Combined antagonists against il-5 / il-5r and il-4 / il-4r or il-13 / il-13r

Abstract

To provide combination therapies and their use in the treatment of chronic airway disease, particularly use in the treatment of asthma.SOLUTION: The combination therapies comprise (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 present invention also provides bispecific antibodies comprising an antigen binding domain that binds to IL-4Rα and an antigen binding domain that binds to IL-5. The bispecific antibodies may be used for the treatment of chronic airway disease, particularly asthma.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to combination therapies and their use in the treatment of chronic airway diseases, particularly asthma. The combination therapy comprises (i) an antagonist of IL-5:IL-5R, and (ii) an antagonist of IL-5R, and (iii) an antagonist of IL-5R, and (iv) an antagonist of IL-5R, and (v) an antagonist of IL-5R, and (vi ... ii) IL-4: Contains an antagonist of IL-4R and / or IL-13: Contains an antagonist of IL-13R. The antagonist may be an antibody molecule, and preferably the combination therapy is directed to an antibody that binds to IL-4Rα. The combination therapy typically includes an antibody molecule that binds to type 2 cytokines and an antibody molecule that binds to IL-5. The present invention also relates to a method for inhibiting signal transduction via IL-4, IL-13, and IL-5. and an antigen-binding domain that binds to IL-5. Bispecific antibodies can be used to treat chronic airway diseases, particularly asthma. [Background technology]

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

[0003] Asthma is a chronic inflammatory disease of the conducting airways that causes symptoms of coughing, wheezing, and chest tightness. It is a disease that affects up to 300 million people worldwide. The obstruction follows a variable course, with asymptomatic periods resulting from environmental allergens and viral infections. Common symptoms include cold and driving. This is bronchial hyperresponsiveness (BHR), a condition in which the airways constrict in response to stimuli such as movement.

[0004] Asthmatics also exhibit symptoms of airway remodeling, whereby the airway walls thicken and the glandular epithelium Or the number of mucus-producing goblet cells in the mucosal glands increases, a phenomenon called goblet cell metaplasia (GCM). Goblet cells produce mucins that control the viscoelasticity and hydration of the mucus that coats the ciliary escalator. In asthmatics, sputum is often very dry, leading to mucus impaction and severe airway obstruction. Currently, there are no therapies to reduce GCM and improve mucus clearance. There are almost no options.

[0005] Historically, asthma treatment approaches have focused on inhaled corticosteroids and beta-2 agonists. This has involved the use of non-specific medications, with varying degrees of success. It is now recognized that multiple phenotypes, each with distinct clinical, physiological, and molecular characteristics, exist. It is generally understood to be a heterogeneous disorder that represents a range of conditions (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] Each type of asthma is caused by a distinct pathogenic molecular mechanism and therefore is considered a different disease. Clinical subtypes of asthma, recognizing that each has different causes and will respond differently to treatment Attempts have been made to group sets (Gauthier et al., (2015) American Jou rnal of Respiratory and Critical Care Medicine; 192(6): 660-668). The set of examples includes early-onset or late-onset asthma, depending on the age at which symptoms appear; Depending on location and type, it can be classified as eosinophilic, neutrophilic, or non-inflammatory asthma; exercise-induced asthma; or obesity-related asthma. Allergic sensitization to inhaled allergens such as house dust mites (HDM) and serum allergen characteristics Atopic asthma characterized by increased levels of allergic IgE; expressed as corticosteroid responsiveness mild or moderate asthma; severe asthma; and type 2 asthma, which represents individuals who share a type 2 inflammatory pattern. Importantly, there is a clear demarcation between these groups. Due to the lack of differentiation, many phenotypes overlap, and patients represent multiple groups of clinical or pathological These characteristics may make it difficult to predict individual responses to treatment. That is the thing.

[0007] These mechanistically distinct groups, also called "endotypes," have become better understood and more relevant. Associated cellular or molecular biomarkers are beginning to be identified. In patients with asthma, asthma is often characterized by the accumulation of eosinophils, mast cells, and CD4+ T lymphocytes. These cells express type 2 cytokines IL-4 and / or IL-5 within the epithelium and lamina propria. However, this type 2 inflammatory condition accounts for only 50% of asthma, especially early-onset asthma, It is only detectable in patients with atopic diathesis and high blood eosinophil counts. In some patients, particularly those with poor steroid response, the airway infiltrate is composed primarily of neutrophils. These neutrophils are activated by IL-17-producing cells such as type 17 T helper lymphocytes or γδT cells. and then recruited to the airways (Lambrecht and Hammad, (2015) Nat. Immunol. 16( 1): 45-56).

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

[0009] (Summary of the Invention) The present invention is based on targeting multiple type 2 cytokine signaling pathways. They bind to T helper type 2 cells (T H 2 cells), so that In chronic airway diseases such as asthma, type 2 cytokines are involved in the regulation of T H Not just two cells, They are also released by cells such as basophils, mast cells, and eosinophils. Type 2 cytokines are responsible for the production of cytokines in chronic It plays an important role in the pathogenesis of airway diseases, especially asthma. The cytokines, particularly IL-5, IL-4 and IL-13, play a role in the underlying conditions and symptoms of chronic airway disease. It has been discovered that the combination of the present invention provides an unexpected synergistic effect in alleviating the Combination therapy is particularly suitable for treating chronic airway diseases.

[0010] In a first aspect, the present invention provides (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 IL-4 receptor complex and the IL-13 receptor complex share a common subunit, IL-4Rα Therefore, IL-4Rα antagonists inhibit both the IL-4:IL-4R and IL-13:IL-13R signaling pathways. In the preferred embodiment below, the combination of the present invention can function as an antagonist of both the agonist and antagonist of the other agonist. This includes IL-5:IL-5R antagonists and IL-4Rα antagonists. The antagonist of R is preferably an antagonist of IL-5. It may also inhibit signaling through IL-4 and IL-13.

[0011] The antagonist of the combination may be an antibody molecule. Thus, in one embodiment, the IL-5:IL- The antagonist of 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 an embodiment, the combination comprises an antibody molecule that binds to IL-5, preferably human IL-5, IL-5:IL- and an antibody molecule that binds to IL-4Rα, preferably human IL-4Rα. Includes antagonists of IL-4Rα.

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

[0013] In one embodiment, the antibody molecules of the combination, e.g., antibody molecules that bind to IL-4Rα and / or Alternatively, the antibody molecule that binds IL-5 may be a humanized or germline variant of a non-human antibody. or an antigen-binding fragment thereof, for example, a camelid-derived antibody or an antigen-binding fragment thereof. is.

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

[0015] In one embodiment, the antibody molecules of the combination, e.g., antibody molecules that bind to IL-4Rα and / or Alternatively, the antibody molecule that binds to IL-5 comprises an Fc domain derived from human IgG, preferably IgG1. The Fc domain may be unmodified, but may be modified to have, for example, increased binding affinity to FcRn (fetal Fc receptor). In a preferred embodiment, the amino acid sequence of the present invention is The antibody molecule comprises an Fc domain, preferably comprising the amino acid substitutions: H433K and N434F; or M252Y, S25 The Fc domain may comprise an Fc domain derived from human IgG, including Fc domains 4T, T256E, H433K, and N434F. The main number is assigned according to the EU numbering scheme.

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

[0017] Regarding the formulation of the above combinations, IL-5:IL-5R and IL-4:IL-4R and / or IL-13:IL-13R The antagonists may be co-formulated or provided separately. In embodiments where an agonist is co-formulated, the antagonist may be formulated in a 1:1 ratio, They may be formulated in non-equimolar ratios. For example, IL-5:IL-5R antagonists, preferably IL-5 and an antagonist of IL-4Rα. The instigators may be formulated in a 1:2 or 2:1 ratio, for example.

[0018] The combination comprises an antagonist of IL-4Rα and an antagonist of IL-5, In embodiments where the antagonist is an antibody molecule, the combination may be a multispecific antibody, e.g., a bispecific antibody. It may include antibody molecules that are combined within an antibody.

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

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

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

[0022] Combinatorial and bispecific antibodies targeting multiple type 2 cytokine signaling pathways It has been discovered that the compound is particularly useful in the treatment of chronic airway diseases, particularly asthma. In a further aspect, the first aspect of the present invention is a method for treating a chronic airway disease in a human subject. Combinations of aspects or bispecific antibodies of the second aspect of the invention are provided. There is provided a method of treating a chronic airways disease of the present invention, comprising administering to a subject a combination of Alternatively, it comprises administering to a subject an effective amount of the bispecific antibody according to the second aspect of the present invention.

[0023] In some embodiments, the chronic airway disease is: asthma; chronic rhinosinusitis (CRS); immunoglobulin G4-associated disease Chronic obstructive pulmonary disease (IgG4-RD); chronic obstructive pulmonary disease (COPD); chronic bronchitis; emphysema; chronic angioedema; Barrett's esophagus Diseases characterized by goblet cell metaplasia, including: ongoing eosinophilic esophagitis; nasal polyposis; chronic sinusitis inflammation; Churg-Strauss syndrome; allergic bronchopulmonary aspergillosis (ABPA); eosinophils hyperplasia syndrome; bullous pemphigoid and cystic fibrosis.

[0024] The chronic airway diseases treated by the methods of the present invention include those characterized by increased mucus production or exacerbated bronchial hyperresponsiveness. In a preferred embodiment, the chronic airway disease to be treated is asthma, any Severe asthma, severe intractable asthma, Type II high asthma, atopic or allergic It is chronic asthma.

[0025] The methods described herein include treating chronic airway diseases, preferably asthma, with goblet cell metaplasia (or GCM). Alternatively or additionally, this method may be useful for reducing and treating chronic airway Useful for treating diseases, preferably asthma, by reducing bronchial hyperresponsiveness (BHR). The method may further comprise administering to the patient one or more An additional step of administering an additional therapeutic agent may also be included. [Brief explanation of the drawings]

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0050] (Detailed description) (A.Definition) Unless otherwise defined herein, technical and scientific terms used herein are defined by the It shall have the meaning commonly understood by a person skilled in the art of the invention.

[0051] "Combination Therapy" - As used herein, the term "combination therapy" refers to a combination therapy in which a subject, e.g., a human subject, "Combination" in the present invention is also referred to as "combination therapy." Two or more therapeutic agents are generally intended to be used to treat a single disease, as defined herein. The combination or combination therapy of the present invention is administered to treat multiple Combining antagonists that target the type 2 cytokine signaling pathway. The combination therapy described herein is a combination of cytokine:cytokine receptor:IL-5:IL-5R; IL-4:IL These cytokine-cytokine receptors are described herein. In a preferred embodiment, the antagonist is a compound that inhibits the activity of these cytokines. The antibody molecule specifically binds to its target, either a cytokine or a protease receptor. As described in the literature, the antagonists included in this combination therapy are They may be co-formulated, e.g., presented separately as separate compositions, for administration to a subject or patient. The combination comprising an antibody molecule that binds to IL-4Rα and an antibody molecule that binds to IL-5 may be provided. In some embodiments, the antibody molecules of the combination may be combined into a single antibody, e.g., a bispecific antibody. It may also be included in a multispecific antibody format such as

[0052] "Antagonist" - As used herein, the term "antagonist" refers to an agent that inhibits or inhibits the activity of a target site. It means any agent or molecule that can inhibit the function of a kine or cytokine receptor. As used herein, IL-5:IL-5R antagonists refer to IL-5 receptors that bind to their cognate receptor complex, the IL-5R. means any agent or molecule capable of inhibiting signal transduction initiated by L-5 binding As used herein, an antagonist of IL-4:IL-4R refers to an antagonist of its cognate type I receptor complex, "IL- Any agent or molecule capable of inhibiting signal transduction initiated by binding of IL-4 to IL-4R As used herein, an antagonist of IL-13:IL-13R refers to an antagonist of its cognate receptor complex. Any agent capable of inhibiting signal transduction initiated by binding of IL-13 to the IL-13R or molecules. As described herein, the type 2 cytokines IL-5; IL-4 and IL-1 The receptor complex to which 3 binds usually consists of two receptor subunits. For example, I IL-5:IL-5R antagonists inhibit the association of IL-5 with its receptor complex or inhibit the association of IL-5 with the IL-5R complex. It inhibits the association between the two subunits of the IL-5R (IL-5Rα and βc) and inhibits IL-5-mediated signal transduction. Similarly, an antagonist of IL-4:IL-4R or an antagonist of IL-1 3. IL-13R antagonists also inhibit the interaction between cytokines (IL-4 or IL-13) and their receptor complexes. or inhibit the association between the two subunits of the IL-4R or IL-13R complex, thereby inhibiting IL- The compounds described herein will inhibit or block signaling mediated by IL-4 or IL-13. As shown, the IL-4R and IL-13R complexes share a common receptor subunit, IL-4Rα. Furthermore, the cytokine IL-4 not only acts via its own type I receptor complex, IL-4R, but also It can also signal through the IL-13R complex. Agonists disrupt both the IL-4R and IL-13R complexes, blocking signaling via both IL-4 and IL-13. Furthermore, in some cases, antagonists of the IL-13R complex can inhibit IL-13 signaling. It may also inhibit IL-4-mediated signaling.

[0053] IL-5:IL-5R, IL-4:IL-4R and IL-13:IL-13R antagonists for use in the combinations of the present invention The antagonist may take the form of any suitable drug or molecule. downregulates the expression of a target, e.g., IL-4Rα or IL-5 expression, thereby inhibiting the expression of that target. In another embodiment, the antagonist inhibits the function of a cytokine or receptor. They may also bind directly to receptor subunits and inhibit the function of their targets. As will be seen, IL-5:IL-5R antagonists inhibit the cytokine IL-5 (IL-5 antagonists). antagonists of IL-5Rα or βc) or IL-5R subunits (IL-5Rα antagonists or βc antagonists) Similarly, IL-4:IL-4R antagonists is a cytokine that binds to IL-4 (called an antagonist of IL-4) or the type I IL-4R subunit (IL- It also binds to one of the receptors (called antagonists of γc or γα). be.

[0054] In a preferred embodiment, the antagonist is specific for its target, e.g., IL-4Rα Antagonists of IL-4Rα (or IL-4Rα antagonists) target the function of IL-4Rα compared to other molecular targets. IL-5 antagonists will preferentially inhibit the function of IL-5 compared to other molecular targets. Antagonists usually do not directly interact with their targets. By using the antibody, it is possible to selectively bind to, for example, IL-4Rα or IL-5 mRNA or protein. By combining these two, the desired level of specificity will be achieved. Suitable drugs or molecules that can be used include inhibitory RNA species, such as siRNA or shRNA, small molecule inhibitors, These include, but are not limited to, biological antagonists. In an embodiment, the antagonist of the combination is an antibody molecule.

[0055] "Antibody molecule" - As used herein, the term "antibody molecule" refers to an antibody molecule, including modified antibodies, humanized antibodies, germline antibodies, and the like. Full length antibodies and their variants, including cell line antibodies and antigen binding fragments thereof The term "antibody" generally refers to a molecule comprising two heavy chains and two light chains, and includes antigen-binding fragments thereof. A heterotetrameric immunoglobulin polypeptide having a combination of chains, has significant specific immune response activity to the target antigen (e.g., IL-4Rα or IL-5). As an IgG class antibody, the antibody consists of two identical polypeptides with a molecular weight of approximately 23,000 daltons. It contains a polypeptide light chain and two identical heavy chains with molecular weights of 53,000 to 70,000. The chains are joined by disulfide bonds to form a "Y" configuration, and the light chains are located at the mouth of the "Y". The light chains of an antibody bracket the heavy chains, starting at the base of the kappa chain and continuing to the variable region. Each heavy chain class is classified as either kappa or lambda (κ, λ). The light and heavy chains are generally covalently linked to each other. The "tail" portions of the two heavy chains are joined by covalent disulfide bonds or by immunoglobulins. When antibodies are produced in hybridomas, B cells, or genetically engineered host cells, they are non-covalently linked. Within the heavy chain, the amino acid sequence is branched in a Y configuration. It runs from the N-terminus at each end to the C-terminus at the bottom of each chain.

[0056] Those skilled in the art will appreciate that heavy chains can be gamma, mu, alpha, delta, or epsilon (γ, μ, α , δ, ε), among which there are several subclasses (e.g., The antibody "classes" are IgG, IgM, IgA, IgD, or It is the properties of these chains that determine whether an antibody is IgE. types), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and functional. As used herein, the term "antibody molecule" refers to an antibody molecule. It encompasses full-length antibodies or antigen-binding fragments thereof from any class or subclass.

[0057] The term "antibody molecule" as used herein also encompasses "heavy chain only antibodies" or "VHH antibodies." The term "heavy chain only antibody" or "VHH antibody" refers to antibodies derived from camels, llamas, and ants. This refers to a type of antibody produced exclusively by species of the Camelidae family, including the paca. Heavy chain-only antibodies are It consists of two heavy chains and lacks a light chain. Each heavy chain has a variable domain at its N-terminus. However, these variable domains are the variable domains of the heavy chains of conventional heterotetrameric antibodies, i.e., the VH To distinguish it from the "VHH" domain, it is called a "VHH" domain.

[0058] With respect to antigen-binding fragments encompassed by the general term "antibody molecule," these fragments do not necessarily represent full-length antibodies. An antibody or a compound that contains fewer amino acids than an intact or complete antibody while retaining antigen-binding activity. As used herein, the term "antibody molecule" refers to a portion or part of an antibody chain, including the amino acid residues. , 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; single-arm (monovalent) antibody; diabody, triabody, tetrabody combinations, assemblies or conjugations of antibodies, antibodies, antibodies to ... and any antigen-binding molecule formed by As used herein, the term "antibody molecule" further includes: unibodies; domain antibodies; and The present invention is intended to encompass antibody fragments selected from the group consisting of: For example, via chemical or enzymatic treatment of an intact or complete antibody or antibody chain, or can be obtained by recombinant techniques.

