RU2801531C2 - Methods of treatment or prevention of asthma through administration of il-4r antagonist - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to a method of reducing the incidence of one or more asthma exacerbations and to a method of reducing the incidence of asthma exacerbations or improving one or more asthma-related parameters. A method of reducing the incidence of one or more asthma exacerbations in a subject suffering from moderate-to-severe eosinophilic asthma is proposed, comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to the interleukin 4 receptor (IL-4R), where the said antibody or antigen-binding fragment contains three sequences of heavy chain complementarity determining regions (HCDRs) containing SEQ ID NOs: 148, 150 and 152, and three light chain complementarity determining regions (LCDR) sequences containing SEQ ID NO: 156, 158, and 160, and wherein the said antibody or antigen-binding fragment thereof is an additional maintenance treatment. Also a method is provided for reduction of the incidence of asthma exacerbations or improving one or more asthma-related parameters in a subject suffering from moderate-to-severe eosinophilic asthma, comprising sequentially administering to the subject a single initial dose of an antibody or antigen-binding fragment thereof that specifically binds to the interleukin 4 receptor (IL-4R), followed by one or more secondary doses of the said antibody or antigen-binding fragment thereof, wherein the said antibody or antigen-binding fragment thereof contains three heavy chain complementarity determining regions (HCDR) sequences containing SEQ ID NOs: 148, 150 and 152, and three light chain complementarity determining regions (LCDR) sequences containing SEQ ID NO: 156, 158, and 160, and wherein the said antibody or antigen-binding fragment thereof is an additional maintenance treatment.

EFFECT: above group of inventions provide targeted therapy methods for treating or preventing asthma.

3 cl, 31 dwg, 13 tbl, 4 ex

Description

Related Applications

The present application claims priority of U.S. Provisional Application 61/691,625, filed Aug. 21, 2012; U.S. Provisional Application 61/758097, filed January 29, 2013; U.S. Provisional Application 61/761,279, filed Feb. 6, 2013; U.S. Provisional Application 61/783,796, filed March 14, 2013; U.S. Provisional Application 61/805,797, filed March 27, 2013; and French patent application 1356994, filed on July 16, 2013. The contents of each of the above applications are hereby incorporated by reference in their entirety.

The technical field to which the invention belongs

The present invention relates to the treatment and/or prevention of asthma and related conditions. More specifically, the invention relates to the administration of an interleukin 4 receptor (IL-4R) antagonist for the purpose of treating or preventing asthma in a patient in need thereof.

State of the art

Asthma is a chronic inflammatory disease of the airways characterized by airway hypersensitivity, acute and chronic bronchoconstriction, airway edema and mucus blockage. The inflammatory component of asthma is believed to include several cell types, including mast cells, eosinophils, T-lymphocytes, neutrophils, and epithelial cells, as well as their biological products. Patients with asthma most commonly present with symptoms such as wheezing, shortness of breath, coughing, and chest tightness. For most patients with asthma, a regimen of control therapy and bronchodilator therapy provides adequate long-term control of attacks. Inhaled corticosteroids (ICS) are considered the "gold standard" in controlling asthma symptoms, with inhaled _beta2- agonists being the most effective bronchodilators currently available. Studies have shown that combination therapy with ICS plus a long-acting inhaled _beta2- agonist (LAAA) provides more effective asthma control than high doses of ICS alone. Therefore, combination therapy was the recommended therapy for those patients in whom low-dose ICS alone failed to relieve asthma symptoms.

However, it is estimated that between 5% and 10% of the asthmatic population have symptomatic disease despite maximum recommended therapy with combinations of anti-inflammatory and bronchodilator agents. In addition, this group with severe asthma accounts for up to 50% of total medical costs for hospital care, emergency use, and unscheduled doctor visits. There is an unmet need for new therapy in this group of severe asthma, as many of these patients respond poorly to ICS due to multiple cellular and molecular mechanisms. In addition, long-term adverse effects of systemic and inhaled corticosteroids on bone metabolism, adrenal function, and growth in children lead to attempts to minimize the amount of corticosteroid used. Although a large proportion of asthma patients are reasonably well controlled with current therapies, there are very few therapeutic options available to patients with severe, corticosteroid-refractory asthma that can provide adequate disease control. The consequence of resistance to therapy or insufficient adherence to therapy is loss of asthma control and, ultimately, exacerbation of asthma.

One reason for the low response to treatment in some patients with severe asthma may be the heterogeneity of the disease. There is growing interest in understanding these different phenotypes as targeted therapy is more likely to be successful in patients with similar underlying pathobiological features. Recent therapeutic approaches in the field of asthma have focused on trying to control the type 2 T-helper cell response. Upregulation of interleukin 4 (IL-4) and interleukin 13 (IL-13) has been implicated as an important inflammatory component of asthma progression.

Thus, there is a need in the art for new targeted therapies for the treatment and/or prevention of asthma.

The essence of the invention

According to one aspect of the present invention, methods are provided for reducing the frequency of asthma exacerbations in a subject in need thereof. In a related aspect, methods are provided for improving one or more asthma-related parameters in a subject in need thereof. In another aspect, the present invention provides methods for treating asthma, eg, moderate to severe eosinophilic asthma, in a subject in need thereof.

The methods of the present invention comprise administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an interleukin 4 receptor (IL-4R) antagonist. In some embodiments, an IL-4R antagonist is an antibody, or an antigen-binding fragment thereof, that specifically binds IL-4R. Examples of anti-IL-4R antibodies that can be used in the methods of the present invention are described elsewhere in this application, including working Example 1. For example, in one embodiment, the IL-4R antagonist is an antibody or antigen-binding fragment that specifically binds to IL-4R, and includes the CDR (complementarity determining region) of the heavy chain and light chain sequences from the heavy chain variable region (HCVR) and the light chain variable region (LCVR) - SEQ ID NOS: 162 and 164, respectively.

In one embodiment, a method is provided for reducing the frequency of one or more asthma exacerbations in a subject in need thereof by administering an antibody, or antigen-binding fragment thereof, that specifically binds IL-4R. An asthma exacerbation can be one or more of the following: (a) a 30% or greater decrease from baseline in morning peak expiratory flow (PEF) for two consecutive days; (b) six or more additional puffs of albuterol or levalbuterol aerosol for breathing relief over a 24-hour period (compared to baseline) on two consecutive days; and (c) worsening asthma requiring: (i) systemic (oral and/or parenteral) steroid therapy, or (ii) an increase in inhaled corticosteroids up to at least 4-fold, with the last dose taken before discontinuation or hospitalization.

In various embodiments, methods for improving one or more asthma-related parameters comprise administering to a subject in need thereof a therapeutically effective amount of an IL-4R antagonist, wherein the improvement in the asthma-related parameter is defined as one of the following: an increase in FEV1 from baseline; increase in PSVu relative to the baseline; increase in PSVv relative to the baseline; decreased use of albuterol/levalbuterol relative to baseline; a decrease in nighttime awakenings relative to baseline; and/or a decrease in SNOT-22 score relative to baseline. Examples of asthma-related parameters include: (a) forced expiratory volume in 1 second (FEV1); (b) peak expiratory flow (PEF), including PEF in the morning (PEVu) and PEF in the evening (PEFv); (c) use of an inhaled bronchodilator such as albuterol or levalbuterol; (d) score on the Asthma Control Questionnaire, five items (ACQ5); (d) waking up at night; and (e) a 22-item score in the Sino-Nasal Outcome Test (SNOT-22). In one embodiment, the improvement in an asthma-related parameter is an increase in FEV1 of at least 0.10 L from baseline. In one embodiment, the improvement in an asthma-related parameter is an increase in PEF of at least 10.0 L/min from baseline. In one embodiment, the improvement in an asthma-related parameter is an increase in PEF of at least 1.0 L/min from baseline. In one embodiment, the improvement in the asthma-related parameter is to reduce albuterol/levalbuterol use by at least 1 aerosol breath per day from baseline. In one embodiment, the improvement in an asthma-related parameter is a decrease of at least 0.5 ACQ5 score from baseline. In one embodiment, the improvement in an asthma-related parameter is to reduce nighttime awakenings by at least 0.2 times per night from baseline. In one embodiment, the improvement in an asthma-related parameter is a decrease of at least 5 points in SNOT-22 score from baseline.

The invention also provides methods for reducing the incidence of asthma exacerbations or improving one or more asthma-related parameters in a subject in need thereof, wherein the methods comprise sequentially administering to the subject in need thereof a single initial dose of a pharmaceutical composition comprising an IL-4R antagonist (e.g. , an anti-IL-4R antibody, or an antigen-binding fragment thereof) followed by one or more subsequent doses of a pharmaceutical composition comprising an IL-4R antagonist. A pharmaceutical composition comprising an IL-4R antagonist may be administered subcutaneously, intranasally, or intravenously to a subject in need thereof.

In some embodiments, the invention provides methods for reducing the incidence of asthma exacerbations or improving one or more asthma-related parameters in a subject in need thereof, wherein the methods comprise administering to the subject about 75 to about 300 mg of a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof. that specifically bind IL-4R. In this aspect, the pharmaceutical composition may be administered to a subject at a frequency such as once per week.

The invention also includes methods for treating asthma (e.g., eosinophilic asthma, moderate to severe eosinophilic asthma, etc.) by selecting a subject who exhibits one or more symptoms or signs of asthma and administering to the patient a pharmaceutical composition comprising an IL-4R antagonist (e.g., an anti-IL-4R antibody or antigen-binding fragment thereof) wherein the subject exhibits one or more of the following symptoms or signs of asthma: (1) the subject has been treated with a constant dose of a combination of fluticasone/salmeterol (250/50 mcg twice daily or 500/50 mcg twice a day) or a combination of budesonide/formoterol (160/9 mcg twice a day or 320/9 mcg twice a day) for at least 3 months before the examination; (2) the subject has a blood eosinophil level greater than or equal to 300 cells/µl; (3) the subject has a sputum eosinophil level greater than or equal to 3%; (4) the subject has elevated levels of IgE, thymus and upregulated chemokine (TARC), eotaxin-3, cancer embryonic antigen (CEA), YKL-40, or periostin; (5) the subject has elevated levels of fractional exhaled nitric oxide (FVOA); and/or (6) the subject has an Asthma Control Questionnaire (ACQ5) score greater than or equal to 1.0.

Embodiments of the invention are directed to the methods of treatment as described above and further comprise administering a second therapeutic agent in combination with an IL-4R antagonist. The second therapeutic agent can be administered to a subject in need thereof before, after, or in parallel with administration of the IL-4R antagonist. Examples of second therapeutic agents include, but are not limited to, one or more of the following in combination: IL-1 inhibitors, IL-5 inhibitors, IL-8 inhibitors, IgE inhibitors, tumor necrosis factor (TNF) inhibitors, corticosteroids, beta2 _- agonists long-acting and leukotriene inhibitors.

In another aspect of the invention, methods are provided to reduce or eliminate dependence of a patient with asthma on DMARDs for asthma, comprising selecting a patient having moderate to severe asthma that is uncontrolled or partially controlled by DMARDs for asthma; administration of a certain dose of an IL-4R antagonist to the patient while maintaining the basic therapy of the patient; and a gradual decrease in the dosage of one or more components of the basic therapy in the subsequent period of treatment with continued administration of the IL-4R antagonist. In some embodiments, the implementation of the basic therapy includes inhaled corticosteroids (ICS), a long-acting beta-agonist (LABA), or a combination of ICS and BABA. In some embodiments, the implementation of basic therapy is gradually reduced or canceled over 2-8 weeks. In some embodiments, the implementation of one component of the basic therapy is excluded after the initial period of treatment. In one embodiment, the basic therapy is gradually reduced during the subsequent period of treatment.

In yet another aspect of the invention, there is provided a method for identifying a patient and treating moderate to severe asthma by selecting a patient with an elevated thymus biomarker and activation regulated chemokine (TARC), IgE, eotaxin-3, periostin, cancer embryonic antigen (CEA) or YKL-40, or having elevated levels of fractional exhaled nitric oxide (FVOA); and administering to the patient a therapeutically effective amount of the IL-4R antagonist.

In another aspect of the invention, a method is provided for monitoring the effectiveness of treatment for moderate to severe asthma in a subject, for example, by: (a) determining the level of expression of a biomarker, such as TARC and/or eotaxin-3, or the total level of serum IgE in a biological sample obtained from a subject prior to treatment with an IL-4R antagonist; (b) determining the level of expression of a biomarker in a biological sample obtained from a subject after treatment with an IL-4R antagonist; (c) comparing the expression level determined in step (a) with the level in step (b), and (d) concluding that the treatment is effective if the level determined in step (b) is lower than the level determined in step (a), or the conclusion that the treatment is ineffective if the level determined in step (b) is the same as or greater than the level determined in step (a).

In one embodiment, the biomarker is VVOA, and if VVOA levels are reduced after administration of the antagonist, then treatment with the IL-4R antagonist is determined to be effective.

The expression level of the biomarker can be determined, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more after the administration of the IL-4R antagonist and compared with the expression level before the administration of the antagonist. The dose or schedule for administering an IL-4R antagonist (eg, an anti-IL-4R antibody) may be adjusted once determined. For example, if the expression of the biomarker does not decrease within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more after administration of the antagonist, then treatment with the antagonist may be suspended, or the dose of the antagonist may be increased. If the expression of the biomarker after administration of the antagonist is reduced, the dosage of the antagonist may be maintained or reduced, for example, to determine the minimum effective dose. In some embodiments, the implementation of the treatment is maintained at the level of the minimum effective dose.

In another aspect of the invention, a method is provided for monitoring a subject's response to treatment with an IL-4R antagonist, wherein the subject has moderate to severe asthma, for example, by obtaining information regarding the level of expression of a biomarker such as TARC and/or eotaxin-3, or total serum IgE level. in a biological sample of the subject after administration of the IL-4R antagonist to the subject, and obtaining an indication that treatment should be continued if the expression level of the biomarker has decreased compared to the level before treatment with the IL-4R antagonist. In one embodiment, the biomarker is VVOA, and if VVOA levels have been determined to have decreased after administration of the antibody, then an indication is given to continue treatment with the IL-4R antagonist.

The invention also includes an IL-4R antagonist disclosed herein for use in the manufacture of a medicament for the treatment and/or prevention of asthma (e.g., eosinophilic asthma, moderate to severe eosinophilic asthma, etc.) or for the treatment of any of the other indications or conditions disclosed in the present description.

The invention also includes an IL-4R antagonist disclosed herein for use in the treatment and/or prevention of asthma (e.g., eosinophilic asthma, moderate to severe eosinophilic asthma, etc.) or for the treatment and/or prevention of any of the other indications or conditions disclosed in the present description.

The invention includes a pharmaceutical composition comprising an anti-IL-4R antagonist antibody, or an antigen-binding fragment thereof, for use in the treatment and/or prevention of asthma and related conditions.

The invention also includes a pharmaceutical composition comprising an anti-IL-4R antagonist antibody, or antigen-binding fragment thereof, for use in reducing the incidence of one or more asthma exacerbations in a subject in need thereof.

In addition, the invention includes a pharmaceutical composition comprising an anti-IL-4R antagonist antibody, or antigen-binding fragment thereof, for use in improving one or more asthma-related parameters in a subject in need thereof.

The invention includes a pharmaceutical composition comprising an antagonist antibody against IL-4R or its antigen-binding fragment for use in the treatment of asthma and related conditions in a patient having an elevated level of a biomarker selected from the group consisting of thymus and activation of regulated chemokine (TARC), IgE, eotaxin -3, periostin, cancer embryonic antigen (CEA), YKL-40 and fractional exhaled nitric oxide (FVOA).

The invention further includes a pharmaceutical composition comprising an anti-IL-4R antagonist antibody, or antigen-binding fragment thereof, for use in the treatment of asthma or moderate to severe eosinophilic asthma in a subject in need thereof, wherein the treatment comprises testing the patient for the presence of a blood eosinophil level of at least 300 cells per microliter and/or a sputum eosinophil level of at least 3%, and initiating/continuing administration of the pharmaceutical composition if such blood eosinophil level and/or sputum eosinophil level is detected.

Other embodiments of the invention will become apparent upon review of the following detailed description.

Brief description of the figures

Figure 1 is a graph showing a Kaplan-Meier plot of asthma exacerbation dynamics in patients treated with placebo (open circles) compared to patients treated with anti-IL-4R mAb1 (asterisks). The effect of treatment with anti-IL-4R mAb1 persisted for a long time, including after 8 weeks, when patients were at higher risk of developing exacerbations due to steroid withdrawal. Broken vertical lines indicate BAAA cancellation.

Figure 2 is a graph showing the mean change in forced expiratory volume at 1 second (FEV1) from baseline in liters in patients treated with placebo (open triangles) compared with patients treated with anti-IL-4R mAb1 (solid circles) . Broken vertical lines indicate BAAA cancellation.

Figure 3 is a graph showing the mean change in peak morning expiratory flow rate (PSVu) in liters per minute from baseline in patients treated with placebo (open triangles) compared to patients treated with anti-IL-4R mAb1 (solid circles) .

Figure 4 is a graph showing the mean change in peak evening expiratory flow rate (PEFf) in liters per minute from baseline in patients treated with placebo (open triangles) compared with patients treated with anti-IL-4R mAb1 (solid circles) .

Figure 5 is a graph showing the mean change in the number of inhalations per day with albuterol from baseline in patients treated with placebo (open triangles) compared to patients treated with anti-IL-4R mAb1 (solid circles). Broken vertical lines indicate BAAA cancellation.

Figure 6 is a graph showing the mean change in Five Item Asthma Control Questionnaire (ACQ5) score from baseline in patients treated with placebo (open triangles) compared to patients treated with anti-IL-4R mAb1 (solid circles) . Broken vertical lines indicate BAAA cancellation.

Figure 7 is a graph showing the mean change in the number of nighttime awakenings from baseline in patients treated with placebo (open triangles) compared to patients treated with anti-IL-4R mAb1 (solid circles). Broken vertical lines indicate BAAA cancellation.

Figure 8 is a graph showing the mean percent change in TARC from baseline at week 0, 1, 4, 8 and 12 visits in the placebo treated mITT group (solid circles) compared to patients treated with anti-IL-mAb1 antibody. 4R (filled squares). Broken vertical lines indicate BAAA cancellation.

Figure 9 is a graph showing the mean percent change in eotaxin-3 from baseline at visits at weeks 0, 1, 4, 8, and 12 in the placebo-treated mITT group (solid circles) compared to patients treated with anti-mAb1 antibody. IL-4R (filled squares). Broken vertical lines indicate BAAA cancellation.

Figure 10 is a graph showing the mean percent change in total IgE from baseline at week 0, 1, 4, 8 and 12 visits in the placebo treated mITT group (solid circles) compared to patients treated with anti-IL mAb1 -4R (filled squares). Broken vertical lines indicate BAAA cancellation.

Figure 11 is a graph showing the mean percent change in periostin from baseline at visits at weeks 0, 1, 4, 8 and 12 in the placebo treated mITT group (solid circles) compared to patients treated with anti-IL-mAb1 antibody. 4R (filled squares).

Figure 12 is a graph showing the mean percent change in cancer embryonic antigen (CEA) from baseline at week 0, 1, 4, 8, and 12 visits in the mITT placebo group (solid circles) compared to patients treated with anti-IL-4R mAb1 (solid squares).

Figure 13 is a graph showing the mean percent change in YKL-40 from baseline at visits at weeks 0, 1, 4, 8, and 12 in the placebo-treated mITT group (solid circles) compared to patients treated with anti-mAb1 antibody. IL-4R (filled squares).

Figure 14 is a graph showing the mean percentage change in blood eosinophils from baseline at visits at weeks 0, 1, 2, 4, 6, 8, and 12 in the mITT placebo group (solid circles) compared with patients treated with anti-IL-4R mAb1 (solid squares).

Figure 15 is a graph showing the mean percentage change in fractional exhaled nitric oxide (NO) from baseline at week 0, 4, 8, and 12 visits in the placebo-treated mITT group (solid circles) compared to patients treated anti-IL-4R mAb1 antibody (solid squares). Broken vertical lines indicate BAAA cancellation.

Figure 16 is a scatterplot of change in FEV1 (l) from baseline at week 12 compared to baseline exhaled nitric oxide fraction (EFVOA) (bn) in the mITT placebo group (open circles and solid line) compared to patients treated with anti-IL-4R mAb1 (plus sign and dotted line).

Figure 17 - Scatterplot of change in PSVu (L/min) from baseline at week 12 compared to baseline FVOA (bn) in placebo treated mITT group (open circles and solid line) compared to antibody treated patients mAb1 against IL-4R (plus sign and dotted line).

Figure 18 - Scatterplot of change in PEF (L/min) from baseline at week 12 compared to baseline FVOA (bn) in placebo treated mITT group (open circles and solid line) compared to antibody treated patients mAb1 against IL-4R (plus sign and dotted line).

Figure 19 - Scatterplot of FEV1 change from baseline at week 12 (l) versus blood eosinophil count (10 ⁹ /l) in the placebo treated mITT group (open circles and solid line) compared to antibody treated patients mAb1 against IL-4R (plus sign and dotted line).

Figure 20 - Scatterplot of change in ACQ score from baseline at week 12 compared to blood eosinophil count (10 ⁹ /l) in the mITT group treated with placebo (open circles and solid line) compared with patients treated with mAb1 antibody against IL-4R (plus sign and dotted line).

Figure 21 - Scatterplot of change with albuterol/levalbuterol per day from baseline at week 12 versus blood eosinophil count (10 ⁹ /l) in the mITT placebo group (open circles and solid line) compared with patients treated with anti-IL-4R mAb1 (plus sign and dotted line).

Figure 22 is a scatterplot of change in ACQ score from baseline at week 12 compared to baseline periostin in the mITT placebo group (open circles and solid line) compared to patients treated with anti-IL-4R mAb1 (plus sign and dotted line).

Figure 23 is a scatterplot of change in ACQ score from baseline at week 12 versus YKL-40 in the mITT placebo group (open circles and solid line) compared to patients treated with anti-IL-4R mAb1 (plus sign and dotted line). line).

Figure 24 is a schematic representation of a timing and dose regimen for the treatment of patients with asthma.

Figure 25 is a graph describing the distribution of patients in a randomized, placebo-controlled, double-blind, parallel group study conducted with subcutaneous once a week 300 mg mAb1 or placebo for 12 weeks in patients with persistent eosinophilic asthma, from moderate to severe, which partially controlled/uncontrolled by inhaled corticosteroid (ICS) and long-acting beta2-agonist (LAAA) therapy.

