eosinophilic syndrome; bullous pemphigoid and cystic fibrosis.

[0293] In certain embodiments, the methods described herein are used to treat chronic airway diseases characterized by increased mucus production. An example of a disease characterized by increased mucus production is cystic fibrosis. In other embodiments, the methods described herein are used to treat chronic airway diseases characterized by bronchial hyperresponsiveness.

[0294] In preferred embodiments, the methods described herein are used to treat 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, oligogranulocytic or non-inflammatory asthma, early-onset asthma, late-onset asthma, high type II asthma, low type II asthma, and type I / Th17 asthma.

[0295] In a preferred embodiment, the methods described herein are used to treat atopic or allergic asthma. Atopic or allergic asthma is a clinically defined form of asthma as follows: a disease consistent with allergic sensitization defined by the presence of serum IgE antibodies and / or a positive skin prick test for (lipo) proteins of common inhaled or ingested allergens, such as house dust mites (HDM), animal dander, fungal spores, plant or tree pollen or peanuts (Lambrecht and Hammad (2015) supra). The vast majority of early-onset asthma cases are allergic or atopic types. Thus, the methods of the present invention can also be used to treat early-onset asthma.

[0296] In other preferred embodiments, the methods described herein are used to treat high type II (or type II Hi) asthma. High type II asthma is characterized by the presence of type 2 cytokine molecular markers. Compared with low type II asthma patients, subjects with high type II asthma generally express higher levels of IL-13 and IL-5, have a larger number of eosinophils and mast cells, and show more atopy and SBM (subepithelial basement membrane) thickening (see Wenzel (2012) supra and Gauthier et al. (2015) supra). The combinations and bispecific antibodies of the present invention target multiple type 2 cytokine signaling pathways. Therefore, the combinations and bispecific antibodies described herein can be particularly effective in treating human subjects with high type II asthma.

[0297] In certain embodiments, the methods described herein are used to treat severe asthma or severe refractory asthma.

[0298] Almost all forms of asthma are associated with pathological 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. Therefore, in certain embodiments, the methods described herein are used to treat asthma by reducing goblet cell metaplasia. In such embodiments, goblet cell metaplasia is reduced compared to the level of goblet cell metaplasia before treatment.

[0299] Patients with chronic airway disease, including most asthma patients, also suffer from bronchial hyperresponsiveness (BHR). BHR is defined as increased sensitivity to a wide variety of airway narrowing stimuli. Most patients with asthma and chronic obstructive pulmonary disease (COPD) exhibit this increased sensitivity. In certain embodiments, the methods described herein are used to treat chronic airway disease by reducing bronchial hyperresponsiveness. In preferred embodiments, the methods described herein are used to treat asthma by reducing bronchial hyperresponsiveness. In such embodiments, bronchial hyperresponsiveness is reduced relative to the level of bronchial hyperresponsiveness before treatment. Bronchial hyperresponsiveness can be assessed with a bronchial provocation test. This most commonly uses products such as methacholine or histamine. These chemicals also trigger bronchospasm in normal individuals, but people with bronchial hyperresponsiveness have a lower critical value.

[0300] Patients with chronic airway diseases, including most asthmatics, also exhibit excessive mucus production. In certain embodiments, the methods described herein are used to treat chronic airway diseases by reducing mucus production. In preferred embodiments, the methods described herein are used to treat asthma by reducing mucus production. In such embodiments, mucus production is reduced relative to the level of mucus production before treatment. Mucus production can be assessed by measuring the expression and / or activity of mucin, such as the expression and / or activity of muc5AC. In certain embodiments, the methods described herein are used to treat chronic airway diseases by reducing the expression and / or activity of mucin relative to expression and / or activity before treatment. In certain embodiments, the methods described herein are used to treat asthma by reducing the expression and / or activity of mucin relative to expression and / or activity before treatment.

[0301] Chronic airway diseases are also characterized by poor lung function. Forced expiratory volume (FEV) is a measure of lung function and can be assessed using a spirometer. Forced expiratory volume (FEV) measures how much air a person can exhale during forced breathing. Forced vital capacity (FVC) is the total amount of air exhaled during the FEV test. The FEV1 / FVC ratio, also known as the Tiffeneau-Pinelli index, is a calculated ratio representing the proportion of the lung capacity that a person can exhale in the first second of forced exhalation to the entire lung capacity. The normal value of the FEV1 / FVC ratio is about 70-80%. In certain embodiments, the methods described herein are used to treat chronic airway diseases by improving lung function. In preferred embodiments, the methods described herein are used to treat asthma by improving lung function. In such embodiments, lung function is improved relative to lung function before treatment. In certain embodiments, the methods described herein are used to treat chronic airway diseases by increasing the FEV1 / FVC ratio relative to the FEV1 / FVC ratio before treatment. In certain embodiments, the methods described herein are used to treat asthma by increasing the FEV1 / FVC ratio relative to the pre-treatment FEV1 / FVC ratio.

[0302] Methods described herein can also be used to treat coexisting diseases associated with chronic airway diseases, for example, coexisting diseases associated with asthma. Severe asthma is associated with many coexisting diseases, including but not limited to: chronic sinusitis; nasal polyposis; allergic rhinitis; respiratory disability; 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). Methods described herein can be used to treat any one of the asthma coexisting diseases listed above.

[0303] The methods described herein may include administering other therapeutic agents. In certain embodiments, the methods described herein include administering one or more additional therapeutic agents to treat chronic airway disease. The combination or bispecific antibody according to the invention may be administered independently, simultaneously, sequentially or in parallel with the additional therapeutic agent.

[0304] Patients or subjects treated according to the methods described herein may have received treatment, such as corticosteroid treatment. Patients or subjects treated according to the methods described herein may be classified as 'corticosteroid responsive' or 'corticosteroid non-responsive'. Patients or subjects may exhibit one or more symptoms associated with chronic airway disease. In certain embodiments, the patient or subject may be an individual who is under medical care and / or actively seeks medical care for the treatment of chronic airway disease.

[0305] In certain embodiments, the patient or subject treated according to the methods described herein may be a patient suffering from a disease characterized by hypereosinophilia. In patients with high eosinophil levels, certain existing agents targeting IL-4Rα, such as dupilumab, cannot be specified because the patient's circulating eosinophil levels will be at unacceptably high levels. Without wishing to be bound by theory, it is believed that combined treatment with an IL-4Rα antagonist (e.g., IL-4Rα antibody) and an IL-5 antagonist (e.g., IL-5 antibody) will be suitable for patients with high eosinophil levels because IL-5 promotes eosinophil proliferation. Therefore, the combined inhibition of IL-5 together with IL-4Rα can prevent or reduce the increase in eosinophil levels seen using IL-4Rα antibody therapy.

[0306] Incorporation by reference

[0307] In the foregoing description and throughout the following examples, various publications are cited, each of which is incorporated herein by reference in its entirety.

[0308] Example

[0309] The invention will be further understood with reference to the following non-limiting examples.

[0310] Example 1: Generation of monoclonal antibodies neutralizing IL-4Rα and IL-5

[0311] A. Llama immunization and library construction:

[0312] Two llamas raised outdoors in accordance with the French Animal Welfare Law were immunized intramuscularly (R&D Systems) with recombinant mouse IL-4Rα-Fc in one shoulder and recombinant mouse IL-5 in the other shoulder, and weekly booster immunizations continued for six weeks. Briefly, the two llamas received 100 μg IL-4Rα-Fc and 50 μg IL-5 buffered in phosphate buffered saline (PBS) and mixed with incomplete Freund's Adjuvant (Incomplete Freund's Adjuvant) (Sigma-Aldrich) for the first two weeks, and 50 μg IL-4Rα-Fc and 25 μg IL-5 for the remaining four weeks. The Fab library was generated using the proprietary SIMPLE antibody platform as previously described (see WO2010 / 001251, the contents of which are incorporated herein in their entirety). Five days after the last immunization, 400 mL of blood containing peripheral blood lymphocytes was collected from the llamas, purified by centrifugation over a Ficoll-Paque gradient, and used to extract total RNA. Total RNA was then converted into random primed cDNA using reverse transcriptase, and the gene sequences encoding the VH-CH1 region and VL-CL domains (κ and λ) of llama IgG1 were isolated and subcloned into the phagemid vector pCB3. The pCB3 vector allows the expression of recombinant antibodies as Fab fragments fused to the phage pilli envelope protein.