[0059] "Variable region" or "variable domain" - The terms "variable region" and "variable domain" refer to These terms are used interchangeably herein and are intended to have the same meaning. This refers to the fact that certain portions of the variable domains VH and VL differ extensively in sequence between antibodies, This takes advantage of the special binding and specificity of each antibody for its target antigen, except However, the variability is not evenly distributed throughout the variable domains of antibodies. Concentrated in three segments called "hypervariable loops" in each of the main and VH domains The first, second, and third V lambda light chain domains form the antigen-binding site. The third hypervariable loops are referred to herein as L1(λ), ​​L2(λ) and L3(λ), and in the VL domain: Residues 24-33 (L1(λ) consisting of 9, 10 or 11 amino acid residues), 49-53 (L1(λ) consisting of 3 residues) It is sometimes defined as including L2(λ) consisting of 5 residues and 90-96 (L3(λ) consisting of 5 residues). (Morea et al., Methods 20:267-279 (2000)). and the third hypervariable loops, referred to herein as L1(κ), L2(κ) and L3(κ), are located within the VL domain. Residues 25-33 (L1(κ) consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L1(κ) consisting of 3 residues), L2(κ) consisting of 10 residues and L3(κ) consisting of 6 residues. (Morea et al., Methods 20:267-279 (2000)). The third hypervariable loop, referred to herein as H1, H2, and H3, is located within the VH domain at residues 25-33 (7 , H1 consisting of 8 or 9 residues), 52-56 (H2 consisting of 3 or 4 residues) and 91-105 (high It is sometimes defined as containing H3 of variable length (Morea et al., Methods 20: 267-279 (2000)).

[0060] Unless otherwise specified, the terms L1, L2 and L3 refer to the first, second and third VL domains, respectively. The hypervariable loops obtained from both the Vkappa and Vlambda isotypes are referred to as the three hypervariable loops. The terms H1, H2 and H3 refer to the first, second and third hypervariable regions of the VH domain, respectively. refers to a loop and can be obtained from any known heavy chain isotype, including gamma, epsilon, delta, alpha, or mu. It encompasses the hypervariable loops.

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

[0062] The CDRs of the VL and VH domains typically consist of the following amino acids: residues 24-34 in the light chain variable domain (LCD R1), 50-56 (LCDR2) and 89-97 (LCDR3), and residues 31-35 or 31-3 in the heavy chain variable domain 5b (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Servi ce, National Institutes of Health, Bethesda, MD. (1991)). Therefore, HV is the corresponding CD R, when referring herein to the "hypervariable loops" of the VH and VL domains Unless otherwise specified, a sequence should be construed to encompass the corresponding CDR, and vice versa. It seems that

[0063] The more highly conserved portions of variable domains are the framework regions (F Each native heavy and light chain variable domain contains four FRs (respectively FRs). FR1, FR2, FR3, and FR4) and connected by three hypervariable loops in a β-sheet configuration The hypervariable loops of each chain are held together in close proximity by the FRs. These, together with the hypervariable loops of the remaining chains, contribute to the formation of the antigen-binding site of the antibody. Structural analysis of the complementarity-determining regions (CDRs) reveals a relationship between the sequence and shape of the binding site formed by the CDRs. However, it was revealed (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); J. Mol. Biol. 215:175-182 (1990)). Despite their high sequence variability, Five of the six loops are "canonical structures" that fit into a very small repertoire. These conformations are determined primarily by the loop length and secondarily by , determined by the presence of key residues at specific positions within loops and framework regions. The residues are selected based on their packing, hydrogen bonding, or ability to assume unusual main-chain conformations. The three-dimensional structure is determined by

[0064] "CDR" - As used herein, the term "CDR" or "complementarity determining region" refers to a region of a heavy or light chain polypeptide. This refers to the discrete antigen-binding sites found within the variable regions of both polypeptides. These particular regions are described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al. (1991), and Chothia et al., Sequences of proteins of immunological interest. et al., J. Mol. Biol. 196:901-917 (1987) and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and when these definitions are compared with each other, The term "anti-cancer agent" refers to an agent that contains overlapping or subsets of the acid residues defined by each of the above-mentioned documents. The amino acid residues encompassing the CDRs are shown for comparison. Preferably, the term "CDR" refers to the sequence The CDRs are those defined by Kabat based on a comparison.

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

[0066] "Framework region" - As used herein, the term "framework region" or "FR region" refers to a "A region" refers to an amino acid sequence that is part of a variable region but not part of a CDR (e.g., using the Kabat CDR definition). Thus, the variable region framework is between about 100-120 amino acids in length. As a special case of the heavy chain variable domain, and Kab As defined by et al., framework region 1 is a variable region encompassing amino acids 1-30. framework region 2 corresponds to the domain of the variable region encompassing amino acids 36-49; framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94; framework region 4 corresponds to the variable region domain from amino acid 103 to the end of the variable region; Similarly, the light chain framework regions correspond to the CDRs of each light chain variable region. Similarly, using the CDR definitions of Chothia et al. or McCallum et al. The framework region boundaries are separated by the ends of the respective CDRs, as described above. In a preferred embodiment, the CDRs are as defined by Kabat.

[0067] In natural antibodies, the six CDRs present on each monomeric antibody are short, discontinuous sequences of amino acids. It is a sequence that forms the antigen-binding site when the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the heavy and light chain variable domains are specifically positioned to form amino acids. The amino acid sequence is less variable between molecules and is called the framework region. The region mainly adopts a β-sheet conformation, and the CDRs form loops to connect the β-sheet structure. These framework regions therefore provide the necessary interchain connectivity. Non-covalent interactions form a scaffold that orients the six CDRs in the correct direction The antigen-binding site formed by the arranged CDRs binds to the target antigen on the immunoreactive antigen. This complementary surface defines a surface that is complementary to the epitope of the antigen. The CDR configuration can be readily identified by one skilled in the art.

[0068] "Constant Region" - As used herein, the term "constant region" refers to a region of a variable domain or region. Refers to the outer part of an antibody molecule. Immunoglobulin light chains usually have a "CL or CL1 domain" It has a single domain called the "constant region." This domain is located at the C-terminus of the VL domain. The immunoglobulin heavy chains are divided into three classes according to the immunoglobulin class (γ, μ, α, δ, ε). Their constant regions differ. Heavy chains γ, α, and δ are divided into CH1 and CH2 domains. It consists of three immunoglobulin domains (termed CH1, CH2, and CH3) with a flexible hinge region that The heavy chains μ and ε each have a constant region consisting of four domains (CH1-CH4). The heavy chain constant domain is located at the C-terminus of the VH domain. do.

[0069] The numbering of amino acids in heavy and light chain immunoglobulins is based on the N at each branched end of the Y configuration. The constant domains of immunoglobulin heavy and light chains extend from the end to the C-terminus at the bottom of each chain. A different numbering scheme is used to define the The heavy chain constant domains of an IgG molecule are identified as follows: CH1 - amino acid residues 118-215; CH2 - amino acid residues 231-340; CH3 - amino acid residues 341-446. IgG components are numbered according to the Kabat numbering scheme. The heavy chain constant domains are identified as follows: CH1 - amino acid residues 114-223; CH2 - amino acid residues 114-223; CH2 - amino acid residues 244-360; CH3 - amino acid residues 361-477. An "Fc domain" or "Fc region" is typically defined as a region comprising the CH2 The Fc region also defines the constant region portion of the heavy chain, which comprises the hinge region, the CH3 domain, and the CH4 domain. The "hinge region" connects the CH1 domain to the CH2 domain. The "hinge region" comprises the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal The hinge region is made up of three distinct domains: the upper and lower domains, allowing the antigen-binding regions to move independently. , which can be subdivided into a middle and lower hinge domain (Roux KH et al., J. Immun ol. 161:4083-90 1998). Antibodies of the present invention that contain a "fully human" hinge region are shown in Table 2 below. It may also contain one of the hinge region sequences.

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

[0071] "Specific" and "Multispecific" Antibodies - Antibodies for use in the combination therapies described herein Antibody molecules bind to specific target antigens. Antibody molecules "specifically bind" to those target antigens. It is preferred that the term "specifically binds" refers to a specific target, e.g., IL-4Rα and The term "antibody" refers to the ability of any antibody molecule to preferentially immunoreact with IL-5 and IL-6. The molecule is monospecific, containing one or more binding sites that specifically bind to a particular target. The antibody molecules of the present combinations and methods may be in a "multispecific antibody" format, e.g. These may be incorporated into multispecific antibodies, which bind to two or more target antigens. For example, in one embodiment, the combination of the present invention comprises an antibody that specifically binds to IL-4Rα. and a second antibody molecule that specifically binds to IL-5. To achieve multiple specificities, "multispecific antibodies" typically contain different VH-VL pairs. The multiple polypeptides are designed to contain different combinations or pairs of heavy and light chain polypeptides having the following structure: Specificity, especially bispecific antibodies, depends on the overall conformation of the native antibody, e.g., the Fc region. To employ Y-shaped antibodies, which have Fab arms of different specificities that are conjugated Other multispecific antibodies, e.g., bispecific antibodies, may be designed using non-native isomers. variable domains with different specificities or variable domain pairs opposite the Fc region The structure may be designed to be disposed at the end of the side.

[0072] "Modified Antibody" - As used herein, the term "modified antibody" refers to an antibody that has been modified so as not to occur in nature. Synthetic forms of modified antibodies, e.g., containing at least two heavy chain portions but not two complete heavy chains. antibodies that do not contain two or more different antigens (such as domain-deleted antibodies or minibodies); Multispecific forms of antibodies engineered to bind to different epitopes on an antigen (e.g., scFv molecules include heavy chain molecules connected to scFv molecules. It is well known in the art and described, for example, in U.S. Pat. No. 5,892,019. "Antibody" refers to a multivalent form of antibody (e.g., trivalent, tetravalent, etc., binding to three or more copies of the same antigen). In another embodiment, the modified antibodies of the invention comprise at least one antibody that lacks a CH2 domain. a polypeptide comprising both a heavy chain portion and a binding portion of a member of a receptor-ligand pair. It is a fusion protein comprising the binding domain of

[0073] "Humanizing substitution" - As used herein, the term "humanizing substitution" refers to a substitution of the VH or VL domain of an antibody. amino acid residues present at particular positions in a reference human VH or VL domain The amino acid substitution refers to the amino acid residue that is replaced by the resulting amino acid residue in the reference human VH or VL domain. may be a VH or VL domain encoded by human germline. As defined herein, modifications may be made to the framework regions and / or CDRs of an antibody. Cut.

[0074] "Humanized variant" - As used herein, the term "humanized variant" or "humanized antibody" refers to a humanized variant of a humanized antibody. "Variant" refers to a variant antibody that contains one or more "humanizing substitutions" compared to a reference antibody; A portion of an antibody (e.g., a VH domain and / or a VL domain or at least one CDR) Some of them (including some of them) have amino acids derived from non-human species, and "humanizing substitutions" are those derived from non-human species. This occurs within the amino acid sequence.

[0075] "Germline variant" - The terms "germline variant" or "germline antigen" are used interchangeably. The term "humanized variant" is used herein specifically to refer to a "humanized variant," "Isotropic substitutions" refer to one or more specific substitutions at one or more specific positions within the VH or VL domain of an antibody. The amino acid residues are the same as those in the reference human VH or VL domain encoded by the human germline. All " For "germline variants," the substitutions made within the germline variant The amino acid residues are derived only from a single human germline-encoded VH or VL domain, or The terms "humanized variant" and "germline" are usually adopted from these. "Sequence variants" are often used interchangeably. When introduced into a camelid (llama) derived VH or VL domain, This results in a "humanized variant" of the VL domain, in which the substituted amino acid residues are derived primarily or exclusively from human germline-encoded VH or VL domain sequences If this occurs, the result is a "human germline" clone of the camelid (llama)-derived VH or VL domain. It might be called "variant."

[0076] "Affinity variant" - As used herein, the term "affinity variant" refers to a variant of a reference antibody. This refers to a variant antibody that exhibits one or more changes in the amino acid sequence compared to the antibody. The antibody exhibits an altered affinity for the target antigen compared to the reference antibody. The variants exhibit a specific activity against a target, e.g., IL-4Rα or IL-5, compared to a reference IL-4Rα or IL-5 antibody. Preferably, the affinity variants will exhibit altered affinity for the target antigen. Affinity variants will typically exhibit improved affinity compared to the reference antibody. The amino acid sequence of the CDRs may be changed by one or more substitutions compared to the original. A naturally occurring amino acid is a peptide in which the original amino acid at a given position is replaced by a different amino acid residue. The amino acid substitutions may be conservative or non-conservative. good.

[0077] "IL-5:IL-5R" - As used herein, the term "IL-5" refers to the interleukin-5 cytokine The term "IL-5R" refers to the receptor complex to which the interleukin-5 cytokine binds. The term "IL-5:IL-5R" is used herein to refer to the IL-5 cytokine / cytokine receptor system. The cytokine IL-5 also acts as a signaling complex for B-cell differentiation factor I, Also known as eosinophil differentiation factor and T cell replacement factor (TRF), the human homolog of IL-5 is It is 134 amino acids long (http: / / www.uniprot.org / uniprot / P05113). The term "IL-5" is intended to encompass all splice variants of the protein. Monomeric IL-5 has no activity and requires homodimers for function. The IL-5 receptor consists of two subunits. The first subunit is the "IL-5 receptor subunit alpha" or "IL-5R α", also known as IL-5R-alpha, IL-5RA, CDw125 and CD antigen, CD125, The subunits form the ligand-binding portion of the receptor complex. Human homolog of IL-5Rα is 420 amino acids long (http: / / www.uniprot.org / uniprot / Q01344). IL-5R complex The second subunit of the β-subunit is a non-liganded common signal transduction beta subunit or IL-5 is secreted by a limited number of mesenchymal cell types. Cells known to express eosinophils include eosinophils, NK cells, TC2CD8+ T cells, mast cells, and CD4 These include IL-5+ CD4+ T cells, gamma delta T cells, and IL-1 beta-activated endothelial cells. Genes involved in proliferation, cell survival, and maturation of B cells and eosinophils, as well as effector functions It is known to regulate gene expression.

[0078] "IL-4:IL-4R" - As used herein, the term "IL-4" refers to the interleukin-4 cytokine The term "IL-4R" refers to the type I receptor complex to which the interleukin-4 cytokine binds. The term "IL-4:IL-4R" refers to the IL-4 cytokine / type I cytokine receptor signaling complex. The cytokine IL-4 is also known as B cell stimulating factor 1 (BSF-1), a B cell stimulating factor (BSF) that stimulates the immune system. Binetrakin, also known as lymphocyte stimulatory factor 1. The human homolog of IL-4 is 1 It is 53 amino acids long (http: / / www.uniprot.org / uniprot / P05112). The term "IL-4" is intended to encompass all splice variants of the protein. The type I receptor for IL-4 consists of two subunits. The first subunit is called IL-4Rα. ", Interleukin-4 receptor subunit alpha, Interleukin-4 binding subunit IL-4Rα is a member of the class I cytokine receptor family. It is a 140 kDa transmembrane glycoprotein that is widely expressed in the human IL-4Rα. The amino acid length is (http: / / www.uniprot.org / uniprot / P24394). The term "IL-4Rα" is intended to encompass all splice variants of the protein. Within the type I IL-4 receptor complex, IL-4Rα binds to a second subunit, the common gamma chain (γc ), and this γc subunit enhances the affinity of IL-4Rα for IL-4, leading to IL-4-mediated The IL-4 receptor complex mediates downstream signal transduction, e.g., in B cells, T cells, Present in monocytes, eosinophils, and fibroblasts, IL-4Rα binds to the JAK1 / 2 / 3-STAT6 pathway. The L-4 response promotes Th2 differentiation and IgE production at sites of allergic inflammation and chemokines. It is involved in regulating the production of insulin and mucus.

[0079] "IL-13:IL-13R" - As used herein, the term "IL-13" refers to an interleukin-13 receptor 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. :IL-13R" to indicate the IL-13 cytokine / cytokine receptor signaling complex. The human homolog of IL-13 is 146 amino acids long (http: / / www.uniprot.org / As used herein, the term "IL-13" refers to all splices of the protein. The receptor for IL-13 is composed of two subunits. The first subunit is "IL-4Rα", which is an interleukin-4 receptor subunit. Also known as CD124, interleukin-4 binding subunit, and CD124. As described, this subunit is common to both the IL-4 and IL-13 receptors. Within the IL-13 receptor complex, IL-4Rα binds to a second subunit, the interleukin-13 receptor. receptor subunit alpha 1, or "IL-13R alpha 1," or "IL-13Rα1," or "IL- This second subunit associates with IL-13RA1, the ligand-binding subunit for IL-13. The IL-13R complex, which is composed of the IL-4Rα and IL-13Rα1 subunits, acts as a In response to IL-13 (through binding to IL-13Rα1) and IL-4 (through binding to IL-4Rα), Cytokines that bind to the receptor complex initiate signal transduction via the JAK1 / 2 / 3-STAT6 pathway. Similar to the IL-4:IL-4R complex, it is composed of IL-4Rα and IL-13Rα1 subunits. Signaling through the IL-13R complex mediates IgE production and chemoattractant production at sites of allergic inflammation. Involved in the regulation of cytokine and mucus production. IL-4 and IL-13 cytokine-receptor signaling For additional information on the cytokine signaling pathway, see, for example, McCormick and Heller (2015) Cytokine 7. 5(1):38-50, the contents of which are incorporated herein in their entirety.

[0080] "Chronic Airway Disease" - As used herein, the term "chronic airway disease" refers to a chronic respiratory disease ( It is sometimes used interchangeably with "CRD" and refers 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). These are chronic obstructive pulmonary disease (COPD), asthma, occupational lung disease and pulmonary hypertension. Risk factors for rheumatoid arthritis include air pollution, occupational chemicals and dust, and frequent lower respiratory tract infections during childhood. Other chronic airway diseases include chronic rhinosinusitis (CRS); immunoglobulin G4-related disease (IgG 4-RD); chronic bronchitis; emphysema; chronic angioedema; diseases characterized by goblet cell metaplasia such as Barrett's esophagus; ongoing eosinophilic esophagitis; nasal polyposis; chronic sinusitis; Churg-Strauss syndrome; allergies pulmonary aspergillosis (ABPA); hypereosinophilic syndrome; bullous pemphigoid, and cysts These include, but are not limited to, fibroids.