Figure 26A and 26B are scatterplots of asthma symptoms in the morning (A) and evening (B) measured over 12 weeks after placebo (open triangles) or mAb1 (solid circles) administration.

Figure 27 is a graph showing serum IgE levels in humanized IL-4/IL-4R mice (IL-4 ^hu/hu IL-4Rα ^hu/hu ) after dust mite (HDM) allergen challenge and administration of either anti-IL- 4R, or IL-13Ra2-Fc decoy receptor, or after sham therapy. Measurements were made on samples taken on Day 40 (24 hours before the first dose of therapy) and at the end of the experiment, on Day 85.

Figure 28 is a graph showing serum IgE levels in wild-type (Balb/c) mice after challenge with dust mite allergens (HDM) and administration of either isotype control, anti-IL-4R antibody, or decoy receptor IL-13Ra2-Fc, or after sham therapy.

Figure 29 is a graph showing the collagen content (expressed in μg/share) in the lungs of humanized IL-4/IL-4R mice after HDM challenge and therapy.

Figure 30 is a graph showing the content of collagen (expressed in μg/share) in the lungs of wild-type mice after HDM challenge and prescribed therapy.

Figure 31A is a graph showing the levels of eosinophils and neutrophils in humanized IL-4/IL-4R mice after HDM challenge and therapy, and Figure 31B is a graph showing the levels of resident dendritic cells and inflammatory dendritic cells in humanized IL- 4/IL-4R mice after HDM challenge and therapy.

Detailed description

Before the present invention is described, it should be understood that this invention is not limited to the specific methods and experimental conditions described, as the methods and conditions may vary. It is also to be understood that the terminology used herein is for the sole purpose of describing particular embodiments, and is not to be limiting as the scope of the present invention will only be limited in accordance with the appended claims.

Unless otherwise defined, all technical and scientific terms used in the present description have the same meaning, by which they are usually understood by those of ordinary skill in the art to which the present invention pertains.

When used in the present description, the term "approximately", when used in relation to a particular specified numerical value, means that the value may deviate from the specified value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "treat", "treatment" or the like means to relieve symptoms, eliminate the etiology of symptoms on a temporary or permanent basis, or prevent or slow the onset of symptoms of the named disorder or condition.

While any methods and materials similar or equivalent to those described herein may be used in the practice of the present invention, preferred methods and materials will now be described. All publications cited in this application are incorporated herein by reference in their entirety.

Ways to reduce the frequency of asthma exacerbations

The invention includes methods for reducing the incidence of asthma exacerbations in a subject in need thereof, comprising administering a pharmaceutical composition comprising an interleukin 4 receptor (IL-4R) antagonist to the subject. When used in the present description, the expression "exacerbation of asthma" means an increase in the severity and/or frequency and/or duration of one or more symptoms or signs of asthma. "Asthma exacerbation" also includes any deterioration in a subject's respiratory health that requires and/or is treatable with therapeutic intervention for asthma (eg, steroid therapy, inhaled corticosteroid therapy, hospitalization, etc.). According to some embodiments of the invention, an asthma exacerbation is defined as one or more of the following: (a) a 30% or greater decrease in peak morning expiratory flow rate ("PEV" as defined elsewhere herein) from baseline over two consecutive days ; (b) six or more additional puffs of albuterol or levalbuterol aerosol for breathing relief over a 24-hour period (compared to baseline) on two consecutive days; and (c) worsening asthma requiring: (i) systemic (oral and/or parenteral) steroid therapy, or (ii) an increase in inhaled corticosteroids to at least 4 times baseline, or (iii) hospitalization.

In some cases, an asthma exacerbation can be categorized as "severe asthma exacerbation". A severe exacerbation of asthma means an incident requiring immediate intervention in the form of treatment with either systemic corticosteroids or inhaled corticosteroids at a dose four or more times the dose used before the incident. The general expression "asthma exacerbation" thus includes and encompasses the more specialized subcategory "severe asthma exacerbations". Accordingly, the invention includes methods for reducing the incidence of severe asthma exacerbations in a patient in need thereof.

"Reduced frequency of occurrence" of asthma exacerbations means that a subject who has already received the pharmaceutical composition of the present invention experiences fewer asthma exacerbations (i.e., at least one less exacerbation) after treatment than before treatment, or does not experience any asthma exacerbations during at least 4 weeks (eg 4, 6, 8, 12, 14 or more weeks) after initiation of treatment with the pharmaceutical composition of the present invention. "Reducing the incidence" of asthma exacerbations alternatively means that after administration of the pharmaceutical composition of the present invention, the likelihood that a subject will experience an asthma exacerbation is reduced by at least 10% (e.g., 10%, 15%, 20%, 25% , 30%, 35%, 40%, 45%, 50% or more) compared to a subject who did not receive the pharmaceutical composition of the present invention.

Ways to improve asthma-related parameters

The invention also includes methods for improving one or more asthma-related parameters in a subject in need thereof, wherein the methods comprise administering a pharmaceutical composition comprising an interleukin 4 receptor (IL-4R) antagonist to the subject. Within the scope of the invention, a decrease in the frequency of asthma exacerbations (as described above) may be correlated with an improvement in one or more asthma-related parameters, however, such a correlation need not be observed in all cases.

Examples of "asthma-related parameters" include: (a) forced expiratory volume in 1 second (FEV1); (b) peak expiratory flow (PEF), including PEF in the morning (PEVu) and PEF in the evening (PEFv); (c) use of an inhaled bronchodilator such as albuterol or levalbuterol; (d) score on the Asthma Control Questionnaire, five items (ACQ5); (d) waking up at night; and (e) a score on the 22-item Nose and Sinus Outcome Test (SNOT-22). "Improvement in an asthma-related parameter" means an increase from baseline in one or more of FEV1, PEF or PSVv, and/or a decrease from baseline in one or more of daily albuterol/levalbuterol, ACQ5 score, average number of nighttime awakenings, or score SNOT-22. As used herein, the term "baseline", in relation to an asthma-related parameter, means the numerical value of an asthma-related parameter in a patient before or during administration of a pharmaceutical composition of the present invention.

To determine if an asthma-related parameter has improved, the parameter is quantified at baseline and at the time following administration of the pharmaceutical composition of the present invention. For example, an asthma-related parameter can be measured on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 14, or at week 3, week 4, week 5, week 6, week 7, week 8, week 9, week 10, week 11, week 12, week 13, week 14, week 15, week 16, week 17, week 18, week 19 , week 20, week 21, week 22, week 23, week 24 or later, after initial therapy with the pharmaceutical composition of the present invention. The difference between the parameter value at a specific time point after the start of treatment and the parameter value at baseline is used to determine whether there has been an "improvement" in the asthma-related parameter (eg, an increase or decrease, as appropriate, depending on the specific parameter being measured).

The terms "obtain" or "obtain" as used herein refer to the acquisition of a physical object or value, such as a numerical value, by "directly obtaining" or "indirectly obtaining" a physical object or value, such as an asthma-related parameter. "Direct acquisition" means performing a process (eg, performing a synthesis or analysis method) to obtain a physical object or value. "Indirect acquisition" refers to the receipt of a physical object or value from another party or from a source (for example, from a third party laboratory that directly obtained the physical object or value). Directly obtaining a physical object involves performing a process that involves physically changing a physical substance, such as the original substance. Examples of change include creating a physical object from two or more starting substances, cutting or grinding a substance, separating or refining a substance, combining two or more separate objects into a mixture, performing a chemical reaction that involves breaking or forming a covalent or non-covalent bond. Direct value acquisition includes performing a process that involves physically modifying a sample or other substance, such as performing an analytical process that involves physically modifying a substance, such as a sample, analyte, or reagent (sometimes referred to herein as "physical analysis").

The information obtained indirectly may be in the form of a message, for example in paper or electronic form, such as from an electronic database or as an application ("App"). The message or information could be provided, for example, by a health care provider such as a hospital or clinic, or a health care provider such as a doctor or nurse.

Forced expiratory volume in 1 second (FEV1) . In some embodiments, administration of an IL-4R antagonist to a patient results in an increase from baseline in forced expiratory volume in 1 second (FEV1). Methods for measuring FEV1 are known in the art. For example, a spirometer that complies with the 2005 American Thoracic Society (ATS)/European Respiratory Society (ERS) guidelines can be used to measure FEV1 in a patient. The standardization of ATS/ERS spirometry can be used as a guide. Spirometry is usually performed between 06:00 and 10:00, at least 6 hours after the suspension of albuterol. Lung function tests are usually performed in the sitting position and the maximum FEV1 value (in liters) is recorded.

The invention includes therapeutic methods that result in an increase in FEV1 from baseline of at least 0.05 L per week 12 after initiation of therapy with a pharmaceutical composition comprising an antagonist against IL-4R. For example, according to the invention, administration of an IL-4R antagonist to a subject in need thereof causes an increase in FEV1 from baseline of approximately 0.05 L, 0.10 L, 0.12 L, 0.14 L, 0.16 L, 0. 18 l, 0.20 l, 0.22 l, 0.24 l, 0.26 l, 0.28 l, 0.30 l, 0.32 l, 0.34 l, 0.36 l, 0, 38 L, 0.40 L, 0.42 L, 0.44 L, 0.46 L, 0.48 L, 0.50 L or more per week 12.

Peak expiratory flow in the morning and in the evening (PSVu and PSVv) . In some embodiments, administering an IL-4R antagonist to a patient results in an increase in morning and/or evening peak expiratory flow rate (PSVu and/or PSVv) relative to baseline. Methods for measuring PSV are known in the art. For example, in one method of measuring PSV, patients are given an electronic PSV meter to record morning (PSVv) and evening (PSVv) PSV (as well as daily albuterol use, assessments of morning and evening asthma symptoms, and the number of nighttime awakenings due to asthma symptoms that require use of emergency medicines). Patients are instructed in the use of the device and receive written instructions on how to use the electronic PSV meter. In addition, the healthcare professional may instruct patients on how to record the appropriate measured values on the electronic PSV meter. PSVu measurement is usually taken within 15 minutes after getting up (from 6:00 to 10:00) before taking albuterol. PSVv measurement is usually taken in the evening (from 18:00 to 22:00) before taking albuterol. Subjects should attempt to withhold albuterol for at least 6 hours prior to measuring their PSV. The patient makes three attempts to measure PSV and registers all 3 values on an electronic PSV meter. Typically, the maximum value is used for evaluation. Baseline PSVv can be calculated as the average measurement taken in the morning recorded within 7 days prior to administration of the first dose of a pharmaceutical composition comprising an IL-4R antagonist, and baseline PSVv can be calculated as the average measurement performed in the evening recorded over 7 days prior to administration the first dose of a pharmaceutical composition comprising an IL-4R antagonist.

The invention includes therapeutic methods that result in an increase in PEF and/or PEF from baseline by at least 1.0 L/min at week 12 after initiation of treatment with a pharmaceutical composition comprising an antagonist against IL-4R. For example, according to the invention, administration of an IL-4R antagonist to a subject in need thereof causes an increase in PSV relative to the baseline of approximately 0.5 l/min, 1.0 l/min, 1.5 l/min, 2.0 l/min. min, 2.5 l/min, 3.0 l/min, 3.5 l/min, 4.0 l/min, 4.5 l/min, 5.0 l/min, 5.5 l/min , 6.0 L/min, 6.5 L/min, 7.0 L/min, 7.5 L/min, 8.0 L/min, 8.5 L/min, 9.0 L/min, 9.5 L/min, 10.0 L/min, 10.5 L/min, 11.0 L/min, 12.0 L/min, 15 L/min, 20 L/min or more per week 12.

Use of albuterol/levalbuterol . In some embodiments, administration of an IL-4R antagonist to a patient results in a decrease in daily albuterol or levalbuterol use from baseline. The number of inhalations of albuterol/levalbuterol can be recorded by patients daily in a diary, on a PSV meter or other recording device. In the course of treatment with the pharmaceutical composition of the invention, the use of albuterol/levalbuterol can usually be carried out as needed in case of symptoms, on an occasional basis or prophylactically. The base number of albuterol/levalbuterol inhalations per day can be calculated from the average of the 7 days prior to the first dose of a pharmaceutical composition comprising an IL-4R antagonist.

The invention includes therapeutic methods that result in a decrease in albuterol/levalbuterol use from baseline of at least 0.25 aerosol breaths per day at week 12 after initiation of treatment with a pharmaceutical composition comprising an antagonist against IL-4R. For example, according to the invention, the administration of an IL-4R antagonist to a subject in need thereof causes a decrease in the use of albuterol/levalbuterol from baseline by approximately 0.25 aerosol breaths per day, 0.50 aerosol breaths per day, 0.75 aerosol breaths per day , 1.00 aerosol breaths/day, 1.25 aerosol breaths/day, 1.5 aerosol breaths/day, 1.75 aerosol breaths/day, 2.00 aerosol breaths/day, 2.25 aerosol breaths/day, 2 .5 aerosol breaths per day, 2.75 aerosol breaths per day, 3.00 aerosol breaths per day or more per week 12.

Five-item Asthma Control Questionnaire (ACQ) score . In some embodiments, administering an IL-4R antagonist to a patient results in a decrease in Asthma Control Questionnaire five-item (ACQ5) score from baseline. ACQ5 is an approved Asthma Control Questionnaire.

The invention includes therapeutic methods that result in a reduction in ACQ5 score from baseline of at least 0.10 point per week 12 after initiation of treatment with a pharmaceutical composition comprising an antagonist against IL-4R. For example, according to the invention, administration of an IL-4R antagonist to a subject in need thereof causes a reduction in ACQ score from baseline of approximately 0.10 points, 0.15 points, 0.20 points, 0.25 points, 0.30 pt, 0.35 pt, 0.40 pt, 0.45 pt, 0.50 pt, 0.55 pt, 0.60 pt, 0.65 pt, 0.70 pt, 0.75 pt, 0.80 points, 0.85 points or more per week 12.

Waking up at night . In some embodiments, administration of an IL-4R antagonist to a patient results in a decrease in the average number of nighttime awakenings from baseline.

The invention includes therapeutic methods that result in a decrease in the average number of awakenings during the night compared to baseline by at least about 0.10 times a night at week 12 after the start of treatment with a pharmaceutical composition comprising an antagonist against IL-4R. For example, according to the invention, the administration of an IL-4R antagonist to a subject in need thereof causes a decrease in the average number of awakenings during the night compared to the baseline by approximately 0.10 times per night, 0.15 times per night, 0.20 times per night. night, 0.25 times a night, 0.30 times a night, 0.35 times a night, 0.40 times a night, 0.45 times a night, 0.50 times a night, 0.55 times a night , 0.60 times a night, 0.65 times a night, 0.70 times a night, 0.75 times a night, 0.80 times a night, 0.85 times a night, 0.90 times a night, 0.95 times per night, 1.0 times per night, 2.0 times per night or more per week 12.

Score in the 22-item Nose and Sinus Outcomes Test (SNOT-22) . In some embodiments, administering an IL-4R antagonist to a patient results in a decrease in score on the 22-Point Nose and Sinus Outcome Test (SNOT-22) from baseline. SNOT-22 is a validated questionnaire to assess the impact of chronic rhinosinusitis on quality of life (Hopkins et al., 2009, Clin. Otolaryngol., 34:447-454).

The invention includes therapeutic methods that result in a reduction in SNOT-22 score from baseline of at least 1 point per week 12 after initiation of treatment with a pharmaceutical composition comprising an antagonist against IL-4R. For example, according to the invention, administration of an IL-4R antagonist to a subject in need thereof causes a decrease in SNOT-22 score relative to baseline by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13 points or more per week 12.

Ways to treat asthma

The invention, in some embodiments, provides methods for treating asthma, including, for example, eosinophilic asthma, in a subject in need thereof, wherein the methods comprise administering to the subject a pharmaceutical composition comprising an interleukin 4 receptor (IL-4R) antagonist. In some embodiments, the methods of the invention may be used to treat moderate to severe eosinophilic asthma in a subject (eg, persistent, moderate to severe eosinophilic asthma).

According to the invention, a subject is identified as having moderate to severe eosinophilic asthma if the subject exhibits a blood eosinophil level of at least 300 cells per microliter and/or a sputum eosinophil level of at least 3%. Any methods known and available in the art for measuring blood and/or sputum levels of eosinophils can be used within the scope of the invention to identify a subject having moderate to severe eosinophilic asthma, who thus becomes a suitable subject for application of the therapeutic methods of the invention.

According to a related aspect of the invention, methods for treating asthma are provided, comprising: (a) selecting a patient that exhibits a blood eosinophil level of at least 300 cells per microliter and/or a sputum eosinophil level of at least 3%; and (b) administering to the patient a pharmaceutical composition comprising an IL-4R antagonist.

In another aspect, methods are provided to reduce or eliminate the dependence of an asthmatic patient on the use of inhaled corticosteroids (ICS) and/or long acting beta agonists (LAAAs) in the treatment of moderate to severe asthma. In some embodiments, the methods include: selecting a patient having moderate to severe asthma that is not controlled or partially controlled by basic therapy; administering to the patient a specific dose of an IL-4R antagonist, preferably an anti-IL-4R antibody, during the initial treatment period while maintaining the patient's basal therapy during the initial treatment period; and gradually reducing the dosage of one or more components of the basic therapy during the subsequent treatment period, with continued administration of the IL-4R antagonist. The term "baseline therapy" refers to standard or conventional therapies known in the art that are used to treat asthma. In some embodiments, the basic therapy includes ICS, LABA, or a combination of both. In some embodiments, the dose of ICS and/or LABA is eliminated or canceled immediately after the initial period of treatment. For example, a dietary supplement, such as salmeterol or formoterol, is administered during the initial period of treatment and completely stopped or canceled during the subsequent period of treatment.

An exemplary treatment regimen for a patient with moderate to severe asthma is shown in Figure 24, where an IL-4R antagonist is administered to a patient with moderate to severe asthma. During the initial period of treatment (also referred to as the "stable phase"), BABA and ICS are administered to the patient as basic therapy. During the subsequent treatment period (also referred to as the "withdrawal phase"), the administration of BABA is suspended, ie BABA is withdrawn or eliminated. The dose of ICS is gradually reduced during the subsequent period of treatment, until complete elimination.

In a related aspect, methods for treating asthma are provided, including adjunctive therapy to basic therapy with systematic withdrawal of basic therapy. In some embodiments, an IL-4R antagonist is administered as add-on therapy to an asthma patient who has been on baseline therapy for a period of time (e.g., 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 5 months, 12 months, 18 months, 24 months or longer) (also called "stable phase"). In some embodiments, the basic therapy includes ICS and/or LABA. The stable phase is followed by a withdrawal phase of the baseline, where one or more components of the baseline are withdrawn or reduced or eliminated while additional therapy is continued. In some embodiments, baseline therapy may be reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, or more during the withdrawal phase. The withdrawal phase can last 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or more. In a preferred embodiment, baseline therapy may be reduced by approximately 5% during the withdrawal phase, with the withdrawal phase being 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks , 10 weeks, 11 weeks, 12 weeks or more. In a preferred embodiment, baseline therapy may be reduced by approximately 10% during the withdrawal phase, with the withdrawal phase being 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks , 10 weeks, 11 weeks, 12 weeks or more. In a preferred embodiment, baseline therapy may be reduced by approximately 20% during the withdrawal phase, with the withdrawal phase being 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks , 10 weeks, 11 weeks, 12 weeks or more. In a preferred embodiment, baseline therapy may be reduced by approximately 30% during the withdrawal phase, with the withdrawal phase being 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks , 10 weeks, 11 weeks, 12 weeks or more. In a preferred embodiment, baseline therapy may be reduced by approximately 40% during the withdrawal phase, with the withdrawal phase being 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks , 10 weeks, 11 weeks, 12 weeks or more. In a preferred embodiment, baseline therapy may be reduced by approximately 50% or more during the withdrawal phase, wherein the withdrawal phase may last 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or more.

In some other embodiments, the invention encompasses methods of treating or reducing the severity of conditions or complications associated with asthma, such as chronic rhinosinusitis, allergic rhinitis, allergic fungal rhinosinusitis, allergic bronchopulmonary aspergillosis, united airway disease, Churg-Strauss syndrome, vasculitis, chronic obstructive lung disease (COPD) and exercise-induced bronchospasm.

The invention also includes methods for treating persistent asthma. As used herein, the term "persistent asthma" means that the subject has symptoms at least once a week, day and/or night, with symptoms lasting from several hours to several days. In some alternative embodiments, persistent asthma is "mildly persistent" (e.g., more than twice a week, but not daily, and the symptoms are severe enough to interfere with daily activities or sleep, and/or where lung function is normal or may return to noma with an inhaled bronchodilator), "moderately persistent" (eg, symptoms occur daily, with sleep interrupted at least once a week and/or lung function is moderately impaired), or "severely persistent" (eg, persistent symptoms despite on the correct use of approved drugs and/or where pulmonary function is severely impaired).

Interleukin-4 receptor antagonists

The methods of the invention include administering to a subject in need thereof a therapeutic composition comprising an interleukin 4 receptor (IL-4R) antagonist. As used herein, an "IL-4R antagonist" is any agent that binds or interacts with IL-4R and inhibits the normal biological function of IL-4R signaling when IL-4R is expressed on a cell in vitro or in vivo . Non-limiting examples of classes of IL-4R antagonists include small molecule IL-4R antagonists, anti-IL-4R aptamers, peptide-based IL-4R antagonists (e.g., peptibody or peptibody molecules), and antibodies or antigen-binding antibody fragments that specifically bind IL. -4R human.

The term "human IL-4R" (hIL-4R) refers to a human cytokine receptor that specifically binds to interleukin 4 (IL-4), such as IL-4Rα (SEQ ID NO: 274).

The term "antibody" refers to immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains, which are linked by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain includes a heavy chain variable region (abbreviated as HCVR or V _H ) and a heavy chain constant region. The heavy chain constant region includes three domains: C _H 1, C _H 2 and C _H 3. Each light chain includes a light chain variable region (abbreviated as LCVR or V _L ) and a light chain constant region. The light chain constant region includes one domain - (C _L 1). The V _H and V _L regions can be further divided into hypervariable regions called complementarity determining regions (CDRs) which alternate with more conserved regions called framework regions (FRs). Each V _H and V _L consists of three CDRs and four FRs, arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, the FR regions of an anti-IL-4R antibody (or antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or artificially modified. The consensus amino acid sequence can be determined based on an alignment analysis of two or more CDRs.

