Method for reducing eosinophil count

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely immunology, and may be used for reducing eosinophil count in a human body. That is ensured by the parenteral administration of approximately 0.01 mg/kg to approximately 0.25 mg/kg of a monoclonal chimeric humanised antibody or a human antibody which binds to IL-5P and contains an immunoglobulin Fc fragment and is fucose-free; the administered antibody reduces the peripheral blood circulating eosinophil count below the detection threshold, and the circulating eosinophil calculation level remains below the detection threshold for approximately 28 days after antibody dosing.

EFFECT: using the given method leads to fast and prolonged reduction of peripheral blood eosinophils.

9 cl, 14 ex, 31 dwg, 1 tbl

The scope of the invention

The present invention relates to a method of reducing the number of eosinophils in the body.

Background of invention

Eosinophils participate in the development of various diseases, including allergic diseases, and is thought to play an important role in the rising incidence of allergic diseases such as chronic bronchial asthma and atopic dermatitis (Adv. Immunol., 39, 177 (1986), Immunol. Today, 13, 501 (1992)). In addition, eosinophils are also involved in the development of diseases, which are usually hypereosinophilia syndrome (HPS), such as eosinophilia, eosinophilic enterogastritis, eosinophilic leukemia, eosinophilic granuloma and disease Kimura (Ann. Intern. Med., 97, 78 (1982)).

Eosinophilic granuloma is a benign cryptogenic defeat, which is osteolytic and alopecia, and is known to be associated with marked tissue eosinophilia (U.S. Armed Forces Med. J., 2, 1085 (1951)). According to the register of patents of Japan, related to cancer of the bone tissue (1972-1984) 379 of 404 (93,8%) patients suffer from eosinophilic granuloma. Eosinophilic granuloma at an early stage mainly includes eosinophils and histiocytes, and granuloma at a later stage includes fibrosis or may lead to the development of pneumosclerosis. Therefore, along with inflammatory is entrusted diseases, such as allergies, eosinophils can cause various other diseases.

Interleukin-5 (hereinafter IL-5), interleukin-3 (hereinafter IL-3) and granulocyte/macrophage colony-stimulating factor (hereinafter KM-CSF), which are members of a family of cytokines that participate in the regulation of differentiation, proliferation and activation of eosinophils. It is established that one of the cytokines, namely IL-5-specific effect on eosinophils and induces specific terminal differentiation (Proc. Natl. Acad. Sci. USA., 85, 2288 (1988)).

In vitro IL-3 and/or GM-CSF can activate eosinophils or to prolong their survival (J. Clin. Invest., 81, 1986 (1988)). Moreover, IL-3 and/or GM-CSF are also mainly on the induction of immature eosinophils from myeloid stem cells (Blood, 76, 1956 (1990)). In addition, chemokines, such as eotaxin and RANTES (normal controls activation of expressed and secreted T-cell), induce the chemotaxis of eosinophils to the inflammation (Clin. Exp. Allergy, 26, 1005 (1996)). Factors stem cells (hereinafter referred to FGC) participate in the accumulation of eosinophils in allergic bronchitis. Along with IL-5 there are many factors that affect the function of eosinophils.

Eosinophils were divided into subgroups, granular, eosinophils and degranulating eosinophils. Found that eosinophils into granuloma the data after activation (Immunology, 47, 531 (1982)). Degranulating eosinophils also belong to the activated eosinophils. It is reported that in eosinophils in the peripheral blood of patients suffering from hydro, along with qualitative and quantitative changes occur changes (Clin. Exp.ImmunoL, 24, 423 (1976)). Activated eosinophils participate in the progression of symptom HPP (Am. J. Cardiol., 52, 321 (1983)). In addition to patients suffering from HPP, activated eosinophils are also present in the peripheral blood and in bronchoalveolar aspirates (BASS) patients with a diagnosis of bronchial asthma (Am. Rev. Respir. Dis, 132, 981 (1985)). By activated eosinophils expressed various receptors, such as receptors of cytokines (J. Immunol., 142, 4416 (1989)). Compared with pelletised eosinophils specified degranulating eosinophils have increased sensitivity to IL-5 (Clin. Exp. Immunol., 85, 312 (1991), J. Exp. Med., 172, 1347 (1990)).

It was also established that these activated eosinophils survive in vitro in the absence of cytokines, inducing differentiation and proliferation of eosinophils (J. Exp. Med., 170, 343 (1989)). Thus, the properties of activated eosinophils are similar to properties of eosinophils, which infiltrate tissue, such as the alveoli (Int. Arch. Allergy ImmunoL, 120, 91 (1999)). Detailed reasons for the insensitivity of activated eosinophils to the action of cytokines is unknown, but their degranulation is prolonged survival is probably due to the different functionally important compounds other than IL-5.

Compounds that inhibit the activity of cytokines or chemokines, which are involved in the differentiation or proliferation of eosinophils, can be seen as agents that inhibit the function of eosinophils. However, in most cases, these agents have no effect on cytokine-insensitive eosinophils that are activated and infiltrated in inflammation. Therefore, eosinophil-specific inhibition and induction of death of activated eosinophils are required for the suppression of the functions of any eosinophila. However, hitherto unknown anti-inflammatory agent, which is able to induce apoptosis of activated eosinophils.

Currently, the treatment of patients suffering from eosinophilic diseases, includes the introduction of steroids. However, the introduction of steroids is often associated with side effects. In particular, such treatment is accompanied by several other problems, for example, during a temporary cessation of steroids pathological condition of a patient can return to the original level, and the continuous introduction of steroids induces resistance to anticancer agent. Therefore, there is a need to develop safe and effective methods of treating diseases and disorders mediated eosinophil is I.

Summary of the invention

The invention proposes a method of reducing the number of eosinophils in the body, which is that a given patient is administered the compound to bind to the IL-5P, which includes (a) a segment-specific binds to IL-5P, and (b) Fc fragment of immunoglobulin.

Brief description of figures

Some embodiments of the invention are illustrated in the following figures, without limiting its scope.

Figure 1. The reduction of cationic protein of eosinophils (CBA) in serum. CBA is a marker produced by eosinophils. In patients from group 1 indicated a decrease in the content of CBA (see figure 1) corresponds to the reduction in the number of eosinophils. On the y-axis shows the concentration of CBA (ng/ml)on the x-axis indicates the time (days).

Figure 2. Reversible reduction in the number of basophils in the peripheral blood. Basophils in blood flow was measured in patients of group 1. On the y-axis is the number of basophils (basophils/mm), and the x-axis indicates the time (days). The rapid decline in the number of basophils in the peripheral blood was observed after 24 h after injection.

Figure 3. Increased (reversible) number of hsCRP (C-reactive protein, which is associated with high sensitivity) in subjects with eosinophilia in the initial phase (baseline). The analysis given marker in patients of the group 1 suggests, observed expected immune response to cells expressing IL-5P. On the y-axis is the concentration of hsCRP (mg/DL), and on the x-axis indicates the time (days).

Figure 4. The minimum increase in the content of IL-6 in serum. The figure shows the results of the analysis of the cytokine IL-6 in patients of group 1. On the y-axis shows the concentration of IL-6 (PG/ml), and the x-axis indicates the time (h).

Figure 5. Variable reduction in the number of neutrophils in the bloodstream. On both panels the results of the analysis of the number of neutrophils in patients of group 1.

6. Variable reduction in the number of lymphocytes in the bloodstream. On both panels the results of the analysis of the number of lymphocytes in patients of group 1.

7. Steady and moderate decrease in the number of NK-cells (%) on day 1. The number of NK-cells in patients of group 1 were measured before treatment, at day 1 after injection and at day 28 after injection.

Fig. Reduced feno in subjects with higher baseline. Fraction of exhaled nitric oxide was measured in patients of group 1. The specified analysis is a noninvasive method for diagnosing pneumonia, and the data indicate a trend of decrease in the severity of the disease.

Fig.9. Analysis of cytotoxicity in vitro. The number of MEDI-563 was measured using the analysis of cytotoxicity in vitro compared with control antibody, which is not associated with the IL-5P (A), and further the additional control fokusirovannym MEDI-563 (B). In the experiment used cells effectors KS 1333 and target cells CTLL2 in the ratio of 5:1. Cytotoxicity was measured after 4 hours On the Y-axis is measured cytotoxicity (%)on the X-axis indicates the concentration of antibodies.

Figure 10. The binding of MEDI-563 with rcil-5α. The affinity of MEDI-563 recombinant IL-5α was measured by the method of plasma-surface resonance in three separate experiments.

11. The binding of MEDI-563 with rhuFcγRs. The figure shows the results of measuring the affinity of MEDI-563 to multiple samples of recombinant human FcγRs when compared with the affinity of izotopicheskii related fokusirovannyi control antibodies. It should be noted that MEDI-563 associated with huFcyRIIIa with higher affinity (5-10 times) than muFcyRIV.

Fig. The figure presents the results of the immunohistochemical analysis of the expression of IL-5α in the lungs of the mouse IL-9tg.

Fig. The figure presents the results of the immunohistochemical analysis of the expression of IL-5α in nasal polyps using MEDI-563, which colors all the eosinophils in nasal polyps.

Fig. Mild transient neutropenia in subjects. The figure shows the absolute number of neutrophils in subjects of group 1. On the Y-axis shows the number of neutrophils (neutrophils/mm³), and on the X-axis indicates the time (days).

Fig. The binding of MEDI-563 with eosinophils solid cu is VI healthy donors. Analysis flow cytometry was performed using samples of whole blood, as described in example 6. In three panels, especially on the third panel, entitled "the binding of MEDI-563 with eosinophils are these cytofluorimetry, indicating the binding of MEDI-563 with eosinophils.

Fig. Analysis of cells from transgenic for IL-5 mice was performed by the method of cytofluorimetry. Analysis of cells from transgenic for IL-5 mice spent running cytometry as described in example 7. On figa shows the results of analysis by the method of cytofluorimetry eosinophils SiglecF+CCR3+. On figb shown that all of eosinophils (SiglecF+CCR3+) from the bone marrow, spleen, blood and lung Express IL-5α+, while in the analysis used MA H7 anti-IL-5α.

Fig. According to SACC in vitro MEDI-563 reduce the number positive for IL-5P mononuclear bone marrow cells. Isolated, not attached to the substrate mononuclear bone marrow cells were subjected to MEDI-563 or izotopicheskii control antibodies (R347) in the presence of cell-effectors, CFSE painted. Positive for IL-5P cells identified using antibodies CM and PE conjugate goat anti-IgG PM. Control staining of samples was performed using izotopicheskii control antibodies A and PE conjugate goat anti-S IgG. The staining profiles of view the rd cells after treatment MEDI-563 or R347 presented in separate graphs KM/PE compared with CFSE or A/PE compared with CFSE. Comparison of charts KM/PE compared with CFSE obtained for samples treated with MEDI-563 and R347, suggests that SACC mediated MEDI-563, from the sample is almost completely removes all cells positive for IL-5P.

Fig. MEDI-563 reversible reduce the number of eosinophils in the peripheral blood of subjects with mild asthma. Six volunteers with mild asthma were administered a single dose of intravenous (A) 0.03 mg/kg or (B) 0.1 mg/kg MEDI-563. The number of eosinophils in the peripheral blood was determined by flow cytometry at day 0 before injection and at regular intervals up to day 84 in the next stage of the survey. On the y-axis is the number of eosinophils (eosinophils/mm³), and on the x-axis indicates the time (days). The rapid decline in the number of eosinophils in the peripheral blood was observed at 24 h after injection. Eosinopenia induced MEDI-563, is reversible.

Fig. According to the results of immunohistochemical analysis using MEDI-563 all the eosinophils from the lungs of a healthy person Express the receptor for IL-5α.

Fig. According to the results of immunohistochemical analysis using MEDI-563 all eosinophils from samples of lung biopsy in patients with a diagnosis of asthma Express the receptor for IL-5α.

Fig. The expression of the receptor for IL-5α primary basophils and eosinophils obtained from healthy the donor, analyzed by flow cytometry. The figure shows the profiles of the staining obtained using antibodies anti-IL-5α MEDI-563 and izotopicheskii control antibodies with different specificity. As a positive control used cells CTLLh5r (tumor cells, transfection IL-5α/(3).

Fig. Analysis of antibody-dependent cellular cytotoxicity (SACC) in vitro. Activity deforsirovannym and fokusirovannyi MEDI-563 compared according to SACC in vitro. As cell effector and target cells used isolated primary NK cells and eosinophils, respectively, with a ratio of 5:1. The analysis was carried out in the presence of 1 ng/ml IL-2. Cell death was detected by flow cytometry with staining dye Annexin V On the Y-axis shows the cytotoxicity (% of maximum cytotoxicity), and on the X-axis indicates the concentration of antibodies. The EC₅₀deforsirovannym antibody MEDI-563 is 0,965 PM.

Fig. Analysis of antibody-dependent cellular cytotoxicity (SACC) in vitro. Activity deforsirovannym and fokusirovannyi MEDI-563 compared according to SACC in vitro. As cell effector and target cells used isolated primary cells NK-cells and basophils, respectively. On the Y-axis shows the cytotoxicity (% of maximum cytotoxicity), which is similar to the X-axis indicates the concentration of antibodies. The EC₅₀deforsirovannym antibody MEDI-563 is 0,561 PM.

Fig. Analysis of degranulation of eosinophils according to antibody-dependent cellular cytotoxicity (SACC) in vitro. Release produced by eosinophils neurotoxin (PAN) was analyzed by the method SACC in vitro in the presence of different concentrations fokusirovannyi (MEDI-563F) and deforsirovannym (MEDI-563), anti-IL-5α antibodies. In the analysis, as target cells and cells of the effector used svezhesobrannye eosinophils and NK-cells or cells RVMS respectively. For reference, here is a maximal degranulation of eosinophils in the processing of 1% Triton X-100.

Fig. MEDI-563-specific binds to the epitope in domain D1 extracellular portion of the receptor IL-5α person. The binding of antibodies from transgenic cells, temporarily expressing hybrid proteins IL-5α, were analyzed by flow cytometry. The figure shows the profiles stained with a fluorescent dye. "Polyclonal antibodies" and "MEDI-563" refers profiles staining observed when using polyclonal antibodies against IL-5α person and MEDI-563, respectively. Double staining" refers to the profile fluorescent staining using polyclonal antibodies (x-axis) and MEDI-563 (y-axis). (A) For a series of transgenic hybrid IL-5α the brow of the ESA/mouse there is an unstable expression. "Knock-out" transgenic represent hybrid constructs IL-5α, including one extracellular domain of the receptor of the mouse in the main sequence (frame) receptor of human rights. "Nominee" transgenes are hybrid constructs IL-5α, including one extracellular domain of the receptor member main sequence (frame) receptor mouse. (B) MEDI-563 associated with specific transgenic cells expressing IL-5α person. MEDI-563 is not associated with transgenic cells expressing IL-5α mouse. (B) MEDI-563 is not associated with transgenic cells expressing the hybrid IL-5α, including D1 receptor mouse and extracellular domains D2-D3 receptor ("knock-out by D1"). MEDI-563 associated with specific transgenic cells expressing the hybrid IL-5α, including extracellular domains D2 or D3 receptor mouse in the main sequence (frame) receptor ("knock-out by D2 or D3). (D) MEDI-563 associated with specific transgenic cells expressing the hybrid IL-5α, including domain D1 receptor human and extracellular domains D2-D3 receptor mouse ("nominee on D1"). MEDI-563 is not associated with transgenic cells expressing the hybrid IL-5α mouse, comprising the extracellular domain D2 or D3 receptor ("nominee on D2 or D3).

IG. MEDI-563-specific binds to the epitope in the composition of the segment In the extracellular domain D1 receptor IL-5α person. Antibodies that bind to the transgenic cells expressing the hybrid IL-5α, analyzed by flow cytometry. The figure shows the profiles stained with a fluorescent dye. "Polyclonal antibodies" and "MEDI-563" refers profiles staining observed when using polyclonal antibodies against IL-5α person and MEDI-563, respectively. Double staining" refers to the profile fluorescent staining using polyclonal antibodies (x-axis) and MEDI-563 (y-axis). (A) Amino acid sequence of the extracellular domain D1 receptor IL-5α mouse 75% identical to a similar amino acid sequence of the receptor IL-5α person. The extracellular domain D1 receptor IL-5α is divided into segments a, b and C. In the figure are the amino acid sequences of IL-5α human and mouse, SEQ ID No. 5 and 6 (1-102), respectively. (B) For series hybrid transgenes IL-5α human/mouse was observed unstable expression. "Knock-out" transgenic represent hybrid constructs IL-5α, including one segment of the extracellular domain of the receptor D1 mouse in the main sequence (frame) receptor of human rights. "Nominee" transgenes are hybrid the constructs IL-5α, includes one segment of the extracellular domain D1 receptor member main sequence (frame) receptor, mouse "D1/D2 mouse/D3-TM mouse". (B) MEDI-563 associated with specific transgenic cells expressing (1) IL-5α person, or (2) the hybrid IL-5α mouse, comprising the extracellular domain D1 of the person (nobunny on Dl). MEDI-563 is not associated with transgenic cells expressing (1) receptor IL-5α mouse, or (2) the hybrid IL-5α person, including the extracellular domain D1 receptor mouse. (D) MEDI-563 is not associated with transgenic cells expressing the receptor, including the segment In the extracellular domain D1 receptor mouse in the frame of the receptor ("knock-out software"). MEDI-563 associated with specific transgenic cells expressing the hybrid IL-5α, including the segment a or With the extracellular domain D1 mouse in the main sequence (frame) receptor ("knock-out on a or. (D) MEDI-563 associated with specific transgenic cells expressing the hybrid IL-5α, including the segment In the extracellular domain D1 in the main sequence (frame) receptor, mouse "D1/D2 mouse/D3-TM mouse" ("nobunny In"). MEDI-563 is not associated with transgenic cells expressing the hybrid IL-5α, including segment And or receptor of a man stood in the e main sequence (frame) receptor, mouse "D1/D2 mouse/D3 mouse", ("nobunny on a or C").