[0081] "Asthma" - As used herein, "asthma" refers to any disease associated with inflammation of the airways in the lungs. Inflammation is intended to mean a disease or condition that affects the sensitivity of nerve endings in the airways. During an asthma attack, the lining of the airways swells, narrowing the airways. Asthma has different clinical and physiological characteristics. Asthma is a heterogeneous disease with multiple phenotypes that exhibit both clinical and molecular characteristics. Examples include severe asthma, severe refractory asthma, mild or moderate asthma, obesity-related asthma, and exercise-related asthma. induced asthma, aspirin-induced asthma, atopic or allergic asthma, eosinophilic asthma, Neutrophilic asthma, paucigranulocytic or non-inflammatory asthma, early-onset asthma, late-onset asthma These include type II asthma, type II high asthma, type II low asthma, and type I / Th17 asthma.

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

[0083] In a first aspect, the present invention provides (i) antagonists of IL-5:IL-5R, and (ii) antagonists of IL-4:IL-4R. and / or an antagonist of IL-13:IL-13R. In one embodiment, the combination comprises an IL-5:IL-5R antagonist and an IL-4:IL-4R antagonist. In a further embodiment, the combination comprises an antagonist of IL-5:IL-5R and and an antagonist of IL-13:IL-13R. In an even further embodiment, the combination comprises an IL-5 : IL-5R antagonist, IL-4: IL-4R antagonist, and IL-13: IL-13R antagonist In one embodiment, the combination includes a combination of IL-5 and IL-4 inhibitors that inhibit signaling through IL-5 and IL-4. In one embodiment, the combination inhibits signaling through IL-5 and IL-13. In a preferred embodiment, the combination inhibits signal transduction via IL-5, IL-4 and IL-13. hinders the progress of

[0084] Without being bound by theory, the combinations of the present invention block type 2 cytokine activity. Due to the combined effects of these compounds, they are believed to be particularly effective in the treatment of chronic airway diseases, especially asthma. IL-4, IL-13 and IL-5 are type 2 cytokines, and the combination of the present invention inhibits all three of these cytokines. The "type 2 immune response" is a type 2 immune response that inhibits all cytokine-mediated signaling. H2) Refers to an immune response that is primarily regulated by a subpopulation of CD4+ T cells known as CD4+ T cells. However, the airway type 2 immune response also involves eosinophils, mast cells, basophils, and T H 2-cell, group 2 nature It is also mediated by lymphocytes (ILC2) and IgE-producing B cells.

[0085] IL-4, IL-13, and IL-5 mediate IgE production by B cells, eosinophil activation and recruitment, and mucus production. Type 2 cytokines activate airway epithelial cells and induce the production of effector cells (mammary Chemoattraction of lymphocytes (e.g., eosinophils, and basophils) and the epithelial and subepithelial matrix triggering a cascade of downstream events, including chronic airway remodeling, e.g., asthma In disease, aberrant signaling through IL-4, IL-13, and IL-5 leads to airway eosinophilia, Remodeling and bronchial hyperresponsiveness may occur (Lambrecht and Hammad, 201 5) Ibid.) Associated remodeling changes include smooth muscle cell changes (hyperplasia and hypertrophy). These may include changes in mucous cells (goblet cell metaplasia), changes in mucous cells (goblet cell metaplasia), and ductal remodeling. In addition, inflammatory and pathological changes in these airways may contribute to the response of subjects to inhaled exacerbating agents. This increases the likelihood of a worse reaction.

[0086] The present inventors have demonstrated that blocking signal transduction via IL-4, IL-13, and IL-5 results in surprising This effect was observed when only IL-4 and IL-13 were blocked. This was confirmed when compared with treatment with IL-4Rα monotherapy or IL-5 monotherapy, which blocks only IL-5. The data provided herein demonstrate that antagonists targeting the IL-5:IL-5R signaling axis and antagonists targeting the IL-4:IL-4R and / or IL-13:IL-13R signaling axes. This shows that the combination therapy of the present invention, which includes In an in vivo model of the disease, decreased mucin production levels (an indicator of goblet cell metaplasia) and decreased Importantly, the combination therapy of the present invention inhibits type 2 cytokines. These compounds contain antagonists that target steroids and completely prevent bronchial hyperresponsiveness in an in vivo model. This shows that...

[0087] The combinations of the present invention target (i) IL-5:IL-5R; and (ii) IL-4:IL-4R and / or IL-13:IL-13R. The term "antagonist" as used herein includes antagonists that target: It is used broadly to mean any agent or molecule that can inhibit the function of its target. For example, IL-5:IL-5R antagonists inhibit IL-5-mediated signaling, leading to the production of IL-5 cytokines. Similarly, IL-4:IL-4R antagonists will inhibit the function of the IL- It inhibits signal transduction via IL-4, thereby inhibiting the function of the IL-4 cytokine-IL-4 receptor complex. Similarly, IL-13:IL-13R antagonists may block IL-13-mediated signaling. This will inhibit the function of the IL-13 cytokine-IL-13 receptor complex.

[0088] Antagonists of the type 2 cytokine-receptor pair described herein, i.e., IL-5:IL-5R, Antagonists of IL-4:IL-4R and IL-13:IL-13R typically inhibit cytokine-receptor complexes. For example, antagonists of IL-5:IL-5R It interacts with IL-5 and inhibits signal transduction through the IL-5:IL-5R signaling pathway. Alternatively, IL-5:IL-5R antagonists interact with the receptor subunit IL-5Rα. They may also interact to inhibit signaling through the IL-5:IL-5R signaling pathway. In a preferred embodiment, the antagonist of IL-5:IL-5R is an antagonist of IL-5. Similarly, IL-4:IL-4R antagonists and IL-13:IL-13R antagonists are also shown to be effective against IL-4 and IL-13R, respectively. It interacts with the cytokines IL-4 and IL-13 or with receptor subunits, which Each may inhibit IL-4 and / or IL-13 signaling.

[0089] Particularly preferred is a combination of the present invention which is 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 that targeting IL-4Rα is preferred is that this receptor subunit The nits form part of the type I receptor complex for IL-4 (together with γc), as well as the IL-13 receptor. It forms part of a complex (together with IL-13Rα1) and therefore acts as an antagonist of IL-4Rα. The inhibitor can simultaneously act as an antagonist of IL-4:IL-4R and IL-13:IL-13R. , thereby inhibiting signal transduction mediated by both IL-4 and IL-13. IL-5: an antagonist of IL-5R, preferably an antagonist of IL-5, and an antagonist of IL-4Rα Combinations containing antagonists can inhibit signaling through IL-5, IL-4, and IL-13. The antagonists of the combinations described herein preferably bind to their respective human targets. Combine with.

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

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

[0092] In one embodiment, the combination comprises an antibody that binds to IL-4 as an IL-4:IL-4R antagonist. In one embodiment, the combination comprises an IL-13:IL-13R antagonist, an IL-13R antagonist, and an IL-13R antagonist. In a preferred embodiment, the combination comprises an antibody molecule that binds to IL-4Rα. As previously described, this antagonist is an IL-4Rα subunit inhibitor. Because the IL-4 and IL-13 receptor complexes share the same receptor, IL-4:IL-4R and IL-13:IL-13R The compounds that can be incorporated into the combinations described herein act as antagonists of both. Antibody molecules that target the IL-4 and IL-13 signaling pathways can be prepared using any suitable antibody molecule known in the art. These antibodies include IL-4, IL-13, IL-4Rα, and IL-13Rα1 antibodies. Dupilumab (Dupixent, Inc.), a fully humanized monoclonal antibody against Similarly, Sheridan C. (2018) Nat. Biotechnol. 36(1): 3- See 5.

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

[0094] The antibodies in the combinations described herein are intended for human therapeutic use and therefore typically contain IgA, IgD, IgE, IgG, IgM, often IgG, which in this case has four subclasses: IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19 ... It can belong to either IgG2a and IgG2b, IgG3 or IgG4. The antibody molecules of the combination are IgG antibodies, optionally IgG1 antibodies. Monoclonal, polyclonal, or other antibodies may be used if they exhibit the appropriate immunospecificity for the target. Monoclonal antibodies may be highly specific antibodies (e.g., bispecific antibodies). These are preferred because they are highly potent and directed against a single antigenic site.

[0095] The antigen-binding fragments of the combinations described herein are typically portions of a full-length antibody, typically its Examples of antibody fragments include the Fab fragment, the antigen-binding domain, and the variable domain. fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab' fragments and Fv fragments, linear antibodies, single chain antibodies These include antibodies, single-chain variable fragments (scFv), and multispecific antibodies formed from antibody fragments ( Holliger and Hudson, (2005) Nature Biology, incorporated herein by reference. (See Otechnol. 23:1126-36).

[0096] The antibody molecules of the combinations described herein may exhibit high human homology. These antibody molecules exhibit a sufficiently high percent sequence identity to the human germline sequence. In addition, it may include antibodies that contain the VH and VL domains of a native non-human antibody. In embodiments, the antibody molecule is a humanized or germline variant of a non-human antibody. It's Ant.

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

[0098] For example, the antibody molecules can be produced by a method comprising immunizing a camelid with a target of interest. The camelid may be selected from an immune library obtained by the above method. Immunization with a polypeptide fragment thereof, or with the protein or polypeptide thereof Immunization with mRNA or cDNA molecules expressing the fragments may also be used to raise antibodies within the camelid species. The method for producing and selecting antibodies against preferred targets from camelid immune libraries is e.g. For example, see International Patent Application No. WO2010 / 001251, which is incorporated herein by reference. It is being done.

[0099] In one embodiment, the antibody molecule comprises a VH domain or a VL domain of a Camelidae species. in that it comprises at least one hypervariable (HV) loop or complementarity determining region obtained from In particular, the antibody molecule may be derived from an outbred camelid, e.g., a llama, e.g. For example, VH and / or VL domains obtained by active immunization with IL-4Rα and IL-5, or may also include their CDRs.

[0100] In this context, the term "derived from" means that the HV or CDR of the antibody molecule is derived from an immunoglobulin of the Camelidae family. The amino acid sequence originally encoded by the globin gene (or its minor variants) However, this is not the case when preparing antibody molecules. This does not necessarily imply any special relationship with respect to the fabrication process used to .

[0101] Antibody molecules from camelids are particularly well known for their ability to bind to llamas, dromedaries, alpacas, and vikings. The animal may be from any camelid species, including the guanaco, camel or camel.

[0102] Antibody molecules containing VH and VL domains, or their CDRs, from camelids are usually recombinantly produced. The expressed polypeptide may be a chimeric polypeptide. A "peptide" is created by the juxtaposition of two or more peptide fragments that are not otherwise contiguous. This definition refers to artificial (non-naturally occurring) polypeptides that are produced by, for example, camelids and produced by juxtaposition of peptide fragments encoded by two or more species, such as humans and The "seed" chimeric polypeptide is

[0103] In one embodiment, the complete VH domain and / or the complete VL domain is from the Camelidae family The camelid-derived VH domain and / or the camelid-derived VL domain can be obtained from any of the following species: The amino acid may then be subjected to protein engineering, resulting in one or more amino acid substitutions, insertions or deletions. These engineered changes preferably include Such changes include amino acid substitutions relative to the VH sequences encoded by camelids. or one or more amino acid residues of the VL domain are homologous to a human-encoded VH or VL domain. "Humanized" or "germlined" includes "humanized" or "germlined" variants in which the amino acid sequence is replaced with the same residue from the original amino acid sequence.

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

[0105] In another embodiment, VH and VL domains from camelids (or engineered barrier domains thereof) are constant domains) and one or more constant domains from a non-camelid antibody, e.g., a human

[0013] Providing "chimeric" antibody molecules containing common domains (or engineered variants thereof) In such embodiments, for example (prior to the introduction of designed amino acid sequence variations): Both VH and VL are derived from llama (Lama glama), or both VH and VL are derived from alpaca (Lama pacos). As previously mentioned, it is preferred that both VH and VL domains are obtained from the same species of camelid. In such an embodiment, both VH and VL domains are expressed in a single animal, particularly a mammal of interest. The antigens may be derived from a single animal that has been actively immunized with the antigen.

[0106] Alternatives may be designed to alter the primary amino acid sequence of the Camelidae VH and / or VL domains. When the hypervariable loops or CDRs from individual camelids, or a combination thereof, are used as the The camel VH / VL domains can be isolated and integrated into other (i.e., non-camelid) frameworks, e.g., human It can also be grafted onto a VH / VL framework by CDR grafting.

[0107] In a non-limiting embodiment, the antibody molecules of the combination comprise a CH1 domain (from each of the heavy and light chains). The amino acid sequence may be fully human or may be a recombinant protein. For antibody molecules intended for human therapeutic use, the antibody will usually be fully human. The entire constant region, 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 The CL domain and the CH4 domain (and CH5 domain, if present) have the following amino acid sequences: The antibody may be fully or substantially human with respect to the CH1 domain, hinge region, CH2 domain , the CH3 domain and / or the CL domain (and / or the CH4 domain, if present) A human antibody, preferably a human IgG antibody, more preferably an IgG1, IgG2, IgG3 or The antibody may be derived from a human IgG1 antibody or an IgG4 antibody.

[0108] Advantageously, the CH1 domain, the hinge region, the CH2 domain, the CH3 domain and the CL domain (and and, if present, the CH4 domain) all have a completely or substantially human amino acid sequence. In the context of the constant region of a humanized or chimeric antibody or antibody fragment, the term "humanized" may be used. "Qualitatively human" refers to a polypeptide having at least 90%, or at least 92%, or at least It refers to 95%, or at least 97%, or at least 99% amino acid sequence identity. The term "human amino acid sequence" as used herein refers to a germline, rearranged, and somatically mutated amino acid sequence. The term "human immunoglobulin" refers to the amino acid sequences encoded by human immunoglobulin genes, including genes encoding the human immunoglobulins. The present invention also relates to human hinge regions, except in embodiments where the presence of a "fully human" hinge region is expressly required. of a "human" sequence, altered by one or more amino acid additions, deletions or substitutions with respect to the sequence. Polypeptides containing constant domains are also contemplated.

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

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

[0111] In a preferred embodiment, one or more amino acid residues in the Fc region are replaced with a different amino acid. It is also possible to increase FcRn binding by increasing FcRn binding, thereby improving the pharmacokinetics of the antibody. Several Fc substitutions have been reported that improve inflammatory responses. Such substitutions are described, for example, by 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. Biotech Nol. 23(10):1283-88, the contents of which are incorporated herein in their entirety. It can be enjoyed.

[0112] In a preferred embodiment, one or more of the antibody molecules of the combinations described herein contain an amino acid substitution A modified human IgG Fc domain comprising or consisting of H433K and N434F. In a further preferred embodiment, the Fc domain numbering is according to EU numbering. One or more antibody molecules of the combinations described herein may contain the amino acid substitutions M252Y, S254T, T256E, H433K, and a modified human IgG Fc domain comprising or consisting of N434F, The Fc domain numbering follows EU numbering.

[0113] In one embodiment, the antibody molecules of the combination, e.g., the IL-4Rα and / or IL-5 antibody molecules, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, relative to the corresponding wild-type IgG sequence A modified human IgG Fc consisting of 9 or less, 10 or less, 12 or less, 15 or less, or 20 or less substitutions. Includes the domain.

[0114] These antibody molecules can also be modified to bind chemotherapeutic drugs, toxins (e.g., bacterial, fungal, plant enzymatically active toxins of human or animal origin, or fragments thereof, or radioactive isotopes Immunoconjugates comprising antibodies conjugated to cytotoxic agents such as ribozymes (i.e., radioconjugates) are used. The Fc region may also be formed as described by Chan and Carter, which is incorporated herein by reference. (2010) Nature Reviews: Immunology 10:301-316, the half-life extension It may be designed for

[0115] In particular embodiments, the Fc region can be engineered to lack effector function. In one embodiment, the antibody molecule of the present invention comprises a naturally occurring IgG antibody with reduced effector function. The Fc region of IgG4 can be further modified. modified, for example, by the introduction of modifications that minimize arm exchange between IgG4 molecules in vivo. The Fc region from IgG4 may be modified to include the S228P substitution, which may increase therapeutic utility. So that's fine.

[0116] In some embodiments, the antibody molecules of the combination may be modified with respect to glycosylation. For example, aglycoslated antibodies can be made (i.e., deglycosylated antibodies). Glycosylation can be altered, for example, to increase the affinity of the antibody for the target antigen. Suitable glycosylation modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions may be made to modify one or more variable region framework glycoproteins. This can result in the elimination of a cosylation site, thereby eliminating glycosylation at that site. Such deglycosylation may increase the affinity of the antibody for the antigen.

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

[0118] Antigen-bound antibodies are taken up by cells and transported to the endosomal-lysosomal degradation pathway Antibodies that may be separated from their antigens in early endosomes are recycled back to the cell surface. Antibodies that bind with high affinity to their antigens in the endosomal compartment are usually If the antibody molecule has pH-dependent antigen-binding activity, it will be transported to the lysosome for resolution. At early endosomal pH, it has a lower binding affinity for its antigen compared to plasma pH. It has previously been shown that when the antibody is bound to the cell surface, it is recycled to the cell surface more efficiently. This extends the plasma half-life of the antibody and allows the same antibody to bind to multiple antigens. Therefore, the antibody molecules of the combinations described herein, e.g., IL-4Rα and / or IL-5 antibody molecules, may exhibit pH-dependent It is advantageous to have the antibody exhibit dependent antigen binding.

[0119] Methods for engineering pH-dependent antigen binding activity in antibody molecules are described, for example, in the literature, which is incorporated herein by reference. The invention is described in European Patent Application No. EP2275443, which is incorporated herein by reference. Methods for designing pH-dependent antigen binding are also described in International Publication No. WO 2005 / 023990, which is incorporated herein by reference. The antibody molecules described herein are described in European Patent Application No. WO2018 / 206748. According to the method described in application no. EP2275443 or international patent application no. WO2018 / 206748, they are pH The antibody may be modified to exhibit dependent antigen binding.