The term "antibody" also includes antigen-binding fragments of full-length antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any natural, enzymatic, synthetic, or engineered polypeptide or glycoprotein that specifically binds to an antigen, forming a complex. Antigen-binding fragments of an antibody can be obtained, for example, from full-length antibody molecules using any suitable standard methods, such as proteolytic cleavage or recombinant genetic engineering methods, involving the manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and/or can be readily obtained, for example, from commercial sources, DNA libraries (including, for example, antibody phage libraries), or can be synthesized. DNA can be sequenced and manipulated using chemical or molecular biology techniques, such as placing one or more variable and/or constant domains in the desired configuration or introducing codons, creating cysteine residues, modifying, adding or deleting amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab') ₂ fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic an antibody hypervariable region (e.g., a distinguished complementarity determining region (CDR) such as a CDR3 peptide), or a conformationally restricted FR3-CDR3-FR4 peptide. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tribodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.) , small modular immunopharmaceuticals (SMIPs), and shark immunoglobulin IgNAR variable domains are also encompassed by the expression "antigen binding fragment".

The antigen-binding fragment of an antibody typically includes at least one variable domain. The variable domain may be of any size or amino acid composition, and typically includes at least one CDR that is adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments in which the V _H domain is connected to the V _L domain, the V _H and V _L domains can be located relative to each other in any suitable configuration. For example, the variable region may be dimeric and contain V _H -V _H , V _H -V _L or V _L -V _L dimers. In the alternative, the antigen-binding fragment of an antibody may contain a monomeric V _H or V _L domain.

In some embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be present in an antigen-binding fragment of an antibody of the present invention include: (i) V _{H -CH} 1; (ii) V _H -C _H 2; (iii) V _H -C _H 3; (iv) V _H -C _H 1 -C _H 2; (V) V _H -C _H 1 -C _H 2 -C _H 3; (vi) V _H -C _H 2 -C _H 3; (vii) V _H -C _L ; (viii) V _L -C _H 1; (ix) V _{L -CH} 2; (x) V _L -C _H 3; (xi) V _L -C _H 1 -C _H 2; (xii) V _L -C _H 1 -C _H 2 -C _H 3; (xiii) V _L -C _H 2 -C _H 3; and (xiv) V _L -C _L . In any configuration of the variable and constant domains, including any of the above exemplary configurations, the variable and constant domains may either be directly linked to each other, or may be linked through a full or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible bond between conjugated variable and/or constant domains in a single polypeptide molecule, preferably a hinge region may be 2-60 amino acids, preferably 5-50 or preferably 10-40 amino acids. In addition, an antigen-binding fragment of an antibody of the present invention may include a homodimer or heterodimer (or other multimers) with any of the variable and constant domain configurations mentioned above in non-covalent association with each other and/or with one or more monomeric V _H or V _L domains. (eg via disulfide bond(s)).

As with full-length antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody typically includes at least two distinct variable domains, where each variable domain is capable of specifically binding to a single antigen or to a specific epitope on a single antigen. Any multispecific antibody format can be adapted for use within the antigen-binding fragment of an antibody of the invention using standard techniques available in the art.

The constant region of an antibody is important in relation to the ability of the antibody to bind complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotype can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

The term "human antibody" includes antibodies having variable and constant regions that are derived from human germline immunoglobulin sequences. Human antibodies of the invention may, however, include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or in vivo somatic mutation), e.g., in the CDR and in particular CDR3. However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of a mammalian species of another species, such as a mouse, have been grafted onto human framework sequences.

The term "recombinant human antibody" includes all human antibodies that are made, expressed, generated, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial library of human antibodies (described further below), antibodies isolated from an animal (e.g. mouse) that is transgenic for human immunoglobulin genes (see e.g. Taylor et al., (1992) Nucl. Acids Res. 20: 6287 -6295), or antibodies obtained, expressed, created or isolated by any other method, which includes combining sequences of human immunoglobulin genes with other DNA sequences. Such recombinant human antibodies contain variable and constant regions that are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, in the case of an animal transgenic for human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the V _H and V _L regions of the recombinant antibodies are sequences that, although derived from and linked to human germline V _H and V _L sequences, may not exist in the in vivo human germline antibody repertoire.

Human antibodies can exist in two forms, which are associated with the heterogeneity of the hinge region. In one form, the immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by a disulfide bond between the heavy chains. In the second form, the dimers are not linked by interchain disulfide bonds, thus forming a molecule weighing approximately 75-80 kDa, consisting of covalently linked light and heavy chains (semi-antibody). Such forms were extremely difficult to separate, even after affine purification.

The frequency of formation of the second form in various intact IgG isotypes is due, without limitation, to structural differences associated with the hinge region of the antibody isotype. A single amino acid substitution in the hinge region of human IgG4 can significantly reduce the production of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels normally seen with the human IgG1 hinge. The present invention encompasses antibodies having one or more mutations in the hinge, C _H 2 or C _H 3 region, which may be desirable, eg upon preparation, to increase the yield of the desired antibody form.

"Isolated antibody" means an antibody that has been identified and separated and/or isolated from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of the body, or from a tissue or cell in which the antibody exists or is naturally produced, is an "isolated antibody" within the meaning of the present invention. An isolated antibody also includes an antibody in situ in a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In some embodiments, the isolated antibody may be substantially free of other cellular material and/or chemical compounds.

The term "specifically binds" or the like means that the antibody, or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds" IL-4R, as used herein, includes antibodies that bind IL-4R, or a portion thereof, with a K _D of less than about 1000 nM, less than about 500 nM, less than about 300 less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than approximately 0.5 nM, as measured by surface plasmon resonance analysis. An isolated antibody that specifically binds human IL-4R may, however, be cross-reactive with other antigens, such as IL-4R molecules from non-human species.

Anti-IL-4R antibodies useful in the methods of the invention may include one or more amino acid substitutions, insertions, and/or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 inserts and/or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 deletions) in frame and/or CDRs regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, in common antibody sequence databases. The invention includes methods involving the use of antibodies and antigen-binding fragments thereof, which are derived from any amino acid sequence disclosed in the present description, where one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) in one or more backbones and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 for a tetrameric antibody, or 1, 2, 3 , 4, 5, or 6 for HCVR and LCVR antibodies) the CDRs of the regions are mutated with the corresponding residue(s) of the germline sequence from which the antibody was derived, or with the corresponding residue(s) of another human germline sequence, or subjected to a conservative amino acid substitution for the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germ mutations"). One of ordinary skill in the art, starting from the heavy and light chain variable region sequences disclosed herein, can readily generate numerous antibodies and antigen-binding fragments that include one or more individual germline mutations, or combinations thereof. In some embodiments, all framework and/or CDR residues in the V _H and/or V _L domains are mutated back to residues present in the original germline sequence from which the antibody was derived. In other embodiments, only some residues are mutated back to residues from the original germline sequence, e.g., only mutated residues present within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only mutated residues present in CDR1, CDR2 or CDR3. In other embodiments, one or more framework and/or CDR residues are mutated with the corresponding residue(s) of a different germline sequence (ie, a germline sequence that differs from the germline sequence from which the antibody was originally derived). In addition, the antibodies of the present invention may contain any combination of two or more germline mutations in the framework and/or CDR regions, for example, where certain individual residues are mutated with the corresponding residue of a particular germline sequence, while some other residues that differ from the original germline sequence are retained or mutated to the corresponding residue of another germline sequence. Once produced, antibodies and antigen-binding fragments that contain one or more germline mutations can be readily tested for one or more of the desired properties, such as increased binding specificity, increased binding affinity, increased or improved antagonistic or agonistic biological properties (depending on circumstances), reduced immunogenicity, etc. The use of antibodies and antigen-binding fragments prepared in this general manner is within the scope of the present invention.

The invention also includes methods involving the use of antibodies against IL-4R, including variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed in this application, which contain one or more conservative substitutions. For example, the invention includes the use of anti-IL-4R antibodies having the amino acid sequences of HCVR, LCVR, and/or CDR, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions compared to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

The term "surface plasmon resonance" refers to an optical phenomenon that allows real-time interaction analysis to detect changes in protein concentrations in a biosensor array, such as when using the BIAcore™ system (Biacore Life Sciences, a division of GE Healthcare, Piscataway, NJ).

The term "K _D " refers to the equilibrium dissociation constant of a particular antigen-antibody interaction.

The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, which is known as a paratope. One antigen may have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can produce different biological effects. Epitopes may be conformational or linear. The conformational epitope is formed by spatially contiguous amino acids from different segments of the linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues in the polypeptide chain. Under some circumstances, an epitope on an antigen may include saccharide groups, phosphoryl groups, or sulfonyl groups.

Obtaining human antibodies

Methods for producing human antibodies in transgenic mice are known in the art. Any such known methods may be used within the scope of the invention to generate human antibodies that specifically bind to human IL-4R.

Using VELOCIMMUNE™ technology (see, for example, US 6596541, Regeneron Pharmaceuticals) or any other known method for producing monoclonal antibodies, high affinity anti-IL-4R chimeric antibodies are initially isolated that have a human variable region and a mouse constant region. VELOCIMMUNE ^® technology involves the creation of a transgenic mouse, the genome of which includes the variable regions of the human heavy and light chains, functionally associated with endogenous mouse constant region loci in such a way that, in response to antigen stimulation, the mouse produces an antibody containing a human variable region and a mouse constant region. The DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to the DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

Typically, a ^{VELOCIMMUNE®} mouse is stimulated with a target antigen, after which lymphatic cells (such as B cells) are isolated from mice that express antibodies. Lymphatic cells can be fused with a myeloma cell line to obtain immortalized hybridoma cell lines, after which such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the target antigen. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the desired isotypic heavy chain and light chain constant regions. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

Initially, high-affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. Antibodies are analyzed and selected for the desired properties, including affinity, selectivity, epitope, etc., using standard techniques known to those skilled in the art. The murine constant regions are replaced with the desired human constant region, resulting in a fully human antibody of the invention, eg wild-type or modified IgG1 or IgG4. Although the constant region chosen may vary depending on the particular application, characteristics such as high affinity antigen binding and target specificity are inherent in the variable region.

Typically, antibodies that can be used in the methods of the invention have high affinities, as described above, when measured by binding to an antigen that is either immobilized on the solid phase or is in the solution phase. Mouse constant regions are replaced with the desired human constant regions to produce fully human antibodies of the invention. Although the constant region chosen may vary depending on the particular application, characteristics such as high affinity antigen binding and target specificity are inherent in the variable region.

Specific examples of human antibodies or antigen-binding antibody fragments that specifically bind IL-4R that can be used in the methods of the present invention include any antibody or antigen-binding fragment that includes the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained in the variable region. heavy chain (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 22, 26, 42, 46, 50, 66, 70, 74, 90, 94, 98, 114, 118 , 122, 138, 142, 146, 162, 166, 170, 186, 190, 194, 210, 214, 218, 234, 238, 242, 258, and 262. An antibody or antigen binding fragment may include three light chain CDRs (LCVR1, LCVR2, LCVR3) contained in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 20, 24, 34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120, 130, 140, 144, 154, 164, 168, 178, 188, 192, 202, 212, 216, 226, 236, 240, 250, 260 and 264. Identification methods and techniques The CDRs in the HCVR and LCVR amino acid sequences are known in the art and can be used to identify CDRs in the HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary rules that may be used to define CDR boundaries include, for example, the definition of Kabat, the definition of Chothia, and the definition of AbM. In general terms, Kabat's definition is based on sequence variability, Chothia's definition is based on the location of structural loop regions, and AbM's definition is a compromise between Kabat's and Chothia's approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); and Martin et al., Proc. Natl. Acad. sci. USA 86: 9268-9272 (1989). Generic databases are also available for identifying CDR sequences in an antibody.

In some embodiments, the antibody or antigen-binding fragment thereof comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) from heavy and light chain variable region (HCVR/LCVR) amino acid sequence pairs selected from the group consisting of SEQ ID NO: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106 114/116 118/120 122/130 138/140 142/144 146/154 162/164 166/168 170/178 186/188 190/192 194/ 202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260 and 262/264.

In some embodiments, the antibody or antigen-binding fragment thereof comprises six CDRs (HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3) having amino acid sequences that are selected from the group consisting of SEQ ID NO: 4/6/8/12/ 14/16; 28/30/32/36/38/40; 52/54/56/60/62/64; 76/78/80/84/86/88; 100/102/104/108/110/112; 124/126/128/132/134/136; 148/150/152/156/158/160; 172/174/176/180/182/184; 196/198/200/204/206/208; 220/222/224/228/230/232; and 244/246/248/252/254/256.

In some embodiments, the antibody or antigen-binding fragment thereof comprises HCVR/LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NO: 2/10, 18/20, 22/24, 26/34, 42/44, 46/ 48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/ 250, 258/260 and 262/264.

Pharmaceutical compositions

The invention includes methods that include administering an IL-4R antagonist to a patient, wherein the IL-4R antagonist is contained in a pharmaceutical composition. Pharmaceutical compositions of the invention are formulated with suitable carriers, excipients, and other means that provide suitable transfer, delivery, tolerability, and the like. Many suitable formulations can be found in the reference book known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipid containing lipid (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water emulsions, and water-in-oil, carbovax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbovax. See also Powell et al., "Compendium of excipients for parenteral formulations", PDA (1998) J. Pharm. sci. Technol., 52: 238-311.

The dose of an antibody administered to a patient according to the methods of the invention may vary depending on the patient's age and weight, symptoms, conditions, route of administration, and the like. The preferred dose is usually calculated according to body weight or body surface area. Depending on the severity of the condition, the frequency and duration of therapy may be adjusted. Effective dosages and administration schedules for pharmaceutical compositions comprising anti-IL-4R antibodies can be determined empirically; for example, the change in the patient's condition can be monitored during periodic examination and, depending on this, adjust the dose. In addition, cross-species conversion of dosages can be performed using known methods (eg, Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, for example, incorporation into liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987 , J Biol Chem 262: 4429-4432). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intratracheal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with others. biologically active agents.

The pharmaceutical composition of the invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with respect to subcutaneous delivery, the pen can be easily used in the delivery of the pharmaceutical composition of the invention. Such a syringe pen can be reusable or disposable. A refillable pen typically uses a replaceable cartridge that contains a pharmaceutical composition. After all of the pharmaceutical composition in the cartridge has been fully administered, the empty cartridge can be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The syringe pen can then be reused. There is no replaceable cartridge in the disposable syringe pen. Instead, the disposable pen is supplied with a pre-filled pharmaceutical composition that is contained in a reservoir within the pen. After the pharmaceutical composition in the tank ends, the entire device is discarded.

A variety of refillable pens and autoinjector devices may be used in subcutaneous delivery of the pharmaceutical composition of the invention. Examples include, without limitation, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ handle (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ handle, HUMALOG™ handle, HUMALIN 70/30 handle ™ (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ handle (Becton Dickinson, Franklin Lakes , NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany) to name but a few. Examples of disposable pens that can be used in subcutaneous delivery of the pharmaceutical composition of the present invention include, without limitation, SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), SURECLICK™ autoinjector (Amgen , Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and the HUMIRA™ pen (Abbott Labs, Abbott Park IL), to name but a few.

In the case of direct sinus administration, the pharmaceutical compositions of the invention may be administered using, for example, a microcatheter (eg, endoscope and microcatheter), aerosol generator, powder dispenser, nebulizer, or inhaler. The methods include administering an IL-4R antagonist to a subject in need thereof in aerosol form. For example, aerosolized anti-IL-4R antibodies can be administered to treat asthma in a patient. Antibodies in the form of an aerosol can be manufactured as described, for example, US 8178098, which is fully included in the present description.

In some situations, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials may be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed near the target of the composition, requiring only a fraction of the systemic dose to be administered (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, page 115 -138). Other controlled release systems are reviewed by Langer, 1990, Science 249: 1527-1533.

Formulations for injection may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, drip infusion, and the like. These injectable preparations can be manufactured by known methods. For example, injectable preparations can be made, for example, by dissolving, suspending or emulsifying an antibody or salt thereof, as described above, in a sterile aqueous or oily medium commonly used for injection. As an aqueous injection medium, for example, physiological saline, isotonic solution containing glucose and other excipients, etc., which can be used in combination with an appropriate solubilizer such as alcohol (for example, ethanol), polyalcohol ( e.g. propylene glycol, polyethylene glycol), non-ionic surfactant [e.g. polysorbate 80, HCO-50 (adduct of polyoxyethylene (50 mol) with hydrogenated castor oil)], etc. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizer such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled into a suitable ampoule.

Preferably, the oral or parenteral pharmaceutical compositions described above are formulated into unit dose dosage forms suitable for adjusting the dosage of the active ingredients. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like.

Examples of pharmaceutical compositions comprising an anti-IL-4R antibody that can be used in the context of the invention are disclosed, for example, in US Patent Application Publication 2012/0097565.

Dosage

The amount of an IL-4R antagonist (eg, anti-IL-4R antibody) administered to a subject according to the methods of the invention is generally a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" means an amount of an IL-4R antagonist that results in one or more of the following: (a) a reduction in the incidence of asthma exacerbations; (b) improvement in one or more asthma-related parameters (as defined elsewhere herein); and/or (c) a detectable improvement in one or more symptoms or signs of an inflammatory disease of the upper respiratory tract. A "therapeutically effective amount" also includes that amount of an IL-4R antagonist that inhibits, prevents, reduces, or delays the progression of asthma in a subject.

In the case of an anti-IL-4R antibody, a therapeutically effective amount may be from about 0.05 mg to about 600 mg, for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2 .0 mg, approximately 3.0 mg, approximately 5.0 mg, approximately 7.0 mg, approximately 10 mg, approximately 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, approximately 210 mg, approximately 220 mg, approximately 230 mg, approximately 240 mg, approximately 250 mg, approximately 260 mg, approximately 270 mg, approximately 280 mg, approximately 290 mg, approximately 300 mg, approximately 310 mg, approximately 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, approximately 460 mg, approximately 470 mg, approximately 480 mg, approximately 490 mg, approximately 500 mg, approximately 510 mg, approximately 520 mg, approximately 530 mg, approximately 540 mg, approximately 550 mg, approximately 560 mg, approximately 570 mg, about 580 mg, about 590 mg, or about 600 mg of anti-IL-4R antibody. In some embodiments, 300 mg of an anti-IL-4R antibody is administered.

The amount of IL-4R antagonist contained in individual doses can be expressed as milligrams of antibody per kilogram of patient body weight (ie, mg/kg). For example, an IL-4R antagonist may be administered to a patient at a dose of about 0.0001 to about 10 mg/kg of the patient's body weight.

Combination Therapies

Methods of the invention, in some embodiments, include administering to a subject one or more additional therapeutic agents in combination with an IL-4R antagonist. When used in the present description, the expression "in combination with" means that additional therapeutic agents are administered before, after or in parallel with a pharmaceutical composition comprising an IL-4R antagonist. In some embodiments, the term "in combination with" includes the sequential or parallel administration of an IL-4R antagonist and a second therapeutic agent. The present invention includes methods for treating asthma or an associated condition or complication or amelioration of at least one exacerbation comprising administering an IL-4R antagonist in combination with a second therapeutic agent for additive or synergistic activity.

For example, when administered "before" a pharmaceutical composition comprising an IL-4R antagonist, the additional therapeutic agent may be administered about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes before administration of a pharmaceutical composition comprising an IL-4R antagonist. When administered "after" a pharmaceutical composition comprising an IL-4R antagonist, the additional therapeutic agent may be administered at about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after administration of a pharmaceutical composition comprising an IL-4R antagonist. Administration "in parallel" with a pharmaceutical composition comprising an IL-4R antagonist means that the additional therapeutic agent is administered to a subject in a separate dosage form within less than 5 minutes (before, after, or at the same time) of administration of a pharmaceutical composition comprising an IL-4R antagonist -4R, or administered to a subject as a single combination dosage form comprising both an additional therapeutic agent and an IL-4R antagonist.

The additional therapeutic agent may be, for example, another IL-4R antagonist, an IL-1 antagonist (including, for example, an IL-1 antagonist as set forth in US Pat. No. 6,927,044), an IL-6 antagonist, an IL-6R antagonist (including, for example, anti-IL-6P antibody as set forth in US Pat. No. 7,582,298), TNF antagonist, IL-8 antagonist, IL-9 antagonist, IL-17 antagonist, IL-5 antagonist, IgE antagonist, CD48 antagonist, leukotriene inhibitor, antifungal agent, NSAID , a long-acting _beta2 -agonist (eg, salmeterol or formoterol), an inhaled corticosteroid (eg, fluticasone or budesonide), a systemic corticosteroid (eg, oral or intravenous), methylxanthine, nedocromil sodium, cromolyn sodium, or combinations thereof. For example, in some embodiments, a pharmaceutical composition comprising an IL-4R antagonist is administered in combination with a combination comprising a long acting beta ₂ agonist and an inhaled corticosteroid (eg, fluticasone + salmeterol [eg, Advair® (GlaxoSmithKline)]); or budesonide + formoterol [eg, Symbicort® (Astra Zeneca))]).

Introduction schemes

In some embodiments, multiple doses of an IL-4R antagonist may be administered to a subject over a period of time. Such methods include sequential administration of multiple doses of an IL-4R antagonist to a subject. As used herein, "sequential administration" means that each dose of an IL-4R antagonist is administered to a subject at different time points, eg, on different days separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention includes methods that include sequentially administering to a patient one initial dose of an IL-4R antagonist, followed by one or more secondary doses of an IL-4R antagonist, and optionally followed by one or more tertiary doses of an IL-4R antagonist.