Fig. MEDI-563-specific binds to the receptor epitope of human IL-5α, including amino acid residue Ile61 in the extracellular domain D1. Antibodies that bind to the transgenic cells, espressioni variant of IL-5α, analyzed by flow cytometry. The figure shows the profiles stained with a fluorescent dye. "Polyclonal antibodies" and "MEDI-563" refers profiles staining observed when using polyclonal antibodies against IL-5α person and MEDI-563, respectively. Double staining" refers to the profile fluorescent staining using polyclonal antibodies (x-axis) and MEDI-563 (y-axis). (A) In the figure given amino acid sequence 50-61 extracellular domain D1 receptor IL-5α person (40-61 SEQ ID No. 5). Residues in italics are different from the corresponding amino acid residues in the composition of the receptor IL-5α mouse. In transgenic cells expressed a series of variants of the receptor for IL-5α comprising at least one mutated amino acid residue. "Knock-out" versions of the IL-5α represent mutant proteins of human, comprising at least one substitution of an amino acid in the protein of the person at the corresponding amino acid of the protein of the mouse. For example, knockout DE" represents the IL-5α include the s replacement of amino acids D56E and E58D. "Nominee" variants of IL-5α are hybrid proteins, including D1 mouse, D2 person, D3 mouse and TM domain of the mouse, where the domain D1 mouse includes mutant segment containing at least one substitution of amino acid residue in the protein sequence of the mouse on the corresponding residue from the sequence of the receptor is human. For example, "nobunny DE" option is a hybrid IL-5α, including mutant segment In receptor mouse containing substitutions of amino acids E56D and D58E. (B) MEDI-563 is not associated with transgenic cells expressing mutant IL-5α person, including replacement of amino acids K53Q, D56E, E58D, I61K ("knock-out by KDEI"). MEDI-563 associated with specific transgenic cells expressing mutant IL-5α person, including replacement of amino acids N40H, N42D, Q46H ("knock-out by NNQ") or D56E, E58D ("knock-out on DE") or N40H, N42D, D56E, E58D ("knock-out by NNDE"). (B) MEDI-563 associated with specific transgenic cells expressing the hybrid IL-5α, including mutant segment In receptor mouse containing substitutions of amino acids Q53K, E56D, D58E, K61I ("nobunny on KDEI"). (D) MEDI-563 is not associated with transgenic cells expressing mutant IL-5α person, which includes the replacement of I61K ("knock-out on I61"). MEDI-563 associated with specific transgenic cells expressing mutant IL-5α person, which includes the replacement of K53Q ("NOC is cozy on K"). (D) MEDI-563 associated with specific transgenic cells expressing the hybrid IL-5α, including mutant segment In receptor mouse containing a replacement KB II ("nobunny on I61"). MEDI-563 is not associated with transgenic cells expressing the hybrid IL-5α, including mutant segment In receptor mouse containing replacement Q53K ("nobunny on K").

Fig. The binding of an antibody H7 against hybrid IL-5α mouse with mouse FcγRs. The figure presents the affinity of the hybrid anti-IL-5α mouse (H7) recombinant mouse FcγRs when compared with the affinity of izotopicheskii related fokusirovannyi control antibodies. Dissociation constants are given in nm. Analysis was performed by the method of plasma-surface resonance.

Fig. (A) Eosinophils were identified by flow cytometry as cells with high lateral scattering, which are positively stained for CCR3 and Siglec-F. (B) Eosinophils from the bone marrow, blood, spleen and lung tissue of mice IL-STg selectively Express the IL-5P.

Fig. Deoksigenirovanii and fokusirovannyi H7 antibodies reduce the number of eosinophils in the spleen (A), lung tissue (A) and blood (B) mice IL-STg. There was no reduction in the number of eosinophils in the bone marrow (B). Deoksigenirovanii H7 antibodies more effective at removing eosinophils compared with fokusirovannyi H7, especially when using low doses of antibodies. Data are presented as mean values ±WITH the number of mice in group n=6-8, in the analysis using the indicated antibodies compared with the control IgG (p<0.05, the criterion U Mann-Whitney U.

Fig. Deoksigenirovanii H7 also reduce the number of eosinophils in the model allergen stimulation. Deoksigenirovanii H7 reduce the number of eosinophils in the lumen of the respiratory tract, in the lung tissue, blood and bone marrow. The decrease was maximum in all parts of the body through 72 h after the end of stimulation (96 h after injection of antibodies). Data are presented as mean values ±WITH the number of mice in group n=6, in the analysis using the indicated antibodies compared with the control IgG*p<0.05, the criterion U Mann-Whitney U.

Detailed description of the invention

As described above without the involvement of a particular hypothesis or theory, eosinophils participate in the pathogenesis of many diseases and disorders. Many of these diseases or disorders characterized by high levels of eosinophils (eosinophilia) and called hypereosinophilic syndromes.

Examples of diseases and disorders involving eosinophils, are asthma, immunoglobulin (IgE)-mediated food Allergy, eosinophilic esophagitis (inflammation of the esophagus), inflammatory bowel disease, X IS PLN, allergic colitis, gastro-oesophageal reflux, eosinophilic gastrointestinal disease (ASCS), eosinophilic gastroenteritis, endomyocardial fibrosis, endocarditis Leffler, disease Denis, episodic angioedema associated with eosinophilia syndrome eosinophilia-myalgia/toxic oil syndrome, registered in Spain, cirrhosis, dermatitis herpetiformis, bullous pemphigoid syndrome charge-Strauss, acute myelogenous eosinophilic leukemia, acute lymphocytic eosinophilic leukemia, systemic mastocytosis with eosinophilia, allergic rhinitis, eczema, Wegener's granulomatosis, Nowotny polyarteritis, eosinophilic fasciitis, and rheumatoid arthritis.

Thus, the invention proposes a method of reducing the number of eosinophils in the body, which is that a given patient is administered the compound to bind to the IL-5P, which includes (a) a segment-specific binds to IL-5P, and (b) Fc fragment of immunoglobulin.

In one embodiment, the invention provides methods of reducing the number of eosinophils in the body, which lies in the fact that a given patient is administered the compound to bind to the IL-5P, which includes (a) a segment-specific binds to IL-5P, and (b) Fc fragment of immunoglobulin. In a specific embodiment, the method according to image meniu reduces the number of eosinophils in the blood, the bone marrow, gastrointestinal tract (e.g., esophagus, stomach, small intestine and colon or lung. In another specific embodiment, the method according to the invention reduces the number of eosinophils in the blood. In another specific embodiment, the method according to the invention reduces the number of eosinophils in the lungs. In a specific embodiment, the method according to the invention reduces the number of progenitor cells eosinophils.

In another embodiment, the method according to the invention reduces the number of eosinophils at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99%. In a specific embodiment, the method according to the invention reduces the number of eosinophils below the detection limit.

In another embodiment, the method according to the invention reduces the number of predecessors of eosinophils at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, less the th least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99%. In a specific embodiment, the method according to the invention reduces the number of predecessors of eosinophils below the detection limit.

In another embodiment, the method according to the invention eliminates all of eosinophils detected after a single injection of compounds that bind to the IL-5P. In a specific embodiment, a single injection of compounds that bind to the IL-5P, eliminates all detected eosinophils at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least at about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, IU is greater at least about 16 weeks, at least about 20 weeks or at least about 25 weeks.

In another embodiment, the method according to the invention eliminates all detected predecessors eosinophils after a single injection of compounds that bind to the IL-5P. In a specific embodiment, a single injection of compounds that bind to the IL-5P, eliminates all detected eosinophils at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least at about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 20 weeks or at least about 25 weeks.

In a specific embodiment, the method p the invention includes an introduction to the subject a single dose of 0.03 mg/kg connection binding to the IL-5P, which includes (a) a segment-specific binds to IL-5P, and (b) Fc fragment of immunoglobulin, and the introduction of compounds to bind to the IL-5P, leads to a decrease in the number of eosinophils in the bloodstream of the subject at least about 99%, and the lower ends 24 h after injection and lasts for at least about 28 days after injection.

In a specific embodiment, the method according to the invention includes an introduction to the subject a single dose of 0.1 mg/kg of the compounds to bind to the IL-5P, which includes (a) a segment-specific binds to IL-5P, and (b) Fc fragment of immunoglobulin, and the introduction of compounds to bind to the IL-5P, leads to a decrease in the number of eosinophils in the bloodstream of the subject at least about 99%, and the lower ends 24 h after injection and lasts for at least about 84 days after injection.

In one embodiment, the compounds of the present invention that are associated with IL-5P include hybrid proteins. In some embodiments, the hybrid protein includes a polypeptide fragment which is specific binds to IL-5P, and Fc fragment of immunoglobulin. Examples polypeptide fragment which is specific binds to IL-5P, are given in US 7109299, 5677280 and in the application US 2006/0014680 A1. In drugexperience polypeptide, the specific data associated with IL-5P, represents IL-5 person (see, for example, Tanabi and others the Journal of Biological Chemistry, 262 (34), 16580-16584 (1987)), or fragments, derivatives or variants (see, for example, US 6465616).

In one embodiment, the compounds of the present invention that are associated with IL-5P include antibodies. Antibodies of the present invention include, but are not limited to, monoclonal antibodies, synthetic antibodies, polyspecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, hybrid antibodies, single-chain Fvs (scFv) (including bispecific scFvs), single-chain antibodies, Fab fragments, F(ab')fragments linked by a disulfide bond Fvs (sdFv), and epitope-binding fragments of any of the above antibodies. Antibodies of the present invention primarily include antibodies and immunologically active fragments of molecules of immunoglobulins, i.e. compounds which contain the antigen-binding site that is specific binds to the antigen. The immunoglobulins of the invention include proteins of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

Antibodies that can be used in the present invention include antibodies that are formed in the body of any animal, including birds and mlec the supply (for example, but not limited to, humans, mice, monkeys, sheep, rabbits, goats, Guinea pigs, camels, horses and chickens). In particular embodiments use human antibodies or humanized monoclonal antibodies.

Antibodies that can be used in the present invention, are monospecific, bispecific, trapezitinae or polyspecific antibodies. Polyspecific antibodies specific contact polypeptide, as well as with heterologous epitope, such as a heterologous polypeptide or solid material of the substrate. See, for example, WO 93/17715, WO 92/08802, WO 91/00360 and WO 92/05793, Tutt, etc., J. Immunol., 147, 60-69 (1991), US 4474893, 4714681, 4925648, 5573920 and 5601819, and Kostelny, etc., J. Immunol., 148, 1547-1553 (1992).

Antibodies for use in the present invention can be a single-chain antibodies. The scheme and design of single-chain antibodies are described in article Marasco and others, Proc. Natl. Acad. Sci., 90, 7889-7893 (1993).

Examples of antibodies according to the invention are given in US 7179464, 6538111, 6018032 and bids US 2004/0136996 A1 and 2005/0226867 A1.

In one embodiment, the compounds of the present invention that are associated with IL-5P include antibodies. In another embodiment, compounds of the present invention that are associated with IL-5P, are antibodies comprising any of the amino acid sequence SEQ ID No. 1-4. In a specific embodiment, connection is, binding to the IL-5P, according to the present invention is an antibody comprising amino acid sequence SEQ ID No. 1 and 3. In a specific embodiment, the compound to bind to the IL-5P, according to the present invention is an antibody comprising amino acid sequence SEQ ID No. 2 and 4.

In one embodiment, the compound of the present invention, which binds to the IL-5P is an antibody that is specific linked with the same epitope as MEDI-563. In a specific embodiment, the antibody is MEDI-563. In another specific embodiment, the compound to bind to the IL-5P, according to the present invention is an antibody that is specific linked with the same epitope as MEDI-563, provided that the antibodies are not MEDI-563.

In one embodiment, the compound of the present invention, which binds to the IL-5P is an antibody that is specific contact epitope comprising amino acid residues 1-102 SEQ ID No. 5. In a specific embodiment, the antibody is MEDI-563. In another specific embodiment, antibodies of the present invention that are associated with IL-5P is an antibody that is specific contact epitope comprising amino acid residues 1-102 SEQ ID No. 5, provided that the antibodies are not MEDI-563.

In one embodiment, the connection is present and is finding, which binds to the IL-5P is an antibody that is specific contact epitope comprising amino acid residues 40-67 SEQ ID No. 5. In a specific embodiment, antibodies are MEDI-563. In another specific embodiment, antibodies of the present invention that are associated with IL-5P, are antibodies that are specific contact epitope comprising amino acid residues 40-67 SEQ ID No. 5, provided that the antibodies are not MEDI-563.

In one embodiment, the compound of the present invention, which binds to the IL-5P is an antibody that is specific contact epitope comprising amino acid residues 52-67 SEQ ID No. 5. In a specific embodiment, the antibody is MEDI-563. In another specific embodiment, antibodies of the present invention that are associated with IL-5P, are antibodies that are specific contact epitope comprising amino acid residues 52-67 SEQ ID No. 5, provided that the antibodies are not MEDI-563.

In one embodiment, the compound of the present invention, which binds to the IL-5P is an antibody that is specific contact epitope comprising amino acid residue 61 of SEQ ID No. 5. In a specific embodiment, antibodies are MEDI-563. In another specific embodiment, antibodies of the present invention that are associated with IL-5P, predstavlyaet an antibody, which specific contact epitope comprising amino acid residue 61 of SEQ ID No. 5, provided that the antibodies are not MEDI-563.

In one embodiment, the compound of the present invention, which binds to the IL-5P is an antibody that is specific linked with the first antigen comprising amino acid residues 1-102 SEQ ID No. 5, but not specific contact with a second antigen comprising the variant residues 1-102 SEQ ID No. 5, the variant comprises the substitution 161 K. In a specific embodiment, the antibody is MEDI-563. In another specific embodiment, antibodies of the present invention that are associated with IL-5P, are antibodies that are specific contact with the first antigen comprising amino acid residues 1-102 SEQ ID No. 5, but not specific contact with a second antigen comprising the variant residues 1-102 SEQ ID No. 5, the variant comprises the substitution 161, provided that the antibodies are not MEDI-563.

In one embodiment, the compound of the present invention, which binds to the IL-5P is an antibody that is specific linked with the first antigen comprising amino acid residues 40-67 SEQ ID No. 5, but not specific contact with a second antigen comprising the variant residues 40-67 SEQ ID No. 5, the variant comprises the substitution I61K. In a specific embodiment, the antibody is MEDI-563. In the other is specific embodiment, antibodies of the present invention, associated with the IL-5P, are antibodies that are specific contact with the first antigen comprising amino acid residues 40-67 SEQ ID No. 5, but not specific contact with a second antigen comprising the variant residues 40-67 SEQ ID No. 5, the variant comprises the substitution I61K, provided that the antibodies are not MEDI-563.

In one embodiment, the compound of the present invention, which binds to the IL-5P is an antibody that is specific linked with IL-5α human (SEQ ID No. 5), but not specific contact with a mutant IL-5α human (SEQ ID No. 5), comprising the substitution I61K. In a specific embodiment, the antibody is MEDI-563. In another specific embodiment, the compound of the present invention, which binds to the IL-5P is an antibody that is specific bound IL-5α human (SEQ ID No. 5), but not specific contact a mutant IL-5α human (SEQ ID No. 5), comprising the substitution I61K, provided that the antibodies are not MEDI-563.

The present invention provides compounds that bind to IL-5P and have increased effector function. Examples of ways to improve effector functions are given in US 5624821, 6602684, 7029872, in applications 2006/0067930 A1, 2005/0272128 A1, 2005/0079605 A1, 2005/0123546 A1, 2004/0072290 A1, 2006/0257399 A1, 2004/0261148 A1, 2007/0092521, 2006/0040325 A1 and 2006/0039904 A1 and in WO 04/029207, W003011878, W005044859, WO 06071856 and WO 06071280.

Methods g is kinetic engineering Fc regions of antibodies for changes effector functions known in the art (see, for example, Koenig and others, US 20040185045 and PCT WO 2004/016750, which describes the change in the Fc-region to improve the affinity to FcγRIIB as compared with the affinity to FCγRIIA, see also Armour and others, PCT WO 99/58572, Idusogie and others, WO 99/51642 and Deo and others, US 6395272, which are included in the description of the application in full as references). Methods of modification of the Fc region for the purpose of lowering the affinity to FcγRIIB is also known (see, for example, Ravetch and other, US 20010036459 and WO 01/79299 included in the description of the application in full as references). Described in the literature, the modified antibodies containing a variant Fc region with an enhanced affinity for FcγRIIIA and/or FcγRIIA compared to the native Fc region (see, for example, Stavenhagen and others, WO 2004/063351 included in the description of the application in full by reference).

The effector function of antibodies can also be modified by obtaining antibodies with altered type of glycosylation. For example, it is possible to obtain antibodies with altered type of glycosylation, such as defilirovali/hepatocanalicular antibodies with reduced content of fucose residues or antibodies with a high content biasotti structures based on N-acetylglucosamine (GlcNac). These types of glycosylation are used to enhance antibody-dependent cellular cytotoxicity (SACC). Such modifications of polysaccharides can be achieved, for example, due to expire is these antibodies in host cells with a modified mechanism of glycosylation. Cells with a modified mechanism of glycosylation are described in the literature and can be used as host cells for expression of recombinant antibodies according to the invention with altered type of glycosylation. For example, in EP 1176195 (Hanai and others) described cell line with a functionally dissected FUT8 gene, which encodes fucosyltransferase, and therefore, antibodies expressed in such cells, are characterized by hypoparathyroidism. In WO 03/035835 (Presta) describes a variant Cho cell line, cells Lee 13, which have a reduced ability to attach fucose to Asn(297)-polysaccharides, which leads to hepatocanalicular antibodies expressed in such cells (see also Shields R.L., and others, J. Biol. Chem., 277, 26733-26740 (2002)). In WO 99/54342 (Umana and others) described the obtaining by means of genetic engineering of cell lines for expression of the glycosyltransferases involved in the biosynthesis of glycoproteins (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)). Antibodies expressed in such cells, are characterized by a high content of biasotti structures based on N-acetylglucosamine (GlcNac) and have a high SACC (see also Umana, etc., Nat. Biotech., 17, 176-180 (1999)).

Methods for producing antibodies with altered type of glycosylation is known in the field of machinery and shall include, but not limited to, the methods described in EBUSY publications: Umana and others, Nat. Biotechnol,, 17, 176-180 (1999), Davies and others, Biotechnol. Bioeng, 74, 288-294 (20017), Shields, etc., J. Biol. Chem., 277, 26733-26740 (2002), Shinkawa, etc., J. Biol. Chem., 278, 3466-3473 (2003), US 6602684, US 10/277,370, US 10/113,929, PCT WO 00/61739 A1, PCT WO 01/292246 A1, PCT WO 02/311140 A1, PCT WO 02/30954 A1, technology Potillegent™ (firm Biowa, Inc. Princeton, N.J.), genetic engineering GlycoMAb™ (firm GLYCART biotechnology AG, Zurich, Switzerland). Cm. also WO 00061739, EA 01229125, US 20030115614, Okazaki and others, J.M.B., 336, 1239-1249 (2004). Antibodies with a modified type fokusirovanie can also be obtained through post-translational removal of fucose (for example, using fucosidase),

The present invention provides antibodies and fragments thereof that are specific associated with IL-5P and are characterized by an increased half-life in vivo. In the present invention primarily provides antibodies and fragments thereof, which are characterized by half-lives in the body of a mammal (for example, but not limited to them) more than 3 days, 7 days, 10 days, 15 days, 25 days, 30 days, 35 days, 40 days, 45 days, 2 months, more than 3 months more than 4 months or more than 5 months.

