Human il-13 antibody molecules

FIELD: medicine.

SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.

EFFECT: applicable in medicine for preparing a vaccine.

52 cl, 32 dwg, 7 tbl, 29 ex

 

The present invention relates to specific binding partners, in particular the molecules of the anti-IL-13 antibodies of human rights and especially the molecules that neutralize the activity of IL-13. The invention also relates to methods of using the molecules of anti-IL-13 antibodies in the diagnosis or treatment associated with IL-13 disorders, including asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease and Hodgkin's lymphoma.

In preferred embodiments, the implementation of the present invention use the domain VH and/or VL of the antibody molecule antibodies, referred to in this description BAK502G9, and other molecules antibodies lines BAK502G9 and line BAK278D6 that are defined in this specification. In the following preferred embodiments use a complementarity determining region (CDR) line BAK278D6 and preferably BAK502G9, in particular CDR3 VH, in frame areas other antibodies. The following aspects of the present invention relate to compositions containing specific binding partners according to the invention, and their use in methods of inhibiting or neutralizing IL-13, including methods of therapeutic treatment of the human or animal.

The present invention relates to antibody molecules having particular importance in binding and neutralization of IL-13 and, therefore, having PR is a change in any of a variety of therapeutic treatments as demonstrated in the experimental work presented in this description, and, moreover, confirmed by the technical literature.

Interleukin (IL) 13 is a cytokine of the 114 amino acids with unmodified molecular weight of about 12 KD [1, 2]. IL-13 is closest IL-4, with which it has a 30% sequence similarity at the amino acid level. Gene IL-13 person located in chromosome 5q31 near genome IL-4 [1], [2]. The specified region of chromosome 5q contains sequences of other genes originating from lymphocytes of the Th2 cytokines, including GM-CSF and IL-5 levels which together with IL-4, as shown, correlate with disease severity in patients with asthma and models of allergic inflammation in rodents[3], [4], [5], [6], [7], [8].

Although IL-13 is initially identified as produced by lymphocytes Th2CD4+ cytokine, he also is produced by T-cells Th1CD4+, T-lymphocytes CD8+ NK-cells and populations of cells, non-T cells, such as mast cells, basophils, eosinophils, macrophages, monocytes and smooth muscle cells of the respiratory tract.

It is reported that IL-13 mediates its effects through a system of receptors, which includes the α-chain of the IL-4 receptor (IL-4Rα), which in itself can bind IL-4, but not IL-13, and at least two other proteins of cell surface IL-13Rα1 and IL-13Rα2 [9], [10]. IL-13Rα1 can bind IL-13 with low the affinity, then mobilising IL-4Rα with the formation of functional high-affinity receptor, which transmits the signal [11], [12]. In the database of Genbank registered amino acid sequence and the nucleic acid sequence of IL-13Rα1 in the form NP_001551 and Y10659 respectively. Research is deficient in STAT6 (signal transducer and activator of transcription 6) mice showed that IL-13 is similar to IL-4 gives the signal, using the path JAK-STAT6 [13], [14]. IL-13Rα2 has 37% sequence identity with IL-13Rα1 at the amino acid level and binds IL-13 with high affinity [15], [16]. However, IL-13Rα2 has a shorter cytoplasmic tail, in which there are no known motives for signal transmission. Cells expressing IL-13Rα2, do not respond to IL-13 even in the presence of IL-4Rα [17]. Therefore, it is assumed that IL-13Rα2 acts as a receptor-traps, regulating IL-13, but not IL-4. This is confirmed by studies deficient in IL-13Rα2 mice, the phenotype of which corresponds to increased sensitivity to IL-13 [18], [19]. In the database of Genbank are given amino acid sequence and the nucleic acid sequence of IL-13Rα2 in the form NP_000631 and Y08768 respectively.

Transmitting signals from the receptor complex of IL-13Rα1/IL-4Rα is expressed on B-cells, mast cells, monocytes/macrophages, dendritic cells, e is siniyah, the basophils, fibroblasts, endothelial cells, epithelial cells of the respiratory tract and the smooth muscle cells of the respiratory tract.

Bronchial asthma is a common persistent inflammatory lung disease characterized by hypersensitivity of the respiratory tract, excessive mucus formation, fibrosis, and elevated levels of IgE in serum. Hypersensitivity of the respiratory tract (AHR) is a excessive airway narrowing in response to nonspecific stimuli such as cold air. Consider that both AHR and excess mucus formation is responsible for the temporary obstruction of the respiratory tract, which leads to difficulty breathing, a characteristic of asthma attacks (exacerbations), and which is the cause of the mortality associated with this disease (about 2000 deaths/year in the UK).

The incidence of asthma along with other allergic diseases has increased significantly in recent years [20], [21]. For example, currently about 10% of the population in the UK are diagnosed as asthma patients.

Current recommendations of the British thoracic society (BTS) and the Global initiative for asthma (GINA) propose a stepwise approach to asthma treatment [22, 23]. Weakly and moderately expressed asthma, you can usually contact airavat, using inhalation corticosteroids in combination with beta-agonists or leukotriene inhibitors. However, because of the documented side effects of corticosteroids patients tend not to follow the treatment regimen, which reduces the effectiveness of the treatment [24-26].

There is an obvious need for new methods of treatment of subjects with more severe disease, which often get very limited use or high doses of inhalation or oral corticosteroids, offered recommendations on asthma. Long-term treatment with oral corticosteroids is associated with side effects such as osteoporosis, slow growth rate in children, diabetes, and oral candidiasis [88]. As a healing and adverse effects of corticosteroids are mediated by the same receptor, and treatment is a balance between safety and efficiency. Hospitalization of these patients, who constitute about 6% of the population of asthmatics in the UK, as a result of severe exacerbations determines the major part of a significant economic burden associated with asthma, health authorities [89].

It is assumed that the pathology of asthma is caused by ongoing inflammation, mediated by Th2 lymphocytes, which occurs as a result of inadequate totalimages system to harmless antigens. Accumulate evidence about the involvement of IL-13, but not a classic produced by the cells of the Th2 cytokine IL-4, as a key mediator in the pathogenesis developed diseases of the respiratory tract.

The introduction of recombinant IL-13 in airway native desensibilisation rodents caused a lot of phenotype features of asthma, including airway inflammation, mucus formation and AHR[27], [28], [29], [30]. A similar phenotype was observed in transgenic mice in which IL-13-specific was sverkhekspressiya in the lungs. In this model, the additional impact of IL-13 has also led to fibrosis [31].

In addition, in models of allergic diseases in rodents many signs of asthma phenotype were associated with IL-13. It is shown that murine IL-13Rα2, strong neutralizing IL-13 inhibited AHR, increased mucus secretion and the influx of inflammatory cells, which are characteristic features of this model in rodents[27], [28], [30]. Additional studies in mice that have been delegated gene IL-13, did not develop induced by allergen AHR. AHR can be restored from the specified deficient in IL-13 mice by introduction of recombinant IL-13. In contrast, mice deficient in IL-4, in the model developed airway disease[32], [33].

Using long-term model of allergen induced inflammation of the lungs, Tube et al. demonstrated the effectiveness of soluble murine IL-13Rα2 developed against diseases of the respiratory tract [34]. Soluble murine IL-13Rα2 inhibited AHR, increased mucus formation and to a lesser extent the inflammation of the Airways. In contrast, soluble IL-4Rα, which binds and antagonisitic IL-4, had little impact on AHR or airway inflammation in this system [35]. These data confirmed Blease et al., who has developed a model of chronic fungal asthma Genesis, in which polyclonal antibodies against IL-13, but not against IL-4, were able to reduce excessive mucus formation, AHR and subepithelial fibrosis [36].

Several types of genetic polymorphism in the gene for IL-13 was also associated with allergic disease. In particular, the version of the gene IL-13, in which the arginine residue at amino acid position 130 is replaced by glutamine (R130Q), was associated with bronchial asthma, atopic dermatitis and elevated levels of IgE in serum[37], [38], [39], [40]. A specific variant of IL-13 also called option Q110R (arginine residue at amino acid position 110 is replaced by glutamine) some groups that excluded the 20-amino acid signal sequence of the numbering of the amino acids. Arima et al. [41] reported that this variant is associated with elevated levels of IL-13 in serum. A variant IL-13 (R130Q) and antibodies against this is arianto discussed in WO 01/62933. A polymorphism in the promoter of IL-13, which changes the production of IL-13, was also associated with allergic asthma [42].

Elevated levels of IL-13 was measured in people with asthma, atopic rhinitis (hay fever), allergic dermatitis (eczema) and chronic sinusitis. For example, found that levels of IL-13 is higher in bronchial biopsy, sputum cells and bronchoalveolar lavage (BAL) of asthmatic patients compared with control subjects[43], [44], [45], [46]. In addition, levels of IL-13 in BAL samples was increased in patients with asthma in the case of provocation with allergen [47], [48]. In addition, it is shown that the ability of T-cells and CD4(+) to produce IL-13 is a useful marker of risk of the subsequent development of allergic disease in infants [49].