[0313] Parallel studies were performed in order to generate recombinant antibodies binding to human IL-4Rα and human IL-5.Llamas were immunized intramuscularly with recombinant human IL-4Rα-Fc and recombinant human IL-5 following the same protocol as described above.

[0314] B. Selection of Fabs Binding to IL-4Rα and IL-5:

[0315] The recombinant phagemids were used to transform E. coli strain TG1 (Netherlands Culture Collection of Bacteria) to generate phage libraries expressing Fab (one λ and one κ library per immunized llama). Because the llamas were immunized with IL-4Rα coupled to the fragment crystallizable (Fc) portion, phage expressing Fc-binding antigen-binding fragments (Fab) were first counter-selected with an irrelevant human Ab. The diversity was then increased to 10 8 -10 9 The resulting Fab-expressing phages within the range were adsorbed to 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 phage expressing IL-4Rα or IL-5 specific Fab. Finally, TG1 E. coli were infected with the selected phages, and individual colonies were isolated. Fab secretion into the periplasm of E. coli strain TG1 was induced using isopropyl β-D-1-thiogalactoside (Sigma-Aldrich) at low glucose concentration (0.1% w / v), and the bacterial periplasmic fraction containing Fab was collected.

[0316] C.Fab screening, characterization and production:

[0317] The binding of Fab to its corresponding mouse or human target and the ability of Fab to neutralize the target were determined by surface plasmon resonance (SPR) using a Biacore 3000 device (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). Periplasmic extracts containing Fab were loaded at a flow rate of 30 μL / min. The Fab dissociation rate was measured over a 90 second period.

[0318] D. Monospecific Ab production, purification and characterization:

[0319] The cDNAs encoding the VH and VL (λ or κ) domains of the selected potent neutralizing IL-4Rα and IL-5 specific Fab fragments were engineered into two independent pUPE mammalian expression vectors containing cDNAs encoding the CH1, CH2 and CH3 domains of mouse IgG2a (or human IgG1) and containing mutations that eliminate Ab effector functions mediated by Fc receptors or CL (λ or κ). The production (by transient transfection of mammalian cells) and purification (by protein A affinity chromatography) of the resulting optimal potent neutralizing anti-IL-4Rα and anti-IL-5 IgG2a (or IgG1) molecules were then performed as previously described (Basilico et al. (2014) Journal of Clinical Investigation 124: 3172).

[0320] The CDR, VH and VL sequences of selected antibodies are shown in Tables 3-14 below.

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330] Example 2: In vitro characterization of IL-4Rα and IL-5 mAbs

[0331] Mouse IL-4Rα and IL-5 mAbs (m4RMP36B7 and m5MP95G7) were tested for their ability to bind to their respective targets in vitro and inhibit cellular effects mediated by IL-4Rα and IL-5 signaling.

[0332] A. Inhibition of IL4- and IL5-induced proliferation of HT-2 and TF-1 cells:

[0333] The neutralizing activity of both IL-4Rα and IL-5 monospecific Abs was evaluated in an in vitro cellular assay, in which mouse IL-4 and mouse IL-5 induced the proliferation of HT-2 and TF-1 cells, respectively.

[0334] Human TF-1 cells (erythroblasts, erythroblasts) were cultured at 37°C with 5% (v / v) CO2 in growth medium containing RPMI 1640 (Sigma), 10% (v / v) heat-inactivated fetal bovine serum (Sigma), 1X gentamicin (Sigma), and 2 ng / mL human granulocyte-macrophage colony-stimulating factor (R&D Systems) or human IL-2 (R&D Systems). CRL-2003 TM ) and mouse HT-2 clone A5E cells (IL-2-dependent T lymphocytes, CRL-1841 TM ). The assay medium was growth medium without human GM-CSF for TF-1 cells or human IL-2 for HT-2 cells. 0.5 ng / mL of 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. Abs were serially diluted 10-fold for TF-1 cells or 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, then washed and incubated at 1.1×10 6 cells / mL or 0.2 × 10 for HT-2 cells 6 Cells were then added to each well, followed by CellTiter Aqueous One Solution Reagent (Promega) After 3 hours of incubation, the absorbance was measured.

[0335] like Figure 1 As shown in, IL-4Rα (squares) and IL-5 (triangles) monospecific Abs potently inhibited mouse IL-4 and IL-5-induced proliferation of HT-2 and TF-1 cells, respectively. Specifically, IL-4Rα and IL-5Abs can block cell proliferation induced by mouse IL-4 and IL-5 with EC50s equal to 0.2 nM and 0.6 nM, respectively.

[0336] B. Binding of IL-4RαmAb and IL-5mAb to IL-4Rα and IL-5, respectively:

[0337] Figure 2Representative surface plasmon resonance (SPR) sensorgrams of the interaction between mAbs (IL-4RαAb, IL-5Ab, or irrelevant IgG2aAb) and immobilized targets (IL-4Rα or IL-5) at varying concentrations (0-20 μg / mL) are shown. Both mAbs bind to their targets, while irrelevant IgG2a does not bind to either target. IL-4Rα and IL-5 monospecific Abs bind to their targets with affinities of 8E-11M and 2E-12M, respectively.

[0338] Additionally, the ability of monospecific Abs to compete with their targets was tested on SPR. Figure 3 Representative SPR sensorgrams of competitive interactions between a mixture consisting of mAbs (IL-4Rα, IL-5, or irrelevant IgG2a) and their targets (IL-4Rα or IL-5) and immobilized proteins (IL-4, IL-13Rα, or IL-5Rα) are shown. A mixture consisting of IL-4Rα mAb and IL-4Rα inhibits the binding of IL-4Rα to the immobilized IL-4 ligand. A mixture consisting of IL-4Rα mAb and IL-13 inhibits the binding of IL-13 to immobilized IL-13Rα. Similarly, a mixture consisting of IL-5 mAb and IL-5 inhibits the binding of IL-5 to immobilized IL-5Rα.

[0339] C. Inhibition of IL-4-induced MHC class II antigens in purified B cells by FACS analysis:

[0340] FACS analysis was used to test whether the IL-4Rα monospecific Ab could compete with mouse IL-4, which is known to induce MHC class II translocation to the surface of B cells.

[0341] For FACS analysis, B cells were collected using magnetic activated cell sorting mouse anti-CD19 microbeads (Miltenyi Biotec) following the manufacturer's protocol. Purified B cells (5 × 10 ) were plated in 24-well plates (Costar) in the presence of 500 ng / mL of anti-IL-4Rα or irrelevant IgG2a Ab or medium controls with or without inhibitors, with or without IL-4 (0.1 ng / mL). 5Cells were cultured for 16 hours with 4% paraformaldehyde (400 μg / ml) of 1% paraformaldehyde (400 μg / ml). The cells were washed and pre-incubated on ice with rat IgG2b anti-mouse FcγR monospecific Ab 2.4G2 (Bioceros) for 20 minutes to block IgG Fc receptors. The cells were then stained with Abs for MHC class II (M5 / 114.15.2, eBioscience) and CD19 (1D3, eBioscience), then incubated at 4°C for 30 minutes, and then washed. Dead cells were excluded from the analysis by using a fixable viability dye (eFluor506, eBioscience). The stained cells were analyzed on an LSR Fortessa flow cytometer (BD Biosciences). Final analysis and graphical output were performed using FlowJo 10.0.7 software (Tree Star).

[0342] like Figure 4 As shown in , MHC class II antigen expression on B cells was significantly higher in the presence of IL-4 than in the absence of IL-4. IL-4Rα mAb potently inhibited IL-4-induced MHC class II antigen expression on purified B cells.

[0343] Example 3: In vivo characterization of anti-mouse IL-4Rα and IL-5 mAbs in the murine house dust mite (HDM) model

[0344] House dust mites (HDM - Dermatophagoides sp.) are one of the most common allergens worldwide. 50-85% of asthma patients are allergic to HDM. Chronic exposure to HDM leads to airway remodeling with increased mucus cell density and airway hyperresponsiveness that remains elevated after cessation of HDM exposure. The murine HDM model is an effective in vivo model for studying asthma.

