EA046656B1 - ANTIGEN-BINDING PROTEINS THAT BIND TO IL-5 AND ARE USED TO TREAT DISEASES - Google Patents

Description

The present invention relates to compositions for the treatment of diseases mediated by interleukin 5 (IL-5) and related methods.

State of the art

IL-5 is a secreted protein. IL-5 plays a role in a number 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 polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis. These serious diseases affect hundreds of millions of people around the world.

This means that there is a need for compositions suitable for the treatment of IL-5-mediated disease. Such compositions and associated methods are provided in the present invention.

The essence of the invention

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

Another aspect of the invention 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.

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

Another aspect of the invention 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.

Another aspect of the invention 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.

Another aspect of the invention is a peptide chain containing the amino acid sequence shown in SEQ ID NO: 3.

Another aspect of the invention is a peptide chain containing the amino acid sequence shown in SEQ ID NO: 1.

Another aspect of the invention 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.

Another aspect of the invention 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.

Another aspect of the invention is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 3.

Another aspect of the invention is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 1.

Another aspect of the invention 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.

Another aspect of the invention 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.

Another aspect of the invention is a composition comprising a nucleic acid having

- 1046656 the sequence shown in SEQ ID NO: 13, and a nucleic acid having the amino acid sequence shown in SEQ ID NO: 14.

Another aspect of the invention is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO: 15.

Another aspect of the invention is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO: 17.

Another aspect of the invention is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO: 13.

Another aspect of the invention is a method of producing a peptide chain containing the amino acid sequence shown in SEQ ID NO: 3, which method includes the step of culturing a recombinant host cell containing a nucleic acid encoding the amino acid sequence shown in SEQ ID. No. 3; and extraction of the peptide chain.

Another aspect of the invention is a method of producing an antibody, comprising the step of: a) culturing a recombinant host cell containing an expression vector containing 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; and b) recovering the antibody; thus obtaining the antibody.

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

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

Another aspect of the invention is a pharmaceutical composition comprising: a) an antibody comprising a heavy chain and a light chain, the heavy chain comprising 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.

Another aspect of the invention 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.

Another aspect of the invention is a method of treating mild to moderate asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with mild to moderate asthma; and b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 a CDR amino acid sequence shown in SEQ ID NO: 8, a CDR amino acid sequence shown in SEQ ID NO: 9, and a CDR amino acid sequence shown in SEQ ID NO: 10; thereby providing treatment for mild to moderate asthma in the subject.

Another aspect of the invention is a method of treating mild to moderate asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with mild to moderate asthma; and b) administering to the subject a therapeutically effective amount of 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: 1; thereby providing treatment for mild to moderate asthma in the subject.

Another aspect of the invention is a method of treating mild to moderate asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with mild to moderate asthma; and b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising 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,

- 2046656 shown in SEQ ID NO: 1, and a pharmaceutically effective carrier; thereby providing treatment for mild to moderate asthma in the subject.

Another aspect of the invention is a method of treating severe asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with severe asthma; and b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 a CDR amino acid sequence shown in SEQ ID NO: 8, a CDR amino acid sequence shown in SEQ ID NO: 9, and a CDR amino acid sequence shown in SEQ ID NO: 10; thereby providing treatment for severe asthma in the subject.

Another aspect of the invention is a method of treating severe asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with severe asthma; and b) administering to the subject a therapeutically effective amount of 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; thereby providing treatment for severe asthma in the subject.

Another aspect of the invention is a method of treating severe asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with severe asthma; and b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising 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 a pharmaceutically effective carrier ; thereby providing treatment for severe asthma in the subject.

Another aspect of the invention is a method of treating a subject with moderate to severe atopic dermatitis, comprising the steps of: a) identifying a subject having at least one of the characteristics selected from the group consisting of: i) diagnosing atopic dermatitis in accordance with the Hanifin criteria; Raika, revised by Eichenwald; ii) a provisional diagnosis of atopic dermatitis made more than two years ago or approximately two years before the start of treatment; iii) an overall health care provider score of three or more; iv) atopic dermatitis lesions greater than or equal to approximately 10% of the body surface area; v) an index of severity and area affected by eczema greater than or equal to 16; vi) an absolute blood eosinophil count greater than or equal to 150 cells per μL, greater than or equal to 200 cells per μL, greater than or equal to 300 cells per μL, or greater than or equal to 350 cells per μL; and vii) at least one pre-treatment condition selected from the group consisting of: 1) an inadequate response for six months or more to a topical atopic dermatitis medication; 2) poor tolerance to topical medications for atopic dermatitis; 3) a side effect from taking topical atopic dermatitis medication; and 4) inadequate response to non-pharmacological treatment for atopic dermatitis; and b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 a CDR amino acid sequence shown in SEQ ID NO: 8, a CDR amino acid sequence shown in SEQ ID NO: 9, and a CDR amino acid sequence shown in SEQ ID NO: 10; thereby providing treatment for atopic dermatitis in the subject.

Another aspect of the invention is a method of treating a subject with moderate to severe atopic dermatitis, comprising the steps of: a) identifying a subject having at least one of the characteristics selected from the group consisting of: i) diagnosing atopic dermatitis in accordance with the Hanifin criteria; Raika, revised by Eichenwald; ii) a provisional diagnosis of atopic dermatitis made more than two years ago or approximately two years before the start of treatment; iii) an overall health care provider score of three or more; iv) atopic dermatitis lesions greater than or equal to approximately 10% of the body surface area; v) an index of severity and area affected by eczema greater than or equal to 16; vi) an absolute blood eosinophil count greater than or equal to 150 cells per μL, greater than or equal to 200 cells per μL, greater than or equal to 300 cells per μL, or greater than or equal to 350 cells per μL; and vii) at least one pre-treatment condition selected from the group consisting of: 1) an inadequate response for six months or more to a topical atopic dermatitis medication; 2) poor tolerance to topical medications for atopic dermatitis; 3) a side effect from taking topical atopic dermatitis medication; and 4) inadequate response to non-pharmacological treatment for atopic dermatitis; and b) administering to the subject a therapeutically effective amount of 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: 1; thus providing treatment

- 3 046656 atopic dermatitis in the subject.

Another aspect of the invention is a method of treating a subject with moderate to severe atopic dermatitis, comprising the steps of: a) identifying a subject having at least one of the characteristics selected from the group consisting of: i) diagnosing atopic dermatitis in accordance with the Hanifin criteria; Raika, revised by Eichenwald; ii) a provisional diagnosis of atopic dermatitis made more than two years ago or approximately two years before the start of treatment; iii) an overall health care provider score of three or more; iv) atopic dermatitis lesions greater than or equal to approximately 10% of the body surface area; v) an index of severity and area affected by eczema greater than or equal to 16; vi) an absolute blood eosinophil count greater than or equal to 150 cells per μL, greater than or equal to 200 cells per μL, greater than or equal to 300 cells per μL, or greater than or equal to 350 cells per μL; and vii) at least one pre-treatment condition selected from the group consisting of: 1) an inadequate response for six months or more to a topical atopic dermatitis medication; 2) poor tolerance to topical medications for atopic dermatitis; 3) a side effect from taking topical atopic dermatitis medication; and 4) inadequate response to non-pharmacological treatment for atopic dermatitis; and b) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising 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: 1, and a pharmaceutically effective carrier ; thereby providing treatment for atopic dermatitis in the subject.

Another aspect of the invention is a method of reducing the absolute number of eosinophils in the blood of a subject, comprising the steps of: a) identifying a subject having 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 granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis and atopic dermatitis; and b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 a CDR amino acid sequence shown in SEQ ID NO: 8, a CDR amino acid sequence shown in SEQ ID NO: 9, and a CDR amino acid sequence shown in SEQ ID NO: 10; thereby reducing the subject's absolute blood eosinophil count.

Another aspect of the invention is a method of reducing the absolute number of eosinophils in the blood of a subject, comprising the steps of: a) identifying a subject having 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 granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis ita ; and b) administering to the subject a therapeutically effective amount of 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: 1; thereby providing treatment for atopic dermatitis in the subject.

Another aspect of the invention is a method of reducing the absolute number of eosinophils in the blood of a subject, comprising the steps of: a) identifying a subject having 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 granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis ita ; and b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising 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: 1, and a pharmaceutically effective carrier ; thereby reducing the subject's absolute blood eosinophil count.

Brief description of drawings

Fig. 1. Both 28Y042-7F11-1 and mepolizumab caused dose-dependent inhibition of human IL-5-induced TF-1 cell proliferation (IC ₅₀ 4 pM to 105 pM, respectively) when tested over a concentration range of 1 nM to 0.042 pM.

- 4 046656

Fig. 2. Inhibition of recombinant IL-5-mediated eosinophil shape changes by 28Y042-7F11-1 and mepolizumab in human whole blood. The data shown is from 6 donors.

Fig. 3. Binding of native IL-5 to 28Y042-7F11-1 and mepolizumab in ELISA assay.

Fig. 4. Comparison of the stability of 28Y042-7F11-1 incubated at 37°C for 6 weeks in pooled human or cynomolgus serum. The data are plotted in comparison with previous results obtained for mepolizumab and the IL-13-specific control antibody.

Fig. 5. Graphs of proportional changes in animals relative to the mean eosinophil response before dosing. Levels of eosinophil response in animals relative to the geometric mean of their values before dosing (from -12 to -26 days). Values were calculated for each animal, with overall geometric means calculated across groups added as horizontal bars. The solid horizontal line corresponds to 20% of the animal's initial eosinophil count; n=4 per group [2? and 2sG]; single (intravenous) administration of the drug on day 1 of the study.

Fig. 6. Cynomolgus monkey serum total IL-5 obtained in the 9-month PK/PD study (data at day 267/week 38) for animals treated with 28Y042-7F11-1, mepolizumab, or vehicle. For 28Y042-7F11-1, an increased period of time during which the total IL-5:antibody complex remained in the serum, as well as an increased amount of this complex. The lowest level of IL-5 quantity (LLOQ) in the cynomolgus monkey was 9.77 pg/ml.

Fig. 7. Mean serum concentrations of 28Y042-7F11-1 and mepolizumab (SB-240563) (ng/mL) in cynomolgus monkeys after a single intravenous (IV) or subcutaneous (SC) injection.

Fig. 8. Module 1-1. Flowchart of diagnosis in clinical practice - an initial presentation.

Fig. 9. Module 3-5. A step-by-step approach to managing symptoms and minimizing future risk.

Detailed Description of the Invention

The present invention relates to compositions for the treatment of diseases mediated by interleukin 5 (IL-5), and related objects of the invention.

As used herein, the term asthma means an inflammatory disease of the airways characterized by reversible airflow limitation and bronchospasm. Common symptoms include wheezing, coughing, chest tightness and shortness of breath. Asthma is a heterogeneous disease that is typically characterized by chronic inflammation of the airways. It is defined by a history of respiratory symptoms such as wheezing, shortness of breath, chest tightness and cough, the severity of which varies over time, as well as variable expiratory airflow limitation.

In the methods of the invention, a diagnosis of asthma in a subject may be made in accordance with the guidance provided by the Global Initiative for Asthma (GINA) in the Global Strategy for Asthma Management and Prevention, updated in 2016). Those skilled in the art are familiar with the GINA Diagnostic Scheme for Clinical Practice (FIG. 8) and the diagnostic criteria for asthma in adults, adolescents, and children 6-11 years of age (Table 1) below, as well as other aspects of the guidelines (eg, for pregnant women women, etc.). See also table. 2 and table. 3.

Table 1

Module 1-2. Diagnostic criteria for asthma in adults, adolescents and children aged 6-11 years Bronchial asthma is a heterogeneous disease, which is usually characterized by the presence of chronic inflammation of the airways. It is defined by a history of respiratory symptoms such as wheezing, shortness of breath, chest tightness and cough, the severity of which varies over time, as well as variable expiratory airflow limitation. DIAGNOSTIC SIGNS DIAGNOSTIC SIGNS 1. History of variable respiratory symptoms Wheezing, shortness of breath, chest tightness and cough. Description of features may change depending on - Typically more than one type of respiratory symptom (in adults, isolated cough is rarely associated with

- 5 046656

Depending on culture and age, for example, a symptom in children may be described as difficulty breathing asthma) - Symptoms vary in time and intensity - Symptoms often worsen at night or immediately after waking up - Symptoms are often triggered by exercise, laughter, allergens, cold air - Symptoms often appear or worsen due to viral infections 2. Confirmed variable expiratory airflow limitation Registered increased Respiratory function variability* (as measured by one or more of the tests below) AND recorded airflow limitation* The greater the variability or the more often it is detected, the greater the confidence in the diagnosis. At least once during the diagnostic process when FEV is low! it is necessary to confirm that the FEV|/FVC ratio is reduced (normally >0.75-0.80 in adults, >0.90 in children) Positive test result for reversibility of bronchial obstruction with using BL* (probability obtaining a positive result is higher if the use of BL before the test is delayed: K DB A - for >4 hours, LABA - for >15 hours) Adults', increase in FEV| by >12% and >200 ml of the initial value 10-15 minutes after administration of 200-400 mcg albuterol or equivalent drug (an increase of >15% and >400 ml is considered more reliable) Children', increase in FEV| by >12% of the expected value Increased variability in PEF measured twice daily for >2 weeks* Adults', average daily variability of PEF >10%** Children'. average daily daily PEF variability >13%** Significant increase in respiratory function indicators after 4 weeks. anti-inflammatory treatment Adults', increase in FEV| by >12% and by >200 ml (or PSV*** by >20%) from the initial value after 4 weeks. treatment

- 6 046656

in the absence of respiratory infections Positive exercise test* Adults', decreased FEV| by >10% and by >200 ml from initial value Children', decreased FEV| by >12% of the expected value or PSV by >15% Positive result bronchoprovocation test (usually performed only in adults) Decrease in FEV| by >20% of baseline when using standard doses of methacholine or histamine, or by >15% when using standardized hyperventilation, use hypertonic sodium chloride solution or performing a bronchoprovocation test with mannitol Increased variability in pulmonary function tests between visits* (less reliable sign) Adults', change in FEV by >12% and by >200 ml between visits in the absence of respiratory infections Children'. change in FEV| by >12% or PEF*** by >15% between visits (possible presence of respiratory infections) BL - bronchodilator (CDBA or LABA with rapid onset of action); FEV|: forced expiratory volume in 1 second; LABA - long-acting 32-agonist; PEF - peak expiratory flow (the highest of the three measured values); CDBA - short-acting β2 agonist; Diagnosis in patients already receiving treatment to control the disease is described in modules 1-4. *These tests may be repeated during symptoms or early in the morning. ** Daily diurnal variability of PEF is calculated from PEF determined twice per day as ([highest daily value minus smallest daily value]/average of the highest and lowest daily values), and calculating the 1-week average. f Use the same device each time to determine PEF, as PEF values obtained on different devices can vary by up to 20%. Reversibility of bronchial obstruction when using BL may disappear during severe exacerbations or viral infection. If reversibility of bronchial obstruction with BL use is not observed on initial evaluation, then the next step depends on the availability of other tests and how urgently treatment is needed. In emergency clinical In this situation, asthma treatment can be started and diagnostic testing can be performed over the next few weeks (see Modules 1-4), but other conditions that may mimic asthma should be considered (see Modules 1-3) and the diagnosis of asthma confirmed as soon as possible .

- 7 046656

table 2

Module 1-3. Differential diagnosis of asthma in adults, adolescents and children aged 6-11 years Age State Symptoms 6-11 years Chronic cough syndrome of the upper respiratory tract Foreign body inhalation Bronchiectasis Primary ciliary dyskinesia Congenital heart disease Bronchopulmonary dysplasia Cystic fibrosis Sneezing, itching, nasal congestion, need to clear throat Sudden onset symptoms, unilateral wheezing Recurrent infections, productive cough, recurrent infections, productive cough, sinusitis Heart murmur Premature birth, symptoms observed from birth Excessive cough and mucus production, gastrointestinal symptoms 12-39 years Chronic cough syndrome of the upper respiratory tract Vocal cord dysfunction Hyperventilation, violation respiratory tract Bronchiectasis Cystic fibrosis Congenital heart disease α-antitrypsin deficiency 1 Foreign body inhalation Sneezing, itching, nasal congestion, need to cough up Shortness of breath, inspiratory wheezing (stridor) Dizziness, paresthesia, sighing Productive cough, recurrent infections Excessive cough and mucus production Heart murmur Difficulty breathing, early

- 8 046656

family history of emphysema Sudden occurrence symptoms 40+ years Vocal cord dysfunction Hyperventilation, violation breathing COPD* Bronchiectasis Heart failure Drug-associated cough Disease caused by damage to the lung parenchyma Pulmonary embolism Central obstruction respiratory tract Dyspnea, inspiratory wheezing (stridor) Dizziness, paresthesia, sighing Cough, sputum, shortness of breath on exertion, smoking or exposure Productive cough recurrent infections Shortness of breath on exertion, nocturnal symptoms Treatment with an angiotensin-converting enzyme (ACE) inhibitor Shortness of breath when exerting, nonproductive cough, thickening of the terminal phalanges of the fingers Sudden onset of shortness of breath, chest pain Shortness of breath, unresponsiveness to bronchodilators *In patients with established asthma, any of the above conditions may also contribute to respiratory symptoms.

Table 3

Module 1-4. Confirming the diagnosis of asthma in a patient who is already receiving treatment to control the disease Variable respiratory symptoms and variable speed limit air flow The diagnosis of asthma is confirmed. Assess asthma control and review therapy to control the disease Variable respiratory symptoms, but without Repeat the test for reversibility of bronchial obstruction using BL after discontinuation of BL (SABA: 4 hours; LABA:

- 9 046656

airflow limit variability >12 hours) or during symptom onset. If the result is normal, consider alternative diagnoses (see Module 1-3). If ΟΦΒχ >70% of predicted value: consider performing a bronchoprovocation test. If the result is negative, consider the possibility of reducing the intensity of therapy aimed at controlling the disease and re-evaluating after 2-4 weeks. If ΟΦΒχ <70% of predicted value: consider increasing the intensity of disease control therapy for 3 months and then reassessing symptoms and pulmonary function tests. If no response is observed, resume previously used therapy and refer the patient for evaluation to establish a diagnosis. A small amount of respiratory symptoms, normal values lung function and lack of variability in airflow limitation Repeat the test for reversibility of bronchial obstruction using BL after discontinuation of BL (SABA: 4 hours; LABA: >12 hours) or during the onset of symptoms. If the result is normal, consider alternative diagnoses (see Module 1-3). Consider reducing the intensity of disease control therapy: - if symptoms appear and lung function tests decrease: asthma is confirmed. Increase the intensity of therapy aimed at controlling the disease to the lowest dose previously effective; - If there is no change in symptoms or pulmonary function at the lowest level of disease control therapy: consider discontinuing the disease control drug and closely monitor the patient for at least 12 months.

- 10 046656

Persistent difficulty breathing and fixed bronchial obstruction Consider increasing the intensity of disease control therapy for 3 months. and subsequent reassessment of symptoms and pulmonary function tests. If no response is observed, resume previously used therapy and refer the patient for evaluation to establish a diagnosis. Consider the possibility of asthma-COPD overlap syndrome. Designations: BL - bronchodilator, LABA - long-acting β-agonist, CDBA - short-acting β-agonist.

In the methods of the invention, asthma may be mild asthma, moderate asthma or severe asthma. In the methods of the invention, the severity of asthma can be assessed according to the GINA guidelines. In particular, the severity of asthma can be assessed retrospectively by the level of therapy required to control symptoms and exacerbations. For example, evaluation may be performed after several months of disease control therapy and, if possible, after an attempt to reduce the intensity of therapy to determine the minimum level that is effective for the patient. The severity of asthma is not a constant characteristic and can change over time, over several months or years.

Asthma severity can be assessed if the patient has been receiving regular disease control therapy for several months.

Mild asthma is asthma that is well controlled by treatment steps 1 and 2 (see Fig. 9, module 3-5), i.e. when using rescue medications only (as needed) or when using low-intensity disease control medications such as low-dose ICS, leukotriene receptor antagonists, or cromones.

Moderate asthma is asthma that is well controlled with Tier 3 therapy (see FIG. 9; Module 3-5), such as low dose ICS/LABA.

Severe asthma is asthma that requires the use of grade 4 or 5 therapy (see Fig. 9; module 3-5), such as high dose ICS/LABA, to prevent it from progressing to uncontrolled asthma, or asthma that remains uncontrolled despite receiving such therapy. Although the treatment of patients with uncontrolled asthma may in many cases be challenging due to inadequate or inappropriate treatment, persistent problems with adherence or comorbidities such as chronic rhinosinusitis or obesity, the European Respiratory Society's Severe Asthma Working Group The American Thoracic Society believes that the definition of severe asthma should be used for patients with refractory asthma and insufficient response to treatment for comorbid conditions. When assessing the severity of asthma, you can also use the table. 4.

- 11 046656

Table 4

Module 3-6. Low, medium, and high daily doses of inhaled corticosteroids Medicine Daily dose (mcg) low average high Beclomethasone dipropionate (CFC)* 200-500 >500-1000 >1000 Beclomethasone dipropionate (BPA) 100-200 >200-400 >400 Budesonide (DLI) 200-400 >400-800 >800 Cyclesonide (HFA) 80-160 >160-320 >320 Fluticasone furoate (DNI) 100 No data 200 Fluticasone propionate (DNI) 100-250 >250-500 >500 Fluticasone propionate (HFPA) 100-250 >250-500 >500 Mometasone furoate 110-220 >220-440 >440 Triamcinolone acetonide 400-1000 >1000-2000 >2000 Children aged 6-11 years (information relevant to children 5 years and younger) Beclomethasone dipropionate (CFC)* 100-200 >200-400 >400 Beclomethasone dipropionate (BFA) 50-100 >100-200 >200 Budesonide (DAYS) 100-200 >200-400 >400 Budesonide (spray) 250-500 >500-1000 >1000 Cyclesonide 80 >80-160 >160 Fluticasone furoate (DNI) No data No data No data Fluticasone propionate (DNI) 100-200 >200-400 >400 Fluticasone propionate (HFPA) 100-200 >200-500 >500 Mometasone furoate 110 >220- <440 >440 Triamcinolone acetonide 400-800 >800-1200 >1200 CFC - chlorofluorocarbon propellant; DNI - metered dose powder inhaler; HFA - hydrofluoroalkane propellant; p.a. - Not applicable; * Beclomethasone dipropionate, CFC, included for comparison with earlier sources literary

In the methods of the invention, asthma may be mild eosinophilic asthma, moderate eosinophilic asthma, or severe eosinophilic asthma.