The invention includes methods comprising administering to a subject a pharmaceutical composition comprising an IL-4R antagonist at an administration frequency of approximately four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks or less until a therapeutic response is achieved. In some embodiments involving the administration of a pharmaceutical composition comprising an anti-IL-4R antibody, a once weekly dose of approximately 75 mg, 150 mg, or 300 mg may be used. In other embodiments involving the administration of a pharmaceutical composition comprising an anti-IL-4R antibody, a biweekly administration of a dose of approximately 75 mg, 150 mg, or 300 mg may be used. In other embodiments involving the administration of a pharmaceutical composition comprising an anti-IL-4R antibody, a once every three week dose of approximately 75 mg, 150 mg, or 300 mg may be used. In other embodiments involving the administration of a pharmaceutical composition comprising an anti-IL-4R antibody, administration of a once every four week dose of approximately 75 mg, 150 mg, or 300 mg may be used. In other embodiments involving the administration of a pharmaceutical composition comprising an anti-IL-4R antibody, administration of a once every five week dose of approximately 75 mg, 150 mg, or 300 mg may be used. In other embodiments involving the administration of a pharmaceutical composition comprising an anti-IL-4R antibody, a once every six week dose of approximately 75 mg, 150 mg, or 300 mg may be used. In other embodiments involving the administration of a pharmaceutical composition comprising an anti-IL-4R antibody, administration of an once every eight week dose of approximately 75 mg, 150 mg, or 300 mg may be used. In other embodiments involving the administration of a pharmaceutical composition comprising an anti-IL-4R antibody, a twelve weekly dose of approximately 75 mg, 150 mg, or 300 mg may be used. The preferred route of administration is subcutaneous.

The term "week" or "weeks" refers to a period of (n×7 days) ± 2 days, preferably (n×7 days) ± 1 day, more preferably (n×7 days), where "n" specifies the number of weeks, e.g. , 1, 2, 3, 4, 5, 6, 8, 12 or more.

The terms "initial dose", "secondary dose" and "tertiary dose" refer to the time sequence of administration of the IL-4R antagonist. Thus, the "starting dose" is the dose that is administered at the beginning of the treatment regimen (also referred to as the "initial dose"); "secondary doses" are doses that are administered after the initial dose; and "tertiary doses" are doses that are administered after secondary doses. The initial, secondary, and tertiary doses may contain the same amount of IL-4R antagonist, but may generally differ from each other in frequency of administration. However, in some embodiments, the amount of IL-4R antagonist contained in the initial, secondary, and/or tertiary doses varies (eg, increased or decreased as needed) during treatment. In some embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the start of the treatment regimen as "loading doses" followed by subsequent doses that are administered at a lesser frequency (e.g., "maintenance doses"). In one embodiment, the maintenance dose may be less than the loading dose. For example, one or more 600 mg loading doses of the IL-4R antagonist may be administered, followed by maintenance doses of about 75 mg to about 300 mg.

In one embodiment, each secondary and/or tertiary dose is administered at 1-14 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8 , 8½, 9, 9½, 10, 10½, 1 1, 11½, 12, 12½, 13, 13½, 14, 14½ or more) weeks after the immediately preceding dose. The phrase "immediately preceding dose" means, in a multiple dose sequence, the dose of the IL-4R antagonist that is administered to the patient prior to the next subsequent dose in the sequence, without intermediate doses being administered.

The methods may include administering to the patient any number of secondary and/or tertiary doses of the IL-4R antagonist. For example, in some embodiments, only one secondary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in some embodiments, only one tertiary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to a patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to a patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to a patient may vary during treatment. The frequency of administration during treatment can also be adjusted by the physician depending on the needs of the individual patient after clinical examination.

The invention includes methods comprising sequentially administering an IL-4R antagonist and a second therapeutic agent to a patient for the treatment of asthma or a related condition. In some embodiments, the methods include administering one or more doses of an IL-4R antagonist followed by one or more doses (eg, 2, 3, 4, 5, 6, 7, 8 or more) of the second therapeutic agent. For example, one or more doses of about 75 mg to about 300 mg of the IL-4R antagonist may be administered, followed by one or more doses (e.g., 2, 3, 4, 5, 6, 7, 8 or more) of the second therapeutic means (for example, an inhaled corticosteroid or beta ₂ agonist, or any other therapeutic agent, as described elsewhere in this application) for the treatment, partial relief, alleviation or reduction in the intensity of one or more symptoms of asthma. In some embodiments, the IL-4R antagonist is administered at one or more doses (e.g., 2, 3, 4, 5, 6, 7, 8 or more) resulting in an improvement in one or more asthma-related parameters, followed by administration of a second a therapeutic agent for preventing recurrence of at least one symptom of asthma. Alternative embodiments relate to the concurrent administration of an IL-4R antagonist and a second therapeutic agent. For example, one or more doses (e.g., 2, 3, 4, 5, 6, 7, 8 or more) of an IL-4R antagonist are administered, followed by the second therapeutic agent being administered at a separate dose, at the same or different frequency as compared to IL-4R antagonist. In some embodiments, the second therapeutic agent is administered before, after, or simultaneously with the IL-4R antagonist.

Treatment groups

The methods of the invention include administering to a subject in need thereof a therapeutic composition comprising an IL-4R antagonist. The expression "subject in need thereof" means a human or non-human animal that exhibits one or more symptoms or signs of asthma (eg, eosinophilic asthma, including moderate-severe eosinophilic asthma), or has been diagnosed with asthma. For example, "subject in need thereof" may include, for example, subjects who, prior to treatment, exhibit (or exhibit) one or more asthma-related parameters such as, for example, an impaired FEV1 (e.g., less than 2.0 L), an impaired PEF (eg, less than 400 L/min), impaired PEF (eg, less than 400 L/min), ACQ5 score of at least 2.5, at least 1 awakening per night, and/or SNOT-22 score of at least 20. In various embodiments, methods for treating moderate, moderate-severe, and severe asthma in patients in need thereof may be used.

In a related embodiment, the "subject in need" may be a subject who was prescribed, prior to taking the IL-4R antagonist, or who is currently taking, an inhaled corticosteroid (ICS)/long-acting _beta2- adrenergic antagonist (LAAA) combination. . Examples of ICS/LAAA therapy include fluticasone/salmeterol combination therapy and budesonide/formoterol combination therapy. For example, the invention includes methods that include administering an IL-4R antagonist to a patient who has been on a regular course of ICS/LAAA for two or more weeks immediately prior to administration of the IL-4R antagonist (such prior therapies are referred to herein as "baseline treatments") . The invention includes therapeutic methods in which baseline treatments are suspended at or just before (eg, 1 day to 2 weeks before) the first administration of an IL-4R antagonist. Alternatively, baseline treatments may be continued in combination with administration of an IL-4R antagonist. In other embodiments, the amount of the ICS component, the BABA component, or both components is gradually reduced before or after the start of administration of the IL-4R antagonist. In some embodiments, the invention includes methods for treating patients with persistent asthma for at least ≥12 months. In one embodiment, a patient with persistent asthma may be refractory to treatment with a therapeutic agent such as a corticosteroid while being administered an IL-4R antagonist according to the present methods.

In some embodiments, the "subject in need thereof" may be a subject with elevated levels of an asthma-associated biomarker. Examples of asthma-related biomarkers include, but are not limited to, IgE, thymus-activated and regulated chemokine (TARC), eotaxin-3, CEA, YKL-40, and periostin. In some embodiments, the "subject in need thereof" may be a subject with a blood eosinophil level of ≥300/µl or a sputum eosinophil level of ≥3%. In one embodiment, the "subject in need" may be a subject with elevated levels of bronchial or airway inflammation as measured by fractional exhaled nitric oxide (FVOA).

Within the scope of the invention, normal IgE levels in healthy subjects are less than about 100 kU/L (eg, as measured using the ^ImmunoCAP® test [Phadia, Inc. Portage, MI]). Thus, the invention includes methods comprising selecting a subject that exhibits an elevated serum IgE level, which is a serum IgE level of greater than about 100 kU/L, greater than about 150 kU/L, greater than about 500 kU/L, greater than about 1000 kU/L, greater than about 1500 kU/L, greater than about 2000 kU/L, greater than about 2500 kU/L, greater than about 3000 kU/L, greater than about 3500 kU/L, greater than about 4000 kU /L, greater than about 4500 KU/L or more than about 5000 KU/L, and administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an IL-4R antagonist.

TARC levels in healthy subjects range from 10 ⁶ ng/l to 431 ng/l, with a mean value of approximately 239 ng/l (an example of an assay system for measuring TARC levels is the TARC quantification ELISA kit available under part number DDN00 on R&D Systems catalog, Minneapolis, MN). Thus, the invention includes methods comprising selecting a subject that exhibits an elevated TARC level, which is a serum TARC level of greater than about 431 ng/L, greater than about 500 ng/L, greater than about 1000 ng/L, greater than about 1500 ng/L, greater than about 2000 ng/L, greater than about 2500 ng/L, greater than about 3000 ng/L, greater than about 3500 ng/L, greater than about 4000 ng/L, greater than about 4500 ng /L or greater than about 5000 ng/L, and administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an IL-4R antagonist.

Eotaxin-3 belongs to a group of chemokines secreted by airway epithelial cells and is regulated by the Th2 cytokines IL-4 and IL-13 (Lilly et al., 1999, J. Allergy Clin. Immunol., 104: 786-790). The invention includes methods comprising administering an IL-4R antagonist to treat patients with elevated levels of eotaxin-3, e.g., greater than about 100 pg/ml, greater than about 150 pg/ml, greater than about 200 pg/ml, greater than about 300 pg/ml or greater than about 350 pg/ml. Serum levels of eotaxin-3 can be measured, for example, by ELISA.

Periostin is an extracellular matrix protein involved in Th2-mediated inflammatory processes. Periostin levels have been found to be elevated in patients with asthma (Jia et al., 2012 J. Allergy Clin. Immunol., 130:647-654.e10. doi: 10.1016/j.jaci.2012.06.025. Epub 2012 Aug 1). The present invention includes methods comprising administering an IL-4R antagonist for the treatment of patients with elevated periostin levels.

Fractional exhaled NO (FVOA) is a biomarker for bronchial or airway inflammation. VVOA is produced by airway epithelial cells in response to inflammatory cytokines, including IL-4 and IL-13 (Alwing et al., 1993, Eur. Respir. J., 6: 1368-1370). FVOA levels in healthy adults range from 2-30 parts per billion (ppb). An example of an assay for measuring FVOA is an assay using the NIOX apparatus (Aerocrine AB, Solna, Sweden). Assessment can be done before spirometry and after fasting for at least one hour. The invention includes methods comprising administering an IL-4R antagonist to patients with elevated levels of exhaled NO (EVOA), e.g., greater than about 30 billion, greater than about 31 billion, greater than about 32 billion, greater than about 33 billion, more than about 34 billion. or more than about 35 bn.

Cancer embryonic antigen (CEA) is a tumor marker that has been shown to correlate with non-neoplastic lung diseases (Marechal et al 1988, Anticancer Res. 8: 677-680). Serum CEA levels can be measured by ELISA. The invention includes methods comprising administering an IL-4R antagonist to patients with elevated levels of CEA, for example, greater than about 1.0 ng/ml, greater than about 1.5 ng/ml, greater than about 2.0 ng/ml, greater than about 2.5 ng/ml, greater than about 3.0 ng/ml, greater than about 4.0 ng/ml, or greater than about 5.0 ng/ml.

YKL-40 [named for its N-terminal amino acids tyrosine (Y), lysine (K), and leucine (L), and its molecular weight of 40 kDa] is a chitinase-like protein whose elevation was found to correlate with exacerbation of asthma, IgE and eosinophils (Tang et al., 2010, Eur. Respir. J., 35: 757-760). Serum levels of YKL-40 are measured, for example, by ELISA. The invention includes methods comprising administering an IL-4R antagonist to patients with elevated levels of YKL-40, for example, greater than about 40 ng/ml, greater than about 50 ng/ml, greater than about 100 ng/ml, greater than about 150 ng/ml ml, greater than about 200 ng/ml, or greater than about 250 ng/ml.

Eosinophils and neutrophils in induced sputum are known direct markers of airway inflammation (Djukanovic et al., 2002, Eur. Respire. J., 37: 1S-2S). Sputum separation is induced by inhalation over hypertonic sodium chloride solution, and then the cells in the sputum are counted in accordance with known methods, for example, according to the guidelines of the European Respiratory Society. The invention includes methods comprising administering an IL-4R antagonist to patients with elevated sputum eosinophil levels, eg, greater than about 2.5% or greater than about 3%.

Methods for assessing asthma-related pharmacodynamic parameters

The invention also includes methods for assessing one or more asthma-related pharmacodynamic parameters in a subject in need thereof elicited by administration of a pharmaceutical composition comprising an interleukin 4 receptor (IL-4R) antagonist. A decrease in the incidence of an asthma exacerbation (as described above) or an improvement in one or more asthma-related parameters (as described above) may be correlated with an improvement in one or more asthma-related pharmacodynamic parameters, however, such a correlation need not be observed in all cases.

Examples of "asthma-related pharmacodynamic parameters" include, for example, the following: (a) expression levels of biomarkers; (b) serum protein and RNA analysis; (c) eosinophil and neutrophil levels in induced sputum; (d) exhaled nitric oxide (EFNO); and (e) the content of eosinophils in the blood. "Improvement in an asthma-related pharmacodynamic parameter" means, for example, a decrease from baseline in one or more biomarkers such as TARC, eotaxin-3 or IgE, a decrease in sputum eosinophils or neutrophils, VVOA, or blood eosinophils. As used herein, the term "baseline", in relation to an asthma-related pharmacodynamic parameter, means the numerical value of an asthma-related pharmacodynamic parameter in a patient before or during administration of a pharmaceutical composition of the invention.

To evaluate an asthma-related pharmacodynamic parameter, the parameter is quantified at baseline and at time after administration of the pharmaceutical composition of the present invention. For example, an asthma-related pharmacodynamic parameter can be measured on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 14, or per week 3, week 4, week 5, week 6, week 7, week 8, week 9, week 10, week 1-1, week 12, week 13, week 14, week 15, week 16, week 17, week 18, week 19, week 20, week 21, week 22, week 23, week 24 or more after the initial use in the treatment of the pharmaceutical composition of the present invention. The difference between the parameter value at a specific time point after the start of treatment and the parameter value at baseline is used to determine whether there has been a change, such as an "improvement", in an asthma-related pharmacodynamic parameter (e.g., an increase or decrease, as appropriate, in depending on the specific measured parameter).

In some embodiments, administration of an IL-4R antagonist to a patient causes a change, such as a decrease or increase, in the expression of a particular biomarker. Asthma-related biomarkers include the following: (a) total IgE; (b) thymus and upregulated chemokine (TARC) activation; (c) YKL-40 and (d) cancer embryonic antigen (CEA, also known as CEA cell adhesion molecule 5 [CEACAM5]) in serum and (e) eotaxin-3 in plasma. For example, administration of an IL-4R antagonist to a patient with asthma may cause one or more of a decrease in TARC or eotaxin-3 levels, or a decrease in serum total IgE levels. A decrease can be detected at week 1, week 2, week 3, week 4, week 5 or more after administration of the IL-4R antagonist. The expression of a biomarker can be analyzed using methods known in the art. For example, protein levels can be measured using ELISA (enzyme-linked immunosorbent assay), or RNA levels can be measured using reverse transcription polymerase chain reaction (RT-PCR).

Expression of a biomarker, as discussed above, can be analyzed by detecting protein or RNA in serum. Serum samples can also be used to monitor additional protein or RNA biomarkers associated with response to IL-4R antagonist therapy, IL-4/IL-13 signaling, asthma, atopic or eosinophilic diseases (eg, by measuring soluble IL-4Rα, IL- 4, IL-13, periostin). In some embodiments, RNA samples are used to determine RNA levels (non-genetic analysis), such as RNA levels of biomarkers; and in other embodiments, the RNA samples are used for transcriptome sequencing (eg, genetic analysis).

EXAMPLES

The following examples are provided to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compositions of the invention may be made and used, and should not limit the scope of what the present inventors consider to be their invention. Efforts have been made to ensure accuracy with respect to the numbers used (eg values, temperatures, etc.), however, some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or about atmospheric pressure.

Example 1 Generating Human Anti-Human IL-4R Antibodies

Human anti-hIL-4R antibodies were designed as described in US Pat. No. 7,608,693. Table 1 lists the sequence identifiers for the amino acid sequence pairs of the heavy and light chain variable regions and the amino acid sequences of the CDRs, selected anti-IL-4R antibodies, and designations of their respective antibodies. .

Table 1 SEQID NO: Antibody designation HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 H1H095-a 2 4 6 8 10 12 14 16 H1H095-b 18 4 6 8 20 12 14 16 H1H095-c 22 4 6 8 24 12 14 16 H1H097-a 26 28 thirty 32 34 36 38 40 H1H097-b 42 28 thirty 32 44 36 38 40 H1H097-c 46 28 thirty 32 48 36 38 40 H1H093-a 50 52 54 56 58 60 62 64 H1H093-b 66 52 54 56 68 60 62 64 H1H093-c 70 52 54 56 72 60 62 64 H1H093-d 74 76 78 80 82 84 86 88 H1H093-e 90 76 78 80 92 84 86 88 H1H093-f 94 76 78 80 96 84 86 88 H1H094-a 98 100 102 104 106 108 110 112 H1H094-b 114 100 102 104 116 108 110 112 H1H094-c 118 100 102 104 120 108 110 112 H1H096-a 122 124 126 128 130 132 134 136 H1H096-b 138 124 126 128 140 132 134 136 H1H096-c 142 124 126 128 144 132 134 136 H1H098-a 146 148 150 152 154 156 158 160 H1H098-b 162 148 150 152 164 156 158 160 H1H098-c 166 148 150 152 168 156 158 160 H1H099-a 170 172 174 176 178 180 182 184 H1H099-b 186 172 174 176 188 180 182 184 H1H099-c 190 172 174 176 192 180 182 184 H4H083-a 194 196 198 200 202 204 206 208 H4H083-b 210 196 198 200 212 204 206 208 H4H083-c 214 196 198 200 216 204 206 208 H4H121-a 218 220 222 224 226 228 230 232 H4H121-b 234 220 222 224 236 228 230 232 H4H121-c 238 220 222 224 240 228 230 232 H4H118-a 242 244 246 248 250 252 254 256 H4H118-b 258 244 246 248 260 252 254 256 H4H118-c 262 244 246 248 264 252 254 256

An example of an IL-4R antagonist used in the following Examples is a human anti-IL-4R antibody designated in Table 1 as H1H098-b (also referred to herein as "mAb1").

Example 2 Clinical Study of Subcutaneous Anti-IL-4R Antibody (mAb1) In Patients with Persistent Moderate-Severe Eosinophilic Asthma, Including Asthma Patients with Chronic Hyperplastic Eosinophilic Sinusitis

A. Objectives and summary of the study

A randomized, placebo-controlled, double-blind, parallel-group study was performed with once-weekly subcutaneous administration of either 300 mg mAb1 or placebo for 12 weeks in patients with persistent moderate to severe eosinophilic asthma that was partially controlled/uncontrolled by therapy. inhaled corticosteroids (ICS) and long-acting _beta2 -agonists (LAAAs). The primary aim of the study was to investigate the effects of mAb1 administered subcutaneously once a week for 12 weeks compared with placebo in reducing the incidence of asthma exacerbations in patients with persistent moderate to severe eosinophilic asthma. Additional study objectives were to assess the safety and tolerability of mAb1 administered subcutaneously once a week for 12 weeks in patients with persistent moderate-severe eosinophilic asthma, and to evaluate serum concentrations of mAb1 after subcutaneous administration once a week in for 12 weeks, in patients with persistent moderate to severe eosinophilic asthma.

Prior to screening, patients were required to continuously take any of the following doses and formulations of ICS/LAAA combination therapy (also referred to as "baseline therapy") for at least 1 month:

Combination therapy with fluticasone/salmeterol

- Advair® Diskus - dry powder inhaler (DPI): 250/50 mcg twice a day or 500/50 mcg twice a day; or

- Advair® HFA - metered dose inhaler (MDI): 230/42 mcg twice a day or 460/42 mcg twice a day; or

Combination therapy with budesonide/formoterol (Symbicort® 160/9 mcg twice daily or 320/9 mcg twice daily); or

Combination therapy with mometasone/formoterol (Dulera® 200/10 mcg twice daily or 400/10 mcg twice daily).

Patients treated with budesonide/formoterol or mometasone/formoterol were switched to an equivalent dose of fluticasone/salmeterol at randomization (Day 1), and patients treated with fluticasone/salmeterol continued as baseline therapy.

Patients who met the inclusion and exclusion criteria (see below) were randomized to receive one of the following therapies: 300 mg mAb1 administered subcutaneously once a week for 12 weeks; or placebo administered subcutaneously once a week for 12 weeks.

The study included a 2-week screening period, a 12-week treatment period including a stable 4-week baseline phase, and an 8-week baseline withdrawal phase after randomization followed by an 8-week post-treatment follow-up period.

Algorithm for the cancellation of basic therapy (ICS / BADD) :

Patients continued baseline therapy with fluticasone/salmeterol, twice daily, for 4 weeks after initial add-on therapy or treatment with 300 mg mAb1 (or placebo). 4 weeks after randomization, patients were switched from fluticasone/salmeterol twice daily combination therapy to an equivalent dose of fluticasone ICS monotherapy (comprising either Flovent® Diskus - DPI formulation, 250 µg or 500 µg twice daily; or Flovent® HFA - MDI formulation, 220 μg or 440 μg twice daily). The BADD component (i.e. salmeterol) was cancelled. At follow-up visits starting at week 6, the fluticasone dose was reduced by approximately 50%, provided that the patient did not meet any of the criteria for an asthma exacerbation (as defined below). If asthma exacerbations did not occur, ICS was discontinued according to the following regimen:

Stable phase of basic therapy Phase of withdrawal of basic therapy Week 4 Week 6 Week 7 Week 8 Week 9 Fluticasone/salmeterol (DPI): 250/50mcg BID Fluticasone (DPI): 250 mcg BID 100 mcg BID 50 mcg BID 0 µg BID 0 µg BID Fluticasone/salmeterol (DPI): 500/50mcg BID Fluticasone (DPI): 500 mcg BID 250 mcg BID 100 mcg BID 50 mcg BID 0 µg BID Fluticasone/salmeterol (MDI): 230/42 mcg BID Fluticasone (MDI): 220 mcg BID 110 mcg BID 44 mcg BID 0 µg BID 0 µg BID Fluticasone/salmeterol (MDI): 460/42 mcg BID Fluticasone (MDI): 440 mcg BID 220 mcg BID 110 mcg BID 44 mcg BID 0 µg BID BID - twice a day

After completion of 12 weeks of study product treatment (or after abortive discontinuation), patients were switched to their baseline dose of fluticasone/salmeterol, budesonide/formoterol, or mometasone/formoterol (dose at entry into the study) and albuterol or levalbuterol, if necessary, to monitor their symptoms for an additional 8 weeks of drug use in the study prior to the final safety assessment.