[0345] Experimental configuration 1:

[0346] Female C57Bl / 6J wild-type mice were obtained from The Jackson Laboratory. The experiments were approved by the ethics committee of the VIB-UGent Center for Inflammation Research. All mice used were between 6 and 8 weeks of age. Age-matched groups were used for the experiments. Figure 5As shown in A, the experimental design involves Ab treatments administered throughout the sensitization and challenge period. On day 0, mice were lightly anesthetized with isoflurane (2.5% in air) and received 1 μg HDM (Greer Laboratories) intratracheally. On days 6-10, mice were lightly anesthetized with isoflurane (2.5% in air) and challenged intranasally with 10 μg HDM daily. On days -1, 1, 6, 8, and 10, mice received either of the following treatments: (i) IL-4Rα monospecific Ab (m4RMP36B7); (ii) IL-5 monospecific Ab (m5MP95G7); (iii) a combination of IL-4Rα and IL-5 monospecific Abs (m4RMP36B7 and m5MP95G7); or (iv) an unrelated IgG2a Ab. Days -1 and 1 represent the sensitization period and days 6, 8, and 10 represent the challenge period. At least n=6 mice were treated in each group.

[0347] A. Potent neutralizing IL-4Rα mAb and IL-5 mAb injected in combination reduce eosinophilia in the murine HDM asthma model:

[0348] Collection and analysis of BAL fluid. On day 14, mice were euthanized. Bronchoalveolar lavage (BAL) was performed into the cannulated trachea using 3×1 mL of PBS containing EDTA, and the cell 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).

[0349] Figure 5 B shows differential cell counts of bronchoalveolar lavage fluid from mice receiving IL-4Rα mAb, IL-5 mAb, a combination of IL-4Ra mAb and IL-5 mAb or an unrelated IgG2a Ab. Cells were counted by FACS analysis. Eosinophil counts were increased by allergen challenge in HDM sensitized mice treated with IgG2a. A significant reduction in eosinophil counts was observed after treatment with a combination of IL-4α / IL-5 monospecific Abs compared to treatment with a control IgG2a Ab.

[0350] Experimental configuration 2:

[0351] A second experimental protocol was designed to further test the effects of IL-4Rα and IL-5 antibodies in a murine HDM model in a clinically relevant therapeutic setting. Figure 6 The experimental design shown in Figure 5 The experimental configuration in A differs in that the procedure involves Ab treatments administered only during the challenge phase and not during the sensitization phase. Thus, mice received their treatments (IL-4Rα monospecific Ab; IL-5 monospecific Ab; IL-4Rα / IL-5 monospecific Ab or irrelevant IgG2a Ab) only 4 hours before challenge on days 6, 8, and 10. Mice received 150 μg of each monospecific Ab, 150 μg of each monospecific antibody injected in combination, or 150 μg of irrelevant mouse IgG2a mAb. At least n=6 mice were treated per group.

[0352] Mice treated according to this protocol were subsequently evaluated for the following: total inflammatory cell counts in BAL fluid ( Figure 7 ); Cytokine production in mediastinal lymph nodes ( Figure 8 ); serum immunoglobulin production ( Fig. 9 ); Indications of goblet cell metaplasia ( Fig.10 ); and bronchial hyperresponsiveness ( Fig.11 ).

[0353] A. Potent neutralizing IL-4Rα mAb and IL-5 mAb injected in combination reduce eosinophilia in the murine HDM asthma model:

[0354] Collection and analysis of BAL fluid. On day 14, mice were euthanized. Bronchoalveolar lavage (BAL) was performed into the cannulated trachea using 3×1 mL of PBS containing EDTA, and the cell 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).

[0355] Figure 7Shown is BAL differential cell counts of HDM treated mice receiving IL-4RαmAb, IL-5mAb, IL-4Rα / IL-5 combination mAb or irrelevant IgG2a Ab as analyzed by FACS. In mice treated with irrelevant IgG2a Ab, airway HDM exposure increased the total count of inflammatory cells in BAL fluid compared with mice stimulated by PBS. More specifically, this increased the number of eosinophils, lymphocytes and macrophages. Interestingly, compared with treatment with control IgG2a Ab, the number of eosinophils was significantly reduced after treatment with a single therapy. The minimum number of eosinophils can be seen in mice treated with combination therapy.

[0356] B. IL-4Rα mAb and IL-4Rα / IL-5 mAb combination reduce cytokine production:

[0357] The effects of IL-4Rα and IL-5 mAbs on cytokine production by mesenteric lymph node (MLN) cells restimulated with allergens were tested in vitro. Single cell suspensions (2 × 10 6 cells / mL). Cells were restimulated ex vivo with 15 μg / mL HDM for 3 days in 96-well round-bottom plates and supernatants were collected for analysis by using Cytokine production was determined by ELISA kit (eBioscience).

[0358] like Figure 8 As shown in, allergen challenge in HDM-sensitized mice treated with IgG2a increased the in vitro production of effector cytokines IL-5 and IL-13 in allergen-restimulated MLN cell cultures. However, this response was significantly reduced after treatment with IL-4Rα monospecific Ab and the combination of IL-4Rα mAb and IL-5 mAb, while IL-5 monospecific Ab had no significant effect.

[0359] C. IL-4Rα mAb and IL-4Rα / IL-5 mAb combination reduce HDM-specific IgE and IgG1 production:

[0360] Blood was obtained from the iliac vein, and serum was prepared and the amount of HDM-specific IgG1 and IgE was determined as previously described (Schuijs et al. (2015) Science, 349: 1106-10).

[0361] like Fig. 9As shown in , serum concentrations of HDM-specific IgG1 and IgE were increased by allergen challenge in IgG2a Ab-treated mice. IL-4RαmAb and IL-4Rα / IL-5mAb combination were able to significantly reduce this increase in allergen-induced IgG. IL-4RαmAb, IL-5mAb, and IL-4Rα / IL-5mAb combination were able to significantly reduce the increase in allergen-induced IgE.

[0362] D. IL-4Rα and IL-5 mAb combination reduces the expression of Muc5AC and Agr2:

[0363] Mucin expression in the lungs was assessed by immunostaining. PBS / OCT (1:1) solution was injected into the lungs, snap frozen in liquid nitrogen and kept at -80°C until further processed for Muc5AC immunofluorescence staining as previously described (Deckers et al. (2017) Journal of Allergy and Clinical Immunology 140(5), 1364-1377).

[0364] Goblet cell metaplasia (GCM) is driven 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 Ab-treated and HDM-sensitized mice, HDM challenge upregulated Muc5AC expression in the airway epithelium compared with PBS challenge ( Fig.10 A). Increased Muc5AC staining was not affected by IL-4RαAb or IL-5Ab monotherapy. However, interestingly, lungs from mice treated with the combination of IL-4RαAb / IL-5Ab had greatly reduced Muc5AC staining intensity compared to the other HDM sensitized and challenged groups.

[0365] To confirm and quantify the effects of IL-4Rα and IL-5 mAbs on GCM, the mRNA expression levels of Muc5ac and Agr2, another IL-13 / STAT6 downstream target gene involved in GCM, were then measured in lung tissues by qRT-PCR.

[0366] The 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 using TriPure separation reagent (Roche, Mannheim, Germany) and isolated according to the manufacturer's instructions. RNA was reverse transcribed using a transcription factor high-fidelity cDNA synthesis kit (Roche), and samples were analyzed for reference genes (Rpl13a, Hprt, and Sdha) using SYBR green-based qRT-PCR with a LightCycler 480 system (Roche).

[0367] like Fig.10 As shown in B, the mRNA expression levels of these two genes were induced by HDM challenge in mice. IL-4Rα antibody and IL-5 antibody alone did not significantly reverse this increase in Muc5ac or Agr2 mRNA levels. However, the combination of IL-4Rα mAb and IL-5 mAb significantly reduced the HDM-mediated increase in Muc5ac or Agr2 mRNA levels.

