Biopharmaceutical compositions and related methods

Abstract

The present disclosure relates to antigen binding compositions, including antibodies, and related methods for treating interleukin 5 (IL-5) mediated diseases. IL-5 is a secreted protein. IL-5 plays a role in a variety of different diseases, such as asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis. The disclosed compositions are suitable for treating these severe IL-5 mediated diseases.

Description

[0001] This application is a divisional application of a patent application filed on May 24, 2018, with a priority date of May 26, 2017, application number 201880046323.0, entitled "Biopharmaceutical Compositions and Related Methods". Invention Field

[0002] This disclosure relates to compositions and related methods for treating interleukin-5 (IL-5) mediated diseases. Background of the Invention

[0003] IL-5 is a secreted protein. IL-5 plays a role in a variety of diseases, such as asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis. These serious diseases affect hundreds of millions of people worldwide.

[0004] This implies a need for compositions suitable for treating IL-5-mediated diseases. This disclosure provides such compositions and related methods. Invention Overview

[0005] One aspect of this disclosure is an antigen-binding protein comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO:5, the CDRH2 amino acid sequence shown in SEQ ID NO:6, and the CDRH3 amino acid sequence shown in SEQ ID NO:7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO:8, the CDRL2 amino acid sequence shown in SEQ ID NO:9, and the CDRL3 amino acid sequence shown in SEQ ID NO:10.

[0006] Another aspect of this disclosure is an antigen-binding protein comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0007] Another aspect of this disclosure is an antibody comprising a heavy chain and a light chain, wherein a) the heavy chain comprises a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO:5, the CDRH2 amino acid sequence shown in SEQ ID NO:6, and the CDRH3 amino acid sequence shown in SEQ ID NO:7; and b) the light chain comprises a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO:8, the CDRL2 amino acid sequence shown in SEQ ID NO:9, and the CDRL3 amino acid sequence shown in SEQ ID NO:10.

[0008] Another aspect of this disclosure is an antibody comprising a heavy chain and a light chain, wherein a) the heavy chain comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and b) the light chain comprises a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0009] Another aspect of this disclosure is an antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO:1 and a light chain having the amino acid sequence shown in SEQ ID NO:2.

[0010] Another aspect of this disclosure is a peptide chain comprising the amino acid sequence shown in SEQ ID NO:3.

[0011] Another aspect of this disclosure is a peptide chain comprising the amino acid sequence shown in SEQ ID NO:1.

[0012] Another aspect of this disclosure is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:3 and a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:4.

[0013] Another aspect of this disclosure is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:1 and a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:2.

[0014] Another aspect of this disclosure is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:3.

[0015] Another aspect of this disclosure is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:1.

[0016] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:15 and a nucleic acid having the sequence shown in SEQ ID NO:16.

[0017] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:17 and a nucleic acid having the amino acid sequence shown in SEQ ID NO:18.

[0018] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:13 and a nucleic acid having the amino acid sequence shown in SEQ ID NO:14.

[0019] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:15.

[0020] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:17.

[0021] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:13.

[0022] Another aspect of this disclosure is a method for generating a peptide chain comprising the amino acid sequence shown in SEQ ID NO:3, the method comprising the steps of: culturing a recombinant host cell comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:3; and recovering the peptide chain.

[0023] Another aspect of this disclosure is a method for generating antibodies, comprising the steps of: a) culturing a recombinant host cell containing an expression vector, the expression vector containing nucleic acids having the sequence shown in SEQ ID NO:17 and nucleic acids having the sequence shown in SEQ ID NO:18; and b) recovering the antibody; thereby generating the antibody.

[0024] Another aspect of this disclosure is a pharmaceutical composition comprising: a) an antigen-binding protein comprising a heavy chain variable region having the amino acid sequences CDRH1 shown in SEQ ID NO:5, CDRH2 shown in SEQ ID NO:6, and CDRH3 shown in SEQ ID NO:7; and a light chain variable region having the amino acid sequences CDRL1 shown in SEQ ID NO:8, CDRL2 shown in SEQ ID NO:9, and CDRL3 shown in SEQ ID NO:10; and b) a pharmaceutically acceptable carrier.

[0025] Another aspect of this disclosure is a pharmaceutical composition comprising: a) an antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO:5, the CDRH2 amino acid sequence shown in SEQ ID NO:6, and the CDRH3 amino acid sequence shown in SEQ ID NO:7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO:8, the CDRL2 amino acid sequence shown in SEQ ID NO:9, and the CDRL3 amino acid sequence shown in SEQ ID NO:10; and b) a pharmaceutically acceptable carrier.

[0026] Another aspect of this disclosure is a pharmaceutical composition comprising: a) an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and the light chain comprises a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4; and b) a pharmaceutically acceptable carrier.

[0027] Another aspect of this disclosure is a pharmaceutical composition comprising: a) an antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO:1 and a light chain having the amino acid sequence shown in SEQ ID NO:2; and b) a pharmaceutically acceptable carrier.

[0028] Another aspect of this disclosure is a method for treating mild to moderate asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of mild to moderate asthma; and (b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:5, a CDR amino acid sequence as shown in SEQ ID NO:6, and a CDR amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:8, a CDR amino acid sequence as shown in SEQ ID NO:9, and a CDR amino acid sequence as shown in SEQ ID NO:10; thereby treating the subject with mild to moderate asthma.

[0029] Another aspect of this disclosure is a method for treating mild to moderate asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of mild to moderate asthma; and (b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating the subject with mild to moderate asthma.

[0030] Another aspect of this disclosure is a method for treating mild to moderate asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of mild to moderate asthma; and (b) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating the subject with mild to moderate asthma.

[0031] Another aspect of this disclosure is a method for treating moderate-to-severe asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of severe asthma; and (b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:5, a CDR amino acid sequence as shown in SEQ ID NO:6, and a CDR amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:8, a CDR amino acid sequence as shown in SEQ ID NO:9, and a CDR amino acid sequence as shown in SEQ ID NO:10; thereby treating the subject with moderate-to-severe asthma.

[0032] Another aspect of this disclosure is a method for treating moderate to severe asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of severe asthma; and (b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating the subject with moderate to severe asthma.

[0033] Another aspect of this disclosure is a method for treating moderate to severe asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of severe asthma; and (b) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2.

[0034] This treatment was used to treat moderate to severe asthma in the subjects.

[0035] Another aspect of this disclosure is a method for treating moderate to severe atopic dermatitis in subjects with this need, comprising the steps of: a) identifying subjects having at least one of the following groups: i) a diagnosis of atopic dermatitis according to the Eichenfield-modified Hanifin and Rajka criteria; ii) a prior diagnosis of atopic dermatitis for more than or equal to about two years prior to treatment; iii) an overall assessment score of more than or equal to about 3 from a healthcare professional; iv) atopic dermatitis involvement of more than or equal to about 10% of the body surface area; v) an eczema area and severity index score of more than or equal to 16; vi) more than per μL The absolute blood eosinophil count is 150 cells or more, 200 cells or more per μL, 300 cells or more per μL, or 350 cells or more per μL; and vii) at least one condition selected from the group consisting of: 1) inadequate response to topical medication for atopic dermatitis for 6 months or more; 2) poor tolerance to topical medication for atopic dermatitis; 3) side effects from topical medication for atopic dermatitis; and 4) inadequate response to nonpharmacological treatment for atopic dermatitis; and b) administration of a therapeutically effective amount of an antibody to the subject, said antibody comprising a heavy chain variable region having as shown in SEQ The CDR amino acid sequence shown in ID NO:5, the CDR amino acid sequence shown in SEQ ID NO:6, and the CDR amino acid sequence shown in SEQ ID NO:7; and a light chain variable region having the CDR amino acid sequence shown in SEQ ID NO:8, the CDR amino acid sequence shown in SEQ ID NO:9, and the CDR amino acid sequence shown in SEQ ID NO:10; thereby treating atopic dermatitis in a subject.

[0036] Another aspect of this disclosure is a method for treating moderate to severe atopic dermatitis in subjects with this need, comprising the steps of: a) identifying subjects having at least one of the following groups: i) a diagnosis of atopic dermatitis according to the Eichenfield-modified Hanifin and Rajka criteria; ii) a prior diagnosis of atopic dermatitis for more than or equal to about two years prior to treatment; iii) an overall evaluation score of more than or equal to about 3 from a healthcare professional; iv) atopic dermatitis involvement of more than or equal to about 10% of the body surface area; v) an eczema area and severity of more than or equal to 16. Index score; vi) absolute blood eosinophil count of ≥150 cells per μL, ≥200 cells per μL, and ≥350 cells per μL; and vii) at least one condition selected from the group consisting of: 1) inadequate response to topical medication for atopic dermatitis for ≥6 months; 2) poor tolerance to topical medication for atopic dermatitis; 3) side effects from topical medication for atopic dermatitis; and 4) inadequate response to nonpharmacological treatment for atopic dermatitis; and b) administration of a therapeutically effective amount of an antibody to the subject, said antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating atopic dermatitis in the subject.

[0037] Another aspect of this disclosure is a method for treating moderate to severe atopic dermatitis in subjects, comprising the steps of: a) identifying subjects having at least one of the following groups: i) a diagnosis of atopic dermatitis according to the Eichenfield-modified Hanifin and Rajka criteria; ii) a prior diagnosis of atopic dermatitis for more than or equal to about two years prior to treatment; iii) an overall assessment score of more than or equal to about 3 from a healthcare professional; iv) atopic dermatitis involvement of more than or equal to about 10% of the body surface area; v) an eczema area and severity index score of more than or equal to 16; vi) more than or equal to [missing information - likely referring to a specific metric or percentage]. The absolute blood eosinophil count is equal to 150 cells, 200 cells per μL or more, and 350 cells per μL or more; and vii) at least one condition selected from the group consisting of: 1) inadequate response to topical medication for atopic dermatitis for 6 months or more; 2) poor tolerance to topical medication for atopic dermatitis; 3) side effects from topical medication for atopic dermatitis; and 4) inadequate response to nonpharmacological treatment for atopic dermatitis; and b) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating the subject with atopic dermatitis.

[0038] Another aspect of this disclosure is a method for reducing absolute blood eosinophil count in a subject, comprising the steps of: (a) identifying a subject with a condition selected from the group consisting of: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, and atopic dermatitis; and (b) administering to the subject a therapeutically effective amount of an antibody, the antibody comprising a heavy chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:5, a CDR amino acid sequence as shown in SEQ ID NO:6, and a CDR amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:8, a CDR amino acid sequence as shown in SEQ ID NO:9, and a CDR amino acid sequence as shown in SEQ ID NO:7. The CDR amino acid sequence shown in ID NO:10; thereby reducing the absolute blood eosinophil count in the subject.

[0039] Another aspect of this disclosure is a method for reducing absolute blood eosinophil count in a subject, comprising the steps of: (a) identifying a subject with a condition selected from the group consisting of: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and (b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating atopic dermatitis in the subject.

[0040] Another aspect of this disclosure is a method for reducing absolute blood eosinophil count in a subject, comprising the steps of: (a) identifying a subject with a condition selected from the group consisting of: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and (b) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby reducing absolute blood eosinophil count in the subject.

[0041] The present invention includes the following embodiments:

[0042] Implementation Scheme 1. An antigen-binding protein comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO:5, the CDRH2 amino acid sequence shown in SEQ ID NO:6, and the CDRH3 amino acid sequence shown in SEQ ID NO:7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO:8, the CDRL2 amino acid sequence shown in SEQ ID NO:9, and the CDRL3 amino acid sequence shown in SEQ ID NO:10.

[0043] Implementation Scheme 2. The antigen-binding protein of Implementation Scheme 1, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0044] Implementation Scheme 3. The antigen-binding protein of Implementation Scheme 2, comprising a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0045] Implementation Scheme 4. An antigen-binding protein comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0046] Implementation Scheme 5. The antigen-binding protein of Implementation Scheme 4, comprising a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0047] Implementation Scheme 6. An antibody, comprising a heavy chain and a light chain, wherein

[0048] a) The heavy chain includes a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO:5, the CDRH2 amino acid sequence shown in SEQ ID NO:6, and the CDRH3 amino acid sequence shown in SEQ ID NO:7; and

[0049] b) The light chain includes a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO:8, the CDRL2 amino acid sequence shown in SEQ ID NO:9, and the CDRL3 amino acid sequence shown in SEQ ID NO:10.

[0050] Implementation Scheme 7. The antibody of Implementation Scheme 6, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0051] Implementation Scheme 8. The antibody of Implementation Scheme 7, wherein the heavy chain comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256.

[0052] Implementation Scheme 9. An antibody, comprising a heavy chain and a light chain, wherein

[0053] a) The heavy chain comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and

[0054] b) The light chain comprises a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0055] Implementation Scheme 10. The antibody of Implementation Scheme 9, wherein the heavy chain comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256.

[0056] Implementation Scheme 11. An antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO:1 and a light chain having the amino acid sequence shown in SEQ ID NO:2.

[0057] Implementation scheme 12. A peptide chain comprising the amino acid sequence shown in SEQ ID NO:3.

[0058] Implementation Scheme 13. A peptide chain comprising the amino acid sequence shown in SEQ ID NO:1.

[0059] Implementation Scheme 14. A composition comprising a nucleic acid encoding the heavy chain variable region of Implementation Scheme 1 and a nucleic acid encoding the light chain variable region of the antigen-binding protein of Implementation Scheme 1.

[0060] Implementation Scheme 15. A composition comprising a nucleic acid encoding the heavy chain variable region of Implementation Scheme 2 and a nucleic acid encoding the light chain variable region of Implementation Scheme 2.

[0061] Implementation Scheme 16. A composition comprising a nucleic acid encoding a heavy chain Fc domain of Implementation Scheme 3 linked to the carboxyl terminus of the heavy chain variable region and a nucleic acid encoding the light chain variable region of Implementation Scheme 3.

[0062] Implementation Scheme 17. A composition comprising a nucleic acid encoding the heavy chain variable region of Implementation Scheme 6 and a nucleic acid encoding the light chain variable region of the antigen-binding protein of Implementation Scheme 6.

[0063] Implementation Scheme 18. A composition comprising a nucleic acid encoding the heavy chain variable region of Implementation Scheme 7 and a nucleic acid encoding the light chain variable region of Implementation Scheme 7.

[0064] Implementation Scheme 19. A composition comprising a nucleic acid encoding a heavy chain Fc domain of Implementation Scheme 8 linked to the carboxyl terminus of the heavy chain variable region and a nucleic acid encoding the light chain variable region of Implementation Scheme 8.

[0065] Implementation Scheme 20. A composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:3 and a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:4.

[0066] Implementation Scheme 21. A composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:1 and a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:2.

[0067] Implementation Scheme 22. A composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:3.

[0068] Implementation Scheme 23. A composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:1.

[0069] Implementation Scheme 24. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:15 and a nucleic acid having the sequence shown in SEQ ID NO:16.

[0070] Implementation Scheme 25. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:17 and a nucleic acid having the sequence shown in SEQ ID NO:18.

[0071] Implementation Scheme 26. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:13 and a nucleic acid having the sequence shown in SEQ ID NO:14.

[0072] Implementation Scheme 27. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:15.

[0073] Implementation Scheme 28. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:17.

[0074] Implementation Scheme 29. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:13.

[0075] Implementation Scheme 30. A composition comprising at least one selected from the group consisting of: a) a nucleic acid encoding the heavy chain variable region of Implementation Scheme 1; b) a nucleic acid encoding the heavy chain variable region of Implementation Scheme 2; and c) a nucleic acid encoding a heavy chain Fc domain connected to the carboxyl terminus of the heavy chain variable region as in Implementation Scheme 3. Brief description of the attached diagram

[0076] Figure 1 When tested in the concentration range of 1 nM–0.042 pM, both 28Y042-7F11-1 and mepolizumab caused dose-dependent inhibition (IC50) of human IL-5-induced TF-1 cell proliferation. 50 (4pM and 105pM respectively).

[0077] Figure 2 Inhibition of recombinant IL-5-mediated eosinophil morphology changes in human whole blood by .28Y042-7F11-1 and mepolizumab. Data shown represent 6 donors.

[0078] Figure 3 The binding of natural IL-5 to 28Y042-7F11-1 and mepolizumab was determined by ELISA.

[0079] Figure 4 Stability comparison of 28Y042-7F11-1 cells in pooled human or cynomolgus monkey serum incubated at 37°C for 6 weeks. Data plotted by comparison with previous results obtained from mepolizumab and IL-13-specific control antibody.

[0080] Figure 5Animal variation in proportion of eosinophilic response relative to pre-drug administration. The ratio of animal eosinophilic response to the geometric mean of its pre-drug values ​​(days -12 to -26). Values ​​calculated for each animal, with the overall geometric mean grouped values ​​added as horizontal bars. The horizontal solid line corresponds to 20% of the animal's original eosinophil count. n = 4 per group [2♀ & 2♂]; drug administered once intravenously (IV) on day 1 of the study.

[0081] Figure 6 Serum total IL-5 levels in cynomolgus monkeys from a 9-month PK / PD study (data up to day 267 / week 38) were measured in animals treated with 28Y042-7F11-1, mepolizumab, or a mediator. 28Y042-7F11-1 demonstrated a prolonged duration and increased amplitude of the serum total IL-5:antibody complex. The lowest level of IL-5 quantification (LLOQ) in cynomolgus monkeys was 9.77 pg / ml.

[0082] Figure 7 Mean serum concentrations (ng / ml) of 28Y042-7F11-1 and mepolizumab (SB-240563) in cynomolgus monkeys following a single intravenous (IV) or subcutaneous (SC) injection.

[0083] Figure 8 Box 1-1. Diagnostic Flowchart in Clinical Practice - Initial Visit.

[0084] Figure 9 Boxes 3-5. Methods for gradually controlling symptoms and minimizing future risks. Invention Details

[0085] This disclosure provides compositions for treating interleukin-5 (IL-5) mediated diseases, and related topics.

[0086] As used in this article, the term "asthma" refers to an inflammatory disease of the airways characterized by reversible airflow obstruction and bronchospasm. Common symptoms include wheezing, coughing, chest tightness, and shortness of breath. Asthma is a heterogeneous disease typically characterized by chronic airway inflammation. It is defined by a history of respiratory symptoms such as wheezing, shortness of breath, chest tightness, and coughing that vary in time and intensity, as well as variable expiratory airflow limitation.

[0087] In the methods disclosed herein, asthma can be diagnosed in subjects according to the guidelines provided in the Global Initiative for Asthma (GINA) Global Asthma Management and Prevention Strategy (2016 Update) document. Those skilled in the art will be familiar with the GINA clinical practice diagnostic flowchart shown below. Figure 8Diagnostic criteria for asthma in adults, adolescents, and children aged 6–11 years (Table 1) and other aspects of the guidelines (e.g., for pregnant women, etc.). See also Tables 2 and 3.

[0088] Table 1.

[0089]

[0090]

[0091] Table 2.

[0092]

[0093]

[0094] Table 3.

[0095]

[0096]

[0097] In the methods disclosed herein, "asthma" can be "mild asthma," "moderate asthma," or "severe asthma." In the methods disclosed herein, asthma severity can be assessed according to GINA guidelines. Specifically, asthma severity can be retrospectively assessed from the level of treatment required to control symptoms and acute exacerbations. For example, it can be assessed once a patient has been on controller therapy for several months, and, where appropriate, slowing treatment can be attempted to find the patient's lowest effective treatment level. Asthma severity is not a static characteristic and may change over months or years.

[0098] Asthma severity can be assessed when a patient has been receiving regular controller therapy for several months:

[0099] • "Mild asthma" refers to asthma that is well controlled with treatment in step 1 or step 2 (see [link]). Figure 9 (See boxes 3-5), the treatment is to use a single, on-demand relief medication or a low-intensity control agent, such as a low-dose ICS, leukotriene receptor antagonist, or cromolyn.

[0100] • "Moderate asthma" refers to asthma that is well controlled with step 3 treatment (see [link]). Figure 9 (See boxes 3-5), the treatment being, for example, low-dose ICS / LABA.

[0101] • “Severe asthma” refers to asthma that requires treatment in step 4 or step 5 (see [link to treatment]). Figure 9(Boxes 3-5) The treatments described include, for example, high-dose ICS / LABA to prevent it from becoming “uncontrolled,” or asthma that remains “uncontrolled” despite such treatment. While many patients with uncontrolled asthma may be difficult to treat due to inadequate or inappropriate treatment, or persistent adherence or comorbidities (such as chronic rhinosinusitis or obesity), the European Respiratory Society / American Thoracic Society Task Force on Severe Asthma considers the definition of “severe asthma” to be reserved for patients with refractory asthma and those who do not respond adequately to treatment of comorbidities. Table 4 may also be referenced during the assessment of asthma severity.

[0102] Table 4.

[0103]

[0104]

[0105] In the methods disclosed herein, "asthma" can be "mild eosinophilic asthma", "moderate eosinophilic asthma" or "severe eosinophilic asthma".