Li et al. [114] recently reported the effects of neutralizing antibodies against IL-13 mouse model of chronic asthma in mice. Chronic such asthmatic response (such as AHR, severe inflammation of the Airways, increased mucus formation) induced in OVA sensitized mice. Li et al. reported that the administration of IL-13 antibodies during each OVA provocation suppresses AHR, infiltration of eosinophils, IgE levels in serum levels of proinflammatory cytokines/chemokines and remodeling of the Airways [14].

In conclusion, our data present evidence that IL-13, and n is IL-4 is a more attractive target for the treatment of allergic diseases of the person.

IL-13 may play a role in the pathogenesis of inflammatory bowel disease. Heller et al. [116] reported that neutralization of IL-13 by the introduction of soluble IL-13Rα2 reduced inflammation of the colon in a mouse model of ulcerative colitis [116]. Accordingly, the expression of IL-13 was higher in the biopsy samples of the rectum from patients with ulcerative colitis compared with controls [117].

In addition to asthma, IL-13 was associated with other fibrotic conditions. Elevated levels of IL-13 to levels 1000 times higher than that of IL-4 measured in the serum of patients with systemic sclerosis [50] and in BAL samples from patients infected with other forms of pulmonary fibrosis [51]. Accordingly, the overexpression of IL-13 but not IL-4 in the lungs of mice resulted in a pronounced fibrosis [52], [53]. The contribution of IL-13 in fibrosis in other tissues other than the lungs, widely studied in the mouse model induced by parasites of pulmonary fibrosis. Specific inhibition of IL-13 by the introduction of soluble IL-13Rα2 or violation of the gene IL-13, but not the exclusion of education IL-4 prevented fibrogenesis in the liver[54], [55], [56].

Chronic obstructive pulmonary disease (COPD) covers a population of patients with varying degrees of chronic bronchitis, diseases of the small Airways and emphysema and is characterized by progressive, irreversible decline in lung function, which is difficult modern therapy, based on asthma treatment [90]. The incidence of COPD has increased dramatically in recent years, and the disease was the fourth among the leading causes of death worldwide (world health organization). Therefore, the treatment of COPD represents a large unmet need for medicine.

The reasons underlying COPD, remain poorly understood. "Dutch hypothesis" assumes that there is a General sensitivity to COPD and asthma and, therefore, such similar mechanisms may contribute to the pathogenesis of both disorders [57].

Zheng et al. [58] showed that overexpression of IL-13 in the lungs of the mouse caused emphysema, increased formation of mucus and inflammation, reflecting signs of a COPD person. In addition, it is shown that the AHR, IL-13-dependent response in murine models of allergic inflammation, is a prognostic sign of decline of lung function in smokers [59]. Also found an Association between promoter polymorphism of IL-13 and sensitivity to the development of COPD [60].

Therefore, there is evidence that IL-13 plays an important role in the pathogenesis of COPD, particularly in patients with similar asthma signs, including AHR and eosinophilia. It is shown that the mRNA levels of IL-13 is higher in autopsy tissue samples from subjects with COPD in the history of the disease compared with lung samples from subjects who did not register the but pulmonary disease (J. Elias, Oral communication at American Thoracic Society Annual Meeting, 2002). In another study, elevated levels of IL-13 is shown by immunohistochemistry in the peripheral sections of the lungs from patients with COPD [91].

Hodgkin's disease is a common type of lymphoma, which is approximately 7,500 cases per year in the United States. Hodgkin's disease is unusual among malignant tumors, as neoplastic cells, reed-Sternberg, often originating from B-cells make up only a small fraction of clinically detectable mass. Obtained in the case of Hodgkin's disease cell line and primary cells, reed-Sternberg often Express IL-13 and its receptor [61]. Because IL-13 stimulates cell viability and proliferation of normal B-cells, we hypothesized that IL-13 can act as a growth factor for cells, reed-Sternberg. Skinnider et al. showed that neutralizing antibodies against IL-13 can inhibit the growth obtained in the case of Hodgkin's disease cell lines in vitro [62]. These observations indicate that cells, reed-Sternberg can improve their own sustainability through autocrine and paracrine cytokine loops IL-13. In accordance with this hypothesis, elevated levels of IL-13 detected in the serum of some patients with Hodgkin's disease compared with normal control of the s [63]. Therefore, inhibitors of IL-13 can prevent progression of the disease as a result of inhibition of proliferation of malignant cells, reed-Sternberg.

Many malignant tumors of the person Express immunogenic tumor-specific antigens. However, despite the fact that many tumors spontaneously regress, some avoid the immune system (immune surveillance), suppressing mediated T-cell immunity. Terabe et al. [64] demonstrated the role of IL-13 in immunosuppression in murine models in which tumors spontaneously regress after the initial growth and then recur. Specific inhibition of IL-13 soluble IL-13Rα2 protected these mice from relapse occurrence of the tumor. Terabe et al. [64] further showed that IL-13 inhibits the differentiation of tumor-specific cytotoxic lymphocytes CD8+, which mediate the antitumor immune responses.

Therefore, inhibitors of IL-13 can be used therapeutically to prevent tumor recurrence or metastases. It is shown that the inhibition of IL-13 enhances antiviral vaccines in animal models and may be useful in the treatment of HIV and other infectious diseases [65].

It should be noted that, as a rule, in this description, the indication of interleukin-13 or IL-13 except where the context dick is the duty to regulate otherwise, belongs to the IL-13 human. In some cases it is also called "antigen". The present invention relates to antibodies to IL-13 human rights, particularly the human antibodies that cross-react with IL-13 Primate other than humans, including IL-13 macaques-Griboedov and rhesus. Antibodies according to some variants of implementation of the present invention recognize the variant IL-13, in which the arginine residue at amino acid position 130 is replaced by glutamine. In other aspects and embodiments of implementation of the present invention offers specific binding partners against IL-13 mice and rats, in particular against murine IL-13.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows the efficiency of neutralization (% inhibition) in the case BAK167A11 (shaded squares) and its derivative BAK615E3 (where there's no shading squares) in the form of scFv against 25 ng/ml IL-13 man in the analysis of cell proliferation, TF-1. Triangles indicate irrelevant scFv. Data represent mean values with lines standard errors based on the definitions in three repetitions in the same experiment.

Figure 2 shows the efficiency of neutralization (% inhibition) in the case BAK278D6 (shaded squares) and its derivative BAK502G9 (where there's no shading squares) in the form of scFv against 25 ng/ml IL-13 man in the analysis of cell proliferation, TF-1. Tree olenici mean irrelevant scFv. Data represent mean values with lines standard errors based on the definitions in three repetitions in the same experiment.

Figure 3 shows the efficiency of neutralization (% inhibition) in the case BAK209B11 (shaded squares) in the form of scFv against 25 ng/ml murine IL-13 in the analysis of cell proliferation, TF-1. Triangles indicate irrelevant scFv. Data represent mean values with lines standard errors based on the definitions in three repetitions in the same experiment.

Figure 4 shows the efficiency of neutralization (% inhibition) in the case BAK278D6 (shaded squares) in the form of scFv against IL-13 in the analysis of cell proliferation, TF-1. Triangles indicate irrelevant scFv. Data represent mean values with lines standard errors based on the definitions in three repetitions in the same experiment.

On figa shown effective against 25 ng/ml IL-13 human.

On FIGU shown effective against 25 ng/ml variant IL-13 human.

On figs shown effective against 50 ng/ml IL-13 Primate other than humans.

Figure 5 shows a comparison of the effectiveness of anti-IL-13 antibodies of the man in the analysis of the proliferation of TF-1. Data represent average values of the efficiency of neutralization with lines standard errors on the basis of 5-7 experiments against 25 is g/ml IL-13 human. The relative efficiency of commercially available antibodies B-B13 evaluated statistically, through one way ANOVA using the criterion of Dunnet. *P<0,05, **P<0,01 compared to B-B13.

Figure 6 shows the efficiency of neutralization (% inhibition) in the case of BAK502G9 (shaded squares), BAK1167F2 (shaded triangles) and BAK1183H4 (shaded inverted triangles) in the form of a human IgG4 against the target of IL-13 in the analysis of cell proliferation, TF-1. Where there's no shading triangles mean irrelevant IgG4. Data represent mean values with lines standard errors based on three separate experiments.

On figa shown effective against 25 ng/ml IL-13 human.

On FIGU shown effective against 25 ng/ml variant IL-13 human.

On figs shown effective against 50 ng/ml IL-13 Primate other than humans.

7 shows the efficiency of neutralization (% inhibition) in the case of BAK502G9 (shaded squares), BAK1167F2 (shaded triangles), BAK1183H4 (shaded inverted triangles) in the form of a human IgG4 and commercial anti-IL-13 antibodies of man (B-B13 - where there's no shading squares; JES10-5A2 - where there's no shading inverted triangles) in the analysis dependent on the native IL-13 cell proliferation HDLM-2. Where there's no shading triangles mean irrelevant IgG4 Data represent mean values with lines standard errors based on the definitions in three repetitions in the same experiment.