For prolonging the circulation of antibodies in the bloodstream (such as, but not limited to, monoclonal antibodies and single-chain antibodies) or fragments of antibodies (such as, but not limited to, Fab fragments) in vivo, for example, antibodies (or to the x fragments) attach inert polymers, such as high molecular weight polyethyleneglycol (PEG), with (or without) a polyfunctional linker or through site-specific conjugation of the PEG to the N - or C-terminal residues of the antibody or ε-amino groups of lysine. It is necessary to use a linear or branched polymers, which provide minimal loss of biological activity. The degree of conjugation is determined by electrophoresis of the LTO-page and mass spectrometry in order to achieve optimal PEG conjugation with antibodies. Unreacted PEG conjugates are separated from the antibody/PEG methods exclusion or ion exchange chromatography. The effectiveness of modified antibodies (or fragments) appreciate binding activity, as well as on the effectiveness of in vivo known methods, such as immunological analysis described in the text of the application.

Antibodies having an increased half-life in vivo can also be obtained by modification of one or more amino acids (i.e. due to substitutions, insertions or deletions) in the conservative domain of IgG or its binding fragment FcRn (e.g., a fragment of the Fc domain or hinge-Fc fragment). See, for example, WO 98/23289, WO 97/34631 and US 6277375 (these works are included in the description of the application in full as a reference).

In addition, antibodies (or ragment) can be konjugierte with albumin to generate antibodies (or fragments), more stable in vivo or have a longer half-life in vivo. Methods of conjugation are known in the art, see, for example, WO 93/15199, WO 93/15200 and WO 01/77137 and EP 413622 (these works are included in the description of the application in full as a reference).

The present invention provides antibodies and fragments thereof that are specific associated with IL-5P, in the form of a recombinant hybrid proteins or conjugates (by covalent or non-covalent binding) to a heterologous protein or polypeptide (or fragment of the polypeptide containing at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids). The invention primarily serves medicines hybrid proteins comprising an antigen-binding fragment of the antibody described in the proposal (for example, but not limited to, Fab fragment, Fd fragment, Fv fragment, F(ab)₂-a fragment, domain, VH, of the criminal code of VH, VL domain, or of the criminal code VL), and a heterologous protein, polypeptide or peptide.

Methods of hybridization or conjugation of proteins, polypeptides or peptides with antibodies (or fragments) are known in the art. See, for example, US 5336603, 5622929, 5359046, 5349053, 5447851 and 5112946, EP 307434 and EP 367166, WO 96/04388 and WO 91/06570,Ashkenazi, etc.. Proc. Natl. Acad. Sci. USA, 88, 10535-10539 (1991), Zheng and others, J. Immunol., 154, 5590-5600 (1995) and Vil and others, Proc. Natl. Acad. Sci. USA. 89, 11337-11341 (1992) (these works are included in the description of the application in full as a reference).

Additional hybrid proteins get methods of gene rearrangement, alteration of sequence motifs, realignment of exons and/or alteration of codons (collectively referred to as "realignment DNA"). The rearrangement of DNA can be used to modify the activity of the antibodies according to the invention or their fragments (for example, to generate antibodies or their fragments with higher affinitiy and lower dissociation rate). See, for example, US 5605793, 5811238, 5830721, 5834252 and 5837458, Patten and others, Curr. Opinion Biotechnol., 8, 724-733 (1997), Harayama, Trends BiotechnoL, 16 (2), 76-82 (1998), Hansson and others, J. Mol. Biol., 287, 265-276 (1999); and Lorenzo and Blasco, Biotechniques, 24 (2), 308-313 (in Russian) (1998) (these works are included in the description of the application in full as references). The antibodies (or fragments thereof) or to encode antibodies or fragments thereof can be changed due to non-specific mutagenesis, PCR low-precision, non-specific insertion of nucleotides or other methods before conducting subsequent recombination. Polynucleotide encoding the antibody (or fragment)can recombine with one or more components, motifs, segments, parts, domains, fragments, etc. of one or more heterologic the x molecules.

Moreover, antibodies (or fragments thereof) can be hybridizat to marker sequences, such as peptide that facilitates purification. The marker amino acid sequence is, for example, hexastylis, such as the token supplied in the vector PoE (firm QIAGEN Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), and others, many of which are commercial products. For example, as described in Gentz and others, Proc. Natl. Acad. Sci. USA, 86, 821-824 (1989), hexastylis provides a simple cleaning of the hybrid protein. Other peptide tags that can be used for purification of such proteins include, but are not limited to, a fragment of the hemagglutinin ("HA"), which corresponds to the epitope derived from the hemagglutinin of influenza virus (Wilson et al., Cell 37, 767 (1984)) and the marker flag.

In other embodiments, the antibodies according to the present invention or fragments thereof kongugiruut with diagnostic or detektivami agent. Such antibodies can be used for monitoring or forecasting attack, development, progression and/or severity of disease or disorders (such as, but not limited to them, autoimmune disorders), and the specified monitoring is an integral part of the methodology of clinical trials, such as determining the effectiveness of specific treatment. Such diagnosis and detection carried out bliod the OC interaction of antibodies with detectivesyme compounds including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase, a prosthetic group, such as, but not limited to, streptavidin/Biotin and avidin/Biotin, fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorofluorescein, ancillary or phycoerythrin, luminescent materials, such as, but not limited to, luminal, bioluminescent materials, such as, but not limited to, luciferase, luciferin, and acorin, radioactive materials, such as, but not limited to, iodine (¹³¹I¹²⁵I¹²³I¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In¹¹³In¹¹²In and¹¹¹In), technetium (⁹⁹TC), thallium (²⁰¹Ti), gallium (⁶⁸Ga⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm¹⁷⁷Lu,¹⁵⁹Gd¹⁴⁹Pm,¹⁴⁰La,¹⁷⁵Yb,¹⁶⁶Ho,⁹⁰Y⁴⁷Sc,¹⁸⁶Re,¹⁸⁸Re,¹⁴²Pr¹⁰⁵Rh,⁹⁷EN,⁶⁸Ge⁵⁷Co.,⁶⁵Zn⁸⁵Sr³²P,¹⁵³Gd¹⁶⁹Yb,⁵¹Cr⁵⁴Mn⁷⁵Se¹¹³Sn and¹¹⁷Sn, a positron is emitted is either metals using various methods of positron emission tomography and nonradioactive paramagnetic metal ions.

In another embodiment, antibodies can be konjugierte with the second antibody with the formation of heteroconjugate, as described in US 4676980 (Segal), which is included in the description of the application in full by reference.

Drug group or the drug is conjugated with the test antibodies (e.g., specific for IL-5P) or their fragments, can be used to provide the desired prophylactic or therapeutic effect on a specific disease or disorders in a subject, for example a disease or disorder characterized by or associated with abnormal expression and/or activity of α-interferon, a disease or disorder characterized by or associated with abnormal expression and/or activity of the receptor α-interferon or one or more of its subunits, autoimmune disease, graft rejection, disease graft versus host disease, or one or more symptoms of the above diseases or disorders. The decision about the choice of drug for conjugation with the specified antibodies, such as antibodies that are specific contacted with α-interferon or its fragment should be the practitioner or other medical personnel, taking into account such factors as the nature of the disease, disease severity and health status of the su is the target.

The antibodies (or fragments thereof)that have been specifically bind to the antigen, can be obtained by any known method for the synthesis of antibodies, primarily by chemical synthesis or recombinant methods of expression (see, US 2007/0014724 A1).

Polyclonal antibodies specific to the antigen, can be obtained by the known methods. For example, the antigen person can enter into the body of various animals, including, but not limited to, rabbits, mice, rats, etc. with the aim to induce the production of sera containing polyclonal antibodies specific to the antigen of human rights. To increase the immunological response, depending on the type of animal you can use various adjuvants, including, but not limited to, complete and incomplete adjuvant's adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, polyols-pluronic, polyanion, peptides, oil emulsions, hemocyanine fissurella, dinitrophenol, and adjuvants that are used for immunization of a person, such as BCG (Bacillus of Colmet-guérin (BCG) and dipterigena bacterium parvum. Such adjuvants well known in the field of engineering.

Monoclonal antibodies obtained using a variety of known methods, including the use of hybrid, recombinant technology and met the Dov phage display technique, or combinations thereof. For example, monoclonal antibodies obtained using a hybrid, including known and described in the literature methods, for example Harlow and others, Antibodies:

A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988), Hammerling, and others, Monoclonal Antibodies and T-Cell Hybridomas, 563-681 (Elsevier, N.Y. (1981), and Harlow and others, Using Antibodies: A laboratory Manual, Cold Spring Harbor Laboratory Press (1999) (these works are included in the description of the application in full as references). The term "monoclonal antibody"used in the description of the application is not limited to antibodies produced hybridoma technology. The term "monoclonal antibody" refers to antibodies that are derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the way in which they are received.

Methods of obtaining and analysis of antibody specificity to the antigen using a hybrid known in the art. For example, mice are subjected to immunization namelennym antigen and in the presence of an immune response, for example, antibodies in serum, specific to the antigen, remove the spleen and isolated splenocytes. This is followed by the fusion of splenocytes known techniques to any suitable myeloma cells, for example cell line SP20, obtained from the collection of ATS. Hybridoma select and clone a limited breeding. In addition, to immunize an animal, you can use the method PIMMS (repetitive immunization at multiple sites), see Kilpatrack and others, Hybridoma, 16, 381-389 (1997), included in the description by reference. Then clones hybridoma analyze known methods on the ability of cells to secrete antibodies that bind to a polypeptide according to the invention. Ascitic fluid, which typically contains high levels of antibodies, receive, immunosera mice positive clones of hybridoma.

The present invention provides methods for obtaining monoclonal antibodies, and antibodies by the method comprising culturing hybridomas secreting antibodies according to the invention, where hybridoma receive the merger of splenocytes isolated from the body of the mouse immunized with namelennym the antigen with myeloma cells and then select obtained by hybridization of clones of hybridoma, secreting antibodies which are able to bind to the antigen.

Antibody fragments that are specific recognize specific epitopes get any well-known to a person skilled methods. For example, Fab and F(ab')2-fragments according to the invention receive proteolytic cleavage of immunoglobulin molecules by enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, a conserved region of a light chain and SN domain of the heavy chain. In addition, antibodies of the present invention can also on ucati using various known methods of phage display.

Methods of phage display include a presentation of the functional domains of the antibodies on the surface of phage particles that contain coding them polynucleotide sequence. More specifically, DNA sequences encoding domains VH and VL, amplified from cDNA libraries animals (e.g., cDNA libraries damaged tissues of human or mouse). DNA encoding the domains VH and VL, hybridized with scFv linker by PCR and clone in formigny vector. The vector is introduced into E. coli by electroporation and infect E. coli by phage-helper. Used in these methods phages are typically filamentous phage including fd and M13 and the domains VH and VL is typically hybridize recombinant method with the gene III or gene VIII phage. Phage expressing an antigen binding domain that binds to a particular antigen, can be selected or identified with the use of an antigen, such as labeled antigen or antigen immobilized or fixed on a solid substrate or granules. Examples of methods of phage display, which can be used to generate antibodies of the present invention include the methods described in the literature (See, for example, Brinkman and others, J. Immunol. Methods, 182, 41-50 (1995), Ames and others, J. Immunol. Methods, 184, 177-186 (1995), Kettleborough and others, Eur. J. Immunol., 24, 952-958 (1994), Persic, and others, Gene. 187, 9-18 (1997), Burton and others, Advances in Immunology 57, 191-280 (1994), PCT/GB91/01 134, WO 90/02809, WO 91/10737, WO 9/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401 and WO 97/13844, and US 5698426, 5223409, 5403484, 5580717, 5427908, 5750753, 5821047, 5571698, 5427908, 5516637, 5780225, 5658727, 5733743, 5969108, 633187, 5824520 and 5702892 (these works are included in the description of the application in full as a reference).

As described in the above works, after selection of phage encoding region of the antibody can be isolated from phage and used to construct a full-sized antibodies, including human antibodies, or any other antigen-binding fragment, and to Express in any desired organism, the host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described below. You can also use the well-known recombinant methods of producing Fab-, Fab'and F(ab')2 fragments, described in WO 92/22324 and articles Mullinax and others, BioTechniques, 12 (6), 864-869 (1992), Sawai and others, AJRI, 34, 26-34 (1995) and Better etc., Science 240, 1041-1043 (1988) (these works are included in the description of the application in full as a reference).

To obtain full-length antibodies using PCR primers comprising the nucleotide sequence of VH or VL, the restriction site and flanking sequence to protect the restriction site, and then amplified sequence of VH or VL in the scFv clones. Using cloning techniques known to the person skilled in the art, amplificatoare PCR domains VH clone in the vector ex is desiroush constant region VH, for example, conservative region of the antibodies of the human γ4, and amplificatoare PCR VL domains can be cloned into vectors expressing a constant region VL, for example conserved region of human antibodies κ (Kappa) or λ (lambda). Vectors for expression domains VH or VL can include a promoter EF-1α, a secretion signal, a cloning site variable domain, conservative domains and marker secretion, such as neomycin. Domains VH and VL can also be cloned in the same vector expressing the necessary conservative area. Then the vectors of the conversion of the heavy chain and the vector conversion light chain together transfection cell line for propagation of the stable or unstable cell lines, which Express full-length antibodies, for example, but not limited to them, IgG, using known methods.

In some cases, including the use of antibodies in vivo for the treatment of human or in vitro, can be used humanized antibodies or hybrid antibodies. Full-length human antibodies and humanized antibodies are primarily useful for treatment of humans. Human antibodies can be obtained using a variety of known methods, including the above-described methods of phage display libraries use the antibodies is, derived from the sequences of human immunoglobulins. Cm. also US 4444887, 4716111 and WO 98/46645, WO 98/50433, WO 98/24893, W098/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (these works are included in the description of the application in full as a reference).

Human antibodies can also be generated using transgenic mice that fail to Express functional endogenous immunoglobulins, but which can Express the genes of the human immunoglobulin. For example, complexes of the gene of the heavy and light chains of human immunoglobulin can be entered randomly or by homologous recombination in embryonic stem cells of the mouse. In another embodiment, variable region, conserved region and wherein the antibody fragment of the person can be entered into embryonic stem cells of a mouse in addition to the genes of the heavy and light chain of a human. The genes of the heavy and light chain immunoglobulin mouse you can turn each separately or simultaneously in a non-functional genes by introducing a locus of human immunoglobulin method of homologous recombination. For example, homozygous deletion of the fragment JH prevents the production of endogenous antibodies. Modified embryonic stem cells multiply and microinjected in plasticity to obtain chimeric mice. Then chimeric mice were bred to obtain homozygote offspring, which expresses human antibodies. Transgenic mice subjected to immunization appropriate antigen in the usual way, for example, using the polypeptide of the invention or part thereof. Monoclonal antibodies to the antigen can be obtained from immunized, transgenic mice by standard hybridoma technology. The transgenes of the human immunoglobulin are accumulated in the process of perestroika in the body transgenic mice during differentiation In cells, and then switches synthesis class (immunoglobulin), and somatic mutation. Thus, by employing such a method can be used to produce antibodies of IgG, IgA, IgM and IgE for therapeutic applications. An overview of this technology to generate antibodies person is defined in article Lonberg and Huszar, Int. Rev. Immunol., 13, 65-93 (1995). A detailed description of the specified methods of obtaining human antibodies, monoclonal human antibodies and methods of producing such antibodies are given in the literature, see, for example, WO 98/24893, WO 96/34096 and WO 96/33735, and US 5413923, 5625126, 5633425, 5569825, 5661016, 5545806, 5814318 and 5939598 (these works are included in the description of the application in full as references). In addition, human antibodies to selected antigens are produced by this technology such companies as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA).

Hybrid antibodies are substances in which the various segments derived from different immunoglobulin. Methods of obtaining hybrid antibodies known in the art. See, for example, Morrison, Science, 229, 1202 (1985), 01 and others, Bio.Techniques. 4, 214 (1986), Gillies and others, J. Immunol. Methods, 125, 191-202 (1989) and US 5807715, 4816567, 4816397 and 6331415 (these works are included in the description of the application in full as a reference).

Humanized antibodies are antibodies or variants or fragments, which are able to communicate with the pre-selected antigen and which includes a frame region containing principally amino acid sequence of a human immunoglobulin and the criminal code, which generally contain amino acid sequence of an antibody of another species. Humanized antibodies include basically all or at least one, and typically two, variable domains (Fab, Fab', F(ab')₂, Fabc, Fv)in which all or almost all of the criminal code corresponding sections contained in the immunoglobulin another type (i.e. donor antibody)and all or substantially all of the frame regions correspond to the consensus sequence of human immunoglobulin. In one embodiment, the humanized antibodies also include at least part of the conservative region of the immunoglobulin (Fc), typically of a human immunoglobulin. Typically, the antibody must contain both light chain and at least the variable domain of the heavy chain. Antibodies can also VK is ucati region SN, the hinged section, CH2, CH3 and CH4 of the heavy chain. Humanized antibodies are selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁, IgG₂, IgG₃and IgG₄. Usually conservative domain is the presence of complement fixing constant domain, to humanized antibodies possessed cytotoxic activity, and usually correspond to the class IgG₁. If the cytotoxic activity is not necessary, conservative domain corresponds to the class IgG₂. Humanized antibodies may include a series of multiple classes or isotypes, and the choice of a specific conservative domains to optimize the necessary effector functions can make the person skilled in the art. Frame section (CU) and areas of complementarity (CC) antibodies person does not have to exactly match the original sequences, for example, the criminal code or the donor consensus KU may include mutation by substitution, insertion or deletion of at least one residue, and therefore, the balance in the respective website of the criminal code of the CU or may not match the consensus sequence or imported antibodies. However, these mutations should be limited. Usually in the humanized antibodies of at least 75%, more than 90% and more than 95 residues correspond to the sequences in the original KU and UK. Humanized antibodies can be obtained by the known methods, including, but not limited to, inoculation of the criminal code (EP 239400, WO 91/09967 and US 5225539, 5530101 and 5585089), disguise or demeterova (EP 592106 and EP 519596, Padlan, Molecular Immunology, 28 (4/5), 489-498 (1991), Studnicka, etc., Protein Engineering, 7(6), 805-814 (1994) and Roguska and others, PNAS, 91, 969-973 (1994), a permutation circuit (US 5565332) and the methods described, for example, in the US 6407213, US 5766886, WO 9317105, Tan, etc., J. Immunol., 169, 1119-1125 (2002), Caldas and others, Protein Eng., 13(5), 353-360 (2000), Morea and others, Methods, 20(3), 267-279 (2000), Vasa and others, J. Biol. Chem, 272(16), 10678-10684 (1997), Roguska, etc., Protein Eng., 9(10), 895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp), 5973-5977 (1995), Couto and others, Cancer Res., 55 (8), 1717-1722 (1995), Sandhu J.S., Gene, 150 (2), 409-410 (1994) and Pedersen and others, J. Mol. Biol., 235 (3), 959-973 (1994). Often the amino acids in frame areas must be replaced with the corresponding residue from the criminal code of the donor antibody to alter, preferably improve binding to the antigen. These substitutions in the framework region identify known methods, for example but not limited to, modeling interactions of the criminal code and framework residues to identify the frame of residues important for binding to the antigen, and sequence comparison to identify unusual frame residues in specific positions (see, for example, Queen and others, US 5585089 and Riechmann and others, Nature, 332, 323 (1988), included in the description of the application in full as a reference).