[0120] For the pH-dependent antibody molecules of the combinations described herein, the antigen binding activity is determined by the antigen binding activity at plasma pH. The activity is lower at endosomal pH compared to the normal pH, which is usually acidic. However, plasma pH is typically neutral. Therefore, the antibody molecules of the present combination, e.g., those described herein, The IL-4Rα and / or IL-5 antibody molecules have antigen-binding activity at neutral pH. It may also exhibit pH-dependent antigen binding, such that the activity is lower at acidic pH compared to endosomal activity. Acidic pH or "acidic pH" is about pH 4.0 to about pH 6.5, preferably about pH 5.5 to about pH 6.5, preferably about pH 6.5 to about pH 4.0. The pH may be about pH 5.5 to about pH 6.0, preferably pH 5.5, pH 5.6, pH 5.7 or pH 5.8. "Neutral pH" refers to a range of about pH 6.9 to about pH 8.0, preferably about pH 7.0 to about pH 8.0, more preferably about pH 7.0 to about pH 8.0. The pH may be about pH 7.4, preferably pH 7.0 or pH 7.4.

[0121] In one embodiment, the antibody molecule, e.g., the IL-4Rα antibody molecule and / or the IL-5 antibody molecule, is pH-dependent binding, such that the antigen-binding activity at pH 5.8 is lower than that at pH 7.4 This pH-dependent antibody molecule, for example, an IL-4Rα antibody molecule and / or an IL-5 antibody molecule, exhibits the following pH-dependent activity: The dissociation constant (KD) of the antibody-antigen interaction at neutral pH or pH 5.8 is In some embodiments, the antibody may exhibit a higher than normal dissociation constant (KD) of the interaction. The molecule, e.g., the IL-4Rα antibody molecule and / or the IL-5 antibody molecule, has a KD for the antigen at pH 5.8 and a KD for the antigen at pH 5.8. The ratio of KD to antigen at pH 7.4 (KD(pH5.8) / KD(pH7.4)) is 2 or more, 4 or more, 6 or more, 8 or more, or 10 or more. It exhibits pH-dependent binding such that its binding is 12 or greater.

[0122] The pH-dependent antigen-binding activity of the antibody molecule impairs its antigen-binding ability at acidic pH and / or was designed by modifying the antibody molecule to increase its antigen-binding ability at neutral pH. For example, the antibody molecule may 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. The site of such histidine mutation (substitution or insertion) is not particularly limited. , the antigen binding at endosomal pH (e.g., pH 5.8) compared to before the mutation or insertion. Any site is acceptable as long as the combined activity is lower than that at plasma pH (e.g., pH 7.4). .

[0123] In one embodiment, the antibody molecule, e.g., the IL-4Rα antibody molecule and / or the IL-5 antibody molecule, The variable domains may be engineered to exhibit pH-dependent antigen binding by introducing one or more substitutions into the variable domains. In a preferred embodiment, the antibody molecule, e.g., an IL-4Rα antibody molecule and / or an IL-5 antibody, The molecule exhibits pH-dependent antigen binding by introducing one or more substitutions into the CDRs of the antibody molecule. The substitution may be designed to replace one or more His residues in the variable domain, preferably the heavy The non-terminal amino acid sequence may be introduced into one or more sites in the high and / or low chain CDRs to confer pH-dependent antigen binding. Histidine substitutions may also be incorporated into the variable domains, particularly the CDRs, of the pH-dependent antibodies described herein. The antibody molecule, e.g., an IL-4Rα antibody molecule and / or an IL-5 antibody molecule, may also be incorporated into the may be designed according to the methods described in International Patent Application No. WO2018 / 206748.

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

[0125] In particularly preferred embodiments, the pH-dependent antibodies of the combinations described herein, e.g., IL-4R The IL-1α and / or IL-5 antibody molecules contain an Fc domain with increased binding affinity to the fetal receptor FcRn. Substitutions that may increase the binding affinity of the Fc domain to FcRn are also included in the present invention. Such substitutions are described elsewhere in the specification and may be incorporated into the pH-dependent antibody molecules of the present combination. It can be incorporated.

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

[0127] Exemplary IL-4Rα and IL-5 Antibodies As noted above, in a preferred embodiment, the combination comprises an antibody molecule that binds IL-5 and an antibody molecule that binds IL-4R. In such an embodiment, the combination comprises an antibody molecule that binds to all three type 2 cytokines. It can inhibit signaling mediated by the cytokines IL-5, IL-4, and IL-13.

[0128] Antibody molecules that bind to human IL-4Rα and that can be incorporated into the combinations described herein include the variable heavy chain CDRs 3 (HCDR3), variable heavy chain CDR2 (HCDR2) and variable heavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3), variable and antibody molecules comprising a combination of a light chain CDR2 (LCDR2) and a variable light chain CDR1 (LCDR1), the combination being as follows: Note: (i) HCDR3 comprising or consisting of SEQ ID NO: 3; HCDR2 consisting of SEQ ID NO: 1 or HCDR1 consisting of SEQ ID NO: 12 LCDR3 comprising or consisting of SEQ ID NO: 11; LCDR4 comprising or consisting of SEQ ID NO: 12 2; LCDR1 comprising or consisting of SEQ ID NO: 10; (ii) HCDR3 comprising or consisting of SEQ ID NO: 6; HCDR2 comprising or consisting of SEQ ID NO: 4; HCDR1 comprising SEQ ID NO: 15; LCDR3 comprising or consisting of SEQ ID NO: 14; LCDR4 comprising or consisting of SEQ ID NO: 15 2; LCDR1 comprising or consisting of SEQ ID NO: 13; (iii) HCDR3 comprising or consisting of SEQ ID NO: 9; HCDR2 comprising or consisting of SEQ ID NO: 7; HCDR1 comprising or consisting of SEQ ID NO: 18 LCDR3 comprising or consisting of SEQ ID NO: 17; DR2; LCDR1 comprising or consisting of SEQ ID NO: 16; (iv) HCDR3 comprising or consisting of SEQ ID NO: 91; HCDR2 comprising or consisting of SEQ ID NO: 7; HCDR1 comprising or consisting of SEQ ID NO: 18 LCDR3 comprising or consisting of SEQ ID NO: 17; DR2; LCDR1 comprising or consisting of SEQ ID NO: 16; (v) HCDR3 comprising or consisting of SEQ ID NO: 92; HCDR2 consisting of SEQ ID NO: 7 or HCDR1 consisting of SEQ ID NO: 97 LCDR3 comprising or consisting of SEQ ID NO: 17; LCDR4 comprising or consisting of SEQ ID NO: 18 2; LCDR1 comprising or consisting of SEQ ID NO: 16; (vi) HCDR3 comprising or consisting of SEQ ID NO: 93; HCDR2 comprising or consisting of SEQ ID NO: 7; HCDR1 comprising or consisting of SEQ ID NO: 98 LCDR3 comprising or consisting of SEQ ID NO: 96; DR2; LCDR1 comprising or consisting of SEQ ID NO: 16; (vii) HCDR3 comprising or consisting of SEQ ID NO: 94; HCDR2 comprising or consisting of SEQ ID NO: 7; HCDR1 comprising or consisting of SEQ ID NO: 98 LCDR3 comprising or consisting of SEQ ID NO: 17; DR2; LCDR1 comprising or consisting of SEQ ID NO: 16; and (viii) HCDR3 comprising or consisting of SEQ ID NO: 95; HCDR2 comprising or consisting of SEQ ID NO: 7; HCDR1 comprising or consisting of SEQ ID NO: 98 LCDR3 comprising or consisting of SEQ ID NO: 17; LCDR2; LCDR1 comprising or consisting of SEQ ID NO:16.

[0129] Antibody molecules that bind to IL-4Rα and that can be incorporated into the combinations described herein also include variable heavy chain CDRs 3 (HCDR3), variable heavy chain CDR2 (HCDR2) and variable heavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3), variable and antibody molecules comprising a combination of a light chain CDR2 (LCDR2) and a variable light chain CDR1 (LCDR1), the combination being as follows: Note: (i) HCDR3 comprising or consisting of SEQ ID NO: 27; HCDR2 comprising or consisting of SEQ ID NO: 25; HCDR1 comprising or consisting of SEQ ID NO: 36 LCDR3 comprising or consisting of SEQ ID NO: 35; LCDR4 comprising or consisting of SEQ ID NO: 36; DR2; LCDR1 comprising or consisting of SEQ ID NO: 34; (ii) HCDR3 comprising or consisting of SEQ ID NO: 30; HCDR2 comprising or consisting of SEQ ID NO: 28; HCDR1 comprising or consisting of SEQ ID NO: 39 LCDR3 comprising or consisting of SEQ ID NO: 38; DR2; LCDR1 comprising or consisting of SEQ ID NO: 37; and (iii) HCDR3 comprising or consisting of SEQ ID NO: 33; HCDR2 comprising or consisting of SEQ ID NO: 31; HCDR1 comprising or consisting of SEQ ID NO: 42 LCDR3 comprising or consisting of SEQ ID NO: 41; LCDR2; LCDR1 comprising or consisting of SEQ ID NO:40.

[0130] In one embodiment, the antibody molecule that binds to human IL-4Rα comprises a variable heavy domain (VH) and and a variable light domain (VL), and the VL domain is as follows: (i) an amino acid sequence comprising or consisting of SEQ ID NO: 19, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence with 9% identity to SEQ ID NO: 20; or , or an amino acid sequence consisting of at least 70%, at least 80%, or at least Amino acid sequences that share 90%, at least 95%, at least 98%, or at least 99% identity with VL domain containing; (ii) an amino acid sequence comprising or consisting of SEQ ID NO: 21, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence with 9% identity to SEQ ID NO: 22; or , or an amino acid sequence consisting of at least 70%, at least 80%, or at least Amino acid sequences that share 90%, at least 95%, at least 98%, or at least 99% identity with VL domain containing; (iii) an amino acid sequence comprising or consisting of SEQ ID NO: 23, or at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least a VH domain comprising an amino acid sequence having at least 99% identity with SEQ ID NO: 24, or an amino acid sequence consisting of, or at least 70%, at least 80%, or at least Amino acids with at least 90%, at least 95%, at least 98%, at least 99% identity VL domain containing sequence; (iv) an amino acid sequence comprising or consisting of SEQ ID NO: 99, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence having 9% identity to SEQ ID NO: 100, or is an amino acid sequence consisting of, or at least 70%, at least 80%, or at least amino acid sequences having at least 90%, at least 95%, at least 98%, or at least 99% identity VL domain containing columns; (v) an amino acid sequence comprising or consisting of SEQ ID NO: 101, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence having 9% identity to SEQ ID NO: 102, or is an amino acid sequence consisting of, or at least 70%, at least 80%, or at least amino acid sequences having at least 90%, at least 95%, at least 98%, or at least 99% identity VL domain containing columns; (vi) an amino acid sequence comprising or consisting of SEQ ID NO: 103, or at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least a VH domain comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 104, or an amino acid sequence consisting of, or at least 70%, at least 80%, or at least Amino acids with at least 90%, at least 95%, at least 98%, at least 99% identity VL domain containing sequence; (vii) an amino acid sequence comprising or consisting of SEQ ID NO: 105, or at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least a VH domain comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 106, or an amino acid sequence consisting of, or at least 70%, at least 80%, or at least Amino acids with at least 90%, at least 95%, at least 98%, at least 99% identity a VL domain comprising the sequence (viii) an amino acid sequence comprising or consisting of SEQ ID NO: 107, or at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least a VH domain comprising an amino acid sequence having 99% identity with SEQ ID NO: 108; or an amino acid sequence consisting of it, or at least 70%, at least 80%, Amino acids with at least 90%, at least 95%, at least 98%, at least 99% identity a VL domain comprising a VL sequence.

[0131] In one embodiment, the antibody molecule that binds to IL-4Rα comprises a variable heavy domain (VH) and a variable The antibody molecule is selected from antibody molecules comprising or consisting of a variable light chain domain (VL), and VL domains are as follows: (i) an amino acid sequence comprising or consisting of SEQ ID NO: 43, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence which is 9% or less of the VH domain and which comprises or is SEQ ID NO: 44; an amino acid sequence consisting of, or at least 70%, at least 80%, at least 90%, VL comprising an amino acid sequence having at least 95%, at least 98%, or at least 99% identity domain; (ii) an amino acid sequence comprising or consisting of SEQ ID NO: 45, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 9% of a VH domain comprising an amino acid sequence of SEQ ID NO: 46, an amino acid sequence consisting of, or at least 70%, at least 80%, at least 90%, VL comprising an amino acid sequence having at least 95%, at least 98%, or at least 99% identity domain; and (iii) an amino acid sequence comprising or consisting of SEQ ID NO: 47, or at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least a VH domain comprising an amino acid sequence which is at least 99% identical to SEQ ID NO: 48; or an amino acid sequence consisting of at least 70%, at least 80%, at least 90% thereof %, at least 95%, at least 98%, or at least 99% identity VL domain.

[0132] Antibody molecules that bind human IL-5 and that can be incorporated into the combinations described herein include those having a variable heavy chain CDR3 (H CDR3), variable heavy chain CDR2 (HCDR2) and variable heavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3), variable light chain and antibody molecules comprising a combination of a variable light chain CDR2 (LCDR2) and a variable light chain CDR1 (LCDR1), wherein the combination is: (i) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 54 LCDR3 comprising or consisting of SEQ ID NO: 53; LCDR4 comprising or consisting of SEQ ID NO: 54 DR2; LCDR1 comprising or consisting of SEQ ID NO: 52; (ii) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 57 LCDR3 comprising or consisting of SEQ ID NO: 56; DR2; LCDR1 comprising or consisting of SEQ ID NO: 55; (iii) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 59 LCDR3 comprising or consisting of SEQ ID NO: 56; LCDR2; LCDR1 comprising or consisting of SEQ ID NO: 58; (iv) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 61 LCDR3 comprising or consisting of SEQ ID NO: 60; DR2; LCDR1 comprising or consisting of SEQ ID NO: 58; and (v) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 62 LCDR3 comprising or consisting of SEQ ID NO: 56; DR2; LCDR1 comprising or consisting of SEQ ID NO:58.

[0133] Antibody molecules that bind IL-5 and can be incorporated into the combinations described herein include those having variable heavy chain CDR3 (HCDR3 ), variable heavy chain CDR2 (HCDR2) and variable heavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3), variable light chain CDR2 and a variable light chain CDR1 (LCDR2) and a variable light chain CDR1 (LCDR1) in combination as follows: (i) HCDR3 comprising or consisting of SEQ ID NO: 72; HCDR2 comprising or consisting of SEQ ID NO: 70; HCDR1 comprising or consisting of SEQ ID NO: 74 LCDR3 comprising or consisting of SEQ ID NO: 38; DR2; LCDR1 comprising or consisting of SEQ ID NO: 73; and (ii) HCDR3 comprising or consisting of SEQ ID NO: 72; HCDR2 comprising or consisting of SEQ ID NO: 70; HCDR1 comprising or consisting of SEQ ID NO: 75 LCDR3 comprising or consisting of SEQ ID NO: 38; DR2; LCDR1 comprising or consisting of SEQ ID NO: 73.

[0134] In one embodiment, an antibody molecule that binds human IL-5 comprises a variable heavy domain (VH) and a variable light domain (VL). The antibody molecule is selected from antibody molecules comprising or consisting of a VH and VL chain domains. The domain is: (i) an amino acid sequence comprising or consisting of SEQ ID NO: 63, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence with 9% identity to SEQ ID NO: 64; or , or an amino acid sequence consisting of at least 70%, at least 80%, or at least Amino acid sequences that share 90%, at least 95%, at least 98%, or at least 99% identity with VL domain containing; (ii) an amino acid sequence comprising or consisting of SEQ ID NO: 63, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence with 9% identity to SEQ ID NO: 65; or , or an amino acid sequence consisting of at least 70%, at least 80%, or at least Amino acid sequences that share 90%, at least 95%, at least 98%, or at least 99% identity with VL domain containing; (iii) an amino acid sequence comprising or consisting of SEQ ID NO: 63, or at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least a VH domain comprising an amino acid sequence having at least 99% identity with SEQ ID NO: 66, or an amino acid sequence consisting of, or at least 70%, at least 80%, or at least Amino acids with at least 90%, at least 95%, at least 98%, at least 99% identity VL domain containing sequence; (iv) an amino acid sequence comprising or consisting of SEQ ID NO: 63, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence with 9% identity to SEQ ID NO: 67; or , or an amino acid sequence consisting of at least 70%, at least 80%, or at least Amino acid sequences that share 90%, at least 95%, at least 98%, or at least 99% identity with VL domain containing; (v) an amino acid sequence comprising or consisting of SEQ ID NO: 63, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence with 9% identity to SEQ ID NO: 68; or , or an amino acid sequence consisting of at least 70%, at least 80%, or at least Amino acid sequences that share 90%, at least 95%, at least 98%, or at least 99% identity with a VL domain comprising: (vi) an amino acid sequence comprising or consisting of SEQ ID NO: 63, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence with 9% identity to SEQ ID NO: 69; or , or an amino acid sequence consisting of at least 70%, at least 80%, or at least Amino acid sequences that share 90%, at least 95%, at least 98%, or at least 99% identity with a VL domain comprising:

[0135] In one embodiment, an antibody molecule that binds IL-5 comprises a variable heavy domain (VH) and a variable light domain (VL). The antibody molecule is selected from antibody molecules comprising or consisting of a VH and VL domain. The inn is as follows: (i) an amino acid sequence comprising or consisting of SEQ ID NO: 76, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence with 9% identity to SEQ ID NO: 77; or , or an amino acid sequence consisting of at least 70%, at least 80%, or at least Amino acid sequences that share 90%, at least 95%, at least 98%, or at least 99% identity with VL domain containing; (ii) an amino acid sequence comprising or consisting of SEQ ID NO: 76, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence with 9% identity to SEQ ID NO: 78; or , or an amino acid sequence consisting of at least 70%, at least 80%, or at least Amino acid sequences that share 90%, at least 95%, at least 98%, or at least 99% identity with a VL domain comprising: (iii) an amino acid sequence comprising or consisting of SEQ ID NO: 76, or at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least a VH domain comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 79, or an amino acid sequence consisting of, or at least 70%, at least 80%, or at least Amino acids with at least 90%, at least 95%, at least 98%, at least 99% identity A VL domain comprising the sequence

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

[0137] A particular antibody molecule has a VH domain or heavy chain that is defined in relation to a particular amino acid sequence. , which also include VL domain or light chain combinations defined in relation to specific amino acid sequences. For each particular VH / VL or heavy chain / light chain combination listed, This definition (unless otherwise stated) defines a VH / heavy chain sequence that has at least 70% identical amino acid sequence to the listed VH / heavy chain sequence. , at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% VH domain / heavy chain having identity with the described VL / light chain amino acid sequence at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least antibody molecules formed by a combination of VL domains / light chains with at least 99% identity It will be recognized that it is.