On Fig shows a comparison of the effectiveness of anti-IL-13 antibodies of the man in the analysis NHLF. Data represent average values of the efficiency of neutralization (IC50PM) with lines standard errors on the basis of 4-5 experiments versus 10 ng/ml IL-13 human analysis release eotaxin NHLF. Efficiency in comparison with the commercially available antibody B-B13 evaluated statistically, through one way ANOVA using the criterion of Dunnet. *P<0,05, **P<0,01 compared to B-B13.

Figure 9 shows the efficiency of neutralization (% inhibition) in the case of BAK502G9 (shaded squares), BAK1167F2 (shaded triangles), BAK1183H4 (shaded inverted triangles) in the form of a human IgG4 against increasing regulation of VCAM-1 on the surface of HUVEC in response to 10 ng/ml IL-13 human. Where there's no shading triangles mean irrelevant IgG4. Data represent mean values with lines standard errors based on the definitions in three repetitions in the same experiment.

Figure 10 shows the efficiency of neutralization (% inhibition) in the case of BAK502G9 (shaded squares), BAK1167F2 (shaded triangles), BAK1183H4 (shaded inverted triangles) in the form of a human IgG4 against release eotaxin of HUVEC with increased regulation of VCAM-1 on the surface in response to either 1 ng/ml IL-4 (IgA), or 0.5 ng/ml IL-1β person (pigv). Where there's no shading triangles mean irrelevant IgG4. Data represent mean values with lines standard errors based on the definitions in three repetitions in the same experiment.

Figure 11 shows the efficiency of neutralization (% inhibition) in the case BAK209B11 (squares) in the form of a human IgG4 against 1 ng/ml murine IL-13 in the analysis dependent on factors proliferation of B9 cells. Where there's no shading triangles mean irrelevant IgG4. Data represent mean values with lines standard errors based on the definitions in three repetitions in the same experiment.

On Fig shows the relative level of IL-13 in lung homogenates from sensitized (s) (right column) and desensibilisation (ns) (left column) mice after provocation in a murine model of acute allergic inflammation of the lungs. The effect of sensitization was evaluated based on the t-student criterion, using quantitative data on IL-13. *<0,05, **<0,01 compared to insensitive control animals (n=5-6 mice). Data represent mean values with standard lines errors.

Fig illustrates the effects on/in the BAK209B11 in the form of a human IgG4 in different quantities compared with matched isotype IgG4 irrelevant control antibody on induzione the s-ovalbumin mobilization of leukocytes in the lungs of sensitized with ovalbumin mice. Shows the number of cells (×104). The treatment effect of the antibodies was evaluated statistically, through one way ANOVA using the criterion of Dunnet, using the data of the differential counting of cells. *<0,05, **<0,01 compared to the control animals, which were subjected to provocations by ovalbumin and was administered PBS (=0% inhibition; n=5-8 mice). Data represent mean values with standard lines errors.

Fig illustrates the effects on/in the BAK209B11 in the form of a human IgG4 in different quantities compared with matched isotype IgG4 irrelevant control antibody on ovalbumin-induced mobilization of eosinophils in the lungs of sensitized with ovalbumin mice. Shows the number of eosinophils (×104). The treatment effect of the antibodies was evaluated statistically, through one way ANOVA using the criterion of Dunnet, using the data of the differential counting of cells. *<0,05, **<0,01 compared to the control animals, which were subjected to provocations by ovalbumin and was administered PBS (=0% inhibition; n=5-8 mice). Data represent mean values with standard lines errors.

Fig illustrates the effects on/in the BAK209B11 in the form of a human IgG4 in different quantities compared with matched isotype IgG4 irrelevant control antibody is and ovalbumin-induced mobilization of neutrophils into the lungs of sensitized with ovalbumin mice. Shows the number of neutrophils (×104). The treatment effect of the antibodies was evaluated statistically, through one way ANOVA using the criterion of Dunnet, using the data of the differential counting of cells. *<0,05, **<0,01 compared to the control animals, which were subjected to provocations by ovalbumin and was administered PBS (=0% inhibition; n=5-8 mice). Data represent mean values with standard lines errors.

Fig illustrates the effects on/in the BAK209B11 in the form of a human IgG4 in different quantities compared with matched isotype IgG4 irrelevant control antibody on ovalbumin-induced mobilization of lymphocytes in the lungs of sensitized with ovalbumin mice. Induction of lymphocyte-dependent dose way inhibited BAK209B11 with maximum inhibition at 3 mg/ml BAK209B11. The treatment effect of the antibodies was evaluated statistically, through one way ANOVA using the criterion of Dunnet, using the data of the differential counting of cells. *<0,05, **<0,01 compared to the control animals, which were subjected to provocations by ovalbumin and was administered PBS (=0% inhibition; n=5-8 mice). Data represent mean values with standard lines errors.

Fig illustrates the effects on/in the BAK209B11 in the form of a human IgG4 in different quantities compared the structure with matched isotype IgG4 irrelevant control antibody on ovalbumin-induced mobilization of monocytes/macrophages in the lungs of sensitized with ovalbumin mice. No significant increase in levels of monocytes/macrophages in sensitized animals compared to control animals. However, such background levels of these cells was decreased when >36 μg/ml BAK209B11 in sensitized animals. The treatment effect of the antibodies was evaluated statistically, through one way ANOVA using the criterion of Dunnet, using the data of the differential counting of cells. *<0,05, **<0,01 compared to the control animals, which were subjected to provocations by ovalbumin and was administered PBS (=0% inhibition; n=5-8 mice). Data represent mean values with standard lines errors.

On Fig shows the influence of commercial neutralizing anti-IL-13 antibodies JES10-5A2 on the flow cell shows the number of cells (×104)) to the air bag mouse caused by the introduction obtained in bacteria, recombinant IL-13 human. The treatment effect of the antibodies was evaluated statistically, through one way ANOVA using the criterion of Dunnet, using the data of the differential counting of cells. *<0,05, **<0,01 compared to control animals CMC (=0% inhibition; n=11-13 mice). Data represent mean values with standard lines errors.

On Fig shows the sequence alignment of IL-13 macaques-Griboedov compared with the amino acid and the sequences of IL-13 human. Hatched seven amino acid residues that are different in IL-13 human and macaque-Griboedov. IL-13 rhesus and macaques-Griboedov had identical amino acid sequence.

Fig illustrates the effect of a single bolus dose of 10 mg/kg/BAK502G9 in the form of a human IgG4 to IgE levels in serum at 4 with allergies, but is not subjected to provocations primates macaques-Griboedov (2 males/2 females) for 29 days. The concentration of IgE in serum significantly decreased from 100% (prior to dose) to 66±10% of control values (p<0,05) on 4 and 5 day post dose. The aforementioned decrease in the concentration of IgE in the serum is recovered to 88±8% of control levels on day 22. *=p<0.05 compared with levels of IgE to dose, repeated measurement ANOVA with subsequent use of the criterion of multiple comparisons of Dunnet (n=4 animals).

On FIGU shows the relative levels of IgE in the serum of male and female primates macaques-Griboedov versus time after a single intravenous dose of 10 mg/kg BAK502G9. Relative data for IgE in serum is expressed as the arithmetic average ± SEM as a percentage of baseline values.

Fig illustrates the effect of intraperitoneal administration BAK209B11 in different quantities (H=237 mcg/day, M=23,7 mcg/day and L=2,37 g/day) compared with matched isotype IgG1 irrelevant control is the major antibody in the lungs of sensitized with ovalbumin and subjected to provocations mice. On figa pulmonary function presents log PC50(log concentration methacholine required to increase PenH initial level 50%) before any treatment (day 0) and after sensitization, provocation and processing of medicinal product (25 day). On figa shows the source data used to calculate the final result of the study shown in figv (difference log PC50). Data represent mean values with lines standard errors for n=8.

On FIGU change of lung function shown in the change log PC50individual mouse (difference log PC50). The difference of log PC50defined as the change log PC50individuals on the 25th day compared with 0 day. Data represent the average for the group difference log PC50(individual changes, averaged over the treatment groups) with lines of standard errors. The treatment effect of the antibodies was evaluated statistically, through one way ANOVA using the criterion of Dunnet, using data about the difference between log PC50. **<0,01 compared to sensitized with ovalbumin and subjected to provocations control animals (n=8 mice).

Fig illustrates the effect of local (i. po.) and the system (in/in) the introduction of BAK502G9 in the form of a human IgG4 in different quantities compared with matched isotype IgG4 irrelevant control the first antibody on the mobilization of total leukocytes (figa) and the mobilization of eosinophils (pigv) in the air pouch of mice BALB/C. Data represent mean values with lines the standard error for n=10. The treatment effect of the antibodies was evaluated statistically, through one way ANOVA using the criterion of Dunnet, using transformed as log data. *<0,05, **<0,01 compared to mice subjected to provocations huIL-13 (n=10).