[0368] E. IL-4RαAb and IL-5mAb combination reduces lung resistance:

[0369] Bronchial hyperresponsiveness (BHR) is the tendency of the airways to constrict in response to small amounts of bronchoconstrictors such as the muscarinic receptor agonist methacholine.Lung function analysis was performed using invasive measurement of dynamic resistance (Flexivent, Scireq).

[0370] 24 hours after the last HDM challenge, nonspecific airway responsiveness was measured by exposing the awakened mice to aerosolized PBS to set the baseline value, followed by increasing the concentration of aerosolized methacholine (0-400 μg / kg-1) using an ultrasonic nebulizer. For invasive measurement of dynamic resistance, mice were anesthetized with urethane, the trachea was cut, and an 18-G catheter was inserted, followed by mechanical ventilation with a Flexivent device (SCIREQ). The respiratory rate was set to 120 breaths / minute and the tidal volume was 0.2 mL, and a positive end-expiratory pressure of 2 mL H2O was applied. Dynamic resistance was recorded after a standardized inhalation strategy was given every 10 seconds for 2 minutes. Baseline resistance was restored before subsequent doses of methacholine were administered.

[0371] like Fig.11As shown in , in control IgG2a Ab treated mice, HDM challenged and sensitized mice, methacholine increased airway resistance compared to PBS challenged HDM sensitized mice. Treatment with IL-5 antibody and IL-4Rα antibody did not significantly reduce BHR. However, mice treated with a combination of IL-4Rα mAb and IL-5 mAb were completely protected from developing BHR. These results demonstrate the synergistic effect of the IL-4Rα / IL-5 antibody combination in the treatment of essential aspects of chronic airway disease.

[0372] Taken together, these results show that the combination of αIL-5 monospecific Ab and αIL-4Rα monospecific Ab given during challenge in HDM-treated mice has a synergistic effect on key aspects of asthma, including GCM and BHR.

[0373] Statistical analysis was performed using GraphPad Prism software version 7.01 and Genstat software version 19. For all experiments, results were expressed as mean ± standard error of mean (SEM) and one-way ANOVA was used to calculate the differences between groups. Contrast the mouse groups sensitized with HDM and stimulated and treated with control IgG2 Ab, when *P≤0.05, **P≤0.01, ***P≤0.001 and ****P≤0.0001, the differences between groups were considered significant.

[0374] Example 4: Generation and in vitro characterization of IL-4Rα / IL-5 bispecific antibodies

[0375] The sequences of 4RMP36B7 IL-4Rα antibody and 5MP95G7 IL-5 antibody were used to generate IL-4Rα / IL-5 bispecific antibodies. The Fc portion of the antibody was modified to include "knob-in-hole" mutations, which promote correct chain pairing in the case of bispecific antibodies (Ridgway et al. (1996) Protein Engineering, Design and Selection, 9: 617-21). Specifically, the IL-4Rα antibody 4RMP36B7 was engineered in the CH3 domain of the murine Fc region to introduce substitutions T366S, L368A and Y407V. The IL-5 antibody 5MP95G7 was engineered in the CH3 domain of the murine Fc region to introduce substitutions T366W.

[0376] As described by Godar et al. (2016, Scientific Reports, 6:31621), two mutant heavy chains and two light chains of IL-4Rα and IL-5 antibodies were co-expressed and bispecific antibodies containing the correct heavy chain-light chain pairing were purified using anti-idiotypic VHH antibodies. This purification process was Fig.12 The diagram is shown in FIG.

[0377] The purity of the IL-4Rα / IL-5 bispecific antibody was confirmed using high-resolution spectroscopy and the dual targeting properties of this antibody were confirmed by BIAcore.

[0378] A. Binding of IL-4Rα / IL-5 bispecific Ab to IL-4Rα and IL-5:

[0379] like Fig.13 As shown in , the bispecific properties of IL-4Rα / IL-5Ab were confirmed using SPR. The bispecific Ab specifically bound to the chip-immobilized IL-4Rα-Fc, and an increased signal was observed when IL-5 was then added, indicating that the bispecific Ab was able to simultaneously bind to the coated IL-4Rα and IL-5 in solution ( Fig.13 A). The experiment was also performed in the reverse order and confirmed that Fig.13 Bispecific nature of the molecule depicted in B.

[0380] In conclusion, these results show that the dual anti-idiotypic approach allows the isolation of pure and functional IL-4Rα / IL-5 bispecific Abs.

[0381] Example 5: In vivo characterization of anti-mouse IL-4Rα / IL-5 bispecific antibody in a murine HDM model

[0382] The IL-4Rα / IL-5 bispecific antibody generated in Example 4 was tested in the murine HDM model described in Example 3 above. Fig.14 Mice were subjected to a HDM sensitization and challenge protocol and were treated with Ab only during the challenge period.

[0383] The in vivo activity of the IL-4Rα / IL-5 bispecific Ab was compared with monotherapy (IL-4Rα monospecific Ab or IL-5 monospecific Ab) and combination (αIL-4Rα / αIL-5 monospecific Ab). To compare equimolar inhibition of the target and differences in the total amount of Ab eliminated, the antibodies were dosed as follows: 75 μg of each monospecific Ab combined with 75 μg of an irrelevant IgG2a Ab for testing individual Abs; 75 μg of each monospecific Ab injected in combination; and 150 μg of the bispecific Ab.

[0384] Mice treated according to this protocol were subsequently evaluated for total inflammatory cell counts in BAL fluid ( Fig.15 ); Cytokine production in mediastinal lymph nodes ( Fig.16 ); serum immunoglobulin production ( Fig.17 ); Indications of goblet cell metaplasia ( Fig.18 ); and bronchial hyperresponsiveness ( Fig.19 ). The experimental protocol was performed as described in Example 3 above.

[0385] A. Injected IL-4Rα / IL-5 bispecific Ab reduces eosinophilia in the murine HDM asthma model:

[0386] like Fig.15 As shown in , HDM challenge in sensitized mice increased the number of eosinophils in BAL fluid. This increase in eosinophil numbers was significantly reduced in mice receiving a combination of IL-4RαAb and IL-5Ab (75μg+75μg). Notably, the bispecific IL-4Rα / IL-5Ab (150μg) was as effective as the combination of both IL-4Rα and IL-5mAb in alleviating airway eosinophilia and lymphocytosis induced by allergen challenge.

[0387] B. IL-4Rα / IL-5 bispecific Ab reduces cytokine production:

[0388] like Fig.16 As shown in , HDM-induced MLN type 2 cytokine (IL-5 and IL-13) levels were significantly reduced after treatment with the combination of IL-4RαAb and IL-5Ab as well as the bispecific Ab.

[0389] C. IL-4Rα / IL-5 bispecific Ab reduces the production of HDM-specific IgE and IgG1:

[0390] like Fig.17 As shown in , HDM-specific IgE and IgG1 were also significantly reduced after treatment with the combination of IL-4Rα Ab and IL-5 Ab as well as the bispecific Ab.

[0391] D. IL-4Rα / IL-5 bispecific Ab reduces the expression of Muc5AC and Agr2:

[0392] To test and compare the effects of bispecific Abs on goblet cell metaplasia (GCM), the mRNA expression levels of Muc5ac and Agr2 were measured in lung tissue. The increased Muc5ac and Agr2 expression observed in HDM-sensitized and challenged mice was significantly reduced by the combination of IL-4RαAb and IL-5Ab as well as the bispecific Abs ( Fig.18 ), while IL-4Rα and IL-5Ab treatment alone had no significant effect.

[0393] E. IL-4Rα / IL-5 bispecific Ab reduces lung resistance:

[0394] Treatment with a combination of IL-4RαAb and IL-5Ab as well as treatment with the bispecific Ab protected the lungs equally well from HDM-induced BHR as assessed by methacholine-induced bronchoconstriction. Fig.19 As shown in, mice treated with a combination of IL-4Rα mAb and IL-5 mAb as well as bispecific Abs were completely protected from developing BHR. In contrast, there was no significant reduction in resistance observed in HDM-sensitized and challenged mice treated with individual IL-4Rα and IL-5 antibodies. This demonstrates the synergistic effect of combination therapy (in the form of co-administration of individual IL-4Rα and IL-5 antibodies or as bispecific antibodies) in the treatment of chronic airway diseases, particularly asthma.