Mild eosinophilic asthma is mild asthma with an eosinophilic phenotype. For example, subjects with mild eosinophilic asthma may have mild asthma with blood eosinophil levels greater than or equal to 150 eosinophils per μL of blood in the past 12 months, greater than or equal to 200 eosinophils per μL of blood in the past 12 months, greater than or equal to 300 eosinophils per μL blood over the past 12 months, or greater than or equal to 350 eosinophils per μl of blood over the past 12 months.

Moderate eosinophilic asthma is mild asthma with an eosinophilic phenotype. For example, subjects with moderate eosinophilic asthma may have moderate asthma with blood eosinophil levels greater than or equal to 150 eosinophils per microliter of blood in the past 12 months, greater than or equal to 200 eosinophils per microliter of blood in the past 12 months, greater than or equal to 300 eosinophils in µl of blood over the past 12 months, or greater than or equal to 350 eosinophils per µl of blood over the past 12 months.

Severe eosinophilic asthma is severe asthma with an eosinophilic phenotype. For example, subjects with severe eosinophilic asthma may have severe asthma with blood eosinophil levels greater than or equal to 150 eosinophils per μL of blood in the past 12 months, greater than or equal to 200 eosinophils per μL of blood in the past 12 months, greater than or equal to 300 eosinophils per μL blood over the past 12 months, or greater than or equal to 350 eosinophils per μl of blood over the past 12 months.

Subjects with severe eosinophilic asthma may also meet one or more criteria

- 12 046656 riyams described in table. 5.

Table 5 Subject suffers from severe eosinophilic asthma if the following criteria are met:

1) The subject has clinical signs of severe refractory asthma similar to those specified by the American Thoracic Society for Refractory Asthma (162 Am. J. Respir. Crit. Care Med. 2341 (2000)) for >12 months.

2) Detailed documented requirements for the subject's regular treatment with high-dose ICS (inhaled corticosteroids) (i.e., >880 mcg/day fluticasone propionate or equivalent daily dose) with or without a maintenance dose of PCS (oral corticosteroids) in the past 12 months.

3) Detailed documented requirements for the subject's supervised treatment with, for example, a long-acting beta-2 agonist, leukotriene receptor antagonist, or theophylline in the past 12 months.

4) The subject has persistent airflow obstruction, as evidenced by the level of FEV| <80% before bronchodilator administration; predictable,

- 13 046656 recorded or maximum daily flow variability >20% for 3 or more days.

5) The subject has airway inflammation, which may be eosinophilic in nature, as indicated by one of the following characteristics currently present or documented in the previous 12 months:

- Elevated peripheral blood eosinophil levels >300/µL associated with asthma or

I- Sputum eosinophil level >3% or

- Exhaled nitric oxide level >50 ppb or

- Rapid deterioration of asthma control (based on documented history or objective measures) following a <25% reduction in regularly administered inhaled or oral corticosteroid maintenance dose in the previous 12 months.

8) The subject has a prior history of two or more exacerbations of asthma requiring treatment with oral or systemic corticosteroids in the previous 12 months, despite the administration of high-dose ICS and an additional controller medication. In subjects receiving a maintenance dose of PCS with a high-dose ICS plus a controller drug to treat exacerbations with PCS, the dose of PCS should be two or more times the dose of the ICS.

9) The subject has asthma documented by either:

- reversible airway obstruction (FEV| >12% and 200 ml) currently or documented in the previous 12 months, or

- airway hyperresponsiveness (provocative concentration of methacholine <8 mg/ml causing a decrease in FEV by 20%, or a challenge dose of histamine <7.8 µmol causing a decrease in FEV by 20%), documented in the previous 12 months, or

- variability of air flow FEV| between two clinical studies by >20%, documented in the previous 12 months (FEV _b recorded during an exacerbation is not valid), or

- airflow variability, as indicated by >20% daily variability in maximum flow observed over 3 or more days.

It is important to note that at the start of treatment, subjects with severe eosinophilic asthma according to these criteria may have less than 150 eosinophils per microliter of blood.

28Y042-7F11-1 is a monoclonal antibody containing 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. 28Y042-7F11-1 and the antigen binding proteins of the invention, in in particular, antibody molecules containing the heavy chain CDR and the light chain CDR 28Y042-7F11-1 can be used to treat severe eosinophilic asthma in accordance with the methods of the invention. For example, 28Y042-7F11-1 or the antigen-binding proteins of the present invention may be prescribed as adjunctive maintenance treatment for severe eosinophilic asthma, as indicated by a blood eosinophil count greater than or equal to 300 cells/μL in the past 12 months and/or blood eosinophil levels greater than or equal to 150 cells/μL at the start of treatment, and/or blood eosinophil levels less than 150 cells/μL at the start of treatment, in patients. Alternatively, 28Y042-7F11-1 or the antigen binding proteins of the present invention may be administered as additional maintenance treatment for severe

- 14 046656 eosinophilic asthma, the presence of which is indicated by a blood eosinophil level greater than or equal to 300 cells/μl in the last 12 months, and/or a blood eosinophil level greater than or equal to 150 cells/μl at the beginning of treatment, in patients. 28Y042-7F11-1 or the antigen-binding proteins of the present invention may be prescribed as adjunctive maintenance treatment for severe eosinophilic asthma as indicated 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 beginning of treatment, in patients. Such patients may be aged 12 years or older. Treatment with 28Y042-7F11-1 or the antigen binding proteins of the invention may alleviate asthma exacerbations in patients (eg, patients with a history of exacerbation). The methods of the invention can be used when administering treatment with 28Y0427F11-1 or the antigen binding proteins of the invention (ie, such treatment with 28Y042-7F11-1 can be combined with the methods of the invention). Treatment with 28Y042-7F11-1 and the antigen binding proteins of the invention allows:

a) reduce the frequency of exacerbations. Compared with placebo, treatment with 28Y042-7F11-1 or the antigen binding proteins of the invention may reduce the incidence of 1) clinically significant exacerbations, 2) exacerbations requiring hospitalization or emergency medical care, and 3) exacerbations requiring hospitalization. This benefit could potentially lead to a reduction in morbidity and mortality due to asthma;

b) reduce the daily dose of PKC: Treatment with 28Y042-7F11-1 or the antigen binding proteins of the invention may allow subjects to reduce the daily dose of a concomitant corticosteroid without loss of asthma control. Subjects treated with 28Y042-7F11-1 or the antigen binding protein compositions of the invention may have a reduction in the median percentage, relative to baseline, of daily oral corticosteroid (OCS) dose compared to patients receiving placebo. In addition, in subjects receiving treatment with 28Y042-7F11-1 or the antigen binding protein compositions of the invention, PCS dose reductions may be as high as 32% compared to subjects receiving placebo;

c) improve lung function: Clinically significant changes in FEV ₁ before and after bronchodilator administration can be demonstrated by treatment with 28Y042-7F11-1 or the antigen binding proteins of the invention compared to placebo. Any improvement in lung function is of particular clinical significance in this group of patients, as most are receiving maximal asthma therapy, including high-dose ICS (inhaled corticosteroids) and/or PCS plus a controller medication;

d) improve asthma control: Statistically significant and clinically relevant improvements can be monitored by the ACQ-5 questionnaire during therapy with 28Y042-7F11-1 or antigen binding proteins of the invention compared to placebo, indicating that subjects achieved asthma control when added to their existing treatment of asthma 28Y042-7F11-1 or antigen-binding proteins of the invention;

e) improve quality of life: Treatment with 28Y042-7F11-1 or the antigen binding proteins of the invention demonstrates statistically significant and clinically relevant changes in SGRQ scores compared to placebo. Subjects may experience noticeable relief of asthma symptoms and improved ability to perform daily activities;

f) provide sustained efficacy and pharmacodynamic effect: During a 32- and/or 52-week course of treatment, a sustained reduction in the frequency of asthma exacerbations and a decrease in eosinophils in the blood, as well as an improvement in lung function, asthma control and quality of life can be observed without the development of tolerance; And

g) reduce the level of eosinophils in the blood. Treatment with compositions containing 28Y042-7F11-1 or antigen-binding proteins of the invention may result in a rapid decrease in blood eosinophils in a subject.

In the methods of the invention, the asthma may be severe asthma. In the methods of the invention, asthma may also be mild asthma, moderate or severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma or severe eosinophilic asthma, as discussed above. Treatment with compositions containing 28Y0427F11-1 or the antigen binding proteins of the invention can be used to treat these conditions in accordance with the methods of the invention.

In the methods of the invention, the asthma may be uncontrolled eosinophilic asthma. Subjects with uncontrolled eosinophilic asthma meet the criteria described in Table. 6.

- 15 046656

Table 6

The subject has uncontrolled eosinophilic asthma if the following criteria are met:

1) The subject has a history of asthma for at least the previous 12 months.

2) Subject has been prescribed daily moderate to high dose ICS (inhaled corticosteroid) plus LABA (long-acting beta agonists) for at least the previous 12 months.

3) The subject's dosage of other asthma control medications must have been stable for at least the previous 30 days.

4) Subject has experienced at least 2 documented asthma exacerbations during the previous 12 months that required systemic corticosteroid pulse therapy.

Treatment with compositions containing 28Y042-7F11-1 or the antigen binding proteins of the invention can be used to treat uncontrolled eosinophilic asthma in accordance with the methods of the invention.

In the methods of the invention, the asthma may be eosinophilic asthma. Subjects with eosinophilic asthma meet the criteria described in Table. 7.

Table 7

A subject has eosinophilic asthma if the following criteria are met:

1) The patient has a previous diagnosis of asthma.

2) The patient has had at least 1 exacerbation of asthma requiring an oral, intramuscular (IM), or intravenous (IV) corticosteroid on at least 3 days in the previous 12 months.

3) Currently, the level of eosinophils in the patient’s blood is at least 400/μl.

4) Prior to beta-agonist administration, the patient had reversible airway obstruction of at least 12%.

5) The patient’s ACQ score is at least 1.5.

6) The patient is taking inhaled fluticasone or equivalent at a daily dose of at least 440 mcg. Long-term oral use of corticosteroids is allowed (no more than 10 mg/day of prednisone or equivalent). The patient's background therapy for asthma (including, but not limited to, inhaled corticosteroids, oral corticosteroids up to a maximum dose of 10 mg prednisone per day or equivalent, leukotriene antagonists, 5-lipoxygenase inhibitors, or cromolyn) must have been stable for the previous 30 days.

In the methods of the invention, the asthma may be sub-eosinophilic asthma. Subjects with sub-eosinophilic asthma meet the criteria described in Table 1. 8.

- 16 046656

Table 8

A subject has sub-eosinophilic asthma if the following criteria are met:

1) The patient has a previous diagnosis of asthma.

2) The patient has had at least 1 exacerbation of asthma requiring an oral, intramuscular (IM), or intravenous (IV) corticosteroid within no time.

Less than 3 days in the previous 12 months.

3) Currently, the patient’s blood eosinophil level is less than 400/μL.

4) Before beta-agonist administration, the patient has reversible airway obstruction of at least 12%.

5) The patient’s ACQ score is at least 1.5.

6) The patient is taking inhaled fluticasone or equivalent at a daily dose of at least 440 mcg. Long-term oral use of corticosteroids is allowed (no more than 10 mg/day of prednisone or equivalent). The patient's background therapy for asthma (including, but not limited to, inhaled corticosteroids, oral corticosteroids up to a maximum dose of 10 mg prednisone per day or equivalent, leukotriene antagonists, 5-lipoxygenase inhibitors, or cromolyn) must have been stable for the previous 30 days.

Treatment with compositions containing 28Y042-7F11-1 or the antigen binding proteins of the invention can be used to treat eosinophilic asthma as well as sub-eosinophilic asthma in accordance with the methods of the invention.

As used herein, the term bullous pemphigoid (BP) refers to an acute or chronic autoimmune skin disease involving the formation of blisters, more commonly known as bullae, in the space between the layers of skin, the epidermis and the dermis. PD is the most common autoimmune blistering skin disease. It is common in older adults (>70 years) with an annual incidence of 5 to 35 per million. The incidence of PD is sharply increasing by an average of 17% per year. PD often begins with intensely itchy skin lesions, similar to eczema or hives, before blisters and blisters appear. In 10-30% of patients, PD also affects the oral mucosa. The severity of the disease can be determined using the Autoimmune Bullous Skin Severity Index (ABSIS), which evaluates the affected area as well as disease activity. The disease occurs due to an autoimmune response to structural components of junctional adhesion complexes, leading to damage to the dermal-epidermal junction with the formation of a subepidermal blister. In particular, autoreactive B and T cell responses to the hemidesmosomal antigens BP180 and BP230 were identified. Serum levels of BP180 autoantibodies reflect the severity and activity of the disease. T cells are memory CD4+ cells that produce Th1 and Th2 cytokines, mainly IL-4, IL-5 and IL-13. IL-5, as well as eotaxin, were found in abundance in the contents of the bladder. IL-5 production is indeed associated with blood eosinophilia and significant infiltration of the skin of PD patients with eosinophils. It is believed that eosinophils take an active part in the formation of blisters, releasing toxic granular proteins (ESP, MBP) and proteolytic enzymes.

As used herein, the term eosinophilic esophagitis (EoE) refers to an allergic inflammatory condition of the esophagus involving eosinophils. Symptoms include difficulty swallowing, difficulty eating, and heartburn. EoE is characterized by a dense infiltrate of eosinophilic leukocytes in the epithelial layer of the esophageal mucosa. EO is thought to be an allergic reaction to food consumed, given the important role that eosinophils play in allergic reactions. To diagnose EoE, you can use the EoE diagnostic panel. EoE may also be diagnosed if gastroesophageal reflux does not respond to a 6-week trial of high-dose proton pump inhibitors (PPI) twice daily or if a negative result on an outpatient pH test rules out gastroesophageal reflux disease (GERD). Endoscopically, ridges, grooves, or rings may be visible in the esophageal wall. Occasionally, numerous rings may appear in the esophagus, giving rise to the term corrugated esophagus or cat esophagus due to the rings' resemblance to a cat's esophagus. The presence of white exudate in the esophagus also confirms the diagnosis. In biopsies taken during endoscopy, numerous eosinophils can usually be seen in the surface epithelium. For regulations

- 17 046656 ki diagnosis requires a minimum of 15 eosinophils per field of view at high power. Eosinophilic inflammation is not limited to the esophagus and extends to the entire gastrointestinal tract. Severely degranulated eosinophils may also be present, as well as microabscesses and expansion of the basal layer. Radiologically, the term annular esophagus has been used to refer to eosinophilic esophagitis in barium swallow studies to visualize with contrast material the transitional transverse folds sometimes seen in esophageal reflux (called feline esophagus).

Treatment with compositions containing 28Y042-7F11-1 or antigen binding proteins of the invention can be used to treat COPD in accordance with the methods of the invention.

Subjects with chronic obstructive pulmonary disease (COPD) may meet one or more of the following criteria: a) previously diagnosed COPD: subjects with a clinically documented history of COPD for at least 1 year according to the American Thoracic Society/European Respiratory Society definition; b) severity of COPD: Subjects may have the following: measured forced expiratory volume one second before and after salbutamol inhalation/forced vital capacity ( _FEV1 /FEF) ratio <0.70 to confirm the diagnosis of COPD; measured percentage of FEV ₁ after inhaled salbutamol >20 percent and <= 80 percent of predicted normal values calculated using the National Health and Nutrition Examination Survey III (NHANES) reference equations; c) history of exacerbations: a well-documented history (e.g., review of medical records) over a 12-month period of at least two exacerbations of moderate COPD. Moderate severity refers to the use of systemic corticosteroids (intramuscular, intravenous, or oral) and/or antibiotic treatment, or at least one severe exacerbation of COPD. Severe is defined as the need for hospitalization. Note: There must have been at least one exacerbation while the subject was taking an inhaled corticosteroid (ICS) plus a long-acting beta-agonist (LABA) plus a long-acting muscarinic antagonist (LAMA). Note: Previous use of antibiotics alone does not qualify as a mild exacerbation unless it was done specifically to treat worsening COPD symptoms; and d) concomitant therapy for COPD: well-documented requirements for use of optimized standards of background therapy (SoC), which includes ICS plus 2 additional COPD drugs (ie, triple therapy) for 12 months and meets the following criteria: immediately before the visit physician, use of an inhaled corticosteroid for at least 3 months (at a dose of >=500 micrograms (mcg)/day plus an equivalent dose of fluticasone propionate); or LABA and MDMA.

Treatment with compositions containing 28Y042-7F11-1 or antigen binding proteins of the invention can be used to treat COPD in accordance with the methods of the invention.

As used herein, the term eosinophilic granulomatosis with polyangiitis (EGPA) refers to an autoimmune condition that causes inflammation of small and medium-sized blood vessels (vasculitis) in individuals with allergic airway hypersensitivity (atopy). EGPA may also be referred to as Churg-Strauss syndrome (CSS) or allergic granulomatosis. EGPA usually manifests itself in three stages. The early (prodromal) stage is characterized by inflammation of the airways; Almost all patients experience asthma and/or allergic rhinitis. The second stage is characterized by an abnormally high number of eosinophils (hypereosinophilia), which causes tissue damage, most often the lungs and digestive tract. The third stage consists of vasculitis, which can eventually lead to cell death and can be life-threatening.

Subjects with EGPA may meet one or more of the following criteria:

a) asthma; b) the level of eosinophils in the blood exceeds 10% of the differential number of leukocytes; c) the presence of mononeuropathy or polyneuropathy;

d) unfixed pulmonary infiltrates; f) the presence of anomalies of the paranasal sinuses; And

e) histological evidence of the presence of extravascular eosinophils. For classification purposes, a patient is considered to have EGPA if at least four of the six previous criteria are positive.

Treatment with compositions containing 28Y042-7F11-1 or antigen-binding proteins of the invention can be used to treat EGPA in accordance with the methods of the invention. The compositions of the invention can be administered to a patient with EGPA in an amount of 300 mg once every 4 weeks.

As used herein, the term hypereosinophilic syndrome (HES) means a disorder characterized by a persistently elevated number of eosinophils (>1500 eosinophils/mm ³ ) in the blood for at least six months without any apparent cause, involving either the heart, nervous system, or bone marrow.

Subjects with hypereosinophilic syndrome may meet one or more of the following criteria: a) documented history of hypereosinophilic syndrome; b) the number of eosinophils in the blood is more than 1500 cells for 6 months; c) signs and symptoms of organ damage; and d) no evidence of parasitic, allergic or other causes of eosinophilia after

- 18 046656 comprehensive assessment.

Treatment with compositions containing 28Y042-7F11-1 or antigen binding proteins of the invention can be used to treat hypereosinophilic syndrome in accordance with the methods of the invention.

As used herein, the term nasal polyps refers to a disease characterized by the presence of polyps in the nasal cavity. Such polyps may be located in the upper part of the nasal cavity and/or may arise within the ostiomeatal complex.

Subjects with nasal polyps may meet one or more of the following criteria: a) documented history of nasal polyps; or b) nasal polyps detected during examination (eg, endoscopic examination).

Treatment with compositions containing 28Y042-7F11-1 or antigen binding proteins of the invention can be used to treat nasal polyps in accordance with the methods of the invention.

As used herein, the term atopic dermatitis means an inflammatory skin condition characterized by chronic itching, lichenification, xerosis, erythematous papules and plaques.

In the methods of the invention, the atopic dermatitis may be moderate or severe atopic dermatitis. Subjects with moderate to severe atopic dermatitis may also meet one or more of the criteria described in Table 1. 9.

- 19 046656

Table 9 A subject has moderate to severe atopic dermatitis if he meets the following criteria (eg, all or “one or more”):

1. Diagnosis of atopic dermatitis according to the Hanifin and Reick criteria as revised by Eichenwald (Eichenfield et al., 70 J Am Acad Dermatol 338 (2014)). See Table 10.

2. Diagnosis of atopic dermatitis >2 years before treatment.

3. Healthcare Provider Global Assessment (HGA; sometimes also called Investigator Global Assessment or IGA) >3 before treatment. See Table 11.

14. Atopic dermatitis involving >10% BSA before treatment. See Table 12.

5. Eczema Extension and Severity Index (EASI) score >16 before treatment. See Table 13.

6. The absolute level of eosinophils in the blood is >350 cells/μl before treatment.

7. Optionally, use an over-the-counter, non-drug (no active ingredient) emollient twice daily for at least 7 days immediately before starting treatment.

8. Before starting treatment, there is at least one of the following: a) inadequate response <6 months to a stable regimen of prescription topical treatment for atopic dermatitis; b) poor tolerability of the prescribed topical drug for atopic dermatitis; c) concerns about potential side effects from prescribed topical atopic dermatitis medications, such as thinning of the skin or increased risk of hypothalamic-pituitary-adrenal [HPA] axis suppression; and/or d) inadequate response to optimization of non-pharmacological measures for atopic dermatitis, such as moisturizers. “Inadequate response” to a stable regimen of prescribed topical medications for the treatment of atopic dermatitis (such as topical corticosteroids or moderate to highly effective topical calcineurin inhibitors) is defined as failure to achieve and maintain remission or a state of low disease activity (equivalent to an HGA score=ot 0 [clear] to 2 [moderate]) despite treatment for the recommended label duration or the maximum duration recommended for treatment of the subject, whichever is shorter.

Subjects with moderate to severe atopic dermatitis may be children under 18 years of age, adults 18 years of age or older, or adults 18 to 70 years of age, inclusive. Subjects can be men or women. It is advisable that women taking treatment are not pregnant, breastfeeding and/or unable to become pregnant.

The diagnosis of atopic dermatitis is based on the Hanifin and Reick criteria, as revised by Eichenwald. See table. 10 and Eichenfield et al., 70 J Am Acad Dermatol 338 (2014).

- 20 046656

Table 10

Criteria for diagnosing atopic dermatitis

MANDATORY MANIFESTATIONS - Must be present:

• Itching • Eczema (acute, subacute, chronic) o Typical morphology and age-specific patterns* * o Chronic or relapsing course * Patterns include:

1. Involvement of the face, neck and flexural areas in infants and children

2. Current or previous lesions on the flexor surfaces in any age group

3. The groin and armpit areas are not involved

IMPORTANT MANIFESTATIONS - Visible in most cases and are additional confirmation of the diagnosis:

• Onset of the disease at an early age • Atopy o Personal history of the patient and/or family history o Increased levels of immunoglobulin E • Xerosis

DISEASE-RELATED MANIFESTATIONS These clinical associations help suggest the diagnosis of atopic dermatitis, but are too nonspecific to be used to define or identify atopic dermatitis and are used for scientific and epidemiological studies:

• Atypical vascular response (eg, facial pallor, white dermographism, blanche retention reaction) • Keratosis pilaris/seborrheic eczema/palmar striation/ichthyosis • Ocular/periorbital changes • Other local changes (eg, perioral changes/periouricular lesions) • Perifollicular accentuation/lichenification/pruritic lesions

SPECIAL CONDITIONS It should be noted that the diagnosis of atopic dermatitis depends on the exclusion of conditions such as:

• Scabies • Seborrheic dermatitis • Contact dermatitis (irritant or allergy) • Ichthyosis • Cutaneous T-cell lymphoma • Psoriasis • Photodermatosis • Primary immunodeficiency • Erythroderma of other etiologies

The Global Assessment (HGA) is a clinical tool to assess the current status/severity of atopic dermatitis in a subject. See Rehal et al, 6 PLos ONE e17520 (2011) and table. 11. This is a static 5-point morphological assessment of the overall severity of the disease, determined by a qualified healthcare professional, using the clinical manifestations of erythema, infiltration, papular formation and crusting as a guide. The HGA assessment is performed without reference to previous scores. Each time the assessment should be carried out in the form of a visual assessment at the current time of the average severity of all affected areas.