Fig illustrates the effects in b/W the introduction of BAK502G9 in the form of a human IgG4 compared with matched isotype IgG4 irrelevant control antibody on the development of AHR after intratracheal injection of IL-13 human rights in the respiratory tract of mice. The treatment effect of the antibodies was evaluated statistically, through one way ANOVA using the criterion of Dunnet, using data for methacholine PC200. *<0,05, **<0,01 compared with positive control group with IL-13 human (n=6-8 mice). Data represent mean values with standard lines errors.

On Fig the efficiency of neutralization (% of maximum response) in the case of BAK502G9 (shaded squares) in the form of IgG4 against 30 ng/ml IL-13 in the analysis of IgE production by B-cells. Where there's no shading squares mean irrelevant IgG4. Data represent mean values with lines standard errors for six donors from separate experiments.

On Fig shows the influence of BAK502G9 on induced IL-13 induced strengthening of agony is that signaling Ca2+ in smooth muscle cells of the bronchi. The area under the curve (AUC) of the response to histamine in the form of signal Ca2+ was determined for each antibody +/- conditions pre-treatment IL-13. The combined data from three independent experiments are shown for irrelevant antibody CAT-001 (a) and BAK502G9 (b) in the form of differences in percent in AUC±SD with untreated cells (ns=not significant (p>0,05), *p<0,05, **p<0,01). The results were evaluated statistically using one-factor analysis of variance (ANOVA) and a posteriori criterion for multiple comparisons, Bonferroni.

On Fig shows the effect introduced in phase II BAK502G9.

On figa shows the effect on AHR, as measured by the change in area under the curve of the dose dependence of the response of histamine (n=14).

On FIGU shows the effect on AHR, as measured by the change PC30(n=18).

On figs shows the effect of premirovany antigen (n=20).

On fig.26D shows the effect of inflammation BAL (n=21).

On Fig shows the influence of BAK502G9 on induced IL-13 expression of CD23. Data represent the response at the percentage of IL-13 alone (100%) and expressed as mean ± SEM % of control for 6 separate experiments from 6 individual donors (three repetitions).

On Fig shows the influence of BAK502G9 and irrelevant IgG4 on induced IL-13 and/or IL-4 expression of CD23 in PBMC. The data presented in the form of answer in percentage by IL-4 alone (100%) and expressed in terms of the environments is its ± SEM % of control for 4 separate experiments from 4 individual donors (three repetitions).

On figa shows the influence of BAK502G9 products eotaxin-1 NHLF induced 48-hour cultivation in a medium containing IL-13/TNF-α/TGF-β1. Data are shown as the mean ± SEM of determinations in three repetitions for environments used in this study in order to induce a change in the shape of cells.

On FIGU shows the influence of BAK502G9 on the shape change of human eosinophils induced by the conditioned media at a dilution of 1:16. The point of the presented data indicate mean ± SEM % compared with the shape change in control environments for individual experiments from four separate donors.

On Fig shows the alignment of IL-13 human and murine IL13 marked with mutations that were introduced in the IL-13 person to get the first panel of chimeras IL-13. Four alpha-helix is highlighted by rectangles and marked loop 1 and loop 3. Received five chimeric proteins in which the spiral B, C and D and the loop 1 and loop 3 was replaced with the murine sequence. Received four additional chimeric protein and are numbered according to the amino acids in the pre-human protein (but not numbered multiple alignment presented above), in which arginine at position residue 30 (position 34 above) mutant, residues 33 and 34 (regulations 37 and 38 above) motivovany, residues 37 and 38 (VH) motivovany (the situation is s 41 and 42 above) and residues 40 and 41 (TQ) motivovany (position 44 and 45 above).

On Fig shows the alignment of IL-13 human and murine IL-13 with marked mutations that were introduced in the IL-13 person to receive the second panel of chimeras IL-13. Received six chimeras, in which the residue(kami) of the person replaced the murine residue(s) (highlighted by rectangles). Received the following four chimeric protein (numbering according to the provisions of amino acids in a pre-protein of human), in which the leucine at the position of residue 58 (62 in the figure above) mutated, leucine in position residue 119 (residue 123 above) mutated lysine in position 123 (remainder 127 above) mutated and arginine at position residue 127 (residue 132 above) mutated.

On Fig shown mutations made in IL-13 human. Mutations are indicated by dark gray, reduced the binding of BAK502G9, mutations are marked in light gray, did not modify the binding. Shows a linear sequence of pre-IL-13 human with mutant residues.

In various aspects and embodiments of the invention offers the subject invention according to the claims below.

The present invention relates to specific partners that bind IL-13, in particular IL-13 human and/or Primate, and/or IL-13 (R130Q) and IL-13 mouse. Preferred variants of the present invention are antibody molecules or antibody (e.g., IgG, such as IgG4), or fragm the options antibodies (for example, scFv, Fab, dAb). Offers antigennegative region of antibodies, which represent the domains VH and VL of the antibody. Within domains, VH and VL are offered complementarity determining region CDR, which can be represented in various frame fields, FR, to form domains VH or VL depending on the circumstances. Antigennegative area can consist of a VH domain and/or VL domain of the antibody.

Antigennegative plot can be obtained by placing the CDR in the frames of proteins that are not antibodies, such as fibronectin or cytochrome B, etc. [115, 116]. A detailed overview of frameworks for design of novel binding sites in proteins are shown in Nygren et al. [116]. Protein cages for mimetics of antibodies described in WO/0034784, where the inventors describe proteins (mimetics of antibodies)that contain the domain of fibronectin type III having at least one randomised loop. Suitable frame, which impart one or more CDRs, such as a set of HCDR may be represented by any representative of the domains of the superfamily of immunoglobulin genes.

Preferred variants of the present invention are variants, referred to in this description of "line BAK278D6". The line is determined with the following set of CDR sequences BAK278D6: HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2), HCDR3 (SEQ ID NO: 3), LCDR1 (SEQ ID NO: 4), LCDR2 (SQ ID NO: 5) and LCDR3 (SEQ ID NO: 6). In one aspect the present invention relates to a specific partner that binds IL-13 human containing antigennegative plot antibodies, which consists of a VH domain of the antibody is human and domain VL of human antibodies and which contains a set of CDRs in which the VH domain contains HCDR1, HCDR2 and HCDR3 domain and VL contains LCDR1, LCDR2 and LCDR3, where HCDR1 has amino acid sequence SEQ ID NO: 1, HCDR2 has amino acid sequence SEQ ID NO: 2, and HCDR3 has amino acid sequence SEQ ID NO: 3, LCDR1 has amino acid sequence SEQ ID NO: 4, LCDR2 has amino acid sequence SEQ ID NO: 5 and LCDR3 has amino acid sequence SEQ ID NO: 6; or in which a set of CDR contains one or two amino acid substitutions compared with a set of CDRs in which HCDR1 has amino acid sequence SEQ ID NO: 1, HCDR2 has amino acid sequence SEQ ID NO: 2, and HCDR3 has amino acid sequence SEQ ID NO: 3, LCDR1 has amino acid sequence SEQ ID NO: 4, LCDR2 has amino acid sequence SEQ ID NO: 5 and LCDR3 has amino acid sequence SEQ ID NO: 6.

A set of CDRs in which HCDR1 has amino acid sequence SEQ ID NO: 1, HCDR2 has amino acid sequence SEQ ID NO: 2, and HCDR3 has amino acid sequence SEQ ID NO: 3, LCDR1 has amino acid sequence SEQ ID NO: 4, LCDR2 them is no amino acid sequence of SEQ ID NO: 5 and LCDR3 has amino acid sequence SEQ ID NO: 6, in this description referred to as "CDR set BAK278D6". HCDR1, HCDR2 and HCDR3 in the set of CDR BAK278D6 called the "set of HCDR BAK278D6" and LCDR1, LCDR2 and LCDR3 in the set of CDR BAK278D6 called "set LCDR BAK278D6". Set CDR set CDR BAK278D6, a set of HCDR BAK278D6 or LCDR BAK278D6 or with one or two substitutions are called line BAK278D6.

As indicated in one aspect, the invention relates to the partner of the specific binding of IL-13 human containing antigennegative plot antibodies, which consists of a VH domain of the antibody is human and domain VL of human antibodies and which contains a set of CDR, the CDR set is a set of CDR BAK278D6 or set of CDRs containing one or two substitutions compared with a set of CDR BAK278D6.

In preferred embodiments, one or two substitutions are at one or two of the following residues in the CDRs of the VH domains and/or VL, numbered using the standard numbering according to Kabat [107].

31, 32, 34 in HCDR1

52, 52A, 53, 54, 56, 58, 60, 61, 62, 64, 65 in HCDR2

96, 97, 98, 99, 101 in HCDR3

26, 27, 28, 30, 31 in LCDR1

56 in LCDR2

95A, 97 in LCDR3

Preferred embodiments have two substitutions compared with a set of CDR BAK278D6, HCDR3 residue 99 and LCDR1 residue 27. Of these options the preferred options are replacing N by S at residue 99 in HCDR3 and/or replacement of N I in the residue 27 in LCDR1. Additional variants have replacement at residue 99 in HCDR3 selected from the group consisting of S, A, I, R, P and K, and/or a substitution at residue 27 LCDR1, selected from the group consisting of I, L, M, C, V, K, Y, F, R, T, S, A, H, and G.