[0106] "Mild eosinophilic asthma" is mild asthma with an eosinophilic phenotype. For example, a subject with mild eosinophilic asthma may have mild asthma and the following blood eosinophil counts: ≥150 eosinophils per μL of blood in the past 12 months, ≥200 eosinophils per μL of blood in the past 12 months, ≥300 eosinophils per μL of blood in the past 12 months, or ≥350 eosinophils per μL of blood in the past 12 months.

[0107] "Moderate eosinophilic asthma" is moderate asthma with an eosinophilic phenotype. For example, a subject with moderate eosinophilic asthma may have moderate asthma and the following blood eosinophil counts: ≥150 eosinophils per μL of blood in the past 12 months, ≥200 eosinophils per μL of blood in the past 12 months, ≥300 eosinophils per μL of blood in the past 12 months, or ≥350 eosinophils per μL of blood in the past 12 months.

[0108] “Severe eosinophilic asthma” is severe asthma with an eosinophilic phenotype. For example, a subject with severe eosinophilic asthma may have severe asthma and the following blood eosinophil counts: ≥150 eosinophils per μL of blood in the past 12 months, ≥200 eosinophils per μL of blood in the past 12 months, ≥300 eosinophils per μL of blood in the past 12 months (preferably), or ≥350 eosinophils per μL of blood in the past 12 months.

[0109] Subjects with severe eosinophilic asthma may also meet one or more of the criteria described in Table 5.

[0110] Table 5.

[0111]

[0112] Importantly, according to these criteria, subjects with severe eosinophilic asthma can have less than 150 eosinophils per μL of blood at the start of treatment.

[0113] 28Y042-7F11-1 is a monoclonal antibody comprising the heavy chain amino acid sequence shown in SEQ ID NO:1 and the light chain amino acid sequence shown in SEQ ID NO:2. According to the methods of this disclosure, 28Y042-7F11-1 and antigen-binding proteins (particularly antibody molecules) comprising the heavy chain CDR and light chain CDR of 28Y042-7F11-1 of this disclosure can be used to treat severe eosinophilic asthma. For example, 28Y042-7F11-1 or the antigen-binding protein of this disclosure can be indicated for adjunctive maintenance therapy for severe eosinophilic asthma, as identified by: a blood eosinophil count greater than or equal to 300 cells / μL in the past 12 months and / or a blood eosinophil count greater than or equal to 150 cells / μL at the start of treatment and / or a blood eosinophil count less than 150 cells / μL at the start of treatment. Alternatively, 28Y042-7F11-1 or the antigen-binding protein of this disclosure may be indicated for additional maintenance therapy in severe eosinophilic asthma, as identified by: a blood eosinophil count greater than or equal to 300 cells / μL in the past 12 months and / or a blood eosinophil count greater than or equal to 150 cells / μL at the start of treatment. 28Y042-7F11-1 or the antigen-binding protein of this disclosure may also be indicated for additional maintenance therapy in severe eosinophilic asthma, as identified by: a blood eosinophil count greater than or equal to 300 cells / μL in the past 12 months and / or a blood eosinophil count less than 150 cells / μL at the start of treatment. Such patients may be 12 years of age or older. Treatment with 28Y042-7F11-1 or the antigen-binding protein of this disclosure may reduce acute exacerbations of asthma in patients (e.g., patients with a history of acute exacerbations). When instructed to be treated with 28Y042-7F11-1 or the antigen-binding protein of this disclosure, the method of the present invention can be used (i.e., such treatment with 28Y042-7F11-1 can be combined with the method of this disclosure). Treatment with 28Y042-7F11-1 and the antigen-binding protein of this disclosure can:

[0114] a) Reduction in the frequency of acute exacerbations. Compared with placebo, treatment with 28Y042-7F11-1 or the antigen-binding protein disclosed herein can reduce the following rates: 1) clinically significant acute exacerbations, 2) acute exacerbations requiring hospitalization or ED presentation, and 3) acute exacerbations requiring hospitalization. This benefit may potentially lead to a reduction in morbidity and fatal events related to asthma.

[0115] b) Reduction in daily OCS dose: Treatment with 28Y042-7F11-1 or the antigen-binding protein of this disclosure allows subjects to reduce their daily dose of concomitant corticosteroids without loss of asthma control. Subjects treated with 28Y042-7F11-1 or the antigen-binding composition of this disclosure achieved a median percentage reduction in daily oral corticosteroid (OCS) dose from baseline compared to subjects treated with placebo. Additionally, subjects treated with 28Y042-7F11-1 or the antigen-binding composition of this disclosure achieved a reduction in OCS dose compared to 32% of subjects treated with placebo.

[0116] c) Improvement in lung function: Clinically relevant changes in FEV1 before and after treatment with bronchodilators, compared to placebo, can be demonstrated by treatment with 28Y042-7F11-1 or the antigen-binding protein disclosed herein. Any improvement in lung function in this subject population is of particular clinical importance, as most subjects are on maximal asthma therapy, including high-dose ICS (inhaled corticosteroids) and / or OCS plus controller medication.

[0117] d) Improved asthma control: Statistically significant and clinically relevant improvements were observed in ACQ-5 with 28Y042-7F11-1 or the antigen-binding protein of this disclosure compared with placebo, indicating that subjects could achieve asthma control by adding 28Y042-7F11-1 or the antigen-binding protein of this disclosure to their existing asthma treatment.

[0118] e) Improvement in quality of life: Statistically significant and clinically relevant changes in SGRQ scores were demonstrated with 28Y042-7F11-1 or the antigen-binding protein of this disclosure compared to placebo. Subjects experienced significant improvements in asthma symptoms and their ability to perform daily activities.

[0119] f) Duration of efficacy and pharmacological effects: During treatment durations of 32 weeks and / or 52 weeks, sustained reductions in acute asthma exacerbations and blood eosinophils, as well as improvements in lung function, asthma control, and quality of life, were observed without the development of tolerance.

[0120] as well as

[0121] g) Decrease in blood eosinophils. Treatment with a composition containing 28Y042-7F11-1 or the antigen-binding protein of this disclosure can result in a rapid decrease in blood eosinophils in the subject.

[0122] In the methods of this disclosure, "asthma" can be "severe asthma". In the methods of this disclosure, "asthma" can also be "mild asthma", "moderate asthma", "severe asthma", "mild eosinophilic asthma", "moderate eosinophilic asthma", or "severe eosinophilic asthma" as discussed above. According to the methods of this disclosure, treatment with a composition comprising 28Y042-7F11-1 or the antigen-binding protein of this disclosure can be used to treat these conditions.

[0123] In the methods disclosed herein, "asthma" can be "uncontrolled eosinophilic asthma". Subjects with uncontrolled eosinophilic asthma meet the criteria described in Table 6.

[0124] Table 6.

[0125]

[0126] According to the method of this disclosure, treatment with a composition containing 28Y042-7F11-1 or the antigen-binding protein of this disclosure can be used to treat uncontrolled eosinophilic asthma.

[0127] In the methods disclosed herein, "asthma" can be "eosinophilic asthma". Subjects with eosinophilic asthma meet the criteria described in Table 7.

[0128] Table 7.

[0129]

[0130] In the methods disclosed herein, "asthma" can be "eosinophilic asthma". Subjects with eosinophilic asthma meet the criteria described in Table 8.

[0131] Table 8.

[0132]

[0133]

[0134] According to the method of this disclosure, treatment with a composition containing 28Y042-7F11-1 or the antigen-binding protein of this disclosure can be used to treat eosinophilic asthma and can also be used to treat subeosinophilic asthma.

[0135] As used herein, the term “bullous pemphigoid” (BP) refers to an acute or chronic autoimmune skin disease involving the formation of vesicles, more appropriately called bullae, in the space between the epidermis and dermis. BP is the most common autoimmune bullous skin disease. It is characterized by affecting older adults (>70 years of age), with an annual incidence of 5 to 35 per million. The incidence of BP increases dramatically at an average rate of 17% per year. BP typically begins with extremely itchy skin lesions, resembling eczema or urticaria, before the appearance of vesicles and bullae. In 10–30% of patients, BP also involves the oral mucosa. Disease severity can be determined by the Autoimmune Bullous Dermatitis Intensity Score (ABSIS), which assesses the area of ​​involvement and disease activity. The disease is attributed to an autoimmune response to structural components of the junctional adhesion complex, resulting in lesions at the dermal-epidermal junction with subepidermal bullous formation. Specifically, autoreactive B and T cell responses to hemidesmosome antigens BP180 and BP230 have been identified. The level of serum autoantibodies against BP180 reflects the severity and activity of the disease. T cells are memory CD4+ cells that produce both Th1 and Th2 cytokines, primarily IL-4, IL-5, and IL-13. High levels of IL-5 and eotaxin were found in vesicular fluid. IL-5 production is indeed associated with blood eosinophilia and significant eosinophilic infiltration in the skin of BP patients. Eosinophils are believed to be closely associated with vesicle formation by releasing toxic granulocyte protein (ESP, MBP) and proteolytic enzymes.

[0136] As used herein, the term “eosinophilic esophagitis” (EoE) refers to an allergic inflammatory condition of the esophagus involving eosinophils. Symptoms include dysphagia, food impaction, and heartburn. EoE is characterized by a dense infiltration of eosinophilic leukocytes into the inner lining of the esophagus. Based on the important role of eosinophils in allergic reactions, EoE is believed to be an allergic reaction to ingested food. An EoE diagnostic panel can be used to diagnose EoE. EoE can also be diagnosed if gastroesophageal reflux does not respond to a 6-week trial of high-dose proton pump inhibitors (PPIs) twice daily, or if a negative dynamic pH study rules out gastroesophageal reflux disease (GERD). Endoscopically, ridges, grooves, or rings can be seen on the esophageal wall. Sometimes, multiple rings may be present in the esophagus, leading to the terms “corrugated esophagus” or “cat esophagus” (due to the similarity of the rings to the cat esophagus). The presence of white exudate in the esophagus also suggests this diagnosis. A biopsy is performed during endoscopy, and numerous eosinophils are typically found in the superficial epithelium. At least 15 eosinophils per high-power field are required for diagnosis. Eosinophilic inflammation is not limited to the esophagus but extends throughout the gastrointestinal tract. Deeply degranulated eosinophils may also be present, such as microabcesses and dilatation of the basal layer. Radiographically, the term "ringed esophagus" has been used in barium swallow studies to contrast with transient transverse folds (called "cat esophagus") sometimes accompanied by esophageal reflux.

[0137] According to the method of this disclosure, treatment with a composition comprising 28Y042-7F11-1 or the antigen-binding protein of this disclosure can be used to treat COPD.

[0138] Subjects with chronic obstructive pulmonary disease (COPD) must meet one or more of the following criteria: a) Previous COPD diagnosis: Subjects with a clinically documented history of COPD for at least one year, as defined by the American Thoracic Society / European Respiratory Society; b) COPD severity: Subjects may have: a forced expiratory volume in one second (FEV1) / forced vital capacity (FVC) ratio <0.70 before and after salbutamol to confirm the diagnosis of COPD; or an FEV1 >20 percent and <= 80 percent after salbutamol, calculated using the National Health and Nutrition Examination Survey (NHANES) III reference equation; c) History of acute exacerbations: A well-documented history (e.g., medical record verification) of at least two moderate COPD acute exacerbations within 12 months. Moderate is defined as treatment with systemic corticosteroids (IM, intravenous, or oral) and / or antibiotics, or at least one severe COPD acute exacerbation. Severe is defined as requiring hospitalization. Note: Subjects taking inhaled corticosteroids (ICS) plus a long-acting beta-2 agonist (LABA) plus a long-acting muscarinic antagonist (LAMA) must have experienced at least one acute exacerbation. Note: This use is not considered a moderate acute exacerbation unless the previous use of an antibiotic alone was specifically for the treatment of symptoms of a COPD exacerbation. d) Concomitant COPD therapy: A well-documented need for optimized standard of care (SoC) background therapy, which includes ICS plus two additional COPD medications (i.e., triple therapy) for the previous 12 months and meets the following criteria: use of inhaled corticosteroids (at a dose >= 500 mcg / day equivalent of fluticasone propionate) for at least 3 months prior to a healthcare provider visit; or LABA and LAMA.

[0139] According to the method of this disclosure, treatment with a composition comprising 28Y042-7F11-1 or the antigen-binding protein of this disclosure can be used to treat COPD.

[0140] As used in this article, the term "eosinophilic granuloma with polyangiitis" (EGPA) refers to an autoimmune condition that causes inflammation (vasculitis) of small and medium-sized blood vessels in individuals with a history of airway allergic hypersensitivity (atopic). EGPA can also be referred to as Churg-Strauss syndrome (CSS) or allergic granuloma. EGPA typically presents in three phases. The early (prodromal) phase is characterized by airway inflammation; almost all patients experience asthma and / or allergic rhinitis. The second phase is characterized by an abnormally high eosinophil count (hypereoeosinophilic syndrome), which causes tissue damage, most commonly affecting the lungs and digestive tract. The third phase is vasculitis, which can ultimately lead to cell death and can be life-threatening.

[0141] Subjects with EGPA may meet one or more of the following criteria: a) asthma; b) serum eosinophil levels greater than 10% of the white blood cell differential count; c) presence of mononeuropathy or polyneuropathy; d) unfixed pulmonary infiltrates; e) presence of paranasal sinus abnormalities; f) histological evidence of extravascular eosinophils. For classification purposes, a patient should be diagnosed with EGPA if at least four of the above six criteria are positive.

[0142] According to the method of this disclosure, treatment with a composition comprising 28Y042-7F11-1 or the antigen-binding protein of this disclosure can be used to treat EGPA. The composition of this disclosure can be administered to EGPA patients at a dose of 300 mg once every 4 weeks.

[0143] As used herein, “hypereoeosinophilic syndrome” (HES) refers to a condition characterized by a persistently elevated eosinophil count (≥1500 eosinophils / mm3) in the blood for at least six months without any identifiable cause, accompanied by involvement of the heart, nervous system or bone marrow.

[0144] Subjects with hypereosinophilic syndrome can meet one or more of the following criteria: a) a documented history of hypereosinophilic syndrome; b) a blood eosinophil count greater than 1500 cells for 6 months; c) signs and symptoms of organ system involvement; and d) no evidence of parasitic, allergic or other causes of eosinophilia after comprehensive evaluation.

[0145] According to the method of this disclosure, treatment with a composition comprising 28Y042-7F11-1 or the antigen-binding protein of this disclosure can be used to treat hypereosinophilic syndrome.

[0146] As used herein, the term "nasal polyposis" refers to a condition characterized by the presence of polyps in the nasal cavity. Such polyps may be located in the superior nasal cavity and / or may originate from the ostiomeatal complex.

[0147] Patients with nasal polyposis may meet one or more of the following criteria: a) a documented history of nasal polyposis; or b) nasal polyps that are clearly visible on examination (e.g., endoscopy).

[0148] According to the method of this disclosure, treatment with a composition comprising 28Y042-7F11-1 or the antigen-binding protein of this disclosure can be used to treat nasal polyps.

[0149] As used herein, the term “atopic dermatitis” refers to an inflammatory skin condition characterized by chronic itching, lichenification, xerosis, erythematous papules and plaques.

[0150] In the methods disclosed herein, "atopic dermatitis" can be "moderate to severe atopic dermatitis". Subjects with moderate to severe atopic dermatitis may meet one or more of the criteria described in Table 9.

[0151] Table 9.

[0152]

[0153]

[0154] Subjects with moderate to severe atopic dermatitis may be children under 18 years of age, adults at least 18 years of age or older, or adults between 18 and 70 years of age (inclusive). Subjects may be male or female. Female subjects to be treated are preferred as they are not pregnant, not breastfeeding, and / or unlikely to become pregnant.

[0155] The diagnosis of atopic dermatitis is based on the Eichenfield-revised Hanifin and Rajka criteria. See Table 10 and Eichenfield et al., 70 J Am Acad Dermatol 338 (2014).

[0156] Table 10.

[0157]

[0158]

[0159] The Healthcare Professional Overall Assessment (HGA) is a clinical tool used to evaluate the current status / severity of atopic dermatitis in a subject. See Rehal et al, 6PLos ONE e17520 (2011) and Table 11. It is a static 5-point morphological assessment of overall disease severity, determined by a trained healthcare professional using clinical features of erythema, infiltration, papule formation, exudation, and crusting as criteria. No previous scores are referenced in the HGA. Each assessment should be considered as a visual “mean” of the severity of all affected areas at the time of assessment.

[0160] Table 11.

[0161]

[0162]

[0163] The percentage of body surface area (%BSA) is an estimate of the percentage of total affected skin with atopic dermatitis. See Table 12. %BSA can be evaluated by looking separately at the inflamed areas within each of the four body surface sites (head and neck, upper extremities, trunk, and lower extremities), and each of these body sites can potentially have up to 100% involvement. To obtain the %BSA area score, the evaluator (e.g., a healthcare professional) estimates the percentage of affected skin in each region and then multiplies it by an appropriate scaling factor to arrive at a %BSA regional involvement value (0.1 for head, 0.2 for upper extremities, 0.3 for trunk, and 0.4 for lower extremities for subjects ≥8 years of age). The regional %BSA involvement values ​​are summed to obtain the total affected %BSA. The regional %BSA area score is also used as part of a matrix to calculate the EASI score.

[0164] Table 12.

[0165]

[0166] The EASI scoring system is a standardized clinical tool for evaluating atopic dermatitis, considering the overall range of affected % body surface area (%BSA) and a severity score for each clinical sign: erythema, induration / papule formation, epidermal exfoliation, and lichenification. See Hanifin et al., 10Exp Dermatol 11 (2001); Rullo et al., 36Allergolet Immunopathol 201 (2008) and Table 13. The %BSA area score from the %BSA assessment is used as part of a matrix to calculate the EASI score. The severity score for each clinical sign (erythema, induration / papule formation, epidermal exfoliation, and lichenification) is graded on a 4-point scale (0 to 3) for each of the four body sites (head and neck, upper extremities, lower extremities, and trunk). The severity scores for each sign at each site are summed, then multiplied by the %BSA area score and by an appropriate scaling factor (0.1 for head, 0.2 for upper extremities, 0.3 for trunk, and 0.4 for lower extremities) to obtain a regional EASI score. These regional EASI scores are then summed to obtain the final EASI score. The EASI score is a static assessment and is not based on previous scores.

[0167] Table 13.

[0168]

[0169]

[0170] A therapeutically effective amount of 28Y042-7F11-1 or the antigen-binding protein disclosed herein can be used to treat patients with atopic dermatitis or to reduce the absolute blood eosinophil count in such patients. This type of atopic dermatitis can be moderate or severe.

[0171] According to the method of the present invention, treatment of atopic dermatitis, such as moderate or severe atopic dermatitis, with 28Y042-7F11-1 or the antigen-binding protein of the present invention can produce at least one result selected from the group consisting of:

[0172] a) The HGA score is 0 or 1, and the HGA has improved by at least 2 levels (e.g., relative to the starting HGA score);

[0173] b) Decreased Eczema Area and Severity Index (EASI) score (e.g., relative to the initial EASI score);

[0174] c) The percentage of total surface area affected (%BSA) decreases (e.g., relative to the initial %BSA); and / or

[0175] d) Healthcare professionals determined that the subjects did not have atopic dermatitis according to the revised Hanifin and Rajka criteria by Eichenfield (Eichenfield et al., 70 J Am Acad Dermatol 338 (2014)).

[0176] The EASI score after treatment according to the methods of this disclosure can be less than 16, such as, for example, from about 0 to less than 16. The EASI score after treatment can also range from about 0 to about 15, about 0 to about 14, about 0 to about 13, about 0 to about 12, about 0 to about 11, about 0 to about 10, about 0 to about 9, about 0 to about 8, about 0 to about 7, about 0 to about 6, about 0 to about 5, about 0 to about 4, about 0 to about 3, about 0 to about 2, about 0 to about 1, from about 1 to less than 16, from about 2 to less than 16, from about 3 to less than 16, from about 4 to less than 16, from about 5 to less than 16, from about 6 to less than 16, from about 7 to less than 16, from about 8 to... Less than 16, from about 9 to less than 16, from about 10 to less than 16, from about 11 to less than 16, from about 12 to less than 16, from about 13 to less than 16, from about 14 to less than 16, from about 15 to less than 16, from about 2 to about 15, from about 3 to about 14, from about 4 to about 13, from about 5 to about 12, from about 6 to about 11, from about 7 to about 10, from about 8 to about 9, from about 0 to about 8, from about 8 to less than 16, from about 0 to about 4, from about 4 to about 8, from about 8 to about 12, and from about 12 to less than 16.