Adult patients were included in the study based on the following criteria: (1) a physician's diagnosis of persistent asthma for at least ≥12 months, based on the Global Initiative on Asthma (GINA) 2009 guidelines, with probable eosinophilic airway inflammation; and (2) asthma is partially controlled or not controlled with long-acting inhaled corticosteroid/beta-agonist combination therapy according to the following criteria: once daily or 500/50 micrograms twice daily, or MDI formulation: 230/42 micrograms twice daily or 460/42 micrograms twice daily), or budesonide/formoterol combination therapy (160/9 micrograms twice daily) day or 320/9 mcg twice daily) or mometasone/formoterol combination therapy (200/10 mcg twice daily or 400/10 mcg twice daily) for at least 1 month prior to screening; (ii) blood eosinophils ≥300 cells/µl or sputum eosinophils ≥3% during the screening phase; (iii) Asthma Control Questionnaire score (5-item version, ACQ, Juniper) ≥1.5 and ≥3.0 at screening; (iv) FEV1 ≥50% of predicted normal during the screening phase (maximum 3 trials) and on the day of randomization prior to the first dose (maximum 3 trials); (v) had, in the 2 years prior to screening or treatment with one or more systemic (oral and/or parenteral) steroids, an episode of worsening asthma or hospitalization or emergency room attendance due to worsening asthma; and (vi) a documented case of reversibility within 12 months of screening that satisfies the criteria of at least 12% and 200 ml in FEV1 after 200 µg to 400 µg (2-4 inhalations) of albuterol during the screening phase (maximum 3 attempts), or a reported case of positive methacholine challenge (methacholine PD20 ≤8 mg) within 12 months prior to screening. Patients with moderate-to-severe asthma that is partially or uncontrollable with moderate-to-high dose combination therapy with inhaled corticosteroids and long-acting beta-agonists (Advair®, Symbicort®, or Dulera®) and blood eosinophils greater than or equal to 300 cells per microliter, or sputum eosinophils greater than or equal to 3% during the screening phase, were included in the study.

Patients who met all inclusion criteria were screened for the following exclusion criteria: (1) patients under 18 years of age or over 65 years of age; (2) clinically significant laboratory abnormalities that may indicate an unknown disease and require additional evaluation; (3) chronic obstructive pulmonary disease (COPD) and/or other lung diseases that interfere with lung function test results; (4) patients requiring beta-adrenergic receptor blockers for any reason; (5) smoking or not smoking within 6 months prior to screening; (6) a history of smoking with a history of smoking >10 pack-years; (7) hospital admissions or emergency room visits due to an asthma exacerbation in the last 2 months prior to screening; (8) plans to initiate allergy vaccination during the study; (9) exposure to another experimental antibody for a pre-screening time period of less than 5 antibody half-lives but not less than 30 days, or, if the antibody half-life is not known, for a pre-screening time period of at least 6 months ; (10) previous registration in the current study; (11) the patient was a researcher, family member, or employee at a research facility; (12) known or suspected non-compliance, alcohol or drug use; (13) inability to follow study procedures (eg, due to language difficulties or psychological disturbances); (14) altered sleep patterns (eg night shift worker); (15) treatment with drugs that prolong the QTc interval; (16) concomitant severe disease(s) in which ICS (eg, active or inactive pulmonary tuberculosis) or LABAs (eg, diabetes, cardiovascular disease, hypertension, hyperthyroidism, thyrotoxicosis, etc.) are contraindicated; (17) use of injectable glucocorticosteroids or oral systemic glucocorticosteroids within 2 months prior to screening or more than 3 courses within 6 months prior to screening; (18) pre-treatment with variable doses of ICS alone or in combination with non-steroidal baseline therapy (other than fluticasone/salmeterol combination therapy, budesonide/formoterol combination therapy, or mometasone/formoterol combination therapy); (19) taking illegal concomitant medications (listed below); (20) known allergy to doxycycline or related compounds; (21) pregnancy or planned pregnancy during the study, breast-feeding or unwillingness to use an effective method of contraception; and (22) recent parasitic infection or travel to an endemic parasite area within 6 months prior to screening.

Patients remained on a constant dose of asthma basal therapy for the first four weeks of the study, after which the basal therapy dose was gradually reduced. Initially, the long-acting beta-agonist, a component of background therapy, was discontinued at week 4, and then the dose of inhaled corticosteroid was halved every 2 weeks until week 12. Patients continued treatment in the study until the end of the study or until they dropped out due to an exacerbation of asthma or for any another reason.

B. Study treatment

Test product: Sterile mAb1 solution 150 mg/ml for s.c. injections, in a 5 ml glass vial. Each vial contained a draw volume of 2 ml. A dose of 300 mg was administered subcutaneously at the study center, once a week, in the morning, for 12 weeks.

Placebo: Sterile placebo for s.c. injection was provided in an equally matched 5 ml glass vial. Each vial contained a draw volume of 2 ml. The placebo was administered subcutaneously at the study center, once a week, in the morning, for 12 weeks.

The following concomitant medications were not allowed throughout the study: any other inhaled steroid other than combination therapy with fluticasone/salmeterol or fluticasone administered in the protocol (either budesonide/formoterol or mometasone/formoterol during the screening period); systemic or ophthalmic steroids; other dietary supplements other than salmeterol, a component of fluticasone/salmeterol combination therapy administered as part of the protocol; any other combined ICS/BADD products other than those listed above; any inhaled anticholinergic (for example, ipratropium bromide or tiotropium); methylxanthines (theophylline, aminophyllines); cromones; anti-IgE therapy; lipoxygenase inhibitors; and leukotriene receptor antagonists or leukotriene synthesis inhibitors.

C. Effectiveness of treatment

The primary endpoint of this study was the occurrence of an asthma exacerbation, defined by any of the following: (1) a 30% or greater decrease from baseline in peak morning expiratory flow (PEF) on two consecutive days; or (2) six or more additional puffs of albuterol or levalbuterol aerosol for breathing relief over a 24-hour period (compared to baseline) on 2 consecutive days; or (3) worsening of asthma, as determined by the investigator, requiring: (a) systemic (oral and/or parenteral) steroid therapy, or (b) an increase in the last dose of ICS received prior to withdrawal from the study by ≥4 times, or (c ) hospitalizations.

Additional study endpoints included mean changes from baseline in the following: (1) Forced expiratory volume in 1 second (FEV1) in liters measured at each visit; (2) Peak expiratory flow in the morning and evening (PEF and PEF) in liters/minute, measured daily; (3) Daily use of albuterol/levalbuterol inhaled/day; (4) Five-item Asthma Control Questionnaire (ACQ5) score at each visit; (5) Nighttime awakenings (once per night) measured daily; and (6) Assessment in the 22-item Nasal and Sinus Outcome Test (SNOT-22), at baseline and at the end of treatment (at Week 12), to assess upper respiratory symptoms. Additional endpoints also included the percentage of patients with a mixed asthma event, defined as a 30% or greater decrease from baseline in the morning of PSV on two consecutive days, in combination with ≥6 additional puffs of albuterol or levalbuterol aerosol to relieve breathing over a 24-hour period compared with baseline for 2 consecutive days. PSV, ACQ5, asthma symptom scores, nighttime awakenings, and use of breathing medications were recorded in an electronic diary. The average number of nighttime awakenings, ranging from 0-10, was averaged over the previous 7 days. Morning and evening asthma symptom scores consisted of an unconfirmed, patient-reported outcome assessed on a 5-point Likert scale, with a higher score corresponding to a poorer outcome (Table 2). Patients recorded total symptom scores twice daily prior to measuring PSV. The data is described as an average of 7 days before the specified time (see, for example, Figures 26A and 26B).

table 2
Assessment of asthma symptoms A) Morning symptom assessment: 0 = No asthma symptoms, sleeping through the night 1 = Good sleep but some complaints in the morning. There were no awakenings at night 2= One awakening due to asthma (including early awakening) 3= Multiple awakenings due to asthma (including early awakenings) 4= Bad night, waking up most of the night due to asthma B) Evening symptom assessment: 0 = Very good, no asthma symptoms 1 = One case of wheezing, cough, or shortness of breath 2= More than one episode of wheezing, coughing, or shortness of breath without affecting daily activities 3= Wheezing, coughing, or shortness of breath for most of the day with some disturbance in daily activities 4= Asthma is very severe. Inability to perform daily activities as usual

D. Control of adverse events

Safety during the study was assessed by monitoring Adverse Events and Serious Adverse Events.

An adverse event (AE) is any undesirable medical event in a subject or subject of a clinical trial who has been administered a pharmaceutical product. Thus, AE can be any undesirable and unintended sign (including abnormal laboratory data), symptom or disease associated in time with the use of the drug, regardless of whether it is considered to be drug (investigational) drug related. AE also includes: any worsening (i.e., any clinically significant change in frequency and/or intensity) of a pre-existing condition that is temporally related to study drug use; abnormal laboratory findings that the investigator considers clinically significant; and any unwanted medical event.

A severe adverse event (SAE) is any adverse medical event that, at any dose, results in death; is life-threatening; requires hospitalization in a hospital or an extension of the current hospitalization; results in permanent or significant incapacity or disability; is a congenital anomaly/congenital defect; or is an important medical event.

E. Statistical methods

For the primary analysis of the proportion of patients experiencing an asthma exacerbation, a logistic regression model was used to compare the SAR group with placebo. The model included treatment duration and stratification factor (previous dose of ICS/LAAA combination therapy). The primary analysis was based on a modified intent-to-treat (mITT) group that included all randomized patients who received at least one dose of investigational drug (ITT). A stratified chi-square test was also used to validate the primary analysis.

With regard to additional endpoints of efficacy, with the exception of SNOT-22, change from baseline was analyzed using a mixed effect model with multiple measures method (MMRM). The model included change from baseline to week 12 as response variables, as well as treatment factors (fixed effects), stratification factor, visits, treatment versus visit, baseline, and baseline versus visit. Statistical inference when comparing treatments for change from baseline at week 12 was derived from a mixed effect model. Change in SNOT-22 from baseline was analyzed using analysis of covariance (ANCOVA), using measurements at the end of treatment to replace missing data. Pharmacodynamic effects were evaluated using the MMRM model in the post hoc method. No multiplicity adjustments were made as only one primary endpoint and analysis was present. Safety variables including AE, laboratory values, vital signs, ECG, clinical laboratory observations and physical examination were summarized using descriptive statistics.

Demographic and clinical indicators were summarized using descriptive characteristics. Charts of additional and pharmacodynamic variables are presented as mean change from baseline over time with standard error. Comparison of treatment effects from MMRM analysis is based on least squares mean change (95% confidence interval [CI]) from baseline at Week 12.

F. Results

The results observed in all 104 randomized patients (out of 491 screened) who completed or stopped the treatment phase in the study are presented below. All randomized patients were treated in the study and were included in the mITT arm. Baseline scores were similar between groups. Demographic and clinical parameters were also similar between the two groups (Table 3). As noted above, patients were treated with either mAb1, 300 mg subcutaneously once a week, or placebo. The treatment period in the study was completed by 86.5% and 67.3% in patients treated with mAb1 and placebo, respectively (Figure 25). The most common reason for early discontinuation was lack of efficacy, which was more common with placebo (21.2%) than mAb1 (1.9%).

Table 3
Baseline Demographics and Clinical Parameters of Treatment Groups* Variable placebo
(N=52) mAb1 300 mg
(N=52) Age (years) 41.6±13.1 37.8±13.2 Male gender, number (%) 26 (50.0) 26 (50.0) Race or ethnic group White 38 (73.1) 45 (86.5) Black or African American 9 (17.3) 5 (9.6) Asians 3 (5.8) 1 (1.9) Other 2 (3.8) 1 (1.9) Body mass index Medium (kg/m ² ) 31.6±7.0 31.3±8.0 ≥30, qty (%) 25 (48.1) 24 (46.2) Duration of asthma (years) 26.9±14.8 24.2±12.6 Number of asthma exacerbations in the last 2 years 1.4±1.3 1.4±1.0 Previous dose of combination therapy ICS / LAAA, number (%) high dose 41 (78.8) 42 (80.8) low dose 11 (21.2) 10 (19.2) Blood eosinophils (×10 ^-9 /l) 0.47±0.21 0.55±0.19 FEV ₁ (l) 2.54±0.66 2.47±0.65 FEV ₁ (% of calculated volume) 72.0±12.7 72.0±12.6 PSV (l/min) In the morning 406.9±110.7 393.0±101.1 In the evening 416.6±116.8 414.6±102.3 ACQ5 score 2.1±0.5 2.1±0.5 Assessment of asthma symptoms In the morning 0.73±0.63 0.75±0.81 In the evening 1.12±0.73 0.92±0.71 Awakenings at night per day 0.21±0.50 0.44±0.80 SNOT-22 26.2±15.6 30.9±14.8 Number of breaths of albuterol or levalbuterol aerosol in a 24-hour period 2.0±1.8 2.2±2.4 FVOA (bn) 35.0±27.1 37.6±28.1 TARC (pg/ml) 470.5±204.7 496.1±342.4 Eotaxin-3 (pg/ml) 117.3±349.2 75.4±44.0 IgE (IU/ml) 694.7±1837.8 657.7±1482.3 *Plus or minus values are mean ± SD, unless otherwise noted. ACQ5 stands for Asthma Control Questionnaire (5-question variant), FVOA - exhaled nitric oxide fraction, _FEV1 - forced expiratory volume in 1 second, IgE - immunoglobulin E, PEF - peak expiratory flow, SNOT-22 - Nasal Outcome Test and paranasal sinuses of 22 points, and TARC - by thymus and regulated chemokine activation.

(i) Main end performance criterion

The frequency of asthma exacerbations in the placebo and mAb1 treatment groups is presented in Table 4.

Table 4
Frequency of asthma exacerbations in the mITT group Placebo (N=52) mAb1 (N=52) Patients without asthma exacerbations 29 (55.8%) 49 (94.2%) Patients with exacerbations of asthma 23 (44.2%) 3 (5.8%) Placebo Disagreement Ratio (95% CI) - 0.077 (0.021, 0.279)

There were a total of 26 asthma exacerbations during the treatment period, and no patients were hospitalized for asthma exacerbations. In the placebo group, 23 patients (44.2%) had an asthma exacerbation, while only 3 patients (5.8%) experienced an asthma exacerbation in the mAb1 treatment group. The disagreement ratio was 0.077 (p<0.0001) and the relative risk reduction was approximately 87%.

Of the 26 asthma exacerbations patients experienced in this study, 9 were considered severe, resulting in the need for immediate intervention in the form of treatment with either systemic corticosteroids or inhaled corticosteroids at 4 or more times the previous dose. A summary of the frequency of severe asthma exacerbations is presented in Table 5.

Table 5
Frequency of severe asthma exacerbations in the mITT group Placebo (N=52) mAb1 (N=52) Patients without asthma exacerbations 29 (55.8%) 49 (94.2%) Patients with severe asthma exacerbations 8 (15.4%) 1 (1.9%) Patients with mild asthma exacerbations 15 (28.8%) 2 (3.8%)

As shown in Table 5, eight severe asthma exacerbations were observed in the placebo group, and only 1 severe asthma exacerbation was observed in the mAb1 treatment group. The remaining 15 asthma exacerbations in the placebo group and 2 in the mAb1 group met the definition of an exacerbation protocol based on decreased morning PEF and/or increased use of albuterol/levalbuterol. As shown in Table 6, in the active treatment groups, sustained improvements from baseline were observed throughout the study across all parameters despite steroid withdrawal.

Table 6
Cases of exacerbations Exodus placebo
(N=52) mAb1
(N=52) ≥30% decrease in PEF from baseline in a 24-hour period for 2 consecutive days 10* (19.2) 1 (1.9) ≥6 additional inhalations of albuterol/levalbuterol within a 24-hour period on 2 consecutive days 10 (19.2) 1 (1.9) Treatment with systemic steroids 5 (9.6) 1 (1.9) ≥4-fold increase in ICS compared to the previous dose 3 (5.8) 0 Hospitalization 0 0 *4 placebo patients were eligible for PEF and systemic steroid treatment, and 1 placebo patient was eligible for PEF and albuterol/levalbuterol escalation.

In the case of mAb1, the time to exacerbation was longer (Figure 1), and the risk of exacerbation was lower compared to placebo (hazard ratio 0.10; 95% CI 0.03, 0.34; P<0.001). Analysis of the time to exacerbation of asthma using the Kaplan-Meier plot showed that the effect of mAb1 treatment was maintained for a long time, including after 8 weeks, when patients were at higher risk of exacerbations due to steroid withdrawal (Figure 1).

Only 1 patient in the placebo group had a mixed episode of asthma. A mixed case of asthma is defined as a 30% or greater decrease from baseline in morning PSV on 2 consecutive days, combined with ≥6 additional puffs of albuterol or levalbuterol aerosol over a 24-hour period (compared to baseline) on 2 consecutive days.

(ii) Other performance endpoints

Lung function parameters (FEV1, PSVu, and PSVv), asthma symptom-based endpoints (ACQ score, nighttime awakenings), and albuterol use were assessed in each patient at each visit. The results observed for these parameters (weekly change from baseline) are presented in Figures 2-7, respectively. In addition, a SNOT-22 score was obtained at baseline and at the end of treatment. For all parameters, mean baseline and means at week 12 (LOCF) and mean difference between treatment groups (ANOVA model for SNOT-22) are shown in Table 7. In Table 7, the column labeled "Difference from placebo" reflects a placebo-adjusted value relative to baseline that takes into account the changes that were observed in relation to the value of the parameter, in comparison with the changes that were observed for the same parameter in the placebo group.

Table 7
Additional lung function and symptom scores N Average base value (SD) RMS change
(SD) Difference with placebo p-value FEV1 (l) placebo 52 2.54 (0.66) -0.22 (0.06) - mAb1 52 2.47 (0.65) 0.05 (0.06) 0.27 (0.11, 0.42) 0.0009 PSVu (l/min) placebo 52 406.9 (110.7) -20.7 (9.1) - mAb1 51 393.0 (101.1) 13.9 (8.8)† 34.6 (10.6, 58.5) 0.0051 PSVv (l/min) placebo 51 416.6 (116.8) -18.4 (8.9)† - mAb1 52 414.6 (102.3) 4.3 (8.5) 22.7 (-0.7, 46.0) 0.0567 Albuterol use (aerosol doses per day) placebo 52 2.0 (1.8) 0.7 (0.3) - mAb1 50 2.2 (2.4) -1.3 (0.3)‡ -2.0 (-2.9, -1.2) <0.0001 ACQ score placebo 52 2.08 (0.52) -0.27 (0.16) - mAb1 52 2.09 (0.46) -1.00 (0.16) -0.73 (-1.15, -0.30) 0.0011 Nighttime awakenings (once per night) placebo 52 0.2 (0.5) 0.1 (0.1) - mAb1 52 0.4 (0.8) -0.2 (0.1) -0.2 (-0.5, -0.0) 0.0518 Average SNOT22 score placebo 51 26.24 (15.62) 0.23 (2.15)† - mAb1 50 30.92 (14.77) -8.26 (2.20)‡ -8.49 (-13.96, -3.03) 0.0027 † 51 patients with at least 1 assessment after baseline.
‡ 50 patients with at least 1 evaluation after baseline.

Treatment with mAb1 resulted in a significant change from baseline in FEV1 values at Week 1 that persisted through Week 12 (Figure 2) despite discontinuation of LABA and ICS, with a slight decrease in FEV1 at Week 5 coinciding with discontinuation of LABA. Similar improvements were observed for PSVv and, to a lesser extent, PSVv (Figures 3 and 4). The mean square (LS) change from baseline to week 12 in FEV1 was -0.22 L for placebo and 0.05 L for the mAb1 group. (p=0.0009).

ACQ5 score improved in both treatment groups at Week 1 (Figure 6). However, despite greater improvement in ACQ5 in the mAb1 group between Weeks 1 and 4, the placebo effect stabilized with the difference remaining up to Week 12.

Morning symptom scores increased from baseline until Week 12 in the placebo group. In the case of mAb1, an initial decrease was observed which remained below baseline until Week 12 (Figure 26A). A similar profile (with greater variability) was observed for evening asthma symptom scores (Figure 26B).

Night awakenings were stable in the placebo group during Week 6, then increased from Week 6 to Week 12. In contrast, night awakenings decreased in the mAb1 group from Week 1 and remained improved from baseline through Week 12 (Figure 7).

Changes in albuterol/levalbuterol use (Figure 5) were consistent with other additional endpoints: initial reduction followed by a return to baseline with placebo. In the case of mAb1, the initial decrease was maintained over time.

There was no significant difference at baseline between SNOT-22 values, with mean placebo score of 26.24 and mean mAb1 score of 39.02. At week 12, the mean LS change was a slight increase of 0.23 points for the placebo group and a mean decrease (improvement) of 8.26 points for the mAb1 group. This corresponded to an improvement value of 8.49 points for the mAb1 group (p=0.0027).

Table 8
Additional endpoints Exodus placebo
(N=52) mAb1
(N=52) Difference with placebo
(95% CI)** p-value Kaplan-Meier score at 12 weeks 46.0
(31.8, 60.2) 5.8
(0.0, 2.1) 0.10
(0.03 - 0.34) <0.001 Change in asthma symptom scores in the morning, from baseline to week 12 0.3±0.1 -0.4±0.1 -0.7
(-0.9 - -0.4) <0.001 Change in asthma symptom scores in the evening, from baseline to week 12 0.1±0.1 -0.6±0.1 -0.7
(-0.9 - -0.4) <0.001

Table 9
Change From Baseline at Week 12 SNOT-22 Evaluation Corresponding to Upper Respiratory Disease Subscale SNOT-22 RMS change ± standard error Difference from placebo (95% CI) p-value placebo
(N=52) mAb1
(N=52) The need to blow your nose -0.25±0.17* 0.95±0.17† -0.70 (-1.13, -0.26) 0.002 Nasal congestion -0.20±0.19* -0.94±0.19† 0.75 (-1.22, -0.28) 0.002 Decreased odor/taste sensitivity 0.04±0.18* -1.13±0.18† -1.16 (-1.62, -0.71) <0.001 *51 and †50 patients with at least 1 assessment after baseline, respectively

For all additional endpoints, measurements at Week 12 were in favor of mAb1 treatment and were significant, except for evening PSV and nighttime awakenings (Tables 7 and 8). Significant improvements with mAb1 were also observed for the three SNOT-22 items corresponding to upper respiratory disease (Table 9).