In preferred embodiments, one or two substitutions carried out in one or two of the following residues in the CDR set BAK278D6 in accordance with known groups of residues possible substitutes:

Preferred options are set CDR BAK278D6 replacing N by S at position residue 99 in HCDR3 and replacing N by I in the position of residue 27 in LCDR1. Thus, characterized by a set of CDR represents the following set: HCDR1 is SEQ ID NO: 7; HCDR2 is SEQ ID NO: 8, HCDR3 is SEQ ID NO: 9; LCDR1 is SEQ ID NO: 10, LCDR2 is SEQ ID NO: 11; LCDR3 is SEQ ID NO: 12. The specified set of CDR in this description referred to as "CDR set BAK502G9".

These preferred options have a set of CDR BAK278D6 with one or two substitutions in the CDR provided that the excluded pair substitutions N by S at position residue 99 in HCDR3 and N I in the position of residue 27 in LCDR1.

Other preferred variants are the following.

BAK1166G2: HCDR1 is SEQ ID NO: 67, HCDR2 is SEQ ID NO: 68, HCDR3 is SEQ ID NO: 69, LCDR1 is SEQ ID NO: 70, LCDR2 is SEQ ID NO: 71; LCDR3 is SEQ ID NO: 72.

BAK1167F2: HCDR1 is SEQ ID NO: 61, HCDR2 is SEQ ID NO: 62, HCDR3 is SEQ ID NO: 63, LCDR1 is SEQ ID NO: 64, LCDR2 is SEQ ID NO: 65; LCDR3 is SEQ ID NO: 66.

BAK1184C8: HCDR1 is SEQ ID NO: 73, HCDR2: SEQ ID NO: 74, HCDR3 is SEQ ID NO: 75. LCDR1 is SEQ ID NO: 76, LCDR2 is SEQ ID NO: 77; LCDR3 is SEQ ID NO: 78.

BAK1185E1: HCDR1 is SEQ ID NO: 79, HCDR2 is SEQ ID NO: 80, HCDR3 is SEQ ID NO: 81. LCDR1 is SEQ ID NO: 82, LCDR2 is SEQ ID NO: 83; LCDR3 is SEQ ID NO: 84.

BAK1167F4: HCDR1 is SEQ ID NO: 85, HCDR2 is SEQ ID NO: 86, HCDR3 is SEQ ID NO: 87. LCDR1 IS SEQ ID NO: 88, LCDR2 IS SEQ ID NO: 9; LCDR3 is SEQ ID NO: 90.

BAK1111D10: HCDR1 is SEQ ID NO: 91, HCDR2 is SEQ ID NO: 92, HCDR3 is SEQ ID NO: 93. LCDR1 is SEQ ID NO: 94, LCDR2 is SEQ ID NO: 95; LCDR3 is SEQ ID NO: 96.

BAK1183H4: HCDR1 is SEQ ID NO: 97, HCDR2 is SEQ ID NO: 98, HCDR3 is SEQ ID NO: 99. LCDR1 is SEQ ID NO: 100, LCDR2 is SEQ ID NO: 101; LCDR3 is SEQ ID NO: 102.

BAK1185F8: HCDR1 is SEQ ID NO: 103, HCDR2 is SEQ ID NO: 104, HCDR3 is SEQ ID NO: 105. LCDR1 is SEQ ID NO: 106, LCDR2 is SEQ ID NO: 107; LCDR3 is SEQ ID NO: 108.

All these options are obtained from the BAK502G9 randomized CDR1 and CDR2 of the heavy chain and, therefore, are line BAK502G9.

Domain VH contains a set of CDRs HCDR1, HCDR2 and HCDR3 of any clone, which are listed in table 1. In this invention, table 1 also presents separately VL domain containing a set of CDRs LCDR1, LCDR2 and LCDR3 of the clones shown in table 1. Preferably specified VH domain is paired with the specified domain VL, and most preferably a pair of domains, VH and VL are the same as in the case of clones, listed in table 1.

In addition, the present invention offers a VH domain containing the set of CDRs HCDR1, HCDR2 and HCDR3, the CDR set corresponds to the set of any clone, shown in table 1, with one or two amino acid substitutions.

In addition, the present invention offers a VL domain that contains a set of CDRs LCDR1, LCDR2 and LCDR3, the CDR set corresponds to the set of any clone, shown in table 1, with one or two amino acid substitutions.

Also in the present invention serves a specific binding partner containing the anti-Christ. envasive domain antibodies, containing such VH and/or VL domain.

The authors of the present invention identified the line BAK278D6 as providing antigennegative domains of human antibodies against IL-13, which are particularly useful. In line identified BAK502G9 as particularly useful. Sets the CDR BAK278D6 and BAK502G9 already identified above.

Following the instructions in the field of computational chemistry on the application of methods of multivariate data analysis of the relationship between the structure/property-activity [94], it is possible to quantify the relationship between activity-property for antibodies using well known mathematical techniques such as statistical regression, recognition and identification of images [95-100]. Properties of antibodies can be obtained on the basis of empirical and theoretical models (for example, the analysis of likely contact residues or calculated physico-chemical property) sequence antibodies, while functional and three-dimensional patterns and these properties can be considered separately and in combination.

Antigennegative plot antibody comprising the VH domain and VL domain, formed by six loops of polypeptide: three of the variable domain of the light chain (VL) and three of the variable domain of the heavy chain (VH). Analysis of antibodies with known atomic structure clarified the relationship between the sequence and the three-dimensional structure of the binding sites of antibodies [101, 102]. These relationships show that, with the exception of the third region (loops) in the VH domains of a loop linking sites are one of a small number of main chain conformations: canonical structures. It is shown that the canonical structure formed in a particular loop is determined by its size and the presence of certain residues at key sites in the loop, and in areas of the skeleton [101, 102].

The study of the relationship between the sequence-structure can be used to predict those residues in the antibody of known sequence but unknown three-dimensional structure, which is important for maintaining the three-dimensional structure of the CDR loops and, therefore, retain the binding specificity. These predictions can be confirmed by comparing the predictions with the result given as examples of experiments for optimization.

In the case of a structural approach can be used to create the model of antibody molecules [103] with the use of readily available commercial software package, such as WAM [104]. You can then use the software package for visualization and analysis of protein, such as Insight II [105] or Deep View [106]to evaluate the possible substitutions at each position in the CDR. The information can then be used to obtain replacements, which, in the o f have a minimal or beneficial effect on activity.

The authors of the present invention analyzed the sequence data for a panel of clones, sets of CDRs are shown in table 1.

The analysis tested the hypothesis that any of the double combinations listed amino acid changes in the CDR of the presented set of variants of scFv leads to a variant scFv, at least with the initial effectiveness of the original scFv BAK278D6.

All variants of scFv on the panel, shown in table 1, were selected in relation to the high affinity, and it was confirmed that they exhibit higher efficiency.

The observed amino acid changes can be either favorable, unfavorable, or neutral in their effect on the initial efficiency of scFv BAK278D6 in the analysis of TF-1 at 44 nm.

Not observed a relationship between any two amino acid changes, which confirmed the absence of synergy as "positive"and "negative"between any two selected changes.

There are four scenarios in which this double combination satisfies the hypothesis, and three scenarios under which the hypothesis is illegal. Synergistic amino acid variants are not considered, as the connection was not observed.

The hypothesis is valid if:

A1: mutation 1 is blahop ethnoi and mutation 2 is favorable,

A2: mutation 1 is favorable, and mutation 2 is neutral,

A3: mutation 1 is neutral and mutation 2 is neutral,

A4: mutation 1 is favorable, and mutation 2 is negative (when the impact exceeds 1 impact 2).

The hypothesis is illegal if:

B1: mutation 1 is negative and mutation 2 is neutral,

B2: mutation 1 is unfavorable and mutation 2 is adverse,

B3: mutation 1 is favorable, and mutation 2 is negative (when the impact exceeds 2 impact 1).

To make it possible for A4, mutation 1 must be highly favorable to balance the negative impact of mutations 2 on efficiency. Since such highly favorable mutation may be present in the library of variants used for selection, it can be selected and therefore can often be found in the panel options. Because synergy can be excluded, this mutation may be useful in any context sequences and therefore must re-occur in different variants of scFv. An example of such frequent replacement of the amino acids is replaced in CDR1 light chain Asn27Ile. However, this mutation itself (clone BAK531E2) has only a modest 2-fold impact on eff is aktivnosti (end IC 50of 23.2 nm). By itself, this mutation could not provide a scenario depicted in A4, as it is a highly favorable mutation. This means that each clone in the presented set of binding IL-13 clones (table 1), which has a replacement Asn27Ile in CDR1 light chain together with one or more additional mutations, is at least as effective as variant having one mutation Asn27Ile in CDR1 light chain. Other mutations are either neutral or positive, but does not have a negative or harmful effect.