[0177] The percentage of BSA after treatment according to the methods of this disclosure can be less than about 10%, such as from about 0% to less than 10%. The percentage of BSA after treatment can also range from about 1% to less than 10%, from about 2% to less than 10%, from about 3% to less than 10%, from about 4% to less than 10%, from about 5% to less than 10%, from about 6% to less than 10%, from about 7% to less than 10%, from about 8% to less than 10%, from about 9% to less than 10%, from about 0% to about 9%, from about 0% to about 8%, from about 0% to about 7%, from about 0% to about 6%, from about 0% to about 5%, from about 0% to about 4%, from about 0% to about 3%, from about 0% to about 3%, from about 0% to about 2%, from about 0% to about 1%, from about 0% to about 5%, from about 5% to less than 10%, from about 0% to about 2.5%, from about 2.5% to about 5%, from about 5% to about 7.5%, and from about 7.5% to less than 10%.

[0178] As used herein, the term “antigen-binding protein” refers to isolated antibodies, antibody fragments (e.g., Fab, etc.) and other antibody-derived protein constructs (e.g., those containing antibody domains (e.g., domain antibodies, etc.)) that are capable of binding to human IL-5 (SEQ ID NO:11).

[0179] As used herein, the term "antibody" refers to a molecule having an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD, or IgE), and includes monoclonal, recombinant, polyclonal, chimeric, human, and humanized molecules of this type. Monoclonal antibodies can be produced by eukaryotic cell clones that express antibodies. Monoclonal antibodies can also be produced by eukaryotic cell lines that can recombinantly express the heavy and light chains of antibodies by introducing nucleic acid sequences encoding the heavy and light chains of the antibody into the cells. Methods for producing antibodies from various eukaryotic cell lines, such as Chinese hamster ovary cells, hybridomas, or immortalized antibody cells derived from animals (e.g., humans), are well known.

[0180] Antibodies can be derived from rats, mice, primates (such as cynomolgus monkeys, Old World monkeys, or apes), humans, or other sources (such as nucleic acids encoding antibody molecules generated using molecular biology techniques).

[0181] Antibodies can contain a constant region, which can be any isotype or subclass. The constant region can be an IgG isotype, such as IgG1, IgG2, IgG3, IgG4, or variants thereof. The constant region of an antigen-binding protein can be IgG1.

[0182] Antigen-binding proteins may contain one or more modifications selected from mutated constant domains, thereby enabling antibodies to have enhanced effector function / ADCC and / or complement activation.

[0183] Antibodies can bind to target antigens. Examples of such target antigens include human IL-5, which contains the amino acid sequence shown in SEQ ID NO:11.

[0184] 28Y042-7F11-1, comprising the heavy chain amino acid sequence shown in SEQ ID NO:1 and the light chain amino acid sequence shown in SEQ ID NO:2, is an example of an antibody. 28Y042-7F11-1, or the antigen-binding protein of this disclosure, binds to human IL-5 and antagonizes its activity.

[0185] 28Y042-7F11-1 is a recombinant humanized monoclonal antibody (IgG1, Kappa). 28Y042-7F11-1 has two light chains and two heavy chains.

[0186] The heavy chain 28Y042-7F11-1 is encoded by the nucleic acid sequence shown in SEQ ID NO:15. The light chain 28Y042-7F11-1 is encoded by the nucleic acid sequence shown in SEQ ID NO:16.

[0187] The heavy and light chains of 28Y042-7F11-1 are covalently linked by a single disulfide bond, and the heavy chain is interconnected by two disulfide bonds, forming a typical IgG molecule.

[0188] The antigen-binding protein 28Y042-7F11-1 or the present disclosure can be provided as a lyophilized powder containing an antibody and excipients reconstituted with a pharmaceutically acceptable carrier (e.g., sterile water). This reconstituted pharmaceutical composition can then be administered subcutaneously or intravenously (e.g., after further dilution). The antigen-binding protein 28Y042-7F11-1 or the present disclosure can also be provided as a liquid formulation containing an antibody, excipients, and a pharmaceutically acceptable carrier. This liquid pharmaceutical composition can then be administered subcutaneously or intravenously (e.g., after further dilution).

[0189] As used herein, the term "antibody variant" means an antibody that differs from the parent antibody by means of at least one amino acid modification (e.g., by having a different amino acid side chain), a post-translational modification, or other modifications in at least one heavy chain, light chain, or a combination thereof (which results in a structural change relative to the parent antibody (e.g., a different amino acid side chain, a different post-translational modification, or other modifications)). 28Y042-7F11-1 is an example of such a parent antibody. Structural changes can be determined directly by a variety of methods known in the art (e.g., LC-MS, direct sequencing) or indirectly by methods such as isoelectric focusing. Such methods are well known to those skilled in the art.

[0190] As used herein, the term "IL-5" refers to human IL-5 containing the amino acid sequence shown in SEQ ID NO:11.

[0191] As used herein, the term "specific binding" in relation to antigen-binding proteins means that the antigen-binding protein binds to the target antigen and discrete domains or discrete amino acid sequences within the target antigen, while having no or negligible binding to other (e.g., unrelated) proteins. However, this term does not preclude the fact that antigen-binding proteins can also cross-react with closely related molecules (e.g., molecules with high sequence homology or from another genus or species). The antigen-binding proteins described herein can bind to human IL-5 or the human IL-5 receptor with an affinity at least 2, 5, 10, 50, 100, or 1000 times greater than that of the closely related molecules they bind to.

[0192] Binding affinity (K) of antigen-binding protein-target antigen interaction D ) can be 1mM or smaller, 100nM or smaller, 10nM or smaller, 2nM or smaller, or 1nM or smaller. Or, K D It can be between 5 and 10 nM; or between 1 and 2 nM. KD It can be between 1 pM and 500 pM; or between 500 pM and 1 nM. The binding affinity of antigen-binding proteins is determined by the association constant (Ka) and dissociation constant (Kd) (KD = Kd / Ka). This can be verified by BIACORE. TM Binding affinity can be measured, for example, by capturing the test antibody onto a protein A-coated sensor surface and allowing the target antigen to flow through that surface. Alternatively, binding affinity can be measured via FORTEBIO, for example, by capturing the test antibody receptor onto a protein A-coated needle and allowing the target antigen to flow through that surface.

[0193] K d It can be 1x10 -3 Ms -1 or smaller, 1x10 -4 Ms -1 or smaller or 1x10 -5 Ms -1 Or smaller. K d It can be 1x10 -5 Ms -1 and 1x10 -4 Ms -1 Between; or 1x10 -4 Ms -1 and 1x10 -3 Ms -1 between.

[0194] As used herein, the term "specific antigen-binding activity" refers to antigen-binding activity as measured by surface plasmon resonance (SPR). IL-5 specific binding activity can be measured using BIACORE. TM The instrument determines this via SPR, for example, in binding mode. It is the binding activity divided by the total protein content (e.g., 28Y042-7F11-1) in the sample.

[0195] As used herein, the term "FcRn binding activity" refers to neonatal Fc (FcRn) receptor binding activity, as measured by surface plasmon resonance (SPR). FcRn binding can be measured using BIACORE. TM The instrument determines this. It is the binding activity to the FcRn receptor divided by the total protein concentration of the sample.

[0196] The SPR method for specific antigen binding and FcRn binding uses the reference standard 28Y042-7F11-1. The 28Y042-7F11-1 reference standard can be used in assays to obtain system suitability and sample comparability data to ensure the method is performed appropriately. The reference standard allows for the establishment of calibration curves and the interpolation of sample concentrations from the curves.

[0197] "Isolated" means the removal of molecules, such as antigen-binding proteins or nucleic acids, from an environment in which they might be found in nature. For example, molecules can be purified from substances that are normally present in nature. For example, the mass of molecules in a sample can be 95% of the total mass. This disclosure also provides isolated nucleic acids and portions thereof comprising SEQ ID NO: 13, 14, 15, 16, 17 and / or 18, and compositions thereof. Importantly, the nucleic acids of this disclosure are generally provided as compositions that may comprise any combination of the nucleic acids of this disclosure, buffers, residual buffers, salts, anti-charge ions, water, alcohols, or carriers, etc. Alternatively, the compositions of the invention may comprise only the nucleic acids of this disclosure.

[0198] The term “V” used in this article H " and "V L "These refer to the heavy chain variable region and the light chain variable region of the antigen-binding protein, respectively."

[0199] “CDR” is defined as the amino acid sequence of the complementarity-determining region of an antigen-binding protein. These are hypervariable regions of the heavy and light chains of immunoglobulins. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Therefore, as used herein, “CDR” means all three heavy chain CDRs, all three light chain CDRs, all heavy chain and light chain CDRs, or at least one CDR, and at least one of these CDRs is CDRH3. Frame regions follow each of these CDR regions. Acceptable heavy chain variable regions and light chain variable regions, frame 1, frame 2, and frame 3 regions, are readily identifiable by those skilled in the art. Acceptable heavy chain constant regions (including hinge regions) and light chain constant regions are also readily identifiable by those skilled in the art. Acceptable antibody isotypes are similarly readily identifiable by those skilled in the art.

[0200] Throughout the specification, amino acid residues in the variable domain sequence and the full-length antibody sequence are numbered according to Kabat numbering conventions. Similarly, the terms “CDR,” “CDRL1,” “CDRL2,” “CDRL3,” “CDRH1,” “CDRH2,” and “CDRH3” used in the specification follow Kabat numbering conventions.

[0201] It will be apparent to those skilled in the art that alternative numbering conventions exist for amino acid residues in variable domain sequences and full-length antibody sequences. Alternative numbering conventions also exist for CDR sequences, such as those listed according to the Chothia numbering convention. The structure and protein folding of the antibody may mean that other residues are considered part of the CDR sequence, and this should be understood by those skilled in the art.

[0202] Other numbering conventions for CDR sequences available to technicians include the "AbM" (University of Bath) and "contact" (University College London) methods. The minimum overlapping area can be determined using at least two of the Kabat, Chothia, AbM, and contact methods to provide a "minimum binding unit." The minimum binding unit can be a sub-part of the CDR.

[0203] Table 14 below presents a definition using each numbering convention for each CDR or binding unit. The Kabat numbering scheme is used in Table 14 to number the variable domain amino acid sequences. It should be noted that some CDR definitions may vary depending on the individual publication used.

[0204] Table 14.

[0205] H1 31-35 / 35A / 35B 26-32 / 33 / 34 26-35 / 35A / 35B 30-35 / 35A / 35B 31-32 H2 50-65 52-56 50-58 47-58 52-56 H3 95-102 95-102 95-102 93-101 95-101 L1 24-34 24-34 24-34 30-36 30-34 L2 50-56 50-56 50-56 46-55 50-55 L3 89-97 89-97 89-97 89-96 89-96

[0206] The percentage identity between the query nucleic acid sequence and the subject nucleic acid sequence is a "identities" value, expressed as a percentage, calculated using the BLASTN algorithm after pairwise BLASTN alignment when the subject nucleic acid sequence has 100% query coverage with the query nucleic acid sequence. Such pairwise BLASTN alignments between the query and subject nucleic acid sequences are performed using the default settings of the BLASTN algorithm available on the website of the National Center for Biotechnology Institute, where filters for low-complexity regions are disabled. Importantly, the query sequence can be described by the nucleic acid sequence identified in one or more claims herein.

[0207] The nucleic acid sequences that may be useful and included in the compositions and related methods of this disclosure may have a similarity of about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 100% to the nucleic acid sequences identified in this disclosure. In this disclosure, the percentage similarity between the nucleic acid sequences may include any discrete subrange of the above percentage similarity ranges (e.g., any range of integer values ​​within a specific range or discrete subvalues ​​within a specific range).

[0208] The percentage consistency between the query amino acid sequence and the subject amino acid sequence is a "consistency" value, expressed as a percentage, calculated using the BLASTP algorithm after pairwise BLASTP alignment when the subject amino acid sequence has 100% query coverage with the query amino acid sequence. Such pairwise BLASTP alignments between the query and subject amino acid sequences are performed using the default settings of the BLASTP algorithm available on the website of the National Center for Biotechnology Institute, where filters for low-complexity regions are disabled. Importantly, the query sequence can be described by the amino acid sequence identified in one or more claims herein.

[0209] The amino acid sequences that may be useful and included in the compositions and related methods of this disclosure may have a similarity of about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 100% with respect to the amino acid sequences identified in this disclosure. In this disclosure, the percentage similarity between the amino acid sequences may include any discrete subrange of the aforementioned percentage similarity ranges (e.g., any range of integer values ​​within a specific range or discrete subvalues ​​within a specific range).

[0210] The terms “peptide,” “polypeptide,” “protein,” and “peptide chain” each refer to a molecule containing two or more amino acid residues. Peptides can be monomeric or polymeric.

[0211] It is well known in the art that certain amino acid substitutions are considered "conserved." Amino acids are grouped into several groups based on common side-chain characteristics, and substitutions within a group that maintain all or substantially all binding affinity of the antigen-binding protein are considered conserved substitutions. See Table 15. The antigen-binding proteins disclosed herein may contain such "conserved" amino acid substitutions.

[0212] Table 15.

[0213]

[0214]

[0215] As used herein, the term "pharmaceutical composition" means a composition suitable for administration to a patient.

[0216] The pharmaceutical compositions described herein may comprise purified formulations of antibodies as described herein.

[0217] For example, a pharmaceutical formulation may comprise a purified formulation of an antibody, as described herein, in combination with a pharmaceutically acceptable carrier.

[0218] Typically, such pharmaceutical compositions contain pharmaceutically acceptable carriers, as known and required by acceptable pharmaceutical practice. Examples of such carriers include sterilized carriers, such as saline, Ringer's solution, or dextran solution, optionally buffered with a suitable buffer to a pH in the range of 5 to 8.

[0219] The pharmaceutical composition may be administered by injection or infusion (e.g., intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, or portal vein). Such compositions appropriately do not contain visible particulate matter. The pharmaceutical composition may contain between 1 mg and 10 g of antigen-binding protein, for example, between 5 mg and 1 g of antigen-binding protein. Alternatively, the composition may contain between 5 mg and 500 mg of antigen-binding protein, for example, between 5 mg and 50 mg.

[0220] Methods for preparing such pharmaceutical compositions are well known to those skilled in the art. The pharmaceutical composition may contain, in unit doses, between 1 mg and 10 g of antigen-binding protein, optionally together with the instructions for use. The pharmaceutical composition may be lyophilized (freeze-dried) according to methods known or obvious to those skilled in the art to reconstitute it prior to administration. In cases where the antibody has an IgG1 isotype, a copper chelating agent such as citrate (e.g., sodium citrate) or EDTA or histidine may be added to the pharmaceutical composition to reduce the extent of copper-mediated degradation of the antibody of that isotype. The pharmaceutical composition may also contain a solubilizer such as arginine, a surfactant / anti-aggregating agent such as polysorbate 80, and an inert gas such as nitrogen to replace oxygen in the headspace of the vial.

[0221] As used herein, the term "therapeutic effective amount" refers to the amount of a medicine (e.g., an antibody or pharmaceutical composition) that provides therapeutic benefit in treating or managing one or more symptoms of a condition to be treated (e.g., asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis). Examples of treatment or management of one or more symptoms of asthma (including asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, and subeosinophilic asthma) include 1) reducing the frequency of acute asthma exacerbations; 2) reducing the time to the first clinically significant acute exacerbation requiring oral or systemic corticosteroids, hospitalization, and / or emergency department (ED) visit; and 3) reducing the number of acute exacerbations requiring hospitalization (including intubation and admission to the intensive care unit) or ED visit. 4) Reduced time to first acute exacerbation requiring hospitalization or ED visit; 5) Change in FEV1 from baseline before clinical bronchodilator administration; 6) Change in FEV1 from baseline after clinical bronchodilator administration; 7) Change in Asthma Control Questionnaire (ACQ) score from baseline; 8) Improved lung function, such as by spirometry (e.g., vital capacity (VC), forced vital capacity (FVC), forced expiratory volume (FEV1) measured at timed intervals of 0.5, 1.0, 2.0, and 3.0 seconds, and forced expiratory flow rate 25-75% (FEF). 25-75) and maximal spontaneous ventilation (MVV) as evaluated as total vital capacity, tidal volume, residual volume, expiratory reserve volume, inspiratory reserve volume, deep inspiratory volume, inspiratory vital capacity, vital capacity, functional residual volume, residual volume as a percentage of total lung capacity, alveolar volume, actual lung volume including the volume of the conduction airways, forced vital capacity, etc.; 9) reduction in asthma exacerbations requiring control with steroids (e.g., oral steroids administered by any route of administration or steroids such as prednisone, prednisolone, etc.). Such reduction in asthma exacerbations requiring control with steroids can be approximately 50% of the reduction in asthma exacerbations requiring steroids (e.g., oral steroids).

[0222] The effective dosage and treatment regimen are usually determined empirically and may depend on factors such as the patient's age, weight, and health condition, as well as the disease or ailment to be treated. These factors are within the authority of the attending physician.

[0223] The dose of antigen-binding protein administered to a subject is typically between 1 μg / kg and 150 mg / kg, between 0.1 mg / kg and 100 mg / kg, between 0.5 mg / kg and 50 mg / kg, between 1 and 25 mg / kg, between about 0.3 mg / kg and about 3 mg / kg, or between 1 and 10 mg / kg, based on the subject's body weight. For example, the dose may be 10 mg / kg, 30 mg / kg, or 60 mg / kg. The dose may also be from 10 mg / kg to 110 mg / kg, 15 mg / kg to 25 mg / kg, or 15 mg / kg to 100 mg / kg. The antigen-binding protein may be administered, for example, parenteral, subcutaneously, intravenously, or intramuscularly. The dose may also be administered based on the individual subject, for example, about 20 mg to about 750 mg per subject, about 75 mg to about 750 mg per subject, or about 20 mg to about 200 mg per subject. The dose may be any discrete subrange having these dose ranges. For example, the dosage can be based on subcutaneous administration per subject, such as about 100 mg per subject (e.g., once every four weeks) or 300 mg per subject (or other administered doses may be subcutaneous, provided that they achieve approximately the same or equivalent bioavailability as intravenous administration—e.g., three doses of 100 mg per subject to achieve a total dose of 300 mg subcutaneously per subject).

[0224] Any range provided in this document includes all values ​​within the specified range as well as values ​​at the endpoints of the specified range.

[0225] If desired, the effective daily dose of the antibody or antigen-binding protein disclosed herein (e.g., as a pharmaceutical composition) may be administered at appropriate intervals throughout the day as two, three, four, five, six, or more separate doses (optionally, in unit dosage form).

[0226] Dosage can be administered by slow, continuous infusion over periods of 2 to 24 hours, such as 2 to 12 hours, or 2 to 6 hours. Such administration can result in fewer side effects.

[0227] Dosage can be repeated once or multiple times as needed, such as three times daily, once daily, every two days, once weekly, once every 14 days, once monthly, once every three months, once every four months, once every six months, or once every 12 months. Antigen-binding proteins can be administered as maintenance therapy, such as once weekly for six months or longer. They can also be administered intermittently, for example, in a cycle of three to six months, followed by three to six months without a dose, then again for three to six months, and so on.

[0228] For example, the dosage can be administered subcutaneously once every 14 or 28 days, or in multiple doses on each day of administration. In one embodiment, the dosage of the composition is 100 mg once every 4 weeks (28 days).

[0229] Antigen-binding proteins can be administered to subjects in a targeted therapy manner that targets specific sites.

[0230] The antigen-binding protein in the method disclosed herein can be used in combination with one or more other therapeutic agents, such as antibodies or small molecule inhibitors.

[0231] As used herein, the term “treatment” and its grammatical variations refer to therapeutic therapy. When referring to a specific condition, treatment means: (1) improving the condition or one or more biological manifestations of the condition; (2) interfering with a) one or more points in the biological cascade that leads to the condition or is responsible for it, or (b) one or more biological manifestations of the condition; (3) alleviating one or more symptoms, effects, or side effects associated with the condition or its treatment; or (4) slowing the progression of the condition or one or more biological manifestations of the condition; or (5) preventing the onset of one or more biological manifestations of the condition. Prophylactic therapy is also considered here. Those skilled in the art will understand that “prevention” is not an absolute term. In medicine, “prevention” should be understood as the prophylactic administration of medicine to substantially reduce the likelihood or severity of a condition or its biological manifestations, or to delay the onset of such a condition or its biological manifestations.

[0232] The terms “individual,” “subject,” and “patient” are used interchangeably herein. A subject is typically a human being. A subject can also be a mammal, such as a mouse, rat, or primate (e.g., a marmoset or monkey). A subject can be a non-human animal. The antigen-binding proteins, compositions, and methods disclosed herein also have veterinary applications. The subject to be treated can be a farm animal, such as a cow or bull, sheep, pig, cattle, goat, or horse, or a domestic animal, such as a dog or cat. The animal can be of any age, or a mature adult animal.

[0233] Treatment can be therapeutic, preventative, or therapeutic. The recipients will be individuals who have this need. In addition to those who may develop the disease in the future, those requiring treatment may also include individuals already suffering from a specific medical condition.