Monodomain the antibodies, for example, antibodies that do not contain light chains, get known methods. Cm. Riechmann, etc., J. Immunol., 231, 25-38 (1999), Nuttall and others, Curr. Pharm. BiotechnoL, 1(3), 253-263 (2000), Muylderman, J. Biotechnol., 74(4), 277302 (2001), US 6005079 and WO 94/04678, WO 94/25591 and WO 01/44301 (these works are included in the description of the application in full as a reference).

In addition, antibodies that are specific contact with the antigen (e.g., IL-5P), can in turn be used to obtain anti-idiotypical antibodies that "mimic" an antigen known methods. (See, for example, Greenspan and Bona, FASEB J. 7(5), 437-444 (1989) and Nissinoff, J. Immunol., 147 8), 2429-2438 (1991).

For recombinant expression of the antibodies of the invention (i.e. heavy or light chain of an antibody according to the invention or their fragments or single-chain antibodies according to the invention), it is necessary to construct the expression vector containing polynucleotide that encodes the antibody. After receiving polynucleotide encoding the antibody heavy or light chain of the antibody or its fragment by known recombinant DNA technology it is possible to obtain the vector necessary for the production of antibodies. Thus, the application describes how to get the protein expression of polynucleotide containing encoding the antibody nucleotide sequence. Construction of expression vectors containing sequences encoding antibodies, and compliance is adequate control signals of transcription and translation, conduct known methods. These methods include, for example, methods of recombinant DNA in vitro, methods of synthesis and methods of genetic recombination in vivo. Thus, the invention offers a replicable vectors comprising a nucleotide sequence encoding the antibody according to the invention, a heavy or light chain antibody variable domain of a heavy or light chain antibodies (including fragments) or CC heavy or light chain associated with the promoter. Such vectors may include the nucleotide sequence encoding a conserved region of the antibody molecules (see, for example, WO 86/05807, WO 89/01036 and US 5122464)and the variable domain of the antibody can be cloned into such a vector for expression of a full-sized heavy, full-length light chain or both full-sized heavy and light chains.

The expression vector is transferred into a cell of the host body by standard methods, then transfection cells cultured by standard methods to generate antibodies according to the invention. Thus, the invention includes cells of the host body containing polynucleotide encoding the antibody according to the invention, or fragments, or a heavy or light chain or a fragment, or single chain antibodies according to the invention, associated with a heterologous promoter. In specific embodiments for the expression of dwohze is targeted antibodies, vectors, encoding the heavy and light chains together Express in cells of the host body for the expression of full-length immunoglobulin molecules, as detailed below.

For expression of the antibodies according to the invention can use a variety of vector systems for the expression in the body of the host (see, for example, US 5807715). Such expression systems are the media through which they receive, and then clear the appropriate coding sequences, and cells, which, after transformation or transfection with the appropriate nucleotide coding sequences can also Express the antibodies according to the invention in situ. These cells include, but are not limited to, microorganisms such as bacteria (for example, but not limited to, E. coli and B. subtilis)transformed with the expression vectors in the form of recombinant DNA bacteriophage, DNA plasmids or DNA of Comedy comprising sequences encoding antibodies, yeast (for example, but not limited to, Saccharomyces Pichia)transformed with recombinant expression vectors of yeast containing sequences encoding antibodies, insect cells infected with recombinant virus expression vectors (for example, but not limited to them, baculovirus), which contains asimi sequence, encoding the antibodies of the system of plant cells infected with recombinant virus expression vectors (for example, but not limited to, the cauliflower mosaic virus (VMCC), tobacco mosaic virus (TDC)or transformed with recombinant plasmid expression vectors (for example, but not limited to, plasmid T1), comprising sequences encoding antibodies, or mammalian cells (such as, but not limited to, COS cells, Cho, KSS, 293, NSO, and T), including recombinant expression constructs containing promoters derived from the genome of cells mammals (such as, but not limited to, promoter of metallothionein)or from mammalian viruses (for example, but not limited to, the late promoter of adenovirus, the promoter of the smallpox virus 7,5K). For expression of recombinant antibodies, primarily for the expression of full-length molecules of recombinant antibodies using bacterial cells such as Escherichia coli, and eukaryotic cells. For example, an effective expression system antibodies are mammalian cells, such as cells of the Chinese hamster ovary (Cho), in conjunction with a vector such as the major intermediate element in the promoter of the early gene of human cytomegalovirus (Foecking and others, Gene, 45, 101 (1986) and Cockett, etc., Bio/Technology, 8,2 (1990)). In a specific embodiment, the expression of the nucleotide sequence that encodes the antibody according to the invention, derivative, analog or fragment, is regulated by a constitutive promoter, inducible promoter or tissue-specific promoter.

In bacterial systems preferably choose a number of expression vectors to generate antibodies that are subject expression, depending on the intended application. For example, if you want to get a large number of such antibodies, for example for the preparation of pharmaceutical compositions of an antibody, it is advisable to use vectors, which stimulate the expression of a high number of products hybrid proteins, which are easy to clean. Such vectors include, but are not limited to, the expression vector of E. coli pUR278 (Ruther and others, EMBO, 12, 1791 (1983)), in which the sequence encoding the antibody, individually Legerova into the vector in frame with the coding region of the lac Z, so that a hybrid protein is produced, and pIN vectors (Inouye and Inouye, Nucleic Acids Res., 13, 3101-3109 (1985), Van Heeke and Schuster, J. Biol. Chem., 24, 5503-5509 (1989)), etc. For the expression of foreign polypeptides in the composition of the hybrid proteins with glutathione-5-transferase (GST) you can also use the pGEX vectors. In General, these hybrid proteins are soluble and can be extracted from the cell lysate by adsorption on the edge of the Ah glutathiones and subsequent elution in the presence of glutathione. The pGEX vectors are designed to include sites that are cleaved by a protease, such as thrombin or factor XA, and hence the product of the cloned gene target can be split from GST.

In the system of cells of the insect Autographa califormca for the expression of foreign genes used nuclear polyhedrosis virus (Aswap). The virus grows in the cells of Spodoptera frugiperda. The sequence encoding the antibody can be cloned individually into interchangeable parts (for example, the polyhedrin gene) of the virus and to cultivate under the control of the promoter Aswap (for example the polyhedrin promoter).

In mammalian cells, you can use a number of systems virus-mediated expression. When used as a vector for the expression of the adenovirus sequence, encoding the antibodies can be ligitamate in the complex control of transcription/translation of adenovirus, for example in the late promoter and three of the leader sequence. Then the specified hybrid gene can be inserted in the adenovirus genome by recombination in vitro or in vivo. Insert into interchangeable plot of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable and can Express the molecule antibodies in the infected cells of the host body (e.g., see Logan and Shenk, Proc. Natl. Acad. Sci. USA, 8, 1, 355-359 (1984)). For effective translat and the inserted sequence, encoding the antibodies may also require specific initiation signals. These signals include the initiation codon ATG and related sequences. Furthermore, the initiation codon must be in the same phase with the reading frame of the desired coding sequence to ensure translation of the complete insert. Using these exogenous signals control broadcast and the initiation codons of different origin, natural or synthetic. The efficiency of expression can be enhanced through inclusion of relevant elements enhance transcription, transcription terminators, etc. (see, for example, Bittner and others, Methods in Enzymol., 153, 51-544 (1987)).

In addition, you can choose the strain of the cells of the host body, which modulates the expression is inserted into the cell sequences, or modifies and processes the gene product required in a specific way. Such modifications (e.g., but not limited to them, glycosylation) and processing (for example, but not limited to, cleavage) of protein products may be important for the function of the protein. Various cells of the host body are characterized by specific and specific mechanisms of post-translational processing and modification of proteins and gene products. To ensure the necessary modifications, processi the ha expressed foreign protein can pick up the appropriate line or system of cells. Finally, you can use eukaryotic cells, which contain cellular mechanisms for the appropriate processing of the primary transcript, glycosylation, and phosphorylation of the gene product. Such mammals include, but are not limited to, cells of Cho, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, VT, Hs578T, HTB2, BT20 and T47D, NSO (line of murine myeloma cells, which are unable to produce endogenous chains of immunoglobulin), cells CRL7030 and HsS78Bst.

For long term production of recombinant proteins with high yield it is necessary to use a stable expression. For example, it is necessary to develop a cell line capable of stably Express the antibody molecules. Rather than using expression vectors which contain viral Replicator, it is advisable to transform cells of the host body by the introduction of DNA containing the appropriate elements of expression (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, and the like), and breeding marker. After the introduction of foreign DNA, engineered cells grow for 1-2 days in an enriched medium, and then transferred to selective medium. Breeding marker comprising recombinant plasmids gives the cell resistance during selection, allows cells stably interest is to illustrate the plasmid into the chromosome and grow with the formation of the clone which in turn can be cloned and duplicated before cell lines. This method has been successfully used to generate cell lines expressing the molecule of antibody. Such lines can primarily be used in screening and evaluation of compositions that directly or indirectly interact with the antibody molecule.

In one embodiment, the cell line used for expression of the compounds to bind to the IL-5P, represents cells that are unable to fokusirovat Fc region of the antibody to bind to the IL-5P. Examples of such cell types are given in US 6946292 and bids US 2006/0078991 A1, 2004/0110282 A1, 2006/0024800 A1, 2005/0216958 A1, 2004/0132140 and 2004/0259150. In a specific embodiment, the compound to bind to the IL-5P is humanitarian, deforsirovannogo α-chain monoclonal IgGI antibody to IL-5P. In another specific embodiment, the antibody is MEDI-563 (also known as BIW-8405). In another specific embodiment, the antibody is not MEDI-563.

As breeding systems can be used, but not limited to, timedancing of herpes simplex virus (Wigler and other Cell, 11, 223 (1977)), hypoxanthineguanine (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA, 48, 202 (1992)) and adrinfo.standortstr (Lowy et al., Cell, 22, 8-17 (1980)), genes which can be used in tk-, hgprt - or aprt-cells COO is responsible. In addition, resistance to antimetabolites can be used as a basis for selection of, for example, dhfr gene, which confers resistance to methotrexate (Wigler, etc., Natl. Acad. Sci. USA, 77, 357 (1980), O'hare and others, Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)), gpt gene, which confers resistance to mycophenolate acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA, 78, 2072 (1981)), the neo gene, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy, 3, 87-95 (1991), Tolstoshev, Ann. Rev. Pharmacol. ToxicoL, 32, 573-596 (1993), Mulligan, Science, 260, 926-932 (1993) and Morgan and Anderson, Ann. Rev. Biochem., 62, 191-217 (1993), May, TIB TECH 11 (5), 155-215 (1993)), and gene hygro, which confers resistance to hygromycin (Santerre and others, Gene, 30, 147 (1984)). The desired recombinant clone selected by standard methods known in recombinant DNA technology and is described in detail in the literature (see, for example, Ausubel and others (as amended), Current Protocols in Molecular Biology, John Wiley and Sons, NY (1993), Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990), Dracopoli and others (as amended). Current Protocols in Human Genetics, John Wiley & Sons, NY, p and 13 (1994), Colberre-Garapin, etc., J. Mol. BioL, 150, 1 (1981) (these works are included in the description of the application in full as a reference).

The expression level of antibodies can be improved by amplification of the vector (for review see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. Academic Press, New York (1987)). If the system vector expressing the antibody, the marker is amplificare, increase level the inhibitor, present in cell culture leads to an increase in the number of copies of a gene marker. Because amplificatory area associated with the gene of the antibody, it also increases the production of antibodies (Crouse and others, Mol. Cell. BioL, 3, 257 (1983)).

The host cell can also be transfectional two expression vectors according to the invention, the first vector encodes a polypeptide heavy chain and a second vector encodes a polypeptide light chain. Both vectors can contain identical breeding markers that provide equal expression of heavy and light polypeptide chains. In another embodiment, it is possible to use a single vector, which encodes and is capable to Express the polypeptides of the heavy and light chains. In such cases, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature, 322, 52 (1986), and Kohler, Proc. Natl. Acad. Sci. USA, 77 (2), 197 (1980)). Coding sequences of the heavy and light chains comprise cDNA or genomic DNA.

After receiving the antibodies according to the invention by recombinant expression product is purified by any known method of purification of immunoglobulins, for example by chromatography (e.g. ion exchange, affinity, primarily due to the affinity for the specific antigen, protein a, and exclusion chromatography), centrifugation, due to the time the primary solubility, or any other standard method of protein purification. In addition, to simplify the methods of purification of the antibodies of the present invention or fragments thereof can be hybreed with heterologous polypeptide sequences described in the text of the application, or you can use other cleaning methods.

The dose of the compounds to bind to the IL-5P (for example antibodies, proteins, polypeptides, peptides and hybrid proteins according to the invention, when administered to a patient is typically from 0.0001 mg/kg to 100 mg/kg body weight, preferably from 0.0001 mg/kg to 20 mg/kg, from 0.0001 mg/kg to 10 mg/kg, from 0.0001 mg/kg to 5 mg/kg, from 0.0001 mg/kg to 2 mg/kg, from 0.0001 mg/kg to 1 mg/kg, from of 0.0001 mg/kg to 0.75 mg/kg, from 0.0001 mg/kg to 0.5 mg/kg, from 0.0001 mg/kg to 0.25 mg/kg, from 0.0001 mg/kg to 0.15 mg/kg, from 0.0001 mg/kg to 0.10 mg/kg, from about 0.001 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 0.25 mg/kg or 0.01 mg/kg to 0.10 mg/kg of body weight. Typically human antibodies are characterized by a longer half-life in the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, it becomes possible to introduce more low doses of human antibodies and less frequent administration. In addition, the dose and frequency of administration of antibodies of the invention or its fragment can be reduced by increasing absorption and penetration into the tissue due to modification the AI antibodies for example lipidization.

In a specific embodiment, the dose of a compound binding to the IL-5P, for the prevention, treatment, control and/or mitigate the disease or one or more of its symptoms is 150 μg/kg or less, preferably 125 μg/kg or less, 100 μg/kg or less, 95 μg/kg or less, 90 μg/kg or less, 85 μg/kg or less, 80 μg/kg or less, 75 μg/kg or less, 70 μg/kg or less, 65 μg/kg or less, 60 μg/kg or less, 55 μg/kg or less, 50 μg/kg or less, 45 μg/kg or less, 40 μg/kg or less, 35 mg/kg or less, 30 μg/kg or less, 25 μg/kg or less, 20 μg/kg or less, 15 μg/kg or less, 10 μg/kg or less, 5 μg/kg or less, 2.5 µg/kg or less, 2 μg/kg or less, 1.5 to μg/kg or less, 1 μg/kg or less, 0.5 μg/kg or less, or 0.5 μg/kg or less. In another embodiment, a standard dose of a compound binding to the IL-5P, for the prevention, treatment, control and/or mitigate the disease or one or more symptoms ranging from 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg

In other embodiments, the subject is administered od is at or more doses, containing an effective amount of one or more medicinal products according to the invention, the dose achieving titers in the serum of at least 0.1 µg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200 μg/ml, at least 225 μg/ml, at least 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375 μg/ml, or at least 400 μg/ml of the medicinal product according to the invention. In other embodiments, the subject is administered a dose containing an effective amount of one compound according to the invention, binding to the IL-5P, to achieve a titer in the serum of at least 0.1 µg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 µg/ml, p is at least 175 μg/ml, at least 200 μg/ml, at least 225 μg/ml, at least 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375 μg/ml, or at least 400 μg/ml of compounds that bind to the IL-5P, and a subsequent dose containing an effective amount of one or more compounds that bind to the IL-5P, to maintain in the serum titer of at least 0.1 µg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200 μg/ml, at least 225 μg/ml, at least 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375 μg/ml, or at least 400 μg/ml In accordance with the specified options subject you can enter 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more consecutive doses.

In a specific embodiment, the invention provides methods of prevention, treatment, control and/or mitigate the disease, mediated by eosinophils, or one or more of its symptoms is s, moreover, these methods are that the entity that needs treatment, a dose containing at least 10 μg, preferably at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, at least 100 μg, at least 105 μg, at least 110 μg, at least 115 μg, or at least 120 μg of one or more drugs (e.g., therapeutic or prophylactic agents), combination of drugs or compositions according to the invention. In another embodiment, the invention provides methods of prevention, treatment, control and/or mitigate the disease, mediated by eosinophils, or one or more symptoms, with these methods is that the entity that needs treatment, a dose containing at least 10 μg, preferably at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg by Myung is our least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, at least 100 μg, at least 105 μg, at least 110 μg, at least 115 μg, or at least 120 μg of one or more compounds according to the invention, binding to the IL-5P, combination of drugs or compositions according to the invention once every 3 day, preferably once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 10 days, once every 2 weeks once every 3 weeks or once a month.