[0138] Each domain is defined by its % sequence identity with the listed domain / chain amino acid sequence. The amino acid sequence variations in the framework regions or other regions outside the CDR regions are not included in the chain. VH / VL domains or heavy / light chain amino acid sequences shown. The CDR sequences may be identical to those of the first or second amino acid sequence.

[0139] In one embodiment, the CDR sequences are defined as above or are specific VH / VL domains. IL-4Rα and / or IL-4Rα are defined as having a specific percentage of identity with the main amino acid sequence. The L-5 antibody molecule may be any antibody or polypeptide from which the CDR, VH and / or VL sequences are derived. The antigen-binding fragment of the present invention is a humanized, germlined, or affinity variant of the antigen-binding fragment of the present invention.

[0140] In a preferred embodiment, exemplary IL-4Rα and IL-5 antibody molecules having the above CDR sequences are For example, antibodies or antigen-binding fragments thereof that exhibit high human homology and from which the CDR sequences are derived. Humanized or germline variants.

[0141] In a non-limiting embodiment, an exemplary IgA antibody having the CDR, VH and / or VL sequences described herein is The L-4Rα and IL-5 antibody molecules contain CH1 domains and / or CL domains (in the heavy and light chains, respectively). (derived from) and their amino acid sequences may be fully or substantially human. The antibody molecule intended for use typically contains the complete constant region of the antibody, or at least a portion thereof. , and have a complete or substantially human amino acid sequence. Thus, the CH1 domain, hinge region, C One or more of the H2 domain, CH3 domain, and CL domain (and CH4 domain, if present) Any combination may be fully or substantially human with respect to its amino acid sequence.

[0142] Advantageously, the CH1 domain, the hinge region, the CH2 domain, the CH3 domain and the CL domain (and and, if present, the CH4 domain) may all have a completely or substantially human amino acid sequence. In the context of the constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially "Human" refers to a polypeptide that is at least 90%, or at least 92%, or at least 95%, or contains a human constant region. refers to at least 97%, or at least 99% amino acid sequence identity. "Human amino acid sequence" refers to a human amino acid sequence, including germline, rearranged, and somatically mutated genes. The present invention also refers to the amino acid sequence encoded by a human immunoglobulin gene. Except for embodiments in which the presence of a hinge region of " is clearly required, one or more amino acids may be added to a human sequence. Polypeptides containing constant domains of "human" sequence, altered by amino acid additions, deletions or substitutions. Any of the exemplary Fc region modifications described herein may be modified in any of the CDRs and / or VH / VL sequences described above. The domain sequences may be incorporated into IL-4Rα and / or IL-5 antibodies. In embodiments, IL-4Rα and / or IL-5 antibodies having the CDR and / or VH / VL domain sequences described above. is a modified human IgG1A comprising or consisting of the amino acid substitutions H433K and N434F. IgG Fc domain, wherein the Fc domain numbering is according to EU numbering. The IL-4Rα and / or IL-5 antibody having the above CDR and / or VH / VL domain sequences is comprising or consisting of the amino acid substitutions M252Y, S254T, T256E, H433K and N434F It contains a modified human IgG Fc domain.

[0143] In a non-limiting embodiment, an IgA antibody having the exemplary CDR, VH and / or VL sequences described herein is The L-4Rα and IL-5 antibody molecules may be modified as described above to exhibit pH-dependent antigen binding. For example, IL-4Rα and IL-5 antibodies having exemplary CDR, VH and / or VL sequences described herein. The molecule is modified so that its antigen-binding activity at pH 5.8 is reduced compared to that at pH 7.4. To engineer antibodies to confer pH-dependent antigen binding, The method of any of the exemplary IL-4Rs described herein having the CDR, VH and / or VL sequences. For example, the variable domains and / or CDR regions may be It may be modified with histidine substitutions or insertions to confer H-dependent binding.

[0144] Unless otherwise specified within this application, the percent sequence identity between two amino acid sequences is expressed in the best possible manner. This can be determined by comparing these two sequences aligned by formula, where the The amino acid sequence is added or deleted relative to the reference sequence for optimal alignment between these two sequences. The percent identity is the percentage of identical amino acid residues between two sequences. Determine the number of identical positions and divide this number by the total number of positions in the comparison window. The result can be calculated by multiplying it by 100 to get the percentage identity between these two sequences. For example, the available online site (http: / / www.ncbi.nlm.nih.gov / gorf / bl2.h tml), the BLAST program, "BLAST2 sequences" (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 1 74:247-250) can be used, and the parameters used in them are the defaults. (especially the "open gap penalty" of 5 and the "extension gap penalty" of 2) The matrix chosen for the parameters is e.g. the matrix recommended by the program "BLOSUM 62"), the percentage of identity between the two sequences being compared is calculated by the program. is calculated directly by

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

[0146] In one embodiment, the antagonist or antibody molecules of the combination are co-formulated, i.e., monoclonal. The antagonist or antibody molecule is a separate molecule formulated as a single pharmaceutical composition. In combined embodiments, the combination or composition is suitable for simultaneous administration of the two components. The compositions may be formulated for single dose administration or for multiple dose administration. In embodiments in which the antibody molecules are co-formulated, the antagonist or antibody molecules are present in equal amounts, e.g., heterologous. and a first and second antagonist or antibody molecule targeting different cytokines or receptors. Alternatively, the antagonist or The antibody molecules may be formulated such that the ratio of different antagonist or antibody molecules is not 1:1. For example, the combination may comprise a first and second antagonist or antibody that binds to different targets. In embodiments comprising or consisting of molecules, the first and second antagonists The ratio of antagonist or antibody molecules may be 2:1, optionally 3:1, or optionally 4:1. The antibody or peptide molecules may be formulated according to a ratio of 1:2, optionally 1:3, optionally 1:4.

[0147] In some embodiments, the antagonist or antibody molecules of the combination are administered separately, e.g., individually. In an embodiment in which the antagonist or antibody molecules are formulated separately, In this case, the possibility exists for administering the different components or compositions simultaneously or separately. When the antagonist or antibody molecule or separate compositions containing them are administered separately, The peptide / antibody molecules or compositions may be administered sequentially in any order. The antibody molecules that bind to IL-5 can be administered first, followed by antibody molecules that bind to IL-5, or vice versa. The interval between administrations of the antagonist / antibody molecule or composition may be any suitable time interval. The administration of the different compositions may be once (for single dose administration) or repeatedly (for multiple dose administration). (Use) may be performed.

[0148] In one embodiment, the antagonist is an antibody molecule, and the antibody molecule of the combination is multispecific. For example, the combination may be a combination of IL-4Rα When the antibody contains an Fab fragment that binds to IL-1 and an Fab fragment that binds to IL-5, the two Fab fragments bind to the IgG Fc region. The two Fab regions are combined into a single bispecific antibody molecule. It's okay to be surrounded.

[0149] The bispecific or multispecific antibodies of the present invention may be any suitable bispecific / multispecific antibody. For example, the antibody molecules of the combination may be configured according to a format, such as Each Fab arm of a Y-shaped antibody has a different binding specificity, allowing it to "transport" to different targets. The antibody can be incorporated into a bispecific or multispecific antibody format, such as a "sense" binding antibody. In another embodiment, the antibody molecule is bispecific, such that the target is bound in a "cis" configuration. can be incorporated into a monospecific or multispecific antibody format. A suitable Fab region or variable domain can be located at the opposite end of the IgG Fc portion.

[0150] Bispecific or multispecific antibodies consist of two Y-shaped Fab fragments each with binding specificity for a first target. The native IgG structure with its C domain and the Fc domain with binding specificity for a second target. It may have one or more additional antigen-binding domains located at its termini. In one embodiment, the bispecific antibodies of the present invention comprise two Fab regions present on the native IgG structure. a classical antigen-binding domain of IL-4Rα, and one or more Fc domains located C-terminal to the Fc domain The VHH domain of the antibody is designed to bind to IL-5. The two classical antigen-binding domains in the b region bind IL-5 and are located C-terminal to the Fc domain. The reverse configuration is also possible, in which more than one VHH domain binds to IL-4Rα.

[0151] Alternatively, bispecific or multispecific antibodies may comprise two antibodies each having a binding specificity for a first target. The native IgG structure with a Y-shaped Fab arm and a 1st Fab domain located at the C-terminus of the Fc domain. It is also possible to have one or more scFv fragments with binding specificity for two targets. For example, in one embodiment Therefore, the bispecific antibody of the present invention utilizes two ancient Fab regions present in the native IgG structure. An exemplary antigen-binding domain binds to IL-4Rα and comprises one or more sc domains located C-terminal to the Fc domain. The Fv domain is designed to bind to IL-5. The Fab region present in the native IgG structure Two classical antigen-binding domains in the region bind IL-5, and one located at the C-terminus of the Fc domain. The reverse configuration is also possible, where the above scFv domains bind to IL-4Rα. In an embodiment, the bispecific antibody comprises two scFv domains located at the C-terminus of an IgG Fc domain. It may also consist of

[0152] Bispecific or multispecific antibodies are antibodies in which one Fab region is composed of different antigen-binding domains, e.g., VH It may also be an asymmetric IgG antibody, with the H domain replaced. For example, in another embodiment, The bispecific antibody of the present invention comprises a Fab region on one complete arm of the antibody that binds to IL-4Rα. and a native VHH domain that binds IL-5 and substitutes for the second Fab region. The Fab region on one complete arm of the antibody may bind to IL-5. The reverse configuration was also available, in which the VHH domain that binds to IL-4Rα is replaced by a second Fab region. It is also possible.

[0153] Regarding the embodiment in which the antagonist is an antibody molecule and the antibody molecule is co-formulated and / or the antibody molecules are provided as separate compositions and / or For embodiments in which the antibody molecule is provided in a multispecific antibody format, the antibody molecule can be formulated using any suitable pharmaceutical carrier or excipient. Techniques for formulating antibodies for this purpose are well known in the art and are described, for example, in their entirety in the literature. Incorporated references: Wang et al. (2007) Journal of Pharmaceutical Sciences, 9 6:1-26. In embodiments in which the antibody molecules are formulated separately, the antibody molecules may be administered in a pharmaceutical carrier or The excipients may be different or the same for the different compositions.

[0154] Pharmaceutically acceptable excipients that can be used to formulate the composition include ion exchangers, alkoxides, and the like. aluminum stearate, lecithin, serum proteins such as human serum albumin buffer substances, e.g., phosphates, glycine, sorbic acid, potassium sorbate, saturated A mixture of partial glycerides of vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, phosphorus Disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, etc., colloidal silica moss, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g., Sodium carboxymethylcellulose), polyethylene glycol, polyacrylate , wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol Coal and wool fat, but are not limited to.

[0155] In some embodiments, the composition is administered intramuscularly, intravenously, intradermally, by intraperitoneal injection, subcutaneously, epidurally, or intravenously. Any of the following routes of administration: nasal, oral, rectal, topical, inhalation, buccal (e.g., sublingual), and transdermal. It is formulated for administration to a subject via an appropriate route of administration, including but not limited to: In embodiments where the antagonist or antibody molecules are formulated separately, The compositions may be formulated to be administered via different routes.

[0156] In some embodiments, the composition comprises one or more additional therapeutic agents. The additional therapeutic agents include: The one or more additional drugs may be drugs suitable for the prevention or treatment of respiratory tract diseases. The compound may be administered via the same route or via a different route than the antagonist or antibody molecule. It can be formulated to

[0157] D. Bispecific IL-4Rα and IL-5 Antibodies As noted above, the antibody molecules of the combinations described herein may be in a multispecific antibody format, e.g. For example, it may be provided in a bispecific antibody format. The present invention provides an antibody molecule that binds to IL-5 and IL-4Rα, provided in a bispecific antibody format. It contains antibody molecules that bind to it.

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

[0159] The bispecific antibodies of the present invention may be prepared using any suitable bispecific / multispecific antibody as described herein. Bispecific antibodies can be constructed according to a multispecific antibody format. For example, bispecific antibodies can be constructed according to a multispecific antibody format. either in a "lance" configuration, i.e., at the same end of the molecule, or in a "cis" configuration, i.e., at opposite ends of the molecule. It may be configured to couple either of them.

[0160] In one embodiment, a bispecific antibody has an antigen-binding region contained within two Fab arms. It has a native IgG structure. The first Fab arm may exhibit binding specificity to IL-4Rα. Alternatively, the second Fab arm may exhibit binding specificity for IL-5. Heterologous antibodies have the native IgG structure and one or more additional Fc domains located at the C-terminus of the Fc region. In such an embodiment, the antibody may further comprise an antigen-binding domain. The two Fab arms of G may bind to the same target, e.g., IL-4Rα, and the Fc region is C-terminal The one or more additional antigen-binding regions may bind to a second target, for example IL-5. The one or more additional antigen-binding regions may include, but are not limited to, a VHH domain or an scFv. The antigen-binding domain may take any suitable antigen-binding form, including a polypeptide.

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

[0162] In one embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention comprises a Fab region that binds to IL-4Rα. In one embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention comprises an scFv that binds to the IL-4Rα / IL-5 domain and an scFv that binds to IL-5. The antibody comprises a Fab region that binds to IL-5 and an scFv that binds to IL-4Rα. In this regard, the IL-4Rα / IL-5 bispecific antibody of the present invention is an IgG antibody that binds to IL-4Rα and an IL-5 In such an embodiment, the antibody comprises or consists of one or more scFvs that bind to The IL-4Rα / IL-5 bispecific antibody is an IgG antibody that binds to IL-4Rα and two antibodies that bind to IL-5. The antibody may comprise or consist of one or more scFv fragments of IL-5. The scFv fragment preferably comprises two Fab fragments that bind to the C-terminus of the Fc region of an IgG antibody, i.e., two Fab fragments that bind to IL-4Rα. It is located at the end of the Fc region opposite the arm.

[0163] The bispecific antibodies of the present invention comprise Fab regions (or Fab arms) each having a different antigen-binding region or domain. Alternatively, the antibody may be constructed as an asymmetric IgG antibody, with the main domain replaced by, for example, a VHH domain. In one embodiment, the IL-4Rα / IL-5 bispecific antibody comprises one complete antibody that binds to IL-4Rα. The VHH domain contains a Fab region on one arm and a VHH domain in place of the second Fab region that binds to IL-5. The reverse configuration is also possible, and In this case, the Fab region on one intact arm of the antibody binds to IL-5 and is replaced by the second Fab region. The VHH domain binds to IL-4Rα.

[0164] IL-4Rα / The IL-5 bispecific antibody may comprise an antigen-binding region that binds to human IL-4R, The binding region can comprise a variable heavy domain (VH) and a variable light domain (VL), The domain is: (i) HCDR3 comprising or consisting of SEQ ID NO: 3; HCDR2 consisting of SEQ ID NO: 1 or HCDR1 consisting of SEQ ID NO: 12 LCDR3 comprising or consisting of SEQ ID NO: 11; LCDR4 comprising or consisting of SEQ ID NO: 12 2; LCDR1 comprising or consisting of SEQ ID NO: 10; (ii) HCDR3 comprising or consisting of SEQ ID NO: 6; HCDR2 comprising or consisting of SEQ ID NO: 4; HCDR1 comprising SEQ ID NO: 15; LCDR3 comprising or consisting of SEQ ID NO: 14; LCDR4 comprising or consisting of SEQ ID NO: 15 2; LCDR1 comprising or consisting of SEQ ID NO: 13; (iii) HCDR3 comprising or consisting of SEQ ID NO: 9; HCDR2 comprising or consisting of SEQ ID NO: 7; HCDR1 comprising or consisting of SEQ ID NO: 18 LCDR3 comprising or consisting of SEQ ID NO: 17; DR2; LCDR1 comprising or consisting of SEQ ID NO: 16; (iv) HCDR3 comprising or consisting of SEQ ID NO: 91; HCDR2 comprising or consisting of SEQ ID NO: 7; HCDR1 comprising or consisting of SEQ ID NO: 18 LCDR3 comprising or consisting of SEQ ID NO: 17; DR2; LCDR1 comprising or consisting of SEQ ID NO: 16; (v) HCDR3 comprising or consisting of SEQ ID NO: 92; HCDR2 consisting of SEQ ID NO: 7 or HCDR1 consisting of SEQ ID NO: 97 LCDR3 comprising or consisting of SEQ ID NO: 17; LCDR4 comprising or consisting of SEQ ID NO: 18 2; LCDR1 comprising or consisting of SEQ ID NO: 16; (vi) HCDR3 comprising or consisting of SEQ ID NO: 93; HCDR2 comprising or consisting of SEQ ID NO: 7; HCDR1 comprising or consisting of SEQ ID NO: 98 LCDR3 comprising or consisting of SEQ ID NO: 96; DR2; LCDR1 comprising or consisting of SEQ ID NO: 16; (vii) HCDR3 comprising or consisting of SEQ ID NO: 94; HCDR2 comprising or consisting of SEQ ID NO: 7; HCDR1 comprising or consisting of SEQ ID NO: 98 LCDR3 comprising or consisting of SEQ ID NO: 17; DR2; LCDR1 comprising or consisting of SEQ ID NO: 16; and (viii) HCDR3 comprising or consisting of SEQ ID NO: 95; HCDR2 comprising or consisting of SEQ ID NO: 7; HCDR1 comprising or consisting of SEQ ID NO: 98 LCDR3 comprising or consisting of SEQ ID NO: 17; LCDR2; LCDR1 comprising or consisting of SEQ ID NO: 16 The CDR sequences are as follows:

[0165] The IL-4Rα / IL-5 bispecific antibody of the present invention may comprise an antigen-binding region that binds to IL-4R. The antigen-binding region can comprise a variable heavy domain (VH) and a variable light domain (VL). wherein the VH and VL domains are: (i) HCDR3 comprising or consisting of SEQ ID NO: 27; HCDR2 comprising or consisting of SEQ ID NO: 25; HCDR1 comprising or consisting of SEQ ID NO: 36 LCDR3 comprising or consisting of SEQ ID NO: 35; LCDR4 comprising or consisting of SEQ ID NO: 36; DR2; LCDR1 comprising or consisting of SEQ ID NO: 34; (ii) HCDR3 comprising or consisting of SEQ ID NO: 30; HCDR2 comprising or consisting of SEQ ID NO: 28; HCDR1 comprising or consisting of SEQ ID NO: 39 LCDR3 comprising or consisting of SEQ ID NO: 38; DR2; LCDR1 comprising or consisting of SEQ ID NO: 37; and (iii) HCDR3 comprising or consisting of SEQ ID NO: 33; HCDR2 comprising or consisting of SEQ ID NO: 31; HCDR1 comprising or consisting of SEQ ID NO: 42 LCDR3 comprising or consisting of SEQ ID NO: 41; LCDR2; LCDR1 comprising or consisting of SEQ ID NO: 40 The CDR sequences are as follows:

[0166] The antigen-binding region that binds to human IL-4Rα consists of a variable heavy chain domain (VH) and a variable light chain domain ( and VH and VL domains, wherein the VH and VL domains are: (i) an amino acid sequence comprising SEQ ID NO: 19, or at least 70%, at least 80%, or at least Amino acid sequences that share 90%, at least 95%, at least 98%, or at least 99% identity with and an amino acid sequence comprising, or at least 70% identical to, SEQ ID NO: 20; At least 80%, at least 90%, at least 95%, at least 98%, at least 99% identical a VL domain comprising an amino acid sequence having the following characteristics: (ii) an amino acid sequence comprising SEQ ID NO: 21, or at least 70%, at least 80%, or at least Amino acid sequences that share 90%, at least 95%, at least 98%, or at least 99% identity with and an amino acid sequence comprising, or at least 70% identical to, SEQ ID NO: 22; At least 80%, at least 90%, at least 95%, at least 98%, at least 99% identical a VL domain comprising an amino acid sequence having the following characteristics: (iii) an amino acid sequence comprising SEQ ID NO: 23, or a sequence identical to it by at least 70%, at least 80%, or at least amino acid sequences having at least 90%, at least 95%, at least 98%, or at least 99% identity a VH domain comprising the sequence SEQ ID NO: 24, or an amino acid sequence at least 70% identical thereto; , at least 80%, at least 90%, at least 95%, at least 98%, at least 99% a VL domain comprising an amino acid sequence having the same identity; (iv) an amino acid sequence comprising or consisting of SEQ ID NO: 99, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence having 9% identity to SEQ ID NO: 100, or is an amino acid sequence consisting of, or at least 70%, at least 80%, or at least amino acid sequences having at least 90%, at least 95%, at least 98%, or at least 99% identity VL domain containing columns; (v) an amino acid sequence comprising or consisting of SEQ ID NO: 101, or at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 9 a VH domain comprising an amino acid sequence having 9% identity to SEQ ID NO: 102, or is an amino acid sequence consisting of, or at least 70%, at least 80%, or at least amino acid sequences having at least 90%, at least 95%, at least 98%, or at least 99% identity VL domain containing columns; (vi) an amino acid sequence comprising or consisting of SEQ ID NO: 103, or at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least a VH domain comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 104, or an amino acid sequence consisting of, or at least 70%, at least 80%, or at least Amino acids with at least 90%, at least 95%, at least 98%, at least 99% identity VL domain containing sequence; (vii) an amino acid sequence comprising or consisting of SEQ ID NO: 105, or at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least a VH domain comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 106, or an amino acid sequence consisting of, or at least 70%, at least 80%, or at least Amino acids with at least 90%, at least 95%, at least 98%, at least 99% identity a VL domain comprising the sequence (viii) an amino acid sequence comprising or consisting of SEQ ID NO: 107, or at least at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least a VH domain comprising an amino acid sequence having 99% identity with SEQ ID NO: 108; or an amino acid sequence consisting of it, or at least 70%, at least 80%, Amino acids with at least 90%, at least 95%, at least 98%, at least 99% identity a VL domain comprising a VL sequence.

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

[0168] The IL-4Rα / IL-5 bispecific antibody of the present invention contains an antigen-binding region that binds to human IL-5. and the antigen-binding region may comprise a variable heavy domain (VH) and a variable light domain (VL). and wherein the VH and VL domains are: (i) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 54 LCDR3 comprising or consisting of SEQ ID NO: 53; LCDR4 comprising or consisting of SEQ ID NO: 54 DR2; LCDR1 comprising or consisting of SEQ ID NO: 52; (ii) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 57 LCDR3 comprising or consisting of SEQ ID NO: 56; DR2; LCDR1 comprising or consisting of SEQ ID NO: 55; (iii) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 59 LCDR3 comprising or consisting of SEQ ID NO: 56; LCDR2; LCDR1 comprising or consisting of SEQ ID NO: 58; (iv) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 61 LCDR3 comprising or consisting of SEQ ID NO: 60; DR2; LCDR1 comprising or consisting of SEQ ID NO: 58; and (v) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 62 LCDR3 comprising or consisting of SEQ ID NO: 56; LCDR2; LCDR1 comprising or consisting of SEQ ID NO: 58; It contains the CDR sequences.

[0169] The IL-4Rα / IL-5 bispecific antibody of the present invention may comprise an antigen-binding region that binds to IL-5. The antigen-binding region may comprise a variable heavy domain (VH) and a variable light domain (VL). , wherein the VH and VL domains are: (i) HCDR3 comprising or consisting of SEQ ID NO: 72; HCDR2 comprising or consisting of SEQ ID NO: 70; HCDR1 comprising or consisting of SEQ ID NO: 74 LCDR3 comprising or consisting of SEQ ID NO: 38; DR2; LCDR1 comprising or consisting of SEQ ID NO: 73; and (ii) HCDR3 comprising or consisting of SEQ ID NO: 72; HCDR2 comprising or consisting of SEQ ID NO: 70; HCDR1 comprising or consisting of SEQ ID NO: 75 LCDR3 comprising or consisting of SEQ ID NO: 38; LCDR2; LCDR1 comprising or consisting of SEQ ID NO: 73; It contains the CDR sequences.

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

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

[0172] Each domain is defined by its % sequence identity with the listed domain / chain amino acid sequence. The amino acid sequence variations in the framework regions or other regions outside the CDR regions are not included in the chain. VH / VL domains or heavy / light chain amino acid sequences shown. The CDR sequences may be identical to those of the first or second amino acid sequence.

[0173] In a preferred embodiment, the IL-4Rα / IL-5 bispecific antibody of the present invention binds to IL-4Rα. and an antigen-binding region that binds to IL-5, and the antigen-binding region that binds to IL-4Rα. The antigen-binding region comprises a first variable heavy domain (VH) and a variable light domain (VL) pair, and The antigen-binding region that binds to IL-5 is a second variable heavy domain (VH) and variable light domain (VL) pair. Including, The first VH-VL domain pair is: (i) HCDR3 comprising or consisting of SEQ ID NO: 3; HCDR2 consisting of SEQ ID NO: 1 or HCDR1 consisting of SEQ ID NO: 12 LCDR3 comprising or consisting of SEQ ID NO: 11; LCDR4 comprising or consisting of SEQ ID NO: 12 2; LCDR1 comprising or consisting of SEQ ID NO: 10; (ii) HCDR3 comprising or consisting of SEQ ID NO: 6; HCDR2 comprising or consisting of SEQ ID NO: 4; HCDR1 comprising SEQ ID NO: 15; LCDR3 comprising or consisting of SEQ ID NO: 14; LCDR4 comprising or consisting of SEQ ID NO: 15 2; LCDR1 comprising or consisting of SEQ ID NO: 13; and (iii) HCDR3 comprising or consisting of SEQ ID NO: 9; HCDR2 comprising or consisting of SEQ ID NO: 7; HCDR1 comprising or consisting of SEQ ID NO: 18 LCDR3 comprising or consisting of SEQ ID NO: 17; LCDR2; LCDR1 comprising or consisting of SEQ ID NO: 16; ,and, the second VH-VL domain pair being: (i) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 54 LCDR3 comprising or consisting of SEQ ID NO: 53; LCDR4 comprising or consisting of SEQ ID NO: 54 DR2; LCDR1 comprising or consisting of SEQ ID NO: 52; (ii) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 57 LCDR3 comprising or consisting of SEQ ID NO: 56; DR2; LCDR1 comprising or consisting of SEQ ID NO: 55; (ii) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising SEQ ID NO: 59 LCDR3 comprising or consisting of SEQ ID NO: 56; DR2; LCDR1 comprising or consisting of SEQ ID NO: 58; (iv) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 61 LCDR3 comprising or consisting of SEQ ID NO: 60; DR2; LCDR1 comprising or consisting of SEQ ID NO: 58; and (v) HCDR3 comprising or consisting of SEQ ID NO: 51; HCDR2 comprising or consisting of SEQ ID NO: 49; HCDR1 comprising or consisting of SEQ ID NO: 62 LCDR3 comprising or consisting of SEQ ID NO: 56; DR2; LCDR1 comprising or consisting of SEQ ID NO: 58. .

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

[0175] In a preferred embodiment, the antigen-binding regions of the bispecific antibodies described herein are highly human For example, the antigen-binding region may have homology with the VH and / or VL domains from which its CDR sequences are derived. The gene may be a humanized or germline variant of the gene.

[0176] The bispecific antibodies of the present invention may also be used in combination with other antibodies having amino acid sequences that are fully or substantially human. For example, the bispecific antibody of the present invention may comprise a human IgG It may also comprise an Fc domain derived from an antibody, such as human IgG1, IgG2, IgG3 or IgG4. .

[0177] The bispecific antibodies of the present invention having an Fc domain may comprise, as described herein, It may be modified to enhance its binding affinity to the fetal receptor FcRn. For example, To increase binding, one or more amino acid residues in the Fc region may be replaced with different amino acid residues. Preferred amino acid substitutions within the Fc region are described herein and are incorporated by reference in their entirety in the context of the present invention. The same applies to bispecific antibodies.

[0178] As described herein, the bispecific antibodies of the present invention are designed to have pH-dependent antigen-binding activity. In particular, the bispecific antibody may be modified to bind IL-4Rα and / or IL-5 at acidic pH. The binding activity may be modified to be lower compared to the binding activity at neutral pH. A specific antibody is one that contains at least one of the IL-4Rα antigen-binding region and / or the IL-5 antigen-binding region of the antibody molecule. by substituting at least one amino acid with histidine, or by substituting at least one histidine The antibody molecule may be modified by inserting a His into one or both antigen-binding regions. The cytosine substitutions and / or insertions may be made in the heavy chain variable domain and / or the light chain variable domain as described herein. It is preferably introduced at one or more sites within the CDRs of the chain variable domain.

[0179] (E. Treatment Method) The combination therapies and bispecific antibodies described herein are useful for treating chronic airway diseases in human subjects. It is used in the following way.

[0180] Accordingly, the present invention provides (i) IL-5:IL-5R antagonists for use in treating chronic airway disease in human subjects. and (ii) an antagonist of IL-4:IL-4R and / or an antagonist of IL-13:IL-13R. The present invention also provides a method for treating chronic airway disease in a human subject, comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of acetaminophen, benzodiazepine ... and an antigen-binding region that binds to IL-5, The present invention provides a bispecific antibody against IL-5 for use in the treatment of chronic airway disease in human subjects. IL-5R antagonists are provided, which are antagonists of IL-4:IL-4R. The present invention provides a method for treating IL-13-associated leukemia, the leukemia, and / or IL-13R inflammatory bowel disease. - IL-4:IL-4R antagonists and / or IL-13: An antagonist of IL-13R is provided, which antagonist is an antagonist of IL-5:IL-5R. In a preferred embodiment, the present invention provides a method for treating chronic airway disease in a human subject. The present invention provides an IL-5 antagonist for use in the treatment of IL-4Rα. In a further preferred embodiment, the present invention provides a method for the treatment of a human subject with steroid hormone receptor agonist (SHR) in combination with a steroid hormone receptor agonist (SHR). The present invention provides an antagonist of IL-4Rα for use in the treatment of chronic airway diseases, and In a further preferred embodiment, the method is administered in combination with an antagonist of IL-5. The antagonist used in the method is an antibody molecule.

[0181] In a still further aspect, the present invention provides a method of treating a chronic airway disease in a human subject, comprising administering to said subject a therapeutically effective amount of ... an effective amount of the combination according to the first aspect of the invention or the bispecific antibody according to the second aspect of the invention, The combination of the first aspect of the invention and the present invention provides a method of treating a rhesus macular degeneration comprising administering to an elephant a compound of formula (I) or (II) of formula (II) or (II). All of the above-described embodiments relating to the bispecific antibodies of the two aspects can be used in combination with the methods described herein. It is equally applicable to

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

[0183] In some embodiments, the methods described herein are directed to treating chronic airway diseases characterized by increased mucus production. An example of a disease characterized by increased mucus production is cystic fibrosis. In a further embodiment, the methods described herein are directed to treating chronic bronchial hyperresponsiveness. It is intended to treat chronic respiratory tract diseases.

[0184] In a preferred embodiment, the methods described herein are for treating asthma. Exemplary asthma subtypes include severe asthma, severe refractory asthma, mild or moderate asthma, obesity-related asthma, and Breath, exercise-induced asthma, aspirin-induced asthma, atopic or allergic asthma, eosinophils neutrophilic asthma, paucigranulocytic or non-inflammatory asthma, early-onset asthma These include, but are not limited to, type 1 asthma, late onset asthma, type 2 high asthma, type 2 low asthma, and type 1 / Th17 asthma. It is not something that can be determined.

[0185] In a preferred embodiment, the methods described herein are directed to the treatment of atopic or allergic asthma. Atopic or allergic asthma is associated with allergic sensitization. Allergic sensitization is a clinically defined form of asthma caused by serum Ig E antibodies and / or may be present in the presence of house dust mites (HDM), animal dander, fungal spores, or plants. of common inhaled or ingested allergens such as plant or tree pollen, or peanuts ( It is defined by a positive skin prick test for lipoproteins (Lambrecht and Ham (Mad, 2015, supra). The majority of early-onset asthma cases are allergic or atopic. Thus, the method of the present invention is also for the treatment of early-onset asthma.

[0186] In a further preferred embodiment, the methods described herein are for treating Type II high (or Type II Hi) asthma. Type II hyperasthma is characterized by the presence of type II cytokine molecules. Patients with type II severe asthma usually have higher levels of IL-13 and IL-14 compared to patients with type II mild asthma. 5 level, had a higher number of eosinophils and mast cells, and had more severe atopy and SBM ( Subcutaneous basement membrane (subcutaneous basement membrane) thickening (Wenzel, (2012) ibid. and Gauthier et al., (2015) ibid.) (See above.) The combinations and bispecific antibodies of the present invention inhibit the signaling of multiple type 2 cytokines. Thus, the combinations and bispecific antibodies described herein target the delivery pathways of type II asthma. It would be particularly effective in treating human subjects with asthma.

[0187] In some embodiments, the methods described herein are for treating severe asthma or severe refractory asthma. It is intended to be used for this purpose.

[0188] Almost all forms of asthma are pathologically associated with goblet cell metaplasia or GCM. Under the influence of IL-4 and / or IL-13 and ligands for the epidermal growth factor receptor, ciliated cells and ciliated Thus, in one embodiment, the present invention provides a method for the transdifferentiation of goblet cells into goblet cells. The methods described herein are for treating asthma by reducing goblet cell metaplasia. In such embodiments, goblet cell metaplasia is reduced compared to the level of goblet cell metaplasia before treatment. do.

[0189] Patients with chronic airway disease, including the majority of asthmatics, also suffer from bronchial hyperresponsiveness (BHR). HR is defined as the increased sensitization to various airway constricting stimuli. Most patients with chronic obstructive pulmonary disease (COPD) exhibit such increased sensitization. Therefore, the methods described herein may be used to treat chronic airway disease by reducing bronchial hyperresponsiveness. In a preferred embodiment, the methods described herein are for treating bronchial hypertension. In such an embodiment, the compound is intended to treat asthma by reducing irritability. , bronchial hyperresponsiveness is reduced compared to the level of bronchial hyperresponsiveness before treatment. The sensitivity of the airway can be assessed by a bronchial challenge test, which involves the use of steroids such as methacholine or histamine. These chemicals can cause bronchospasm in normal individuals. However, people with bronchial hyperresponsiveness have a lower threshold.

[0190] Patients with chronic airway disease, including the majority of asthmatics, also exhibit excessive mucus production. In embodiments, the methods described herein treat chronic airway diseases by reducing mucus production. In a preferred embodiment, the methods described herein are directed to reducing mucus production. In such an embodiment, mucus production is measured prior to treatment. Mucus production is reduced compared to the level of mucus in the normal range. Mucus production is determined by the expression and / or activity of mucins, For example, it can be assessed by measuring the expression and / or activity of muc5AC. The methods described herein can be used to increase mucin expression and / or activity compared to pre-treatment expression and / or activity. or reduced activity to treat chronic airway disease. In aspects, the methods described herein reduce mucin expression and / or activity compared to pre-treatment expression and / or activity. The present invention is intended to treat asthma by reducing the expression and / or activity of steroid hormones.