[0234] Therefore, if specified, the methods, antigen-binding proteins, and compositions of this disclosure can be used for preventative or therapeutic purposes. In this case, the methods, antigen-binding proteins, and compositions of this disclosure can be used to prevent or delay the onset of one or more aspects or symptoms of a disease. The subject may be asymptomatic. The subject may have a genetic susceptibility to the disease. An effective amount of the antigen-binding protein is administered to such individuals. An effective amount is the amount that prevents or delays the onset of one or more aspects or symptoms of the disease described herein.

[0235] The methods, antigen-binding proteins, and compositions of this disclosure do not need to affect every symptom or manifestation of the disease to constitute a viable therapeutic treatment. As is appreciated in the art, a drug used as a therapeutic agent in a treatment method can reduce the severity of a given disease state, but does not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, preventive treatment does not need to completely and effectively prevent the onset of the disease to constitute a viable preventive agent. It is sufficient to simply reduce the effects of the disease (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reduce the likelihood in a subject that the disease will occur (e.g., by delaying the onset of the disease) or worsen.

[0236] One aspect of this disclosure is an antigen-binding protein comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO:5, the CDRH2 amino acid sequence shown in SEQ ID NO:6, and the CDRH3 amino acid sequence shown in SEQ ID NO:7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO:8, the CDRL2 amino acid sequence shown in SEQ ID NO:9, and the CDRL3 amino acid sequence shown in SEQ ID NO:10.

[0237] In one embodiment of the antigen-binding protein disclosed herein, the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0238] In one embodiment, the antigen-binding protein of this disclosure comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, and wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0239] Another aspect of this disclosure is an antigen-binding protein comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0240] Another aspect of this disclosure is an antibody comprising a heavy chain and a light chain, wherein a) the heavy chain comprises a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO:5, the CDRH2 amino acid sequence shown in SEQ ID NO:6, and the CDRH3 amino acid sequence shown in SEQ ID NO:7; and b) the light chain comprises a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO:8, the CDRL2 amino acid sequence shown in SEQ ID NO:9, and the CDRL3 amino acid sequence shown in SEQ ID NO:10.

[0241] In one embodiment of the antibody disclosed herein, the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0242] In one embodiment of the antibody disclosed herein, the heavy chain comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254, and a glutamic acid residue at position 256.

[0243] Another aspect of this disclosure is an antibody comprising a heavy chain and a light chain, wherein a) the heavy chain comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and b) the light chain comprises a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0244] In one embodiment of the antibody disclosed herein, the heavy chain comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254, and a glutamic acid residue at position 256.

[0245] Another aspect of this disclosure is an antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO:1 and a light chain having the amino acid sequence shown in SEQ ID NO:2.

[0246] Another aspect of this disclosure is a peptide chain comprising the amino acid sequence shown in SEQ ID NO:3.

[0247] Another aspect of this disclosure is a peptide chain comprising the amino acid sequence shown in SEQ ID NO:1.

[0248] In one embodiment, the composition of this disclosure comprises a nucleic acid encoding the heavy chain variable region of this disclosure and a nucleic acid encoding the light chain variable region of the antigen-binding protein of this disclosure.

[0249] In one embodiment, the composition of this disclosure comprises a nucleic acid encoding the heavy chain variable region of this disclosure and a nucleic acid encoding the light chain variable region of this disclosure.

[0250] In one embodiment, the composition of this disclosure comprises a nucleic acid encoding a heavy chain Fc domain of the disclosed heavy chain variable region linked to a carboxyl terminus and a nucleic acid encoding a light chain variable region of the disclosed composition.

[0251] Another aspect of this disclosure is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:3 and a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:4.

[0252] Another aspect of this disclosure is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:1 and a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:2.

[0253] Another aspect of this disclosure is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:3.

[0254] Another aspect of this disclosure is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:1.

[0255] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:15 and a nucleic acid having the sequence shown in SEQ ID NO:16.

[0256] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:17 and a nucleic acid having the amino acid sequence shown in SEQ ID NO:18.

[0257] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:13 and a nucleic acid having the amino acid sequence shown in SEQ ID NO:14.

[0258] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:15.

[0259] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:17.

[0260] Another aspect of this disclosure is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO:13.

[0261] In one embodiment, the composition of this disclosure comprises a nucleic acid encoding the heavy chain variable region of this disclosure.

[0262] In one embodiment, the composition of this disclosure comprises a nucleic acid encoding the heavy chain variable region of this disclosure.

[0263] In one embodiment, the composition of this disclosure comprises a nucleic acid encoding a heavy chain Fc domain that is linked to the carboxyl terminus of the heavy chain variable region of this disclosure.

[0264] In one embodiment, the expression vector of this disclosure comprises the composition of this disclosure.

[0265] In one embodiment, the recombinant host cell of this disclosure comprises an expression vector containing the composition of this disclosure. In an alternative embodiment, the recombinant host cell may comprise a first expression vector encoding a first antigen-binding protein peptide chain (e.g., an antibody heavy chain) and a second expression vector encoding a second antigen-binding peptide chain (e.g., an antibody light chain) of this disclosure.

[0266] Another aspect of this disclosure is a method for generating a peptide chain comprising the amino acid sequence shown in SEQ ID NO:3, the method comprising the steps of: culturing a recombinant host cell comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:3; and recovering the peptide chain.

[0267] In one embodiment of the method disclosed herein, the nucleic acid comprises the sequence shown in SEQ ID NO:15.

[0268] In one embodiment of the method disclosed herein, the nucleic acid comprises the sequence shown in SEQ ID NO:13.

[0269] In one embodiment of the method disclosed herein, the nucleic acid comprises the sequence shown in SEQ ID NO:17.

[0270] One embodiment of this disclosure is a method for generating an antigen-binding protein, comprising the steps of: a) culturing a recombinant host cell containing an expression vector comprising the composition of this disclosure; and b) recovering the antigen-binding protein; thereby generating the antigen-binding protein.

[0271] In one embodiment of this disclosure, the antigen-binding protein is generated by the method of this disclosure.

[0272] One embodiment of this disclosure is a method for generating antibodies, comprising the steps of: a) culturing a recombinant host cell containing an expression vector comprising the composition of this disclosure; and b) recovering the antibody; thereby generating the antibody.

[0273] One embodiment of this disclosure is an antibody generated by the method of this disclosure.

[0274] Another aspect of this disclosure is a method for generating antibodies, comprising the steps of: a) culturing a recombinant host cell containing an expression vector, the expression vector containing nucleic acids having the sequence shown in SEQ ID NO:17 and nucleic acids having the sequence shown in SEQ ID NO:18; and b) recovering the antibody; thereby generating the antibody.

[0275] Another aspect of this disclosure is a pharmaceutical composition comprising: a) an antigen-binding protein comprising a heavy chain variable region having the amino acid sequences CDRH1 shown in SEQ ID NO:5, CDRH2 shown in SEQ ID NO:6, and CDRH3 shown in SEQ ID NO:7; and a light chain variable region having the amino acid sequences CDRL1 shown in SEQ ID NO:8, CDRL2 shown in SEQ ID NO:9, and CDRL3 shown in SEQ ID NO:10; and b) a pharmaceutically acceptable carrier.

[0276] In one embodiment of the pharmaceutical composition disclosed herein, the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0277] In one embodiment, the pharmaceutical composition of this disclosure comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, and wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0278] In one embodiment of the pharmaceutical composition disclosed herein, the antigen-binding protein is at a concentration between about 75 mg / ml and about 150 mg / ml.

[0279] In one embodiment of the pharmaceutical composition disclosed herein, the pharmaceutically effective carrier comprises an aqueous liquid formulation at a pH of about 5.5 to about pH 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by weight of polysorbate 80 by volume, and about 0.05 mM EDTA.

[0280] In one embodiment of the pharmaceutical composition disclosed herein, the pH is about 6.0, and the antigen-binding protein is at a concentration of about 150 mg / ml.

[0281] Another aspect of this disclosure is a pharmaceutical composition comprising: a) an antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO:5, the CDRH2 amino acid sequence shown in SEQ ID NO:6, and the CDRH3 amino acid sequence shown in SEQ ID NO:7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO:8, the CDRL2 amino acid sequence shown in SEQ ID NO:9, and the CDRL3 amino acid sequence shown in SEQ ID NO:10; and b) a pharmaceutically acceptable carrier.

[0282] 58. The pharmaceutical composition of claim 57, wherein the antibody is at a concentration between about 75 mg / ml and about 150 mg / ml.

[0283] In one embodiment of the pharmaceutical composition disclosed herein, the pharmaceutically effective carrier comprises an aqueous liquid formulation at a pH of about 5.5 to about pH 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by weight of polysorbate 80 by volume, and about 0.05 mM EDTA.

[0284] In one embodiment of the pharmaceutical composition disclosed herein, the pH is about 6.0, and the antibody is at a concentration of about 150 mg / ml.

[0285] Another aspect of this disclosure is a pharmaceutical composition comprising: a) an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and the light chain comprises a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4; and b) a pharmaceutically acceptable carrier.

[0286] Another aspect of this disclosure is a pharmaceutical composition comprising: a) an antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO:1 and a light chain having the amino acid sequence shown in SEQ ID NO:2; and b) a pharmaceutically acceptable carrier.

[0287] One embodiment of this disclosure is a method for treating a disease in a subject, comprising the steps of: a) identifying a subject suffering from a disease selected from the group consisting of: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and (b) administering a therapeutically effective amount of an antigen-binding protein according to this disclosure to the subject; thereby treating the disease in the subject.

[0288] In one embodiment of the method disclosed herein, the amount of antigen-binding protein is from about 2 mg to about 600 mg. For example, the amount of antigen-binding protein (e.g., antibody) may be a dose of 2 mg, 10 mg, 30 mg, 100 mg, 300 mg, or 600 mg.

[0289] In one embodiment of the method disclosed herein, the antigen-binding protein is administered once every 3 months or once every 6 months.

[0290] In one embodiment of the method disclosed herein, the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0291] One embodiment of this disclosure is a method for treating a disease in a subject, comprising the steps of: a) identifying a subject suffering from a disease selected from the group consisting of: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and (b) administering a therapeutically effective amount of an antibody according to this disclosure to the subject; thereby treating the disease in the subject.

[0292] One embodiment of this disclosure is a method for treating a disease in a subject, comprising the steps of: a) identifying a subject suffering from a disease selected from the group consisting of: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and (b) administering to the subject a therapeutically effective amount of the composition according to this disclosure; thereby treating the disease in the subject.

[0293] Another aspect of this disclosure is a method for treating mild to moderate asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of mild to moderate asthma; and (b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:5, a CDR amino acid sequence as shown in SEQ ID NO:6, and a CDR amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:8, a CDR amino acid sequence as shown in SEQ ID NO:9, and a CDR amino acid sequence as shown in SEQ ID NO:10; thereby treating the subject with mild to moderate asthma.

[0294] One embodiment is the method of this disclosure, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0295] One embodiment is the method of this disclosure, wherein the antibody comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, and wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0296] One embodiment is the method of this disclosure, wherein the antibody comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0297] One implementation is the method disclosed herein, in which antibodies are administered subcutaneously.

[0298] Another aspect of this disclosure is a method for treating mild to moderate asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of mild to moderate asthma; and (b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating the subject with mild to moderate asthma.

[0299] Another aspect of this disclosure is a method for treating mild to moderate asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of mild to moderate asthma; and (b) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating the subject with mild to moderate asthma.

[0300] One embodiment is the method of this disclosure, wherein the pharmaceutically effective carrier comprises an aqueous liquid formulation at a pH of about 5.5 to about pH 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by weight of polysorbate 80 and about 0.05 mM EDTA.

[0301] Another aspect of this disclosure is a method for treating moderate-to-severe asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of severe asthma; and (b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:5, a CDR amino acid sequence as shown in SEQ ID NO:6, and a CDR amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:8, a CDR amino acid sequence as shown in SEQ ID NO:9, and a CDR amino acid sequence as shown in SEQ ID NO:10; thereby treating the subject with moderate-to-severe asthma.

[0302] Another aspect of this disclosure is a method for treating moderate to severe asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of severe asthma; and (b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating the subject with moderate to severe asthma.

[0303] Another aspect of this disclosure is a method for treating moderate to severe asthma in a subject, comprising the steps of: (a) identifying a subject with a diagnosis of severe asthma; and (b) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating the subject with moderate to severe asthma.

[0304] Another aspect of this disclosure is a method for treating moderate to severe atopic dermatitis in subjects with this need, comprising the steps of: a) identifying subjects having at least one of the following groups: i) a diagnosis of atopic dermatitis according to the Eichenfield-modified Hanifin and Rajka criteria; ii) a prior diagnosis of atopic dermatitis for more than or equal to about two years prior to treatment; iii) an overall assessment score of more than or equal to about 3 from a healthcare professional; iv) atopic dermatitis involvement of more than or equal to about 10% of the body surface area; v) an eczema area and severity index score of more than or equal to 16; vi) more than per μL The absolute blood eosinophil count is 150 cells or more, 200 cells or more per μL, 300 cells or more per μL, or 350 cells or more per μL; and vii) at least one condition selected from the group consisting of: 1) inadequate response to topical medication for atopic dermatitis for 6 months or more; 2) poor tolerance to topical medication for atopic dermatitis; 3) side effects from topical medication for atopic dermatitis; and 4) inadequate response to nonpharmacological treatment for atopic dermatitis; and b) administration of a therapeutically effective amount of an antibody to the subject, said antibody comprising a heavy chain variable region having as shown in SEQ The CDR amino acid sequence shown in ID NO:5, the CDR amino acid sequence shown in SEQ ID NO:6, and the CDR amino acid sequence shown in SEQ ID NO:7; and a light chain variable region having the CDR amino acid sequence shown in SEQ ID NO:8, the CDR amino acid sequence shown in SEQ ID NO:9, and the CDR amino acid sequence shown in SEQ ID NO:10; thereby treating atopic dermatitis in a subject.

[0305] One implementation is the method of this disclosure, wherein the antibody is administered intravenously.

[0306] Another aspect of this disclosure is a method for treating moderate to severe atopic dermatitis in subjects with this need, comprising the steps of: a) identifying subjects having at least one of the following groups: i) a diagnosis of atopic dermatitis according to the Hanifin and Rajka criteria revised by Eichenfield; ii) a prior diagnosis of atopic dermatitis for more than or equal to about two years prior to treatment; iii) an overall assessment score of more than or equal to about 3 from a healthcare professional; iv) atopic dermatitis involvement of more than or equal to about 10% of the body surface area; v) an eczema area and severity index score of more than or equal to 16; vi a) Absolute blood eosinophil counts of ≥150 cells per μL, ≥200 cells per μL, ≥300 cells per μL, and ≥350 cells per μL; and vii) at least one condition selected from the group consisting of: 1) inadequate response to topical medication for atopic dermatitis for ≥6 months; 2) poor tolerance to topical medication for atopic dermatitis; 3) side effects from topical medication for atopic dermatitis; and 4) inadequate response to nonpharmacological treatment for atopic dermatitis; and b) administration of a therapeutically effective amount of an antibody to the subject, said antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO:1 and a light chain having the amino acid sequence shown in SEQ ID NO:2; thereby treating atopic dermatitis in the subject.

[0307] Another aspect of this disclosure is a method for treating moderate to severe atopic dermatitis in subjects, comprising the steps of: a) identifying subjects having at least one of the following groups: i) a diagnosis of atopic dermatitis according to the Eichenfield-modified Hanifin and Rajka criteria; ii) a prior diagnosis of atopic dermatitis for more than or equal to about two years prior to treatment; iii) an overall assessment score of more than or equal to about 3 from a healthcare professional; iv) atopic dermatitis involvement of more than or equal to about 10% of the body surface area; v) an eczema area and severity index score of more than or equal to 16; vi) more than or equal to 150 cells per μL. a) Absolute blood eosinophil counts of ≥200 cells per μL, ≥300 cells per μL, and ≥350 cells per μL; and vii) at least one condition selected from the group consisting of: 1) inadequate response to topical medication for atopic dermatitis for ≥6 months; 2) poor tolerance to topical medication for atopic dermatitis; 3) side effects from topical medication for atopic dermatitis; and 4) inadequate response to nonpharmacological treatment for atopic dermatitis; and b) administration of a therapeutically effective amount of a pharmaceutical composition to the subject, the pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating atopic dermatitis in the subject.

[0308] Another aspect of this disclosure is a method for reducing absolute blood eosinophil count in a subject, comprising the steps of: (a) identifying a subject with a condition selected from the group consisting of: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, and atopic dermatitis; and (b) administering to the subject a therapeutically effective amount of an antibody, the antibody comprising a heavy chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:5, a CDR amino acid sequence as shown in SEQ ID NO:6, and a CDR amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:8, a CDR amino acid sequence as shown in SEQ ID NO:9, and a CDR amino acid sequence as shown in SEQ ID NO:7. The CDR amino acid sequence shown in ID NO:10; thereby reducing the absolute blood eosinophil count in the subject.

[0309] One embodiment of the method disclosed herein further includes the following steps: a) performing a first measurement of the absolute blood eosinophil count in the subject; b) performing a second measurement of the absolute blood eosinophil count in the subject after administering a therapeutically effective amount of antigen-binding protein to the subject; and c) comparing the first measurement and the second measurement.

[0310] One embodiment of the method disclosed herein further includes the following steps: a) performing a first measurement of the absolute blood eosinophil count in the subject; b) performing a second measurement of the absolute blood eosinophil count in the subject after administering a therapeutically effective amount of antigen-binding protein to the subject; and c) comparing the first measurement and the second measurement; and wherein the subject has an absolute blood eosinophil count selected from the group consisting of: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0311] Another aspect of this disclosure is a method for reducing absolute blood eosinophil count in a subject, comprising the steps of: (a) identifying a subject with a condition selected from the group consisting of: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and (b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby treating atopic dermatitis in the subject.

[0312] Another aspect of this disclosure is a method for reducing absolute blood eosinophil count in a subject, comprising the steps of: (a) identifying a subject with a condition selected from the group consisting of: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and (b) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2; thereby reducing absolute blood eosinophil count in the subject.

[0313] One embodiment of this disclosure is a composition according to this disclosure used in a therapeutic application.

[0314] One embodiment of this disclosure is a composition according to the disclosure for treating asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis.

[0315] The compositions disclosed herein may further comprise a buffer selected from the group consisting of disodium hydrogen phosphate heptahydrate, phosphate, citric acid, citrate, sodium phosphate, potassium phosphate, sodium citrate, and histidine, at a pH between 6.8 and 7.2, or a pH from pH 6.2 to pH 6.6, preferably a pH of 6.3. The buffer in the compositions disclosed herein may be present in the range of about 10-30 mM, about 10-20 mM, about 20 mM, or about 15.5 mM. For example, the buffer in the compositions disclosed herein is present in about 20 mM or about 15.5 mM disodium hydrogen phosphate heptahydrate.

[0316] The compositions disclosed herein may comprise disodium hydrogen phosphate heptahydrate and a citrate buffer, at a pH of 6.2 to 6.6 (inclusive), preferably 6.3. The disodium hydrogen phosphate heptahydrate buffer may be present in the range of about 15-16.4 mM, and the citrate buffer may be present in the range of about 3.8-4.9 mM. For example, the compositions disclosed herein may comprise about 15.5 mM disodium hydrogen phosphate heptahydrate and about 4.5 mM citrate monohydrate.

[0317] The compositions disclosed herein may further comprise sugars. The compositions disclosed herein may further comprise sucrose. Sucrose may be present in the compositions disclosed in a range from about 5-20%; about 10-15%; about 11-13%; or about 12% by volume.

[0318] The compositions disclosed herein may further comprise polysorbate 80. Polysorbate 80 may be present in a range from about 0.01% to 0.1% by weight (by volume). For example, polysorbate 80 may be present in the compositions disclosed herein at about 0.02% by volume or about 0.05% by volume.

[0319] The compositions disclosed herein may further comprise EDTA. EDTA may be present in the range of about 0.01-0.1 mM. For example, EDTA may be present in the range of about 0.05 mM.

[0320] In one embodiment, the composition disclosed herein further comprises 20 mM disodium hydrogen phosphate heptahydrate, 12% by weight of sucrose by volume, and 0.05% by weight of polysorbate 80 by volume.

[0321] In another embodiment, the composition disclosed herein further comprises 15.5 mM disodium hydrogen phosphate, 3.9 mM citrate monohydrate, 12% by weight of sucrose, 0.02% by weight of polysorbate 80, and 0.05 mM EDTA.

[0322] The compositions disclosed herein may comprise an aqueous liquid formulation at pH 6.2 containing 16.1 mM disodium hydrogen phosphate heptahydrate, 3.9 mM citrate monohydrate, 12% by weight of sucrose, 0.02% by weight of polysorbate 80, and 0.05 mM EDTA.