The present invention provides methods of prevention, treatment, control and/or mitigate disorders or diseases mediated by eosinophils, or one or more symptoms, and these methods are that (a) the entity that needs treatment is administered one or more doses containing a prophylactically or therapeutically effective amount one or more compounds that bind to the IL-5P, combination of drugs or compositions according to the invention, and (b) record the level/concentration in the serum of a subject put these compounds bind to the IL-5P, poslesvecheniya a certain number of doses of these medicines (for example, therapeutic or prophylactic agents). Moreover, preferably the specified number of doses containing a therapeutically or prophylactically effective amount of one or more compounds that bind to the IL-5P, combination of drugs or compositions according to the invention, may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

In a specific embodiment, the invention features a method of prevention, treatment, control and/or mitigate disorders or diseases mediated by eosinophils, or one or more symptoms, and these methods are that (a) the entity that needs treatment, a dose containing at least 10 μg, preferably at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, at least 100 μg) of one or more drugs (e.g., therapeutic or prophylactic agents) according to the invention, and (b) a specified subject is administered one or more doses, if the level of connection that communicates with IL-5P, the serum of the subject is less than 0.1 μg/ml, preferably less than 0.25 μg/ml, less than 0.5 μg/ml, less than 0.75 μg/ml or less than 1 μg/ml In another embodiment, the invention features a method of prevention, treatment, control and/or mitigate disorders or diseases mediated by eosinophils, or one or more symptoms, with this method is that (a) the entity that needs treatment, a dose containing at least 10 μg, preferably at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, at least 100 μg) of one or more compounds according to the invention to bind to the IL-5P, (b) register the specified level of the introduced compounds that bind to the IL-5P, in the serum of a subject after administration of a certain number of doses, and (C) a specified subject is administered an additional dose of the compounds according to the invention, binding to the IL-5P, if the level of the compounds to bind to the IL-5P, in the serum of the specified subject is less than 0.1 μg/ml, preferably less than 0.25 μg/ml, less than 0.5 μg/ml, less than 0.75 mcgml or less than 1 µg/ml In some embodiments, the number of doses containing an effective amount of one or more compounds according to the invention, binding to the IL-5P, may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

Drugs (e.g., prophylactic or therapeutic agents), is not identical with the compounds according to the invention, binding to the IL-5P, which are currently used for the prevention, treatment, control and/or mitigate the hyperproliferative disease or one or more symptoms, you can type in combination with one or more compounds that bind to the IL-5P, according to the methods according to the invention for the treatment, control, prevention and/or mitigation eosinophil-mediated disorders or diseases or one or more of its symptoms. Preferably the dose of prophylactic or therapeutic agents used in combination drug according to the invention below the doses that were used or is currently used for the prevention, treatment, control and/or mitigate eosinophil-mediated disorders or diseases or one or more of its symptoms. The recommended dosages of agents currently used for the prevention, treatment, control and/or mitigate the hyperproliferative disease or one or more of its symptoms can be found in the relevant is literature, including, but not limited to, Hardman and others, as amended, Goodman & Oilman, The Pharmacological Basis of Basis of Therapeutics, 10th ed. (2001), Mc-Graw-Hill, New York; Physician''s Desk Reference (PDR), 58th ed., Medical Economics Co., Inc., Montvale, NJ (2004), which are included in the description of the application in full as references.

In various embodiments, a drug (e.g., prophylactic or therapeutic agents) are administered in 5 minutes or less, 30 minutes or less after 1 h, after approximately 1 h, after approximately 1-2 hours, in about 2-3 hours, after about 3-4 hours, after about 4-5 hours, after approximately 5-6 h in about 6-7 hours, in about 7-8 hours, in about 8-9 hours, in about 9-10 hours, after approximately 10 h-11 h, in about 11-12 hours, after approximately 12-18, after 18-24 h through 24-36 h after 36-48 h, through 48-52 h after 52-60 h, after 60-72 h, through 72-84 h, through 84-96 h or through 96-120 hours In other embodiments, two or more drugs administered during a single patient visit to the doctor.

In some embodiments, one or more compounds according to the invention, binding to the IL-5P, and one or more other drugs (e.g., prophylactic or therapeutic agents) are administered cyclically. Cyclic therapy involves the introduction of the first drug (for example, the first prophylactic or therapeutics the CSOs agent) within a certain period of time, the introduction of a second drug (e.g., a second prophylactic or therapeutic agent) for a certain period of time, and then the optional introduction of a third drug (e.g., prophylactic or therapeutic agent) for a certain period of time and so on, and the repetition of the sequence of administration, i.e. the repetition of the cycle in order to reduce the development of resistance to one drug, excluding or reducing the side effects of one or more drugs and/or increase the efficacy of drugs.

In some embodiments, the introduction of the compounds according to the invention, binding to the IL-5P, you can try and enter through at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or at least 6 months. In other embodiments, the introduction of this drug (e.g., prophylactic or therapeutic agent), is not identical to the connection according to the invention, binding to the IL-5P, you can try and enter through at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or at least 6 months.

In a specific embodiment, the compound according to the invention, binding to the IL-5P, mo is but to be introduced in the form of a single intravenous dose of 0.03 mg/kg

The present invention provides methods of prevention, treatment, control and/or mitigate eosinophil-mediated disorders or diseases or one or more symptoms, with this method is that (a) the entity that needs treatment is administered one or more doses containing a prophylactically or therapeutically effective amount one or more compounds that bind to the IL-5P, combination of drugs, or the composition according to the invention, and (b) the entity has registered at least one sign or symptom of the disease before and after the introduction of one or more doses these drugs (e.g., therapeutic or prophylactic agents).

In one embodiment, the subject suffers from COPD.

In yet another embodiment, the subject suffers from weak stable or mild intermittent asthma attacks in accordance with the definition of the expert Working group NAEPP (2002).

In one embodiment, a sign or symptom of the disease in the subject recorded before and after administration of a single dose of one or more compounds that bind to the IL-5P. In another embodiment, a sign or symptom of the disease in the subject register before and after the introduction of multiple doses of one or more compounds that bind to the IL-5P.

In one embodiment, a sign or symptom of the disease are the two who is the self-assessment symptom of asthma on the scale. An example of a ball-assessment is a daily self-assessment of symptoms of asthma by the subject at home. Assessment of asthma symptoms in points in the last 24 hours based on the severity of the morning, night and daytime symptoms. Symptoms and their scoring are described in table 1. The maximum daily score is 9, the minimum score is 0. Subjects produced a self-assessment and registration in continuous mode.

In one embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P, equal to X, and the symptom of asthma in points after the introduction of one or more doses of one or more compounds that bind to the IL-5P equal to X-Y, where X is equal to 1, 2, 3, 4, 5, 6, 7, 8 or 9, Y is a 1, 2, 3, 4, 5, 6, 7, 8 or 9, and the score after the introduction is never less than 0.

In one embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P equal to from 0 to 9. In yet another embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P equal to from 0 to 3, from 1 to 4, from 2 to 5, 3 to 5, from 4 to 7, 5 to 8 or 6 to 9. In yet another embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P, equal 1, 2, 3, 4, 5, 6, 7, 8 or 9. In yet another embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P equal to 1. In yet another embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P equal to 2. In another od the Ohm version of the index symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds binding to the IL-5P equal to 3. In yet another embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P equal to 4. In yet another embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P, equal to 5. In yet another embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P equal to 6. In yet another embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P equal to 7. In yet another embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P equal to 8. In yet another embodiment, the indicator symptom of asthma in points before the introduction of the subject one or more doses of one or more compounds that bind to the IL-5P, equal to 9.

In one embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that bind to the IL-5P equal to from 0 to 9. In yet another embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that bind to the IL-5P, ranges from 0 to 3, from 1 to 4, or to 5, 3 to 5, from 4 to 7, 5 to 8 or 6 to 9. In yet another embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that bind to the IL-5P, equal 1, 2, 3, 4, 5, 6, 7, 8 or 9. In yet another embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that bind to the IL-5P equal to 1. In yet another embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that bind to the IL-5P equal to 2. In yet another embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that bind to the IL-5P equal to 3. In yet another embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that bind to the IL-5P equal to 4. In yet another embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that bind to the IL-5P, equal to 5. In yet another embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that bind to the IL-5P equal to 6. In yet another embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that communicates with the L-5P, equal 7. In yet another embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that bind to the IL-5P equal to 8. In yet another embodiment, the indicator symptom of asthma in points after administration to a subject one or more doses of one or more compounds that bind to the IL-5P, equal to 9.

In one embodiment, the indicator symptom of asthma in points decreases after administration to a subject one or more doses of one or more compounds that bind to the IL-5P in comparison with the index to introduction to the subject one or more doses of one or more compounds that bind to the IL-5P, and after the introduction of the indicator is never less than 0. In a specific embodiment, the indicator symptom of asthma in points is reduced by 1. In a specific embodiment, the indicator symptom of asthma in points is reduced by 2. In a specific embodiment, the indicator symptom of asthma in points is reduced by 3. In a specific embodiment, the indicator symptom of asthma in points is reduced by 4. In a specific embodiment, the indicator symptom of asthma in points is reduced by 5. In a specific embodiment, the indicator symptom of asthma in points is reduced by 6. In a specific embodiment, the indicator symptom of asthma in points is reduced by 7. In a specific embodiment, the indicator symptom of asthma in points is reduced by 8. In a specific embodiment, the indicator symptom is stmy in points decreases by at least 1. In a specific embodiment, the indicator symptom of asthma in points is reduced by at least 9. In a specific embodiment, the indicator symptom of asthma in points decreases by at least 2. In a specific embodiment, the indicator symptom of asthma in points is reduced by at least 3. In a specific embodiment, the indicator symptom of asthma in points is reduced by at least 4. In a specific embodiment, the indicator symptom of asthma in points is reduced by at least 5. In a specific embodiment, the indicator symptom of asthma in points is reduced by at least 6. In a specific embodiment, the indicator symptom of asthma in points is reduced by at least 7. In a specific embodiment, the indicator symptom of asthma in points is reduced by at least 8.

In one embodiment, a sign or symptom of the disease is the fraction of exhaled nitric oxide (FENO). FENO was measured in accordance with the joint recommendations of the European society for diseases of the Airways and American thoracic society (American Thoracic Society, European Respiratory Society. ATS/ERS Recommendations for Standardized Procedures for the Online and Offline measurement of Exhaled Lower Respiratory Nitric Oxide and Nasal Nitric Oxide (2005), Am. J. Respir. Crit. Care Med., 171, 912-930 (2005)). Measurement of FENO is carried out with the use of the drug NIOX at a flow rate of 50 ml/s (standard ATO).

In one embodiment, the subject is characterized by the value of FENO from 20 to 500 ppm MLR is to introduce one or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO from 20 to 500 ppm billion, from 20 to 400 ppm billion, from 20 to 300 ppm billion, from 20 to 200 ppm billion, from 50 to 500 ppm billion, from 100 to 500 ppm billion, from 150 to 500 ppm billion, from 200 to 500 ppm billion, from 20 to 50 part./billion, from 50 to 100 ppm billion, from 100 to 200 ppm billion, from 200 to 300 ppm billion, from 300 to 500 ppm billion prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO at least 50 part./billion, at least 100 ppm billion, at least 150 ppm billion, at least 200 ppm billion, at least 250 ppm billion, at least 300 ppm billion, at least 350 ppm billion, at least 400 ppm billion prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by the value of FENO 50 part./billion prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P.

In yet another embodiment, the subject is characterized by the value of FENO 100 ppm billion prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO 150 ppm billion prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by ve is ichinoe FENO 200 ppm billion prior to the introduction of one or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO 250 ppm billion prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO 300 ppm billion prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO 350 ppm billion prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO 400 ppm billion prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P.

In one embodiment, the subject is characterized by the value of FENO from 20 to 500 ppm billion after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO from 20 to 500 ppm billion, from 20 to 400 ppm billion, from 20 to 300 ppm billion, from 20 to 200 ppm billion, from 50 to 500 ppm billion, from 100 to 500 ppm billion, from 150 to 500 ppm billion, from 200 to 500 ppm billion, from 20 to 50 part./billion, from 50 to 100 ppm billion, from 100 to 200 ppm billion, from 200 to 300 ppm billion, from 300 to 500 ppm billion after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO not more than 50 ppm billion, not more than 100 ppm m is rd, not more than 150 ppm billion, not more than 200 ppm billion, not more than 250 ppm billion, not more than 300 ppm billion, not more than 350 ppm billion, not more than 400 ppm billion after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO not more than 20 ppm billion after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO not more than 50 ppm billion after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO not more than 100 ppm billion after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO not more than 150 ppm billion after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by FENO value not more than 200 ppm billion after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO not more than 250 ppm billion after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO not more than 300 ppm billion after the introduction one is th or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO not more than 350 ppm billion after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of FENO not more than 400 ppm billion after the introduction of one or more doses of one or more compounds that bind to the IL-5P.

In one embodiment, the FENO level in the subject is reduced after the introduction of one or more doses of one or more compounds that bind to the IL-5P, compared to the level prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P, and the level of FENO never drops below 0 part./billion In a specific embodiment, the FENO value is reduced by at least 50 part./billion In a specific embodiment, the FENO value is reduced by at least 100 ppm billion In a specific embodiment, the FENO value is reduced by at least 150 ppm billion In a specific embodiment, the FENO value is reduced by at least 200 ppm billion In a specific embodiment, the FENO value is reduced by at least 250 ppm billion In a specific embodiment, the FENO value is reduced by at least 300 ppm billion In a specific embodiment, the FENO value is reduced by at least 10%. In a specific embodiment, the FENO value is reduced by at least 20%. In a specific embodiment, the FENO value is reduced by at least 30. In a specific embodiment, the FENO value is reduced by at least 40%. In a specific embodiment, the FENO value is reduced by at least 50%. In a specific embodiment, the FENO value is reduced by at least 60%. In a specific embodiment, the number of FENO is reduced by at least 70%. In a specific embodiment, the FENO value is reduced by at least 80%. In a specific embodiment, the FENO value is reduced by at least 90%. In a specific embodiment, the FENO value is reduced by 10%. In a specific embodiment, the FENO value is reduced by 20%. In a specific embodiment, the FENO value is reduced by 30%. In a specific embodiment, the FENO value is reduced by 40%. In a specific embodiment, the FENO value is reduced by 50%. In a specific embodiment, the FENO value is reduced by 60%. In a specific embodiment, the FENO value is reduced by 70%. In a specific embodiment, the FENO value is reduced by 80%. In a specific embodiment, the FENO value is reduced by 90%.

In one embodiment, a sign or symptom of the disease is eosinophilic cationic protein (CBA). The content of CBA in serum define known to a person skilled ways, for example, but not limited to, ELISA and radioimmunoassay. In addition, the level of CBA can be assessed by any commercial method of analysis.

In one embodiment, the subject is characterized by the level of CBA in serum from 20 to 500 ng/ml prior to the introduction of one or more doses od the CSO or more compounds, binding to the IL-5P. In one embodiment, the subject is characterized by the level of CBA in serum from 20 to 200 ng/ml, from 20 to 150 ng/ml, from 20 to 100 ng/ml, from 20 to 50 ng/ml, from 30 to 200 ng/ml, from 40 to 200 ng/ml, 50 to 200 ng/ml, from 30 to 100 ng/ml, from 30 to 80 ng/ml, from 30 to 70 ng/ml, from 20 to 80 ng/ml, 20 up to 70 ng/ml, from 20 to 60 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by the level of CBA in the serum of at least 20 ng/ml, at least 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 60 ng/ml, at least 100 ng/ml, at least 150 ng/ml, at least 200 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in serum 25 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in the serum of 30 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in the serum of 35 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in the serum of 40 ng/ml prior to the introduction of one the or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in serum and 50 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in the serum of 60 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in the serum of 70 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in the serum of 80 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in serum and 100 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in serum 150 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in serum 200 ng/ml prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P.

In one embodiment, the subject is characterized redetection level CBA in serum after administration of one or more doses is underwater or more compounds, binding to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in serum from 1 to 500 ng/ml after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in serum from 1 to 200 ng/ml, from 1 to 150 ng/ml, 1 to 100 ng/ml, 1 to 50 ng/ml, from 1 to 20 ng/ml, from 10 to 200 ng/ml, from 10 to 100 ng/ml, from 10 to 50 ng/ml, from 20 to 100 ng/ml, from 20 to 50 ng/ml after administration of one or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in the serum of not more than 1 ng/ml, not more than 5 ng/ml, not more than 10 ng/ml, not more than 20 ng/ml, 30 ng/ml, not more than 50 ng/ml after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in the serum of not more than 1 ng/ml after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in the serum of not more than 5 ng/ml after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in serum no more than 10 ng/ml after administration of one or more doses of one or more compounds that bind to the IL-5P. In another VA who iante the subject is characterized by the level of CBA in the serum of not more than 15 ng/ml after administration of one or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in the serum of not more than 20 ng/ml after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in serum no more than 25 ng/ml after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the level of CBA in the serum of not more than 30 ng/ml after administration of one or more doses of one or more compounds that bind to the IL-5P.

In one embodiment, the level of CBA in the serum is reduced after the introduction of one or more doses of one or more compounds that bind to the IL-5P in comparison with the level observed prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P, and the level of CBE never drops below 0 ng/ml In a particular embodiment, the content of CBA in the serum is reduced by at least 50 ng/ml In a particular embodiment, the content of CBA in the serum is reduced by at least 100 ng/ml In a particular embodiment, the content of CBA in the serum is reduced by at least 150 ng/ml In a particular embodiment, the content of CBA in the serum is reduced by at least 200 ng/ml In a particular embodiment, the content of CBA in serum SN is supplied by at least 250 ng/ml In a particular embodiment, the content of CBA in the serum is reduced by at least 300 ng/ml In a particular embodiment, the content of CBA in the serum is reduced by at least 10%. In a particular embodiment, the content of CBA in the serum is reduced by at least 20%. In a particular embodiment, the content of CBA in the serum is reduced by at least 30%. In a particular embodiment, the content of CBA in the serum is reduced by at least 40%. In a particular embodiment, the content of CBA in the serum is reduced by at least 50%. In a particular embodiment, the content of CBA in the serum is reduced by at least 60%. In a particular embodiment, the content of CBA in the serum is reduced by at least 70%. In a particular embodiment, the content of CBA in the serum is reduced by at least 80%. In a particular embodiment, the content of CBA in the serum is reduced by at least 90%. In a particular embodiment, the content of CBA in the serum is reduced by at least 95%. In a particular embodiment, the content of CBA in the serum is reduced by 10%. In a particular embodiment, the content of CBA in the serum is reduced by 20%. In a particular embodiment, the content of CBA in the serum is reduced by 30%. In a particular embodiment, the content of CBA in the serum is reduced by 40%. In a particular embodiment, the content of CBA in serum SN is supplied by 50%. In a particular embodiment, the content of CBA in the serum is reduced by 60%. In a particular embodiment, the content of CBA in the serum is reduced by 70%. In a particular embodiment, the content of CBA in the serum is reduced by 80%. In a particular embodiment, the content of CBA in the serum is reduced by 90%. In a particular embodiment, the content of CBA in the serum is reduced by 95%. In a particular embodiment, the content of CBA in the serum is reduced by 99%.

In one embodiment, the subject is characterized redetection level CBA in serum after administration of one or more doses of one or more compounds that bind to the IL-5P.

In a particular embodiment, the content of CBA in serum remains redetection for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least the roughly 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 20 weeks or at least about 25 weeks.