The following example is Asn99Ser in CDR3 of the heavy chain (see table 1). Because the clone carrying the specified specific change in one amino acid was not identified, the effectiveness of this clone was assessed as 12,0 nm for the following reason:

Efficiency BAK278D6 is 44 nm. Changes N27I CDR1 VL+N99S CDR3 VH leads to BAK502G9 with an efficiency of 8 nm, i.e. a 5.5-fold improvement.

Efficiency BAK278D6 is 44 nm. Change N27I CDR1 VL leads to BAK531E2 with an efficiency of 23 nm, i.e. to 1.9-fold improvement.

Efficiency BAK278D6 is 44 nm. Change N99S VH CDR3 makes possible the clone with the efficiency of 12.2 nm, i.e. a 2.9-fold improvement(5,5/1,9=2,9).

Double combination Asn99Ser CDR3 of the heavy chain with Asn27Ile CDR1 light chain gives scFv BAK0502G9 with an efficiency of 8 nm. As the synergy is excluded, it is, therefore, the contribution of replacement Asn99Ser in CDR3 of the heavy chain in the BAK502G9 is additive.

Therefore, each clone in the presented set of binding IL-13 clones (table 1), which has a replacement AsnH99Ser in CDR3 of the heavy chain together with one or more additional mutations must have efficiency, component, at least 12 nm or more, 2.5 times the permitted interval analysis for n=1-2.

Thus, the inventors have noted that highly favorable amino acid change that is possible it would be preferable to choose, is not observed. As discussed above, two changes, which are highlighted in table 1 variants of scFv was analyzed more thoroughly. Any variant scFv in table 1 with any of these mutations, together with one or more additional mutations have been effective, which was as superior as in the case of a clone containing any one of these two single amino acid changes in the source BAK278D6. Therefore, there is no evidence that highly favorable amino acid change, which could provide a scenario A4, present on the panel.

The specified observation has led the inventors to the conclusion that this set of scFv variants are not present unfavorable mutations. This means that h is about the scenario A4 and B1-B3 are not suitable and the hypothesis is valid.

Accordingly, as already noted, the present invention relates to specific binding partners containing specific sets of CDR, in particular the set of CDR BAK278D6, and sets the CDR line BAK278D6 with one or two substitutions in the CDR set, e.g. a set of CDRs BAK502G9.

Features suitable set of CDR in the framework regions of antibodies or other protein skeleton, such as fibronectin or cytochrome B [115, 116]. Preferably use a frame region of the antibodies, and if used, they preferably represent a frame region of the germ line, more preferably a frame region of the antibody heavy chain may be DP14 from the VH1 family. The preferred frame for light chain can be λ3-3H. For a set of CDR BAK502G9 preferably, the frame region of the antibody represented for VH FR1 SEQ ID NO: 27, for VH FR2 SEQ ID NO: 28, VH FR3 SEQ ID NO 29, light chain FR1 SEQ ID NO: 30, for FR2 light chain SEQ ID NO: 31, for the light chain FR3 SEQ ID NO: 32. In vysokoproizvoitelnoj variant VH domain with amino acid sequence SEQ ID NO: 15, it is called the "domain BAK502G9 VH". In the following vysokoproizvoitelnoj variant VL domain with amino acid sequence SEQ ID NO: 16, it is called the "domain BAK502G9 VL". Vysokoproizvoditelnykh antigennegative plot antibodies, proposed according to astasia the invention, consists of a VH domain BAK502G9, SEQ ID NO: 15, and domain BAK502G9 VL, SEQ ID NO: 16. Specified antigennegative plot antibodies can be provided in any desired form molecules such as scFv, Fab, IgG, IgG4, dAb, etc. that are additionally discussed in this specification.

In the following vysokoproizvoitelnoj embodiment, the present invention relates to a molecule antibodies IgG4 containing domain BAK502G9 VH, SEQ ID NO: 15, and domain BAK502G9 VL, SEQ ID NO: 16. The molecule is referred to in this description of "IgG4 BAK502G9".

In the present invention proposes another IgG4 or other molecules of the antibody containing a VH domain BAK502G9, SEQ ID NO: 15, and/or domain BAK502G9 VL, SEQ ID NO: 16, which represent the other molecules of the antibody containing a set of HCDR BAK502G9 (SEQ ID NO: 7, 8, and 9) in the VH domain of the antibody and/or a set of LCDR BAK502G9 (SEQ ID NO: 10, 11, and 12) in the VL domain of the antibody.

Should indicate that the "and/or" when used in this description should be taken as a specific description of each of the two specific characteristics or components together or separately. For example, "A and/or B" should be understood as a specific description of each option, (i) A (ii) B and (iii) A and B are the same as in the case when separately specified in this specification.

As indicated, the present invention relates to specific binding partner that binds IL-13 person and which contains the VH domain BAK502G9 (SEQ ID NO: 15) and/or VL BAK502G9 (SEQ ID NO: 16).

As a rule, consists of a VH domain with the domain VL to get antigennegative plot antibodies, although, as discussed later, can be used separately VH domain to bind the antigen. In one preferred embodiment, consists of a VH domain BAK502G9 (SEQ ID NO: 15) with the VL domain of BAK502G9 (SEQ ID NO: 16), to form antigennegative plot antibody containing the VH and VL domains BAK502G9. In other embodiments, form a couple of BAK502G9 VH c other VL domain that is different from the BAK502G9 VL. Obtaining a heterogeneous light chains are well developed in this area.

In this way any set of HCDR line BAK278D6 can be represented in the VH domain, which is used as a specific binding partner alone or in combination with VL domain. Can be obtained VH domain with a set of HCDR antibodies lines BAK278D6, for example, shown in table 1, and if such VH domain combined with a VL domain, VL domain can be obtained with a set of LCDR antibodies lines BAK278D6, for example, shown in table 1. The Union of a set of HCDR and set LCDR may be as shown in table 1, with the formation of antigennegative plot antibodies containing a set of CDRs as shown in table 1. Frame region of the VH domains and/or VL can be frames the germ line. Frame region domain of the heavy chain can be selected from the family of VH-1, and preferably the m frame VH-1 is the frame DP-14. Frame region of the light chain can be selected from the family of λ3, and therefore the preferred frame is λ3-3H.

One or more CDR can be taken from a VH domain or VL BAK502G9 and included in the appropriate frame. This is discussed further in this description. HCDR 1, 2 and 3 BAK502G9 shown in SEQ ID NO: 7, 8, and 9, respectively. LCDR 1, 2 and 3 BAK502G9 shown in SEQ ID NO: 10, 11, and 12, respectively.

The same applies for other CDR line BAK278D6 and CDR sets are shown in table 1.

The following embodiments of the invention relate to a specific binding partner that contains the VH domain and/or VL, or antigennegative plot containing the CDRs of the VH domain and/or VL of the antibody molecules, referred to in this description 167A11 (VH: SEQ ID NO: 23, and VL: SEQ ID NO: 24), and its derivatives 615E3 (VH: SEQ ID NO: 33 and a VL: SEQ ID NO: 34), BAK582F7 (VH CDR SEQ ID 141-143) and BAK612B5 (VH CDR SEQ ID 147-149). These partners recognize IL-13 human. Derivatives 167A11 obtained by randomization VH CDR3, are effective scFv molecules (5-6 nm). Line 167A11 can be used in any aspect and embodiment of the present invention, which are described in this description for other molecules, for example in the ways of the mutation engine and selection antigenspecific areas with increased efficiency.

Variants domains VH and VL and CDR according to the present invention, including variants, amino acid pic is egovernance which are shown in this description and which can be used in specific partners, binding of IL-13, can be obtained using the methods of modification or mutation engine sequences and screening. Such methods are also provided in the present invention.

Variants of the amino acid sequence of the variable domain of any of the domains VH and VL sequences which are specifically described in this description may be used according to the present invention, as discussed. Specific options may include one or more changes in amino acid sequence (accession, a deletion, substitution and/or insertion of amino acid balance, may be less than approximately 20 changes, less than approximately 15 changes, less than approximately 10 changes to or less than approximately 5 changes, 4, 3, 2 or 1. Changes can be made in one or more frame regions and/or one or more CDR.

According to further aspects of the present invention serves a specific binding partner, which competes for binding to the antigen with any specific binding partner that binds the antigen and contains a specific binding partner, VH and/or VL domain contained in this description or HCDR3 described herein, or a variant of any of these. Competition between binding partners can easily analyze in vitro, e.g. the using ELISA and/or by one binding partner specific reporter molecule, which can detect the presence of another unlabeled binding partner(ROS)to provide the ability to identify specific binding partners that bind the same epitope or an overlapping epitope.

Thus, another aspect of the present invention relates to specific binding partner containing antigennegative plot of human antibodies, which competes with the antibody molecule BAK502G9, in particular scFv and/or IgG4 BAK502G9, in relation to binding to IL-13. The following aspects of the present invention relates to specific binding partner containing antigennegative plot of human antibodies, which competes with antigennegative plot antibodies for binding to IL-13, while antigennegative plot antibody consists of a VH domain and VL domain, and domains VH and VL contain a set of CDR line BAK278D6.