[0323] The compositions disclosed herein may comprise an aqueous liquid formulation at pH 6.2 containing 15.2 mM disodium hydrogen phosphate heptahydrate, 4.8 mM citrate monohydrate, 12% by weight of sucrose, 0.02% by weight of polysorbate 80, and 0.05 mM EDTA.

[0324] The compositions disclosed herein may comprise an aqueous liquid formulation at pH 6.4 containing 15.8 mM disodium hydrogen phosphate heptahydrate, 4.2 mM citrate monohydrate, 12% by weight of sucrose, 0.02% by weight of polysorbate 80, and 0.05 mM EDTA.

[0325] The compositions disclosed herein may comprise an aqueous liquid formulation at pH 6.6 containing 16.3 mM disodium hydrogen phosphate heptahydrate, 3.7 mM citrate monohydrate, 12% by weight of sucrose, 0.02% by weight of polysorbate 80, and 0.05 mM EDTA.

[0326] The compositions disclosed herein may comprise an aqueous liquid formulation at pH 6.3 containing 15.5 mM disodium hydrogen phosphate heptahydrate, 4.5 mM citrate monohydrate, 12% by weight of sucrose, 0.02% by weight of polysorbate 80, and 0.05 mM EDTA. Importantly, the tangential filtration and ultrafiltration exchange steps of Example 1 below can be modified to produce the compositions of this disclosure, for example, comprising the following compositions of this disclosure: 15.5 mM disodium hydrogen phosphate heptahydrate at pH 6.3, 4.5 mM citrate monohydrate, 12% by weight of sucrose, 0.02% by weight of polysorbate 80, 0.05 mM EDTA, or other such liquid formulations.

[0327] The compositions disclosed herein may comprise purified formulations of monoclonal antibodies and buffers, wherein the compositions are at a pH of 6.8 to 7.2, and wherein the buffers are histidine, phosphate, citric acid, citrate, or salts thereof.

[0328] In the compositions disclosed herein, the buffer may be at least one selected from the group consisting of: disodium hydrogen phosphate heptahydrate, phosphate, citric acid, and citrate.

[0329] In the compositions disclosed herein, the buffer may be sodium phosphate, potassium phosphate, or sodium citrate.

[0330] The compositions disclosed herein may contain sugars, carbohydrates, and / or salts.

[0331] The compositions disclosed herein may also contain sucrose or trehalose.

[0332] The compositions disclosed herein may also comprise purified formulations of monoclonal antibodies and buffers, wherein the compositions are at a pH of 6.8 to 7.2, and wherein the buffers are phosphates or salts thereof.

[0333] The compositions disclosed herein may further comprise a formulation selected from the following: a first formulation: 20 mM disodium hydrogen phosphate heptahydrate, 12% by weight of sucrose and 0.05% by weight of polysorbate 80; a second formulation: 15.5 mM disodium hydrogen phosphate heptahydrate, 3.9 mM citrate monohydrate, 12% by weight of sucrose and 0.02% by weight of polysorbate 80 and 0.05 mM EDTA; and a third formulation: 26 mM disodium hydrogen phosphate heptahydrate, 15% by weight of sucrose and 0.065% by weight of polysorbate 80. The compositions may be at a pH between about 6.8 and about 7.2, about 6.1 and about 6.5, or about 6 and about 6.6.

[0334] The compositions described herein can be produced using many conventional techniques. For example, the compositions can be expressed and purified from a recombinant expression system. In one embodiment, the compositions are produced by culturing host cells under conditions suitable for expressing peptides comprising SEQ ID NO:1 and SEQ ID NO:2, wherein the compositions are expressed, optionally purified, and optionally formulated within a pharmaceutical composition.

[0335] Many different expression systems and purification protocols can be used to generate the composition. Typically, host cells are transformed with a recombinant expression vector encoding the antibody. A wide range of host cells can be used, including mammalian-derived eukaryotic cell lines (e.g., CHO, Perc6, HEK293, HeLa, NS0). Suitable host cells include mammalian cells such as CHO (e.g., CHOK1 and CHO-DG44).

[0336] The host cell can be an isolated host cell. The host cell is typically not part of a multicellular organism (e.g., a plant or an animal). The host cell can be a non-human host cell.

[0337] Suitable cloning and expression vectors for use with eukaryotic or mammalian cell hosts, as well as methods of cloning, are known in the art.

[0338] Cells can be cultured under conditions that promote antibody expression. For example, a production bioreactor can be used to culture the cells. The volume of the production bioreactor can be (i) about 20,000 liters, about 10,000 liters, about 5,000 liters, about 2,000 liters, about 1,000 liters, or about 500 liters; or (ii) between 500 and 20,000 liters; between 500 and 10,000 liters; between 500 and 5,000 liters; between 1,000 and 10,000 liters; or between 2,000 and 10,000 liters. For example, cells can be cultured in a production bioreactor at a pH of about 6.75 to 7.00. Alternatively, cells can be cultured in a production bioreactor for about 12 to about 18 days. This culture step can help control the level of deamidated antibody variants, for example, by reducing the level of deamidated antibody variants.

[0339] The composition can be recovered and purified using standard protein purification procedures. For example, the composition can be harvested directly from the culture medium. Harvesting of cell culture medium can be achieved through clarification, such as by centrifugation and / or deep filtration. The recovery of the composition is followed by purification to ensure adequate purity.

[0340] Purification may employ one or more chromatographic steps, such as one or more chromatographic resins; and / or one or more filtration steps. For example, affinity chromatography using resins such as protein A, G, or L can be used to purify the composition. Alternatively, or in addition, ion exchange resins such as cation exchange resins can be used to purify the composition. Alternatively, or in addition, hydrophobic interaction chromatographic resins can be used to purify the composition. Alternatively, the purification steps may include: an affinity chromatography resin step, followed by a cation exchange resin step, followed by a hydrophobic interaction chromatographic resin step.

[0341] For example, the harvested material is placed in contact with protein A resin. A solution containing the composition can be eluted from the protein A resin and treated at pH 3.3 to 3.7 for 15 to 240 minutes. This protein A resin step can help control the level of aggregated antibody variants, for example, by reducing the level of aggregated antibody variants.

[0342] The solution containing the composition can then be further clarified by depth filtration and / or double filtration.

[0343] Alternatively, or otherwise, anion exchange resins may be used. The solution containing the composition can be placed at a loading pH of 8.3 to 8.7 with anion exchange resin (e.g., Q-SEPHAROSE). TM (Rapid flow anion exchange chromatography) contact. Solutions containing tissue can be eluted from the anion exchange resin and held for 96 hours or less. This anion exchange resin step can help control the levels of deamidated antibody variants, for example, reducing the levels of deamidated antibody variants.

[0344] Optionally, guanidine and / or ammonium sulfate can be added to the solution containing the composition and kept for 15 to 240 minutes.

[0345] Alternatively, or otherwise, a hydrophobically interacting chromatographic resin can be used. The solution containing the composition can be mixed with a hydrophobically interacting chromatographic resin (e.g., phenyl SEPHAROSE) at a loading ratio of 12 to 27 g protein / L resin. TM (Fast flow chromatography) contact. For example, a solution containing the composition can be eluted using an elution gradient volume (bed volume; BV) of about 9 to about 11. Elution peak cutoff (% of maximum peak height) of about 17 to about 23 can be used during elution from the hydrophobic interaction chromatography resin. This hydrophobic interaction chromatography resin step can help control the level of aggregated antibody variants, for example, reduce the level of aggregated antibody variants.

[0346] The solution containing the composition can then be filtered to remove the virus. The solution containing the composition can then be prepared at an antibody concentration of approximately 76 g protein / L to approximately 82 g protein / L, or approximately 100 g protein / L. The solution containing the composition can be filled into a container and frozen. Aliquots of the solution containing the composition can be lyophilized. The lyophilized product can be reconstituted by adding water to produce a composition at a pH of approximately 6.8 to approximately 7.2, containing 75 mg / L protein, monoclonal anti-IL-5 antibody, 20 mM disodium hydrogen phosphate heptahydrate, 12% by weight sucrose by volume, and 0.05% by weight polysorbate 80 by volume.

[0347] In general, this disclosure includes:

[0348] 1. An antigen-binding protein comprising a heavy chain variable region having the amino acid sequences CDRH1 shown in SEQ ID NO:5, CDRH2 shown in SEQ ID NO:6, and CDRH3 shown in SEQ ID NO:7; and a light chain variable region having the amino acid sequences CDRL1 shown in SEQ ID NO:8, CDRL2 shown in SEQ ID NO:9, and CDRL3 shown in SEQ ID NO:10.

[0349] 2.1 is an antigen-binding protein, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0350] 3.2 is an antigen-binding protein comprising a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0351] 4. An antigen-binding protein comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0352] 5.4 is an antigen-binding protein comprising a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0353] 6. Antibodies, which consist of heavy and light chains, among which...

[0354] a) The heavy chain includes a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO:5, the CDRH2 amino acid sequence shown in SEQ ID NO:6, and the CDRH3 amino acid sequence shown in SEQ ID NO:7; and

[0355] b) The light chain includes a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO:8, the CDRL2 amino acid sequence shown in SEQ ID NO:9, and the CDRL3 amino acid sequence shown in SEQ ID NO:10.

[0356] 7.6 antibody, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0357] The antibody of 8.7, wherein the heavy chain comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256.

[0358] 9. Antibodies, which consist of heavy and light chains, among which...

[0359] a) The heavy chain comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and

[0360] b) The light chain comprises a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0361] An antibody of 10.9, wherein the heavy chain comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256.

[0362] 11. An antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO:1 and a light chain having the amino acid sequence shown in SEQ ID NO:2.

[0363] 12. A peptide chain comprising the amino acid sequence shown in SEQ ID NO:3.

[0364] 13. A peptide chain comprising the amino acid sequence shown in SEQ ID NO:1.

[0365] 14. A composition comprising a nucleic acid encoding a heavy chain variable region of 1 and a nucleic acid encoding a light chain variable region of an antigen-binding protein of 1.

[0366] 15. A composition comprising a nucleic acid encoding a heavy chain variable region 2 and a nucleic acid encoding a light chain variable region 2.

[0367] 16. A composition comprising a nucleic acid encoding a heavy chain Fc domain of 3 linked to the carboxyl terminus of a heavy chain variable region and a nucleic acid encoding a light chain variable region of 3.

[0368] 17. A composition comprising a nucleic acid encoding a heavy chain variable region of 6 and a nucleic acid encoding a light chain variable region of an antigen-binding protein of 6.

[0369] 18. A composition comprising a nucleic acid encoding a heavy chain variable region of 7 and a nucleic acid encoding a light chain variable region of 7.

[0370] 19. A composition comprising a nucleic acid encoding a heavy chain Fc domain of 8 linked to the carboxyl terminus of a heavy chain variable region and a nucleic acid encoding a light chain variable region of 8.

[0371] 20. A composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:3 and a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:4.

[0372] 21. A composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:1 and a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:2.

[0373] 22. A composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:3.

[0374] 23. A composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:1.

[0375] 24. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:15 and a nucleic acid having the sequence shown in SEQ ID NO:16.

[0376] 25. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:17 and a nucleic acid having the amino acid sequence shown in SEQ ID NO:18.

[0377] 26. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:13 and a nucleic acid having the amino acid sequence shown in SEQ ID NO:14.

[0378] 27. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:15.

[0379] 28. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:17.

[0380] 29. A composition comprising a nucleic acid having the sequence shown in SEQ ID NO:13.

[0381] 30. A composition comprising a nucleic acid encoding a heavy chain variable region of 1.

[0382] 31. A composition comprising a nucleic acid encoding a heavy chain variable region 2.

[0383] 32. A composition comprising a nucleic acid encoding a heavy chain Fc domain linked to the carboxyl terminus of the heavy chain variable region in 3.

[0384] 33. An expression vector comprising a composition of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32.

[0385] 34. A recombinant host cell comprising an expression vector, said expression vector comprising a composition of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32.

[0386] 35. A method for producing a peptide chain comprising the amino acid sequence shown in SEQ ID NO:3, the method comprising the steps of: culturing a recombinant host cell comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO:3; and recovering the peptide chain.

[0387] The method of 36.35, wherein the nucleic acid comprises the sequence shown in SEQ ID NO:15.

[0388] The method of 37.35, wherein the nucleic acid comprises the sequence shown in SEQ ID NO:13.

[0389] Method 38.35, wherein the nucleic acid comprises the sequence shown in SEQ ID NO:17.

[0390] 39. A method for generating an antigen-binding protein, comprising the following steps:

[0391] a) Culturing recombinant host cells containing an expression vector comprising a composition of 14, 15, or 16;

[0392] and b) recovery of antigen-binding proteins;

[0393] This results in the production of antigen-binding proteins.

[0394] 40. Antigen-binding protein, which is produced by the method described in 39.

[0395] 41. A method for generating antibodies, comprising the following steps:

[0396] a) Culturing recombinant host cells containing an expression vector comprising a composition of 17, 18, or 19; and

[0397] b) Recover antibodies;

[0398] This produces antibodies.

[0399] 42. Antibodies, which are produced by the method described in 41.

[0400] 43. A method for generating antibodies, comprising the following steps:

[0401] a) Culturing recombinant host cells containing an expression vector comprising a composition of 20 or 22; and

[0402] b) Recover antibodies;

[0403] This produces antibodies.

[0404] 44. Antibodies, which are produced by the method described in 43.

[0405] 45. A method for producing antibodies, comprising the following steps:

[0406] a) Culturing recombinant host cells containing an expression vector comprising a composition of 24, 26, or 27; and

[0407] b) Recover antibodies;

[0408] This produces antibodies.

[0409] 46. ​​Antibodies, which are produced by the method described in 45.

[0410] 47. A method for producing antibodies, comprising the following steps:

[0411] a) Culturing recombinant host cells containing an expression vector, said expression vector comprising nucleic acids having the sequence shown in SEQ ID NO:17 and nucleic acids having the sequence shown in SEQ ID NO:18; and

[0412] b) Recover antibodies;

[0413] This produces antibodies.

[0414] 48. Antibodies, which are produced by the method described in 47.

[0415] 49. A pharmaceutical composition comprising:

[0416] a) An antigen-binding protein comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO:5, the CDRH2 amino acid sequence shown in SEQ ID NO:6, and the CDRH3 amino acid sequence shown in SEQ ID NO:7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO:8, the CDRL2 amino acid sequence shown in SEQ ID NO:9, and the CDRL3 amino acid sequence shown in SEQ ID NO:10; and

[0417] b) Pharmaceutically acceptable carriers.

[0418] The pharmaceutical composition of 50.49, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0419] The pharmaceutical composition of 51.50 comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, and wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0420] The pharmaceutical composition of 52.51, wherein the antigen-binding protein is at a concentration between about 75 mg / ml and about 150 mg / ml.

[0421] Pharmaceutical compositions of 53.49, 50, 51, or 52, wherein the pharmaceutically effective carrier comprises an aqueous liquid formulation at a pH of about 5.5 to about pH 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by weight of polysorbate 80 and about 0.05 mM EDTA.

[0422] The pharmaceutical composition of 54.53, wherein the pH is about 6.0 and the antigen-binding protein is at a concentration of about 150 mg / ml.

[0423] 55. A pharmaceutical composition comprising:

[0424] a) An antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO:5, the CDRH2 amino acid sequence shown in SEQ ID NO:6, and the CDRH3 amino acid sequence shown in SEQ ID NO:7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO:8, the CDRL2 amino acid sequence shown in SEQ ID NO:9, and the CDRL3 amino acid sequence shown in SEQ ID NO:10; and

[0425] b) Pharmaceutically acceptable carriers.

[0426] The pharmaceutical composition of 56.55, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0427] The pharmaceutical composition of 57.56 comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, and wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0428] The pharmaceutical composition of 58.57, wherein the antibody is at a concentration between about 75 mg / ml and about 150 mg / ml.

[0429] Pharmaceutical compositions of 59, 55, 56, 57, or 58, wherein the pharmaceutically effective carrier comprises an aqueous liquid formulation at a pH of about 5.5 to about pH 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by weight of polysorbate 80 and about 0.05 mM EDTA.

[0430] The pharmaceutical composition is 60.59, wherein the pH is about 6.0 and the antibody is at a concentration of about 150 mg / ml.

[0431] 61. A pharmaceutical composition comprising:

[0432] a) An antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and the light chain comprises a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4; and

[0433] b) Pharmaceutically acceptable carriers.

[0434] The pharmaceutical composition of 62.61 comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, and wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0435] The pharmaceutical composition of 63.62, wherein the antibody is at a concentration between about 75 mg / ml and about 150 mg / ml.

[0436] Pharmaceutical compositions of 64, 61, 62, or 63, wherein the pharmaceutically effective carrier comprises an aqueous liquid formulation at a pH of about 5.5 to about pH 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by weight of polysorbate 80 and about 0.05 mM EDTA.

[0437] The pharmaceutical composition of 65.64, wherein the pH is about 6.0 and the antibody is at a concentration of about 150 mg / ml.

[0438] 66. A pharmaceutical composition comprising:

[0439] a) An antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO:1 and a light chain having the amino acid sequence shown in SEQ ID NO:2; and

[0440] b) Pharmaceutically acceptable carriers.

[0441] The pharmaceutical composition of 67.66, wherein the antigen-binding protein is at a concentration between about 75 mg / ml and about 150 mg / ml.

[0442] The pharmaceutical composition of 68.67, wherein the pharmaceutically effective carrier comprises an aqueous liquid formulation at a pH of about 5.5 to about pH 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by weight of polysorbate 80 and about 0.05 mM EDTA.

[0443] A pharmaceutical composition of 69.68, wherein the pH is about 6.0 and the antigen-binding protein is at a concentration of about 150 mg / ml.

[0444] 70. A method for treating a disease in a subject, comprising the following steps:

[0445] a) Identify subjects suffering from diseases selected from the following groups: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and

[0446] (b) Administer to the subject a therapeutically effective amount of antigen-binding protein according to 1, 2, 3, 4, 5 or 40;

[0447] This is used to treat the diseases in the subjects.

[0448] Method 71.70, wherein the amount of antigen-binding protein is from about 2 mg to about 600 mg.

[0449] Method 72.71, wherein the antigen-binding protein is administered once every 3 months or once every 6 months.

[0450] Methods 73, 70, 71, or 72, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0451] 74. A method for treating a disease in a subject, comprising the following steps:

[0452] a) Identify subjects suffering from diseases selected from the following groups: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and

[0453] (b) Administering a therapeutically effective amount of the antibodies according to 6, 7, 8, 9, 10, 11, 42, 44, 46 and 48 to the subject;

[0454] This is used to treat the diseases in the subjects.

[0455] Method 75.74, wherein the amount of antibody is from about 2 mg to about 600 mg.

[0456] The method is 76.75, in which antibodies are administered once every 3 months or once every 6 months.

[0457] Methods 77, 74, 75, or 76, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0458] 78. A method for treating a disease in a subject, comprising the following steps:

[0459] a) Identify subjects suffering from diseases selected from the following groups: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and

[0460] (b) Administering to the subject a therapeutically effective amount of the composition according to 49, 50, 51, 52, 53 or 54;

[0461] This is used to treat the diseases in the subjects.

[0462] The method of 79.78, wherein the amount of composition provides a dose of antigen-binding protein of about 2 mg to about 600 mg.

[0463] Method 80.79, wherein the composition is applied once every 3 months or once every 6 months.

[0464] Methods 81, 78, 79, or 80, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0465] 82. A method for treating a disease in a subject, comprising the following steps:

[0466] a) Identify subjects suffering from diseases selected from the following groups: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and

[0467] (b) administering to the subject a therapeutically effective amount of the composition according to 55, 56, 57, 58, 59, 60, 61, 62, 63, 65, 66, 67, 68 or 69;

[0468] This is used to treat the diseases in the subjects.

[0469] The method of 83.82, wherein the amount of composition provides a dose of about 2 mg to about 600 mg of antigen-binding protein.

[0470] Method 84.83, in which antibodies are administered once every 3 months or once every 6 months.

[0471] Methods 85, 82, 83, or 84, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0472] 86. A method for treating mild to moderate asthma in a subject, comprising the following steps:

[0473] (a) Identifying subjects diagnosed with mild to moderate asthma; and

[0474] (b) Administering a therapeutically effective amount of an antibody to a subject, the antibody comprising a heavy chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:5, a CDR amino acid sequence as shown in SEQ ID NO:6, and a CDR amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:8, a CDR amino acid sequence as shown in SEQ ID NO:9, and a CDR amino acid sequence as shown in SEQ ID NO:10;

[0475] This treatment was used to treat mild to moderate asthma in the subjects.

[0476] The method of 87.86, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0477] The method of 88.87, wherein the antibody comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, and wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0478] The method of 89.88, wherein the antibody comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0479] The method of 90.89, wherein the antibody dose is approximately 2 mg to approximately 600 mg.

[0480] The 91.90 method involves administering antibodies every 3 months or every 6 months.

[0481] Method 92.90, in which antibodies are administered subcutaneously.