In one embodiment, the sign or symptom is a provocative test with methacholine (MRP). MRP carried out according to the instructions of the American thoracic society (ATO), Guidelines for Methacholine and Exercise Testing - 1999. Am. J. Respir. Crit. Care Med., 161, 309-329 (2000), in the presence of a doctor who specializes in the treatment of bronchospasm with appropriate fast-acting therapeutic agents. In summary, the spirometer is calibrated according to the instructions ATO. The spray should produce particles with mass median aerodynamic diameter (MMAD) 1-4 microns at a flow rate of 0.13±10% ml/min For analysis is metafolin obtained from manufacturers approved by the Commission for the monitoring of food and drugs (FDA), which is diluted with sterile normal saline. Inhalation is carried out at a breath inhale/exhale for 2 min or method of measurement for five breaths-out, as described in the cited publication. The solution metacholine injected at various concentrations according to the established order in the range of from 0.06 mg/DL to 25.0 mg/DL. FEV (forced expiratory volume in 1 s) was measured after 30 and 90 seconds after completion of injection of each dose and record the higher of the two values. Solutions with a higher concentration was introduced to until FEV₁will not decrease by at least 20% from baseline. RS₂₀denotes the concentration of methacholine, which causes a decrease in FEV₁at least 20% from baseline. After the introduction of the final dose to the subject, you can give albuterol dosing sprinklers or spray at the discretion of the chief specialist.

In one embodiment, the subject is characterized by a value of RS₂₀from 0.06 to 25 mg/DL prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀from 0.06 to 25 mg/DL, from 0.1 to 10 mg/DL, from 0.06 to 3 mg/DL, from 0.06 to 2 mg/DL, from 0.06 to 1 mg/DL, from 0.1 to 3 mg/DL, from 0.1 to 2 mg/DL, from 0.1 to 1 mg/DL, from 0.2 to 10 mg/DL, from 0.5 to 10 mg/DL, from 1 to 10 mg/DL, from 0.1 to 5 mg/DL, from 0.2 to 5 mg/DL, from 0.5 to 5 mg/DL, from 0.1 to 2 mg/DL, from 0.2 to 2 mg/DL, 0.5 to 2 mg/DL, from 0.06 to 0.1 mg/DL, 0.1 to 0.2 mg/DL, 0.2 to 0.5 mg/DL, 0.5 to 1 mg/DL, 1 to 2 mg/DL, 2 to 5 mg/DL, 5 to 10 mg/DL prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀not more than 0.1 mg/DL, < 0.2 mg/DL, not more than 0.4 mg/DL, not more than 0.5 mg/DL, not more than 1 mg/DL, not more than 2 mg/DL, not more than 5 mg/DL, not more than 10 mg/DL prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by a value of RS₂₀10 mg/DL prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀5 mg/DL prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀2 mg/DL prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀1 mg/DL prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value PC₂₀0.5 mg/DL prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀0.2 mg/DL prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value PC₂₀0.1 mg/DL prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P.

In one embodiment, the subject characterized the : amount RS ₂₀from 0.5 to 25 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀from 1 to 25 mg/DL, from 2 to 25 mg/DL, from 5 to 25 mg/DL, 10 to 25 mg/DL, from 1 to 10 mg/DL, from 2 to 10 mg/DL, from 2 to 10 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀at least 1 mg/DL, at least 2 mg/DL, at least 5 mg/DL, at least 10 mg/DL, at least 20 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀at least 0.2 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀at least 0.3 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀at least 0.4 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value PC₂₀at least 0.5 mg/DL after administration of one or more doses of one or more compounds with asiaasia with IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀at least 0.7 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value RS at least 1 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀at least 2 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀at least 5 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀at least 10 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the value of the RS-20 at least 20 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by a value of RS₂₀at least 25 mg/DL after administration of one or more doses of one or more compounds that bind to the IL-5P.

In one embodiment, the value PC₂₀the subject has increased after the introduction of one or more of the oz one or more compounds binding to the IL-5P in comparison with the value of RS₂₀prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In a particular embodiment, the value RS₂₀the subject is increased by at least 0.3 mg/DL. In another specific embodiment, the value RS₂₀the subject is increased by at least 0.5 mg/DL. In another specific embodiment, the value PC₂₀the subject is increased by at least 0.7 mg/DL. In another specific embodiment, the value RS₂₀the subject is increased by at least 1 mg/DL. In another specific embodiment, the value RS₂₀the subject is increased by at least 3 mg/DL. In another specific embodiment, the value RS₂₀the subject is increased by at least 5 mg/DL. In another specific embodiment, the value RS₂₀the subject is increased by at least 10 mg/DL. In another specific embodiment, the value RS₂₀the subject is increased by at least 15 mg/DL. In another specific embodiment, the value RS₂₀the subject is increased by at least 20 mg/DL. In another specific embodiment, the value RS₂₀the subject is increased at least 2 times. In another specific embodiment, the value RS₂₀the subject is increased at least 4 times. In another specific embodiment, the value PC₂₀the subject has increased at least 8 times. In chem is one particular embodiment, the value RS ₂₀the subject has increased at least 10 times. In another specific embodiment, the value RS₂₀the subject has increased at least 12 times. In another specific embodiment, the value RS₂₀the subject has increased at least 15 times. In another specific embodiment, the value RS₂₀the subject has increased at least 20 times. In another specific embodiment, the value RS₂₀the subject has increased in 2 times. In another specific embodiment, the value RS₂₀the subject has increased by 4 times. In another specific embodiment, the value RS₂₀the subject has increased 8 times. In another specific embodiment, the value RS₂₀the subject has increased in 10 times. In another specific embodiment, the value RS the subject has increased 15 times. In another specific embodiment, the value RS₂₀increased by 60%. In another specific embodiment, the value RS₂₀increases 20 times.

In one embodiment, the sign or symptom is the number of eosinophils in the bloodstream. The number of eosinophils in blood flow determined by known methods, for example, but not limited to, histology and flow cytometry. The number of eosinophils in the blood flow can be determined using any commercial set.

In one embodiment, the subject is characterized by the number of eosinophils in krovat the ke from 50 to 1000 cells/µl prior to the introduction of one or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the bloodstream from 50 to 1000 cells/μl, from 100 to 1000 cells/μl, from about 150 to 1000 cells/μl, from 200 to 1000 cells/μl, from 250 to 1000 cells/μl, from 300 to 1000 cells/μl, from 400 to 1000 cells/μl, from 500 to 1000 cells/μl, from 50 to 500 cells/µl, from 100 to 500 cells/µl, from 100 to 400 cells/µl, 150 up to 500 cells/µl, from 200 to 500 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the blood flow of at least 50 cells/µl for at least 100 cells/µl for at least 150 cells/µl for at least 200 cells/µl for at least 250 cells/µl for at least 300 cells/µl for at least 400 cells/ml, at least 500 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by the number of eosinophils in the blood of 50 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P.

In yet another embodiment, the subject is characterized by the number of eosinophils in the blood stream 100 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the bloodstream 150 cells/µl prior to the introduction of odnosili more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the bloodstream 200 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the blood flow of 250 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the blood flow of 300 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the bloodstream 350 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the blood flow of 400 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the bloodstream 500 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P.

In one embodiment, the subject is characterized by the number of eosinophils in the bloodstream from 1 to 400 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils krovatke from 1 to 200 cells/µl, from 1 to 100 cells/µl, from 1 to 50 cells/µl, from 1 to 40 cells/μl, from 10 to 200 cells/µl, from 10 to 100 cells/µl, from 10 to 40 cells/μl, from 20 to 200 cells/µl, from 20 to 100 cells/µl, from 20 to 50 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the blood flow is not more than 1 cell/μl, no more than 5 cells/μl, no more than 10 cells/μl, no more than 20 cells/μl, no more than 30 cells/μl, no more than 40 cells/μl, no more than 50 cells/μl, no more than 60 cells/μl, no more than 80 cells/μl, no greater than 100 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by the number of eosinophils in the blood flow is not more than 1 cell/ml after administration of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the bloodstream no more than 5 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in blood flow does not exceed 10 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the blood flow is not more than 20 cells/µl after suggesting the one or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the blood flow is not more than 30 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the blood flow is not more than 40 cells/ál after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the blood flow is not more than 50 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the blood of 60 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of eosinophils in the blood flow is not more than 80 cells/μl after the introduction of one or more doses of one or more compounds that bind to the IL-5P.

In one embodiment, the number of eosinophils in the blood flow of the subject is reduced after the introduction of one or more doses of one or more compounds that bind to the IL-5P in comparison with the number of eosinophils in the blood stream prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P, and the number of eosinophils in the blood never falls below About cells/µl. In oncletom embodiment, the number of eosinophils in the blood stream is reduced by at least 50 cells/µl. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 100 cells/µl. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 150 cells/µl. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 200 cells/µl. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 250 cells/µl. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 300 cells/µl. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 10%. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 20%. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 30%. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 40%. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 50%. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 60%. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 70%. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 80%. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 90%. In a specific embodiment, the number of eosinophils in blood flow is reduced in men is her least 95%. In a specific embodiment, the number of eosinophils in the blood stream is reduced by at least 99%. In a specific embodiment, the number of eosinophils in the blood is reduced by 10%.

In a specific embodiment, the number of eosinophils in the blood is reduced by 20%. In a specific embodiment, the number of eosinophils in the blood is reduced by 30%. In a specific embodiment, the number of eosinophils in the blood is reduced by 40%. In a specific embodiment, the number of eosinophils in the blood is reduced by 50%. In a specific embodiment, the number of eosinophils in the blood is reduced by 60%. In a specific embodiment, the number of eosinophils in the blood is reduced by 70%. In a specific embodiment, the number of eosinophils in the blood is reduced by 80%. In a specific embodiment, the number of eosinophils in the blood is reduced by 90%. In a specific embodiment, the number of eosinophils in the blood is reduced by 95%. In a specific embodiment, the number of eosinophils in the blood is reduced by 99%.

In one embodiment, the subject is characterized redetection number of eosinophils in the bloodstream after administration of one or more doses of one or more compounds that bind to the IL-5P. In a specific embodiment, the number of eosinophils in the blood is stored on redetection level for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least closer is Ino 5 days at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 20 weeks or at least about 25 weeks.

In one embodiment, a sign or symptom of the disease is the content (%) eosinophils in induced sputum. Content (%) eosinophils in induced sputum can be determined by any known method, for example, but not limited to, the methods described in the article Belda and others, Am. J. Respir. Crit. Care Med. 161, 475-478 (2000). Content (%) eosinophils in induced sputum can be defined using any commercial set.

In one embodiment, the subject is characterized by content (%) eosinophils in induced sputum from 0.1% to 10% prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum from 0.1% to 2%, from 0.1% to 5%, from 0.5% to 2%, from 0.5% to 5%, from 0.5% to 10%, from 1% to 2%, from 1% to 5%, from 1% to 10%, from 2% to 5%, from 2% to 10%, from 3% to 5%, from 3% to 10%, from 1.5% to 5%, from 2.5% to 5% prior to the introduction of one or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum at least 0.1%of at least 0.5%and at least 1%, at least 1.5%, at least 2%, at least 2.5%of at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10% prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum of 0.5% until the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum of 1% prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum of 1.5% before the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum 2% to put the I one or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum of 2.5% prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum 3% prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum 4% prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum 5% prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum 6% prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum 7% prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum 8% prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. Even on the nom embodiment, the subject is characterized by content (%) eosinophils in induced sputum 9% prior to the introduction of one or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum 10% prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P.

In one embodiment, the subject is characterized by content (%) eosinophils in induced sputum from 0.1% to 5% after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum from 0.1% to 3%, from 0.1% to 2%, from 0.1% to 1.5%, from 0.5% to 5%, from 0.5% to 3%, from 0.5% to 1%, from 1% to 5%, from 1% to 3%, from 2% to 5%, from 3% to 5%, from 2.5% to 5% after the introduction of one or more doses of one or more compounds binding to the IL-5P. In one embodiment, the subject is characterized by content (%) eosinophils in induced sputum of not more than 1%not more than 2%not more than 3%, no more than 4%, no more than 5%, no more than 6%not more than 7%, 8%, 9%, no more than 10% after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by content (%) eosinophils in induced sputum of not more than 1% after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum of not more than 2% after the introduction of one or the more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum of not more than 3% after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum of not more than 4% after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum of not more than 5% after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by content (%) eosinophils in induced sputum of not more than 6% after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum of not more than 7% after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by content (%) eosinophils in induced sputum of not more than 8% after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by content (%) eosinophils in induced sputum of not more than 9% after the introduction of the one who does more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by content (%) eosinophils in induced sputum of not more than 10% after the introduction of one or more doses of one or more compounds that bind to the IL-5P.

In one embodiment, the content (%) eosinophils in induced sputum of the subject is reduced after the introduction of one or more doses of one or more compounds that bind to the IL-5P in comparison with content (%) eosinophils prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P, and the content (%) eosinophils in induced sputum never fall below 0%. In a particular embodiment, the content (%) eosinophils in induced sputum is reduced by at least 10%. In a particular embodiment, the content (%) eosinophils in induced sputum is reduced by at least 9%. In a particular embodiment, the content (%) eosinophils in induced sputum is reduced by at least 8%. In a particular embodiment, the content (%) eosinophils in induced sputum is reduced by at least 7%. In a particular embodiment, the content (%) eosinophils in induced sputum is reduced by at least 6%. In a particular embodiment, the content (%) eosinophils in induced sputum is reduced by at least 5%. In a particular embodiment, the content (%) eosinophils in induced wet the te is reduced by at least 4%. In a particular embodiment, the content (%) eosinophils in induced sputum is reduced by at least 3%. In a particular embodiment, the content (%) eosinophils in induced sputum is reduced by at least 2%. In a particular embodiment, the content (%) eosinophils in induced sputum is reduced by at least 1%.

In one embodiment, the subject is characterized redetection content of eosinophils in induced sputum after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In a specific embodiment, the eosinophils in induced sputum stored on redetection level for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at measures is approximately 14 weeks, at least about 16 weeks, at least about 20 weeks or at least about 25 weeks.

In one embodiment, the sign or symptom is the number of basophils in the bloodstream. The number of basophils in the blood flow determined by known methods, for example, but not limited to, histology and flow cytometry. The number of basophils in the blood flow can be determined using any commercial set.

In one embodiment, the subject is characterized by the number of basophils in the bloodstream from 5 to 500 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by the number of basophils in the bloodstream from 50 to 500 cells/µl, from 10 to 500 cells/µl, from 20 to 500 cells/µl, from 30 to 500 cells/µl, from 40 to 500 cells/µl, from 50 to 500 cells/µl, from 10 to 400 cells/μl, from 10 to 300 cells/µl, from 10 to 200 cells/µl, from 10 to 100 cells/µl, from 20 to 100 cells/µl, from 30 to 100 cells/µl, from 10 to 75 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by the number of basophils in the bloodstream for at least 5 cells/ml, at least 10 cells/µl for at least 15 cells/µl for at least 20 cells/µl for at least 30 cells/µl for at least 50 cells/µl, IU is greater least 60 cells/μl, at least 100 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by the number of basophils in the bloodstream 5 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the bloodstream 10 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the blood of 15 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the bloodstream 20 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the blood of 30 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the blood of 50 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the bloodstream 60 cells/µl prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P. In other odnawiane the subject is characterized by the number of basophils in the bloodstream 100 cells/µl prior to the introduction of one or more doses of one or more compounds binding to the IL-5P.

In one embodiment, the subject is characterized by the number of basophils in the bloodstream from 1 to 100 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by the number of basophils in the bloodstream from 1 to 100 cells/µl, from 1 to 50 cells/µl, from 1 to 30 cells/µl, from 1 to 20 cells/µl, from 1 to 10 cells/µl, from 5 to 100 cells/µl, from 5 to 50 cells/µl, from 5 to 20 cells/µl, from 5 to 10 cells/ál, 10 to 30 cells/µl after the introduction of one or more doses of one or more compounds that bind with IL-5P. In one embodiment, the subject is characterized by the number of basophils in the bloodstream no more than 1 cell/μl, no more than 5 cells/μl, no more than 10 cells/μl, no more than 20 cells/μl, no more than 30 cells/μl, no more than 50 cells/μl, no greater than 100 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In one embodiment, the subject is characterized by the number of basophils in the bloodstream no more cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the bloodstream no more than 5 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the bloodstream no more than 1 cell/ml after administration of one or more doses of one or more compounds binding to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the bloodstream no more than 20 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the bloodstream no more than 30 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the bloodstream no more than 40 cells/ál after the introduction of one or more doses of one or more compounds that bind to the IL-5P. In yet another embodiment, the subject is characterized by the number of basophils in the bloodstream is not more than 50 cells/µl after the introduction of one or more doses of one or more compounds that bind to the IL-5P.

In one embodiment, the number of basophils in the bloodstream the body of the subject is reduced after the introduction of one or more doses of one or more compounds that bind to the IL-5P in comparison with the number of basophils in the bloodstream prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P prior to the introduction of one or more doses of one or more compounds that bind to the IL-5P, and the number of basophils in the blood never falls below 0 cells/µl. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 10 cells/µl. In particular VA is iante the number of basophils in the bloodstream is reduced by at least 20 cells/µl. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 30 cells/µl. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 50 cells/µl. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 75 cells/µl. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 100 cells/µl. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 10%. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 20%. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 30%. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 40%. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 50%. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 60%. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 70%. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 80%. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 90%. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 95%. In a specific embodiment, the number of basophils in the bloodstream is reduced by at least 99%. In a specific embodiment, the number of bases is the Filov in the bloodstream is reduced by 10%. In a specific embodiment, the number of basophils in the bloodstream is reduced by 20%. In a specific embodiment, the number of basophils in the bloodstream is reduced by 30%. In a specific embodiment, the number of basophils in the bloodstream is reduced by 40%. In a specific embodiment, the number of basophils in the bloodstream is reduced by 50%. In a specific embodiment, the number of basophils in the bloodstream is reduced by 60%. In a specific embodiment, the number of basophils in the bloodstream is reduced by 70%. In a specific embodiment, the number of basophils in the bloodstream is reduced by 80%. In a specific embodiment, the number of basophils in the bloodstream is reduced by 90%. In a specific embodiment, the number of basophils in the bloodstream is reduced by 95%. In a specific embodiment, the number of basophils in the bloodstream is reduced by 99%.

In one embodiment, the subject is characterized redetection content of basophils in the bloodstream after administration of one or more doses of one or more compounds that bind to the IL-5P. In a particular embodiment, the content of circulating basophils remains redetection for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at measures is approximately 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 20 weeks or at least about 25 weeks.

Specific embodiments of the inventions

1. The way to reduce the number of eosinophils in the body, which is that the specified subject is administered a compound to bind to the IL-5P, which includes (a) a segment-specific binds to IL-5P, and (b) Fc fragment of immunoglobulin.

2. The method according to option 1, where the specified compounds that bind to IL-5P, are antibodies.

3. The method according to option 2, where these antibodies are monoclonal antibodies.

4. The method according to option 3, where these antibodies are hybrid antibodies.

5. The method according to option 3, where these antibodies are humanitarianism antibodies.

6. The method according to option 3, where these antibodies are human antibodies.

7. The method according to option 1, where the specified portion, which is associated with specific IL-5P, includes Amin is acid sequence of IL-5 or its fragments, variants that include substitutions of amino acids, or derivatives.