Table 11

Global Physician Assessment (HGA) Point/degree Description 0 Clean No erythema or induration/papulosity or crusting; Possible residual discoloration 1 Almost clean There may be traces of mild pink erythema, almost no induration/papulosity, and no weeping/crusting 2 Weak Possible mild pink erythema with induration/papulosity with barely noticeable raised areas, no weeping/crusting 3 Average There may be a clearly visible dark red erythema with induration/papulosity with clearly visible elevations, but not pronounced; Possible slight weeping/crusting 4 Heavy Possible presence of deep or bright red erythema with induration/papulosity with pronounced elevations (deep, clear border), with weeping/crusting

The body surface area (%BSA) estimate is an estimate of the total skin surface area (%) affected by atopic dermatitis. See table. 12. Assessment of %BSA can be performed by examining inflamed areas in each of 4 body surface areas separately: head and neck, upper extremities, torso, and lower extremities, and each of these body areas has the potential to be up to 100% involved. Assessors (eg, healthcare professionals) can estimate the percentage of skin involved in each area to obtain that area's %BSA score, which is then multiplied by the appropriate proportionality factor to obtain the %BSA value for the involved area (for subjects aged > 8 years: 0. 1 for the head, 0.2 for the upper limbs, 0.3 for the torso and 0.4 for the lower limbs). The %BSA values obtained for the regions are summed to obtain the total %BSA involved. The %BSA score of a specific area can also be used as part of the matrix to calculate the EASI score.

- 22 046656

Table 12

Body surface area • The %BSA involved area score can be determined by performing the following 3 steps described herein to calculate the total %BSA involvement. • Step 1: Assess the involvement of each body area in % BSA; Step 2: Multiply the % of engagement by the percentage of total body area; Step 3: Calculate total engagement in % BSA. Below is an example of how the % of total BSA involved can be calculated. Body area % of involvement (0-100% of each area) % of involvement x proportionality coefficient Regional involvement in % B.S.A. Head and neck ___x0.1 Upper limbs ___x 0.2 Torso ___x 0.3 Lower limbs ___x 0.4 Total %BSA involved (sum of 4 values) =___

The EASI scoring system is a standardized clinical tool for assessing atopic dermatitis that takes into account the total body surface area (%BSA) involved and a severity score for each of the clinical manifestations: erythema, induration, papularity, excoriation, and lichenification. See Hanifin et al., 10 Exp Dermatol 11 (2001); Rullo et al., 36 Allergol et Immunopathol 201 (2008) and tab. 13. The %BSA lesion area score based on the %BSA score must be used as part of the matrix to calculate the EASI score. Severity scores for each of the clinical manifestations (erythema, induration, papularity, excoriation, and lichenification) are scored on a 4-point scale (0 to 3) for each of the 4 body regions (head and neck, upper extremities, lower extremities, and trunk). Severity scores for each manifestation are summed for each area and multiplied by the %BSA affected area score and the appropriate proportionality factor (for subjects aged >8 years, 0.1 for head, 0.2 for upper extremities, 0.3 for the trunk and 0.4 for the lower extremities) to obtain the EASI score for the regions. The EASI scores for the domains are then summed to produce a final EASI score. The EASI score is a static assessment made without reference to previous scores.

- 23 046656

Table 13

Eczema Severity and Area Index (EASI) After determining the involvement of each area in % BSA, each percentage is converted to an affected area score based on the following definitions: 0=not involved 1 =<10% 2=10%-29% 3=30%-49% 4=50%-69% 5=70%-89% 6=90%-100% Severity of manifestation: Rate the severity of each manifestation on a scale from 0 to 3: - Take the average severity value for the entire involved area. - Half values of integers can be used, for example 2.5 0 - absent 1 - weak 2 - moderate 3 - heavy Evaluation table: Region body Erite ma (o-z) Induration/papularity (o-z) Excoriation (o-z) Lichenification (o-z) Score for area (0-6) Coefficient Body bark score Head/neck ( + + + ) X x0.1 Torso ( + + + ) X x0.3 Upper final tees ( + + + ) X x0.2 Lower tees ( + + + ) X x0.4 The final EASI score is the sum of scores in 4 areas: (0-72)

Therapeutically effective amounts of 28Y042-7F11-1 or the antigen binding proteins of the invention can be used to treat a patient with atopic dermatitis or to reduce the absolute number of eosinophils in the blood of such patients. This atopic dermatitis can be moderate atopic dermatitis or severe atopic dermatitis.

Treatment of atopic dermatitis, such as moderate atopic dermatitis or severe atopic dermatitis, with 28Y042-7F11-1 or antigen binding proteins of the invention in accordance with the methods of the invention can provide at least one result selected from the group consisting of:

a) an HGA score of 0 or 1 and at least a 2-point improvement in the HGA score (e.g. relative to the initial HGA score);

b) reduction in Eczema Severity and Area Score (EASI) (eg relative to baseline EASI);

c) reduction in the percentage of total body surface area affected (%BSA) (eg, relative

- 24 046656 exactly the initial %BSA); and/or

d) a physician's opinion that the subject does not have atopic dermatitis according to the Hanifin and Reick criteria as revised by Eichenwald (Eichenfield et al., 70 J Am Acad Dermatol 338 (2014).

The EASI score after treatment in accordance with the methods of the invention may be less than 16, such as from about 0 to less than 16. The EASI score after treatment may also be from about 0 to about 15, from about 0 to about 14, from about 0 to about 13 , from about 0 to about 12, from about 0 to about 11, from about 0 to about 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, about 0 to about 4, about 0 to about 3, about 0 to about 2, about 0 to about 1, about 1 to less than 16, about 2 to less than 16, 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, about 4 to about 13, about 5 to about 12, about 6 to about 11, about 7 to about 10, about 8 to about 9, about 0 to about 8, about 8 to less 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.

The %BSA after treatment in accordance with the methods of the present invention may be less than about 10%, such as from about 0% to less than 10%. Post-treatment %BSA may also be 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% up 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%.

As used herein, the term antigen binding protein refers to isolated antibodies, antibody fragments (e.g., Fab, etc.) and other protein constructs derived from antibodies, such as antibody-containing domains (e.g., domain antibodies, etc.), which are capable of binding to human IL-5 (SEQ ID NO: 11).

As used herein, the term antibody refers to molecules with an immunoglobulin-like domain (eg, IgG, IgM, IgA, IgD, or IgE) and includes monoclonal, recombinant, polyclonal, monoclonal, recombinant, polyclonal, chimeric, human, and humanized molecules of this type. Monoclonal antibodies can be produced by a clone of a eukaryotic cell expressing the antibody. Monoclonal antibodies can also be produced by a eukaryotic cell line that can recombinantly express the heavy chain and light chain of the antibody due to the presence of nucleic acid sequences encoding them introduced into the cell. Methods for producing antibodies from various eukaryotic cell lines, such as Chinese hamster ovary cells, hybridomas, or antibody-secreting immortal cells obtained from an animal (eg, human), are well known.

The antibody may be derived from a rat, mouse, primate (eg, cynomolgus, Old World monkey, or ape), human, or other sources, such as molecular biology-derived nucleic acids that encode the antibody molecule.

The antibody may contain a constant region, which may be of any isotype or subclass. The constant region may be of the IgG isotype, for example IgGi, IgG2, IgG3, IgG4, or variants thereof. The constant region of the antigen binding protein may be IgG1.

The antigen binding protein may contain one or more modifications selected from the constant domain so mutated that the antibody has improved effector functions/ADCC and/or complement activation.

The antibody may be capable of binding to a target antigen. Examples of such target antigens include human IL-5 containing the amino acid sequence shown in SEQ ID NO: 11.

28Y042-7F11-1 containing 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 antigen binding proteins of the invention bind to human IL-5 and antagonize its activity.

28Y042-7F11-1 is a recombinant humanized monoclonal antibody (IgG1, kappa) 28Y042-7F11-1, which contains two light and two heavy chains.

- 25 046656

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

The heavy and light chains of 28Y042-7F11-1 are covalently linked by one disulfide bond, and the heavy chains are linked to each other by two disulfide bonds, resulting in a typical IgG molecule.

The 28Y042-7F11-1 or antigen binding proteins of the invention may be provided as a lyophilized powder containing the antibody and excipients that can be reconstituted with a pharmaceutically acceptable carrier (eg, sterile water). This reconstituted pharmaceutical composition can then be administered either subcutaneously or intravenously (eg, by further dilution). The 28Y042-7F11-1 or antigen binding proteins of the invention may also be presented in the form of a liquid composition containing the antibody, excipients and a pharmaceutically acceptable carrier. This liquid pharmaceutical composition can then be administered either subcutaneously or intravenously (eg, by further dilution).

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

As used herein, the term IL-5 means human IL-5 containing the amino acid sequence shown in SEQ ID NO: 11.

The term specifically binds, as used herein in relation to antigen-binding proteins, means that the antigen-binding protein binds to a target antigen, as well as to a discrete domain or discrete amino acid sequence in the target antigen with little or no binding to others (e.g., unrelated) proteins. However, this term does not exclude the fact that antigen-binding proteins may also be cross-reactive with closely related molecules (eg, molecules with a high degree of sequence identity, or molecules from other genera or species). The antigen binding proteins described herein can bind to human IL-5 or the human IL-5 receptor with an affinity of at least 2, 5, 10, 50, 100 or 1000 times greater than their binding affinity to closely related molecules.

The binding affinity (KD) for the interaction of an antigen binding protein with a target antigen may be 1 mM or less, 100 nM or less, 10 nM or less, 2 nM or less, or 1 nM or less. Alternatively, the KD may be from 5 to 10 nM; or 1 to 2 nM. KD can range from 1 pM to 500 pM; or from 500 pM to 1 nM. The binding affinity of an antigen binding protein is determined by the association constant (Ka) and dissociation constant (Kd) (K _D =Kd/Ka). Binding affinity can be measured using BIACORE™, for example, by capturing a test antibody on a Protein A-coated sensing surface and allowing the target antigen to flow over that surface. Alternatively, binding affinity can be measured using FORTEBIO, for example, using the test antibody receptor immobilized on a protein A-coated needle and flowing the target antigen over this surface.

Kd may be 1x10 ^-3 Ms ^-1 or less, 1x10 ^-4 Ms ^-1 or less, or 1x 10 ^-5 Ms ^-1 or less. Kd can range from 1x 10 ^-5 Ms ^-1 to 1x10 ^-4 Ms ^-1 ; or from 1x10 ^-4 Ms ^-1 to 1x10 ^-3 Ms ^-1 . Slow Kd may result in slow dissociation of the antigen-binding protein-target antigen complex and improved neutralization of the target antigen.

As used herein, the term specific antigen binding activity means antigen binding activity measured by surface plasmon resonance (SPR). IL-5 specific binding activity can be determined by SPR using the BIACORE™ instrument, for example, in binding mode. This is the binding activity divided by the total protein content (e.g. 28Y042-7F11-1) in the sample.

As used herein, the term FcRn binding activity means neonatal Fc receptor (FcRn) binding activity measured by surface plasmon resonance (SPR). FcRn binding can be determined using the BIACORE™ instrument. This is the FcRn receptor binding activity divided by the total protein concentration in the sample.

The SPR method uses reference standard 28Y042-7F11-1 for specific antigen binding and FcRn binding. Reference Standard 28Y042-7F11-1 can be used in assays for

- 26 046656 obtaining data on the suitability of the system and the comparability of the sample to ensure proper implementation of the methods. A reference standard can allow one to obtain a calibration curve, and interpolate sample concentrations from that curve.

By isolated it is meant that a molecule, such as an antigen-binding protein or nucleic acid, has been removed from the environment in which it would be naturally found. For example, a molecule can be purified from substances with which it normally exists in nature. For example, the mass of a molecule in a sample may be 95% of the total mass. The disclosure also provides isolated nucleic acids containing SEQ ID NO: 13, 14, 15, 16, 17 and/or 18 and parts thereof, as well as compositions thereof. It is important that the nucleic acid of the invention is typically provided in the form of a composition that may contain any combination of nucleic acids of the invention, buffer, residual buffer, salts, counterions, water, alcohols or vectors and the like. Alternatively, the composition of the present invention may contain only the nucleic acids of the invention.

The terms VH and VL are used herein to refer to the heavy chain variable region and the light chain variable region, respectively, of an antigen binding protein.

CDRs are defined as the amino acid sequences of the complementarity determining region of an antigen binding protein. They are hypervariable regions of the heavy and light chains of immunoglobulin. The variable portion of an immunoglobulin has three heavy chain CDRs and three light chain CDRs (or CDR regions). Thus, a CDR as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least one CDR, wherein at least one CDR is CDRH3. Each of these CDR regions is followed by frame regions. Suitable variable heavy chain and variable light chain framework regions 1, 2 and 3 are readily recognized by those skilled in the art. Suitable heavy chain constant regions (including hinge regions) and light chain constant regions will also be readily recognized by those skilled in the art. Likewise, those skilled in the art will readily recognize acceptable antibody isotypes.

In the present description, amino acid residues in the variable domain sequences and full-length antibody sequences are numbered in accordance with the Kabat numbering convention. Likewise, the terms CDR, CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, CDRH3 used in the description follow the Kabat numbering convention.

It will be apparent to those skilled in the art that there are alternative conventions for numbering amino acid residues in full-length variable domain sequences and antibody sequences. There are also alternative numbering conventions for CDR sequences, such as those set out according to the Chotia numbering convention. The structure and folding of the antibody protein may mean that other residues are considered part of the CDR sequence and should be understood by one of ordinary skill in the art.

Other CDR sequence numbering conventions available to one skilled in the art include the AbM (University of Bath) and Contact (University College London) methods. The minimum area of overlap by applying at least two methods from Kabat, Chotia, AbM and contact can be determined to obtain the minimum binding unit. The minimum linking unit may be part of the CDR.

In the table below. 14 provides one definition using each numbering convention for each CDR or linking unit. The Kabat numbering scheme is used in Table. 14 for numbering the amino acid sequence of the variable domain. It should be noted that some definitions of CDR may differ depending on the individual publication used.

Table 14

CDR for Kabat CDR by Chotia CDR by method ABM CDR by contact method Minimum Linking Unit H1 31-35/35A/35V 26-32733/34 26-35/35A/35V 30-35/35A/35V 31-32 H2 50-65 52-56 50-58 47-58 52-56 NZ 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

The percent identity between the query nucleic acid sequence and the searched nucleic acid sequence is the identity value, expressed as a percentage, which is calculated by the BLASTN algorithm when the searched sequence

- 27 046656 nucleic acid has 100% requested coverage with the requested nucleic acid sequence after pairwise alignment using the BLASTN algorithm. This pairwise BLASTN alignment between the query nucleic acid sequence and the target nucleic acid sequence is performed using the default BLASTN settings available on the National Biotechnology Institute Center website with the low complexity filter disabled. It is important that the requested sequence may be described by a nucleic acid sequence defined in one or more claims.

Nucleic acid sequences that may be useful and included in the compositions and corresponding methods of the invention may be from about 85% to about 100%, from about 90% to about 100%, from 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% identity to the nucleic acid sequences defined herein (e.g., nucleic acids , encoding an antibody heavy chain or an antibody light chain). According to the invention, the percent identity between the described nucleic acid sequences may include any discrete subrange of the percent identity ranges specified above (eg, any range of integer values within a specified range or discrete sub-values within a specific range).

The percent identity between the query amino acid sequence and the target amino acid sequence is the percent identity value calculated by the BLASTP algorithm when the subject's amino acid sequence has 100% query coverage with the query amino acid sequence after pairwise alignment using the BLASTP algorithm. These pairwise BLASTP alignments between the query amino acid sequence and the target amino acid sequence are performed using the default BLASTP settings available on the National Biotechnology Institute Center website with the low complexity filter disabled. It is important that the requested sequence may be described by an amino acid sequence defined in one or more claims.

Amino acid sequences that may be useful and included in the compositions and corresponding methods of the invention may be from about 85% to about 100%, from about 90% to about 100%, from about 95% to about 100%, about 91% from about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 100% identity with the amino acid sequences specified in the description (for example, for the heavy chain of an antibody or light chain of the antibody). According to the invention, the percent identity between the described amino acid sequences may include any discrete subrange of the above percent identity ranges (eg, any range of integer values within a particular range or discrete sub-values within a particular range).

The terms peptide, polypeptide, protein and peptide chain each refer to a molecule containing two or more amino acid residues. The peptide may be monomeric or polymeric.

It is well known in the art that certain amino acid substitutions are considered conservative. Amino acids are divided into groups based on the general properties of their side chains, and substitutions in groups that retain all or almost all of the binding affinity of the antigen-binding protein are considered conservative substitutions. See table. 15. The antigen binding proteins disclosed herein may contain such conservative amino acid substitutions.

Table 15

Side chain Members Hydrophobic met, ala, val, leu, ile Neutral hydrophilic cys, ser, thr Sour asp,glu Alkaline asn, gin, his, lys, arg Residues that affect chain orientation gly,pro Aromatic trp, tyr, phe

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

The pharmaceutical compositions described herein may contain purified antibody preparations as described herein.

For example, the pharmaceutical preparation may contain a purified antibody preparation described

- 28 046656 specified in this application, in combination with a pharmaceutically acceptable carrier.

Typically, such pharmaceutical compositions contain a pharmaceutically acceptable carrier that is known and expected to be acceptable for pharmaceutical use. Examples of such vehicles include sterilized vehicles such as saline, Ringer's solution or dextrose solution, optionally buffered with suitable buffers to a pH in the range of 5 to 8.

The pharmaceutical compositions can be administered by injection or infusion (eg, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, or intraportal). Such compositions do not contain visible particles. Pharmaceutical compositions may contain from 1 mg to 10 g of antigen binding protein, for example from 5 mg to 1 g of antigen binding protein. Alternatively, the composition may contain from 5 to 500 mg of antigen binding protein, for example from 5 to 50 mg.

Methods for preparing such pharmaceutical compositions are well known to those skilled in the art. Pharmaceutical compositions may contain from 1 mg to 10 g of antigen binding protein in unit dosage form, optionally along with instructions for use. Pharmaceutical compositions can be lyophilized (sublimated) for reconstitution before administration in accordance with methods well known or obvious to those skilled in the art. When antibodies are of the IgG ₁ isotype, a copper chelator, such as citrate (eg, sodium citrate) or EDTA or histidine, can be added to the pharmaceutical composition to reduce the extent of copper-mediated degradation of antibodies of that isotype. Pharmaceutical compositions may also include a solubilizer such as arginine, a surfactant/aggregation-inhibiting agent such as polysorbate 80, and an inert gas such as nitrogen to replace oxygen in the headspace of the vial.

As used herein, the term therapeutically effective amount means an amount of an agent (such as an antibody or pharmaceutical composition) that provides a therapeutic benefit in treating or treating one or more symptoms of the condition being treated (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 polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis it , moderate atopic dermatitis and severe atopic dermatitis). Examples of such treatment or therapy for 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 sub-eosinophilic asthma include : 1) reduction in the frequency of asthma exacerbations; 2) reducing the time to the first clinically significant exacerbation requiring the administration of oral or systemic corticosteroids, hospitalization and/or calling an ambulance; 3) reduction in the frequency of exacerbations requiring hospitalization (including intubation and delivery to the intensive care unit) or calling a physician; 4) reducing the time until the first exacerbation requiring hospitalization or calling a specialist; 5) a clinically significant change in FEV1 relative to the baseline level before the administration of a bronchodilator; 6) clinically significant change in FEV1 relative to baseline after administration of a bronchodilator; 7) change in Asthma Control Questionnaire (ACQ) score from baseline; 8) improved pulmonary function, assessed by spirometry (eg, vital capacity (VC), forced vital capacity (FVC), forced expiratory volume (FEV) at time intervals of 0.5, 1.0 (FEV, 2.0, and 3.0 seconds at forced expiratory flow rate 25-75% (FEF 25-75) and level of maximum voluntary ventilation (MVV), total lung volume, tidal volume, residual volume, expiratory reserve volume, inspiratory reserve volume, inspiratory capacity, vital capacity inspiratory capacity, vital capacity, functional residual capacity, residual volume expressed as a percentage of total lung capacity, alveolar gas volume, actual lung volume including the volume of the conducting airways, forced vital capacity, etc.); and 9) reducing the administration of steroids needed to control asthma exacerbations (such as oral steroids or steroids such as prednisone, prednisolone, etc., administered by any route). This reduction in the administration of steroids needed to control asthma exacerbations could be approximately a 50% reduction in the administration of steroids needed for exacerbations (eg, oral steroids).

Therapeutically effective amounts and treatment regimens are generally determined empirically and may depend on factors such as the age, weight and health status of the patient and the disease or disorder being treated. Such factors are within the competence of the attending physician.

The dose of antigen binding protein administered to a subject is typically 1 μg/kg to 150 mg/kg, 0.1 mg/kg to 100 mg/kg, 0.5 mg/kg to 50 mg/kg, 1 to 25 mg/kg, from about 0.3 mg/kg to about 3 mg/kg, or from 1 to 10 mg/kg of 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 10 mg/kg to 110 mg/mg, 15 mg/kg to 25 mg/kg, or 15 mg/kg to 100 mg/kg. The antigen binding protein can be administered, for example, parenterally, subcutaneously, intravenously

- 29 046656 but or intramuscularly. Doses may also be administered based on the individual subject, for example, from about 20 mg/subject to about 750 mg/subject, from about 75 mg/subject to about 750 mg/subject, from about 20 mg/subject to about 200 mg/subject. The dose may be any subrange within these dose ranges. For example, the dosage may also be administered subcutaneously to suit the individual subject, e.g., about 100 mg/subject (e.g., once every four weeks) or 300 mg/subject (or other doses may be administered subcutaneously to achieve bioavailability approximately the same or comparable to that of intravenous administration - for example, three doses of 100 mg/subject, resulting in a total subcutaneous dose of 300 mg/subject).