[0482] Methods 93, 86, 87, 88, 89, 90, 91, or 92, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0483] 94. A method for treating mild to moderate asthma in a subject, comprising the following steps:

[0484] (a) Identifying subjects diagnosed with mild to moderate asthma; and

[0485] (b) administering a therapeutically effective amount of an antibody to a subject, said antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2;

[0486] This treatment was used to treat mild to moderate asthma in the subjects.

[0487] The method of 95.94, wherein the antibody dose is from about 2 mg to about 600 mg.

[0488] The method is 96.95, in which antibodies are administered once every 3 months or once every 6 months.

[0489] The method is 97.96, in which antibodies are administered subcutaneously.

[0490] Methods 98, 94, 95, 96, or 97, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0491] 99. A method for treating mild to moderate asthma in a subject, comprising the following steps:

[0492] (a) Identifying subjects diagnosed with mild to moderate asthma; and

[0493] (b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2;

[0494] This treatment was used to treat mild to moderate asthma in the subjects.

[0495] The method of 100.92, wherein the pharmaceutically effective carrier comprises an aqueous liquid formulation at a pH of about 5.5 to about pH 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by weight of polysorbate 80 by volume, and about 0.05 mM EDTA.

[0496] Method 101.100, wherein the antibody dose is from about 2 mg to about 600 mg.

[0497] The method of 102.101, wherein the pharmaceutical composition is administered once every 3 months or once every 6 months.

[0498] The method of 103.102, wherein the pharmaceutical composition is administered subcutaneously.

[0499] Methods 104.99, 100, 101, 102, or 103, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0500] 105. A method for treating moderate to severe asthma in a subject, comprising the following steps:

[0501] (a) Identifying subjects diagnosed with severe asthma; and

[0502] (b) Administering a therapeutically effective amount of an antibody to a subject, the antibody comprising a heavy chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:5, a CDR amino acid sequence as shown in SEQ ID NO:6, and a CDR amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:8, a CDR amino acid sequence as shown in SEQ ID NO:9, and a CDR amino acid sequence as shown in SEQ ID NO:10;

[0503] This treatment was used to treat moderate to severe asthma in the subjects.

[0504] The method of 106.105, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0505] The method of 107.106, wherein the antibody comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, and wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0506] The method of 108.107, wherein the antibody comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0507] The method of 109.108, wherein the antibody dose is from about 2 mg to about 600 mg.

[0508] Method 110.109, in which antibodies are administered once every 3 months or once every 6 months.

[0509] Method 111.110, in which antibodies are administered subcutaneously.

[0510] Methods 112, 105, 106, 107, 108, 109, 110, or 111, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0511] 113. A method for treating moderate to severe asthma in a subject, comprising the following steps:

[0512] (a) Identifying subjects diagnosed with severe asthma; and

[0513] (b) administering a therapeutically effective amount of an antibody to a subject, said antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2;

[0514] This treatment was used to treat moderate to severe asthma in the subjects.

[0515] The method of 114.113, wherein the antibody dose is from about 2 mg to about 600 mg.

[0516] Method 115.114, in which antibodies are administered once every 3 months or once every 6 months.

[0517] Method 116.115, in which antibodies are administered subcutaneously.

[0518] Methods 117, 113, 114, 115, or 116, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0519] 118. A method for treating moderate to severe asthma in a subject, comprising the following steps:

[0520] (a) Identifying subjects diagnosed with severe asthma; and

[0521] (b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2;

[0522] This treatment was used to treat moderate to severe asthma in the subjects.

[0523] The method of 119.118, wherein the pharmaceutically effective carrier comprises an aqueous liquid formulation at a pH of about 5.5 to about pH 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by weight of polysorbate 80 and about 0.05 mM EDTA.

[0524] Method 120.119, wherein the antibody dose is approximately 2 mg to approximately 600 mg.

[0525] Method 121.120, wherein the pharmaceutical composition is administered once every 3 months or once every 6 months.

[0526] The method of 122.121, wherein the pharmaceutical composition is administered subcutaneously.

[0527] Methods 123, 118, 119, 120, 121, or 122, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0528] 124. A method for treating moderate to severe atopic dermatitis in subjects with this need, comprising the following steps:

[0529] a) Identify subjects who have at least one of the following groups:

[0530] i) Diagnosis of atopic dermatitis according to the Hanifin and Rajka criteria revised by Eichenfield;

[0531] ii) Prior diagnosis of atopic dermatitis for a period of two years or more prior to treatment;

[0532] iii) Overall evaluation score of approximately 3 for healthcare professionals;

[0533] iv) Atopic dermatitis affecting more than or equal to about 10% of the body surface area;

[0534] v) Eczema area and severity index score greater than or equal to 16;

[0535] vi) Absolute blood eosinophil counts of 150 or more cells per μL, 200 or more cells per μL, and 350 or more cells per μL;

[0536] (vii) Prior to treatment, patients must have at least one of the following conditions: 1) inadequate response to topical medication for atopic dermatitis for six months or more; 2) poor tolerance to topical medication for atopic dermatitis; 3) side effects from topical medication for atopic dermatitis; and 4) inadequate response to nonpharmacological treatments for atopic dermatitis.

[0537] b) Administering a therapeutically effective amount of an antibody to a subject, the antibody comprising a heavy chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:5, a CDR amino acid sequence as shown in SEQ ID NO:6, and a CDR amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:8, a CDR amino acid sequence as shown in SEQ ID NO:9, and a CDR amino acid sequence as shown in SEQ ID NO:10;

[0538] This treatment was used to treat atopic dermatitis in the subjects.

[0539] The method of 125.124, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0540] The method of 126.125, wherein the antibody comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, and wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0541] The method of 127.126, wherein the antibody comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0542] Method 128.127, wherein the antibody dose is approximately 2 mg to approximately 600 mg.

[0543] Method 129.128, in which antibodies are administered once every 3 months or once every 6 months.

[0544] Method 130.128, in which antibodies are administered subcutaneously.

[0545] Method 131.128, in which antibodies are administered intravenously.

[0546] 132. A method for treating moderate to severe atopic dermatitis in subjects with this need, comprising the following steps:

[0547] a) Identify subjects who have at least one of the following groups:

[0548] i) Diagnosis of atopic dermatitis according to the Hanifin and Rajka criteria revised by Eichenfield;

[0549] ii) Prior diagnosis of atopic dermatitis for a period of two years or more prior to treatment;

[0550] iii) Overall evaluation score of approximately 3 for healthcare professionals;

[0551] iv) Atopic dermatitis affecting more than or equal to about 10% of the body surface area;

[0552] v) Eczema area and severity index score greater than or equal to 16;

[0553] vi) Absolute blood eosinophil counts of 150 or more cells per μL, 200 or more cells per μL, and 350 or more cells per μL;

[0554] (vii) Prior to treatment, patients must have at least one of the following conditions: 1) inadequate response to topical medication for atopic dermatitis for six months or more; 2) poor tolerance to topical medication for atopic dermatitis; 3) side effects from topical medication for atopic dermatitis; and 4) inadequate response to nonpharmacological treatments for atopic dermatitis.

[0555] b) Administering a therapeutically effective amount of an antibody to a subject, said antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2;

[0556] This treatment was used to treat atopic dermatitis in the subjects.

[0557] The method of 133.132, wherein the antibody dose is from about 2 mg to about 600 mg.

[0558] Method 134.133, in which antibodies are administered once every 3 months or once every 6 months.

[0559] Method 135.134, in which antibodies are administered subcutaneously.

[0560] Methods 136, 132, 133, 134, or 135, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 150 cells per μL, greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0561] 137. A method for treating moderate to severe atopic dermatitis in a subject, comprising the following steps:

[0562] a) Identify subjects who have at least one of the following groups:

[0563] i) Diagnosis of atopic dermatitis according to the Hanifin and Rajka criteria revised by Eichenfield;

[0564] ii) Prior diagnosis of atopic dermatitis for a period of two years or more prior to treatment;

[0565] iii) Overall evaluation score of approximately 3 for healthcare professionals;

[0566] iv) Atopic dermatitis affecting more than or equal to about 10% of the body surface area;

[0567] v) Eczema area and severity index score greater than or equal to 16;

[0568] vi) Absolute blood eosinophil counts of 150 or more cells per μL, 200 or more cells per μL, and 350 or more cells per μL;

[0569] (vii) Prior to treatment, patients must have at least one of the following conditions: 1) inadequate response to topical medication for atopic dermatitis for six months or more; 2) poor tolerance to topical medication for atopic dermatitis; 3) side effects from topical medication for atopic dermatitis; and 4) inadequate response to nonpharmacological treatments for atopic dermatitis.

[0570] b) Administering a therapeutically effective amount of a pharmaceutical composition to a subject, the pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2;

[0571] This treatment was used to treat atopic dermatitis in the subjects.

[0572] The method of 138.137, wherein the pharmaceutically effective carrier comprises an aqueous liquid formulation at a pH of about 5.5 to about pH 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by weight of polysorbate 80 by volume, and about 0.05 mM EDTA.

[0573] The method of 139.138, wherein the antibody dose is from about 2 mg to about 600 mg.

[0574] Method 140.139, wherein the pharmaceutical composition is administered once every 3 months or once every 6 months.

[0575] The method of 141.140, wherein the pharmaceutical composition is administered subcutaneously.

[0576] Methods 142, 137, 138, 139, 140, or 141, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0577] 143. A method for reducing the absolute blood eosinophil count in a subject, comprising the following steps:

[0578] (a) Identify subjects with conditions selected from the following groups: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, and atopic dermatitis; and

[0579] (b) Administering to the subject a therapeutically effective amount of an antibody, the antibody comprising a heavy chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:5, a CDR amino acid sequence as shown in SEQ ID NO:6, and a CDR amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having a CDR amino acid sequence as shown in SEQ ID NO:8, a CDR amino acid sequence as shown in SEQ ID NO:9, and a CDR amino acid sequence as shown in SEQ ID NO:10;

[0580] This reduces the absolute blood eosinophil count in the subjects.

[0581] The method of 144.143, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:21.

[0582] The method of 145.144, wherein the antibody comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254 and a glutamic acid residue at position 256, and wherein the amino terminus of the heavy chain Fc domain is connected to the carboxyl terminus of the heavy chain variable region.

[0583] The method of 146.145, wherein the antibody comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:3; and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:4.

[0584] Method 147.146, wherein the antibody dose is from about 2 mg to about 600 mg.

[0585] Method 148.147, in which antibodies are administered once every 3 months or once every 6 months.

[0586] Method 149.148, in which antibodies are administered subcutaneously.

[0587] Methods 150, 142, 144, 145, 146, 147, 148, or 149, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0588] Methods 151, 142, 144, 145, 146, 147, 148, or 149 further include the following steps:

[0589] a) Perform the first measurement of the absolute blood eosinophil count in the subject;

[0590] b) A second measurement of the absolute blood eosinophil count in the subject was performed after administration of a therapeutically effective amount of antigen-binding protein; and

[0591] c) Compare the first measurement and the second measurement.

[0592] Methods 152, 142, 144, 145, 146, 147, 148, or 149 further include the following steps:

[0593] a) Perform the first measurement of the absolute blood eosinophil count in the subject;

[0594] b) A second measurement of the absolute blood eosinophil count in the subject was performed after administration of a therapeutically effective amount of antigen-binding protein; and

[0595] c) Compare the first measurement and the second measurement; and wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0596] 153. A method for reducing the absolute blood eosinophil count in a subject, comprising the following steps:

[0597] (a) Identify subjects with conditions selected from the following groups: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and

[0598] (b) administering a therapeutically effective amount of an antibody to a subject, said antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2;

[0599] This treatment was used to treat atopic dermatitis in the subjects.

[0600] Method 154.153, wherein the antibody dose is from about 2 mg to about 600 mg.

[0601] Method 155.154, in which antibodies are administered once every 3 months or once every 6 months.

[0602] Method 156.155, in which antibodies are administered subcutaneously.

[0603] Methods 157, 154, 155, 156, or 157, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0604] Methods 158, 154, 155, 156, or 157, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0605] Methods 159, 154, 155, 156, or 157 further include the following steps:

[0606] a) Perform the first measurement of the absolute blood eosinophil count in the subject;

[0607] b) A second measurement of the absolute blood eosinophil count in the subject was performed after administration of a therapeutically effective amount of antigen-binding protein; and

[0608] c) Compare the first measurement and the second measurement.

[0609] Methods 160, 154, 155, 156, or 157 further include the following steps:

[0610] a) Perform the first measurement of the absolute blood eosinophil count in the subject;

[0611] b) A second measurement of the absolute blood eosinophil count in the subject was performed after administration of a therapeutically effective amount of antigen-binding protein; and

[0612] c) Compare the first measurement and the second measurement; and wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0613] 161. A method for reducing the absolute blood eosinophil count in a subject, comprising the following steps:

[0614] (a) Identify subjects with conditions selected from the following groups: asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis; and

[0615] (b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody and a pharmaceutically effective carrier, the antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO:1 and a light chain having an amino acid sequence as shown in SEQ ID NO:2;

[0616] This reduces the absolute blood eosinophil count in the subjects.

[0617] The method of 162.161, wherein the pharmaceutically effective carrier comprises an aqueous liquid formulation at a pH of about 5.5 to about pH 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by weight of polysorbate 80 and about 0.05 mM EDTA.

[0618] The method of 163.162, wherein the antibody dose is from about 2 mg to about 600 mg.

[0619] Method 164.163, wherein the pharmaceutical composition is administered once every 3 months or once every 6 months.

[0620] Method 165.164, wherein the pharmaceutical composition is administered subcutaneously.

[0621] Methods 166, 162, 163, 164, 165, or 166, wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0622] Methods 167, 162, 163, 164, 165, or 166 further include the following steps:

[0623] a) Perform the first measurement of the absolute blood eosinophil count in the subject;

[0624] b) A second measurement of the absolute blood eosinophil count in the subject was performed after administration of a therapeutically effective amount of antigen-binding protein; and

[0625] c) Compare the first measurement and the second measurement.

[0626] Methods 168, 162, 163, 164, 165, or 166 further include the following steps:

[0627] a) Perform the first measurement of the absolute blood eosinophil count in the subject;

[0628] b) A second measurement of the absolute blood eosinophil count in the subject was performed after administration of a therapeutically effective amount of antigen-binding protein; and

[0629] c) Compare the first measurement and the second measurement; and wherein the subject has an absolute blood eosinophil count selected from the following groups: greater than or equal to 200 cells per μL and greater than or equal to 350 cells per μL.

[0630] 169. A composition according to any one of 1-11, 40, 42, 44, 46, 47 or 49-69, used in a therapeutic manner.

[0631] 170. A composition according to any one of 1-11, 40, 42, 44, 46, 47 or 49-69, for the treatment of asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, subeosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granuloma with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis. Example

[0632] Example 1

[0633] Kinetic analysis of 28Y042-7F11-1

[0634] Kinetic analyses were performed to compare 28Y042-7F11-1, mepolizumab, and GSK3559090A (a comparative based on IgG1 anti-IL-5 mAb molecules). The results are summarized in Table 16 below. Due to the high affinity of 28Y042-7F11-1, the dissociation rate could not be accurately determined at 25°C using a Biacore 4000; therefore, KINEXA analysis was used to calculate the accurate affinity of 28Y042-7F11-1 for human IL-5 at 25°C. KINEXA solution-phase affinity analysis showed that the affinity of 28Y042-7F11-1 for human IL-5 at 25°C was 10.47 pM (95% confidence range 0.88 pM–31.97 pM). In comparison, the affinity of mepolizumab for binding to human IL-5 was 122.8 pM (measured by BIACORE 4000 at 25°C).

[0635] The binding of the comparative antibody GSK3559090A to human IL-5 at 25°C was analyzed using a BIACORE 4000, yielding an affinity of 16.4 pM (K0.05). D GSK3559090A exhibits a 10-fold higher K+ activating capacity against human IL-5 than mepolizumab. D This is mainly due to the association rate (kJ / kJ) of GSK3559090A. a It is 20 times faster than mepolizumab.

[0636] The affinity of 28Y042-7F11-1 for human and cynomolgus monkey IL-5 was determined using a BIACORE T200 at 37 °C. Higher temperatures increased the dissociation rate (kJ / kcal) of 28Y042-7F11-1. d This ensures that the sample is within the range of the instrument. The affinity of 28Y042-7F11-1 for IL-5 in humans and cynomolgus monkeys at 37℃ is 39.13 pM and 23.93 pM, respectively.

[0637] Competitive assays performed on a FORTEBIO OCTET RED384 BLI instrument showed that 28Y042-7F11-1 competes with mepolizumab for binding to human IL-5. Table 16 Explanation / Description: Kinetics K D Summary of data. (ND not determined)

[0638] Table 16.

[0639]

[0640]

[0641] Binding to human Fc receptor

[0642] At pH 6.0, 28Y042-7F11-1 showed approximately 13-fold increased affinity for human FcRn compared to mepolizumab (157 nM vs. 2082 nM), and bound with low affinity at pH 7.4; the affinity at pH 7 was 16 μM. K, as measured by BIACORE T200 at 37°C, was... D The values ​​are shown in Table 17. Table 17 Explanation / Description: Comparison of binding affinity of 28Y042-7F11-1 and mepolizumab to human FcRn at pH 6.0 and pH 7.4.

[0643] Table 17.

[0644]

[0645] Analysis of the binding of 28Y042-7F11-1 to the human Fc gamma receptor (FcγR) using PROTEON XPR36 demonstrated its comparability with YTE-containing control antibodies. These YTE-containing mAbs exhibited approximately 1.5-fold lower affinity for FcγR compared to the human IgG1 wild-type control. 28Y042-7F11-1 also showed lower affinity for complement component C1q compared to the human IgG1 wild-type control (750 nM and 465 nM, respectively).

[0646] In another experiment, 28Y042-7F11-1 showed an approximately 3-fold increase in affinity for the human neonatal receptor (FcRn) at pH 6.0 compared to the wild-type human IgG1 control (130 nM vs. 359 nM, respectively), and bound with low affinity (2650 nM) at pH 7.4, while the wild-type control did not bind to FcRn at this pH (Table 18). This is comparable to the YTE control used in the experiment. Table 18 Explanation / Description: Binding of 28Y042-7F11-1 with recombinant human neonatal receptor (FcRn) using PROTEON. The wild-type isotype control and the Fc-disabled isotype control were derived from the non-functional (for CDR binding) antibody originally produced against F9 coagulation factor IX. The Fc-disabled isotype control contains two point mutations in the Fc region (L235 and G237, both mutated to alanine), which reduce the interaction with the Fc gamma receptor.

[0647] Table 18.

[0648]

[0649]

[0650] TF-1 Functional Cell Assay

[0651] The ability of 28Y042-7F11-1 cells to inhibit the proliferation of human IL-5-mediated TF-1 cells in a dose-dependent manner was evaluated. TF-1 cells are an erythroleukemia cell line engineered to proliferate in response to human and cynomolgus monkey IL-5 stimulation.

[0652] Analysis of the effects of 28Y042-7F11-1 in a TF-1 cell proliferation assay showed that it is a potent inhibitor of IL-5-mediated TF-1 cell proliferation. When tested in the concentration range of 1 nM–0.042 pM, both 28Y042-7F11-1 and mepolizumab induced dose-dependent inhibition of human IL-5-induced TF-1 cell proliferation (IC50). 50 They are 4pM and 105pM respectively. Figure 1This indicates that the cytological assay efficacy of 28Y042-7F11-1 is approximately 30-fold higher than that of mepolizumab.

[0653] When tested at concentrations ranging from 1 nM to 0.042 pM, both 28Y042-7F11-1 and mepolizumab induced dose-dependent inhibition of human IL-5-induced TF-1 cell proliferation (IC50). 50 The values ​​are 0.004 nM and 0.105 nM, respectively.

[0654] Further analysis of 28Y042-7F11-1 was performed after exposing the molecules to thermal stress by incubation at 40°C for one week in acetate or phosphate buffer. Under these conditions, the IC50 of 28Y042-7F11-1 in the TF-1 cell assay was... 50 The values, in the range of 4 pM to 5 pM, show similar values ​​to those of 28Y042-7F11-1 exposed to “stress-free” control conditions.

[0655] Assay for eosinophil morphology changes and their binding with endogenous IL-5

[0656] The ability of 28Y042-7F11-1 to inhibit IL-5-mediated eosinophil morphology changes in human whole blood was evaluated. Both mepolizumab and 28Y042-7F11-1 (10 μg / ml) showed inhibition of recombinant IL-5 (10 ng / ml)-mediated eosinophil morphology changes, while the control antibody pascolizumab and anti-RSV (Fc-incapacitated) failed to prevent IL-5-mediated eosinophil morphology changes. Figure 2 ).