8. The method according to version 7, where the specified portion, which is associated with specific IL-5P, includes non-functional variant of IL-5.

9. The method according to any of the options 1-8, where the specified connection that binds to IL-5P, binds to the α chain of the IL-5P.

10. The method according to option 1, where the specified Fc fragment of immunoglobulin modified to improve effector functions.

11. The method according to option 1, where the specified Fc fragment of immunoglobulin contains a reduced number of fucose.

12. The method according to version 11, where the specified Fc-fragment of the immunoglobulin does not contain fucose.

13. The method according to option 1, where the specified Fc fragment of immunoglobulin includes replacement of amino acids, which lead to increased effector function.

14. The method according to option 1, where the indicated substitutions of amino acids include inserts in the Fc-fragment of the following amino acid sequence: E, 239D and 330L indicated by the EU index in Kabat.

15. The method according to option 1, where the decrease in the number of eosinophils occurs in the peripheral blood.

16. The method according to option 1, where the number of eosinophils is reduced to less than 50 eosinophils/mm

17. The method according to option 1, where the reduction in the number of eosinophils was observed during the first 48 h after injection.

18. The method according to option 1, where SN is a provision in the number of eosinophils was observed during the first 24 h after injection.

19. The method according to option 1, where the reduction in the number of eosinophils is reversible.

20. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least about 25 eosinophils/mm³.

21. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least about 50 eosinophils/mm³.

22. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least about 75 eosinophils/mm³.

23. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least approximately 100 eosinophils/mm³.

24. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least approximately 125 eosinophils/mm³.

25. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least about 150 eosinophils/mm³.

26. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least about 175 eosinoph the fishing/mm ³.

27. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least approximately 200 eosinophils/mm³.

28. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least approximately 225 eosinophils/mm³.

29. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least about 250 eosinophils/mm³.

30. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least approximately 275 eosinophils/mm³.

31. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least about 300 eosinophils/mm³.

32. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least approximately 325 eosinophils/mm³.

33. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least approximately 325 eosinophils/mm³.

33. The method according to option 1, where there is prolongiro the data (after injection) reduced the absolute number of eosinophils at least approximately 350 eosinophils/mm ³.

34. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least approximately 375 eosinophils/mm³.

35. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least approximately 400 eosinophils/mm³.

36. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least approximately 425 eosinophils/mm³.

37. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least about 450 eosinophils/mm³.

38. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least approximately 475 eosinophils/mm³.

39. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils at least about 500 eosinophils/mm³.

40. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils in approximately and including 50 to about 500 eosinophils/mm³.

41. The method according to option 1, where n is observed prolonged (after injection) reduced the absolute number of eosinophils in approximately and including 75 to about 250 eosinophils/mm ³.

42. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils in approximately and including 100 to about 200 eosinophils/mm³.

43. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils in approximately and including 50 to about 250 eosinophils/mm³.

44. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils in approximately and including 50 to about 200 eosinophils/mm³.

45. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils in approximately and including 50 to about 150 eosinophils/mm³.

46. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils in less than approximately 100 eosinophils/mm³.

47. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils in less than approximately 75 eosinophils/mm³.

48. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils on less than approximately 50 eosinophils/mm^{3 .}

^{49. The method according to option 1, where there is prolonged (after injection) reduced the absolute number of eosinophils in less than approximately 25 eosinophils/mm3.}

50. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) constitutes from approximately 50 to approximately 500 eosinophils/mm³.

51. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is from about 75 to about 475 eosinophils/mm³.

52. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is from about 75 to about 200 eosinophils/mm³.

53. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) ranges from approximately 100 to approximately 200 eosinophils/mm³.

54. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 25 eosinophils/mm³.

55. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 50 eosinophils/mm³.

56. The method according to option 1, where the absolute contents eosinophil is in the body of the subject prior to the introduction of (drug) is approximately 50 eosinophils/mm ³.

57. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 100 eosinophils/mm³.

58. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 125 eosinophils/mm³.

59. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 150 eosinophils/mm³.

60. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 175 eosinophils/mm³.

61. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 200 eosinophils/mm³.

62. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 225 eosinophils/mm³.

63. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 250 eosinophils/mm³.

64. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 275 eosinophils/m is ³.

65. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 300 eosinophils/mm³.

66. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 325 eosinophils/mm³.

67. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 350 eosinophils/mm³.

68. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 375 eosinophils/mm³.

69. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 400 eosinophils/mm³.

70. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 425 eosinophils/mm³.

71. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 450 eosinophils/mm³.

72. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 475 eosinophils/m is ³.

73. The method according to option 1, where the absolute eosinophils count in the body of the subject prior to the introduction of (drug) is approximately 500 eosinophils/mm³.

74. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced since the introduction of the drug) for at least about 5 basophils/mm³.

75. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced since the introduction of the drug) for at least about 20 basophils/mm³.

76. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced since the introduction of the drug) for at least about 15 basophils/mm³.

77. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced since the introduction of the drug) for at least about 20 basophils/mm³.

78. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced after administration (preparation) of at least about 25 basophils/mm³.

79. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced since the introduction of the drug) for at least about 30 buzof the fishing/mm ³.

80. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced after administration (preparation) of at least about 35 basophils/mm³.

81. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced after administration (preparation) of at least about 40 basophils/mm³.

82. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced since the introduction of the drug) for at least about 45 basophils/mm³.

83. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced since the introduction of the drug) for at least approximately 50 basophils/mm³.

84. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced since the introduction of the drug) for at least about 55 basophils/mm³.

85. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced since the introduction of the drug) for at least about 60 basophils/mm³.

86. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced after administration (drug) at IU is at approximately 65 basophils/mm ³.

87. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject is reduced after administration (preparation) of at least about 70 basophils/mm³.

88. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject after administration (drug) is from 0 to about 10 basophils/mm³.

89. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject after administration (drug) is approximately 2 basofil/mm³.

90. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject after administration (drug) is approximately 5 basophils/mm³.

91. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject after administration (drug) is approximately 7 basophils/mm³.

92. The method according to any of the options 1-73, where the absolute level of basophils in the body of the subject after administration (drug) is approximately 9 basophils/mm³.

93. The method according to any of the options 1-73, where the reduction in the number of basophil occurs within 48 h after injection.

94. The method according to any of the options 1-73, where the reduction in the number of basophil occurs within 24 h after injection.

95. The method according to l is the Bohm option 1-94, where the specified connection, binding to the IL-5P impose a specified subject in a dose of from about 0.001 to about 100 mg/kg

96. The method according to version 95, where this dose is approximately 0.03 mg/kg

97. The method according to option 95 where the specified dose of 0.03 mg/kg

98. The method according to any of the options 1-97, where the specified connection, binding to the IL-5P, administered parenterally.

99. The method according to version 98, where the specified connection, binding to the IL-5P, is injected.

100. The method according to any of the options 1-99, provided that the compound to bind to the IL-5P, doesn't mean MEDI-563.

101. The method according to any of the options 1-100, where the decrease in the number of eosinophils leads to reduction of asthma symptoms.

102. The method according to any of the options 1-100, where the decrease in the number of eosinophils leads to reduction of symptoms of COPD.

Examples

The invention is illustrated by the following examples without limiting its scope. Assume that the examples include all variations which become evident when reading the description.

Example 1

MEDI-563, antibodies against the receptor of interleukin-5, are characterized by high tolerance and induce reversible eosinopenia blood in patients with mild asthma according to tests (phase 1)

Introduction

Eosinophils play the main role in the pathogenesis of asthma. Interleukin-5 (IL-5) is a key cytokine in the biology of eosinophils, and expression of its receptor (IL-5R) occurs mainly only on the surface of eosinophils, basophils and mast cells. The low efficiency of treatment of asthma, in which the target is IL-5, due to insufficient reduction of the level of eosinophils in the lung tissue. Complete removal of eosinophils from the lungs will allow you to get more information about the role of these cells in the pathogenesis of asthma and helps to develop new treatment strategy.

Purpose

To assess the safety and biological activity of MEDI-563 (formerly BIW-8405) monoclonal antibody MEDI-563 (deforsirovannym humanitarianly IgG1 anti-IL-5P), which significantly increase antibody-dependent cellular cytotoxicity, was received at the company BioWa Inc. using patented technology Potelligent^®. MEDI-563 inhibit the activity of IL-5 and reduce the level of eosinophils in tissues in preclinical models with an acceptable toxicity profile.

Methods

In the first group (BIW-8405-001) tests MEDI-563 open-label, first performed in humans, have been included six subjects with mild asthma who were not treated with corticosteroids. Patients were introduced to 0.03 mg/kg MEDI-563 in the form of a single intravenous dose, the latter is the abuser introduction was performed at day 84.

Results

MEDI-563 characterized by high tolerance and have no significant side effects. All side effects (PD) is characterized by moderate intensity, most often as PD mentioned fatigue the day after a dose (3 of 6 subjects). All 6 subjects within 24-48 h after the dose, there is a decrease in the number of eosinophils in blood flow below the detection threshold (considering the magnitude of the standard deviation calculated dose). Specified side effects continued for 8-12 weeks, some of the subjects of the eosinophils were detected at day 58 after a dose, and we all studied subjects at day 84 after a dose level of eosinophils reached ≥70% from baseline. The number of basophils in the bloodstream has changed in the same way. As expected, given the mechanism of action of MEDI-563, within 72 hours after a dose level of neutrophils was decreased slightly and gradually, were developed neutropenia moderate in 2 of the 6 subjects, the intensity of which decreased in the course of 3 days. Introduction MEDI-563 was accompanied by an increase in the number of C-reaktivnogo protein in the serum (2 of 6 subjects) and IL-6 (in 2 of 3 subjects) fairly quickly (within 6 h), to an average level (<10× the baseline if the AI) and gradually (within < 1 week).

Conclusions

The introduction of 0.03 mg/kg MEDI-563 in the form of a single intravenous dose induces sustainable eosinopenia blood, and this dose is characterized acceptable approved in the current security profile.

Example 2

Mediated by antibodies cell-mediated cytotoxicity

Cell effectors KS 1333 (NK-cells, sverkhekspressiya FcgRIIIa and FceRIg person) were incubated for 4 h in conjunction with target cells CTLL-2 (cell line modified by genetic engineering of the cells mouse lymphoma with overexpression of IL-5α person) in the presence of MEDI-563 or control antibodies, and the ratio of cells-effector/target cells was 5:1. Mediated by antibodies cytotoxicity was evaluated by the method of staining viable cells using dye calcein AM. The results are presented on figa. This method was used in the analysis of other control drugs (fokusirovannyi MEDI-563). The results are presented on figb.

Example 3

The assessment of the equilibrium binding of MEDI-563 with IL-5P by plasma surface resonance

The experiment used a commercial preparation of soluble extracellular domain of IL-5α person who is not connected with the carrier (R+D Systems). Recombinant chil-5α was immobilized directly on top of the ity of sensor chip by the standard method via the amino group. The dynamics of the binding of MEDI-563 with immobilized chill 5α controlled by changing the refractive index, the values of k_on, k_offand K_Dwas calculated by standard methods. The results are shown in figure 10.

Example 4

The assessment of the equilibrium binding of MEDI-563 with FcγRs by the method of surface plasmon resonance

MEDI-563 was immobilized directly on the surface of sensor chip by the standard method via the amino group. The dynamics of the binding of soluble human FcγRs (firm Medlmmune) with immobilized MEDI-563 controlled by changing the refractive index, the values of k_on, k_offand K_Dwas calculated by standard methods. The results are presented in figure 11.

Example 5

Immunohistochemical analysis of the expression of IL-5α

Isecheno cloth nasal polyp were fixed with formaldehyde for 24 h and embedded in paraffin. Successively, the obtained sections were stained for the detection of IL-5α, IL-9R, CCR3 and c-kit man by standard methods using commercial polyclonal antibodies against IL-5P (R+D Systems, company Santa Cruz Biotechnology). Samples of lung tissue of transgenic IL-9 mice or wild-type strain, corresponding to the samples of the control FVB mice were fixed in formaldehyde for 24 h and embedded in paraffin. To assess the expression of IL-9R (pAb, firm Santa Cruz Biotechnlogy) and IL-5P (pAb, R+D Systems) sections were analyzed using standard immunohistochemical methods. The results are presented on Fig and Fig.

Example 6

The binding of Medi-563 with eosinophils whole blood of healthy donors

Granulocytes were isolated from whole blood of healthy donors by centrifugation in a density gradient. The reagents on the basis of primary antibodies containing the labels, used for analysis of expression of CD 16 (flurochrome FITZ) and MEDI-563 F(Ab)'2 (fluorochrome Alexa-647). The mixture CD16/FITZ+MEDI-563/Alexa-647 or CD16/FITZ+izotopicheskie control antibody tagged with Alexa-647 was added in the preparation of granulocytes in the amount of 1 μg to 10 cells. After incubation in an ice bath for 45 min, the cells washed three times with cold saline and analyzed the binding of the antibody with the surface of the cells by flow cytometry. It was also analyzed the eosinophils, which does not Express CD 16, and also determined the level of binding of MEDI-563 cell populations of granulocytes, not expressing CD 16, compared with the level of binding izotopicheskii control antibodies. The results are presented on Fig.

Example 7

Staining of cells from transgenic for IL-5α mice by flow cytometry

Leukocytes were isolated from blood, bone marrow, lung and spleen of transgenic IL-5 mice. Suspension cells were stained in the morning the nom solution FSB, containing 1% ETS. To reduce nonspecific binding of the cells before staining were incubated for 15 min in the presence of the reagent Fc Block, BD Biosciences). As the antibodies used antibody against mouse CCR3 (R&D systems), against Siglec F mouse (company BD Biosciences) and anti IL-5P mouse (H7). Cells were stained for 30 min, incubation in an ice bath, then washed twice and fixed in cytofix buffer solution (company BD Biosciences). The samples were analyzed by flow cytometry on a LSRII cytometer (firm Becton Dickinson) using software FACS Diva (firm Becton Dickinson). The results were analyzed using FlowJo software (firm TreeStar Inc.). The results are presented on figa and figb.

Example 8

The decrease in the number positive for IL-5α mononuclear cells from bone marrow mediated Medi-563

Frozen mononuclear cells from bone marrow (OCCM, Lonza) were thawed, washed, transferred into tablets for cell culture and incubated for 2 h at 37°C. After incubation of mononuclear cells from bone marrow, not adherent to the surface (NP-OCCM)were removed from the tablets. To assess SACC (antibody-dependent cellular cytotoxicity) 100000 NP-OCCM and 50,000 cells effector KS 1333 together were incubated for 18 h in 96-well tablet for under the project of cells, in each well was added 200 μl of the suspension of these cells (as cultivation media used the medium RPMI 1640 containing 10% ETS and antibody Medi-563 in a concentration of 10 μg/ml). Negative control reactions were performed using control izotopicheskii R347 antibody with a different specificity. To assess SACC cell effectors KS 1333 stained with a dye CFDA SE. After incubation for 18 h, the cells contained in each reaction mixture, washed three times with warm medium was added coloring antibodies and analyzed by flow cytometry. Positive for IL-5α cells were detected by staining with primary antibodies KM/conjugate PE+secondary goat anti-Mu IgG Fcg antibodies. Control staining of samples was performed using a primary izotopicheskii control antibodies A in combination with conjugate PE+secondary goat anti-Mu Fcg antibodies. Staining with antibodies and flow cytometry were performed by standard techniques. The number of cells positive for IL-5α remaining in the sample after evaluating SACC, was evaluated by counting the number of positive cells CM in the lymphocyte gate. Staining with antibodies and flow cytometry was performed using a calibration curve obtained using transgenic cells cell line CTLL-2 expressing IL-5α human is and. The result is dependent cellular cytotoxicity antibody-mediated MEDI-563, occurs in almost complete removal of samples NP-OCCM of cells positive for IL-5α. The results are presented on figa and figb.

Example 9

The reduction in the number of eosinophils in the peripheral blood, mediated MEDI-563

In tests of MEDI-563 open label included two groups of six subjects with mild asthma. Subjects from group 1 and group 2 were injected 0.03 mg/kg and 0.1 mg/kg MEDI-563, respectively, in the form of a single intravenous dose. The levels of eosinophils in the peripheral blood of these subjects were determined at day 0 to dose with equal intervals up to day 84 in the next period of the survey. The number of eosinophils in the blood was determined by flow cytometry. All 6 subjects in both groups the number of eosinophils in blood flow was reduced below the level of detection within 24 h after dose. Eosinopenia mediated MEDI-563, continued for 8-12 weeks. In group 1 after injection of 0.03 mg/kg MEDI-563 in the form of a single dose of five subjects for which trials were completed in 84 days, one of the subject were found on eosinophils detektiruya level at day 58, three subjects at day 84, the fifth subject of the eosinophils is not detected at day 84. In group 2 after administration of 0.1 mg/kg MEDI-563 in the form of one who Ratna dose none of the subject of eosinophils in the blood flow is not detected at day 84. However, during the survey period after testing all six subjects from the second group of peripheral blood eosinophils were found. On figa and 18B shows the change in the levels of eosinophils in the peripheral blood of subjects from the first and second groups depending on the time after the introduction of MEDI-563 in the form of a single dose.

Example 10

Immunohistochemical analysis of cells expressing IL-5α

Samples of lung tissue of a healthy person were stained MEDI-563 standard histochemical methods. The results are presented on Fig (cells expressing IL-5α, colored in black).

Samples of lung tissue obtained by bronchial or transbronchial biopsy of patients with a diagnosis of asthma, were stained with MEDI-563 standard histochemical methods. The results are presented on Fig (cells expressing IL-5α, painted dark grey/black).

Example 11

MEDI-563 effective on isolated basophils and eosinophils according to the analysis dependent cellular cytotoxicity antibody (SACC)

Basophils and eosinophils were obtained from healthy donors, using a commercial kit RoboSep (set for negative selection of NK-cells/eosinophils/basophils, firm Stem Cell Technologies, Vancouver, Canada). The expression of IL-5α in isolated cells were analyzed by the method of flowing qi is hometree. Cells were stained with antibodies MEDI-563 or izotopicheskii control antibodies with different specificity by standard methods. Stained with the antibodies, the cells were analyzed by flow cytometry. The staining profiles presented on Fig. According to the data obtained, the intensity of staining of isolated basophils and eosinophils antibody MEDI-563 higher in comparison with the intensity of staining izotopicheskii control antibodies. As a positive control was used painted transgenic cells expressing IL-5α/β person.