In this area there are various methods of obtaining antibodies against IL-13, and these antibodies can compete with antibody molecule BAK502G9, the antibody molecule with a set of CDR BAK502G9 or antibody molecule with a set of CDR line BAK278D6 for binding to IL-13.

In the following aspect the present invention relates to a method for producing one or more specific binding partners capable of binding antigen, the method which engages in the implementation of contact library specific binding partners according to the invention and the specified antigen and selection of one or more specific binding partners from the library, able to bind the specified antigen.

The library can be presented on the surface of bacteriophage particles, with each particle contains nucleic acid encoding a variable domain VH antibody presented on its surface, and optionally also presents VL domain, if present.

After selection of the specific binding partners capable of binding the antigen and presents on the particles of the bacteriophage, bacteriophage particles representing the specified selected specific binding partner, can be taken nucleic acid. Such nucleic acid may be used in subsequent obtaining specific binding partner or variable domain VH antibody (optional variable domain VL of the antibody) by the expression of the nucleic acid sequence of nucleic acid taken from a bacteriophage particles representing the specified selected specific binding partner.

Variable domain VH of the antibody with the amino acid sequence of the variable domain VH antibody specified selected specific binding partner can be obtained in isolated form, as well as in the form of a specific binding partner containing such VH domain. Can then be tested the ability of the bind IL-13, and the ability to compete with the BAK502G9 (for example, in the form of scFv and/or in the form of IgG, for example, IgG4) for binding to IL-13. Can be tested by the ability to neutralize IL-13, as discussed later.

Specific binding partner according to the present invention can bind IL-13 with an affinity molecule antibodies BAK502G9, such as scFv or preferably IgG4 BAK502G9, or with a higher affinity.

Specific binding partner according to the present invention can neutralize IL-13 with the efficiency of antibody molecules BAK502G9, such as scFv or preferably IgG4 BAK502G9, or with higher efficiency.

Specific binding partner according to the present invention can neutralize the naturally occurring IL-13 with the efficiency of antibody molecules BAK502G9, e.g. scFv, or preferably IgG4 BAK502G9, or with higher efficiency.

The affinity of binding and neutralization efficiency of different specific binding partners can be compared in suitable conditions.

Antibodies according to the present invention have several advantages compared to existing commercial anti-IL-13-antibodies, in particular three commercial rodent antibodies against IL-13 human, namely JES10-5A2 (BioSource), B-B13 (Euroclone) and clone 321166 (R and D Systems). The effectiveness of the antibodies according to the present invented the Yu was compared with commercial antibodies JES10-A2 and B-B13. Clone 321166 not evaluated, because preliminary experiments showed that this clone was significantly less effective than known commercial antibodies.

The effectiveness and application of commercial IL-13 antibodies rodents to humans, likely to be limited due to the increased ability to induce immunogenic responses and, consequently, more rapid clearance from the body. Kinetic analysis of the antibodies according to the present invention in primates other than humans, suggests that these antibodies possess a speed of clearance, which is similar to the rate of clearance of other known human or humanized antibodies.

Antibodies are offered in different variants of implementation of the present invention, recognize IL-13 primates other than man, including IL-13 rhesus and macaques-Griboedov. Defining profiles of efficacy and safety antibodies in primates other than humans, it is extremely important, as it provides the ability to predict the safety, pharmacokinetic and pharmacodynamic profile of antibodies in humans.

In addition, the antibodies according to different variants of implementation of the present invention additionally recognize the variant IL-13 human R130Q, which is associated with asthma. Cross-reactivity with the variant IL-13 allows the use of antibodies is in accordance with the present invention and compositions containing antibodies according to the present invention, for the treatment of patients with IL-13 wild-type and variant IL-13.

The preferred implementation of the present invention includes antibodies that neutralize naturally occurring IL-13 with an efficiency that is equal to or exceeds the efficiency of binding the antigen IL-13 section formed by domain BAK502G9 VH (SEQ ID NO: 15) and domain BAK502G9 VL (SEQ ID NO: 16). For example, the inventors have shown that the typical clones, such as BAK502G9, 1167F2 and N, are significantly more effective against naturally occurring IL-13 than known commercial antibodies (Fig.7).

In addition to the sequences of the antibodies, specific binding partner according to the present invention may contain other amino acids, e.g. forming a peptide or polypeptide, such as subjected to the folding of the domain, or giving the molecule another functional property in addition to ability to bind antigen. Specific binding partners according to the invention can carry the registered label or can be conjugated with a toxin or guides to the target residue, or an enzyme (e.g., via a peptide bond or a linker).

The following aspects the invention relates to an isolated nucleic acid that contains the successor of the awn, encoding a specific binding partner, VH domain and/or VL domains according to the present invention, and to methods for specific binding partner, VH domain and/or VL domain according to the invention, which include the expression of specified nucleic acid under conditions that lead to the receipt of the indicated specific binding partner, VH domain and/or VL domain, and retrieve it.

Specific binding partners according to the invention can be used in the treatment or diagnosis of human or animal, such as a method of treatment (which may include preventative treatment) of a disease or disorder in a patient person, which includes the introduction of a specified patient an effective amount of a specific binding partner according to the invention. Condition amenable to treatment according to the present invention, include any condition in which IL-13 plays a role, in particular asthma, atopic dermatitis, allergic rhinitis, fibrosis, chronic obstructive pulmonary disease, scleroderma, inflammatory bowel disease and Hodgkin's lymphoma. In addition, the antibodies according to the present invention can also be used in the treatment of tumors and viral infections, as these antibodies will be Engibarov is to be mediated by IL-13 immunosuppression [64, 65].

Another aspect of the present invention relates to a nucleic acid, generally isolated, encoding a variable domain VH and/or variable domain VL of the antibodies described in this specification.

Another aspect of the present invention relates to a nucleic acid, generally isolated, encoding described in this description of the sequence of CDR of VH or VL CDR, in particular VH CDR selected from SEQ ID NO: 7, 8, and 9, or VL CDR selected from SEQ ID NO: 10, 11 and 12, most preferably CDR3 BAK502G9 VH (SEQ ID NO: 9). Also, the present invention features nucleic acid encoding the CDR set BAK502G9, nucleic acid encoding a set of HCDR BAK502G9, and nucleic acid encoding a set of LCDR BAK502G9, which are nucleic acids encoding a separate CDR, HCDR, LCDR and CDR sets, HCDR, LCDR line BAK278D6.

Another aspect relates to the cell host transformed by a nucleic acid according to the invention.

Another aspect relates to a method for receiving variable domain VH antibody, the method is to induce the expression of the coding nucleic acid. This method may consist in the cultivation of host cells under certain conditions, to obtain the specified variable domain VH antibody.

Also offered similar ways of getting vari is belnyj domains VL and specific binding partners, containing VH and/or VL domain, as the following aspects of the present invention.

The method of obtaining may include the extraction and/or purification of the product.

The method of obtaining may be the cooking product in the form of a composition containing at least one additional component, such as a pharmaceutically acceptable excipient.

These and other aspects of the invention are described in more detail below.

TERMINOLOGY

Specific binding partner

This term describes a member of a pair of molecules which have binding specificity with the other member of the pair. Partners in specific binding pair can be of natural origin or fully or partially obtained by synthesis. One partner in the pair of molecules has an area on its surface or recess, which is specific bound and, therefore, is complementary to a particular spatial and polar organization of another member of the pair of molecules. Thus, the members of the pair have the property specific to communicate with each other. Examples of types of specific binding pairs are antigen-antibody, Biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The present invention relates to reactions of the type a antigen-antibody.

Molecule antibodies

This term describes an immunoglobulin, either natural or produced wholly or partly by means of a synthesis. The term also covers any polypeptide or protein containing the binding domain of the antibody. Antibody fragments that contain antigennegative domain, represent such molecules as Fab, scFv, Fv, dAb, Fd, and dianthicola.

You can get monoclonal and other antibodies and to use the methods of recombinant DNA technology to get other antibodies or chimeric molecules which retain the specificity of the original antibody. Such methods can involve the introduction of DNA encoding the variable region of the immunoglobulin or complementarity determining region (CDR) of the antibody constant region or constant region plus frame the scope of another immunoglobulin. See, for example, EP-A-184187, GB2188638A or EP-A-239400, and a large number of later literature. Hybridoma or other cell producing the antibody may be subject to genetic mutation or other changes that can alter or not to alter the binding specificity of the produced antibodies.

As antibodies can be modified in a number of ways, the term "antibody molecule" should be regarded as covering any specific binding partner or the substance with teensnaturalway domain antibodies of the desired specificity. Thus, the term encompasses fragments and derivatives of antibodies, including any polypeptide containing the binding domain of an immunoglobulin, either natural or wholly or partially obtained through synthesis. Therefore enabled chimeric molecule containing the binding domain of the immunoglobulin or equivalent, fused to another polypeptide. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and in a large number of later literature.