(iii) Security

Overall, mAb1 was safe and well tolerated. Treatment-emergent adverse events (TEAE) were also reported by 40 (76.9%) patients treated with placebo and 42 (80.8%) patients treated with mAb1 (Table 10). TEAE events were non-specific, usually mild to moderate in intensity, with most occurring at the end of the study. An increase in subsequent TEAE reports was observed for mAb1 compared with placebo: injection site reactions were reported by 15 (28.8%) mAb1 patients and 5 placebo patients (9.6%); nasopharyngitis was reported by 7 (13.5%) mAb1 patients and 2 placebo patients (3.8%); headache was reported by 6 (11.5%) mAb1 patients and 3 (5.85%) placebo patients, and nausea was reported by 4 (7.7%) mAb1 patients and 1 (1.9%) placebo patient.

Table 10
Adverse events Adverse event Placebo (N=52) mAb1 (N=52) Number of patients (%) Any unwanted event 40 (76.9) 42 (80.8) Any severe adverse event 3 (5.8) 1 (1.9) Early termination of participation in a study due to an adverse event 3 (5.8) 3 (5.8) Death 0 0 Most common AE* Injection Site Reactions† 5 (9.6) 15 (28.8) Nasopharyngitis 2 (3.8) 7 (13.5) Upper respiratory tract infection 9 (17.3) 7 (13.5) Headache 3 (5.8) 6 (11.5) Nausea 1 (1.9) 4 (7.7) Reactions resembling arthropod bites 0 3 (5.8) Muscle spasms 0 3 (5.8) Nasal congestion 1 (1.9) 3 (5.8) Rash 1 (1.9) 3 (5.8) Hives 0 3 (5.8) Viral infection of the upper respiratory tract 0 3 (5.8) * ≥3 patients in any treatment group by term of preferred use
† Injection site reaction includes: injection site pain, injection site reaction, injection site redness, injection site rash, injection site hematoma, injection site urticaria, injection site dermatitis, injection site inflammation, nodule at the injection site, itching at the injection site and swelling at the injection site.

No deaths were recorded during the study. Of the 4 reported SAEs, 1 mAb1 patient had bipolar disorder and 3 placebo patients experienced SAEs typical of asthma due to pneumonia, gunshot wound with left pneumothorax, and right ankle fracture. None of these SAEs were considered to be associated with IMP and all but a recent ankle fracture recovered by the end of the study. There were no deaths.

A total of 6 patients withdrew from the study due to TEAE: 3 patients in the mAb1 group (bipolar disorder, wheezing asthma and angioedema) and 3 patients in the placebo group (upper respiratory infection, psoriasis and asthma). TEAE angioedema occurred in a 42-year-old African American woman after the ninth dose of treatment in the study and presented as an pruritic, papular rash observed at and some distance from the injection site. The edema persisted for one week and resolved after discontinuation of treatment in the study and therapy with prednisone and diphenhydramine. This phenomenon was considered treatment related. This AE occurred after the onset of a mild rash at the injection site after the first and sixth doses of treatment in the study.

Among the most common AEs seen in ≥3 patients in any treatment group (Table 10), injection site reactions, nasopharyngitis, nausea, and headache occurred more frequently with mAb1 than with placebo. No clinically significant changes in vital signs, physical examination, clinical laboratory findings, or ECG were reported in either group.

G. Conclusion

Significant improvements in lung function and other parameters of asthma control were observed. Efficiency was observed from the early stages and persisted despite the abolition of basic therapy. A relative reduction of approximately 87% (p<0.0001) in the primary endpoint of asthma exacerbations in patients with persistent, moderate-to-severe asthma with eosinophilia was observed after 12 weeks of treatment with 300 mg mAb1 once a week (5.8%) compared with placebo (44.2%). As shown in Table 7, clinically and statistically significant (non-multiplicity adjusted) improvements with treatment compared with placebo were observed in lung function measures (FEV1, PSVu), asthma symptom scores (ACQ), and albuterol use. Positive trends were observed for PSVv (p=0.0567) and awakenings at night (p=0.0518). A statistically significant (no multiplicity adjustment) improvement was also observed for the SNOT-22 score. In the main treatment group, a long-term improvement relative to baseline was observed for all parameters during the study, despite the abolition of ICS and BAAA. Overall, mAb1 was safe and well tolerated.

Example 3: Biomarker studies

Biomarker analysis was performed on samples obtained from subjects who participated in clinical trials mAb1 (see Example 2, above). In particular, serum/plasma biomarkers associated with TH2 inflammation such as thymus and upregulated chemokine (TARC; CCL17), immunoglobulin E (IgE), eotaxin-3, periostin, cancer embryonic antigen (CEA), YKL-40 and blood eosinophils were measured in patient samples at baseline and at various time points after treatment initiation in the study. Baseline levels of these biomarkers were evaluated for potential predictive value for treatment response. In addition, exhaled NO fraction (EFFA) and eosinophils and neutrophils in induced sputum were measured as a biomarker of bronchial inflammation. Exhaled nitric oxide was assessed before spirometry and after fasting for at least 1 hour using the NIOX device (Aerocrine AB, Solna, Sweden). Biomarkers were analyzed using a mixed model, below are the average values obtained by the method of least squares based on this model.

Subjects with asthma (N=104) received either mAb1 (300 mg) or placebo subcutaneously on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, and 78 of the study (i.e., 12 weekly doses) (see Example 2). Samples for biomarker analysis were taken from subjects treated with antibody and placebo at weeks 0, 1, 4, 8 and 12. Antigen-specific IgE was detected using the ^Phadiatop® test.

TARC, eotaxin-3 and IgE remained unchanged in response to placebo (Figures 8, 9 and 10). In contrast, a rapid decline in TARC (mean % change -22.7% vs. +0.3%; p=0.0003) (Figure 8) and eotaxin-3 (mean % change -39.62% vs. 12 .69%; p<0.0001) (Figure 9) observed for one week in patients treated with mAb1 and persisted until week 12: TARC: -26.0% vs. +7.6% placebo (p= 0.0005); Eotaxin-3: -45.67% vs. +5.13% placebo (p<0.0001).

TARC levels changed over the course of a week in response to exposure to mAb1, which was administered subcutaneously at a dose of 300 mg. TARC levels plateaued at approximately 50% of baseline in mAb1-treated subjects despite ICS withdrawal. The data suggest that TARC expression is more directly related to IL-4R signaling than to changes in FEV1 (which decreases in parallel with ICS withdrawal [after Week 4]), and that blocking IL-4R causes a change in the TH1 signature, as observed, for example, with the introduction of IFN-gamma. It would be possible to titrate the mAb1 dose using TARC (and, for example, CXCL10), especially in patients requiring long-term treatment and at risk of developing TH1-type immune diseases.

Total serum IgE also decreased after mAb1 treatment. The total serum IgE response was more heterogeneous and delayed compared to the TARC response. The mean (SD) baseline IgE levels were 694.68 IU/L (1837.82) for the placebo group (n=52) and 657.66 (1482.25) for the mAb1 group (n=52), while the median was 169.95 for the placebo group and 206.15 for the mAb1 group. Despite this heterogeneity, there was a trend towards a decrease in IgE in patients treated with mAb1 compared with placebo, however, only starting at week 4. Serum IgE decreased significantly in the mAb1 group compared with placebo (mean % change -10.1% compared c +13.5%; p=0.0325) from week 4 and continued to decline until week 12 (mean % change -36.8% for REGN668/SAR231893 vs -5.5% for placebo; p <0.0001) (Figure 10).

All changes from baseline and placebo at Week 12 for FVOA, TARC, eotaxin-3, and IgE were in favor of mAb1 (all P<0.001) (Table 11). No differences regarding baseline or between treatment groups were observed for YKL-40 or CEA.

Table 11
Percent Change from Baseline at Week 12 by Pharmacodynamic Endpoints Exodus RMS percent change ± standard error p-value placebo
(N=52) mAb1
(N=52) FVOA 35.0±10.8 28.7±11.2 <0.001 TARC 7.6±6.9 -26.0±6.9 <0.001 Eotaxin-3 5.1±4.7 -45.7±4.7 <0.001 IgE 5.5±3.6 -36.8±3.6 <0.001 Blood eosinophils 2.7±15.8 41.6±15.7 0.078

There was a transient decrease in periostin levels, followed by an increase with the abolition of BADD/ICS (Figure 11). The introduction of mAb1 delayed the increase, but did not prevent the increase above the baseline. No consistent treatment effect was observed for CEA (Figure 12) and YKL-40 (Figure 13). The content of eosinophils in the blood remained unchanged during Week 6, but then increased in Weeks 8 and 12 (Figure 14). The content of eosinophils in peripheral blood did not change to placebo during the entire treatment. The difference between treatments was not significant, with a borderline increase that was caused by a more significant increase in blood eosinophils in only a few patients treated with mAb1. Most patients showed little or no increase (Table 12).

Table 12
Percentage of Patients Achieving Threshold Changes in Blood Eosinophil Levels Change in eosinophils Number (%) of patients Placebo (n=52) mAb1 (n=52) >15% increase 13 (30.2) 21 (47.7) 15% increase - 0% change 7 (16.3) 6 (16.6) 0% - 15% increase 8 (18.6) 4 (9.1) 15% - 100% increase 13 (30.2) 6 (13.6) 100% - 200% increase 2 (4.7) 3 (6.8) >200% increase 0 4 (9.1)

Because only 3 mAb1 patients experienced an asthma exacerbation during the study, no conclusion can be drawn regarding the association between baseline biomarker levels and asthma exacerbations.

mAb1 treatment was also associated with a significant decrease from baseline in VVOA values at Week 4, with VVOA remaining below baseline until Week 12, regardless of ICS discontinuation (mean % change at week 12: -28.7 for mAb1 and 35, 0 for placebo, p<0.0001) (Figure 15). On the contrary, the values of FVOA in the placebo group remained stable until Week 8, with a subsequent increase in Week 12, coinciding with the withdrawal of ICS.

Improvement in forced expiratory volume in 1 second (FEV1) was significantly correlated with a decrease in FVOA (r=-0.408, p=0.009) at week 12 (Figure 16). Similarly, improvements in PSVu and PSVv correlated with a decrease in FVOA (Figures 17 and 18). Other correlations with FVOA were not significant. See Table 13.

Table 13
Correlation between FEV1 and PD endpoints Exodus Correlation p-value FVOA -0.408 <0.009 TARC -0.248 0.10 Eotaxin-3 -0.146 0.34 IgE -0.279 0.06 Blood eosinophils 0.165 0.28

Scatterplot analysis of baseline eosinophils versus change in FEV1 from baseline at week 12 did not appear to link baseline eosinophils and treatment effect as measured by change in FEV1 from baseline at week 12 in the study arm (baseline eosinophils ≥0 ,3×10 ⁹ /l) (Figure 19). Baseline eosinophils correlated with a decrease in ACQ (Figure 20) and a decrease in albuterol/levalbuterol use (Figure 21). Periostin and YKL-40 at baseline correlated with a decrease in ACQ (Figures 22 and 23).

The change in FEV1 from baseline at week 12 was associated with discontinuation of ICS (starting at week 4). Similar studies failed to link baseline TARC or IgE levels to change in FEV1 from baseline at week 12 in the study group (baseline eosinophils ≥0.3×10 ⁹ /L).

conclusions

These results indicate that mAb1 induced a significant decrease in serum biomarkers associated with Th2 inflammation (TARC, eotaxin-3 and IgE) and bronchial inflammation (BVOA) in adult patients with asthma. The correlation between a decrease in FVOA and an improvement in FEV1 indicates a relationship between IL-4/IL-13 mediated anti-inflammatory activity and improvement in lung function in moderate-to-severe, uncontrolled asthma.

The scope of the present invention should not be limited to the specific embodiments described in this application. Moreover, various modifications, in addition to those described in this application, will become apparent to those skilled in the art from the previous description and accompanying figures. Such modifications are to be included within the scope of the appended claims.

Example 4 Blockade of the IL-4/IL-13 Signaling Pathway Inhibits IgE Production and Airway Remodeling in a Mouse Model of Eosinophilic Asthma Induced by Dust Mite Allergens

INTRODUCTION

The dust mite allergen (HDM) has been shown to induce a Th2 immune response, including an influx of Th2 cells into the lungs, and an IL-4-induced trans-endothelial migration of eosinophils into the lungs. Eosinophils, or eosinophilic granulocytes, are the main effector cells in allergic reactions, and the release of the contents of the granules (containing IL-4) from eosinophils contributes to the development of inflammation. In asthmatics, Th2-regulated production of IL-4 promotes the migration of eosinophils from the blood to the lungs via eotaxin, a potent eosinophil chemoattractant (Mochizuki et al., J. Immunol., 1998, 160(1):60-68). In addition, when located at the site of inflammation, eosinophils produce and secrete IL-4, thus contributing to the continuation of Th2-regulated inflammation (Bjerke et al., Respir. Med., 1996, 90(5):271-277). In patients with allergic asthma, HDM induction increased serum IgE and Th2 cytokines for 5 weeks after allergen induction (van de Pol et al., Allergy, 2012, 67(7):67-73).

In this Example, an HDM-induced chronic asthma model was used to evaluate the pharmacodynamic effects of anti-IL-4R antibodies on markers of airway inflammation in mice. In addition, the effect of anti-IL-4R antibodies on airway collagen deposition was assessed in this model, as collagen deposition correlates with the degree of airway remodeling.

MATERIALS AND METHODS

two different anti-IL-4Rα antibodies were used in the experiments of this Example: "mAb1", a fully human monoclonal antibody specific for human IL-4Rα (ie, the anti-IL-4R antibody used in the other working examples provided herein); and "anti-mIL-4Rα", a mouse monoclonal antibody specific for mouse IL-4Rα protein. mAb1 does not cross-react with mouse IL-4Rα; therefore, mAb1 was evaluated in humanized mice in which engineered human IL-4 and IL-4Rα ectodomain were introduced to replace their respective mouse sequences in mice (IL-4 ^hu/hu IL-4Rα ^hu/hu ). The mouse anti-mouse IL-4Rα antibody "anti-mIL-4Rα", on the other hand, was tested in wild-type (Balb/c) mice. Also tested in these experiments is a murine IL-13Rα2-mFc fusion protein that acts as a decoy receptor, blocking IL-13 signaling by shutting down the IL-13 cytokine.

In relation to the model of HDM-induced asthma, mice were sensitized with daily intranasal application of HDM (50 μg per mouse in 20 μl PBS) for 10 days, followed by a break (resolution period 2 weeks). Allergen c was administered by intranasal application of HDM (50 μg per mouse in 20 μl PBS) three times a week for 8 weeks. For each HDM administration, during the sensitization or challenge period, mice were lightly anesthetized with isoflurane.

Mice were allowed to acclimatize in the experimental laboratory for a minimum of 5 days before starting the experimental procedure. Throughout the duration of the experiment, the animals remained in the experimental laboratory under standard conditions on a 12-hour day/night cycle with free access to food and water. The number of mice per cage was limited to a maximum of 5 mice.

A total of 48 humanized mice were used in two experiments in which the human IL-4 ligand and the human IL-4Rα ectodomain were genetically engineered to replace their respective mouse sequences (IL-4 ^hu/hu IL-4Rα ^hu/hu ) . IL-4 ^hu/hu IL-4Rα ^{hu/hu mice} had a mixed background of C57BI/6NTac(75%)/129S6SvEvTac(25%). In addition, 20 wild-type littermates with an identical mixed background were used in one of the three experiments. In each experiment, mice were sensitized with HDM (or PBS in the control group) daily for ten days, followed by a resolution period from day 11 to day 29. From day 30, animals were stimulated with HDM three times a week, for 8 weeks, until day 81 and then euthanized on day 85 for analysis. Mice were divided into six experimental groups as follows:

(1) Non-sensitized, untreated : PBS was administered intranasally during periods of sensitization and provocation. Mice were not injected with antibodies (IL-4 ^hu/hu IL-4Rα ^hu/hu mice n=9, wild-type littermates n=5).

(2) HDM-sensitized, untreated : HDM was administered intranasally during periods of sensitization and provocation. Mice were not treated with antibodies (IL-4 ^hu/hu IL-4Rα ^hu/hu mice n=7; wild-type littermates n=5);

(3) HDM-sensitized treated with anti-mIL-4Rα : HDM was administered intranasally during periods of sensitization and provocation. Mice were treated IP with anti-mIL-4Rα at 50 mg/kg, twice a week, from week 7 to week 12, for a total of 12 doses, over a period of 6 weeks (wt littermates n=5 );

(4) HDM-sensitized treated with anti-human IL-4R mAb1 : HDM was administered intranasally during periods of sensitization and challenge. Mice were injected i.p. with mAb1 at 50 mg/kg, twice a week, from week 7 to week 12, for a total of 12 doses, over a period of 6 weeks (IL-4 ^hu/hu IL-4Rα ^{hu/ hu} mice n=12).

(5) HDM-sensitized treated with mouse IL-13Rα2-mFc fusion protein : HDM was administered intranasally during periods of sensitization and challenge. Mice were injected ip with IL-13Rα2-mFc at 25 mg/kg, twice a week, from week 7 to week 12, for a total of 12 doses, over a period of 6 weeks (IL-4 ^hu/hu IL-4Rα ^hu/hu mice n=7, wild-type littermates n=5);

(6) HDM-sensitized, isotype-control antibody-treated : HDM was administered intranasally during periods of sensitization and challenge. Mice were injected i.p. with isotype control Ab at 50 mg/kg, twice a week, from week 7 to week 12, for a total of 12 doses over a period of 6 weeks (IL-4 ^hu/hu IL-4Rα ^hu/hu mice n=7).

Mice were euthanized on day 85, bled for analysis of serum immunoglobulin levels, after which the lung (one lobe) was used to obtain one of the following: i) bronchoalveolar lavage fluid (BAL), ii) an enzymatically processed single cell suspension sample for flow cytometric analysis, iii) a formalin-fixed sample for staining and histological analysis, or iv) a sample for analysis using the Sircol™ Collagen Assay Kit to determine the amount of collagen contained in a lung lobe.

BAL fluid was obtained from euthanized animals by first opening the trachea and inserting a 23G lavage tube through a small incision in the tracheal wall. Sterile PBS (1 ml) was then injected into the lung and BAL fluid was withdrawn through a lavage tube using a syringe. 100 μl of BAL was applied to a Cytospin column, which was centrifuged for 5 minutes at 500 rpm to isolate the cells and apply them to glass slides. The preparations were dried and stained with hematoxylin-eosin (HE) to visualize eosinophils.

Serum IgE levels were quantified using a commercial ELISA kit. Briefly, serially diluted serum samples were incubated with anti-IgE capture antibody in 96-well plates and IgE was detected with a biotinylated anti-mouse IgE secondary antibody. Purified mouse IgE labeled with HRP was used as a standard.

Serum HDM-specific IgG1 levels were quantified by ELISA. Briefly, HDM-coated plates were incubated with serially diluted serum samples, followed by incubation with HRP-conjugated anti-mouse IgG1 antibody. Relative serum IgG1 levels were reported as units of titer (OD450 multiplied by the dilution factor required to achieve OD450≤0.5). The harvested lung lobes were flash frozen in liquid nitrogen and stored at -80° C. until the isolation step. For collagen isolation, the lung was homogenized in ice-cold NaCl/NaHCO ₃ solution and centrifuged at 9000×g for 10 min. This step was repeated three times, after which the resulting precipitate was treated with pepsin in acetic acid for 18 hours at 4°C.

The samples were centrifuged, the supernatant was removed and mixed with the Sircol Dye Reagent to stain the collagen contained in the samples. Samples were washed with Acid-Salt Wash to remove unbound Sircol Dye and then mixed with Alkali. 200 μl of each sample was transferred to a 96-well plate and the OD was measured at 555 nm. A collagen standard was used for the final quantification of the collagen contained in each sample.

Lungs were harvested from euthanized mice and kept in complete DMEM on ice until treated with a mixture of collagenase and DNase in HBSS buffer for 20 minutes at 37°C. Collagenase activity was neutralized by adding 0.5M EDTA, samples were centrifuged and erythrocytes were lysed with ACK buffer. Cell suspensions obtained for each sample were divided into three separate pools and stained with antibody mix 1 (anti-CD11c-APC, anti-SiglecF-PE, anti-F4/80-FITC, anti-CD45-PerCp-Cy5.5) or antibody mixture 2 (anti-CD11c-APC, anti-CD11b-PerCp-Cy5.5, anti-CD103-FITC, anti-MHCII-PE), or antibody mixture 3 (anti-CD19-PE, anti-Ly6G-APC, anti-CD3-FITC, anti-CD11b-PerCp-Cy5.5) for 25 minutes at 4°C. Stained cells were fixed in Cytofix/Cytoperm solution for 30 minutes at 4°C and stored in PBS until flow cytometric analysis on FACSCanto (BD Biosciences).

In the HDM-induced chronic eosinophilic asthma (EA) model, left lung lobes were harvested from 4 mice per group for microarray analysis of gene expression using ^GeneChip® technology. Gene expression levels in mice sensitized and challenged with HDM and then treated with isotype control Abs were compared to gene expression levels in mice sham (PBS) sensitized and challenged and not treated with antibody. The threshold for changes in gene expression was set to >1.5-fold. A set of genes identified as differentially expressed in mice sensitized and challenged with HDM were then also analyzed in the anti-IL-4Rα treated group compared to the isotype control treated group. The threshold for gene expression change in the anti-IL-4Rα treated group compared to the isotype control group was set to >2-fold.

RESULTS

Sensitization and HDM provocation resulted in increased levels of IgE and HDM-specific IgG1. The increase in IgE was completely blocked by both antibodies against IL-4Rα, but not by the treatment of IL-13Rα2-Fc (Figures 27A and 27B); HDM-specific IgG1 levels were not affected by either treatment (data not shown).

Sensitization and challenge with HDM also caused an increase in the collagen contained in the lungs of mice. The collagen content in the lungs of mice treated with AT to IL-4Rα and protein IL-13Rα2-Fc decreased to levels observed in mock-sensitized and challenged mice (Figures 28A and 28B).