[0395] Taken together, these results show that bispecific Abs simultaneously targeting IL-4Rα and IL-5 are effective in reducing all prominent asthma features when administered during the challenge phase in a HDM-driven asthma model.

[0396] Example 6: In vitro characterization of IL-4Rα mAb and IL-5 mAb

[0397] As described in Example 2, human IL-4Rα mAbs and IL-5 mAbs (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 cellular effects mediated by IL-4Rα and IL-5 signaling. The proliferation assay was modified to use hIL-4 and hIL-5 at concentrations that provided suboptimal cell proliferation. The results are shown in the table below.

[0398] Table 15

[0399]

[0400] Table 16

[0401]

[0402] Example 7: Generation and in vitro characterization of a second IL-4Rα / IL-5 bispecific antibody

[0403] A second IL-4Rα / IL-5 bispecific antibody was generated using the sequences of the 4RMP36B7 IL-4Rα antibody and the 5MP95G7 IL-5 antibody. The VH-VL domains of the 5MP95G7 antibody were generated as single-chain Fv (scFv) fragments using a 15 or 20 amino acid linker (15GS = (GGGS) 3 or 20GS = (GGGS) 4) and two of these scFv fragments were attached to the C-terminus of the Fc domain of the 4RMP36B7 IgG antibody via a (GGGS) 3 linker (15GS). In addition, in order to stabilize the scFv, two mutations were introduced, one in VH95G7 (G44C) and one in VL97G7 (G100C). A schematic diagram of this second IL-4Rα / IL-5 bispecific antibody is shown in Fig. 20 Shown in.

[0404] 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.

[0405] The assay was performed generally as described above in Example 2. Briefly, a fixed concentration of mIL-5 (R&D Systems) was pre-incubated with a 5-fold serial dilution of the antibody (starting at 100 nM) and added to 500.000 human TF-1 cells (erythroblasts, CRL-2003 TM ; mouse IL-5 (R&D Systems) at a final concentration of 0.25 ng / mL) and incubated the cells at 37°C in 5% CO2 for 48 hours. Add 20 μl CellTiter Aqueous One Solution Reagent (Promega) and incubate cells for 3 hours at 37° C. Absorbance was measured at A490 vs. A655.

[0406] The results are Fig.21 and are shown in Table 17 below.

[0407] Table 17

[0408]

[0409] As expected, the IL-4Ra antibody had no effect on TF-1 cell proliferation, while the 95A7 antibody constructed as either a human IgG1 or a murine 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 domains of the 95A7 antibody constructed as a scFv fragment at the C-terminus of the Fc region of the IL-4Rα IgG antibody were much more potent than when constructed as Fab arms in the native IgG structure.

[0410] Example 8: In vivo expression of anti-mouse IL-4Rα / IL-5 IgG-scFv bispecific antibody in a murine HDM model Levy

[0411] 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. Fig. 22 Mice were subjected to a HDM sensitization and challenge protocol and were treated with Ab only during the challenge period.

[0412] The in vivo activity of the IL-4Rα / IL-5 bispecific Ab (4Rsc5) was compared to a combination of anti-mIL-4Rα (36B7) and anti-mIL5 (95G7). Each injection was either 150 μg or 75 μg of the bispecific antibody (4Rsc5) or the combination (in that case 75 μg of each antibody to achieve a total of 150 μg or 36.75 μg of each antibody to achieve a dose of 75 μg per injection).

[0413] Mice treated according to this protocol were subsequently evaluated for total eosinophil and lymphocyte counts in BAL fluid ( Fig.23 ) and the expression of muc5a, spdef and agr2 ( Fig.24 ). The experimental protocol was performed as described in Example 3 above.

[0414] Example 9: Engineering Human IL-4Rα Antibody

[0415] Human IL-4Rα mAb-4RMP3D6 was engineered to cross-react with macaque / rhesus IL-4Rα. Variants of 4RMP3D6 were generated using an error-prone PCR method by random mutagenesis. To this end, the GeneMorphII EZClone domain mutagenesis kit (Agilent) was used. According to the supplier's recommendation, mutations were introduced into the VH and VK of 4RMP3D6. The mutated VH (VHm*) and VK (VKm*) were further cloned into a phagemid vector (PCB13) containing CH1 and CK constant domains to generate a mutated Fab library.

[0416] Sequencing confirmed that this procedure produces approximately 3 to 5 amino acid substitutions in each V domain (VH and VK). Several libraries (VHm* / VKwt, VHwt / VKm*, and VHm* / VKm*) were made and used for several rounds of phage display selections for macaque recombinant IL-4Rα (agroBioscience catalog number ILR-C52H8). After 3 rounds of selection, clones (from rounds 2 and 3) were selected, and Fabs were produced from periplasmic extracts and tested by SPR. For SPR, Biacore3000 was used in combination with a CM5 chip coated with human IL-4Rα or macaque IL-4Rα. Binding (R0) and dissociation (kd, s) were recorded as shown in Table 18 below. -1 ).

[0417] Table 18

[0418]

[0419] The CDR, VH and VL sequences of selected antibodies are shown in Tables 19-21 below.

[0420]

[0421]