[0191] Chronic airway disease is also characterized by a decline in lung function. Forced expiratory volume (FEV) is a measure of lung function. Forced expiratory volume (FEV) is the amount of air a person expels during a forced breath. Forced expiratory volume (FVC) is the amount of air exhaled during the FEV test. The FEV1 / FVC ratio, also called the Tiffeneau-Pinelli index, is the ratio of the total lung capacity to the total vital capacity. , a calculated ratio that represents the fraction of vital capacity that the subject can exhale during the first second of a forced exhalation. A normal FEV1 / FVC ratio is about 70-80%. The method is for treating chronic airway disease by improving lung function. In a preferred embodiment, the methods described herein treat asthma by improving lung function. In such embodiments, pulmonary function is improved compared to pulmonary function before treatment. In certain embodiments, the methods described herein provide an improved FEV1 / FVC ratio compared to the FEV1 / FVC ratio before treatment. In one embodiment, the present invention is for treating chronic airway disease by increasing the FVC / FVC ratio. In such cases, the methods described herein increase the FEV1 / FVC ratio compared to the FEV1 / FVC ratio before treatment. and thereby treating asthma.

[0192] The methods described herein also include treating comorbidities associated with chronic airway disease, such as those associated with asthma. Used to treat comorbidities such as: severe asthma; chronic sinusitis; nasal polyposis; allergic Allergic rhinitis; respiratory dysfunction; vocal cord dysfunction; anxiety and depression; obesity; sleep apnea syndrome (O SAS); Gastroesophageal reflux disease (GERD); Bronchiectasis; Allergic bronchopulmonary aspergillosis (ABPA) and eosinophilic granulomatosis with polyangiitis (EGPA) (Porsbjerg and Menzies-Gow, 2017). It is associated with many comorbidities, including, but not limited to, pulmonary embolism (see Espirology 22:651-661). The methods described herein are not intended to be used to treat any of the above-mentioned asthma comorbidities. It can be used.

[0193] The methods described herein may further comprise the administration of a therapeutic agent. The methods described herein include the administration of one or more additional therapeutic agents for the treatment of chronic airway disease. The disclosed combination or bispecific antibodies may be administered with additional therapeutic agents separately, simultaneously, sequentially, or in combination with other therapeutic agents. , and can be administered simultaneously in duplicate.

[0194] The patient or subject to be treated with the methods described herein may be a patient receiving a treatment such as corticosteroid treatment. The patient or subject to be treated with the methods described herein may have already received These may be classified as "corticosteroid responsive" or "corticosteroid non-responsive." The patient or subject may also exhibit one or more symptoms associated with a chronic airway disease. In the present invention, the patient or subject is receiving medical care for the treatment of a chronic airway disease, and / or They may also be individuals who are actively seeking medical care for the treatment of a chronic airway disease.

[0195] In some embodiments, the patient or subject treated with the methods described herein has elevated eosinophilia. In patients with high eosinophil levels, dupilumab ( Certain existing drugs that target IL-4Rα, such as IL-4Rα-targeting agent dupilumab, have been shown to increase circulating eosinophil levels in patients. It cannot be prescribed because it would be unacceptably high. The range is not limited by theory. However, IL-4Rα antagonists, such as IL-4Rα antibodies, and IL-5 antagonists, such as IL-5 Combination treatment with antibodies is appropriate for patients with high eosinophil levels, as IL-5 promotes eosinophil proliferation. The combined inhibition of IL-5 and IL-4Rα is thought to be a key factor in the development of IL-4Rα. This may prevent or reduce the increase in eosinophil levels that may occur during treatment with steroids.

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

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

[0198] Example 1: Production of neutralizing IL-4Rα and IL-5 monoclonal antibodies A. Llama immunization and library construction: Two llamas, raised outdoors in accordance with French animal welfare laws, were given transgenic mice on one shoulder. The mice were immunized intramuscularly with IL-4Rα-Fc in one shoulder and recombinant mouse IL-5 (R&D Systems) in the other. They were vaccinated and boosted weekly for 6 weeks. Briefly, for the first 2 weeks, they received phosphate-buffered saline. Buffered saline (PBS) and mixed with incomplete Freund's adjuvant (Sigma-Aldrich) For the remaining 4 weeks, mice were given 100 μg of IL-4Rα-Fc and 50 μg of IL-5, and for the remaining 4 weeks, mice were given 50 μg of IL-4Rα-Fc and 25 μg of IL-5. The Fab library was constructed using the previously described SIMPLE antibody library, which is proprietary to the applicant. The experiments were carried out using a 3D printer platform (the contents of which are incorporated herein in their entirety). (See International Patent Application No. WO2010 / 001251.) Five days after the last immunization, 400 mL of blood containing 1000 kcal was collected from a llama and purified by centrifugation on a Ficoll-Paque gradient. The total RNA was then randomly primed using reverse transcriptase. The VH-CH1 region and VL-CL domains (kappa and lambda) of llama IgG1 were then converted into cDNA. The gene sequence encoding was isolated and subcloned into the phagemid vector pCB3. The pCB3 vector expresses the recombinant Fab fragment as a Fab fragment fused to the phage pIII envelope protein. This allows for the expression of recombinant antibodies.

[0199] Parallel studies were conducted to generate recombinant antibodies that bind to human IL-4Rα and human IL-5. Following the same protocol as described above, llamas were injected with recombinant human IL-4Rα-Fc and recombinant human IL-4Rα-Fc. Mice were immunized intramuscularly with IL-5.

[0200] B. Selection of Fabs that bind to IL-4Rα and IL-5 E. coli strain TG1 (Netherlands Fungal Culture Collection, Netherlands Culture Co.) A collection of bacteria was transformed with the recombinant phagemid to generate Fab-expressing phages. Libraries (one lambda and one kappa library per immunized llama) were generated. The mice were immunized with IL-4Rα bound to the crystallizable fragment (Fc) portion, allowing for Fc-bound antigen binding. Counterselection against phage expressing fragments (Fab) was first performed using an unrelated human antibody. The resulting Fab-expressing phages were collected at 10 8 ~10 9 With a range of diversity, then Adsorbed onto immobilized recombinant biotinylated IL-4Rα-Fc or IL-5 as described Elution was performed using trypsin (De Haard et al., (1999) Journal of Biological Chemistry Publication, 274: 18218-30). After three rounds of selection, phages expressing IL-4Rα or IL-5 specific Fab were isolated. Finally, TG1 E. coli was infected with the selected phages and individual colonies were isolated. The secretion of Fab into the periplasm of E. coli strain TG1 was investigated under low glucose concentrations (0.1% w / v). and derivatized using isopropyl β-D-1-thiogalactopyranoside (Sigma-Aldrich). The Fab-containing periplasmic fraction of the bacteria was collected.

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

[0202] D. Monospecific Antibody Generation, Purification, and Characterization Potently neutralizing IL-4Rα- and IL-5-specific selected Fab fragments (VH and VL (lambda or cDNA encoding the Fc receptor or CL (lambda or kappa) domain was modified to encode the Fc receptor or CL (lambda or kappa) domain, respectively. ) containing mutations that abolish antibody effector functions mediated by murine IgG2a (or human IgG1). Two separate pUPE mammalian expression vectors containing cDNAs encoding the CH1, CH2, and CH3 domains of gG1 Next, we used the anti-IL-4Rα and anti-IL-5 IgG2a vectors with the most potent neutralizing activity. (or IgG1) molecules (by transient transfection of mammalian cells) and purification ( Protein A affinity chromatography) was performed as previously described. (Basilico et al. (2014) Journal of Clinical Investigation 124:3172). The CDR, VH, and VL sequences of the selected antibodies are shown in Tables 3 to 14 below.

[0203] [Table 3]

[0204] [Table 4]

[0205] [Table 5]

[0206] [Table 6]

[0207] [Table 7]

[0208] [Table 8]

[0209] [Table 9]

[0210] [Table 10]

[0211] [Table 11]

[0212] [Table 12]

[0213] [Table 13]

[0214] [Table 14]

[0215] Example 2: In vitro characterization of IL-4Rα and IL-5 monoclonal antibodies The target antibodies of mouse IL-4Rα and IL-5 monoclonal antibodies (m4RMP36B7 and m5MP95G7), respectively their ability to bind to targets in vitro and to mediate IL-4Rα and IL-5 signaling The compounds were tested for their ability to inhibit the cellular effects of steroids.

[0216] A. Inhibition of IL4- and IL5-induced HT-2 and TF-1 cell proliferation The neutralizing activity of both the IL-4Rα and IL-5 monospecific antibodies was assessed against mouse IL-4 and mouse IL-5, respectively. This was assessed by in vitro cell assays, where it induces proliferation of HT-2 and TF-1 cells.

[0217] Human TF-1 cells (erythroblasts, ATCC® CRL-2003™) and murine HT-2 clone A5E cells Cells (IL-2 dependent T lymphocytes, ATCC® CRL-1841™) were incubated at 37°C in 5% (v / v) CO2 and RPMI1640 (Sigma), 10% (v / v) heat-inactivated fetal bovine serum (Sigma), 1X gentamicin gentamycin (Sigma), and 2 ng / mL human granulocyte-macrophage colony-stimulating factor (R The cells were cultured in a growth medium containing either human IL-2 (R&D Systems) or human IL-2 (R&D Systems). The assay medium was growth medium without human GM-CSF for TF-1 cells and without human IL-2 for HT-2 cells. 0.5 ng / mL mouse IL-5 (R&D Systems) for TF-1 cells or mouse IL-5 (HT-2 cells) IL-4 (R&D Systems) was added to the assay medium. Antibodies were administered at 1 ng of mouse I for TF-1 cells. Assay medium containing IL-5 or 0.75 ng of mouse IL-4 for HT-2 cells, and 0.75 ng of mouse IL-4 for TF-1 cells. The cells were serially diluted 10-fold for the HT-2 cells and 5-fold for the HT-2 cells. After incubating the cells at 37°C for 1 hour, Wash and place TF-1 cells at 1.1 x 10 6 cells / mL, HT-2 cells were 0.2 × 10 6 Resuspend in a final volume of 100 cells / mL Cells were then added to each well, followed by CellTiter 96® AQueous One Solution Reagent (P After 3 hours of incubation, the absorbance was measured.

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

[0219] B. Binding of IL-4Rα and IL-5 Monoclonal Antibodies to IL-4Rα and IL-5, Respectively Figure 2 shows the results of the monoclonal antibodies (IL-4Rα antibody, IL-5 antibody, or The interaction between the antibody (or an irrelevant IgG2a antibody) and the immobilized target (IL-4Rα or IL-5) Representative surface plasmon resonance (SPR) sensorgrams are shown. Both monoclonal antibodies IL-4R binds to these targets, whereas irrelevant IgG2a does not bind to either target. The α and IL-5 monospecific antibodies bind to their targets with affinities of 8E-11M and 2E-12M, respectively. did.

[0220] Furthermore, the ability of the monospecific antibodies to compete with their respective targets was tested by SPR. Monoclonal antibodies and their targets (IL-4Rα or IL-5, or unrelated IgG2a) ) and the immobilized protein (IL-4, IL-13Rα, or IL-5Rα). Representative SPR sensorgrams of competitive interactions are shown. A mixture composed of IL-4Rα inhibited the binding of IL-4Rα to immobilized IL-4 ligand. A mixture consisting of IL-4Rα monoclonal antibody and IL-13 binds to immobilized IL-13Rα. Similarly, a mixture of IL-5 monoclonal antibody and IL-5 inhibited the binding of IL-13. The compounds inhibited the binding of IL-5 to immobilized IL-5Rα.

[0221] C. Inhibition of IL-4-induced MHC class II antigen expression in purified B cells analyzed by FACS. Using FACS analysis, we demonstrated that IL-4Rα monospecific antibodies inhibit the translocation of MHC class II to the surface of B cells. We tested whether this compound competes with mouse IL-4, which is known to induce IL-1.

[0222] For FACS analysis, B cells were sorted by magnetic activated cell sorting according to the manufacturer's protocol. Purified B cells (5 × 10) were collected using anti-CD19 microbeads (Miltenyi Biotec). 5 cells / mL) in 24-well plates (Costar) with or without IL-4 (0.1 ng / mL). , in the presence of 500 ng / mL anti-IL-4Rα or irrelevant IgG2a antibody, or with or without inhibitors The cells were then cultured for 16 hours in a control medium. The cells were washed and incubated on ice with rat IgG2b anti-mouse Fc Preincubation with γR monospecific Ab 2.4G2 (Bioceros) for 20 min allowed the IgG Fc receptor The cells were then transfected with MHC class II (M5 / 114.15.2, eBioscience) and CD19. After staining with an antibody against 1D3 (eBioscience), the cells were incubated at 4°C for 30 minutes, and then Dead cells were detected using a fixable viability stain (eFluor 506, eBioscience). The stained cells were analyzed using an LSR Fortessa flow cytometer (BD Biosciences). The final analysis and image output were performed using FlowJo v10.0.7 software (Tree Station). The test was performed using a fluororesin (Company r).

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

[0224] Example 3: Anti-mouse IL-4Rα and IL-5 monoclonal antibodies in a murine house dust mite (HDM) model In vivo characterization of antibody House dust mites (HDM Dermatophagoides species) are the most common airborne dust mites in the world. HDM is one of the allergens. 50-85% of asthma patients are allergic to HDM. Prolonged exposure to HDM Dew leads to airway remodeling with increased mucus cell density and airway hyperresponsiveness, which may contribute to HD. The murine HDM model is a useful in vivo model for studying asthma. It's Dell.

[0225] (Experimental setting 1) Female C57Bl / 6J wild-type mice were obtained from The Jackson Laboratory. The study was approved by the Ethics Committee of the VIB-UGent Center. All mice were 6–8 weeks old. The experiments were performed using age-matched groups. Additionally, the experimental design included antibody treatment given throughout the full sensitization and challenge period. On day 0, mice were intratracheally inoculated with isoflurane (2.5% in air). The mice were lightly anesthetized with acetaminophen (HDM) and administered 1 μg of HDM (Greer Laboratories). The mice were lightly anesthetized with intranasal isoflurane (2.5% in air) and challenged with 10 μg of HDM. The treatment was performed daily. On days 1, 1, 6, 8, and 10, mice were treated with either: (i) IL- (ii) IL-4Rα monospecific antibody (m4RMP36B7), (iii) IL-5 monospecific antibody (m5MP95G7), a combination of IL-4Rα and IL-5 monospecific antibodies (m4RMP36B7 and m5MP95G7); or (iv) an unrelated The day before and day 1 represent the sensitization period, and days 6, 8, and 10 represent the challenge period. At least n=6 mice were treated per group.

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

[0227] Figure 5B shows the results of the IL-4Rα monoclonal antibody, the IL-5 monoclonal antibody, and the IL-4Ra monoclonal antibody. Mice receiving a combination of IL-5 monoclonal antibody and IL-5 monoclonal antibody, or an unrelated IgG2a antibody Figure 1 shows the different cell counts in the bronchoalveolar lavage fluid of the bronchial tumor. Cells were analyzed by FACS. In IgG2a-treated HDM-sensitized mice, the number of eosinophil cells was significantly increased by 100%. The IL-4α / IL-5 monoclonal antibody response was significantly increased compared to treatment with the control IgG2a antibody. A significant decrease in eosinophil cell count was observed after treatment with the specific antibody combination.

[0228] (Experimental setting 2) The second experimental protocol examines the effects of IL-4Rα and IL-5 antibodies in a murine HDM model. The experimental design shown in Figure 6 was designed for further testing in a clinically relevant therapeutic setting. The procedure involving treatment is performed only during the challenge phase and not during the sensitization phase. Therefore, mice were given 100 mg of 10 ... , and their treatments (IL-4Rα monospecific antibody; IL-5 monospecific antibody; IL-4Rα / IL-5 monospecific antibody) Mice received either monospecific antibody or an irrelevant IgG2a antibody. 150 μg of each monospecific injected in combination, or 150 μg of an unrelated mouse At least n = 6 mice per group were administered 150 μg of IgG2a monoclonal antibody. The problem was treated.

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

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

[0231] Figure 7 shows the results of IL-4Rα monoclonal antibody, IL-5 monoclonal antibody, and IL-4Rα / IL-5 combination. HDM-treated mice administered monoclonal antibodies or an irrelevant IgG2a antibody Differential cell counts in BAL analyzed by FACS. In the study, airway HDM exposure significantly increased inflammatory cells in the BAL fluid compared with PBS-challenged mice. More specifically, it increased the numbers of eosinophils, lymphocytes, and macrophages. Interestingly, compared with treatment with a control IgG2a antibody, monotherapy The lowest number of eosinophils was observed in patients treated with this combination therapy. This was observed in mice that had been exposed to the drug.

[0232] (B. IL-4Rα monoclonal antibody and IL-4Rα / IL-5 monoclonal antibody combination are (Decreases kine production.) Allergen-restimulated mesenteric lymph nodes (IL-4Rα and IL-5 monoclonal antibodies) The effect of IL-1 on cytokine production in MLN (multi-cellular lymphoid tissue) cells was examined in vitro. (2×10 6 cells / mL) by homogenizing the organ through a 100 μm cell sieve Cells were restimulated in vitro with 15 μg / mL HDM in 96-well round-bottom plates for 3 days and then cultured. The supernatant was collected and analyzed using a Read-SET-Go!® ELISA kit (eBioscience). Kine production was determined.

[0233] As shown in Figure 8, effector cytotoxicity in cultures of allergen-restimulated MLN cells was significantly increased. In vitro production of cytokines IL-5 and IL-13 in IgG2a-treated HDM-sensitized mice However, this response was boosted by allergen challenge. The IL-4Rα and IL-5 monoclonal antibody combination significantly reduced the incidence of IL-4Rα and IL-5 deficiency after treatment with the monoclonal antibody In contrast, IL-5 monospecific antibody did not show any significant effect.

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

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

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

[0237] Goblet cell metaplasia (GCM) is induced by IL-13 and is present in the airways of asthmatic mice and humans. It is characterized by increasing the production of Muc5AC, a gel-forming mucin present in the blood. In treated, HDM-sensitized mice, HDM challenge significantly increased the risk of HIV infection compared with PBS challenge. , upregulated Muc5AC expression in the airway epithelium (Fig. 10A). was not affected by IL-4Rα or IL-5 antibody monotherapy. Furthermore, lungs of mice treated with the IL-4Rα / IL-5 antibody combination were significantly higher than those sensitized and challenged with other HDMs. The staining intensity of Muc5AC was significantly reduced compared with the control group.