In addition, mAb1 treatment prevented the influx of eosinophils, neutrophils, and inflammatory dendritic cells into the lung (Figure 29, panels A and B).

Microarray analysis of mRNA isolated from lung tissue of HDM-induced IL-4 ^hu/hu IL-4Rα ^hu/hu mice injected with isotype control antibody revealed differential expression of 1468 genes (826 genes upregulated and 642 downregulated) compared to sham-sensitized and challenged mice. mAb1 treatment of HDM-induced hu/ ^hu IL-4Rα hu ^/hu IL-4Rα mice resulted in changes in the expression of only 521 genes (compared to sham-sensitized/challenged mice), effectively blocking approximately 65% of the genes affected by HDM sensitization/challenging ( >1.5-fold change, p<0.05). Of particular interest is the fact that mAb1 mediated the downregulation of gene expression of several members of the IL-1 cytokine family, in particular IL-1α (2.9-fold), IL-33 (2.6-fold) and IL-18 binding protein ( 1.5 times). IL-1β gene expression was not increased in the HDM-induced isotype control treated group (compared to sham-sensitized/challenged mice), but was reduced (1.5-fold) in the mAb1 treated group. Expression of the Th1 gene for the inflammatory cytokines, IL-12β and IFN-γ, was also downregulated by mAb1 administration compared to the isotype control treated group. In particular, eight genes encoding chemokine ligands involved in cell homing and directed cell migration were downregulated in the mAb1-treated group compared to the isotype control group: Ccl11 (~9-fold reduction), Ccl8 and Cxcl2 (both ~5-fold reduction), Cxcl1, Ccl7, Ccl6 (all ~3-fold reduction), Ccl2 and Ccl9 (about 2-fold reduction).

CONCLUSIONS

This Example shows that blockade of IL-4 signaling through type I and type II receptors by anti-IL-4Rα antibodies suppresses inflammatory and fibrotic changes in the lungs of HDM-provoked mice, as well as changes in the gene signature that HDM causes.

Other embodiments are presented in the claims.

--->

SEQUENCE LIST

<110> Marius Ardeleanu

Namita Gandhi

Neil Graham

Stephane C. Kirkesseli

Sudeep Kundu

Allen Radin

Ross E. Rocklin

Steve Weinstein

Jennifer Davidson Hamilton

Jeffrey Ming

<120> METHODS OF TREATMENT OR PREVENTION OF ASTHMA THROUGH ADMINISTRATION

IL-4R ANTAGONIST

<130> US2012/080-WO-PCT

<140> To be assigned

<141> Filed herewith

<150> US 61/691,625

US 61/758,097

US 61/761,279

US 61/783,796

US 61/805,797

FR 1356994

<151> 2012-08-21

2013-01-29

2013-02-06

2013-03-14

2013-03-27

2013-07-16

<160> 275

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 1

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttccgc tcttatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcggtc atatcatatg atggaagtaa taaatattat 180

atagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgaat 240

ctgcaaatga acagcctgag acttgaggac acggctgtat attactgtgc gaaagagggg 300

agggggggat ttgactactg gggccaggga atcccggtca ccgtctcctc a 351

<210> 2

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 2

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ile Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Asn

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Leu Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Glu Gly Arg Gly Gly Phe Asp Tyr Trp Gly Gln Gly Ile Pro

100 105 110

Val Thr Val Ser Ser

115

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 3

ggattcacct tccgctctta tggc 24

<210> 4

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 4

Gly Phe Thr Phe Arg Ser Tyr Gly

15

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 5

atatcatatg atggaagtaa taaa 24

<210> 6

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 6

Ile Ser Tyr Asp Gly Ser Asn Lys

15

<210> 7

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 7

gcgaaagagg ggaggggggg atttgactac 30

<210> 8

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 8

Ala Lys Glu Gly Arg Gly Gly Phe Asp Tyr

1 5 10

<210> 9

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 9

gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca ggtcataaac aattatttag cctggtttca gcagaaacca 120

gggaaagtcc ctaagtccct gatccatgct gcatccagtt tacaaagtgg ggtcccatca 180

aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgccaacag tataatagtc acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa acga 324

<210> 10

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Ser Leu Ile

35 40 45

His Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser His Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 11

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 11

caggtcataa acaattat 18

<210> 12

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 12

Gln Val Ile Asn Asn Tyr

15

<210> 13

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 13

gctgcatcc 9

<210> 14

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 14

Ala Ala Ser

1

<210> 15

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 15

caacagtata atagtcaccc gtggacg 27

<210> 16

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 16

Gln Gln Tyr Asn Ser His Pro Trp Thr

15

<210> 17

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 17

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttccgc tcttatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcggtc atatcatatg atggaagtaa taaatattat 180

atagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgaat 240

ctgcaaatga acagcctgag acttgaggac acggctgtat attactgtgc gaaagagggg 300

agggggggat ttgactactg gggccaggga accctggtca ccgtctcctc a 351

<210> 18

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 18

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ile Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Asn

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Leu Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Glu Gly Arg Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 19

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 19

gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca ggtcataaac aattatttag cctggtttca gcagaaacca 120

gggaaagtcc ctaagtccct gatccatgct gcatccagtt tacaaagtgg ggtcccatca 180

aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgccaacag tataatagtc acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 20

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Ser Leu Ile

35 40 45

His Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser His Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 21

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 21

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttccgc tcttatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gaaagagggg 300

agggggggat ttgactactg gggccaggga accctggtca ccgtctcctc a 351

<210> 22

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 22

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Glu Gly Arg Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 23

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 23

gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca ggtcataaac aattatttag cctggtttca gcagaaacca 120

gggaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgccaacag tataatagtc acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 24

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser His Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 25

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 25

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcaga agctatggca tacactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacactgtat 240

ctgcaaatga acagcctgat aactgaggac acggctgtgt attattgtgt gaaagagggg 300

aggggggggt ttgactactg gggccaggga accacggtca ccgtctcctc a 351

<210> 26

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 26

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Ile Thr Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Glu Gly Arg Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 27

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 27

ggattcacct tcagaagcta tggc 24

<210> 28

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 28

Gly Phe Thr Phe Arg Ser Tyr Gly

15

<210> 29

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 29

atatcatatg atggaagtaa taaa 24

<210> 30

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 30

Ile Ser Tyr Asp Gly Ser Asn Lys

15

<210> 31

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 31

gtgaaagagg ggaggggggg gtttgactac 30

<210> 32

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 32

Val Lys Glu Gly Arg Gly Gly Phe Asp Tyr

1 5 10

<210> 33

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 33

gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca ggtcattaat aattatttag cctggtttca gcagaaacca 120

gggaaagtcc ctaagtccct gatccatgct gcatccagtt tgcaaagagg ggtcccatca 180

aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcaacag cctgcagcct 240

gaagattttg caacttatta ctgccaacaa tataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa acga 324

<210> 34

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Ser Leu Ile

35 40 45

His Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 35

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 35

caggtcatta ataattat 18

<210> 36

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 36

Gln Val Ile Asn Asn Tyr

15

<210> 37

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 37

gctgcatcc 9

<210> 38

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 38

Ala Ala Ser

1

<210> 39

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 39

caacaatata atagttaccc gtggacg 27

<210> 40

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 40

Gln Gln Tyr Asn Ser Tyr Pro Trp Thr

15

<210> 41

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 41

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcaga agctatggca tacactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacactgtat 240

ctgcaaatga acagcctgat aactgaggac acggctgtgt attattgtgt gaaagagggg 300

aggggggggt ttgactactg gggccaggga accctggtca ccgtctcctc a 351

<210> 42

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 42

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Ile Thr Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Glu Gly Arg Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 43

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 43

gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca ggtcattaat aattatttag cctggtttca gcagaaacca 120

gggaaagtcc ctaagtccct gatccatgct gcatccagtt tgcaaagagg ggtcccatca 180

aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcaacag cctgcagcct 240

gaagattttg caacttatta ctgccaacaa tataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 44

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 44

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Ser Leu Ile

35 40 45

His Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 45

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcaga agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgt gaaagagggg 300

aggggggggt ttgactactg gggccaggga accctggtca ccgtctcctc a 351

<210> 46

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 46

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Glu Gly Arg Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 47

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 47

gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca ggtcattaat aattatttag cctggtttca gcagaaacca 120

gggaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgccaacaa tataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 48

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 49

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 49

caggtgcagc tggtggagtc tgggggaggc ttggaacagc cgggggggtc cttgagactc 60

tcctgtgcag gctctggatt cacgtttaga gactatgcca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtcgcatcg attagtggtt ccggtggtaa cacatacttc 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattat ggtttggacg tctggggcca agggtccacg 360

gtcaccgtct cctca 375

<210> 50

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 50

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Phe Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Ser Thr Val Thr Val Ser Ser

115 120 125

<210> 51

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 51

ggattcacgt ttagagacta tgcc 24

<210> 52

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 52

Gly Phe Thr Phe Arg Asp Tyr Ala

15

<210> 53

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 53

attagtggtt ccggtggtaa caca 24

<210> 54

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 54

Ile Ser Gly Ser Gly Gly Asn Thr

15

<210> 55

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 55

gcgaaagatc gactctctat aacaattcgc ccacgctatt atggtttgga cgtc 54

<210> 56

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 56

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

1 5 10 15

Asp Val

<210> 57

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 57

gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgttggaga cagagtcacc 60

atcacttgtc gggcgagtca ggccattaac aatcatttag cctggtttca gcagaaacca 120

gggaaagccc ctaagtccct gatctttgct gtatccagtt tgcaaagtgg ggtcccatca 180

aagttcagcg gcagtggatc tgggacagac ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgccaacag tataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa acga 324

<210> 58

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 58

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Asn Asn His

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Phe Ala Val Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 59

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 59

caggccatta acaatcat 18

<210> 60

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 60

Gln Ala Ile Asn Asn His

15

<210> 61

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 61

gctgtatcc 9

<210> 62

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 62

Ala Val Ser

1

<210> 63

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 63

caacagtata atagttaccc gtggacg 27

<210> 64

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 64

Gln Gln Tyr Asn Ser Tyr Pro Trp Thr

15

<210> 65

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 65

gaggtgcagc tggtggagtc tgggggaggc ttggaacagc cgggggggtc cttgagactc 60

tcctgtgcag gctctggatt cacgtttaga gactatgcca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtcgcatcg attagtggtt ccggtggtaa cacatacttc 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattat ggtttggacg tctggggcca agggaccacg 360

gtcaccgtct cc 372

<210> 66

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 66

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Phe Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 67

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 67

gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgttggaga cagagtcacc 60

atcacttgtc gggcgagtca ggccattaac aatcatttag cctggtttca gcagaaacca 120

gggaaagccc ctaagtccct gatctttgct gtatccagtt tgcaaagtgg ggtcccatca 180

aagttcagcg gcagtggatc tgggacagac ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgccaacag tataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 68

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 68

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Asn Asn His

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Phe Ala Val Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 69

<211> 373

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 69

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacgtttaga gactatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggtt ccggtggtaa cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattat ggtttggacg tctggggcca agggaccacg 360

gtcaccgtct cct 373

<210> 70

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 70

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 71

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 71

gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca ggccattaac aatcatttag cctggtttca gcagaaacca 120

gggaaagccc ctaagtccct gatctatgct gtatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgccaacag tataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 72

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 72

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Asn Asn His

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Val Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 73

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 73

caggtgcagc tggtggagtc tgggggaggc ttggaacagc cgggggggtc cttgagactc 60

tcctgtgcag gctctggatt cacgtttaga gactatgcca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtcgcatcg attagtggtt ccggtggtaa cacatacttc 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattat ggtttggacg tctggggcca agggtccacg 360

gtcaccgtct cctca 375

<210> 74

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 74

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Phe Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Ser Thr Val Thr Val Ser Ser

115 120 125

<210> 75

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 75

ggattcacgt ttagagacta tgcc 24

<210> 76

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 76

Gly Phe Thr Phe Arg Asp Tyr Ala

15

<210> 77

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 77

attagtggtt ccggtggtaa caca 24

<210> 78

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 78

Ile Ser Gly Ser Gly Gly Asn Thr

15

<210> 79

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 79

gcgaaagatc gactctctat aacaattcgc ccacgctatt atggtttgga cgtc 54

<210> 80

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 80

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

1 5 10 15

Asp Val

<210> 81

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 81

gaaatagtgt tgacgcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg tatagtattg gatacaacta tttggattgg 120

tacctgcaga agtcagggca gtctccacag ctccttatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttgggttt tattactgca tgcaagctct acaaactccg 300

tacacttttg gcccggggac caagctggag atcaaacga 339

<210> 82

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 82

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ile Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ser Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Tyr Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 83

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 83

cagagcctcc tgtatagtat tggatacaac tat 33

<210> 84

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 84

Gln Ser Leu Leu Tyr Ser Ile Gly Tyr Asn Tyr

1 5 10

<210> 85

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 85

ttgggttct 9

<210> 86

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 86

Leu Gly Ser

1

<210> 87

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 87

atgcaagctc tacaaactcc gtacact 27

<210> 88

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 88

Met Gln Ala Leu Gln Thr Pro Tyr Thr

15

<210> 89

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 89

gaggtgcagc tggtggagtc tgggggaggc ttggaacagc cgggggggtc cttgagactc 60

tcctgtgcag gctctggatt cacgtttaga gactatgcca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtcgcatcg attagtggtt ccggtggtaa cacatacttc 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattat ggtttggacg tctggggcca agggaccacg 360

gtcaccgtct cc 372

<210> 90

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 90

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Phe Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 91

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 91

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg tatagtattg gatacaacta tttggattgg 120

tacctgcaga agtcagggca gtctccacag ctccttatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttgggttt tattactgca tgcaagctct acaaactccg 300

tacacttttg gcccggggac caagctggag atcaaa 336

<210> 92

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 92

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ile Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ser Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Tyr Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 93

<211> 373

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 93

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacgtttaga gactatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggtt ccggtggtaa cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattat ggtttggacg tctggggcca agggaccacg 360

gtcaccgtct cct 373

<210> 94

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 94

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 95

<211> 337

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 95

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg tatagtattg gatacaacta tttggattgg 120

tacctgcaga agccaggggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300

tacacttttg gccaggggac caagctggag atcaaac 337

<210> 96

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 96

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ile Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 97

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 97

caggtgcagc tggtggagtc tgagggactc ttggaacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caactttaga gactttgcca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct attagtggta gtggtagtaa tacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaacca cacgctgtat 240

ctgcgaatga acagcctgag agccgaagac acggccgtgt attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattac ggtctggacg tctggggcca agggtccacg 360

gtcaccgtct cctca 375

<210> 98

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 98

Gln Val Gln Leu Val Glu Ser Glu Gly Leu Leu Glu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Arg Asp Phe

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn His Thr Leu Tyr

65 70 75 80

Leu Arg Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Ser Thr Val Thr Val Ser Ser

115 120 125

<210> 99

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 99

ggattcaactttagagactttgcc 24

<210> 100

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 100

Gly Phe Asn Phe Arg Asp Phe Ala

15

<210> 101

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 101

attagtggta gtggtagtaa taca 24

<210> 102

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 102

Ile Ser Gly Ser Gly Ser Asn Thr

15

<210> 103

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 103

gcgaaagatc gactctctat aacaattcgc ccacgctatt acggtctgga cgtc 54

<210> 104

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 104

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

1 5 10 15

Asp Val

<210> 105

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 105

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcgagtca ggacattagc aattattttg cctggtatca gcagaagcca 120

gggaaagttc ctaagctcct gatctttgct gcatccactt tgcatccagg ggtcccatct 180

cggttcagtg gcagtggatc tgggacagat ttcactctca ccattcgcag cctgcagcct 240

gaagatgttg caacttatta ctgtcaaaaa tatgacagtg ccccgtacac ttttggccag 300

gggaccaagg tggaaatcaa acga 324

<210> 106

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 106

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Phe Ala Ala Ser Thr Leu His Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 107

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 107

caggacatta gcaattat 18

<210> 108

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 108

Gln Asp Ile Ser Asn Tyr

15

<210> 109

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 109

gctgcatcc 9

<210> 110

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 110

Ala Ala Ser

1

<210> 111

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 111

caaaaatatg acagtgcccc gtacact 27

<210> 112

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 112

Gln Lys Tyr Asp Ser Ala Pro Tyr Thr

15

<210> 113

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 113

gaggtgcagc tggtggagtc tgagggactc ttggaacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caactttaga gactttgcca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct attagtggta gtggtagtaa tacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaacca cacgctgtat 240

ctgcgaatga acagcctgag agccgaagac acggccgtgt attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattac ggtctggacg tctggggcca agggaccacg 360

gtcaccgtct cc 372

<210> 114

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 114

Glu Val Gln Leu Val Glu Ser Glu Gly Leu Leu Glu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Arg Asp Phe

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn His Thr Leu Tyr

65 70 75 80

Leu Arg Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 115

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 115

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcgagtca ggacattagc aattattttg cctggtatca gcagaagcca 120

gggaaagttc ctaagctcct gatctttgct gcatccactt tgcatccagg ggtcccatct 180

cggttcagtg gcagtggatc tgggacagat ttcactctca ccattcgcag cctgcagcct 240

gaagatgttg caacttatta ctgtcaaaaa tatgacagtg ccccgtacac ttttggccag 300

gggaccaagc tggagatcaa a 321

<210> 116

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 116

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Phe Ala Ala Ser Thr Leu His Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 117

<211> 373

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 117

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caactttaga gactttgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtagtaa tacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattac ggtctggacg tctggggcca agggaccacg 360

gtcaccgtct cct 373

<210> 118

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 118

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Arg Asp Phe

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 119

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 119

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcgagtca ggacattagc aattatttag cctggtatca gcagaaacca 120

gggaaagttc ctaagctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct 180

cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagatgttg caacttatta ctgtcaaaaa tatgacagtg ccccgtacac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 120

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 120

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 121

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 121

caggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgtag cttctggatt cacccttaac aactttgtca tgaactgggt ccgccaggtt 120

ccagggaagg gactggagtg ggtctctttt attagtgcta gtggtggtag tatatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca cttccaagaa cacattatat 240

ctgcaaatga acagcctgag agccgacgac acggccgtct attactgtgc gaaatccccg 300

tataactgga acccctttga ctattggggc cagggaacca cggtcaccgt ctcctca 357

<210> 122

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 122

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asn Asn Phe

20 25 30

Val Met Asn Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Phe Ile Ser Ala Ser Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Pro Tyr Asn Trp Asn Pro Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 123

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 123

ggattcaccc ttaacaactt tgtc 24

<210> 124

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 124

Gly Phe Thr Leu Asn Asn Phe Val

15

<210> 125

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 125

attagtgcta gtggtggtag tata 24

<210> 126

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 126

Ile Ser Ala Ser Gly Gly Ser Ile

15

<210> 127

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 127

gcgaaatccc cgtataactg gaaccccttt gactat 36

<210> 128

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 128

Ala Lys Ser Pro Tyr Asn Trp Asn Pro Phe Asp Tyr

1 5 10

<210> 129

<211> 327

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 129

gacatccagt tgacccagtc tccagccacc ctgtctgtgt ctccagggga acgagccacc 60

ctctcctgca gggccagtct gagtgttagc agcaaattag cctggtacca gcagacacct 120

ggccaggctc ccagactcct catctatagt gcctccaccc gggccactgg tatcccagtc 180

aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240

gaagattttg cggtttatta ctgtcagcag tataatcatt ggcctccgta cacttttggc 300

caggggacca aggtggagat caaacga 327

<210> 130

<211> 109

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 130

Asp Ile Gln Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Leu Ser Val Ser Ser Lys

20 25 30

Leu Ala Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn His Trp Pro Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 131

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 131

ctgagtgtta gcagcaaa 18

<210> 132

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 132

Leu Ser Val Ser Ser Lys

15

<210> 133

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 133

agtgcctcc 9

<210> 134

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 134

Ser Ala Ser

1

<210> 135

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 135

cagcagtata atcattggcc tccgtacact 30

<210> 136

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 136

Gln Gln Tyr Asn His Trp Pro Pro Tyr Thr

1 5 10

<210> 137

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 137

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgtag cttctggatt cacccttaac aactttgtca tgaactgggt ccgccaggtt 120

ccagggaagg gactggagtg ggtctctttt attagtgcta gtggtggtag tatatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca cttccaagaa cacattatat 240

ctgcaaatga acagcctgag agccgacgac acggccgtct attactgtgc gaaatccccg 300

tataactgga acccctttga ctattggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 138

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 138

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asn Asn Phe

20 25 30

Val Met Asn Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Phe Ile Ser Ala Ser Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Pro Tyr Asn Trp Asn Pro Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 139

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 139

gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga acgagccacc 60

ctctcctgca gggccagtct gagtgttagc agcaaattag cctggtacca gcagacacct 120

ggccaggctc ccagactcct catctatagt gcctccaccc gggccactgg tatcccagtc 180

aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240

gaagattttg cggtttatta ctgtcagcag tataatcatt ggcctccgta cacttttggc 300

caggggacca agctggagat caaa 324

<210> 140

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 140

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Leu Ser Val Ser Ser Lys

20 25 30

Leu Ala Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn His Trp Pro Pro

85 90 95

Tyr Thr Phe Gly Glyn Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 141

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 141

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacccttaac aactttgtca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtgcta gtggtggtag tatatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaatccccg 300

tataactgga acccctttga ctattggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 142

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 142

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asn Asn Phe

20 25 30

Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Ala Ile Ser Ala Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Pro Tyr Asn Trp Asn Pro Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 143

<211> 325

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 143

gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60

ctctcctgca gggccagtct gagtgttagc agcaaattag cctggtacca gcagaaacct 120

ggccaggctc ccaggctcct catctatagt gcctccacca gggccactgg tatcccagcc 180

aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240

gaagattttg cagtttatta ctgtcagcag tataatcatt ggcctccgta cacttttggc 300

caggggacca agctggagat caaac 325

<210> 144

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 144

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Leu Ser Val Ser Ser Lys

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn His Trp Pro Pro

85 90 95

Tyr Thr Phe Gly Glyn Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 145

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 145

caggtgcagc tggtggagtc tgggggaggc ttggaacagc cgggggggtc cctgagactc 60

tcctgtgcag gctctggatt cacctttaga gactatgcca tgacctgggt ccgccaggct 120

ccagggaagg gactggagtg ggtctcatct attagtggtt ccggtggtaa cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattat ggtttggacg tctggggcca agggtccacg 360

gtcaccgtct cctca 375

<210> 146

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 146

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Ser Thr Val Thr Val Ser Ser

115 120 125

<210> 147

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 147

ggattcacct ttagagacta tgcc 24

<210> 148

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 148

Gly Phe Thr Phe Arg Asp Tyr Ala

15

<210> 149

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 149

attagtggtt ccggtggtaa caca 24

<210> 150

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 150

Ile Ser Gly Ser Gly Gly Asn Thr

15

<210> 151

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 151

gcgaaagatc gactctctat aacaattcgc ccacgctatt atggtttgga cgtc 54

<210> 152

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 152

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

1 5 10 15

Asp Val

<210> 153

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 153

gacatcgtgt tgacccagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg tatagtattg gatacaacta tttggattgg 120

tacctgcaga agtcagggca gtctccacag ctccttatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttgggttt tattactgca tgcaagctct acaaactccg 300

tacacttttg gccaggggac caagctggag atcaaacga 339

<210> 154

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 154

Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ile Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ser Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 155