Ranges of any type presented herein include all values within the particular range described and the values of the boundaries of the particular range.

If desired, an effective daily dose of an antibody or antigen-binding protein of the invention (e.g., in the form of a pharmaceutical composition) can be administered in two, three, four, five, six or more doses administered separately throughout the day at appropriate intervals, optionally in single doses. dosage forms.

Dosing may be a slow continuous infusion over 2 to 24 hours, such as 2 to 12 hours or 2 to 6 hours. Such administration may result in a reduction in side effects.

The dosage may be repeated as necessary one or more times, for example, three times a day, once a day, once every 2 days, once a week, once every 14 days, once a month, once every 3 months , once every 4 months, once every 6 months, or once every 12 months. Antigen-binding proteins can be administered as maintenance therapy, for example, once a week for 6 months or more. Antigen-binding proteins can be administered as intermittent therapy, for example, for 3 to 6 months, followed by a break for 3-6 months, followed by repeated administration of antigen-binding proteins for 3 to 6 months, and so on, in a cycle.

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

The antigen binding protein can be administered to a subject in such a way that therapy is directed to a specific site.

The antigen binding protein in the methods of the invention can be used in combination with one or more other therapeutically active agents, such as antibodies or small molecule inhibitors.

The term treatment and its grammatical variants as used herein mean therapeutic treatment. For a particular condition, treatment means: (1) improving one or more biological manifestations of the condition, (2) blocking a) one or more points in the biological cascade that lead to or cause the development of that condition, or b) one or more more biological manifestations of the condition, (3) alleviating one or more symptoms, effects or side effects associated with the condition or its treatment, (4) slowing the progression of the condition or one or more biological manifestations, or (5) preventing the occurrence of one or more biological manifestations of the condition . This also includes preventive therapy. It will be apparent to one skilled in the art that prevention is not an absolute term. In medicine, prophylaxis means the prophylactic administration of a drug to significantly reduce the likelihood or severity of a condition or its biological manifestation, or to delay the onset of such a condition or its biological manifestation.

The terms individual, subject and patient are used interchangeably herein. The subject is usually a human. The subject may also be a mammal such as a mouse, rat, or primate (eg, a monkey or monkey). The subject may be a non-human animal. Antigen-binding proteins, compositions and methods of the invention also find use in veterinary medicine. The subject to be treated may be a farm animal, such as a cow or bull, sheep, pig, ox, goat or horse, or may be a domestic animal, such as a dog or cat. The animal may be of any age or may be a mature adult animal.

Treatment may be therapeutic, prophylactic, or preventive. The subject is a subject in need of this treatment. Those who need treatment may include people who already have a particular medical condition as well as people who may develop the condition in the future.

Thus, the methods, antigen-binding proteins and compositions of the invention disclosed herein can be used for prophylactic treatment or preventive treatment, if necessary. In this case, the methods, antigen binding proteins and compositions of the invention 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

- 30 046656 predisposition to the disease. Such an individual is administered a prophylactically effective amount of antigen binding protein. A prophylactically effective amount is an amount that prevents or delays the onset of one or more aspects or symptoms of a disease disclosed herein.

The methods, antigen-binding proteins and compositions of the invention do not need to result in complete cure or elimination of every symptom or manifestation of a disease to be useful as a therapeutic treatment. As is known in the art, in order for drugs used as therapeutic agents in methods of treatment to be considered therapeutic agents, they do not have to eliminate every manifestation of a disease, and may reduce the severity of a given disease state. Likewise, a treatment administered for prophylactic purposes does not necessarily have to be effective in completely preventing the occurrence of a disease to be considered as a useful prophylactic agent. Simply reducing the impact of the disease (for example, by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood of the disease occurring (for example, by delaying the onset of the disease) or weakening it in the subject is sufficient .

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

In one embodiment of the antigen binding protein of the invention, the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21.

In one embodiment, the antigen binding protein of the invention 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, wherein the amino terminus of the heavy chain Fc domain is connected to the carboxy terminus of the variable region heavy chain.

Another aspect of the invention 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.

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

In one embodiment of the antibody of the invention, the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21.

In one embodiment of the antibody of the invention, 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.

Another aspect of the invention 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.

In one embodiment of the antibody of the invention, 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.

Another aspect of the invention 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.

Another aspect of the invention is a peptide chain containing the amino acid sequence shown in SEQ ID NO: 3.

Another aspect of the invention is a peptide chain containing the amino acid sequence shown in SEQ ID NO: 1.

- 31 046656

In one embodiment, the composition of the invention comprises a nucleic acid encoding a heavy chain variable region of the invention and a nucleic acid encoding a light chain variable region of an antigen binding protein of the invention.

In one embodiment, the composition of the invention contains a nucleic acid encoding a heavy chain variable region of the invention and a nucleic acid encoding a light chain variable region of the invention.

In one embodiment, a composition of the invention comprises a nucleic acid encoding a heavy chain Fc domain coupled to the carboxy terminus of a heavy chain variable region of the invention and a nucleic acid encoding a light chain variable region of the invention.

Another aspect of the invention 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.

Another aspect of the invention 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.

Another aspect of the invention is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 3.

Another aspect of the invention is a composition comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 1.

Another aspect of the invention 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.

Another aspect of the invention 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.

Another aspect of the invention 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.

Another aspect of the invention is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO: 15.

Another aspect of the invention is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO: 17.

Another aspect of the invention is a composition comprising a nucleic acid having the sequence shown in SEQ ID NO: 13.

In one embodiment, the composition of the invention contains a nucleic acid encoding a heavy chain variable region of the invention.

In one embodiment, the composition of the invention contains a nucleic acid encoding a heavy chain variable region of the invention.

In one embodiment, the composition of the invention comprises a nucleic acid encoding a heavy chain Fc domain linked to the carboxy terminus of a heavy chain variable region of the invention.

In one embodiment, the expression vector of the invention contains a composition of the invention.

In one embodiment, the recombinant host cell of the invention contains an expression vector containing a composition of the invention. In an alternative embodiment, the recombinant host cell may comprise a first expression vector encoding a first peptide chain of an antigen binding protein (e.g., an antibody heavy chain) and a second expression vector encoding a second peptide chain of an antigen binding protein of the invention (e.g., an antibody light chain).

Another aspect of the invention is a method of producing a peptide chain containing the amino acid sequence shown in SEQ ID NO: 3, the method comprising the step of culturing a recombinant host cell containing a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 3; and extraction of the peptide chain.

In one embodiment of the method of the invention, the nucleic acid contains the sequence shown in SEQ ID NO: 15.

In one embodiment of the method of the invention, the nucleic acid contains the sequence shown in SEQ ID NO: 13.

In one embodiment of the method of the invention, the nucleic acid contains the sequence shown in SEQ ID NO: 17.

One embodiment of the invention is a method for producing an antigen-binding protein, which includes the steps of: a) cultivating a recombinant host cell containing a vector

- 32 046656 expression containing the composition according to the invention; and b) recovering the antigen binding protein; thereby obtaining antigen binding protein.

In one embodiment of the invention, the antigen binding protein is produced by the method of the invention.

One embodiment of the invention is a method for producing an antibody, comprising the steps of: a) cultivating a recombinant host cell containing an expression vector containing a composition according to the invention; and b) recovering the antibody; thus obtaining the antibody.

One embodiment of the present invention is an antibody produced by the method of the invention.

Another aspect of the invention is a method of producing an antibody, comprising the steps of: a) culturing a recombinant host cell containing an expression vector containing 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; and b) recovering the antibody; thus obtaining the antibody.

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

In one embodiment of the pharmaceutical composition of the invention, the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21.

In one embodiment, the pharmaceutical composition of the invention 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, wherein the amino terminus of the heavy chain Fc domain is connected to the carboxy terminus of the variable region heavy chain.

In one embodiment of the pharmaceutical composition of the invention, the antigen binding protein is at a concentration of from about 75 mg/ml to about 150 mg/ml.

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

In one embodiment of the pharmaceutical composition of the invention, the pH is about 6.0 and the antigen binding protein is at a concentration of about 150 mg/ml.

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

58. The pharmaceutical composition of claim 57, wherein the antibody has a concentration of from about 75 to about 150 mg/ml.

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

In one embodiment of the pharmaceutical composition of the invention, the pH is about 6.0 and the antibody is at a concentration of about 150 mg/ml.

Another aspect of the invention is a pharmaceutical composition comprising: a) an antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3; 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.

Another aspect of the invention 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) pharma

- 33 046656 Ceutically acceptable carrier.

One embodiment of the invention is a method of treating a disease in a subject, comprising the steps of: a) identifying a subject having 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, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, 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 antigen binding protein of the invention; thereby providing treatment for the disease in the subject.

In one embodiment of the methods of the invention, the amount of antigen binding protein is from about 2 to about 600 mg. For example, the amount of antigen binding protein (eg, antibody) may be a dose of 2 mg, 10 mg, 30 mg, 100 mg, 300 mg, or 600 mg.

In one embodiment of the method of the present invention, the antigen binding protein is administered once every 3 months or once every 6 months.

In one embodiment of the method of the invention, the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

One embodiment of the invention is a method of treating a disease in a subject, comprising the steps of: a) identifying a subject having 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, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, 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 of the invention; thereby providing treatment for the disease in the subject.

One embodiment of the invention is a method of treating a disease in a subject, comprising the steps of a) identifying a subject having a disease selected from the group consisting of asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, 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 composition of the invention; thereby providing treatment for the disease in the subject.

Another aspect of the invention is a method of treating mild to moderate asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with mild to moderate asthma; and b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 a CDR amino acid sequence shown in SEQ ID NO: 8, a CDR amino acid sequence shown in SEQ ID NO: 9, and a CDR amino acid sequence shown in SEQ ID NO: 10; thus providing treatment for mild to moderate asthma.

One embodiment is the method of the invention wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21.

One embodiment is a method of the invention 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, wherein the amino terminus of the heavy chain Fc domain is connected to carboxy terminus of the heavy chain variable region.

One embodiment is the method of the invention, 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.

One embodiment is the method of the invention in which the antibody is administered subcutaneously.

Another aspect of the invention is a method of treating mild to moderate asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with mild to moderate asthma.

- 34 046656 moderate severity; and b) administering to the subject a therapeutically effective amount of 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: 1; thus providing treatment for mild to moderate asthma.

Another aspect of the invention is a method of treating mild to moderate asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with mild to moderate asthma; and b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising 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: 1, and a pharmaceutically effective carrier ; thus providing treatment for mild to moderate asthma.

One embodiment is a method of the invention wherein the pharmaceutically effective carrier contains an aqueous liquid composition at a pH of from about 5.5 to about 6.0 containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02 by volume polysorbate 80 and about 0.05 mM EDTA.

Another aspect of the invention is a method of treating severe asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with severe asthma; and b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 amino acid sequence

the CDR shown in SEQ ID NO: 8, the amino acid sequence of the CDR shown in SEQ ID NO: 9, and the amino acid sequence of the CDR shown in SEQ ID NO: 10; thus providing treatment for severe asthma.

Another aspect of the invention is a method of treating severe asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with severe asthma; and b) administering to the subject a therapeutically effective amount of 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: 1; thus providing treatment for severe asthma.

Another aspect of the invention is a method of treating severe asthma in a subject, comprising the steps of: a) identifying a subject diagnosed with severe asthma; and b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising 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: 1, and a pharmaceutically effective carrier ; thus providing treatment for severe asthma.

Another aspect of the invention is a method of treating a subject with moderate to severe atopic dermatitis, comprising the steps of: a) identifying a subject having at least one of the characteristics selected from the group consisting of: i) diagnosing atopic dermatitis in accordance with the Hanifin criteria; Raika, revised by Eichenwald; ii) a provisional diagnosis of atopic dermatitis made more than two years ago or approximately two years before the start of treatment; iii) an overall health care provider score of three or more; iv) atopic dermatitis lesions greater than or equal to approximately 10% of the body surface area; v) an index of severity and area affected by eczema greater than or equal to 16; vi) an absolute blood eosinophil count greater than or equal to 150 cells per μL, greater than or equal to 200 cells per μL, greater than or equal to 300 cells per μL, or greater than or equal to 350 cells per μL; and vii) at least one pre-treatment condition selected from the group consisting of: 1) an inadequate response for six months or more to a topical atopic dermatitis medication; 2) poor tolerance to topical medications for atopic dermatitis; 3) a side effect from taking topical atopic dermatitis medication; and 4) inadequate response to non-pharmacological treatment for atopic dermatitis; and b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 a CDR amino acid sequence shown in SEQ ID NO: 8, a CDR amino acid sequence shown in SEQ ID NO: 9, and a CDR amino acid sequence shown in SEQ ID NO: 10; thereby providing treatment for atopic dermatitis in the subject.

One embodiment is the method of the invention in which the antibody is administered intravenously.

Another aspect of the invention is a method of treating a subject with moderate to severe atopic dermatitis, comprising the steps of: a) identifying a subject having at least

- 35 046656 one of the signs selected from the group consisting of: i) a diagnosis of atopic dermatitis in accordance with the Hanifin and Rajk criteria, revised by Eichenwald; ii) a provisional diagnosis of atopic dermatitis made more than two years ago or approximately two years before the start of treatment; iii) an overall health care provider score of three or more; iv) atopic dermatitis lesions greater than or equal to approximately 10% of the body surface area; v) an index of severity and area affected by eczema greater than or equal to 16; vi) an absolute blood eosinophil count greater than or equal to 150 cells per μL, greater than or equal to 200 cells per μL, greater than or equal to 300 cells per μL, or greater than or equal to 350 cells per μL; and vii) at least one pre-treatment condition selected from the group consisting of: 1) an inadequate response for six months or more to a topical atopic dermatitis medication; 2) poor tolerance to topical medications for atopic dermatitis; 3) a side effect from taking topical atopic dermatitis medication; and 4) inadequate response to non-pharmacological treatment for atopic dermatitis; and b) administering to the subject a therapeutically effective amount of 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: 1; thereby providing treatment for atopic dermatitis in the subject.

Another aspect of the invention is a method of treating a subject with moderate to severe atopic dermatitis, comprising the steps of: a) identifying a subject having at least one of the characteristics selected from the group consisting of: i) diagnosing atopic dermatitis in accordance with the Hanifin criteria; Raika, revised by Eichenwald; ii) a provisional diagnosis of atopic dermatitis made more than two years ago or approximately two years before the start of treatment; iii) an overall health care provider score of three or more; iv) atopic dermatitis lesions greater than or equal to approximately 10% of the body surface area; v) an index of severity and area affected by eczema greater than or equal to 16; vi) an absolute blood eosinophil count greater than or equal to 150 cells per μL, greater than or equal to 200 cells per μL, greater than or equal to 300 cells per μL, or greater than or equal to 350 cells per μL; and vii) at least one pre-treatment condition selected from the group consisting of: 1) an inadequate response for six months or more to a topical atopic dermatitis medication; 2) poor tolerance to topical medications for atopic dermatitis; 3) a side effect from taking topical atopic dermatitis medication; and 4) inadequate response to non-pharmacological treatment for atopic dermatitis; and b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising 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: 1, and a pharmaceutically effective carrier ; thereby providing treatment for atopic dermatitis in the subject.

Another aspect of the invention is a method of reducing the absolute number of eosinophils in the blood of a subject, comprising the steps of: a) identifying a subject having 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 granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis and atopic dermatitis; and b) administering to the subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 a CDR amino acid sequence shown in SEQ ID NO: 8, a CDR amino acid sequence shown in SEQ ID NO: 9, and a CDR amino acid sequence shown in SEQ ID NO: 10; thereby reducing the subject's absolute blood eosinophil count.

One embodiment of the method of the invention further comprises the steps of: a) taking a first measurement of the absolute number of eosinophils in the blood of a subject; b) performing a second measurement of the absolute number of eosinophils in the blood of the subject after administering to the subject a therapeutically effective amount of antigen binding protein; and c) comparing the first dimension and the second dimension.

One embodiment of the method of the invention further comprises the steps of: a) taking a first measurement of the absolute number of eosinophils in the blood of a subject; b) performing a second measurement of the absolute number of eosinophils in the blood of the subject after administering to the subject a therapeutically effective amount of antigen binding protein; and c) comparing the first measurement and the second measurement; wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

- 36 046656

Another aspect of the invention is a method of reducing the absolute number of eosinophils in the blood of a subject, comprising the steps of: a) identifying a subject having 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 granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis ita ; and b) administering to the subject a therapeutically effective amount of 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: 1; thereby providing treatment for atopic dermatitis in the subject.

Another aspect of the invention is a method of reducing the absolute number of eosinophils in the blood of a subject, comprising the steps of: a) identifying a subject having 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 granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis ita ; and b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising 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: 1, and a pharmaceutically effective carrier ; thereby reducing the subject's absolute blood eosinophil count.

One embodiment of the invention is a composition of the invention for use in therapy.

One embodiment of the invention is a composition of the invention for use in the treatment 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 granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis.

The compositions of the invention may further contain a buffering agent selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate, citric acid, citrate, sodium phosphate, potassium phosphate, sodium citrate and histidine, providing a pH of 6.8 to 7.2 or pH from 6.2 to 6.6, with a pH value of 6.3 being preferred. The buffer in the compositions of the invention may be about 10-30 mM, about 10-20 mM, about 20 mM, or about 15.5 mM. For example, the buffer in the compositions of the invention may be dibasic sodium phosphate heptahydrate at a concentration of about 20 mM or about 15.5 mM.

The compositions of the invention may contain the buffering agents dibasic sodium phosphate heptahydrate and citric acid to provide a pH of 6.2 to 6.6, inclusive, with a pH of 6.3 being preferred. The sodium phosphate heptahydrate buffering agent may be present in the range of about 15-16.4 mM, and the citric acid buffering agent may be present in the range of about 3.8-4.9 mM. For example, the compositions of the invention may contain about 15.5 mM dibasic sodium phosphate heptahydrate and about 4.5 mM citric acid monohydrate.

The compositions of the invention may additionally contain sugar. The compositions of the invention may additionally contain sucrose. Sucrose may be present in the compositions of the invention in the range of about 5-20%; about 10-15%, about 11-13%, or about 12% by volume.

The compositions of the invention may additionally contain polysorbate 80. Polysorbate 80 may be present in the range of about 0.01-0.1% by volume. For example, polysorbate 80 may be present in the compositions of the invention in an amount of about 0.02% by volume or about 0.05% by volume.

The compositions of the invention may additionally contain EDTA. EDTA may be present in the range of about 0.01-0.1 mM. For example, EDTA may be present at a concentration of approximately 0.05 mM.

In one embodiment, the compositions of the invention further contain 20 mM dibasic sodium phosphate heptahydrate, 12% by volume sucrose and 0.05% by volume polysorbate 80.

In another embodiment, the compositions of the present invention further contain 15.5 mM dibasic sodium phosphate, 3.9 mM citric acid monohydrate, 12% vol.

- 37 046656 mass of sucrose, 0.02% by volume of polysorbate 80 and 0.05 mM EDTA.

The compositions of the invention may comprise an aqueous liquid composition with a pH of 6.2 containing 16.1 mM dibasic sodium phosphate heptahydrate, 3.9 mM citric acid monohydrate, 12% by volume sucrose, 0.02% by volume polysorbate 80 and 0.05 mM EDTA.

The compositions of the invention may comprise an aqueous liquid composition with a pH of 6.2 containing 15.2 mM dibasic sodium phosphate heptahydrate, 4.8 mM citric acid monohydrate, 12% by volume sucrose, 0.02% by volume polysorbate 80 and 0.05 mM EDTA.

The compositions of the invention may comprise an aqueous liquid composition with a pH of 6.4 containing 15.8 mM dibasic sodium phosphate heptahydrate, 4.2 mM citric acid monohydrate, 12% by volume sucrose, 0.02% by volume polysorbate 80 and 0.05 mM EDTA.

The compositions of the invention may comprise an aqueous liquid composition with a pH of 6.6 containing 16.3 mM dibasic sodium phosphate heptahydrate, 3.7 mM citric acid monohydrate, 12% by volume sucrose, 0.02% by volume polysorbate 80 and 0.05 mM EDTA.

The compositions of the invention may comprise an aqueous liquid composition with a pH of 6.3 containing 15.5 mM dibasic sodium phosphate heptahydrate, 4.5 mM citric acid monohydrate, 12% by volume sucrose, 0.02% by volume polysorbate 80 and 0.05 mM EDTA. It is important to note that the tangential flow ultrafiltration and exchange/ultrafiltration step in Example 1 below can be adjusted to produce compositions of the invention, such as a composition of the invention containing 15.5 mM dibasic sodium phosphate heptahydrate, 4.5 mM citric acid monohydrate acid, 12% by volume sucrose, 0.02% by volume polysorbate 80, 0.05 mM EDTA at pH 6.3 or other such liquid compositions.

The compositions of the invention may contain a purified monoclonal antibody preparation and a buffering agent, the composition having a pH of 6.8 to 7.2, wherein the buffering agent is histidine, phosphate, citric acid, citrate or a salt thereof.

In the compositions of the invention, the buffering agent may be at least one selected from the group consisting of dibasic sodium phosphate heptahydrate, phosphate, citric acid and citrate.

In the compositions of the invention, the buffering agent may be sodium phosphate, potassium phosphate or sodium citrate.

The compositions of the invention may contain sugar, carbohydrate and/or salt.

The compositions of the present invention may also contain sucrose or trehalose.

The compositions of the invention may also contain a purified monoclonal antibody preparation and a buffering agent, wherein the composition has a pH of 6.8 to 7.2, wherein the buffering agent is a phosphate or a salt thereof.

The composition of the invention may also contain a composition selected from: a first composition containing 20 mM dibasic sodium phosphate heptahydrate, 12% by volume sucrose and 0.05% by volume polysorbate 80; and a second composition containing 15.5 mM dibasic sodium phosphate heptahydrate, 3.9 mM citric acid monohydrate, 12% by volume sucrose, 0.02% by volume polysorbate 80 and 0.05 mM EDTA; and a third composition containing 26 mM dibasic sodium phosphate heptahydrate, 15% by volume sucrose and 0.065% by volume polysorbate 80.

The composition may have a pH of from about 6.8 to about 7.2, from about 6.1 to about 6.5, or from about 6 to about 6.6.

The compositions described herein can be prepared by any number of conventional methods. For example, the compositions can be expressed in recombinant expression systems and purified from these systems. In one embodiment, the composition is prepared by culturing host cells under conditions suitable for expressing a polypeptide comprising SEQ ID NO: 1 and SEQ ID NO: 2, wherein the composition is expressed, and optionally purified, and optionally formulated into a pharmaceutical composition.