[0422] Sequence Listing <110> Argens <120> Combination antagonists targeting IL-5 / IL-5R and IL-4 / IL-4R or IL-13 / IL-13R <130> P158919WO00 <150> GB1802487.7 <151> 2018-02-15 <160> 108 <170> PatentIn Version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 1 Ser Tyr Ile Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 2 Gly Ile Ser Ser Gly Gly Gly Ile Thr Ala Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 8 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 3 Gly Leu Leu Arg Val Glu Gly Tyr 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 4 Ser Tyr Asp Met Thr 1 5 <210> 5 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 5 Ala Ile Asn Ser Gly Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 6 <211> 16 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 6 Ala Leu Arg Thr Val Val His Asp Arg Arg Leu Phe Tyr Ile Asp Ser 1 5 10 15 <210> 7 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 7 Ser Tyr Ser Leu Thr 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 8 Thr Ile Lys Ala Arg Gly Gly Thr Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Asp <210> 9 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 9 Pro Leu Tyr Asn Asn Phe Ala Gly Asp Phe Gly Ser 1 5 10 <210> 10 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 10 Gly Leu Ser Ser Gly Ser Val Thr Ser Ser Asn Tyr Pro Gly 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 11 Ala Thr Ala Ser Arg His Ser 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 12 Ala Leu His Lys Gly Thr Tyr Val 1 5 <210> 13 <211> 11 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 13 Gly Gly Asn Asn Ile Gly Ser Lys Ser Ala Gln 1 5 10 <210> 14 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 14 Ala Asp Ser Arg Arg Pro Ser 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 15 Gln Val Trp Asp Thr Ser Ala Val Val 1 5 <210> 16 <211> 11 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 16 Gln Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala 1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 17 Gly Gly Ser Arg Leu Gln Thr 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 18 Leu Gln Asp Tyr Ser Trp Pro Leu Thr 1 5 <210> 19 <211> 117 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 19 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Gly Gly Gly Ile Thr Ala Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ala Gly Leu Leu Arg Val Glu Gly Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 20 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 20 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Thr Ser Ser 20 25 30 Asn Tyr Pro Gly Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Ser 35 40 45 Leu Ile Tyr Ala Thr Ala Ser Arg His Ser Gly Val Pro Ser Arg Tyr 50 55 60 Ser Gly Ser Ile Ser Gly Asn Lys Ala Thr Leu Thr Ile Thr Gly Ala 65 70 75 80 Glu Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu His Lys Gly Thr 85 90 95 Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 21 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 21 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ala Leu Arg Thr Val Val His Asp Arg Arg Leu Phe Tyr Ile 100 105 110 Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 22 <211> 106 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 22 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asp Ser Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Val Ser Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Ala Val Val 85 90 95 Phe Gly Gly Gly Thr Arg Leu Thr Val Leu 100 105 <210> 23 <211> 121 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 23 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Lys Ala Arg Gly Gly Thr Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Leu Tyr Asn Asn Phe Ala Gly Asp Phe Gly Ser Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 24 <211> 107 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 24 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Gly Ser Arg Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Ser Phe Thr Leu Thr Ile Ser Gly Leu Glu Thr 65 70 75 80 Glu Asp Leu Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Leu Lys 100 105 <210> 25 <211> 5 <212> PRT <213> artificially <220> <223> Synthetic peptides <400> 25 Asp Tyr Ala Met Ser 1 5 <210> 26 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 26 Ile Ile Ser Trp Asn Gly Glu Ile Thr His Tyr Ala Glu Ser Met Lys 1 5 10 15 Gly <210> 27 <211> 11 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 27 Asn His Tyr Thr Leu Thr Trp Gly Tyr Asp Tyr 1 5 10 <210> 28 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 28 Ser Tyr Tyr Met Ala 1 5 <210> 29 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 29 His Ile His Ala Gly Gly Ser Leu Thr Gln Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 30 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 30 Ile Tyr Gly Asp Ala Thr Ser Tyr Asp Tyr 1 5 10 <210> 31 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 31 Ser Tyr Ala Met Asn 1 5 <210> 32 <211> 16 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 32 Ala Ile Ser Gly Gly Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 33 <211> 16 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 33 Ser Gly Phe Tyr Ser Asp Tyr Glu Arg Arg Tyr Arg Tyr Leu Glu Val 1 5 10 15 <210> 34 <211> 11 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 34 Gln Gly Gly Asn Phe Gly Arg Tyr Tyr Ala Ser 1 5 10 <210> 35 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 35 Gly Ser Ser Asn Ser Pro Ser 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 36 Gln Val Trp Asp Ser Ser Gly Tyr Lys 1 5 <210> 37 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 37 Ala Gly Thr Ser Ser Asp Ile Gly Gly Tyr Asn Tyr Val Ser 1 5 10 <210> 38 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 38 Glu Val Asn Lys Arg Ala Ser 1 5 <210> 39 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 39 Ala Ser Tyr Arg Ser Asn Asn Asn Ile Val 1 5 10 <210> 40 <211> 11 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 40 Gln Ala Ser Gln Ser Ile Arg Pro Glu Ile Ser 1 5 10 <210> 41 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 41 Gly Ala Ser Arg Leu Gln Thr 1 5 <210> 42 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 42 Leu Gln Asp Tyr Ser Trp Pro Tyr Ser 1 5 <210> 43 <211> 120 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ile Ile Ser Trp Asn Gly Glu Ile Thr His Tyr Ala Glu Ser Met 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Thr Leu Lys Ser Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn His Tyr Thr Leu Thr Trp Gly Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 44 <211> 106 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 44 Asn Phe Met Leu Thr Gln Pro Ser Ala Val Ser Val Ser Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gln Gly Gly Asn Phe Gly Arg Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Gln Val Ile Tyr 35 40 45 Gly Ser Ser Asn Ser Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asp Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Gly Tyr Lys 85 90 95 Phe Gly Gly Gly Thr Thr Leu Thr Val Leu 100 105 <210> 45 <211> 119 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile His Ala Gly Gly Ser Leu Thr Gln Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Val Arg Ile Tyr Gly Asp Ala Thr Ser Tyr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Ser Val Ser Ser 115 <210> 46 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 46 His Ser Ala Val Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Ala Gly Thr Ser Ser Asp Ile Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Glu Val Asn Lys Arg Ala Ser Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Ser Ile Ser Gly Leu 65 70 75 80 Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Tyr Arg Ser Asn 85 90 95 Asn Asn Ile Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110 <210> 47 <211> 124 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 47 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Gly Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Ser Gly Phe Tyr Ser Asp Tyr Glu Arg Arg Tyr Arg Tyr Leu Glu 100 105 110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 48 <211> 107 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Arg Pro Glu 20 25 30 Ile Ser Trp Tyr Gln Gln Lys Pro Gly Gln Thr Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Ser Phe Thr Leu Thr Ile Ser Gly Leu Glu Ala 65 70 75 80 Glu Asp Leu Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Ser Trp Pro Tyr 85 90 95 Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 49 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 49 Ser Ser Phe Met Ser 1 5 <210> 50 <211> 16 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 50 Thr Ile Asn Gly Asn Gly Gly Thr Tyr Tyr Tyr Ala Asp Ser Met Lys Gly 1 5 10 15 <210> 51 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 51 Asp Trp Ile Ser Gly Gly Tyr Tyr Tyr Phe Pro Ala Leu Gly Tyr 1 5 10 <210> 52 <211> twenty two <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 52 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val Ser Val Ala Gln Thr Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys 20 <210> 53 <211> 15 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 53 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Ala Ile Tyr 1 5 10 15 <210> 54 <211> 32 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 54 Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr 1 5 10 15 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 <210> 55 <211> twenty two <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 55 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Leu Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Lys Met Thr Cys 20 <210> 56 <211> 15 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 56 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 1 5 10 15 <210> 57 <211> 32 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 57 Gly Ile Pro Glu Arg Phe Ala Gly Ser Asn Ala Gly Asn Thr Ala Thr 1 5 10 15 Leu Thr Ile Ser Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 <210> 58 <211> twenty two <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 58 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys 20 <210> 59 <211> 32 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 59 Gly Ile His Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr 1 5 10 15 Leu Thr Ile Gly Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 <210> 60 <211> 15 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 60 Trp Tyr Gln Gln Ser Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 1 5 10 15 <210> 61 <211> 32 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 61 Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr 1 5 10 15 Leu Thr Ile Ser Gly Ala Arg Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 <210> 62 <211> 32 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 62 Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr 1 5 10 15 Leu Thr Ile Ser Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 <210> 63 <211> 122 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 63 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Ser 20 25 30 Phe Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asn Gly Asn Gly Gly Thr Tyr Tyr Ala Asp Ser Met Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Ser Thr Val Thr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Asp Trp Ile Ser Gly Gly Tyr Tyr Phe Pro Ala Leu Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 64 <211> 108 <212> PRT <213> Artificial <220> <223> synthetic peptide <400> 64 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val Ser Val Ala Gln Thr Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys Gly Gly Asp Asn Ile Gly Ser Lys Ser Val 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Ala Ile Tyr 35 40 45 Ala Asp Thr Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Thr Asn Val 85 90 95 Ala Val Phe Gly Gly Gly Thr Arg Leu Thr Ile Leu 100 105 <210> 65 <211> 108 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 65 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Leu Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Lys Met Thr Cys Gly Gly Asn Lys Ile Gly Ser Lys Ser Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asp Ser Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ala Gly Ser 50 55 60 Asn Ala Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ile Ser Ala Asn Ala 85 90 95 Ala Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 <210> 66 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 66 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Leu Ser Thr 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asp Asn Arg Arg Pro Ser Gly Ile His Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Gly Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Ser Ala Asn Ala 85 90 95 Leu Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 <210> 67 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 67 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys Gly Gly Asp Asn Ile Gly Ser Lys Ser Ala 20 25 30 Gln Trp Tyr Gln Gln Ser Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asp Ala Ser Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Ala Arg Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Thr Asn Ala 85 90 95 Ala Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 <210> 68 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 68 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asp Ser Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Val Ser Thr Asn Ala 85 90 95 Ala Val Phe Gly Gly Gly Thr His Leu Ser Val Leu 100 105 <210> 69 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 69 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Arg Lys Ser Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asp Ser Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ala Asn Ala 85 90 95 Ala Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 <210> 70 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 70 Ser Tyr Gly Met Ser 1 5 <210> 71 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 71 Thr Ile Ser Arg Gly Gly Asp Ser Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 72 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 72 Leu Gly Arg Leu Gly Phe Gly 1 5 <210> 73 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 73 Ala Gly Thr Ser Ser Asp Ile Gly Asp Trp Asn Tyr Val Ser 1 5 10 <210> 74 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 74 Thr Ser Tyr Arg Ser Gly Asn Asn Trp Val 1 5 10 <210> 75 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 75 Val Ser Tyr Arg Ser Gly Asn Asn Trp Val 1 5 10 <210> 76 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 76 Glu Leu Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Arg Gly Gly Asp Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Leu Gly Arg Leu Gly Phe Gly Arg Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 77 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 77 Ser Ser Ala Leu Thr Gln Pro Pro Ser Met Ser Gly Thr Leu Gly Lys 1 5 10 15 Thr Leu Thr Ile Ser Cys Ala Gly Thr Ser Ser Asp Ile Gly Asp Trp 20 25 30 Asn Tyr Val Ser Trp Phe Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Ser Glu Val Asn Lys Arg Ala Ser Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Ser Ile Ser Gly Leu 65 70 75 80 Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Thr Ser Tyr Arg Ser Gly 85 90 95 Asn Asn Trp Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110 <210> 78 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 78 Gln Ala Gly Leu Thr Gln Pro Pro Ser Met Ser Gly Thr Leu Gly Lys 1 5 10 15 Thr Leu Thr Ile Ser Cys Ala Gly Thr Ser Ser Asp Ile Gly Asp Trp 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Glu Val Asn Lys Arg Ala Ser Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Ser Ile Ser Gly Leu 65 70 75 80 Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Val Ser Tyr Arg Ser Gly 85 90 95 Asn Asn Trp Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110 <210> 79 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 79 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Lys 1 5 10 15 Thr Leu Thr Ile Ser Cys Ala Gly Thr Ser Ser Asp Ile Gly Asp Trp 20 25 30 Asn Tyr Val Ser Trp Phe Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Ser Glu Val Asn Lys Arg Ala Ser Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Ser Ile Ser Gly Leu 65 70 75 80 Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Thr Ser Tyr Arg Ser Gly 85 90 95 Asn Asn Trp Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 110 <210> 80 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 80 Glu Pro Lys Ser Cys Asp Lys Thr His Thr 1 5 10 <210> 81 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 81 Cys Pro Pro Cys Pro 1 5 <210> 82 <211> 8 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 82 Ala Pro Glu Leu Leu Gly Gly Pro 1 5 <210> 83 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 83 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr 1 5 10 <210> 84 <211> 50 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 84 Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys 1 5 10 15 Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro 20 25 30 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 35 40 45 Cys Pro 50 <210> 85 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 85 Glu Ser Lys Tyr Gly Pro Pro 1 5 <210> 86 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 86 Cys Pro Ser Cys Pro 1 5 <210> 87 <211> 8 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 87 Ala Pro Glu Phe Leu Gly Gly Pro 1 5 <210> 88 <211> 3 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 88 Glu Arg Lys 1 <210> 89 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 89 Cys Cys Val Glu Cys Pro Pro Pro Cys Pro 1 5 10 <210> 90 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 90 Ala Pro Pro Val Ala Gly Pro 1 5 <210> 91 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 91 Pro Leu Tyr Ser Asn Leu Ala Gly Asp Leu Gly Ser 1 5 10 <210> 92 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 92 Pro Leu Tyr Ser Asn Leu Ala Gly Asp Phe Gly Ser 1 5 10 <210> 93 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 93 Pro Leu Tyr Asn Asn Leu Ala Gly Asp Phe Gly Ser 1 5 10 <210> 94 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 94 Pro Leu Tyr Ser Asn Phe Ala Gly Asp Phe Gly Ser 1 5 10 <210> 95 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 95 Pro Leu Tyr Asn Asn Phe Ala Gly His Phe Gly Ser 1 5 10 <210> 96 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 96 Gly Gly Ser Arg Leu Gln Ala 1 5 <210> 97 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 97 Leu Gln Val Tyr Ser Trp Pro Leu Thr 1 5 <210> 98 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 98 Gln Gln Asp Tyr Ser Trp Pro Leu Thr 1 5 <210> 99 <211> 121 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 99 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Lys Ala Arg Gly Gly Thr Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Leu Tyr Ser Asn Leu Ala Gly Asp Leu Gly Ser Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 100 <211> 107 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 100 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Gly Ser Arg Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Ser Phe Thr Leu Thr Ile Ser Gly Leu Glu Thr 65 70 75 80 Glu Asp Leu Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 101 <211> 121 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 101 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Lys Ala Arg Gly Gly Thr Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Leu Tyr Ser Asn Leu Ala Gly Asp Phe Gly Ser Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 102 <211> 107 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 102 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Ser Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Gly Ser Arg Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Ser Phe Thr Leu Thr Ile Ser Gly Leu Glu Thr 65 70 75 80 Glu Asp Leu Ala Thr Tyr Tyr Cys Leu Gln Val Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 103 <211> 121 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 103 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Lys Ala Arg Gly Gly Thr Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Leu Val Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Leu Tyr Asn Asn Leu Ala Gly Asp Phe Gly Ser Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 104 <211> 107 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 104 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Gly Ser Arg Leu Gln Ala Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Ser Phe Thr Leu Thr Ile Gly Gly Leu Glu Thr 65 70 75 80 Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 105 <211> 121 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 105 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Lys Ala Arg Gly Gly Thr Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Lys Pro Leu Tyr Ser Asn Phe Ala Gly Asp Phe Gly Ser Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 106 <211> 107 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 106 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Gly Ser Arg Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Ser Phe Thr Leu Thr Ile Ser Gly Leu Glu Thr 65 70 75 80 Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 107 <211> 121 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 107 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Lys Ala Arg Gly Gly Thr Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Leu Tyr Asn Asn Phe Ala Gly His Phe Gly Ser Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 108 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 108 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Gly Ser Arg Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Ser Phe Thr Leu Thr Ile Ser Gly Leu Glu Thr 65 70 75 80 Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105