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 155

cagagcctcc tgtatagtat tggatacaac tat 33

<210> 156

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 156

Gln Ser Leu Leu Tyr Ser Ile Gly Tyr Asn Tyr

1 5 10

<210> 157

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 157

ttgggttct 9

<210> 158

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 158

Leu Gly Ser

1

<210> 159

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 159

atgcaagctc tacaaactcc gtacact 27

<210> 160

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 160

Met Gln Ala Leu Gln Thr Pro Tyr Thr

15

<210> 161

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 161

gaggtgcagc tggtggagtc tgggggaggc ttggaacagc cgggggggtc cctgagactc 60

tcctgtgcag gctctggatt cacctttaga gactatgcca tgacctgggt ccgccaggct 120

ccagggaagg gactggagtg ggtctcatct attagtggtt ccggtggtaa cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattat ggtttggacg tctggggcca agggaccacg 360

gtcaccgtct cc 372

<210> 162

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 162

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 163

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 163

gacatcgtga tgacccagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg tatagtattg gatacaacta tttggattgg 120

tacctgcaga agtcagggca gtctccacag ctccttatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttgggttt tattactgca tgcaagctct acaaactccg 300

tacacttttg gccaggggac caagctggag atcaaa 336

<210> 164

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 164

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ile Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ser Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 165

<211> 373

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 165

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttaga gactatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggtt ccggtggtaa cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga 300

ctctctataa caattcgccc acgctattat ggtttggacg tctggggcca agggaccacg 360

gtcaccgtct cct 373

<210> 166

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 166

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 167

<211> 337

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 167

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg tatagtattg gatacaacta tttggattgg 120

tacctgcaga agccaggggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300

tacacttttg gccaggggac caagctggag atcaaac 337

<210> 168

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 168

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ile Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 169

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 169

caggtgcagc tggtggagtc tgggggagtc ttggagcagc ctggggggtc cctgagactc 60

tcctgtacag cctctggatt cacctttaga gactatgcca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct attagtggta gtggtggtaa tacatactac 180

gcagactccg tgaggggccg gttcaccatc tccagagaca actccaacca cacgctgtat 240

ctgcaaatga acagcctgag agccgaagac acggccgtat attactgtgc gaaagatcga 300

ctctccataa caattcgccc acgctattac ggtttggacg tctggggcca agggtccacg 360

gtcaccgtct cctca 375

<210> 170

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 170

Gln Val Gln Leu Val Glu Ser Gly Gly Val Leu Glu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn His Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Ser Thr Val Thr Val Ser Ser

115 120 125

<210> 171

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 171

ggattcacct ttagagacta tgcc 24

<210> 172

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 172

Gly Phe Thr Phe Arg Asp Tyr Ala

15

<210> 173

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 173

attagtggta gtggtggtaa taca 24

<210> 174

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 174

Ile Ser Gly Ser Gly Gly Asn Thr

15

<210> 175

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 175

gcgaaagatc gactctccat aacaattcgc ccacgctatt acggtttgga cgtc 54

<210> 176

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 176

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

1 5 10 15

Asp Val

<210> 177

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 177

gatattgtga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

attacttgcc gggcgagtca ggacattagc aattattttg cctggtatca gcagaagcca 120

gggaaagttc ctaaactcct gatctttgct gcatccactt tgcatccagg ggtcccatct 180

cggttcagtg gcagtggatc tgggacagat ttcactctca ccattagtag cctgcagcct 240

gaagatgttg caacttatta ctgtcaaaag tataacagtg ccccgtacac ttttggccag 300

gggaccaagg tggaaatcaa acga 324

<210> 178

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 178

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Phe Ala Ala Ser Thr Leu His Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 179

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 179

caggacatta gcaattat 18

<210> 180

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 180

Gln Asp Ile Ser Asn Tyr

15

<210> 181

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 181

gctgcatcc 9

<210> 182

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 182

Ala Ala Ser

1

<210> 183

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 183

caaaagtata acagtgcccc gtacact 27

<210> 184

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 184

Gln Lys Tyr Asn Ser Ala Pro Tyr Thr

15

<210> 185

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 185

gaggtgcagc tggtggagtc tgggggagtc ttggagcagc ctggggggtc cctgagactc 60

tcctgtacag cctctggatt cacctttaga gactatgcca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct attagtggta gtggtggtaa tacatactac 180

gcagactccg tgaggggccg gttcaccatc tccagagaca actccaacca cacgctgtat 240

ctgcaaatga acagcctgag agccgaagac acggccgtat attactgtgc gaaagatcga 300

ctctccataa caattcgccc acgctattac ggtttggacg tctggggcca agggaccacg 360

gtcaccgtct cc 372

<210> 186

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 186

Glu Val Gln Leu Val Glu Ser Gly Gly Val Leu Glu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn His Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 187

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 187

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

attacttgcc gggcgagtca ggacattagc aattattttg cctggtatca gcagaagcca 120

gggaaagttc ctaaactcct gatctttgct gcatccactt tgcatccagg ggtcccatct 180

cggttcagtg gcagtggatc tgggacagat ttcactctca ccattagtag cctgcagcct 240

gaagatgttg caacttatta ctgtcaaaag tataacagtg ccccgtacac ttttggccag 300

gggaccaagc tggagatcaa a 321

<210> 188

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 188

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Phe Ala Ala Ser Thr Leu His Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 189

<211> 373

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 189

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttaga gactatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtaa tacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga 300

ctctccataa caattcgccc acgctattac ggtttggacg tctggggcca agggaccacg 360

gtcaccgtct cct 373

<210> 190

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 190

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 191

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 191

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcgagtca ggacattagc aattatttag cctggtatca gcagaaacca 120

gggaaagttc ctaagctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct 180

cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagatgttg caacttatta ctgtcaaaag tataacagtg ccccgtacac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 192

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 192

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 193

<211> 355

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 193

gaagtgcacc tggtggaatc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60

tcctgtgagg cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct 120

ccggggaagg gcctggaatg ggtctcaggt cttagtcgga caagtgtcag tataggctat 180

gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctttat 240

ttggaaatga acagtctgag acctgaggac acggccttat attactgtgc aaaatggggg 300

acccgggggt attttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355

<210> 194

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 194

Glu Val His Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Leu Ser Arg Thr Ser Val Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Glu Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Trp Gly Thr Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 195

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 195

ggattcacct ttgatgatta tgcc 24

<210> 196

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 196

Gly Phe Thr Phe Asp Asp Tyr Ala

15

<210> 197

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 197

cttagtcgga caagtgtcag tata 24

<210> 198

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 198

Leu Ser Arg Thr Ser Val Ser Ile

15

<210> 199

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 199

gcaaaatggg ggacccgggg gtattttgac tac 33

<210> 200

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 200

Ala Lys Trp Gly Thr Arg Gly Tyr Phe Asp Tyr

1 5 10

<210> 201

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 201

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagt atttggttag cctggtatca gcagagtcca 120

gggaaagccc ctaaactcct gatcaatgtt gcatcccgtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcaacag tctgcagcct 240

gaagattttg taacttacta ttgtcaacag gctaacagtt tcccgatcac cttcggccaa 300

gggacacgac tggcgaccaa ac 322

<210> 202

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 202

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ile Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Ser Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Asn Val Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Val Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile

85 90 95

Thr Phe Gly Glyn Gly Thr Arg Leu Ala Thr Lys

100 105

<210> 203

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 203

caggatatta gtatttgg 18

<210> 204

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 204

Gln Asp Ile Ser Ile Trp

15

<210> 205

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 205

gttgcatcc 9

<210> 206

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 206

Val Ala Ser

1

<210> 207

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 207

caacaggcta acagtttccc gatcacc 27

<210> 208

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 208

Gln Gln Ala Asn Ser Phe Pro Ile Thr

15

<210> 209

<211> 355

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 209

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60

tcctgtgagg cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct 120

ccggggaagg gcctggaatg ggtctcaggt cttagtcgga caagtgtcag tataggctat 180

gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctttat 240

ttggaaatga acagtctgag acctgaggac acggccttat attactgtgc aaaatggggg 300

acccgggggt attttgacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355

<210> 210

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 210

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Leu Ser Arg Thr Ser Val Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Glu Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Trp Gly Thr Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 211

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 211

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagt atttggttag cctggtatca gcagagtcca 120

gggaaagccc ctaaactcct gatcaatgtt gcatcccgtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcaacag tctgcagcct 240

gaagattttg taacttacta ttgtcaacag gctaacagtt tcccgatcac cttcggccaa 300

gggacacgac tggagattaa ac 322

<210> 212

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 212

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ile Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Ser Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Asn Val Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Val Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 213

<211> 355

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 213

gaagtgcagc tggtggagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct 120

ccagggaagg gcctggagtg ggtctcaggt cttagtcgga caagtgtcag tataggctat 180

gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240

ctgcaaatga acagtctgag agctgaggac acggccttgt attactgtgc aaaatggggg 300

acccgggggt attttgacta ctggggccaa ggaaccctgg tcaccgtctc ctcag 355

<210> 214

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 214

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Leu Ser Arg Thr Ser Val Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Trp Gly Thr Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 215

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 215

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagt atttggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgtt gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaacagtt tcccgatcac cttcggccaa 300

gggacacgac tggagattaa ac 322

<210> 216

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 216

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ile Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Val Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 217

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 217

gaggtgcagc tgttggagtc tgggggaggc ttgctacagc cgggggggtc cctgagactc 60

tcctgtgcag ccctctggaat cacctttagc acctatgcca tgagctggggt ccgtcaggct 120

ccagggaggg ggctggagtg ggtctcagct attagtggta gtggtgatag cacatcctac 180

gcagactccg tgaagggccg gttcaccagc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagtcata 300

gcagctcgtc ctcactggaa cttcgatctc tggggccgtg gcaccctggt cactgtctcc 360

tca 363

<210> 218

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 218

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Leu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Thr Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Asp Ser Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ser Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Val Ile Ala Ala Arg Pro His Trp Asn Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 219

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 219

ggaatcacct ttagcaccta tgcc 24

<210> 220

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 220

Gly Ile Thr Phe Ser Thr Tyr Ala

15

<210> 221

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 221

attagtggta gtggtgatag caca 24

<210> 222

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 222

Ile Ser Gly Ser Gly Asp Ser Thr

15

<210> 223

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 223

gcgaaagtca tagcagctcg tcctcactgg aacttcgatc tc 42

<210> 224

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 224

Ala Lys Val Ile Ala Ala Arg Pro His Trp Asn Phe Asp Leu

1 5 10

<210> 225

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 225

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagt agatatttag cctggtatca acagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg gagtttatta ctgtcagcag cgtagtgact ggccgctcac tttcggcgga 300

gggaccaagg tggagatcaa acgg 324

<210> 226

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 226

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Gly Val Tyr Tyr Cys Gln Gln Arg Ser Asp Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 227

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 227

cagagtgtta gtagatat 18

<210> 228

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 228

Gln Ser Val Ser Arg Tyr

15

<210> 229

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 229

gatgcatcc 9

<210> 230

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 230

Asp Ala Ser

1

<210> 231

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 231

cagcagcgta gtgactggcc gctcact 27

<210> 232

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 232

Gln Gln Arg Ser Asp Trp Pro Leu Thr

15

<210> 233

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 233

gaggtgcagc tgttggagtc tgggggaggc ttgctacagc cgggggggtc cctgagactc 60

tcctgtgcag ccctctggaat cacctttagc acctatgcca tgagctggggt ccgtcaggct 120

ccagggaggg ggctggagtg ggtctcagct attagtggta gtggtgatag cacatcctac 180

gcagactccg tgaagggccg gttcaccagc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagtcata 300

gcagctcgtc ctcactggaa cttcgatctc tggggccgtg gcaccctggt cactgtctcc 360

tca 363

<210> 234

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 234

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Leu Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Thr Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Asp Ser Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ser Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Val Ile Ala Ala Arg Pro His Trp Asn Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 235

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 235

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagt agatatttag cctggtatca acagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg gagtttatta ctgtcagcag cgtagtgact ggccgctcac tttcggcgga 300

gggaccaagg tggagatcaa acgg 324

<210> 236

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 236

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Gly Val Tyr Tyr Cys Gln Gln Arg Ser Asp Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 237

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 237

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc cgggggggtc cctgagactc 60

tcctgtgcag ccctctggaat cacctttagc acctatgcca tgagctggggt ccgtcaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtgatag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagtcata 300

gcagctcgtc ctcactggaa cttcgatctc tggggccgtg gcaccctggt cactgtctcc 360

tca 363

<210> 238

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 238

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Thr Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Val Ile Ala Ala Arg Pro His Trp Asn Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 239

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 239

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagt agatatttag cctggtatca acagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg cagtttatta ctgtcagcag cgtagtgact ggccgctcac tttcggcgga 300

gggaccaagg tggagatcaa acgg 324

<210> 240

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 240

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asp Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 241

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 241

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

acctgtgcag cctctggatt caccttcagt agtaatggca tgcactggggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcaatt atatcatatg atggaaataa tcaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagca cacgctgtat 240

ctggaaatga acagcctgag agctgaggac acggctgtgt attactgtac aaaagccatc 300

tctataagtg gaacttacaa ctggttcgat tcctggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 242

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 242

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ile Ile Ser Tyr Asp Gly Asn Asn Gln Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys His Thr Leu Tyr

65 70 75 80

Leu Glu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Lys Ala Ile Ser Ile Ser Gly Thr Tyr Asn Trp Phe Asp Ser Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 243

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 243

ggattcacct tcagtagtaa tggc 24

<210> 244

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 244

Gly Phe Thr Phe Ser Ser Asn Gly

15

<210> 245

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 245

atatcatatg atggaaataa tcaa 24

<210> 246

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 246

Ile Ser Tyr Asp Gly Asn Asn Gln

15

<210> 247

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 247

acaaaagcca tctctataag tggaacttac aactggttcg attcc 45

<210> 248

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 248

Thr Lys Ala Ile Ser Ile Ser Gly Thr Tyr Asn Trp Phe Asp Ser

1 5 10 15

<210> 249

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 249

gaaattgtat tgacacagtc tccagccatc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc aggtacttag cctggtacca acagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg cagtttatta ctgtcaacag cgtagcaact ggccgctcac tttcggcgga 300

gggaccaagg tggagatcaa acgg 324

<210> 250

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 250

Glu Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 251

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 251

cagagtgtta gcaggtac 18

<210> 252

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 252

Gln Ser Val Ser Arg Tyr

15

<210> 253

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 253

gatgcatcc 9

<210> 254

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 254

Asp Ala Ser

1

<210> 255

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 255

caacagcgta gcaactggcc gctcact 27

<210> 256

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 256

Gln Gln Arg Ser Asn Trp Pro Leu Thr

15

<210> 257

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 257

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

acctgtgcag cctctggatt caccttcagt agtaatggca tgcactggggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcaatt atatcatatg atggaaataa tcaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagca cacgctgtat 240

ctggaaatga acagcctgag agctgaggac acggctgtgt attactgtac aaaagccatc 300

tctataagtg gaacttacaa ctggttcgat tcctggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 258

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 258

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ile Ile Ser Tyr Asp Gly Asn Asn Gln Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys His Thr Leu Tyr

65 70 75 80

Leu Glu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Lys Ala Ile Ser Ile Ser Gly Thr Tyr Asn Trp Phe Asp Ser Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 259

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 259

gaaattgtat tgacacagtc tccagccatc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc aggtacttag cctggtacca acagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg cagtttatta ctgtcaacag cgtagcaact ggccgctcac tttcggcgga 300

gggaccaagg tggagatcaa acgg 324

<210> 260

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 260

Glu Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 261

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 261

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agtaatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaaataa tcaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtac aaaagccatc 300

tctataagtg gaacttacaa ctggttcgat tcctggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 262

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 262

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Asn Asn Gln Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Lys Ala Ile Ser Ile Ser Gly Thr Tyr Asn Trp Phe Asp Ser Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 263

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 263

gaaattgtat tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc aggtacttag cctggtacca acagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg cagtttatta ctgtcaacag cgtagcaact ggccgctcac tttcggcgga 300

gggaccaagg tggagatcaa acgg 324

<210> 264

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 264

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 265

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> OPTION

<222> (1)...(8)

<223> Xaa=Any amino acid

<400> 265

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

15

<210> 266

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> OPTION

<222> (1)...(8)

<223> Xaa=Any amino acid

<400> 266

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

15

<210> 267

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> OPTION

<222> (1)...(18)

<223> Xaa=Any amino acid

<400> 267

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa

<210> 268

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> OPTION

<222> (1)...(11)

<223> Xaa=Any amino acid

<400> 268

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10

<210> 269

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> OPTION

<222> (1)...(3)

<223> Xaa=Any amino acid

<400> 269

Xaa Xaa Xaa

1

<210> 270

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> OPTION

<222> (1)...(9)

<223> Xaa=Any amino acid

<400> 270

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

15

<210> 271

<211> 330

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 271

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 272

<211> 327

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 272

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 273

<211> 327

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic

<400> 273

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 274

<211> 207

<212> PROTEIN

<213> Homo sapiens

<400> 274

Met Lys Val Leu Gln Glu Pro Thr Cys Val Ser Asp Tyr Met Ser Ile

1 5 10 15

Ser Thr Cys Glu Trp Lys Met Asn Gly Pro Thr Asn Cys Ser Thr Glu

20 25 30

Leu Arg Leu Leu Tyr Gln Leu Val Phe Leu Leu Ser Glu Ala His Thr

35 40 45

Cys Ile Pro Glu Asn Asn Gly Gly Ala Gly Cys Val Cys His Leu Leu

50 55 60

Met Asp Asp Val Val Ser Ala Asp Asn Tyr Thr Leu Asp Leu Trp Ala

65 70 75 80

Gly Gln Gln Leu Leu Trp Lys Gly Ser Phe Lys Pro Ser Glu His Val

85 90 95

Lys Pro Arg Ala Pro Gly Asn Leu Thr Val His Thr Asn Val Ser Asp

100 105 110

Thr Leu Leu Leu Thr Trp Ser Asn Pro Tyr Pro Pro Asp Asn Tyr Leu

115 120 125

Tyr Asn His Leu Thr Tyr Ala Val Asn Ile Trp Ser Glu Asn Asp Pro

130 135 140

Ala Asp Phe Arg Ile Tyr Asn Val Thr Tyr Leu Glu Pro Ser Leu Arg

145 150 155 160

Ile Ala Ala Ser Thr Leu Lys Ser Gly Ile Ser Tyr Arg Ala Arg Val

165 170 175

Arg Ala Trp Ala Gln Cys Tyr Asn Thr Thr Trp Ser Glu Trp Ser Pro

180 185 190

Ser Thr Lys Trp His Asn Ser Tyr Arg Glu Pro Phe Glu Gln His

195 200 205

<210> 275

<211> 231

<212> PROTEIN

<213> Macaca fascialis

<400> 275

Met Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val

1 5 10 15

Leu Leu Gln Val Ala Ser Ser Gly Ser Met Lys Val Leu Gln Glu Pro

20 25 30

Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met

35 40 45

Gly Gly Pro Thr Asn Cys Ser Ala Glu Leu Arg Leu Leu Tyr Gln Leu

50 55 60

Val Phe Gln Ser Ser Glu Thr His Thr Cys Val Pro Glu Asn Asn Gly

65 70 75 80

Gly Val Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Met

85 90 95

Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys

100 105 110

Gly Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn

115 120 125

Leu Thr Val His Thr Asn Val Ser Asp Thr Val Leu Leu Thr Trp Ser

130 135 140

Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn Asp Leu Thr Tyr Ala

145 150 155 160

Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Tyr Ser Arg Ile His Asn

165 170 175

Val Thr Tyr Leu Lys Pro Thr Leu Arg Ile Pro Ala Ser Thr Leu Lys

180 185 190

Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln His Tyr

195 200 205

Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp Tyr Asn Ser

210 215 220

Tyr Arg Glu Pro Phe Glu Gln

225 230

<---

Claims (13)

1. A method of reducing the incidence of one or more asthma exacerbations in a subject suffering from moderate-severe eosinophilic asthma, comprising administering to the subject an antibody or antigen-binding fragment that specifically binds to the interleukin 4 receptor (IL-4R), wherein said antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining region (HCDR) sequences containing SEQ ID NOs: 148, 150 and 152 and three light chain complementarity determining region (LCDR) sequences containing SEQ ID NOs: : 156, 158 and 160, and wherein said antibody or antigen-binding fragment thereof is an additional maintenance treatment. 2. The method of claim 1, wherein the asthma exacerbation is selected from the group consisting of the following: (a) 30% or greater reduction from baseline in peak morning expiratory flow (PEF) on two consecutive days; (b) six or more additional puffs of albuterol or levalbuterol aerosol to facilitate breathing over a 24-hour period compared to baseline on two consecutive days; And (c) worsening asthma requiring: (i) systemic steroid treatment, or (ii) increasing the use of inhaled corticosteroids by at least 4 times the last dose taken before treatment was stopped, or (iii) hospitalizations. 3. A method of reducing the incidence of asthma exacerbations or improving one or more asthma-related parameters in a subject suffering from moderate-severe eosinophilic asthma, comprising sequentially administering to the subject a single initial dose of an antibody or antigen-binding fragment thereof that specifically binds to the interleukin 4 receptor (IL- 4R), followed by one or more secondary doses of said antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining region (HCDR) sequences containing SEQ ID NOs: 148, 150 and 152 and three light chain complementarity determining region (LCDR) sequences containing SEQ ID NOs: : 156, 158 and 160, and wherein said antibody or antigen-binding fragment thereof is an additional maintenance treatment.