Several different expression systems and purification regimes can be used to obtain the compositions. Host cells are typically transformed with a recombinant expression vector encoding the antibody. A wide range of host cells can be used, including mammalian eukaryotic cell lines (eg, CHO, Perc6, HEK293, HeLa, NS0). Suitable host cells include mammalian cells such as CHO (eg, CHOK1 and CHO-DG44).

The host cell may be an isolated host cell. The host cell is usually not part of a multicellular organism (such as a plant or animal). The host cell may be a host cell other than a human cell.

Suitable cloning and expression vectors for use with eukaryotic host cells or mammalian host cells and cloning methods are known in the art.

Cells can be cultured under conditions that promote antibody expression. For example, a production bioreactor is used to cultivate cells. The production bioreactor volume may be: (i) about 20,000 L, about 10,000 L; approximately 5000 l; approximately 2000 l; approximately 1000 l; or approximately 500 l; or (ii) from 500 to 20,000 l; from 500 to 10000 l; from 500 to

- 38 046656

5000 l; from 1000 to 10000 l or from 2000 to 10000 l. For example, cells can be cultured in a production bioreactor at a pH of about 6.75 to pH 7.00. Alternatively, the cells can be cultured in a production bioreactor for about 12 to about 18 days. Alternatively, the cells can be cultured in a production bioreactor at a pH of about 6.75 to pH 7.00 for about 12 to about 18 days. This culture step can help control the level of deamidated antibody variants, for example, to reduce the level of deamidated antibody variants.

The composition can be isolated and purified using conventional protein purification procedures. For example, the composition can be collected directly from the culture medium. Collection of cell culture media can be accomplished by clarification, such as centrifugation and/or deep filtration. Removal of the composition is followed by cleaning to ensure adequate purity.

For purification, one or more chromatography steps may be used, for example, one or more chromatography resins; and/or one or more filtering steps. For example, affinity chromatography using resins such as protein A, G or L can be used to purify the composition. Alternatively or additionally, an ion exchange resin such as a cation exchange resin can be used to purify the composition. Alternatively or additionally, a hydrophobic interaction chromatography resin can be used to purify the composition. Alternatively, the purification steps include: an affinity chromatography resin step, followed by a cation exchange resin step, followed by a hydrophobic interaction chromatography resin step.

For example, the collected medium is placed in contact with a Protein A resin. The solution containing the composition can be eluted from the Protein A resin and treated at a pH of 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, to reduce the level of aggregated antibody variants.

The solution containing the composition can then be further clarified by deep filtration and/or double-layer filtration.

Alternatively or additionally, an anion exchange resin can be used. The solution containing the composition can be contacted with an anion exchange resin (eg, anion exchange chromatography on a Q-SEPHAROSE™ Fast Flow column) at a pH of 8.3 to 8.7. The solution containing the composition may be eluted from the anion exchange resin and incubated for 96 hours or less. This anion exchange resin step can help control the level of deamidated antibody variants, for example, to reduce the level of deamidated antibody variants.

Optionally, guanidine and/or ammonium sulfate may be added to the solution containing the composition, followed by stabilization for 15 to 240 minutes.

Alternatively or additionally, a hydrophobic interaction chromatography resin can be used. The solution containing the composition can be contacted with a hydrophobic interaction chromatography resin (eg, fast flow phenylSEPHAROSE™ chromatography) at a loading ratio of 12 to 27 g protein/L resin. For example, a solution containing the composition can be eluted with a gradient elution volume (bed volumes; BV) of about 9 to about 11. During elution from a hydrophobic interaction chromatography resin, the elution peak maximum (% of maximum peak height) can be stopped from decreasing from about 17 to about 23. This hydrophobic interaction resin chromatography step can help control the level of aggregated antibody variants, for example, to reduce the level of aggregated antibody variants.

The solution containing the composition can then be filtered to remove the virus. Thereafter, a solution can be prepared containing the composition with an antibody concentration of from about 76 g protein/L to about 82 g protein/L or up to about 100 g protein/L. The solution containing the composition can be poured into containers and frozen. Aliquots of the solution containing the composition may be lyophilized. The lyophilisate can be reconstituted by adding water to obtain a composition containing 75 mg/L protein, anti-HE-5 monoclonal antibody, and 20 mM dibasic sodium phosphate heptahydrate, 12% by volume sucrose and 0.05% by volume polysorbate 80 at a pH of approximately 6.8 to approximately 7.2. Thus, the invention includes:

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

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

- 39 046656

3. Antigen-binding protein of 2, containing 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 carboxy terminus of the variable region heavy chain.

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.

5. Antigen binding protein of 4, containing 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 carboxy terminus of the variable region heavy chain.

6. An antibody containing a heavy chain and a light chain, wherein

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

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

7. Antibody according to 6, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21.

8. Antibody according to 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.

9. An antibody containing a heavy chain and a light chain, wherein

a) the heavy chain contains 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.

10. Antibody according to 9, wherein the heavy chain contains 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.

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.

12. A peptide chain containing the amino acid sequence shown in SEQ ID NO: 3.

13. A peptide chain containing the amino acid sequence shown in SEQ ID NO: 1.

14. A composition containing a nucleic acid encoding the heavy chain variable region of 1 and a nucleic acid encoding the light chain variable region of an antigen binding protein of 1.

15. A composition containing a nucleic acid encoding a heavy chain variable region of 2 and a nucleic acid encoding a light chain variable region of 2.

16. A composition containing a nucleic acid encoding a heavy chain Fc domain connected to the carboxy terminus of a heavy chain variable region at 3, and a nucleic acid encoding a light chain variable region at 3.

17. A composition containing a nucleic acid encoding a heavy chain variable region at 6 and a nucleic acid encoding a light chain variable region of an antigen binding protein at 6.

18. A composition containing a nucleic acid encoding a heavy chain variable region of 7 and a nucleic acid encoding a light chain variable region of 7.

19. A composition containing a nucleic acid encoding a heavy chain Fc domain connected to the carboxy terminus of an 8 heavy chain variable region, and a nucleic acid encoding an 8 light chain variable region.

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.

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.

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

23. A composition containing a nucleic acid encoding an amino acid sequence

- 40 046656 item shown in SEQ ID NO: 1.

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.

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.

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.

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

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

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

30. A composition containing a nucleic acid encoding a heavy chain variable region of 1.

31. A composition containing a nucleic acid encoding a heavy chain variable region of 2.

32. A composition containing a nucleic acid encoding a heavy chain Fc domain linked to the carboxy terminus of the heavy chain variable region at 3.

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

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

35. A method of producing a peptide chain containing the amino acid sequence shown in SEQ ID NO: 3, the method comprising the step of culturing a recombinant host cell containing a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 3; and extraction of the peptide chain.

36. The method of 35, wherein the nucleic acid contains the sequence shown in SEQ ID NO: 15.

37. The method of 35, wherein the nucleic acid contains the sequence shown in SEQ ID NO: 13.

38. The method of 35, wherein the nucleic acid contains the sequence shown in SEQ ID NO: 17.

39. A method for producing an antigen-binding protein, comprising the steps of:

a) culturing a recombinant host cell containing an expression vector containing the composition of 14, 15 or 16; And

b) extracting the antigen binding protein; thereby obtaining antigen binding protein.

40. Antigen binding protein produced by the method of 39.

41. A method for producing an antibody, comprising the step of:

a) culturing a recombinant host cell containing an expression vector containing the composition of 17, 18 or 19; And

b) recovering the antibody;

thus obtaining an antibody.

42. Antibody produced by the method of 41.

43. A method for producing an antibody, comprising the step of:

a) culturing a recombinant host cell containing an expression vector containing the composition of 20 or 22; And

b) recovering the antibody;

thus obtaining an antibody.

44. Antibody produced by the method of 43.

45. A method for producing an antibody, comprising the step of:

a) culturing a recombinant host cell containing an expression vector containing the composition of 24, 26 or 27; And

b) recovering the antibody;

thus obtaining the antibody.

46. Antibody produced by the method of 45.

47. A method for producing an antibody, comprising the step of:

a) culturing a recombinant host cell containing an expression vector containing 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; And

- 41 046656

b) recovering the antibody;

thus obtaining an antibody.

48. Antibody produced by the method of 47.

49. Pharmaceutical composition containing:

a) an antigen binding protein comprising a heavy chain variable region having the amino acid sequence CDRH1 shown in SEQ ID NO: 5, the amino acid sequence CDRH2 shown in SEQ ID NO: 6, and the amino acid sequence CDRH3 shown in SEQ ID NO: 7; and a light chain variable region having the amino acid sequence of CDRL1 shown in SEQ ID NO: 8, the amino acid sequence of CDRL2 shown in SEQ ID NO: 9, and the amino acid sequence of CDRL3 shown in SEQ ID NO: 10; and b) a pharmaceutically acceptable carrier.

50. The pharmaceutical composition of 49, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21.

51. Pharmaceutical composition according to 50, containing 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 carboxy terminus of the variable region heavy chain.

52. The pharmaceutical composition of 51, wherein the antigen binding protein is at a concentration of from about 75 mg/ml to about 150 mg/ml.

53. The pharmaceutical composition of claim 49, 50, 51, or 52, wherein the pharmaceutically effective carrier contains an aqueous liquid composition at a pH of from about 5.5 to about 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine , about 8 mM methionine, about 0.02% by volume polysorbate 80 and about 0.05 mM EDTA.

54. The pharmaceutical composition of 53, wherein the pH is about 6.0 and the antigen binding protein is at a concentration of about 150 mg/ml.

55. Pharmaceutical composition containing:

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

b) a pharmaceutically acceptable carrier.

56. The pharmaceutical composition of 55, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21.

57. Pharmaceutical composition according to 56, containing 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 carboxy terminus of the variable region heavy chain.

58. The pharmaceutical composition of 57, wherein the antibody has a concentration of from about 75 mg/ml to about 150 mg/ml.

59. The pharmaceutical composition of claim 55, 56, 57, or 58, wherein the pharmaceutically effective carrier contains an aqueous liquid composition at a pH of from about 5.5 to about 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine , about 8 mM methionine, about 0.02% by volume polysorbate 80 and about 0.05 mM EDTA.

60. The pharmaceutical composition of 59, wherein the pH is about 6.0 and the antibody is at a concentration of about 150 mg/ml.

61. Pharmaceutical composition containing:

a) an antibody comprising a heavy chain and a light chain, the heavy chain comprising 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.

62. Pharmaceutical composition according to 61, containing 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 carboxy terminus of the variable region heavy chain.

63. The pharmaceutical composition of 62, wherein the antibody has a concentration of from about 75 mg/ml to about 150 mg/ml.

64. The pharmaceutical composition of claim 61, 62, or 63, wherein the pharmaceutically effective carrier contains an aqueous liquid composition at a pH of from about 5.5 to about 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine,

- 42 046656 approximately 0.02% by volume polysorbate 80 and approximately 0.05 mM EDTA.

65. The pharmaceutical composition of 64, wherein the pH is about 6.0 and the antibody is at a concentration of about 150 mg/ml.

66. Pharmaceutical composition containing:

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.

67. The pharmaceutical composition of 66, wherein the antigen binding protein is at a concentration of from about 75 mg/ml to about 150 mg/ml.

68. A pharmaceutical composition in which the pharmaceutically effective carrier contains an aqueous liquid composition at a pH of from about 5.5 to about 6.0, containing about 40 mm histidine, about 180 mm trehalose, about 100 mm arginine, about 8 mm methionine, about 0 .02% by volume polysorbate 80 and approximately 0.05 mM EDTA.

69. The pharmaceutical composition of 68, wherein the pH is about 6.0 and the antigen binding protein is at a concentration of about 150 mg/ml.

70. A method of treating a disease in a patient, including the steps:

a) identification of a subject having 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, sub-eosinophilic asthma , chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis; And

b) administering to the subject a therapeutically effective amount of 1, 2, 3, 4, 5, or 40 antigen binding protein;

thereby providing treatment for the disease in the subject.

71. The method of 70, wherein the amount of antigen binding protein is from about 2 to about 600 mg.

72. The method of 71, wherein the antigen binding protein is administered once every 3 months or once every 6 months.

73. The method of 70, 71, or 72, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

74. A method of treating a disease in a patient, including the steps:

a) identification of a subject having 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, sub-eosinophilic asthma , chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis; And

b) administering to the subject a therapeutically effective amount of antibody 6, 7, 8, 9, 10, 11, 42, 44, 46, and 48;

thereby providing treatment for the disease in the subject.

75. The method of 74, wherein the amount of antibody is from about 2 to about 600 mg.

76. The method of 75, wherein the antibody is administered once every 3 months or once every 6 months.

77. The method of 74, 75, or 76, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

78. A method of treating a disease in a patient, including the steps:

a) identifying a subject having 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, sub-eosinophilic asthma , chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis; And

b) administering to the subject a therapeutically effective amount of composition 49, 50, 51, 52, 53, or 54;

thereby providing treatment for the disease in the subject.

79. The method of 78, wherein the amount of the composition provides a dose of from about 2 mg to about 600 mg of antigen binding protein.

80. The method of 79, wherein the composition is administered once every 3 months or once every

- 43,046,656 months.

81. The method of claim 78, 79, or 80, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

82. A method of treating a disease in a patient, including the steps:

a) identifying a subject having 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, sub-eosinophilic asthma , chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis; And

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

thereby providing treatment for the disease in the subject.

83. The method of 82, wherein the amount of the composition provides a dose of the antigen binding protein from about 2 mg to about 600 mg.

84. The method of 83, wherein the antibody is administered once every 3 months or once every 6 months.

85. The method of 82, 83, or 84, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

86. A method of treating mild to moderate asthma in a subject, comprising the steps of:

a) identification of a subject diagnosed with mild to moderate asthma; And

b) administering to a subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 a CDR amino acid sequence shown in SEQ ID NO: 8, a CDR amino acid sequence shown in SEQ ID NO: 9, and a CDR amino acid sequence shown in SEQ ID NO: 10;

thus providing treatment for mild to moderate asthma.

87. The method of 86, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21.

88. The method of 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, wherein the amino terminus of the heavy chain Fc domain is connected to the carboxy terminus variable region of the heavy chain.

89. The method of 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.

90. The method of 89, wherein the dose of the antibody is from about 2 mg to about 600 mg.

91. The method of 90, wherein the antibody is administered once every 3 months or once every 6 months.

92. The method of 90, wherein the antibody is administered subcutaneously.

93. The method of claim 86, 87, 88, 89, 90, 91, or 92, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

94. A method of treating mild to moderate asthma in a subject, comprising the steps of:

a) identification of a subject diagnosed with mild to moderate asthma; And

b) administering to a subject a therapeutically effective amount of 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;

thus providing treatment for mild to moderate asthma.

95. The method of 94, wherein the dose of the antibody is from about 2 mg to about 600 mg.

96. The method of 95, wherein the antibody is administered once every 3 months or once every 6 months.

97. The method of 96, wherein the antibody is administered subcutaneously.

98. The method of claim 94, 95, 96, or 97, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

99. A method of treating mild to moderate asthma in a subject, comprising the steps of:

a) identification of a subject diagnosed with mild to moderate asthma; And

b) administering to the subject a therapeutically effective amount of a pharmaceutical composition, with

- 44 046656 containing 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 a pharmaceutically effective carrier;

thus providing treatment for mild to moderate asthma.

100. The method of 92, wherein the pharmaceutically effective carrier contains an aqueous liquid composition at a pH of from about 5.5 to about 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by volume polysorbate 80 and approximately 0.05 mM EDTA.

101. The method of 100, wherein the dose of the antibody is from about 2 mg to about 600 mg.

102. The method of 101, wherein the pharmaceutical composition is administered once every 3 months or once every 6 months.

103. The method of 102, wherein the pharmaceutical composition is administered subcutaneously.

104. The method of claim 99, 100, 101, 102, or 103, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

105. A method for treating severe asthma in a patient, comprising the steps of:

a) identification of a subject diagnosed with severe asthma; And

b) administering to a subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 a CDR amino acid sequence shown in SEQ ID NO: 8, a CDR amino acid sequence shown in SEQ ID NO: 9, and a CDR amino acid sequence shown in SEQ ID NO: 10;

thus providing treatment for severe asthma.

106. The method of 105, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21.

107. The method of 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, wherein the amino terminus of the heavy chain Fc domain is connected to the carboxy terminus variable region of the heavy chain.

108. The method of 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.

109. The method of 108, wherein the dose of the antibody is from about 2 mg to about 600 mg.

110. The method of 109, wherein the antibody is administered once every 3 months or once every 6 months.

111. The method of 110, wherein the antibody is administered subcutaneously.

112. The method of claim 105, 106, 107, 108, 109, 110, or 111, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

113. A method for treating severe asthma in a patient, comprising the steps of:

a) identification of a subject diagnosed with severe asthma; And

b) administering to a subject a therapeutically effective amount of 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: 1;

thus providing treatment for severe asthma.

114. The method of 113, wherein the dose of the antibody is from about 2 mg to about 600 mg.

115. The method of 114, wherein the antibody is administered once every 3 months or once every 6 months.

116. The method of 115, wherein the antibody is administered subcutaneously.

117. The method of claim 113, 114, 115, or 116, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

118. A method for treating severe asthma in a patient, comprising the steps of:

a) identification of a subject diagnosed with severe asthma; And

b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising 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: 1, and a pharmaceutically effective carrier;

thus providing treatment for severe asthma.

119. The method of 118, wherein the pharmaceutically effective carrier comprises an aqueous liquid

- 45 046656 composition at a pH of from about 5.5 to about 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by volume polysorbate 80 and about 0 .05 mM EDTA.

120. The method of 119, wherein the dose of the antibody is from about 2 mg to about 600 mg.

121. The method of 120, wherein the pharmaceutical composition is administered once every 3 months or once every 6 months.

122. The method of 121, wherein the pharmaceutical composition is administered subcutaneously.

123. The method of claim 118, 119, 120, 121, or 122, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

124. A method of treating a subject with moderate to severe atopic dermatitis, comprising the steps of:

a) identification of an entity having at least one of the characteristics selected from the group consisting of:

i) diagnosis of atopic dermatitis according to the criteria of Hanifin and Reick, revised by Eichenwald;

ii) a provisional diagnosis of atopic dermatitis made more than two years ago or approximately two years before the start of treatment;

iii) an overall health care provider score of three or more;

iv) atopic dermatitis lesions greater than or equal to approximately 10% of the body surface area;

v) an index of severity and area affected by eczema greater than or equal to 16;

vi) an absolute blood eosinophil count greater than or equal to 150 cells per μL, greater than or equal to 200 cells per μL, greater than or equal to 300 cells per μL, or greater than or equal to 350 cells per μL;

and vii) at least one pre-treatment condition selected from the group consisting of: 1) an inadequate response for six months or more to a topical atopic dermatitis medication; 2) poor tolerance to topical medications for atopic dermatitis; 3) a side effect from taking topical atopic dermatitis medication; and 4) inadequate response to non-pharmacological treatment for atopic dermatitis; And

b) administering to a subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 a CDR amino acid sequence shown in SEQ ID NO: 8, a CDR amino acid sequence shown in SEQ ID NO: 9, and a CDR amino acid sequence shown in SEQ ID NO: 10;

thereby providing treatment for atopic dermatitis in the subject.

125. The method of 124, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21.

126. The method of 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, wherein the amino terminus of the heavy chain Fc domain is connected to the carboxy terminus variable region of the heavy chain.

127. The method of 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.

128. The method of 127, wherein the dose of the antibody is from about 2 mg to about 600 mg.

129. The method of 128, wherein the antibody is administered once every 3 months or once every 6 months.

130. The method of 128, wherein the antibody is administered subcutaneously.

131. The method of 128, wherein the antibody is administered intravenously.

132. A method of treating a subject with moderate to severe atopic dermatitis, comprising the steps of:

a) identification of a subject having at least one of the characteristics selected from the group consisting of:

i) diagnosis of atopic dermatitis according to the criteria of Hanifin and Reick, revised by Eichenwald;

ii) a provisional diagnosis of atopic dermatitis made more than two years ago or approximately two years before the start of treatment;

iii) an overall health care provider score of three or more;

- 46 046656 iv) atopic dermatitis lesions greater than or equal to approximately 10% of the body surface area;

v) an index of severity and area affected by eczema greater than or equal to 16;

vi) an absolute blood eosinophil count greater than or equal to 150 cells per μL, greater than or equal to 200 cells per μL, greater than or equal to 300 cells per μL, or greater than or equal to 350 cells per μL;

and vii) at least one pre-treatment condition selected from the group consisting of: 1) an inadequate response for six months or more to a topical atopic dermatitis medication; 2) poor tolerance to topical medications for atopic dermatitis; 3) a side effect from taking topical atopic dermatitis medication; and 4) inadequate response to non-pharmacological treatment for atopic dermatitis; And

b) administering to a subject a therapeutically effective amount of 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: 1;

thereby providing treatment for atopic dermatitis in the subject.

133. The method of 132, wherein the dose of the antibody is from about 2 mg to about 600 mg.

134. The method of 133, wherein the antibody is administered once every 3 months or once every 6 months.

135. The method of 134, wherein the antibody is administered subcutaneously.

136. The method of claim 132, 133, 134, or 135, wherein the subject has an absolute eosinophil count selected from the group consisting of 200 cells per μL or more and 150 cells per μL or more, 200 cells per μL or more and 350 cells per μL or more. µl or more.

137. A method of treating a subject with moderate to severe atopic dermatitis, comprising the steps of:

a) identification of an entity having at least one of the characteristics selected from the group consisting of:

i) diagnosis of atopic dermatitis according to the criteria of Hanifin and Reick, revised by Eichenwald;

ii) a provisional diagnosis of atopic dermatitis made more than two years ago or approximately two years before the start of treatment;

iii) an overall health care provider score of three or more;

iv) atopic dermatitis lesions greater than or equal to approximately 10% of the body surface area;

v) an index of severity and area affected by eczema greater than or equal to 16;

vi) an absolute blood eosinophil count greater than or equal to 150 cells per μL, greater than or equal to 200 cells per μL, greater than or equal to 300 cells per μL, or greater than or equal to 350 cells per μL;

and vii) at least one pre-treatment condition selected from the group consisting of: 1) an inadequate response for six months or more to a topical atopic dermatitis medication; 2) poor tolerance to topical medications for atopic dermatitis; 3) a side effect from taking topical atopic dermatitis medication; and 4) inadequate response to non-pharmacological treatment for atopic dermatitis; And

b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising 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: 1, and a pharmaceutically effective carrier;

thereby providing treatment for atopic dermatitis in the subject.