[0238] To confirm and quantify the effects of IL-4Rα and IL-5 monoclonal antibodies on GCM We then compared the mRNA expression level of Muc5ac with that of Ag, another IL-13 / STAT6 downstream target gene involved in GCM. As with r2, it was measured by qRT-PCR in lung tissue.

[0239] Lungs were snap-frozen in liquid nitrogen and subjected to real-time quantitative inversion as previously described. The plates were kept at -80°C until further processing for quantitative real-time polymerase chain reaction (qRT-PCR) (Dul Laers et al. (2017) Journal of Allergy and Clinical Immunology, 140:76-88). Simply put, we used TriPure Isolation Reagent (Roche, Mannheim, Germany). RNA was obtained and isolated according to the manufacturer's protocol. RNA was reverse transcribed using a RNA synthesis kit (Roche) and the reference genes (Rpl13a, Hprt, and Sdha) were inserted. ) using SYBR Green-based qRT-PCR on a LightCycler 480 system (Roche). and analyzed the samples.

[0240] As shown in Figure 10B, the mRNA expression levels of these two genes were significantly elevated during HDM challenge in mice. IL-4Rα and IL-5 antibodies alone did not induce Muc5ac mRNA or Agr2 mRNA. However, IL-4Rα monoclonal antibody and IL-5 The monoclonal antibody combination significantly inhibited HDM-mediated increases in Muc5ac or Agr2 mRNA levels. It decreased.

[0241] (E. The combination of IL-4Rα and IL-5 monoclonal antibodies reduces lung resistance.) Bronchial hyperresponsiveness (BHR) is a condition that can be caused by small doses of muscarinic receptor agonists, such as methacholine. The tendency of airways to constrict in response to bronchoconstrictors. Pulmonary function is measured by invasive measurements of dynamic resistance. The experiment was carried out using a Flexivent (Scireq).

[0242] Twenty-four hours after the final HDM challenge, nonspecific airway responsiveness was assessed using an ultrasonic nebulizer. Conscious mice were exposed to aerosolized PBS to establish baseline values, followed by aerosolization. Measurements were performed using increasing concentrations of hydroxylated methacholine (0–400 μg / kg). To perform the procedure, mice were anesthetized with urethane, tracheotomized, and intubated with an 18-G catheter, followed by Flex Mechanical ventilation was performed using a ivent device (SCIREQ). The respiratory rate was 12 s with a tidal volume of 0.2 mL. The dynamic resistance was set at 0 breaths / min and a positive end-expiratory pressure of 2 mL H2O was applied. The entrance movements were recorded every 10 seconds for 2 minutes. Before the next dose of methacholine was administered, Baseline resistance was restored.

[0243] As shown in Figure 11, control IgG2a antibody-treated mice, HDM-challenged mice, and In sensitized mice, methacholine significantly reduced the inflammatory response compared to HDM-sensitized mice that received PBS challenge. Treatment with IL-5 and IL-4Rα antibodies significantly reduced BHR. However, mice treated with a combination of IL-4Rα and IL-5 monoclonal antibodies showed no significant reduction in IL-4Rα. Mice were completely protected from the development of BHR. These results suggest that BHR may be a fundamental mechanism underlying chronic airway disease. These results demonstrate the synergistic effect of the IL-4Rα / IL-5 antibody combination in the treatment of IL-4Rα-like tumors.

[0244] Overall, these results suggest that HDM-treated mice were significantly more susceptible to steroids than nonsteroidal anti-inflammatory drugs (NSAIDs). The monospecific antibody combination of αIL-5 and αIL-4Rα is a promising candidate for the treatment of asthma, including GCM and BHR. It was shown that there is a synergistic effect.

[0245] Statistical analysis was performed using GraphPad Prism software v7.01 and Genstat software v19. In all experiments, results are expressed as mean ± standard error of the mean (SEM) and are group-specific. Differences between groups were calculated using one-way ANOVA test. Differences between groups were *P ≤ 0.05 vs. the group challenged with and treated with control IgG2 antibody. Significance was considered when **P≦0.01, ***P≦0.001, and ****P≦0.0001.

[0246] Example 4: Generation and in vitro characterization of IL-4Rα / IL-5 bispecific antibodies The IL-4Rα / IL-5 bispecific antibody was synthesized by using the sequences of the 4RMP36B7 IL-4Rα antibody and the 5MP95G7 IL-5 antibody. The Fc portion of the antibody was generated using a nucleotide sequence that promotes correct chain pairing in the context of a bispecific antibody. The protein was engineered to contain a "knob-into-hole"-like mutation (Ridgway et al., (1996) Protein Engineering, Design and Selection, 9:617-21). In particular, the IL-4Rα antibody 4RMP36B7 inhibits the IL-4Rα signaling pathway in mice. The antibody was designed to introduce the substitutions T366S, L368A, and Y407V within the CH3 domain of the Fc region. The IL-5 antibody 5MP95G7 was engineered to introduce the substitution T366W within the CH3 domain of the murine Fc region. did.

[0247] As described in Godar et al. (2016, Scientific Reports, 6:31621), IL-4Rα and IL- Co-expression of two mutant heavy chains and two light chains of the 5 antibodies resulted in the creation of bispecific antibodies containing the correct heavy-light chain pairing. The purified antibodies were purified using anti-idiotypic VHH antibodies. The purification process is outlined in Figure 12. Illustrated diagrammatically.

[0248] The purity of the IL-4Rα / IL-5 bispecific antibody was confirmed using high-resolution spectroscopy. The dual target property was confirmed by BIAcore.

[0249] A. Binding of IL-4Rα / IL-5 Bispecific Antibodies to IL-4Rα and IL-5 The dual specificity of the IL-4Rα / IL-5 antibody was confirmed using SPR, as shown in Figure 13. The specific antibody specifically binds to the IL-4Rα-Fc immobilized on the chip, followed by the addition of IL-5. The signal increase was observed when the IL-4Rα was coated with the bispecific antibody. This experiment showed that the antibody was able to bind to IL-5 in solution and also to IL-5 in solution (Fig. 13A). This was done sequentially, confirming the bispecificity of the molecule as shown in Figure 13B.

[0250] Taken together, these results suggest that the dual anti-idiotype approach is both pure and functional. These results demonstrate that it is possible to isolate effective IL-4Rα / IL-5 bispecific antibodies.

[0251] Example 5: In vivo characteristics of anti-mouse IL-4Rα / IL-5 bispecific antibody in a mouse HDM model Sexual Assessment) The IL-4Rα / IL-5 bispecific antibody prepared in Example 4 was used in the mouse HDM model described in Example 3 above. The experimental protocol is shown in Figure 14. Mice were subjected to HDM sensitization and challenge protocol, with antibody treatment only during the challenge period. was carried out.

[0252] The in vivo activity of IL-4Rα / IL-5 bispecific antibodies was evaluated using monotherapy (IL-4Rα or IL-5 monospecific antibodies) Equimolar inhibition of the target was compared with the control (αIL-4Rα / αIL-5 monospecific antibody) and the combination (αIL-4Rα / αIL-5 monospecific antibody). To compare the total antibody dose and eliminate differences in the total antibody dose, the antibodies were dosed as follows: a combination of 75 μg of each monospecific antibody and 75 μg of an irrelevant IgG2a antibody; 75 μg of each monospecific antibody was combined at the time of injection; and 150 μg of bispecific antibody.

[0253] Mice treated according to this protocol were subsequently analyzed for the total number of inflammatory cells in the BAL fluid (Figure 15 ); cytokine production in mediastinal lymph nodes (Figure 16); serum immunoglobulin production (Figure 17); goblet cell metaplasia The experimental protocol was as described above. This was carried out as described in Example 3.

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

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

[0256] C. IL-4Rα / IL-5 bispecific antibodies reduce HDM-specific IgE and IgG1 production. As shown in Figure 17, HDM-specific IgE and IgG1 also interacted with the combination of IL-4Rα and IL-5 antibodies, as well as There was a significant decrease after treatment with the bispecific antibody.

[0257] D. IL-4Rα / IL-5 bispecific antibodies reduce Muc5AC and Agr2 expression. To test and compare the effects of bispecific antibodies on goblet cell metaplasia (GCM), Muc5ac The mRNA expression levels of Agr2 and Agr3 were measured in lung tissue. The increased expression of Muc5ac and Agr2 observed in mice was due to the combination of IL-4Rα and IL-5 antibodies, as well as The expression of IL-4Rα was significantly reduced by the bispecific antibody (Fig. 18), whereas the expression of IL-4Rα and IL-5Rα was significantly reduced by the individual anti-IL-4Rα and anti-IL-5Rα antibodies (Fig. 19). Systemic treatment had no significant effect.

[0258] (E. IL-4Rα / IL-5 bispecific antibodies reduce lung resistance.) Treatment with a combination of IL-4Rα and IL-5 antibodies, as well as treatment with bispecific antibodies, is HDM-induced BHR also resulted in pulmonary hypertension, as assessed by choline-induced bronchoconstriction. As shown in Figure 19, the combination of IL-4Rα and IL-5 monoclonal antibodies, and Mice treated with the bispecific antibody were completely protected from developing BHR. The results were observed in HDM-sensitized and challenged mice treated with IL-4Rα and IL-5 antibodies. The observed tolerance was not significantly reduced, which is a fundamental feature of chronic airway disease, especially asthma. Combination therapy (in the form of co-administration of individual IL-4Rα and IL-5 antibodies, or bispecific antibodies) to demonstrate synergistic effects.

[0259] Taken together, these results suggest that a single bispecific antibody simultaneously targets IL-4Rα and IL-5. When administered during the challenge phase of the HDM-induced asthma model, it prevented all significant asthma symptoms. It has been shown to be effective in reducing the characteristics.

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

[0261] Table 15 [Table 15]

[0262] Table 16 [Table 16]

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

[0264] This second IL-4Rα / IL-5 bispecific antibody was used to inhibit IL-5-induced proliferation of TF-1 cells. The bispecific antibodies were tested for their ability to bind IL-4Rα antibody 36B7hIgG1 and IL-5 antibody 95G7hI. It was tested together with IgG1 and 95G7mIgG2a.

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

[0266] Table 17 [Table 17]

[0267] As expected, IL-4Ra antibody had no effect on the proliferation of TF-1 cells, whereas human Ig The 95A7 antibody formulated as either a G1 or murine IgG2a antibody inhibited IL-5-induced cell proliferation Interestingly, the IL-4Rα / IL-5 bispecific antibody inhibited the expression of IL-4Rα more effectively than the 95A7 IgG antibody. Much higher potency = 100-fold increase in potency was able to inhibit IL-5 induced cell proliferation. The VH-VL domain of the 95A7 antibody, constructed as an scFv fragment at the C-terminus of the Fc region of the IL-4Rα IgG antibody, The antibody was much more potent than when organized as a Fab arm in the native IgG structure. Ta.

[0268] Example 8: Effect of anti-mouse IL-4Rα / IL-5 IgG-scFv bispecific antibody in a mouse HDM model In vivo characterization The IL-4Rα / IL-5 bispecific antibody described in Example 7 was used in combination with the antibody described in Examples 3 and 5. The experimental protocol is shown in Figure 22. Mice were sensitized with HDM and then challenged with chalazioni. A range protocol was applied, with antibody treatment only during the challenge phase.

[0269] The in vivo activity of the IL-4Rα / IL-5 bispecific antibody (4Rsc5) was investigated using anti-mIL-4Rα (36B7) and anti-mIL Each injection contained 150 μg or 75 μg of the bispecific (4Rs c5), or combination (in which case 75 μg of each antibody for a total of 150 μg, or The doses were either 36.75 μg of the antibody or 75 μg of the dose per injection.

[0270] Mice treated according to this protocol were subsequently analyzed for eosinophils and lymphocytes in the BAL fluid. The total number of cells (Fig. 23) and the expression of muc5a, spdef, and agr2 (Fig. 24) were evaluated. The experimental protocol was carried out as described in Example 3 above.

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

[0272] Sequencing has shown that this procedure results in approximately 3–5 amino acid substitutions within each V domain (VH and VK). Several libraries (VHm * / VKwt, VHwt / VKm * and VHm * / VKm * ) A recombinant IL-4Rα vector from cynomolgus monkeys (agroBioscience, cat♯ILR-C52H8) was prepared. ) for several rounds of phage display selection. After three rounds of selection, Clones were selected (from rounds 2 and 3) and Fab was generated from periplasmic extracts and purified by SPR. For SPR, the Biacore 3000 was used with either human IL-4Rα or cynomolgus monkey IL-4Rα. The binding (R0) and dissociation (kd, s- 1) was recorded as shown in Table 18 below.

[0273] Table 18 [Table 18]

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

[0275] [Table 19]

[0276] [Table 20]

[0277] [Table 21]

Claims

1. (i) IL-5 antagonists; and (ii) IL-4Rα's antagonist A pharmaceutical composition containing the following:

2. The pharmaceutical composition according to claim 1, wherein the antagonist (i) and the antagonist (ii) jointly inhibit signal transduction via IL-5, IL-4, and IL-13.

3. The pharmaceutical composition according to claim 1 or 2, wherein the antagonist in (i) is an antibody molecule and / or the antagonist in (ii) is an antibody molecule.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antagonist in (i) is an antibody molecule that binds to IL-5.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the antagonist in (ii) is an antibody molecule that binds to IL-4Rα.

6. The pharmaceutical composition according to any one of claims 3 to 5, wherein the antibody molecule in (i) and / or the antibody molecule in (ii) is independently selected from the group consisting of an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single-chain antibody (scFv), an F(ab')2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, a single-arm antibody, a diabody, a triabody, a tetrabody, a VHH antibody, or any antigen-binding molecule formed by a combination, assembly, or conjugation of these antigen-binding fragments.

7. The pharmaceutical composition according to any one of claims 3 to 5, wherein the antibody molecule in (i) and / or the antibody molecule in (ii) is an IgG antibody.

8. The pharmaceutical composition according to any one of claims 3 to 7, wherein the antibody molecule in (i) and / or the antibody molecule in (ii) comprises a CH1 domain, a hinge region, a CH2 domain and / or a CH3 domain of human IgG.

9. The pharmaceutical composition according to any one of claims 3 to 8, wherein the antibody molecule in (i) and / or the antibody molecule in (ii) comprises a human IgG-derived Fc domain.

10. The pharmaceutical composition according to any one of claims 3 to 9, wherein the antibody molecule in (i) and / or the antibody molecule in (ii) comprises an Fc domain derived from human IgG1.

11. The pharmaceutical composition according to claim 9 or 10, wherein the Fc domain is modified by one or more amino acid substitutions to increase its binding affinity to FcRn.

12. The pharmaceutical composition according to claim 11, wherein the Fc domain comprises amino acid substitutions: H433K and N434F, or M252Y, S254T, T256E, H433K and N434F.

13. The pharmaceutical composition according to any one of claims 3 to 12, wherein the antibody molecule in (i) and / or the antibody molecule in (ii) exhibit lower antigen-binding activity under acidic pH than under neutral pH.

14. The pharmaceutical composition according to claim 13, wherein the ratio of the antigen-binding activity under acidic pH to that under neutral pH is at least 2, as evaluated by KD(acidic pH) / KD(neutral pH).

15. A pharmaceutical composition according to any one of claims 1 to 14, comprising an antibody molecule that binds to IL-4Rα and an antibody molecule that binds to IL-5, wherein the antibody molecules are combined within a multispecific antibody.

16. A multispecific antibody containing an antigen-binding region that binds to IL-4Rα and an antigen-binding region that binds to IL-5.

17. The multispecific antibody according to claim 16, wherein the multispecific antibody is a bispecific antibody.

18. The multispecific antibody according to claim 16 or 17, wherein the antigen-binding region that binds to IL-4Rα includes 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 includes a second variable heavy chain domain (VH) and a variable light chain domain (VL) pair.

19. The multispecific antibody according to any one of claims 16 to 18, wherein the antibody is an IgG antibody having a first VH-VL pair that binds to IL-4Rα and a second VH-VL pair that binds to IL-5.

20. The multispecific antibody according to any one of claims 16 to 19, 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 under acidic pH than under neutral pH.

21. The multispecific antibody according to claim 20, wherein the ratio of the antigen-binding activity under acidic pH to that under neutral pH is at least 2, as evaluated by KD(acidic pH) / KD(neutral pH).

22. Use of a multispecific antibody according to any one of claims 16 to 21 in the manufacture of a pharmaceutical product for the treatment of chronic respiratory diseases in human subjects.

23. The use of an IL-5 antagonist in the manufacture of a pharmaceutical product for the treatment of a chronic airway disease in human subjects, wherein the treatment comprises administering the antagonist in combination with an IL-4Rα antagonist.

24. The use of an IL-4Rα antagonist in the manufacture of a pharmaceutical product for the treatment of a chronic airway disease in human subjects, wherein the treatment comprises administering the antagonist in combination with an IL-5 antagonist.

25. The use according to claim 23 or 24, wherein the IL-5 antagonist is an antibody molecule that binds to IL-5, and the IL-4Rα antagonist is an antibody molecule that binds to IL-4Rα.

26. The use according to any one of claims 22 to 25, wherein the chronic airway disease is asthma.

27. A pharmaceutical composition according to any one of claims 1 to 15, or a pharmaceutical composition comprising a multispecific antibody according to any one of claims 16 to 21, for use in treating chronic respiratory diseases in human subjects.

28. The pharmaceutical composition according to claim 27, wherein the chronic airway disease is selected from: asthma; chronic sinusitis (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; progressive eosinophilic esophagitis; nasal polyposis; chronic sinusitis; Churg-Strauss syndrome; allergic bronchopulmonary aspergillosis (ABPA); eosinophilic syndrome; bullous pemphigoid and cystic fibrosis.

29. The pharmaceutical composition according to claim 27 or 28, wherein the chronic airway disease is asthma.

30. The pharmaceutical composition according to claim 29, for use in treating severe asthma, severe refractory asthma, type II asthma, or atopic or allergic asthma.

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