Activity fokusirovannyi and deforsirovannym MEDI-563 was evaluated in vitro by the method of SACC isolated eosinophils and autologous NK cells. Eosinophils and NK cells were obtained from healthy donors, was used a commercial kit RoboSep (set for negative selection of NK-cells/eosinophils/basophils, firm Stem Cell Technologies, Vancouver, Canada). In the above analysis used the isolated NK cells and eosinophils as cell effector and target cells in a 5:1 ratio. The analysis used the antibody in the concentration range from 10^-15M to 10^-7M. Cells were incubated for 24 h, then cytotoxicity was assessed by flow cytometry staining dye Annexin V. the Level SACC deforsirovannym EDI-563 exceeded the level SACC fokusirovannyi MEDI-563 several orders of magnitude. Value IS deforsirovannym MEDI-563 is 0,965 PM. The results of a typical experiment are presented in Fig.

Activity deforsirovannym MEDI-563 was evaluated in vitro by the method of SACC isolated basophils and autologous NK cells. Basophils and NK cells were obtained from healthy donors, was used a commercial kit RoboSep (set for negative selection of NK-cells/eosinophils/basophils, firm Stem Cell Technologies, Vancouver, Canada). When evaluating SACC selected NK cells and eosinophils were used as cell effector and target cells in a 5:1 ratio. The analysis used the antibody in a concentration range of from 10 M to 10 M. Cells were incubated for 24 h, then cytotoxicity was evaluated by flow cytometry to determine the level of cells positive in respect of the reagent Annexin V. In the above analysis, the value IS deforsirovannym MEDI-563 is 0,561 PM. The results of a typical experiment are presented in Fig.

Example 12

According to the analysis SACC mediated MEDI-563, eosinophils do not release cytotoxic granules

When analyzing SACC mediated MEDI-563, evaluated the degranulation of eosinophils at the level of the PAN (produced by eosinophils W / W)released in the supernatant. SACC was evaluated in vitro as described in example 11. Eosinophils and NK-cells or OKC (ar is yadernye cells from peripheral blood) were obtained from healthy donors and used as target cells and cell-effectors, respectively. The analysis was performed using fokusirovannyi MEDI-563, deforsirovannym MEDI-563 or control izotopicheskii deforsirovannym R347 antibody. The maximum level of degranulation was detected during the processing of eosinophils 1% Triton X-100, and the concentration of PAN was >220 ng/ml. the Results of typical experiments are presented on Fig. In SACC mediated MEDI-563, levels PENG decreased to less than 25 ng/ml (baseline). MEDI-563 in a concentration of 33 μg/ml or 100 μg/ml or above fokusirovannyi antibodies had no significant effect on the levels of degranulation.

Example 13

Mapping of the epitope is associated with MEDI-563

MEDI-563-specific contacted transgenic cells expressing IL-5α person, MEDI-563 was not contacted with cells expressing IL-5α mouse (see figb and figv). Amino acid sequence of IL-5α human and mouse are characterized by a very high degree of homology. The specificity of binding of MEDI-563 epitope was determined by the binding of MEDI-563 with a wide panel of hybrid (mouse/human) IL-5α (see Fig-27). In our experiments we used transgenic cells on which surface expressed hybrid IL-5α. Transgenic constructs were obtained and expressed according to standard methods. Antibody binding to the hybrid IL-5α, which is xpresroute on the surface of transgenic cells, analyzed by flow cytometry. Profiles of fluorescent staining presented on Fig-27. "Polyclonal antibodies" and "MEDI-563" refers profiles staining observed when using polyclonal antibodies against IL-5α person and MEDI-563, respectively. While MEDI-563-specific contacted one epitope comprising IL-5α human polyclonal antibodies was associated with many epitopes as part of the IL-5α person (see figb and figv). Double staining" refers to the profile fluorescent staining using polyclonal antibodies (x-axis) and MEDI-563 (y-axis).

First, the epitope bound MEDI-563, mapped in relation to the fragment of the extracellular domain D1 receptor IL-5α. IL-5α includes 3 extracellular domain (D1, D2, and D3), a transmembrane domain, and intracellular domain (see figa). As MEDI-563 was associated with IL-5α intact cells, epitope with which they are associated, should be placed in one of the extracellular domains. Mapping of the epitope in one of three extracellular domains, which are linked MEDI-563, were obtained transgenic cells expressing the hybrid IL-5α comprising the extracellular domains of the receptors in mouse and human, according to standard methods of molecular cloning. Schematic representation of the hybrid proteins used in the experimental the tah, presented at Figo. "Knock-out" options hybrid IL-5α represent receptors, including one extracellular domain of the receptor of the mouse in the composition of the primary sequence of the receptor is human. "Nominee" options hybrid IL-5α are the receptors of the mouse, including one extracellular domain of the receptor member primary sequence of the receptor of the mouse.

On figb-In presents the results of a typical experiment. MEDI-563 and polyclonal antibodies were stained transgenic cells expressing IL-5α that none of these antibodies were stained transgenic cells expressing IL-5α mouse (see figb). MEDI-563 did not correlate with the transgenic cells expressing the hybrid IL-5α, including D1 receptor domain of the mouse and extracellular domains D2-D3 receptor (see FIGU, "knock-out by D1"). MEDI-563-specific contacted transgenic cells expressing the hybrid IL-5α, including extracellular domains D2 or D3 mouse in the composition of the primary sequence of the receptor (see FIGU, "knock-out by D2 or D3). MEDI-563-specific contacted transgenic cells expressing the hybrid IL-5α comprising the extracellular domain D1 receptor human and extracellular domains D2-D3 receptor mouse (see Figg, "nominee on D1"). MEDI-563 did not correlate with the transgene, the suspended cells, expressing the hybrid IL-5α mouse, comprising the extracellular domain D2 or D3 receptor (see Figg, "nobunny on D2 or D3). All cells expressing the hybrid IL-5α containing at least one extracellular domain of the receptor human, stained polyclonal antibodies against IL-5α person that testified to the fact that the difference in the pattern of staining samples of transgenic cells antibody MEDI-563 is not associated with differences in levels of expression of the hybrid protein.

Secondly, the epitope of MEDI-563 is located in the segment In the extracellular domain D1 receptor IL-5α person (see Fig). The extracellular domain D1 receptor IL-5α is divided into three segments (see figa, segments a, b and C). Received a series of transgenic hybrid IL-5α human/mouse, including various combinations of segments of the extracellular domain D1 between human and mouse. Hybrid proteins are used at this stage include all amino acid sequences of the extracellular domain D1 receptor human. "Knock-out" transgenic represent hybrid constructs IL-5α, including one segment of the extracellular domain D1 receptor mouse in the composition of the primary sequence of the receptor is human. "Nominee" transgenes are hybrid constructs IL-5α, including one segment of the extracellular domain D1 receptor human is in the composition of the primary sequence of the receptor mouse D1/D2 mouse/D3-TM mouse (see figb). On Fig In presents the results of a control experiment. MEDI-563-specific contacted transgenic cells expressing: (1) IL-5α person, or (2) the hybrid IL-5α mouse, comprising the extracellular domain D1 receptor ("IL-5α man" and "nobunny on D1"). MEDI-563 did not correlate with the transgenic cells expressing: (1) IL-5α mouse, or (2) the hybrid IL-5α person, including the extracellular domain D1 receptor mouse (IL-5α mouse", "knock-out by D1"). On Figg and 26D presents the results of a typical experiment for mapping. MEDI-563 did not correlate with the transgenic cells expressing the hybrid IL-5α, including the segment In the extracellular domain D1 receptor mouse in the composition of the primary sequence of the receptor ("knock-out software"). MEDI-563-specific contacted transgenic cells expressing the hybrid IL-5α, including the segment a or With the extracellular domain D1 receptor mouse in the composition of the primary sequence of the receptor ("knock-out on A", "knock-out With"). On Figg presents an example of the results of experiments in which we used "nominee" constructs. MEDI-563-specific contacted transgenic cells expressing the hybrid IL-5α, including the segment In the extracellular domain D1 receptor men in the primary sequence of the receptor m the Chi Dl4enoBeKa/D2MbiniH/D3-TM mouse ("nobunny In"). MEDI-563 did not correlate with the transgenic cells expressing the hybrid IL-5α, including the segment a or With the extracellular domain D1 receptor men in the primary sequence of the receptor mouse D1/D2 mouse/D3-TM mouse ("nobunny on a or C"). All cells expressing the hybrid IL-5α, stained polyclonal antibodies against IL-5α person, which suggests that the difference in the pattern of staining samples of transgenic cells antibody MEDI-563 is not associated with the difference in the level of expression of the hybrid protein.

Thirdly, the epitope of MEDI-563 is located in the segment B1 of the extracellular domain D1 receptor IL-5α person. Received a series of variants of the receptor for IL-5α comprising at least one mutated amino acid residue in the segment B1 of the extracellular domain D1, which expressibility in transgenic cells. The position of the mutated residues were chosen by comparing the amino acid sequence of the protein between human and mouse. Schematic representation of variants of proteins used in the experiments presented in figa. "Knock-out" versions of the IL-5α represent mutant proteins of human, comprising at least one substitution of amino acid residue protein of the person on the appropriate balance of protein mouse. For example, the version of "knock-out by DE, is an IL-5α person, VK is uchumi replacement of amino acids D56E and E58D. "Nominee" variants of IL-5α are hybrid proteins, including D1 mouse, D2 person, D3 mouse and TM domains of the mouse, where the domain D1 receptor mouse includes mutant segment containing at least one substitution of amino acid residue of the receptor of the mouse on the appropriate balance receptor of human rights. For example, option "nobunny by DE", is a hybrid IL-5α, including mutant segment In receptor mouse in the composition of the primary sequence of the receptor mouse D1/D2 mouse/D3-Tmesi, where the mutant segment of the receptor of the mouse comprises a replacement of amino acids E56D and D58E. On figb presents the results of experiments in which we used "knock-out" constructs. MEDI-563 did not correlate with the transgenic cells expressing mutant IL-5α person, including replacement of amino acids K53Q, D56E, E58D, I61K ("knock-out by KDEI"), MEDI-563-specific contacted transgenic cells expressing mutant IL-5α person, including replacement of amino acids N40H, N42D, Q46H ("knock-out by NNQ") or D56E, E58D ("knock-out on DE"), or N40H, N42D, D56E, E58D ("knock-out by NNDE"). On Fig In presents examples of results obtained when using nakinig constructs. MEDI-563-specific contacted transgenic cells expressing a variant IL-5α, including mutant segment In domain D1 receptor mouse, including replacement AMI is ocelot Q53K, E56D, D58E, CBI ("nominee on KDEI"). On Figg presents an example of results obtained when using knock-out constructs. MEDI-563 did not correlate with the transgenic cells expressing mutant IL-5α person, which includes the replacement of amino acids 161 To ("knock-out by 161"). MEDI-563-specific contacted with cells expressing the mutant IL-5α person, which includes the replacement of amino acids K53Q ("knock-out by C"). (D) On Figg presents an example of results obtained when using nakinig constructs. MEDI-563-specific contacted transgenic cells expressing a variant IL-5α, including mutant segment In domain D1 receptor mouse, which includes the replacement of amino acids KB II ("nobunny on 161"). MEDI-563 did not correlate with the transgenic cells expressing a variant IL-5α including mutant segment In domain D1 receptor mouse, which includes the replacement of amino acids Q53K ("nobunny on K"). All cells expressing the hybrid IL-5α, stained polyclonal antibodies against IL-5α person, which suggests that the difference in the pattern of staining samples of transgenic cells antibody MEDI-563 is not associated with differences in levels of expression of the hybrid protein.

Example 14

Estimation of the reduction in the number of eosinophils in various tissues of the body in the analysis of in vivo

The effectiveness of selective reduction is s the number of eosinophils in various tissues, indirect deforsirovannym antibodies against IL-5α mouse (afuc H7), was evaluated in vivo in comparison with fokusirovannyi H7 antibodies (fuc H7).

Methods

Monoclonal antibodies H7

Variable plots H7 antibodies were attached (grafted) to hIgG2 Fc (Fc-fragment IgG1). Antibodies fuc H7 expressed in cells Cho wild type, afuc antibody H7 cells SNO, scarce FUT8 gene.

Evaluation of the affinity of antibodies (KD)

The affinity of the antibodies was assessed by plasma surface resonance.

Mouse

In the tests used transgenic for IL-5 mice, and BALB/c mice aged 6-8 weeks.

The reduction in the number of eosinophils in mice IL-5Tg

The mice were injected intraperitoneally 0.01 to 10 mg/kg afuc H7 and fuc H7 and estimated the number of eosinophils in 48 hours

Induction of allergic airway inflammation

BALB/c mice were senzibilizirani to ovalbumin (OA), included in alum, a response to the introduction of OA was developed in the days 17-22. Day 22 mice were administered 0.1 mg/kg afuc H7 (intraperitoneally) and analysis was performed after 1 h, 24 h and 72 h after the last sensitizing dose. The results were consistent with the condition after treatment using antibodies through 25, 48 and 96 hours

Obtaining leukocytes

1. Obtaining leukocytes from the blood

Blood samples were obtained by puncture of the heart and stored in heparinized samples is rcah. The blood leukocytes were phenotypically on the Hematology analyzer Sysmex (company Sysmex Corp.) or by flow cytometry.

2. Obtaining cells from the lumen of the respiratory tract

Conducted lavage of the respiratory tract (3×0.6 ml buffer solution FSB). Samples of bronchoalveolar lavage (BAL) was centrifuged at 1200 rpm, the supernatant was removed and cells resuspendable in RPMI medium. Cells were counted using a Coulter particle counter Z2 (firm Beckman-Coulter) and sorted depending on the phenotype of cells by flow cytometry.

3. Obtaining cells from lung tissue

Lung lobe were incubated for 1 h at 37°C in RPMI medium containing 10% ETS, in the presence of a reagent for disintegration (18 μg/ml liberase (Blenzyme 2, firm Roche), 25 µg/ml Gnkazy (type 1, the firm Roche)). The obtained cells were separated by filtration through a nylon filter with a pore diameter of 70 μm (firm Falcon), washed twice, counted and sorted depending on the phenotype of the cells, as described for the BALL.

4. Obtaining cells from the bone marrow

In the experiment used the femur of a mouse, from which the bone marrow was washed by a stream buffer solution FSB (not containing calcium/magnesium), using a syringe with needle size 25. The resulting suspension was stirred using a syringe with needle size 22 to obtain a suspension containing discre the data cells in the bone marrow. Bone marrow cells were separated by centrifugation for 5 min at 1200 rpm, washed twice with buffer solution FSB (not containing an additive), resuspendable in medium RPMI, counted and sorted depending on the phenotype of cells by flow cytometry.

5. Obtaining cells from the spleen

The subject was removed spleen and received a suspension containing discrete cells, using a nylon filter with a pore diameter of 70 μm. Leukocytes resuspendable in medium RPMI, counted and sorted depending on the phenotype of the cells, as described for the BALL.

Flow cytometry

Cells were sorted based on their phenotype by flow cytometry, was used antimurine antibodies against CD4, CD19, CD11b, Siglec-F, Gr-1, IL-5P, c-kit, BD Biosciences), FcεR1 (firm Biosciences) and CCR-3 (firm R&D Systems), as a control antibody used appropriate izotopicheskie antibodies. Samples were analyzed on a flow cytometer LSRII, used software FACS DIVA (BD Biosciences). The obtained results were analyzed using FlowJo software (TreeStar Corp.)

Identification of eosinophils

Eosinophils were identified by flow cytometry as cells with high lateral scattering, which are positively stained with antibodies to CCR3 and Siglec-f./p>

Results

IL-5P selectively expressed by eosinophils from bone marrow, blood, spleen and lung tissue of mice KDI-5Tg. IL-5P is expressed only by eosinophils and is not detected in any cells of other types, including mast cells and basophils. Antibodies against IL-5P selectively reduced the number of eosinophils in the spleen, lung tissue and blood of mice IL-5Tg. Deoksigenirovanii (afuc H7) and fokusirovannyi (fuc H7) antibodies against IL-5P did not reduce the number of neutrophils (Gr-1hi), macrophages/monocytes (CD11b+), T cells (CD3+), b cells (CD19+). The afuc antibody H7 and fuc H7 decreased the number of eosinophils in the spleen, lung tissue and blood of mice IL-5Tg. The decrease in the number of these cells in the bone marrow were observed. The afuc antibody H7 is more effective at removing eosinophils compared with antibodies fuc H7, especially when using low doses of antibodies.

The afuc antibody H7 also selectively reduced the number of eosinophils in the model, the induction of allergen. The afuc antibody H7 decreased the number of eosinophils in the lumen of the Airways, lung tissue, blood and bone marrow. The decrease was maximum in all parts of the body through 72 h after the last induction (96 h after delivery of antibodies).

Although the present invention is described with reference to specific variations in its implementation, to a person skilled in the art it is obvious, is that within the essence and scope of the invention various changes to these options.

All publications, patents and patent applications cited in the description, included in the description as a reference in each case to the same extent as if each patent, patent application and publication cited separately. In addition, the patent application US 60/924422 (registered 14 may 2007), US 60/924832 (registered 1 June 2007), US 60/935005 (registered 20 July 2007) and US 61/064612 (registered 14 March 2008) included as references in full.

1. The way to reduce the number of eosinophils in the human body, comprising injecting a specified subject from about 0.01 mg/kg to about 0.25 mg/kg monoclonal, hybrid, gumanitarnogo antibodies or human antibodies, which binds to the IL-5P and includes the Fc fragment of immunoglobulin and does not contain fucose, in which the introduction of antibody reduces the number of peripheral blood eosinophils circulating in the body, below the threshold of detection and counting of circulating eosinophils remains below the detection threshold for at least about 28 days after dosing antibodies.

2. The method according to claim 1, where the indicated specific antibodies bind to the α-chain of the IL-5P.

3. The method according to claim 1, where the reduction in the number of eosinophils occurs within the first 48 h after injection.

4. The method according to claim 1, where the reduction is their number of eosinophils is reversible.

5. The method according to claim 1, where the absolute eosinophils count in the subject prior to the introduction ranges from approximately 50 to approximately 500 eosinophils/mm³.

6. The method according to claim 1, where the absolute number of eosinophils in the subject prior to the introduction is about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 450, about 475, or approximately 500 eosinophils/mm³.

7. The method according to claim 1, where the absolute number of basophils in the subject is reduced after the introduction of at least about 5, at least
about 10, at least about 15, at least
about 20, at least about 25, at least
about 30, at least about 35, at least
about 40, at least about 45, at least
about 50, at least about 55, at least
about 60, at least about 65, at least
approximately 70 basophils/mm³.

8. The method according to claim 1, where the decrease in the number of EO is infolow leads to reduction of asthma symptoms.

9. The method according to claim 1, where the decrease in the number of eosinophils leads to reduction of symptoms of COPD.