138. The method of 137, wherein the pharmaceutically effective carrier contains an aqueous liquid composition at a pH of from about 5.5 to about 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by volume polysorbate 80 and approximately 0.05 mM EDTA.

139. The method of 138, wherein the dose of the antibody is from about 2 mg to about 600 mg.

140. The method of 139, wherein the pharmaceutical composition is administered once every 3 months or once every 6 months.

141. The method of 140, wherein the pharmaceutical composition is administered subcutaneously.

142. The method of 137, 138, 139, 140, or 141, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

143. A method for reducing the absolute number of eosinophils in the blood of a subject, comprising the steps of:

a) identifying a subject having a condition selected from the group consisting of asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, eosinophilic asthma

- 47 046656 moderate, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis and atopic dermatitis; And

b) administering to a subject a therapeutically effective amount of an antibody comprising a heavy chain variable region having the CDR amino acid sequence shown in SEQ 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 a CDR amino acid sequence shown in SEQ ID NO: 8, a CDR amino acid sequence shown in SEQ ID NO: 9, and a CDR amino acid sequence shown in SEQ ID NO: 10;

thereby reducing the subject's absolute blood eosinophil count.

144. The method of 143, wherein the heavy chain variable region further comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21.

145. The method of 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, wherein the amino terminus of the heavy chain Fc domain is connected to the carboxy terminus variable region of the heavy chain.

146. The method of 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.

147. The method of 146, wherein the dose of the antibody is from about 2 mg to about 600 mg.

148. The method of 147, wherein the antibody is administered once every 3 months or once every 6 months.

149. The method of 148, wherein the antibody is administered subcutaneously.

150. The method of 142, 144, 145, 146, 147, 148, or 149, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

151. The method according to 142, 144, 145, 146, 147, 148 or 149, further comprising the steps:

a) taking a first measurement of the absolute number of eosinophils in the subject's blood;

b) performing a second measurement of the absolute number of eosinophils in the blood of the subject after administering to the subject a therapeutically effective amount of antigen binding protein; And

c) comparison of the first measurement and the second measurement.

152. The method according to 142, 144, 145, 146, 147, 148 or 149, further comprising the steps:

a) taking a first measurement of the absolute number of eosinophils in the subject's blood;

b) performing a second measurement of the absolute number of eosinophils in the blood of the subject after administering to the subject a therapeutically effective amount of antigen binding protein; And

c) comparing the first measurement and the second measurement; wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

153. A method for reducing the absolute number of eosinophils in the blood of a subject, comprising the steps of:

a) identifying a subject having 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 granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis; And

b) administering to a subject a therapeutically effective amount of 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: 1;

thereby providing treatment for atopic dermatitis in the subject.

154. The method of 153, wherein the dose of the antibody is from about 2 mg to about 600 mg.

155. The method of 154, wherein the antibody is administered once every 3 months or once every 6 months.

156. The method of 155, wherein the antibody is administered subcutaneously.

157. The method of claim 154, 155, 156, or 157, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

158. The method of claim 154, 155, 156, or 157, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

159. The method according to 154, 155, 156 or 157, further comprising the steps:

- 48 046656

a) taking a first measurement of the absolute number of eosinophils in the subject's blood;

b) performing a second measurement of the absolute number of eosinophils in the blood of the subject after administering to the subject a therapeutically effective amount of antigen binding protein; And

c) comparison of the first measurement and the second measurement.

160. The method according to 154, 155, 156 or 157, further comprising the steps:

a) taking a first measurement of the absolute number of eosinophils in the subject's blood;

b) performing a second measurement of the absolute number of eosinophils in the blood of the subject after administering to the subject a therapeutically effective amount of antigen binding protein; And

c) comparing the first measurement and the second measurement; wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

161. A method for reducing the absolute number of eosinophils in the blood of a subject, comprising the steps of:

a) identifying a subject having 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 granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis; And

b) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising 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: 1, and a pharmaceutically effective carrier;

thereby reducing the subject's absolute blood eosinophil count.

162. The method of 161, wherein the pharmaceutically effective carrier contains an aqueous liquid composition at a pH of from about 5.5 to about 6.0, containing about 40 mM histidine, about 180 mM trehalose, about 100 mM arginine, about 8 mM methionine, about 0.02% by volume polysorbate 80 and approximately 0.05 mM EDTA.

163. The method of 162, wherein the dose of the antibody is from about 2 mg to about 600 mg.

164. The method of 163, wherein the pharmaceutical composition is administered once every 3 months or once every 6 months.

165. The method of 164, wherein the pharmaceutical composition is administered subcutaneously.

166. The method of claim 162, 163, 164, 165, or 166, wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

167. The method according to 162, 163, 164, 165 or 166, further comprising the steps of:

a) taking a first measurement of the absolute number of eosinophils in the subject's blood;

b) performing a second measurement of the absolute number of eosinophils in the blood of the subject after administering to the subject a therapeutically effective amount of the antigen binding protein; And

c) comparison of the first measurement and the second measurement.

168. The method according to 162, 163, 164, 165 or 166, further comprising the steps of:

a) taking a first measurement of the absolute number of eosinophils in the subject's blood;

b) performing a second measurement of the absolute number of eosinophils in the blood of the subject after administering to the subject a therapeutically effective amount of the antigen binding protein; And

c) comparison of the first measurement and the second measurement; wherein the subject has an absolute blood eosinophil count selected from the group consisting of 200 cells per μL or more and 350 cells per μL or more.

169. The composition according to any of 1-11, 40, 42, 44, 46, 47 or 49-69 for use in treatment.

170. Composition according to any of 1-11, 40, 42, 44, 46, 47 or 49-69 for use in the treatment of asthma, mild asthma, moderate asthma, severe asthma, mild eosinophilic asthma, moderate eosinophilic asthma, severe eosinophilic asthma asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis.

Examples

Example 1.

Kinetic analysis of 28Y042-7F11-1.

Kinetic analysis was performed to compare 28Y042-7F11-1, mepolizumab and GSK3559090A (comparator anti-1L-5 mAb IgG1 molecule). The results are summarized in table. 16 below. Due to the high affinity of the 28Y042-7F11-1 antibody, accurate determination of the dissociation rate using the Biacore 4000 apparatus at 25°C was not possible; therefore, to calculate the exact type

- 49 046656 binding of 28Y042-7F11-1 to human IL-5 at 25°C used the KINEXA assay. The affinity of 28Y042-7F11-1 for human IL-5 at 25°C by KINEXA solution phase affinity assay was 10.47 pM (95% confidence interval 0.88 pM - 31.97 pM). This affinity was compared with the binding affinity of mepolizumab to human IL-5 of 122.8 pM (determined using BIACORE 4000 at 25°C).

The reference antibody GSK3559090A was assayed by BIACORE 4000 for binding to human IL-5 at 25°C, yielding a binding affinity (KD) value of 16.4 pM. GSK3559090A has a 10-fold higher KD value for human IL-5 than mepolizumab, driven primarily by the 20-fold higher rate of association (Ka) observed with GSK3559090A compared to mepolizumab.

The affinity for human IL-5 and cynomolgus IL-5 of 28Y042-7F11-1 at 37°C was determined using BIACORE T200. Higher temperature increased the dissociation rate (kd) of 28Y0427F11-1 so much that it was at the limit of the instrument's measurement range. The affinity of 28Y042-7F111 for human and cynomolgus IL-5 at 37°C was 39.13 pM and 23.93 pM, respectively.

Comparative analysis performed on the FORTEBIO OCTET RED384 BLI showed that 28Y0427F11-1 competes with mepolizumab for binding to human IL-5.

Table 16

Brief information on _KD kinetics. (n.a. - not defined)

Mepolizumab 28Y042-7F11-1

GSK3559090A

Biacore 4000 25°С (K _D , pM) Kinexa 25°С (K _D , pM) Biacore T200 37°С (K _D , pM) Biacore T200 37°С (K _D , pM) human IL-5 human IL-5 human IL-5 Cynomolgus IL-5 122.8 But. But. But. But. 10.5 39.1 23.9 16.4 But. But. But.

Binding to human Fc receptors.

28Y042-7F11-1 has approximately 13-fold higher affinity for human FcRn at pH 6.0 compared to mepolizumab (157 nM and 2082 nM, respectively), and also binds with low affinity at pH 7.4; the affinity is 16 µM at pH 7. _KD values determined using

BIACORE T200 at 37°C are given in table. 17. Table 17: Comparison of binding affinities of 28Y0427F11-1 and mepolizumab to human FcRn at pH 6.0 and pH 7.4.

Table17

Analysis of 28Y042-7F11-1 binding to human Fc receptor gamma (FcyR) using the PROTEON XPR36 system demonstrated comparability with a control antibody containing

YTE. The binding affinity of these YTE-containing mAbs to FcyR was approximately 1.5-fold lower than that of control wild-type human IgG1. For complement component C1q, the affinity of 28Y042-7F11-1 was also lower than that of wild-type human IgG1 (750 nM and 465 nM, respectively).

In a separate experiment, 28Y042-7F11-1 demonstrated an approximately 3-fold increase in affinity for the neonatal human receptor at pH 6.0 compared to wild-type human control IgG1 (130 nM and 359 nM, respectively), as well as low binding affinity for FcRn at pH 7.4 (2650 nM), whereas the wild-type control antibody had no binding at this pH (Table 18). These results are comparable to those of the control YTE used in the experiment. Table 18 shows the binding results of 28Y042-7F11-1 to the recombinant human neonatal receptor (FcRn) obtained using the PROTEON device. The wild-type isotype control and Fc-deactivated isotype control are derived from a non-functional (with respect to CDR binding) antibody originally produced against coagulation factor IX(F9). The Fc-deactivated isotype control contains two point mutations in the Fc region (L235 and G237, both mutated to alanine) that reduce interaction with Fc gamma receptors.

- 50 046656

Table 18

_Kd (hM) tion cle Fun Oce Antibodies human FcRn, pH 6.0 human FcRn, pH 7.4 28Y042-7F11-1 GSK2800528 control ISOTYPE IgGi wild type control ISOTYPE IgGi with deactivated Fc functional test for culturing ability 28Y042- 130.0 94.3 359.0 314.0 e TF-1 cells. 7F11-1 inhibit dose 2650.0 1190.0 N.B. N.B. proliferator-dependent manner

TF-1 current mediated by human IL-5. TF-1 cells are an erythroleukemic cell line that was generated to proliferate in response to human and cynomolgus IL-5 stimulation.

Analysis of the effect of 28Y042-7F11-1 in a TF-1 cell proliferation assay showed that this antibody is a potent inhibitor of IL-5-mediated TF-1 cell proliferation. 28Y042-7F11-1 and mepolizumab both caused dose-dependent inhibition of human IL-5-induced TF-1 cell proliferation when tested over a concentration range of 1 nM to 0.042 pM (IC ₅₀ 4 pM and 105 pM, respectively, Fig. 1). This represents an approximately 30-fold improvement in the efficacy of 28Y042-7F11-1 compared to mepolizumab, based on cell-based assays.

28Y042-7F11-1 and mepolizumab both caused dose-dependent inhibition of human IL-5-induced TF-1 cell proliferation when tested over a concentration range of 1 nM to 0.042 pM (IC ₅₀ 0.004 nM and 0.105 nM, respectively).

Further analysis of 28Y042-7F11-1 was performed after subjecting the molecule to thermal stress by incubation for 1 week at 40°C in acetate or phosphate buffer. Under these conditions, the IC ₅₀ value for 28Y042-7F11-1 in the TF-1 cell assay ranged from 4 pM to 5 pM, showing values similar to those obtained for 28Y042-7F11-1 under non-stress control conditions.

Analysis of eosinophil shape changes and binding to endogenous IL-5.

The ability of 28Y042-7F11-1 to inhibit IL-5-mediated eosinophil shape change in human whole blood was assessed. Both mepolizumab and 28Y042-7F11-1 (10 μg/ml) demonstrated inhibition of eosinophil shape changes mediated by recombinant IL-5 (10 ng/ml), while control antibodies, pascolizumab and anti-RSV (with deactivated Fc ), failed to prevent IL-5-mediated eosinophil shape changes (Fig. 2).

The ability of 28Y042-7F11-1 and mepolizumab (both at a concentration of 1 μg/ml) to bind native IL5 from the supernatant of CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) was assessed by ELISA. 28Y042-7F11-1 and mepolizumab bound native IL-5, whereas the control antibody pascolizumab showed no binding. Although the assay demonstrated binding to native IL-5, it was not further optimized to determine accurate EC ₅₀ values (Figure 3).

In vitro stability of 28Y042-7F11-1 in human and cynomolgus macaque serum by immunoassay.

The ability of 28Y042-7F11-1 to bind recombinant IL-5 was assessed after incubation at 37°C for 6 weeks in pooled human or cynomolgus control serum (Fig. 4). After incubation, samples were tested using an MSD immunoassay in which the remaining active 28Y042-7F11-1 was coupled to immobilized biotinylated IL-5 and then detected using a labeled anti-human Fc-specific monoclonal antibody reagent. In both human and cynomolgus serum, the release of active 28Y042-7F11-1 showed a gradual decrease in _T0 concentration over time, which after 6 weeks reached values of 73.1% and 77.1%, respectively. These data are comparable to older data obtained for mepolizumab (51.0% [human] and 64.2% [cynomolgus]) and a control antibody specific for IL-13, which is known to have poor stability in this assay (27.9% [human] and 24.9% [cynomolgus]). The results of this study demonstrated that the in vitro stability of 28Y042-7F11-1 in human and cynomolgus macaque serum compares favorably with values obtained for mepolizumab in past studies and suggests that 28Y042-7F11-1 behaves as expected for a typical monoclonal antibody.

- 51 046656

Identification of PK/PD 28Y042-7F11-1 in the cynomolgus macaque.

A 9-month non-GLP in vivo pharmacokinetic/pharmacodynamic (PK/PD) study was conducted involving 4 groups of cynomolgus monkeys (Macaca flavicularis), each group consisting of 2 males and 2 females. On day 1, animals received an intravenous bolus injection of 28Y042-7F11-1 (0.05 mg/kg or 1 mg/kg), mepolizumab (1 mg/kg), or vehicle. In addition to determining the PK parameters of the injected antibodies, two PD parameters were assessed to compare the in vivo activity of 28Y042-7F11-1 and mepolizumab: a) the level and duration of eosinophil suppression; b) level and time of maintenance of total IL-5 concentration in serum.

Rating PK 28Y042-7F11-1.

PK assessment was performed on cynomolgus serum samples up to 5376 h (week 32). The analysis showed that the rate of clearance of 28Y042-7F11-1 from serum is 1.8 times lower than that of mepolizumab (0.105 ml/hour/kg versus 0.185 ml/hour, respectively), and 28Y042-7F11-1 has an improved period half-life, 24.5 days, compared with 11.5 days for mepolizumab (Table 19). In table Table 19 shows the pharmacokinetic parameters determined in studies to determine PK in cynomolgus monkey samples (*median value).

Table 19

Group Animal AUC AUC _inf G '-chpah _tah Period semi- M.R. C.I. ^VvSS brought out nya T (H'MKg/ml) (H'MKg/ml) (µg/ml) (h) (h) (h) (ml/h/ Kg) (ml/kg) 28Y042- 7F11-1 (0.05 mg/kg) IV 301 493 542 1.30 0.25 598 672 0.092 3 80.1 302 394 467 1.20 3.00 501 447 0.107 74.8 351 427 556 1.23 0.25 649 484 0.089 9 81.1 352 515 573 1.41 0.25 614 630 0.087 3 75.1 average 257 534 1.29 0.25* 590 558 0.094 2 77.8 28Y042- 7F11-1 (1 mg/kg) IV 401 9930 9950 24.1 0.25 543 735 0.100 75.1 402 9400 9430 22.6 0.25 616 748 0.106 81.3 451 9840 9870 23 0.25 558 745 0.101 77.0 452 9020 9070 24 0.25 603 749 0.110 85.3 average 9550 9580 23.4 0.25 580 744 0.105 79.7 Mepolizu mab (1 mg/kg) IV 201 5140 5190 27.7 0.25 304 374 0.193 76.0 202 5790 5850 28.9 0.25 307 400 0.171 71.9 251 5060 5160 30.4 0.25 242 302 0.194 63.8 252 5390 5540 28.8 0.25 253 308 0.181 62.2 average 5350 5430 29.0 0.25 276 346 0.185 68.5

28Y042-7F11-1-mediated suppression of eosinophils.

Reduction in eosinophil count was used as a biomarker for 28Y042-7F11-1-mediated neutralization of IL-5 activity. 28Y042-7F11-1 exhibits in vivo increased duration of eosinophil suppression compared with mepolizumab in cynomolgus monkeys (Fig. 5). At a dose of 28Y042-7F11-1 that is 1/20 the dose of mepolizumab, it exhibits equivalent or slightly improved eosinophil suppression, indicating that 28Y042-7F11-1 is at least 20-fold more active in vivo compared to mepolizumab. At 24 weeks, predose blood eosinophil counts were <50% of predose 28Y042-7F11-1 values at 1 mg/kg compared with 4 to 7 weeks for mepolizumab at the same dose. Eosinophil counts in

- 52 046656 blood began to recover at approximately week 7 for 28Y042-7F11-1 and at week 4 for mepolizumab. These data suggest that 3-monthly dosing in humans is achievable and that dosing every six months is also possible.

Serum total IL-5 levels.

Levels of total IL-5 (IL-5 found almost exclusively in complex with 28Y042-7F11-1 or mepolizumab) were observed to increase post-dose and remained more stable in the two 28Y042-7F11-1 groups compared with the mepolizumab, in which the level of the IL-5 complex began to decrease much earlier (Fig. 6). Reductions were observed after 29 study days (672 hours) in the group receiving 1 mg/kg mepolizumab, after 85 study days (2016 hours) in the group receiving 0.05 mg/kg 28Y042-7F11-1, and after 113 study days ( 2688 hours) in the group receiving 1 mg/kg 28Y042 -7F11-1. This suggests that mepolizumab has a lower affinity for IL-5 and a shorter half-life compared to 28Y042-7F11-1, which is complexed with IL-5 for a longer period of time.

28Y042-7F11-1 was also studied in a non-compartmental pharmacokinetic analysis in which 28Y042-7F11-1 was administered as a single intravenous and subcutaneous dose. When administered intravenously, GSK3511294 demonstrated an increased serum half-life compared with mepolizumab (24 days versus 11.5 days) as well as a 1.8-fold decrease in serum clearance (Figure 4).

Methods: functional test on TF-1 cell culture.

Antibody samples and controls were prepared in 96-well polypropylene plates for initial screening. Antibodies were diluted in cell culture medium to a final assay concentration of 200 nM, then sterile filtered using a filter plate (Pall Corporation Multiwell Plates, ACRO PREP 96 Filter Plate, 3.0 µm glass fiber media/0.2 µm BIO-INERT membrane, No. 5053). Samples were serially diluted 1 in 4 throughout the plate to produce a 10-point series over a concentration range (200 nM-7.63 pM). The goal was to obtain a dose-response curve for a single dose for the initial assessment of IC ₅₀ values for the variants. Human IL-5 cytokine (homodimer molecular weight, 28.5 kDa) for cell stimulation was diluted in cell culture medium and prepared at a final assay concentration of 17.5 pM (0.5 ng/ml), EC ₈₀ for human IL stimulation -5 in analysis. Human IL-5 was added to a plate containing a series of antibody dilutions and incubated for 1 hour at room temperature. TF1 cells were washed 3 times with PBS to successfully remove the growth factor, GMCSF (granulocyte macrophage colony stimulating factor), used for cell propagation. Cells were seeded into white, flat-bottomed 96-well tissue culture plates at a density of 0.2 x 106 cells/well. The pre-incubated antibody and cytokine were then added to the cells and further incubated for 3 days at 37°C, 5% CO ₂ . The plates were removed from the incubator, and 100 μl of CELLTITER-GLO fluorescent reagent (Promega, G7571) was added to the plate wells and incubated for 1 h at room temperature on a plate shaker. The plates were then read on an ENVISION fluorescent plate reader (Biomax 501510).

Alternatively, to evaluate IC ₅₀ values in more detail, the following modifications were made from the method described above. Serial dilutions of antibodies and controls were prepared in 96-well polypropylene plates. Antibodies were diluted to a final assay concentration of 1 nM in cell culture medium and serially diluted 1 in 1.7 throughout the plate to give a concentration range (1 nM - 0.000418 pM). Human cell stimulation cytokine IL-5 was diluted in cell culture medium and prepared at a final assay concentration of 3 pM.

Methods: BIACORE.

Binding to human IL-5 was measured using BIACORE 4000 (GE Healthcare). The sample flow rate in the study was 10 μL/min for binding and regeneration, and 30 μL/min was used for kinetics determinations. Protein A was immobilized on a CM5 S series chip (GE Healthcare, BR-1005-30) via primary amine coupling (GE Healthcare, BR-1000-50). This surface was then used to capture anti-HE-5 antibodies in slots 1 and 5, while slots 2 and 4 were used as comparison. Recombinant human IL-5 was then passed over the captured antibody at 100 nM. A 30-minute dissociation time was used because previous experiments had determined that this time was necessary to accurately determine the dissociation rate of mepolizumab. Two reference curves for the buffer (i.e. 0 nM) were generated and data fitting was performed using BIACORE 4000 software using a 1:1 model for evaluation. The experiment was carried out at 25°C using HBS-EP (Teknova, H8022) as the working buffer and 50 mM NaOH as the regeneration solution.

Analysis of the data showed that the antibody dissociation rates were within the sensitivity of the BIACORE 4000 instrument, and therefore the calculation of accurate dissociation rates at 25°C was not possible. For this reason, the cycle was repeated at 37°C to increase the dissociation rate, allowing accurate dissociation rates to be determined for the antibodies.

- 53 046656

Methods: MSD-SET analysis.

The MSD-SET (MSD Solution Equilibrium Titration) assay was used to determine the affinity of these antibodies for human IL-5 at 25°C because dissociation rates were found to be too slow to be measured by BIACORE at this temperature. MSD-SET determines the solution phase, equilibrium affinity of antibodies. The method is based on the detection of free antigen in an equilibrium state in a titrated series of antibody concentrations.

Biotinylated human IL-5 was used at a constant concentration of 30 pM, while antibody samples were titrated 1 in 3 from 2.5 nM to 0.5 pM with a final dilution of 1:10 at 0.05 pM in a 96-well polypropylene plate. The titrated antibody and IL-5 were incubated for 24 h at room temperature.