Claims

1. A pharmaceutical composition comprising: (i) an antagonist of IL-5:IL-5R, which is an antibody molecule that binds to IL-5; and (ii) IL-4:IL-4R antagonists, which are antibody molecules that bind to IL-4Rα, The antibody molecule of (i) comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH domain and the VL domain comprise CDR sequences selected from the group consisting of: (i) 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:54; LCDR2 consisting of SEQ ID NO:53; LCDR1 consisting of SEQ ID NO:52; (ii) 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:57; LCDR2 consisting of SEQ ID NO:56; LCDR1 consisting of SEQ ID NO:55; (iii) 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; (iv) 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:61; LCDR2 consisting of SEQ ID NO:60; LCDR1 consisting of SEQ ID NO:58; and (v) 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; wherein the antibody molecule of (ii) comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH domain and the VL domain comprise CDR sequences selected from the group consisting of: (i) 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; (ii) 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; (iii) HCDR3 consisting of SEQ ID NO:9; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:18; LCDR2 consisting of SEQ ID NO:17; LCDR1 consisting of SEQ ID NO:16; (iv) HCDR3 consisting of SEQ ID NO:91; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:18; LCDR2 consisting of SEQ ID NO:17; LCDR1 consisting of SEQ ID NO:16; (v) HCDR3 consisting of SEQ ID NO:92; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:97; LCDR2 consisting of SEQ ID NO:17; LCDR1 consisting of SEQ ID NO:16; (vi) HCDR3 consisting of SEQ ID NO:93; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:98; LCDR2 consisting of SEQ ID NO:96; LCDR1 consisting of SEQ ID NO:16; (vii) HCDR3 consisting of SEQ ID NO:94; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:98; LCDR2 consisting of SEQ ID NO:17; LCDR1 consisting of SEQ ID NO:16; and (viii) HCDR3 consisting of SEQ ID NO:95; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:98; LCDR2 consisting of SEQ ID NO:17; LCDR1 consisting of SEQ ID NO:

16. 2 . The pharmaceutical composition according to claim 1 , which inhibits signal transduction via IL-5, IL-4 and IL-13.

3. The pharmaceutical composition according to claim 1, wherein the antagonist of (i) is an antibody molecule that binds to human IL-5. The pharmaceutical composition according to claim 1 , wherein the antagonist of (ii) is an antibody molecule that binds to human IL-4Rα.

5. The pharmaceutical composition according to claim 1, wherein the antibody molecule of (i) and / or the antibody molecule of (ii) is independently selected from the group consisting of: a single-chain antibody; a F(ab')2 fragment; a Fab fragment; a Fv fragment; a one-armed antibody; a double-chain antibody, a three-chain antibody, a four-chain antibody or any antigen-binding molecule formed by the combination, assembly or conjugation of such antigen-binding fragments.

6. The pharmaceutical composition according to claim 1, wherein the antibody molecule of (i) and / or the antibody molecule of (ii) is an IgG antibody.

7. The pharmaceutical composition according to claim 1, wherein the antibody molecule of (i) and / or the antibody molecule of (ii) is a humanized or germlined variant of a non-human antibody.