[0657] The ability of 28Y042-7F11-1 and mepolizumab (both 1 μg / ml) to bind native IL-5 in the supernatant of CD3 / CD28-stimulated peripheral blood mononuclear cells (PBMCs) was evaluated by ELISA. 28Y042-7F11-1 and mepolizumab bound native IL-5, while the control antibody pacolizumab did not. Although the assay demonstrated binding to native IL-5, it was not further optimized for accurate EC measurement. 50 value( Figure 3 ).

[0658] The in vitro stability of 28Y042-7F11-1 in human and cynomolgus monkey serum was determined by immunoassay.

[0659] The ability of 28Y042-7F11-1 to bind recombinant IL-5 was evaluated after incubation at 37°C for 6 weeks in collected human or cynomolgus monkey serum. Figure 4Following incubation, samples were tested using the MSD immunoassay, in which immobilized biotinylated IL-5 was used to capture the remaining active 28Y042-7F11-1, followed by detection using a directly labeled anti-human Fc monoclonal reagent. Recovery of active 28Y042-7F11-1 was observed to gradually decrease over time in both human and cynomolgus monkey sera, reaching T0 concentrations of 73.1% and 77.1%, respectively, after 6 weeks. This was compared with historical data from mepolizumab (51.0% [human] and 64.2% [cynomolgus monkey]) and control antibodies known to have poor stability in this assay and specific for IL-13 (27.9% [human] and 24.9% [cynomolgus monkey]). The results of this study demonstrate that the in vitro stability of 28Y042-7F11-1 in human and cynomolgus monkey serum is favorable compared to historical values ​​obtained with mepolizumab, and indicate that the behavior of 28Y042-7F11-1 is in line with expectations for typical monoclonal antibodies.

[0660] PK / PD determination in cynomolgus monkey 28Y042-7F11-1

[0661] A 9-month non-GLP in vivo pharmacokinetic / pharmacodynamic (PK / PD) study was conducted in four groups of cynomolgus macaques (Macaca fascicularis), each consisting of two males and two females. On day 1, animals were administered 28Y042-7F11-1 (0.05 mg / kg or 1 mg / kg), mepolizumab (1 mg / kg), or a carrier via intravenous bolus injection. In addition to determining the PK parameters of the injected antibody, two PD parameters were evaluated to compare the in vivo activity of 28Y042-7F11-1 with that of mepolizumab: a) the level and duration of eosinophil suppression; b) the level and duration of serum total IL-5 concentration.

[0662] PK evaluation of 28Y042-7F11-1

[0663] Pharmacokinetic (PK) evaluations were performed on cynomolgus monkey serum samples over a period of 5376 hours (week 32). Analysis showed that 28Y042-7F11-1 had a 1.8-fold reduced serum clearance compared to mepolizumab (0.105 ml / hr / kg vs. 0.185 ml / hr / kg, respectively), and an improved half-life of 24.5 days compared to mepolizumab's 11.5 days (Table 19). Table 19 Explanation / Description: Pharmacokinetic parameters determined from the cynomolgus monkey PK study (*median).

[0664] Table 19.

[0665]

[0666]

[0667] 28Y042-7F11-1-mediated eosinophil suppression

[0668] A decrease in eosinophil count was used as a biomarker for 28Y042-7F11-1-mediated IL-5 neutralizing activity. In vivo, compared with mepolizumab in cynomolgus monkeys, 28Y042-7F11-1 exhibited a prolonged duration of eosinophil suppression. Figure 5 When 28Y042-7F11-1 was administered at 1 / 20th the dose of mepolizumab, it showed equivalent or slightly superior inhibition of eosinophils, indicating that 28Y042-7F11-1 has at least 20-fold higher activity than mepolizumab in vivo. When administered at 1 mg / kg, blood eosinophil counts with 28Y042-7F11-1 remained ≤50% of pre-administration levels for up to 24 weeks post-administration, compared to 4–7 weeks with mepolizumab. Recovery of blood eosinophil counts began to be observed around week 7 with 28Y042-7F11-1 and week 4 with mepolizumab. These data demonstrate the feasibility of three-monthly dosing in humans, and also support six-monthly dosing.

[0669] Serum total IL-5 level

[0670] Total IL-5 levels (IL-5 in almost exclusive complex with 28Y042-7F11-1 or mepolizumab) were observed to increase after administration, and both 28Y042-7F11-1 administration groups showed significantly greater persistence compared to the mepolizumab group (where the IL-5 complex began to decrease much earlier). Figure 6 The effects of mepolizumab on IL-5 began to decline after day 29 (672 hours), after day 85 (2016 hours), and after day 113 (2688 hours). This reflects that mepolizumab has a lower affinity for IL-5 and a shorter half-life compared to 28Y042-7F11-1, which maintains the IL-5 complex for a longer duration.

[0671] Non-compartmental pharmacokinetic analyses were performed on the delivered 28Y042-7F11-1 via a single intravenous administration and a single subcutaneous administration. Intravenous administration of GSK3511294 showed an increased serum half-life (24 days vs. 11.5 days) and a 1.8-fold decrease in serum clearance compared to mepolizumab. Figure 4 ). Methods: TF-1 functional cell assay

[0672] Antibody samples and controls were prepared in 96-well polypropylene plates for preliminary screening. Antibodies were diluted in cell culture medium to a final assay concentration of 200 nM and then aseptically filtered using a filter plate (Pall Corporation multi-well plate, ACRO PREP 96 filter plate, 3.0 μm glass fiber media / 0.2 μm BIO-INERT membrane, #5053). Samples were serially diluted 1 / 4 on the plate to generate a 10-point series with a concentration range of (200 nM–7.63 pM). The aim was to generate individual dose-response curves to provide varying IC50 values. 50 Early estimation of the value. The human IL-5 cytokine used to stimulate cells (molecular weight of homodimer, 28.5 kDa) was diluted in cell culture medium and determined to a final assay concentration of 17.5 pM (0.5 ng / ml) (assay for human IL-5-stimulated EC5). 80 Preparation. Human IL-5 was added to plates containing a series of antibody dilutions and incubated at room temperature for 1 hour. TF1 cells were washed three times with PBS to ensure successful removal of GMCSF (granulocyte-macrophage colony-stimulating factor), a growth factor used for cell proliferation. Cells were cultured at 0.2 x 10⁻⁶ cells / mL. 6 Cells were seeded at a density of 100 cells / well in 96-well solid white flat-bottomed tissue culture plates. Pre-incubated antibodies and cytokines were then added to the cells, and the plates were further incubated at 37°C, 5% CO2 for 3 days. The plates were removed from the incubator, and 100 μl of CELLTITER-GLO luminescent reagent (Promega, G7571) was added to each well. The plates were then incubated at room temperature on a shaker for 1 hour. The plates were then read using an ENVISION luminescent plate reader (Biomax 501510).

[0673] Alternatively, to evaluate IC in more detail 50 The values ​​were adjusted using the following variations compared to the methods described above. A series of dilutions of the antibody and control were prepared in 96-well polypropylene plates. The antibody was diluted to a final assay concentration of 1 nM in cell culture medium and serially diluted on plates at 1 / 1.7 ratios to produce a concentration range of (1 nM – 0.000418 pM). The human IL-5 cytokine used to stimulate cells was diluted in cell culture medium and prepared to a final assay concentration of 3 pM.

[0674] Method: BIACORE

[0675] Measurements of binding to human IL-5 were performed using a BIACORE 4000 (GE Healthcare). The sample flow rate used throughout was 10 μl / min for conjugation and regeneration, and 30 μl / min for kinetic assays. Protein A was immobilized on a Series S CM5 chip (GE Healthcare, BR-1005-30) via primary amine conjugation (GE Healthcare, BR-1000-50). This surface was then used to capture anti-IL-5 antibodies on spots 1 and 5, while spots 2 and 4 served as references. Recombinant human IL-5 was then passed through the captured antibody at 100 nM. A 30-minute dissociation time was used, as previous experiments found this necessary for accurate determination of the off-rate of mepolizumab. Binding curves were referenced twice with buffer injection (i.e., 0 nM), and the data were fitted to the BIACORE 4000 evaluation software using a 1:1 model. The operation was performed at 25°C using HBS-EP (Teknova, H8022) as the run buffer and 50 mM NaOH as the regeneration solution.

[0676] Data analysis revealed that the antibody dissociation rate was at the sensitivity limit of the BIACORE 4000 instrument, making it impossible to calculate the accurate dissociation rate at 25°C. Therefore, the test was repeated at 37°C to increase the dissociation rate, thus making it possible to calculate the accurate dissociation rate of the antibody.

[0677] Method: MSD-SET analysis

[0678] The affinity of these antibodies for human IL-5 at 25°C was determined using MSD-SET (MSD solution equilibrium titration) analysis, as the dissociation rate at this temperature was too slow to be measured by BIACORE. MSD-SET determines the solution phase and the equilibrium affinity of the antibodies. This method relies on detecting the free antigen at equilibrium in the antibody concentrations of the titration series.

[0679] Biotinylated human IL-5 was used at a constant concentration of 30 pM, while antibody samples were titrated from 2.5 nM to 0.5 pM in 1 / 3 increments, with a final dilution of 0.05 pM in 96-well polypropylene plates. The titrated antibody and IL-5 were incubated at room temperature for 24 hours. After 24 hours, the antibody (20 nM in PBS) was coated onto standard binding MSD plates (MesoScale Discovery, L15XA) at room temperature for 30 minutes. The plates were then blocked with STARTING BLOCK blocking buffer (ThermoScientific, #37542) for 30 minutes with shaking at 700 rpm, followed by three washes with washing buffer. The incubated solution was added to the MSD plates for 150 seconds with shaking at 700 rpm, followed by one wash. The captured antigen on the plates was detected using SULFOTAG-labeled streptavidin (Meso Scale Discovery, R32AD-1) by incubation on the plates for 3 minutes. The plates were washed three times with washing buffer and then read using an MSD SECTOR IMAGER instrument (Meso Scale Discovery, R92TC-1) with 1x read buffer T containing surfactant. The percentage of free antigen was plotted as a function of antibody titration using GRAPHPAD PRISM software and fitted to a quadratic equation.

[0680] The affinity for cynomolgus monkey IL-5 at 25°C was also determined using MSD-SET analysis. The method used was the same as described above, but with a cynomolgus monkey IL-5 concentration of 62.5 pM.

[0681] Methods: Kinexa analysis of 28Y042-7F11-1

[0682] To generate an accurate affinity determination of 28Y042-7F11-1 for binding to human IL-5 at 25°C, KINEXA (kinetic repulsion assay) was used as an alternative to MSD-SET analysis because the 95% confidence intervals generated for 28Y042-7F11-1 showed poor fit of the data to the model.

[0683] Solution-phase affinity measurements were performed using a Sapidyne KINEX A3200 instrument. This method relies on detecting free antibody at equilibrium in a titration series of antigen concentrations. For the assay, a bead matrix was created using NHS-activated SEPHEROSE beads (GE Healthcare, 17-0906-01) coated with human IL-5. For the affinity assay, a fixed concentration of antibody was incubated with a series of dilutions of human IL-5 concentrations, allowing binding to reach equilibrium before the sample was run on the KINEX A3200 instrument. Each solution was passed through aliquots of antigen beads, where the free antibody bound to the antigen-coated beads, and detected using anti-human IgG antibody (DYLIGHT 649-AFFINIPURE F(ab')2 fragment goat anti-human IgG; Jackoson Immunoresearch, 109-496-170), where fresh antigen beads were used to measure each sample. Data were analyzed using the software inherent in the KINEXA machine, with multiple runs performed using different initial antibody concentrations above and below the expected affinity for the interaction, namely 300 pM and 50 pM (concentration-driven and affinity-driven interactions, respectively). The range of antigen concentrations in a single assay was sufficient to saturate all available antibodies, resulting in almost 100% unbinding (10 nM to 4.88 pM for concentration-driven curves and 1 nM to 0.49 pM for affinity-driven curves). The data from the multiple runs were then combined and analyzed using the KINEXA machine's inherent "n-plot" analysis software to determine K... D And 95% confidence interval.

[0684] Methods: IL-5 binding of 28Y042-7F11-1, a competitor to mepolizumab.

[0685] To determine the identical epitopes on human IL-5 bound by 28Y042-7F11-1 and mepolizumab, a competitive assay was performed using a FORTEBIO OCTET RED384 Biolayer Interferometer (BLI). Since human IL-5 is a dimer, a tandem assay was used, in which biotinylated human IL-5 at 5 μg / mL in PBSF buffer was captured onto a streptavidin surface (FORTEBIO, 18-5019). This surface was saturated with mepolizumab at 100 nM in PBSF, followed by 28Y042-7F11-1 at 100 nM. This process was repeated with the IL-5 surface saturated with 28Y042-7F11-1 followed by mepolizumab, and a self-binding control was also included. Analysis was performed at 25°C with a plate shaker speed of 1000 rpm. Data were analyzed using the instrument's FORTEBIO data analysis.

[0686] Method: SEC analysis

[0687] Analytical size exclusion chromatography (SEC) was performed to assess the purity (% monomer) and retention time of the molecules. Retention time can indicate potential exploitability issues of the molecules. SEC was performed on an AGILENT 1100 HPLC system using a TSK G3000SWXL, 250A, 5 μm, 30 cm x 7.8 mm column. The running conditions were 200 mM NaH₂PO₄, 250 mM NaCl, pH 6.0, and a flow rate of 0.5 mL / min. The loading per sample was 20 μg, with a run time of 30 min. The results were analyzed using peak integration software in CHEMSTATION.

[0688] Methods: The binding of 28Y042-7F11-1 to the human Fc receptor was evaluated using PROTEON.

[0689] The binding of 28Y042-7F11-1 to recombinant soluble human Fc gamma receptor (FcγR) was evaluated using a PROTEON XPR36 (BIORAD) biosensor instrument. Antibodies were analyzed against a positive control antibody containing the wild-type human IgG1 Fc region and a negative control antibody containing two point mutations (L235A / G237A) in the Fc region that reduce interaction with the Fc gamma receptor. Another control antibody containing a YTE mutation in its Fc region was also included (28Y042-7F11-1 also contains a YTE mutation).

[0690] Mouse anti-polyhistidine IgG (ANTI-TETRA-HIS; Qiagen, 34670) was immobilized on a GLM biosensor chip (Bio-Rad, 176-5012) via primary amine conjugation (GE Healthcare, BR-1000-50). This surface served as a capture surface for polyhistidine-tagged human Fc gamma receptors (all internally generated reagents [except CD64-Fc; R&DSystems, 1257-FC]). The antibody to be tested was used as the analyte and passed at 1024 nM, 256 nM, 64 nM, 16 nM, and 4 nM, with 0 nM injection (i.e., a separate buffer) used for dual-reference binding curves. The mouse anti-polyhistidine IgG surface was regenerated with 100 mM phosphate between interactions. Runs were performed at 25 °C using HBS-EP (Teknova, H8022) as the run buffer. Data for each receptor were analyzed separately, with the overall R-max set and the equilibrium model inherent in PROTEON's analysis software used.

[0691] Methods: The binding of 28Y042-7F11-1 to human complement C1q was evaluated using PROTEON.

[0692] Using PROTEON XPR36 (BioRad) TMThe biosensor instrument 28Y042-7F11-1 was evaluated for binding to recombinant human soluble complement C1q. A control antibody containing a YTE variation in its Fc region was included as a control.

[0693] The antibody to be tested was immobilized on a GLC chip (Bio-Rad, 176-5011) via primary amine conjugation (GE Healthcare, BR-1000-50). Recombinant C1q (Sigma, C1740) was passed through the immobilized antibody at 512 nM, 128 nM, 32 nM, 8 nM, 2 nM, and 0 nM (i.e., separate buffers), and blank activation and inactivation flow cells were used for dual-reference binding curves. The running buffer used for binding analysis was HBS-EP (pH 7.4, Teknova, H8022) with 10 mM CaCl2. Data were fitted to PROTEON XPR36 (Bio-Rad) using the overall R-max value. TM The analysis software's inherent equilibrium model.

[0694] Method: FcRn binding

[0695] The binding affinity of 28Y042-7F11-1 and mepolizumab to human FcRn at pH 6.0 and pH 7.4 was evaluated using a BIACORE T200 instrument at 37 °C. Human recombinant IL-5 was diluted in acetate buffer at pH 5.0 and immobilized to a level of 535 RU by amine conjugation onto a CM5 chip (GE, BR100530). 28Y042-7F11-1 or mepolizumab was captured by passing a 100 nM solution of either mAb in HBS-EP (Teknova, H8022) buffer through the chip surface (5 μl / min) for 24 seconds. After antibody capture via immobilized IL-5, different concentrations (0.5 μM to 32 μM) of human FcRn were cycled through the chip surface at a contact time of 120 seconds at 5 μl / min, followed by dissociation for 80 seconds per cycle. At the end of each cycle, the chip surface was regenerated with 10 mM glycine at pH 1.5 at 50 μl / min for 5 seconds, followed by 10 mM NaOH at 50 μl / min for 5 seconds. FcRn was cycled at each concentration at pH 6.0 (HBS-EP+, Teknova, catalog: H8022, pH 6.0) and pH 7.4 (HBS-EP). For human FcRn, a molecular weight of 42929 Da was used for calculation.

[0696] Method: Combining natural IL-5

[0697] The binding capacity of 28Y042-7F11-1, mepolizumab, and negative control antibodies (pacolizumab and anti-RSV) to native IL-5 was determined. 28Y042-7F11-1, mepolizumab, or control antibody were diluted in PBS to a concentration of 1 μg / ml / 200 μl / well and incubated overnight at 4°C on MAXISORP ELISA plates (Nunc, 10394751), followed by washing (all washes were performed using 200 μl / well of PBS supplemented with 0.05% Tween 20). The plates were blocked for 2 hours with 300 μl / well of PBS supplemented with 1% BSA (Sigma, A9576), and then a 1:2 titration of culture supernatant containing 4 ng / ml native IL-5 was added to the plates. The supernatant and antibody were incubated at room temperature for 1 hour, washed, and then 100 μl / well of biotinylated anti-IL-5 antibody (Fisher, MM550CB) diluted at 1 μg / ml in PBS supplemented with 0.5% BSA was added to the plate and incubated at room temperature for 1 hour. Streptavidin-HRP (GE Healthcare, RPN4401V) diluted 1:5500 in PBS supplemented with 0.5% BSA was used for detection (100 μl / well) and incubated at room temperature for 20 minutes, followed by the addition of 100 μl / well of TMB substrate for 5 minutes, and then the reaction was terminated with 100 μl / well of 1M H2SO4. Absorbance was read at 450 nm using a SPECTRAMAX plate reader (Biomax, 088261; all spots were performed in duplicate). The control conditions using culture supernatant lacking IL-5 or without capture antibodies (28Y042-7F11-1, mepolizumab, and negative control antibody) were also evaluated.

[0698] Human-isolated PBMCs were stimulated with anti-CD3 and anti-CD28 antibodies to generate native IL-5. Blood (100 ml) from a healthy volunteer donor was obtained in sodium heparin (1000 IU / 100 ml). Following the manufacturer's instructions, blood was separated to isolate PBMCs using density gradient centrifugation in HISTOPAQUE FICOLL and LEUCOSEP tubes (Greiner, 227290), followed by a 1:1 dilution with RPMI (Gibco, 31870074). After separation, the PBMCs were washed twice with RPMI (rotation at 1200 rpm for 2 x 5 minutes). After the second wash, the cells were resuspended in 50 ml of RPMI (supplemented with 10% fetal bovine serum, penicillin / streptomycin, and L-glutamine), from which 500 μl of sample was taken and mixed 1:1 with TRYPLE EXPRESS (Gibco, 12604-021) and run on a VICELL to obtain cell counts. Pre-coat plates with 1 μg / ml anti-CD3 (internal OKT3) and 3 μg / ml anti-CD28 (internal) at 37°C for 60 min, followed by washing the wells once with PBS. Dilute the cells to 1 x 10⁻⁶. 6 Cells / ml, and 200 μl / well (2 x 10⁻⁶) 5 Cells were added to wells pre-coated with anti-CD3 / CD28 and incubated at 37°C, 5% CO2 for 4 days. After stimulation, the cell supernatant was collected into 50 ml Falcon tubes and rotated (5 min, 1200 rpm). The supernatant (45 ml) was collected into a new Falcon tube and the cell pellet was discarded. BSA (667 μl, from Sigma, A9576) was added to 40 ml of supernatant (0.5% final concentration) and aliquoted between two VIVASPIN 20 columns (Sartorius, VS0112) and centrifuged at 3600 g (4500 rpm) 2 x 10 min to concentrate the supernatant. The concentrated supernatant fractions were collected and stored at 4°C. As a control, 5 ml of unrotated or unadded BSA-rich raw supernatant was also stored at 4°C. The unstimulated supernatant sample was rotated in the same manner as above and stored at 4°C to obtain an IL-5-deficient control supernatant. The concentration of IL-5 was quantified using the QUANTIKINE ELISA kit (R&D Systems, D5000B).