After 24 h, antibodies (20 nM in PBS) were applied to standard MSD binding plates (Meso Scale Discovery, L15XA) for 30 min at room temperature. The plates were then blocked with STARTING BLOCK blocking buffer (Thermo Scientific, #37542) for 30 min with shaking at 700 rpm, followed by three washes with wash buffer. The incubated solutions were added to the MSD plates for 150 seconds while shaking at 700 rpm, followed by washing. The antigen immobilized on the plate was detected with SULFOTAG-labeled streptavidin (Meso Scale Discovery, R32AD-1) by incubating on the plate for 3 minutes. The plates were washed three times with wash buffer and then read on an MSD SECTOR IMAGER using 1x Reading Buffer T with Surfactant (Meso Scale Discovery, R92TC-1). The percentage of free antigen was plotted as a function of titrated antibody using GRAPHPAD PRISM software and fitted to a quadratic equation.

Affinity for cynomolgus IL-5 at 25°C was also determined using the MSD-SET assay. The method used was the same as described above, but using cynomolgus IL-5 at a concentration of 62.5 pM.

Methods: KINEXA analysis 28Y042-7F11-1.

To accurately determine the binding affinity of 28Y042-7F11-1 to human IL-5 at 25°C, KINEXA (kinetic exclusion assay) was used as an alternative to the MSD-SET assay, as the 95% confidence intervals obtained for 28Y042-7F11-1 showed poor fit of the data to the model.

Measurement of solution phase affinity was performed using a Sapidyne KINEXA 3200 instrument. The method was based on the detection of free antibody at equilibrium in a titrated series of antigen concentrations. For detection, a particle array was created using SEPHEROSE NHS-activated particles (GE Healthcare, 17-0906-01) coated with human IL-5. To determine affinity, a fixed concentration of antibody was incubated with a series of dilutions of human IL-5 concentrations and allowed to reach equilibrium binding state before measuring samples on a KINEXA 3200 instrument. Each solution was passed through an aliquot of antigen particles, causing free antibody to bind to the antigen-coated particles. and then using an anti-human IgG antibody (DYLIGHT 649-AFFINIPURE F(ab')2 fragment goat anti-human IgG; Jackoson Immunoresearch, 109-496-170) to detect the newly obtained particles with the antigens used to measure each sample. Data were analyzed using the software built into the KINEXA apparatus in several runs performed with different initial concentrations of antibodies that were above and below the expected affinity of the interaction, i.e. 300 pM and 50 pM (concentration- and affinity-dependent interactions, respectively) and with a range of antigen concentrations within a single assay to saturate all available antibodies, leaving almost 100% unbound (10 nM to 4.88 pM for the concentration curve, from 1 nM to 0.49 pM for the affinity curve). Data from multiple runs were pooled and analyzed using n-plot analysis software built into the KINEXA instrument to determine K _D and 95% confidence intervals.

Methods: competitive binding of 28Y042-7F11-1 to IL-5 with mepolizumab.

To determine whether 28Y042-7F11-1 binds to the same epitope on human IL-5 as mepolizumab, a competition assay was performed using a FORTEBIO OCTET RED384 biolayer interferometric instrument (BLI). Since human IL-5 is a dimer, a tandem assay format was used in which biotinylated human IL-5 at a concentration of 5 μg/ml in PBSF buffer was immobilized on 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. The process was repeated with IL-5-surface saturation of 28Y042-7F11-1 followed by mepolizumab and self-binding controls. The analysis was carried out at 25°C with a shaking speed on a shaker of 1000 rpm. Data were analyzed using FORTEBIO data analysis.

Methods: SEC analysis.

Analytical size exclusion chromatography (SEC) was performed to evaluate the purity (% monomer) and retention time of the molecule. Late retention times may indicate potential

- 54 046656 problems associated with the coloring of molecules. SEC was performed on a TSK G3000SWXL, 250A, 5 μm, 30 cm x 7.8 mm column on an AGILENT 1100 HPLC system. Operating conditions were as follows: 200 mM NaH ₂ PO ₄ , 250 mM NaCl, pH 6.0 at a flow rate of 0. 5 ml/min. The load was 20 μg per sample with a recording time of 30 min. The results were analyzed using CHEMSTATION peak integration software.

Methods: Binding of 28Y042-7F11-1 to human Fc receptors assessed using PROTEON.

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

Mouse anti-polyhistidine IgG (ANTI-TETRA-HIS; Qiagen, 34670) was immobilized onto a GLM biosensor chip (Bio-Rad, 176-5012) by coupling to a primary amine (GE Healthcare, BR-1000-50). This surface was used as a capture surface for polyhistidine-labeled human Fc receptor gamma (all reagents produced in-house [except CD64-Fc; R&D Systems, 1257-FC]). Test antibodies were used as the analyte and run at concentrations of 1024 nM, 256 nM, 64 nM, 16 nM and 4 nM, as well as 0 nM (ie, one buffer) used to generate two reference binding curves. Between interactions, the surface of mouse anti-polyhistidine IgG was regenerated with 100 mM phosphoric acid. The experiment was carried out at 25°C using HBS-EP (Teknova, H8022) as the running buffer. Data were analyzed for each receptor separately by setting the global R-max and using the equilibrium model inherent in the PROTEON analysis software.

Methods: Binding of 28Y042-7F11-1 to human complement C1q assessed by PROTEON.

The binding of 28Y042-7F11-1 to human recombinant soluble complement C1q was assessed using the PROTEOn XPR36 biosensor (BioRad™). A control antibody containing a YTE substitution in the Fc region was included as a control.

Test antibodies were immobilized onto a GLC chip (Bio-Rad, 176-5011) by coupling to a primary amine (GE Healthcare, BR-1000-50). Recombinant C1q (Sigma, C1740) was passed over the immobilized antibody at 512 nM, 128 nM, 32 nM, 8 nM, 2 nM and 0 nM (i.e. in one buffer only), and an empty activated and deactivated flow cell was used as two reference binding curves. The running buffer for the binding assay was HBS-EP (pH 7.4, Teknova, H8022) with 10 mM CaCl ₂ . Data were fitted to the equilibrium model inherent in PROTEON XPR36 software (BioRad™) using the global R-max value.

Methods: FcRn binding.

The binding affinities of 28Y042-7F11-1 and mepolizumab to human FcRn at pH 6.0 and pH 7.4 were assessed using a BIACORE T200 instrument at 37°C. Human recombinant IL-5 was diluted in acetate buffer pH 5.0 and immobilized to 535 RU by amine coupling on a CM5 chip (GE, BR100530). 28Y042-7F11-1 or mepolizumab was immobilized by flowing (5 μL/min) over the chip surface a solution of 100 nM of either mAb in HBS-EP buffer (Teknova, H8022) for 24 seconds. After antibody capture with immobilized IL-5, various concentrations (0.5 μM to 32 μM) of human FcRn were cycled over the chip surface at 5 μL/min for a contact time of 120 seconds followed by dissociation for 80 seconds for each cycle. At the completion of each cycle, the chip surface was regenerated using 10 mM glycine, pH 1.5, for 5 s at 50 μL/min, followed by 10 mM NaOH for 5 s at 50 μL/min. FcRn cycling was performed for each concentration at pH 6.0 (HBSEP+, Teknova, Cat:H8022, pH 6.0) and pH 7.4 (HBS-EP). For human FcRn, a molecular weight of 42929 Da was used in the calculations.

Methods: binding to native IL-5.

The ability of 28Y042-7F11-1, mepolizumab and negative control antibodies (pascolizumab & anti-RSV) to bind to native IL-5 was determined. 28Y042-7F11-1, mepolizumab or control antibodies were diluted to 1 µg/ml/200 µl/well in PBS and incubated on a MAXISORP ELISA plate (Nunc, 10394751) overnight at 4°C, then washed (all During the washing steps, 200 µl/well of PBS supplemented with 0.05% Tween 20 was used. The plate was blocked with 300 μl/well of PBS supplemented with 1% BSA (Sigma, A9576) for 2 h, and a supernatant containing 4 ng/ml native IL-5 was added before a 1:2 titration of the culture. The supernatant was incubated with antibodies for 1 hour at room temperature, washed, then 100 μl/well of biotinylated anti-Gk-5 antibody (Fisher, MM550SV) at a concentration of 1 μg/ml, diluted in PBS supplemented with 0, was added to the mixture in the plate. 5% BSA, for 1 hour at room temperature. Streptavidin-HRP (GE Healthcare, RPN4401V)

- 55 046656 was used for detection, diluted 1:5500 in PBS supplemented with 0.5% BSA (100 µl/well), and incubated for 20 min at room temperature, followed by the addition of 100 µl/well of TMB substrate for 5 min, after which the reaction was stopped using 100 μl/well of 1M H ₂ SO ₄ . The absorbance value was measured at 450 nm using a SPECTRAMAX plate reader (Biomax, 088261; all measurements were performed in duplicate). Control conditions were also evaluated using culture supernatant lacking IL-5 or lacking captive antibody (28Y042-7F11-1, mepolizumab, and negative control antibodies).

Native IL-5 was produced by stimulating isolated PBMCs with human anti-CD3 and anti-CD28 antibodies. Blood (100 ml) from a healthy volunteer donor (human) was obtained in sodium heparin (1000 IU/100 ml). Blood was diluted 1:1 with RPMI (Gibco, 31870074) and then separated by density gradient centrifugation to isolate PBMCs using HISTOPAQUE FICOLL and LEUCOSEP tubes (Greiner, 227290) according to the manufacturer's recommendations. After isolation, PBMCs were washed twice with RPMI (centrifugation 2x5 min at 1200 rpm). After a second wash, cells were resuspended in 50 ml RPMI (supplemented with 10% fetal bovine serum, penicillin/streptomycin and L-glutamine), 500 µl was removed from the sample and mixed 1:1 with TRYPLE EXPRESS (Gibco, 12604-021) and analyzed on VICELL to count cell numbers. The plates were precoated with 1 μg/ml anti-CD3 (OKT3 in-house) and 3 μg/ml anti-CD28 (in-house) at 37°C for 60 min, followed by washing the wells once with PBS. Cells were diluted to 1x10 ⁶ /ml, and 200 μl/well (2x105 cells) were added to wells pre-coated with anti-CD3/CD28 and incubated at 37°C with 5% CO ₂ for 4 days. After stimulation, cell supernatants were pooled in a 50-ml Falcon tube and centrifuged (5 min, 1200 rpm). The supernatant (45 ml) was collected into a fresh Falcon tube and the cell clump was discarded. BSA (667 µl from Sigma, A9576) was added to 40 ml of supernatant (0.5% final concentration) and distributed equally between 2 VIVASPIN 20 columns (Sartorius, VS0112), and centrifuged at 3600 g (4500 rpm) for 2x10 min to obtain concentrated supernatant. Enriched supernatant fractions were pooled and stored at 4°C. As a control in the assay, 5 ml of the original supernatant, which was not centrifuged or where BSA was not added, was also stored at 4°C. Unstimulated supernatant samples were centrifuged as described above and stored at 4°C to obtain a control supernatant lacking IL5. IL-5 concentrations were quantified using the QUANTIKINE ELISA assay kit (R&D Systems, D5000B).

Methods: Inhibition of IL-5-mediated eosinophil shape changes using flow cytometry.

This assay was used to measure the inhibition of recombinant IL-5-mediated eosinophil shape changes by 28Y042-7F11-1 or mepolizumab in human whole blood by (all points were performed in duplicate). Blood from healthy volunteer donors (human; with appropriate consent) was obtained in sodium heparin (1000 IU/100 ml) from the GlaxoSmithKline Stevenage Blood Donation Unit. 28Y042-7F11-1, mepolizumab, and control antibodies (pascolizumab and anti-RSV) were diluted to a final assay concentration of 10 μg/mL. Antibodies were incubated with an equal volume of recombinant IL-5 (R&D Systems, Lot # 091231202) at a final concentration of 10 ng/ml at 37°C for 1 hour. After incubation, each 20 μl sample of the antibody/HE-5 complex was added to 80 μl of whole blood obtained from one of six donors in a 96-well polypropylene deep well plate (Fisher Scientific, 10007621). The plate was incubated at 37°C for 30 minutes, after which it was placed on ice and fixed for 2 minutes by adding 250 μl/well of CELL FIX (BD, 340181) diluted 1:9:30 CELL FIX:water:PBS ( this ratio gives a concentration 4 times weaker than the manufacturer's recommendations, which allowed us to provide conditions that do not damage the integrity of eosinophils). Cells were then lysed with ice-cold PHARM LYSE (BioLegend, RBC lysis buffer, 420301) in quantities according to the manufacturer's protocol. Samples were centrifuged and the supernatant was removed before resuspension in FACS buffer and data were obtained by gating eosinophils with CANTO II for their autofluorescence in the PE channel. The effect of 28Y042-7F11-1, mepolizumab, pascolizumab and anti-RSV in the absence of IL-5 was also tested using PBS alone. The effect of IL-5 on eosinophil shape changes was also tested in the absence of either antibody using PBS alone.

Methods: serum stability study.

28Y042-7F11-1 was diluted in either pure pooled sterile human serum (GSK Stevenage Blood Donation Unit) or pure pooled sterile cynomolgus serum (from SeraLabs) to obtain 4 ml of each sample containing 28Y042-7F11-1 at a target concentration of 120 µg/ml. Each serum sample (human or cynomolgus) was then aliquoted into 5 x 750 μL aliquots into sterile 2 mL microcentrifuge tubes and the caps were tightly sealed. One aliquot of each serum was then immediately placed on dry ice and left to freeze to obtain _T0 samples, and then transferred to -80°C storage conditions. The remaining aliquots were placed

- 56 046656 into a humidified tissue culture incubator set at 37°C with 5% CO2. After 1, 2, 4 and 6 weeks, 1 aliquot for each type of serum was removed, frozen on dry ice as above, then transferred to -80°C storage conditions. In vitro stability analysis was completed when the 6-week samples were removed for storage.

Samples obtained for in vitro serum stability assay were tested using IL-5 capture immunoassay in MSD (Meso Scale Discovery) to quantify 28Y0427F11-1 as a function of serum incubation time. By using biotinylated IL-5 as the capture reagent, only molecules with IL-5 activity were captured and then detected, so any change in recovery detected over time represented a loss of 28Y042-7F11-1 activity. All samples were tested together in one assay after completion of the 6-week incubation.

IL-5 uptake immunoassay was performed in 96-well standard MSD binding plates (MSD, #L15XA-6) that were coated with 50 μl of NEUTRAVIDIN (Thermo-Fisher Scientific, #31000) at a concentration of 2 μg/ml diluted in PBS for culture tissue (Sigma-Aldrich, no. D8537). The plates were left overnight at +4°C. Coated plates were washed using an automatic plate washer (Biotek ELx405), with each well washed 3 times with 300 μl of PBS+0.1% Tween-20. After washing, the plates were tapped over a paper towel to remove any remaining 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 h on a plate shaker (Heidolph TITRAMAX 1000) with a shaking speed of approximately 750 rpm (usually used speed), and then washed as before. Biotinylated human IL5 (GSK reagent) was diluted to 100 ng/ml in assay buffer, and 25 μl of this solution was added to all wells of blocked and washed plates. The plates were then incubated at room temperature for at least 1 hour on a shaker and then washed as previously described. During incubation with biotinylated IL-5, a standard curve was generated for 28Y042-7F11-1 and test samples. The standard curve for 28Y042-7F11-1 was prepared by diluting to a maximum concentration of 250 ng/ml in assay buffer. Serial dilution was then performed at a dilution factor of 2.5 for a total of 11 dilution points, with the 12th point being assay buffer only as a blank control. All serum samples tested for stability were diluted in assay buffer at dilution factors of 2000, 20,000, and 200,000.

Each pure sample was diluted using a minimum of 20 μl between dilutions and using serial dilutions not exceeding a dilution factor of 10 (i.e., a dilution factor of 2000 was obtained from 3 serial 10-fold dilutions followed by a 2-fold dilution). After incubation with biotinylated IL-5 was completed and the plates were washed, 25 μl of 28Y042-7F11-1 was added in triplicate, followed by 25 μl of each test sample at each dilution in duplicate. The plates were then incubated at room temperature for at least 1 hour on a plate shaker and washed as before. To detect bound 28Y042-7F11-1, mouse monoclonal anti-human Fc SULFOTAG (labeled with an 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 at room temperature for at least 1 hour on a plate shaker and washed as before. Buffer T for MSD reading with surfactant was diluted with distilled water to obtain a 1x working solution, 150 μl of which was then added to all wells. The plates were then read using a SECTOR 6000 MSD scanner. The resulting mean concentrations of 28Y042-7F11-1 measured in human or cynomolgus serum were normalized to the % concentration determined in _T0 samples using the formula:

%T ₀ = [concentration in the test sample/concentration in the sample T ₀ ]*100

To compare the serum stability data obtained for 28Y042-7F11-1, they were plotted together with data obtained previously for mepolizumab and for the control antibody specific for IL-13. The established serum stability used for these molecules was identical to that described above, except that time points of 0, 2, 4 and 6 weeks were used and the batch of human and cynomolgus serum used was different. Mepolizumab samples were analyzed similarly to those described above, except that the mepolizumab standard curve was used, which started at 500 ng/mL, and samples were tested using only a dilution factor of 1000. Analysis of control IL-13-specific antibody samples was performed same as described above, except that the antibody standard curve started at 500 ng/ml, samples were tested using only a dilution factor of 1000, and for the data presented herein, an IL-13 capture assay was used in which instead of biotinylated IL-5, biotinylated IL-13 (GSK internal reagent) was used at a concentration of 100 ng/ml in assay buffer. Data were normalized to the value at T0 as described above.

Methods: Cynomolgus monkey PK/PD in vivo.

The study included 4 groups of cynomolgus macaques (Masaca flavicularis, 2-5 years old, weight 2-6 kg, specially bred naive of Mauritian origin), each of which consisted of 2 males and 2 females. Cynomolgus macaques were housed in pens of 4 animals of the same sex per cage with an enriched environment to promote social interaction, play and learning. During the study, each animal had access to an average of 200 g/day of standard diet (PMI Nutrition International Certified Primate Diet No. 5S48 (25% protein) and Special Diets Services (SDS) Mazuri Expanded Short (MP(E)short SQC) ) throughout the study and to water without restriction. Total hematologic cell counts, including eosinophil counts, were performed predose and then every 1 or 2 weeks postdose (for 6 months). Animals were given an intravenous bolus injection of 28Y042-7F11-1 (0.05 mg/kg or 1 mg/kg), mepolizumab (1 mg/kg) or vehicle on day 1. In addition to hematological cell counts, the RA of test substance and total IL were also measured -5.

Methods: data from the Republic of Kazakhstan.

The animals were administered the test substance on day 1 and samples were collected (see Table 20 and Table 21) by drawing blood from the femoral vein without the addition of an anticoagulant. Sample collection was performed as late in the afternoon as possible (between 1 and 3 pm) to coincide with the hematology (eosinophil) blood collection schedule. Samples were allowed to coagulate for at least 1 hour at ambient temperature and then centrifuged at 2500 g for 10 minutes at 4°C. The resulting serum was separated, transferred into standard Sarstedt tubes with unique labeling and immediately frozen on dry ice, then stored at -80°C.

The concentrations of 28Y042-7F11-1 and mepolizumab in cynomolgus serum samples were determined by immunoassay using the GYROLAB Work Station platform (Gyros, P0004943). Biotinylated recombinant human IL-5 for capture (lab-produced reagent) and 28Y042-7F11-1 standards (in cynomolgus serum) were diluted in REXXIP A buffer (Gyros, P0004820), and ALEXA-647-labeled anti-human IgG for detection (clone JDC-10) was diluted in REXXIP F buffer (Gyros, P0004825). The assay was validated in the range of 30-10,000 ng/ml for 28Y0427F11-1 and 100-10,000 ng/ml for mepolizumab on CD BIOAFFY 1000 (Gyros, P0004253). Serum concentration values for 28Y042-7F11-1 were within the expected range. In table Figure 20 shows a graph of cynomolgus monkey blood sampling for PK and total IL-5 analysis. The blood volume for extraction was 0.7 ml (0.5 ml where there is an *). In table 21 shows sample collection schedules for hematological analysis.

Table 20

Time points for blood sampling (Time post-dose in hours) from the end of injection on day 1; [Day No.] 0.25 h [1] 1h [1] Zh[1] 6h [1] 24h [2] 48h [3] 96h [5] 168h [8] 336h [15] 672h [29] 1008h [43] 1344h [57] 1680h [71]* 2016h [85] 2688h [FROM] 3360h [141] 4032h [169] 4368h [183] 4704h [197] 5040h [2I] 5376h [255] 5712h [239] 6048h [253] 6384h [267] Table 21 Time points for blood sampling (Time post-dose in hours) from the end of injection on day 1; [Day No.] 25h [2] 168h [8] 336h [15] 672h [29] 1008h [43] 1176h [50] 1344h [57] 1512h [64] 1680h [71] 1848h [78] 2016h [85] 2184h [92] 2376h [YuO] 2520h [Sub] 2688h [FROM] 3024h [127] 3360h [141] 3696h [155] 4032h [169] 4368h [183] 4704h [197] 5040h [211] 5376h [225] 5712h [239] 6048h [253] 6384h [267]

Methods: Data regarding total IL-5 levels.

Animals were dosed and blood samples were collected and processed as described for PK data collection. Standard curves of cynomolgus IL-5 (final concentration 2.44-10,000 pg/ml) were prepared at a serial dilution of 1 in 4 with ×2 final concentration in pooled cynomolgus serum (SeraLab, S-118-D). Four control QC-labeled IL-5 samples were also prepared at the required 2x final concentration (considering a 1:2 dilution with the antibody cocktail) using pooled cynomolgus monkey serum (5000, 500, 50 and 0 pg/ml IL-5). Each standard QC-labeled/control serum sample was then transferred (50 μl) to a new 96-well polypropylene plate. The capture and detection antibody cocktail was prepared using rat anti-human IL-5 biotin conjugate (Southern Biotech, 10118-08) at a final concentration of 0.5 μg/ml as the capture mAb and rat anti-human IL-5 mAb sulfo-labeled (Southern Biotech, 10118-14 (sulfo-labeled MSD, laboratory prepared)) at a final concentration of 0.5 μg/mL as detection mAb. 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]) with 2x final concentration taking into account a 1:2 dilution in the standard/sample/control. An antibody cocktail (50 μl) was added to each standard/QC-labeled control/test serum sample and incubated for 3 h at room temperature with shaking (600 rpm) in the dark. Streptavidin MSD plate with gold nanoparticles (Meso Scale Discovery, L15SA-1) was blocked with 150 μl/well of MSD blocking buffer (3% MSD Blocker A (Meso Scale Discovery, R93BA-1) in PBS and incubated for 1 hour at room temperature under shaking conditions (600 rpm). After a 3-hour incubation of the antibody cocktail and standard/QC-labeled/test samples, 25 μl/well of serum in duplicate (or in triplicate for QC-labeled controls) was transferred to the blocked and washed (SKAN WASHER 300, Skatron Instruments) MSD plate with streptavidin with gold nanoparticles. The plate was then incubated for 1 hour at room temperature under shaking conditions (600 rpm). After incubation, the plate was washed (SKAN WASHER 300, Skatron Instruments). Reading buffer T (2x) was prepared and 150 μl/well was added to each well of the gold nanoparticle streptavidin MSD plate and then quantitative electrochemiluminescence analysis was performed using an MSD Sector S 600 (Model 1201) for 15 min.