8. The pharmaceutical composition of claim 7, wherein the non-human antibody is derived from Camelidae.

9. The pharmaceutical composition according to claim 1, wherein the antibody molecule of (i) and / or the antibody molecule of (ii) comprises a CH1 domain, a hinge region, a CH2 domain and / or a CH3 domain of human IgG.

10. The pharmaceutical composition according to claim 1, wherein the antibody molecule of (i) and / or the antibody molecule of (ii) exhibits high homology with human IgG. 11 . The pharmaceutical composition according to claim 1 , wherein the antibody molecule of (i) and / or the antibody molecule of (ii) exhibits a high homology with human IgG1.

12. The pharmaceutical composition according to claim 1, wherein the antibody molecule of (i) and / or the antibody molecule of (ii) comprises an Fc domain derived from human IgG. 13 . The pharmaceutical composition according to claim 1 , wherein the antibody molecule of (i) and / or the antibody molecule of (ii) comprises an Fc domain derived from human IgG1.

14. The pharmaceutical composition of claim 12, wherein the Fc domain is modified by one or more amino acid substitutions to increase binding affinity to FcRn.

15. The pharmaceutical composition of claim 14, wherein the Fc domain comprises the amino acid substitutions: H433K and N434F.

16. The pharmaceutical composition of claim 14, wherein the Fc domain comprises the amino acid substitutions: M252Y, S254T, T256E, H433K, and N434F.

17. The pharmaceutical composition according to claim 1, wherein the antibody molecule of (ii) comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from the group consisting of: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that is at least 70% identical thereto; (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that is at least 70% identical thereto; (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that is at least 70% identical thereto; (iv) a VH domain comprising the amino acid sequence of SEQ ID NO: 99, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence that is at least 70% identical thereto; (v) a VH domain comprising the amino acid sequence of SEQ ID NO: 101, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 102, or an amino acid sequence that is at least 70% identical thereto; (vi) a VH domain comprising the amino acid sequence of SEQ ID NO: 103, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence that is at least 70% identical thereto; (vii) a VH domain comprising the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence that is at least 70% identical thereto; and (vii) a VH domain comprising the amino acid sequence of SEQ ID NO: 107, or an amino acid sequence at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 108, or an amino acid sequence at least 70% identical thereto.

18. The pharmaceutical composition of claim 1, wherein the antibody molecule of (i) comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from the group consisting of: (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence that is at least 70% identical thereto; (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that is at least 70% identical thereto; (iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence that is at least 70% identical thereto; (iv) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that is at least 70% identical thereto; (v) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence that is at least 70% identical thereto; and (vi) a VH domain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence at least 70% identical thereto, and a VL domain comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence at least 70% identical thereto.

19. The pharmaceutical composition according to claim 1, wherein the antibody molecule of (i) and / or the antibody molecule of (ii) exhibits lower antigen binding activity at an acidic pH than at a neutral pH.

20. The pharmaceutical composition of claim 19, wherein the ratio of antigen binding activity at acidic pH to at neutral pH, as measured by KD at acidic pH / KD at neutral pH, is at least 2.

21. The pharmaceutical composition according to claim 20, wherein the antagonist of (i) and the antagonist of (ii) are formulated according to a ratio of 1:1 or 1:2 or 2:

1. 22 . The pharmaceutical composition according to claim 1 , wherein the antibody molecule that binds to IL-4Rα and the antibody molecule that binds to IL-5 are combined into a multispecific antibody. 23 . The pharmaceutical composition according to claim 22 , wherein the antibody molecule that binds to IL-4Rα and the antibody molecule that binds to IL-5 are combined into a bispecific antibody.

24. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises one or more additional therapeutic agents.

25. A bispecific antibody comprising 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 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, Wherein the first variable heavy chain domain (VH) and variable light chain domain (VL) pair comprises a CDR sequence selected from the following: (i) 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; (ii) 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; (iii) HCDR3 consisting of SEQ ID NO:9; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:18; LCDR2 consisting of SEQ ID NO:17; LCDR1 consisting of SEQ ID NO:16; (iv) HCDR3 consisting of SEQ ID NO:91; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:18; LCDR2 consisting of SEQ ID NO:17; LCDR1 consisting of SEQ ID NO:16; (v) HCDR3 consisting of SEQ ID NO:92; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:97; LCDR2 consisting of SEQ ID NO:17; LCDR1 consisting of SEQ ID NO:16; (vi) HCDR3 consisting of SEQ ID NO:93; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:98; LCDR2 consisting of SEQ ID NO:96; LCDR1 consisting of SEQ ID NO:16; (vii) HCDR3 consisting of SEQ ID NO:94; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:98; LCDR2 consisting of SEQ ID NO:17; LCDR1 consisting of SEQ ID NO:16; and (viii) HCDR3 consisting of SEQ ID NO:95; HCDR2 consisting of SEQ ID NO:8; HCDR1 consisting of SEQ ID NO:7; LCDR3 consisting of SEQ ID NO:98; LCDR2 consisting of SEQ ID NO:17; LCDR1 consisting of SEQ ID NO:16; and Wherein the second variable heavy chain domain (VH) and variable light chain domain (VL) pair comprises a CDR sequence selected from the following: (i) 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:54; LCDR2 consisting of SEQ ID NO:53; LCDR1 consisting of SEQ ID NO:52; (ii) 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:57; LCDR2 consisting of SEQ ID NO:56; LCDR1 consisting of SEQ ID NO:55; (iii) 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; (iv) 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:61; LCDR2 consisting of SEQ ID NO:60; LCDR1 consisting of SEQ ID NO:58; and (v) 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.

26. The bispecific antibody of claim 25, wherein the antibody is an IgG antibody having at least one scFv fragment attached thereto. 27 . The bispecific antibody according to claim 26 , 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 the at least one scFv fragment.

28. The bispecific antibody of claim 25, wherein the antigen binding region that binds to IL-4Rα and / or the second antigen binding region that binds to IL-5 is a humanized or germlined variant of a non-human antibody or an antigen binding fragment thereof.

29. The bispecific antibody of claim 28, wherein the non-human antibody is a camelid antibody or an antigen-binding fragment thereof. 30 . The bispecific antibody of claim 29 , 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.

31. The bispecific antibody of claim 30, wherein the ratio of antigen binding activity at acidic pH to at neutral pH, as measured by KD at acidic pH / KD at neutral pH, is at least 2.

32. Use of an antagonist of IL-5:IL-5R as an antibody molecule binding to IL-5 in the preparation of a medicament for treating type 2 cytokine-associated chronic airway disease in a human subject, wherein the treatment comprises administering the antagonist in combination with an antagonist of IL-4:IL-4R as an antibody molecule binding to IL-4Rα, wherein the antibody molecule binding to IL-5 and the antibody molecule binding to IL-4Rα are as defined in claim 1.

33. Use of an antagonist of IL-4:IL-4R as an antibody molecule binding to IL-4Rα in the preparation of a medicament for treating type 2 cytokine-associated chronic airway disease in a human subject, wherein the treatment comprises administering a pharmaceutical composition of the antagonist and an antagonist of IL-5:IL-5R as an antibody molecule binding to IL-5, wherein the antibody molecule binding to IL-4Rα and the antibody molecule binding to IL-5 are as defined in claim 1.

34. Use of the pharmaceutical composition according to any one of claims 1-24 or the bispecific antibody according to any one of claims 25-31 in the preparation of a medicament for treating a type 2 cytokine-related chronic airway disease in a human subject.

35. The use according to claim 34, wherein the type 2 cytokine-related chronic airway disease is asthma.

36. Use according to claim 35, wherein the medicament is for the treatment of type II asthma.

37. The use according to claim 35, wherein the medicament is for treating atopic or allergic asthma.

38. The use of claim 34, wherein the treatment reduces goblet cell metaplasia (GCM).

39. The use according to claim 34, wherein the treatment reduces bronchial hyperresponsiveness (BHR).

40. The use of claim 34, wherein the treatment further comprises administering one or more additional therapeutic agents to treat the type 2 cytokine-associated chronic airway disease.

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