[0699] Methods: The inhibition of IL-5-mediated eosinophil morphology changes was determined by flow cytometry.

[0700] This assay was used to measure the inhibition of eosinophil morphology changes mediated by recombinant IL-5 in human whole blood by 28Y042-7F11-1 or mepolizumab (all sites were performed in duplicate). Blood (1000 IU / 100 ml) from healthy volunteer donors (human; with appropriate consent) was obtained from GlaxoSmithKline Stevenage blood donation units in heparin sodium. 28Y042-7F11-1, mepolizumab, and the control antibodies pacolizumab and anti-RSV were each diluted to obtain a final assay concentration of 10 μg / ml. The antibody was incubated with an equal volume of recombinant IL-5 (R&D Systems, lot number 091231202) at a final concentration of 10 ng / ml at 37°C for 1 hour. After incubation, 20 μl of each antibody / IL-5 complex sample was added to 80 μl of whole blood from one of six donors in a 96-well polypropylene plate (Fisher Scientific, 10007621). The plates were incubated at 37°C for 30 min, then placed on ice and fixed at 250 μl / well with CELL FIX (BD, 340181) diluted 1:9:30 with water:PBS (this ratio is 4 times higher than the manufacturer’s recommended dilution to provide conditions that do not compromise eosinophil integrity) for 2 min. Cells were then lysed at 1 ml / well with ice-cold PHARM LYSE (BioLegend, RBC lysis buffer, 420301) according to the manufacturer’s protocol. The samples were rotated and the supernatant was removed, then resuspended in FACS buffer, and data were acquired on CANTO II-gated eosinophils by their autofluorescence in the PE channels. The effects of 28Y042-7F11-1, mepolizumab, pacolizumab, and anti-RSV in the absence of IL-5 were also tested using PBS alone. The effect of IL-5 on eosinophil morphology changes in the absence of any antibody was also tested using PBS alone.

[0701] Methods: Serum stability study

[0702] Dilute 28Y042-7F11-1 to pure, pooled, sterile human serum (GSK Stevenage blood donation units) or pure, pooled, sterile cynomolgus monkey serum (from SeraLabs) to give 4 ml of each containing 28Y042-7F11-1 at a target concentration of 120 μg / ml. Then, aliquot each serum sample (human or cynomolgus monkey) into 5 x 750 μl aliquots, place them in sterile 2 ml microcentrifuge tubes, and seal the tubes. Immediately place one aliquot of each serum species on dry ice and allow it to freeze to produce T0 samples, then transfer to -80°C for storage. Place the remaining aliquots in a humidified tissue incubator set at 37°C, 5% CO2. After 1, 2, 4, and 6 weeks, remove one aliquot of each serum species, freeze it on dry ice as before, and then transfer it to -80°C for storage. In vitro stability studies were performed when samples were removed and stored for 6 weeks.

[0703] To quantify 28Y042-7F11-1 during serum incubation, samples derived from in vitro serum stability studies were tested using the MSD (Meso Scale Discovery) IL-5 capture immunoassay. Because biotinylated IL-5 was used as the capture reagent, only molecules with IL-5 activity were captured and subsequently detected; therefore, any change in recovery detected over time indicates a loss of activity of 28Y042-7F11-1. All samples were tested together in a single assay after completing 6 weeks of incubation.

[0704] The IL-5 capture immunoassay was performed using a 96-well standard-binding MSD plate (MSD, #L15XA-6). The plate was coated with 50 μl of NEUTRAVIDIN (Thermo-Fisher Scientific, #31000) diluted 2 μg / mL to tissue culture-grade PBS (Sigma-Aldrich, #D8537). The plate was incubated overnight at +4°C. The coated plates were washed using an automated plate washer (Biotek ELx405), with each well washed three times with 300 μl of PBS + 0.1% TWEEN-20. After washing, the plates were patted on paper towels to remove residual liquid. All plates were then blocked with 150 μl of assay buffer (PBS + 5% BSA (Sigma-Aldrich, #A7030) + 1% TWEEN-20 (Fisher Scientific, #BP337)). The plates were incubated at room temperature for 1 hour on a plate shaker (Heidolph TITRAMAX 1000) set to approximately 750 rpm (for all use), followed by washing as before. Biotinylated human IL-5 (GSK reagent) was diluted to 100 ng / ml in assay buffer, and 25 μl of this was added to all wells of the sealed and washed plates. The plates were then incubated on the plate shaker at room temperature for at least 1 hour, followed by washing as before. A standard curve for 28Y042-7F11-1 and test samples were prepared during incubation with biotinylated IL-5. The standard curve for 28Y042-7F11-1 was prepared by diluting to the highest concentration of 250 ng / ml in assay buffer. This was then serially diluted at a dilution factor of 2.5 at a total of 11 dilution points, with the 12th point being a separate assay buffer as a assay blank. All test serum stability samples were diluted to the assay buffer using dilution factors of 2000, 20,000, and 200,000. A minimum of 20 μl of each pure sample was used between dilutions, and dilutions were performed using sequential dilutions of no more than 10 (i.e., achieving a dilution factor of 2000 through three consecutive 10-fold dilutions followed by a 2-fold dilution). Once incubation with biotinylated IL-5 and plate washing were complete, 25 μl of the 28Y042-7F11-1 standard was added in triplicate, followed by 25 μl of each test sample at each dilution in duplicate. The plates were then incubated on a plate shaker at room temperature for at least 1 hour, followed by washing as before. To detect the binding of 28Y042-7F11-1, mouse monoclonal anti-human Fc SULFOTAG (labeled with unlabeled antibody, from Southern Biotech, #9040-01) was diluted to 500 ng / ml in assay buffer, and 25 μl was added to all wells.The plates were then incubated on a plate shaker at room temperature for at least 1 hour, followed by washing as before. MSD read buffer T containing surfactant was diluted with distilled H2O to a 1x working solution, and 150 μl was added to all wells. The plates were then read using a SECTOR 6000MSD imager. The mean concentration of 28Y042-7F11-1 measured in human or cynomolgus monkey serum was then normalized to % of the concentration measured in the T0 sample using the following formula:

[0705] T0% = [Concentration in test sample / Concentration in T0 sample] * 100

[0706] To contextualize the serum stability data generated for 28Y042-7F11-1, it was plotted alongside historical data generated for mepolizumab and the IL-13-specific control antibody. The serum stability settings used for these molecules were the same as described above, except that the time points used were 0, 2, 4, and 6 weeks, and the batches of human and cynomolgus sera used differed. Analysis of the mepolizumab samples was as described above, except that the antibody standard curve was tested using a dilution factor of 1000 starting at 500 ng / ml and the samples were tested using only a dilution factor of 1000. Analysis of the IL-13-specific control antibody samples was as described above, except that the antibody standard curve was tested using a dilution factor of 1000 starting at 500 ng / ml and the IL-13 capture assay was used for the data reported here (where biotinylated IL-13 at 100 ng / ml (GSK internal reagent) was used instead of biotinylated IL-5 in the assay buffer). Data were normalized to values ​​at T0 as described above.

[0707] Methods: In vivo PK / PD in cynomolgus monkeys

[0708] The study included four groups of cynomolgus macaques (2-5 years old, weighing 2-6 kg, native to Mauritius for breeding purposes), each group consisting of 2 males and 2 females. The macaques were housed in enclosures of four of the same sex, with an enriched environment to promote social interaction, play, and exploration. Throughout the study, each animal received an average of 200 g / day of a standard diet (PMI Nutrition International Certified Primate Diet No. 5S48 (25% protein) and a Special Diet Service (SDS) Mazuri Expanded Short (MP(E) short SQC)) and free access to water. Complete hematological cell counts (including eosinophil counts) were performed prior to administration and then every 1 or 2 weeks after administration (for a total of 6 months). On day 1, animals were administered 28Y042-7F11-1 (0.05 mg / kg or 1 mg / kg), mepolizumab (1 mg / kg), or a carrier via intravenous bolus. In addition to hematological cell counts, PK assays and total IL-5 measurements were also performed.

[0709] Method: PK data

[0710] On day 1, administer the test substance to the animals and sample by drawing blood from the femoral vein without the addition of an anticoagulant (see Tables 20 and 21). Sampling should be performed as late as possible during the afternoon (between 1 and 3 pm) to conform to the hematological (eosinophilic) blood sampling schedule. Allow the sample to coagulate at ambient temperature for at least 1 hour, then centrifuge at 2500g for 10 minutes at 4°C. The resulting serum should be transferred to uniquely labeled standard Sarstedt tubes and immediately frozen on dry ice, then stored at -80°C.

[0711] The concentrations of 28Y042-7F11-1 and mepolizumab in cynomolgus monkey serum samples were determined by immunoassay using a GYROLAB workstation (Gyros, P0004943) platform. Biotinylated recombinant human IL-5 capture (internal reagent) and 28Y042-7F11-1 standard (from cynomolgus monkey serum) were diluted in REXXIP A buffer (Gyros, P0004820), and the ALEXA-647-labeled anti-human IgG assay (clone JDC-10) was diluted in REXXIP F buffer (Gyros, P0004825). The assay was validated on a BIOAFFY 1000CD (Gyros, P0004253) in the range of 30–10,000 ng / ml for 28Y042-7F11-1 and 100–10,000 ng / ml for mepolizumab. Serum concentrations of 28Y042-7F11-1 were within the expected range. Table 20: Description / Explanation: Cynomolgus monkey blood sampling schedule for PK and total IL-5 assays. Blood extraction volume was 0.7 ml (* indicates 0.5 ml). Table 21: Description / Explanation: Hematological sample collection schedule.

[0712] Table 20.

[0713]

[0714] Table 21.

[0715]

[0716]

[0717] Method: Total IL-5 data

[0718] As described for PK data collection, animals were administered medication, blood samples were collected, and processed. Standard curves for cynomolgus monkey IL-5 (2.44–10,000 pg / ml final concentration) were prepared by serial dilutions at 1 / 4 in pooled cynomolgus monkey serum (SeraLab, S-118-D) at x2 final concentrations. Four QC-spiked IL-5 controls were also prepared using pooled cynomolgus monkey serum (5000, 500, 50, and 0 pg / ml IL-5) at the desired 2x final concentration (resulting in 1:2 dilutions with the antibody mixture). Each standard / QC-spiked control / serum test sample (50 μl) was then transferred to a new 96-well polypropylene plate. A mixture of capture and detection antibodies was prepared using a rat anti-human IL-5-biotin conjugate mAb (Southern Biotech, 10118-08) at a final concentration of 0.5 μg / ml as the capture mAb and a rat anti-human IL-5 sulfonated mAb (Southern Biotech, 10118-14 (internal sulfonated MSD)) at a final concentration of 0.5 μg / ml as the detection mAb. Both the capture and detection antibodies were prepared in assay buffer (RK / CI buffer: [6.4 mM EDTA, 5.1 mM EGTA, 50 mM HEPES, 149.2 mM NaCl, 1% Triton X-100, 1% BSA, pH 7.4]) at 2x the final concentration, which readily resulted in a 1:2 dilution in standards / samples / controls. Add 50 μl of the antibody mixture to each standard / QC-spiked control / test serum sample and incubate at room temperature for 3 hours with shaking (600 rpm) in the dark. Block the streptavidin gold MSD plate (MesoScale Discovery, L15SA-1) with 150 μl / well MSD blocking buffer (3% MSD blocking agent A in PBS (Meso Scale Discovery, R93BA-1)) and incubate at room temperature for 1 hour with shaking (600 rpm). After incubating the antibody mixture and standard / QC-spiked control / test serum samples for 3 hours, transfer 25 μl / well in duplicate (or triplicate, for QC-spiked controls) to the blocked and washed (SKAN WASHER 300, Skatron Instruments) MSD streptavidin gold plate. Then incubate the plate at room temperature for 1 hour with shaking (600 rpm). After incubation, the plates were washed (SKAN WASHER 300, Skatron Instruments). Reading buffer T (2x) was prepared and 150 μl / well was added to each well of the MSD streptavidin gold plate.Electrochemiluminescence was then quantified using an MSD Sector S 600 (model 1201) within 15 minutes.

[0719] Methods: Eosinophil count

[0720] A decrease in eosinophil count was used as a biomarker for 28Y042-7F11-1-mediated IL-5 neutralizing activity. Eosinophil levels in a cohort of 39 animals (days -51 and -44) were pre-screened prior to administration of the test substance, and animals with eosinophil levels (≥180 eosinophils / μL) were selected for the study. Animals with higher eosinophil counts were selected based on the assumption that they would provide a larger assay window to measure the level of eosinophil suppression. Concerningly, because these animals were bred in captivity and lived under cleanroom conditions, they had lower baseline eosinophil counts than wild animals, and these lower eosinophil counts could potentially drop below the minimum quantifiable level when suppressed by the drug. Also evident during the pre-screening phase was the significant fluctuations in eosinophil counts exhibited by some animals. The cause of this variation is unclear but could be attributed to a variety of factors, such as environment, stress, or hormonal changes.

[0721] Once 16 animals were selected for the study, they were re-housed in research enclosures (4 animals of the same sex per enclosure) and allowed to acclimatize to their new environment. During the acclimatization period, three pre-drug hematological counts were performed (days -21, -14, and -7). On day 1, the animals were administered the test substance, and 0.5 ml of blood was drawn from the femoral vein using EDTA as an anticoagulant. Sampling was performed as late as possible during the afternoon (between 1 and 3 pm) to control for diurnal variations in blood eosinophil levels. Following collection, samples were processed within 60 minutes of the final sample collection at each time point and quantified using both the peroxidase method and the basophil / lobularity method on an ADVIA 120 hematology analyzer (Siemens).

[0722] Example 2

[0723] 28Y042-7F11-1 was also evaluated in 4-week single-dose and 26-week repeated-dose GLP toxicity studies (10 and 100 mg / kg / week). In these studies, 28Y042-7F11-1 was administered subcutaneously to cynomolgus monkeys. The off-dose phase of the 26-week study is still ongoing (May 2017), therefore, an interim report is reported here, presenting data generated from pretreatment to the end of the dosing period. Systemic exposures achieved in this study are shown in Table 22. These studies and related analyses were performed using standard methodologies. Table 22 Description: Comparative evaluation of mean systemic exposures following subcutaneous administration of 28Y042-7F11-1 in cynomolgus monkeys. Table 23 Description: Mean pharmacokinetic parameters of 28Y042-7F11-1 in cynomolgus monkeys following a single IV or SC injection. Table 24 Explanation / Description: Safety margin (“safety coverage”) when comparing cynomolgus monkey NOAEL data with predicted human data for subcutaneous injection doses.

[0724] Table 22.

[0725]

[0726] Table 23.

[0727]

[0728] NA = Not applicable

[0729] 1.CL / F

[0730] 2.Vz / F

[0731] Table 24.

[0732]

[0733] 1- Assume a 70kg subject; 2- Dosage (expressed in mg / kg) coverage

[0734] Example 3

[0735] Informal sequence list

[0736] The underlined sections below, according to the Kabat definition of CDRs, identify CDR sequences in the variable heavy and light chain portions of the antibody or in the nucleic acid sequences encoding these CDR sequences. For example, in SEQ ID NO:1, frames and CDRs are shown sequentially from the proximal amino terminus to the carboxyl terminus of the displayed sequence as plain text frame 1, underlined CDR1, plain text frame 2, underlined CDR2, plain text frame 3, underlined CDR3, and plain text frame 4. This scheme is used, for example, in SEQ ID NO:1-4. The N-terminal methionine residues shown in these sequences are cleavable. Therefore, sequences showing N-terminal methionine residues here should also be considered to disclose the cleavage forms of proteins lacking such N-terminal methionine residues. Nucleic acid sequences are represented as DNA nucleic acid sequences and include “t” nucleic acid residues; the corresponding RNA sequences should also be considered disclosed, such that “t” nucleic acid residues can also be considered to disclose “u” nucleic acid residues. In addition, the 5' proximal "atg" start codon and the 3' proximal "taa", "tag" and "tga" stop codons have been omitted from the cDNA nucleic acid sequence below.

[0737] 28Y042-7F11-1 Full-length heavy chain

[0738] SEQ ID NO:1

[0739]

[0740] 28Y042-7F11-1 Full-length light chain

[0741] SEQ ID NO:2

[0742]

[0743] 28Y042-7F11-1 VH

[0744] SEQ ID NO:3

[0745]

[0746] 28Y042-7F11-1 VL

[0747] SEQ ID NO:4

[0748]

[0749] 28Y042-7F11-1 CDRH1

[0750] SEQ ID NO:5

[0751] GSSVH

[0752] 28Y042-7F11-1 CDRH2

[0753] SEQ ID NO:6

[0754] VIWASGGTDYNSALMS

[0755] 28Y042-7F11-1 CDRH3

[0756] SEQ ID NO:7

[0757] DPPSGLLRLDY

[0758] 28Y042-7F11-1 CDRL1

[0759] SEQ ID NO:8

[0760] KSSQSLLNSGNQKNYLA

[0761] 28Y042-7F11-1 CDRL2

[0762] SEQ ID NO:9

[0763] GASTRES

[0764] 28Y042-7F11-1 CDRL3

[0765] SEQ ID NO:10

[0766] QNVHSFPFT

[0767] Human IL-5 (mature protein)

[0768] SEQ ID NO:11

[0769]

[0770] Human IL-5 receptor subunit ALPHA isotype 1 (mature protein) SEQ ID NO:12

[0771]

[0772] DNA encoding the full-length heavy chain 28Y042-7F11-1 with a leader sequence SEQ ID NO:13

[0773]

[0774] DNA encoding the full-length light chain 28Y042-7F11-1 with a leader sequence SEQ ID NO:14

[0775]

[0776] DNA encoding the 28Y042-7F11-1 heavy chain variable region

[0777] SEQ ID NO:15

[0778]

[0779] DNA encoding the 28Y042-7F11-1 light chain variable region

[0780] SEQ ID NO:16

[0781]

[0782] DNA encoding the full-length heavy chain 28Y042-7F11-1

[0783] SEQ ID NO:17

[0784]

[0785] DNA encoding the full-length light chain 28Y042-7F11-1

[0786] SEQ ID NO:18

[0787]

[0788] 28Y042-7F11-1 Heavy Chain Leader Sequence

[0789] SEQ ID NO:19

[0790] MGWSCIILFLVATATGVHS

[0791] 28Y042-7F11-1 Heavy Chain Leader Sequence

[0792] SEQ ID NO:20

[0793] MGWSCIILFLVATATGVHS

[0794] 28Y042-7F11-1 Heavy chain FR4 sequence

[0795] SEQ ID NO:21

[0796] WGRGTLVTVSS

[0797] The invention has now been fully described, and it will be apparent to those skilled in the art that many changes and modifications may be made therein without departing from the spirit or scope of the appended claims.

[0798] Material from an ASCII text file named “PU66209P_US_SeqList[,]”, created on May 26, 2017, and having a size of 21,851 bytes, is incorporated herein by reference in its entirety.

Claims

1. Use of a pharmaceutical composition comprising an antibody that binds to human IL-5 in combination with a pharmaceutically acceptable carrier in the preparation of a medicament for treating chronic obstructive pulmonary disease in a human subject with such need, wherein said antibody comprises a heavy chain and a light chain, wherein a) The heavy chain includes a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and b) The light chain includes a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO:

10. The antibody mentioned therein is a humanized IgG1 antibody.

2. The use of claim 1, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence as shown in SEQ ID NO:

21.

3. The use of claim 1 or claim 2, wherein the antibody comprises a heavy chain and a light chain, wherein a) The heavy chain comprises a heavy chain variable region sequence consisting of the amino acid sequence shown in SEQ ID NO: 3; and b) The light chain comprises a light chain variable region sequence consisting of the amino acid sequence shown in SEQ ID NO:

4.

4. The use of claim 3, wherein the heavy chain comprises a heavy chain Fc domain having a tyrosine residue at position 252, a threonine residue at position 254, and a glutamic acid residue at position 256.

5. The use of claim 3 or claim 4, wherein the antibody comprises a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO:

2.

6. Use according to any one of claims 1-5, wherein the antibody is at a concentration between 75 mg / mL and 150 mg / mL.

7. Use according to any one of claims 1-6, wherein the pharmaceutically acceptable carrier comprises an aqueous liquid formulation at a pH of 5.5 to 6.0, the aqueous liquid formulation containing 40 mM histidine, 180 mM trehalose, 100 mM arginine, 8 mM methionine, 0.02% by weight of polysorbate 80 and 0.05 mM EDTA.

8. The use of claim 7, wherein the pH is 6.0 and the antibody is at a concentration of 150 mg / mL.

9. Use according to any one of claims 1-8, wherein the antibody is administered in an amount from 2 mg to 600 mg.

10. The use of claim 9, wherein the antibody is administered once every 3 months or once every 6 months.

11. Use according to any one of claims 1-10, wherein the antibody is administered subcutaneously.

12. Use according to any one of claims 1-10, wherein the antibody is administered intravenously.

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