Methods: eosinophil counting.

Reduction in eosinophil counts was used as a biomarker of 28Y042-7F11-1-mediated IL-5 neutralization activity. Before test substance administration, a panel of 39 animals was prescreened for eosinophil levels (days -51 and -44), and animals with eosinophil levels (>180 eosinophils/μl) were selected for further study. Animals with higher eosinophil counts were selected on the assumption that they would provide a longer assay window to measure the level of eosinophil suppression. It was hypothesized that because the animals were kept in captivity and lived in clean room conditions, they had lower baseline eosinophil counts than wild animals, and if exposed to the drug, these lower eosinophil counts could fall below the threshold level allowing quantification definition. During the preliminary screening, it also became apparent that some animals exhibited significant fluctuations in eosinophil counts. The reason for this variability is unknown, but can be explained by many factors such as environment, stress or hormonal changes.

After selecting 16 animals for the study, they were returned to the study pens (4 animals of the same sex per pen) and given time to acclimatize to their new environment. During acclimation, 3 hematological studies were performed prior to dosing (days -21, -14 and -7). Animals were administered the test substance on the first day and samples were collected by collecting 0.5 ml of blood from the femoral vein using EDTA as an anticoagulant. Sample collection was performed as late as possible in the afternoon (between 1 and 3 hours) to monitor diurnal changes in blood eosinophil levels. After collection, samples were processed within 60 min of final sample collection at each time point, and quantitation was performed on an ADVIA 120 hematology analyzer (Siemens) using both the peroxidase method and the basophil/lobulation method.

Example 2.

28Y042-7F11-1 was also evaluated in a GLP toxicity assay (10 and 100 mg/kg/week) with a 4-week single dose and 26-week repeat doses. In these studies, 28Y042-7F11-1 was administered subcutaneously to cynomolgus monkeys. The dose reduction phase of the 26-week study is ongoing (May 2017), so this description provides an interim report that presents data obtained during pretreatment performed before the end of the dosing period. Systemic exposures obtained in this study are presented in Table. 22. Standard methods were used to conduct these studies and related analyses. In table Figure 22 provides a comparative assessment of the mean systemic response in cynomolgus monkeys following subcutaneous administration of 28Y042-7F11-1. In table Table 23 shows the average pharmacokinetic parameters of 28Y042-7F111 obtained in cynomolgus monkeys after a single IV or SC administration. In table Figure 24 shows acceptable safety limits when comparing data obtained in the NOAEL analysis for Cynomolgus monkeys with predicted human data for subcutaneously administered doses (safety coverage). Table 22

duration Dose (mg/kg) Floor (M/F) AUC (µg*h/ml) ^a C _tah (mcg/ml) Ttah (h) Day 1 A week 14 Day 1 A week 14 Day 1 A week 14 Single dose 4 ^weeks 10 (M/F) 58900 N.A. 131 N.A. 84 N.A. 100 (M/F) 551000 N.A. 1200 N.A. 84 N.A. Repeat dose 26 weeks ^b > ^s 10 (M/F) 104000 (85100 138000) 116000 0 (40300 185000 ) 125 (113153) 149 (101221) 120 120 100 (M/F) 837000 (67100 0- 983000 ) 112000 0 (89800 0129000 θ) 1200 (1110 1279) 1390 (1090- 1610) 72 96 Explanation: a. AUC(0-t) = area under the plasma concentration-time curve from 0 to 672 hours (4 weeks) after a single dose and from 0 to 2016 hours (12 weeks) after a repeat dose. b. n=3/gender/group; both sexes are combined due to the lack of significant differences between sexes in exposure to 28Y042-7F11-1 s. 5/sex/group at a dose of 10 mg/kg and n=3/sex/group at a dose of 100 mg/kg; both sexes are combined due to no significant differences between sexes in exposure to 28Y042-7F11-1. The first dose of the drug is administered on day 1; the second dose is given at week 14 No Observed Adverse Event Levels (NOAEL) in bold. Values in parentheses represent range. Stzx ^{= maximum} concentration NA - not determined T _max = time to observed maximum concentration

- 60 046656

Table 23

Parameter Intravenous Subcutaneous Dose (mg/kg) 0.05 1 1 Stach (mcg/ml) 1.29 23.4 12.5 AUCo-4 (µg*h/ml) 457 9550 9270 CL (ml/h/kg) 0.0942 0.105 0.0974 ¹ V _ss (ml/kg) 77.8 79.7 74.0 ² T _1/2 (days) 25 24 22 Bioavailability (%) N.A. N.A. 111

NA - not defined l.CL/f

2.Vz/F

Table 24

Predicted _Cmax and AUC in humans Safety cover (vs. NOAEL for monkeys) Dose (mg) Dose (mg/kg) ¹ with ^v -ma\ (µg/ml) AUC (µg*h/ml) Dose coverage ² Coverage US _ah AUC coverage 2 0.03 0.226 323 3500x 6156x 3471x 10 0.14 1.13 1613 700x 1231x 694x thirty 0.43 3.39 4840 233x 410x 231x 100 1.43 11.3 16132 7Oh 123x 69x 300 4.29 33.9 48396 23x 41x 23x

- alleged subject weighing 70 kg;

- dose coverage (expressed in mg/kg)

Example 3.

Information regarding the sequence list.

The underlining below denotes CDR sequences, as defined by Kabat's CDR, in the heavy chain variable regions and light chain variable regions of antibodies or in nucleic acid sequences encoding these CDR sequences. For example, in SEQ ID NO: 1, the wireframe and CDR are represented as plain text for wireframe 1, underlined text for CDR1, plain text for wireframe 2, underlined text for CDR2, plain text for wireframe 3, underlined text for CDR3 and in plain text for framework region 4 in the direction from the amino-proximal part to the carboxy-terminal part of the sequence. This scheme is used, for example, in SEQ ID NO: 1-4. The amino-terminal methionine residues shown in these sequences can be cleaved off. Thus, sequences showing an amino-terminal methionine residue herein should also be considered as cleaved variants of these proteins lacking such an amino-terminal methionine residue. Nucleic acid sequences are represented as DNA nucleic acid sequences and include t nucleic acid residues, the corresponding RNA sequence should also be considered to be disclosed, given that the t nucleic acid residues should also be considered to disclose the nucleic acid residue. In addition, the 5'-proximal start codon atg and the 3'-proximal stop codons taa, tag and tga are omitted from the cDNA nucleic acid sequences below.

- 61 046656

FULL SIZE HEAVY CHAIN 28Y042-7F11-1 SEQIDNO: 1

OVTLRESGPALVKPTOTLTLTCTVSGFSLTGSSVHWVROPPGKGLEWLGVIWASGGTDYN SALMSRLSISKDTSRNOVVLTMTNMDPVDTATYYCARDPPSGLLRLDYWGRGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLY1TREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAP1EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK

FULL SIZE LIGHT CHAIN 28Y042-7F11-1

SEQ ID NO: 2

DIVMTQSPDSLA VSLGERATJ NCKSSOSLLNSGNOKNYLA W YQQKPGQPPKLL [ YGASTRE SGVPDRFSGSGSGTDFTISSLOAEDVAVYYCONVHSFPFTFGGGTKLE1KRTVAAPSVFIF PPSDEQLK.SGTASVVCLLNNFYPREAKVQWK.VDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC

28Y042-7FI1-1 VH

SEQ ID NO: 3

OVTLRESGPALVKPTOTLTLTCTVSGFSLTGSSVHWVROPPGKGLEWLGV1WASGGTDYN SALMSRLSISKDTSRNOWLMTTNMDPVDTATYYCARDPPSGLLRLDYWGRGTLVTVSS

28Y042-7FI1-1 VL

SEQ ID NO: 4

DIVMTOSPDSLAVSLGERADNCKSSOSLLNSGNQKNYLAWYOOKPGOPPKLLIYGASTRE SGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCONVHSFPFTFGGGTKLEIKR

28Y042-7F11-1 CDRH1

SEQ ID NO: 5

GSSVH

28Y042-7F1LI CDRH2

SEQ ID NO: 6

VIWASGGTDYNSALMS

28Y042-7F11-1 CDRH3

SEQ ID NO: 7

DPPSGLLRLDY

28Y042-7FI1-1 CDRL1

SEQ ID NO: 8

KSSQSLLN SGNQKNYL A

28Y042-7FI1-1 CDRL2

SEQ ID NO: 9

GASTRES

28Y042-7FI1-1 CDRL3

SEQ ID NO: 10

QNVHSFPFT

HUMAN IL-5 (MATURE PROTEIN)

SEQ ID NO: 11

[PTEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNHQLCTEEIFQGIGTLESQTVQGGT VERLFKNLSLIKKYIDGQK KKCGEERRR VNQFLDY LQEFLG VMNTE W11ES

ISOFORM 1 ALPHA SUBUNIT OF HUMAN IL 5 RECEPTOR (MATURE PROTEIN)

- 62 046656

SEQ ID NO: 12

DLLPDEKISLLPPVNFTIKVTGLAQVLLQWKPNPDQEQRNVNLEYQVK1NAPKEDDYETRIT ESKCVTILHKGFSASVRTILQNDHSLLASSWASAELHAPPGSPGTSIVNLTCTTNTTEDNYSR LRSYQVSLHCTWLVGTDAPEDTQYFLYYRYGSWTEECQEYSKDTLGRNIACWFPRTF1LSK GRDWLAVLV NGSSKHSA1RPFDQLFALHAIDQ1NPPLNVTAE1EGTRLS1QWEKPVSAFPIHC FDYEVKIHNTRNGYLQIEKLMTNAF1SIIDDLSKYDVQVRAAVSSMCREAGLWSEWSQP1Y VGNDEHKPLREWFVIVIMATJCFILLJLSL1CKICHLW1KLFPPIPAPKSNIKDLFVTTNYEKAG SSETEIEVIC Y1EKPGVETLEDSVF

DNA encoding FULL-LENGTH HEAVY CHAIN 28Y0427F11-1 WITH LEADER SEQUENCE

SEQ1DN0:13 atgggciggtcctgcatcalccigtttctggtggccaccgccaccggtgigcacagccaggtgacccigaggggagagcggccccgccctggtg aagcccacacagaccctcactcigacctgcaccgtgagcggcttcagcctgaccggcictagcgiccactgggigaggcagccccccggcaa gggcctggagtggctgg gcgtgatctgggcaagcggggggacggactacaactcggccctgatgagcaggctctccatcagcaaggacac cagccggaaccaggtggtgctgaccatgaccaacatggaccccgtggacaccgccacctattacigcgccagggaccctccctccggcctgc tgaggctggactggggcaggggaacaclagtgaccgtgtccagcgccagcaccaag ggccccagcgtgttccccctggcccGcagcag caagagcaccagcggcggcacagccgcccigggctgcclggtgaaggactacttccccgagcccgtgaccgtgtcctggaacagcggagc cctgaccagcggcgtgcacaccttccccgccgtgctgcagagcagcggcctgtacagcctgagcagcgtgg tgaccgtgcccagcagcagc clgggcacccagacctacatctgtaaegtgaaccacaagcccagcaacaccaaggtggacaagcgggtggagcccaagagcttgacaaga cccacacctgccccccctgccctgcccccgagctgctgggaggccccagcgtgttcctgttcccccccaagcctaaggacaccctgtacatca agaacccgaggtgacctgtgtgglggtggatgtgagccacgaggaccctgaggtgaagttcaactggtacgtggacggcgtggagglg cacaatgccaagaccaagcccagggaggagcagtacaacagcacctaccgggiggtgiccgtgctgaccgtgctgcaccaggattggctga acggcaaggagtacaagtgtaaggtgtccaacaagg ccctgcctgcccctatcgagaaaaccatcagcaaggccaagggccagcccagaga gccccaggtgtacaccctgccccctagcagagaggagatgaccaagaaccaggtgtccctgacctgcctggtgaagggcttctaccccagcg acatcgccgtggagtgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagc gatggcagcttcttcctg lacagcaagctgaccgtggacaagagcagaiggcagcagggcaacgtgttcagctgctccgigatgcacgaggccclgcacaatcactacac ccagaagagcctgagcctgtcccctggcaag

DNA encoding FULL-LENGTH LIGHT CHAIN 28Y042-7F111 WITH LEADER SEQUENCE

SEQ ID NO: 14 atgggctggtcctgcatcatcclgttlctggtggccaccgccaccggtgtgcacagcgacalcgtgatgacccagtctcccgattcactggccgl gagcctgggcgagagggccaccatcaactgcaagagcagccagagcctcctgaacagcggcaaccagaagaactacctggcctggtacca gcagaaacccggccagcc ccccaagctgctgatctatggcgcctccaccagggagagcggcgtgccagacaggtttagcggcagcggcag cggcaccgacttcaccctgacaatcagcagcctgcaggccgaggacgiggccgigtactactgccagaacgtccacagcttccccttcaccttc ggcgggggaaccaagctggagatcaagcgtocggtggccgcccccag cgtgttcatcttcccccccagcgatgagcagctgaagagcggca ccgccagcgtggigtgtcigctgaacaacttciacccccgggaggccaaggigcagtggaagglggacaatgccctgcagagcggcaacag ccaggagagcgtgaccgagcaggacagcaaggactccacclacagcctgagcagcaccctgaccctgagcaa ggccgactacgagaagc acaaggigtacgcctgtgaggigacccaccagggcctgtccagccccgtgaccaagagcttcaaccggggcgagtgc

DNA ENCODING HEAVY CHAIN VARIABLE REGION

28Y042-7F11-1

SEQ ID NO: 15 caggtgaccctgagggagagcggccccgccctggtgaagcccacacagaccctcactctgacctgcaccgtgagcggcttcagcctgaccg gctctagcgtccactgggtgaggcagccccccggcaagggcclggagtggctgggcgtgatctgggcaagcggggggacggactacaact cggccctgatgagcagg ciciccatcagcaaggacaccagccggaaccaggtggtgctgaccatgaccaacatggaccccgtggacaccgc cacctattactgcgccagggaccctccctccggcctgctgaggctggactactggggcaggggaacactagtgaccgtgtccagc

DNA ENCODING THE VARIABLE REGION OF THE LIGHT CHAIN

28Y042-7F11-1

- 63 046656

SEQ ID NO: 16 gacatcgt galgaccca gtctc ccgat icactggccgt gagcctgggcgagaggggccaccatcaactgcaagagcagccagagccicc tgaa cagcggcaaccagaagaaclacctggcclggtaccagcagaaacccggccagccccccaagclgctgatctatggcgcctccaccagggag agcggcgtgccagacagg tttagcggcagcggcagcggcaccgacttcaccctgacaatcagcagcctgcaggccgaggacgtggccgtg lac tact gccagaacglccaca gel (ccccltcaccltcggcgggggaaccaagclggagatcaagcgl

DNA ENCODING FULL-LENGTH HEAVY CHAIN 28Y0427F11-1

SEQ ID NO: 17 caggtgaccctgagggagagcggccccgccclggigaagcccacacagaccclcactctgacclgcaccgtgagcggcttcagcclgaccg gctctagcgtccacigggigaggcagccccccggcaagggcciggagtggcigggcgigatcigggcaagcggggggacggactacaact cggccclgat ga gca gg clcl CC ggcacagccgccctgggclgcctggtgaaggaclactl ccccgagcccgtgaccgigtcciggaacagcggagccigaccagcggcgtgcacacciiccccgccgigcigcagagcagcggcctgtac agcctgagcagcgtggtgaccgtgcccagcagcagcctgggcacccagacctacatctgtaacglgaacca caagcccagcaacaccaagg iggacaagcggglgga gcccaa gagclglgacaagacccacacctgccccccctgccct gccccc gagetge tgggaggccccagcgtgl t cclgttcccccccaagcctaaggacaccctgtacalcaccagagaacccgaggtgacctgtglggtgglggatgtgagccacgaggaccctga ggt gaagttcaactggtacgtggacggcgtggaggtgcacaatgccaagaccaagcccagggaggagcagtacaacagcacctaccgggt ggtgtccgtgctgaccgtgctgcaccaggattggcigaacggcaaggagtacaagtgiaaggtgtccaacaaggccctgcctgcccctatcga gaaaaccatcagcaaggccaagggccagc ccagagagccccagglgtacaccctgccccclagcagagaggagatgaccaagaaccagg Igtccctgacctgcclggtgaagggcttctaccccagcgacatcgccgtggaglggggagagcaacggccagcccgagaacaactacaagac caccccccctgtgctggacagcgaiggcagcttcitcctgtacagcaagcigaccgtggacaagagcaga tggcagcagggcaacgtgttca gcigctccgtgatgcacgaggccctgcacaaicactacacccagaagagccigagcctgicccciggcaag

DNA ENCODING FULL LENGTH LIGHT CHAIN 28Y042-7F111

SEQ ID NO: 18 gacatcgtgatgacccagtctcccgattcactggccgtgagcctgggcgagagggccaccatcaactgcaagagcagccagagcctcctgaa cagcggcaaccagaagaactacctggcctggtaccagtagaaacccggccagccccccaagctgctgatctatggcgcctccaccagggag agcggcgtgccagacaggt ttagcggcagcggcagcggcaccgacttcaccctgacaatcagcagcctgcaggccgaggacgtggccgtg iactactgccagaacgtccacagcticcccitcaccticggcgggggaaccaagctggagatcaagcglacggtggccgcccccagcgtgiic atcttcccccccagcgatgagcagctgaagagcggcaccgccagcgtggt gtgtctgctgaacaacttctacccccgggaggccaaggtgca gtggaaggtggacaatgccctgcagagcggcaacagccaggagagcgtgaecgagcaggacagcaaggactccacctacagcctgagca gcaccctgaccctgagcaaggccgactacgagaagcacaaggtgtacgcctgtgaggtgacccaccagggcctgtccag ccccgtgaccaa gagcttcaaccggggcgagtgc

LEADER SEQUENCE OF HEAVY CHAIN 28Y042-7F11-1

SEQ ID NO: 19

MGWSCIILFLVATATGVHS

HEAVY CHAIN LEADER SEQUENCE 28Y042-7F11-1 SEQ ID NO: 20 MGWSCIILFLVATATGVHS

SEQUENCE FR4 HEAVY CHAIN 28Y042-7F11-1 SEQ ID NO: 21 WGRGTLVTVSS

Now that the present invention has been fully described, it will be apparent to one skilled in the art that many changes and modifications can be made to the invention without departing from the spirit or scope of the appended claims.

The material in the ASCII text file named PU66209P_US_SeqList[,], created on May 26, 2017 and measuring 21,851 bytes, is incorporated herein by reference in its entirety.

Claims (25)

1. An antigen binding protein that binds to human IL-5, comprising a heavy chain variable region having the amino acid sequence of CDRH1 shown in SEQ ID NO: 5, the amino acid sequence of CDRH2 shown in SEQ ID NO: 6, and the amino acid sequence of CDRH3 shown in in SEQ ID NO: 7; and a light chain variable region having the amino acid sequence of CDRL1 shown in SEQ ID NO: 8, the amino acid sequence of CDRL2 shown in SEQ ID NO: 9, and the amino acid sequence of CDRL3 shown in SEQ ID NO: 10. 2. The antigen binding protein of claim 1, wherein the heavy chain variable region comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21. - 64 046656 3. The antigen-binding protein according to claim 2, containing 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 carboxy terminus variable region of the heavy chain. 4. An antigen binding protein that binds to human IL-5, 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. 5. The antigen-binding protein according to claim 4, containing 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 carboxy terminus variable region of the heavy chain. 6. An antibody that binds to human IL-5, comprising a heavy chain and a light chain, wherein a) the heavy chain comprises a heavy chain variable region having the amino acid sequence CDRH1 shown in SEQ ID NO: 5, the amino acid sequence CDRH2 shown in SEQ ID NO: 6, and the amino acid sequence CDRH3 shown in SEQ ID NO: 7; And b) the light chain comprises a light chain variable region having the amino acid sequence CDRL1 shown in SEQ ID NO: 8, the amino acid sequence CDRL2 shown in SEQ ID NO: 9, and the amino acid sequence CDRL3 shown in SEQ ID NO: 10. 7. The antibody of claim 6, wherein the heavy chain variable region comprises the heavy chain FR4 amino acid sequence shown in SEQ ID NO: 21. 8. The antibody of claim 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. 9. An antibody that binds to human IL-5, comprising a heavy chain and a light chain, wherein a) the heavy chain contains 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. 10. The antibody of claim 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. 11. An antibody that binds to human IL-5, 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. 12. A vector containing 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. 13. A vector 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. 14. A vector containing a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 3. 15. A vector containing a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 4. 16. A vector containing a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 1. 17. A vector containing a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2. 18. A nucleic acid sequence selected from the following: SEQ ID NO: 15, which encodes a heavy chain variable region (VH) sequence, SEQ ID NO: 17, which encodes a heavy chain, and SEQ ID NO: 13, which encodes a heavy chain and leader sequence. 19. A nucleic acid sequence selected from the following: SEQ ID NO: 16, which encodes a light chain variable region (VL) sequence, SEQ ID NO: 18, which encodes a light chain, and SEQ ID NO: 14, which encodes a light chain and leader sequence. 20. A cell for expressing an antigen binding protein that binds to human IL-5, comprising the nucleic acid shown in SEQ ID NO: 17, which encodes a heavy chain, and SEQ ID NO: 18, which encodes a light chain. 21. Pharmaceutical composition for subcutaneous or intravenous administration, containing an antigen-binding protein or antibody according to claims. 1-11 in combination with a pharmaceutically acceptable carrier. - 65 046656 22. Use of a protein, antibody or pharmaceutical composition according to any one of claims 1 to 11 and 21 in therapy. 23. Use according to claim 22 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, sub-eosinophilic asthma, chronic obstructive pulmonary disease , eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, bullous pemphigoid, eosinophilic esophagitis, atopic dermatitis, moderate atopic dermatitis and severe atopic dermatitis. 24. A method of treating a disease in a subject, comprising the steps of: (a) identifying a subject with 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, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyps, 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 protein, antibody, or pharmaceutical composition according to any one of claims 1 to 11 and 21. 25. The method of claim 24, wherein the protein, antibody or pharmaceutical composition is administered once every 3 months or once every 6 months.