The following methods available in the design area antibodies, has made possible the allocation of human and humanized antibodies. For example, hybridoma person can be obtained, as described in Kontermann et al. [107]. Phage display, the other developed a way to create specific binding partners are described in detail in many publications, such as Kontermann et al. [107] and WO 92/01047 (discussed below). Transgenic mice in which the genes of mouse antibodies inactivated and functionally replaced by the genes of antibodies of a person and that left intact the other components of the immune system of mice, can be used for the selection of human antibodies to antigens [108].

Synthetic molecules antibodies can be created through gene expression formed from oligonucleotides synthesized and collected in suitable is xpressway vectors, for example, as described in Knappik et al. J. Mol. Biol.(2000) 296, 57-86 or Krebs et al. Journal of Immunological Methods 254, 2001, 67-84.

It is shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL domains, VH, CL and CH1; (ii) the Fd fragment consisting of the VH domains and CH1; (iii) an Fv fragment consisting of domains VL and VH of a single antibody; (iv) the dAb fragment (Ward, E.S. et al., Nature 341, 544-546 (1989), McCafferty et al. (1990) Nature, 348, 552-554), which consists of a VH domain; (v) isolated CDR field; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv), in which the VH domain and a VL domain are linked by peptide linker which allows the two domains to associate with education antigennegative site (Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single-chain Fv dimers (PCT/US 92/09965) and (ix) "dianthicola", multivalent or polyspecific fragments constructed by gene fusion (WO 94/13804; P.Holliger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993). Molecules, Fv, scFv or dianthicola can be stabilized by the introduction of binding domains VH and VL disulfide bridges (Y.Reiter et al., Nature Biotech, 14, 1239-1245, 1996). Can also be obtained mini-antibodies containing scFv associated with the CH3 domain (S.Hu et al., Cancer Res., 56, 3055-3061, 1996).

In that case, when using bispecific and is tetela, they can be a common bispecific antibodies that can be created in a variety of ways (Holliger, P.and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)), for example obtained by chemical means or from hybrid hybridomas, or can be any of bispecific fragments of the antibodies listed above. Examples bispecific antibodies include antibodies obtained using techniques BiTE™, which can be used binding domains of the two antibodies with different specificity, and directly linked short flexible peptide. Thus unite the two antibodies in one short polypeptide chains. Dianthicola and scFv can be constructed without the Fc region using only the variable domains, which potentially reduces the impact of anti-idiotypical reaction.

Bispecific dianthicola, unlike bispecific whole antibodies, can also be particularly useful because they are easy to construct and Express in E. coli. Dianthicola (and many other polypeptides, such as antibody fragments with appropriate specificnosti binding can easily be selected using phage display technique (WO 94/13804) from libraries. If one shoulder dianthicola need to keep a constant, for example, with a specificity directed against IL-13, can be created in the library cat who swarm the other shoulder varies, and choose the appropriate antibody specificity. Bispecific whole antibodies can be obtained by the designing method "knobs-into-holes" ("head-into the groove" (J.B.B.Ridgeway et al., Protein Eng., 9, 616-621, 1996).

Antigennegative domain

The term describes the portion of the molecule antibodies, which contains a region that is specific bound and is complementary to part or all of the antigen. In the case when the major antigen, the antibody can bind only to a particular part of the antigen, and the specified part is called the epitope. Antigennegative domain may be due to one or more variable domains of antibodies (e.g., the so-called Fd-fragment antibodies comprising the VH domain). Preferably antigennegative domain contains a variable region light chain antibody (VL) and variable region heavy chain antibodies (VH).

Specific

The term may be used, in relation to the situation in which one member of a specific binding pair will not show any significant binding to other molecules other than its specific partner(ROS) in the binding. The term is also applicable, for example, in the case when antigennegative domain is specific to a particular epitope, which have a number of antigens, and in this case, the specification is static binding partner, bearing antigennegative domain, will be able to communicate with various antigens carrying the epitope.

Contain

The term is usually used in the sense include, i.e. in the sense of allowing for the presence of one or more symptoms or features.

Insulated

The term refers to the condition in which, according to the present invention will be specific binding partners according to the invention or nucleic acid encoding such binding partners. Insulated partners and isolated nucleic acid will be free or essentially will not contain substances with which they are associated in the natural environment, such as other polypeptides or nucleic acids with which they occur in their natural environment or in the environment in which they are received (e.g. cell culture)when such preparation is getting in practice on the basis of recombinant DNA in vitro or in vivo. Partners and nucleic acid can be prepared with diluents or adjuvants and still be isolated for practical purposes - for example, partners will generally be mixed with gelatin or other carriers when used to coat the tablets for micrometrology for use of immunoassays, or will be mixed with pharmaceutically acceptable the mi carriers or diluents when used in diagnosis or therapy. Specific binding partners may be glycosylated, either naturally or by systems of heterologous eukaryotic cells (e.g. cells CHO or NSO (ECACC 85110503), or they can be (for example, in the case of receiving the result of the expression in a prokaryotic cell) deglycosylation.

Naturally occurring IL-13

The term usually refers to the condition in which it can meet the protein IL-13 or its fragments. Naturally occurring IL-13 means the protein IL-13, which in natural conditions is produced by the cell without the prior introduction of the coding nucleic acid using recombinant technology. Thus, naturally occurring IL-13 can be produced in vivo, for example T-cells CD4+, and/or may be isolated from a mammal, such as human, Primate, non-human, a rodent such as a rat or mouse.

Recombinant IL-13

The term refers to the condition in which it can meet the protein IL-13 or its fragments. Recombinant IL-13 means the protein IL-13 or its fragments, obtained using recombinant DNA in a heterologous host. Recombinant IL-13 may differ from the naturally occurring IL-13 glycosylation.

Recombinant proteins expressed in prokaryotic bacterial the systems of expression, not glycosylases, whereas proteins expressed in eukaryotic systems, such as mammalian cells or insects, are glycosylated. However, proteins expressed in insect cells, differ in glycosylation of proteins expressed in mammalian cells.

By "essentially the same specified" means that suitable CDRs or VH or VL domain according to the invention will be either identical or highly similar to specific areas of the sequence that are listed in this description. The term "highly similar" means that from 1 to 5, preferably from 1 to 4, for example from 1 to 3, or 1, or 2, or 3, or 4 amino acid substitutions may be made in the CDRs and/or VH or VL domain.

Structure carrying a CDR or set of CDRs according to the invention, typically, will be a sequence of the heavy chain or light chain of the antibody or its main part, in which the CDR or set of CDRs is localized at the position corresponding to the CDR or set of CDRs of naturally occurring variable domains of antibody VH and VL encoded subjected rearrangeable genes of immunoglobulins. The structure and position of the variable domains of immunoglobulins can be obtained for publication (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 187, and its updated version, currently available on the Internet (http://immuno.bme.nwu.edu or search "Kabat" using any search engine).

The media CDR can also be other frameworks, such as fibronectin or cytochrome B [115, 116].

Preferably the amino acid sequence of CDR, essentially such, as specified in this description, in the form of CDR carries a variable domain of a person or its bulk. The HCDR3 sequence, essentially those that are listed in this description represent preferred embodiments of the present invention, and preferably, each of these sequences as HCDR3 was in the variable domain of the heavy chain of a human or its main part.

The variable domains used in the invention can be obtained from any variable domain of the germline human or variable domain of the person subjected to rearrangeable, or can be a synthetic variable domain, based on the consensus sequences of known variable domains of a human. The sequence of the CDR according to the invention (e.g., CDR3) may be introduced into a repertoire of variable domains lacking a CDR (e.g., CDR3), using techniques of recombinant DNA.

For example, Marks et al. (io/Technology, 1992, 10: 779-783) describe methods of producing repertoires of variable domains of antibodies, using consensus primers directed or located close to the 5'-end region of the variable domain, together with consensus primers to the third framework region of VH genes of a person to receive a repertoire of variable domains VH, in which there is no CDR3. Marks et al., in addition, describe how you can combine this repertoire CDR3-specific antibodies. Using a similar method, you can shuffle derived from the CDR3 sequences according to the present invention with repertoires of VH or VL domains lacking CDR3, and subjected to shuffling a full domains VH or VL can be combined with a cognate VL or VH domain to obtain specific binding partners according to the invention. Then, the repertoire can be represented in a suitable system of the host, such as a system of phage display according to WO 92/01047 or under any of the many, many later literature, including Kay, B.K., Winter, J. and McCafferty, J. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, San Diego: Academic Press, in order to choose a suitable specific binding partners. The repertoire may consist of any number from 104individual partners and higher, for example from 106-108 or 1010partners. Other suitable system hosts include yeast display, bacterial display, the display T7, ribosomal display, and the like. The review on ribosomal display, see Lowe and D. L. Jermutus, 2004, Curr. Pharm, Biotech, 517-27, and WO 92/01047.

Similar ways of shuffling or combinatorial methods described by Stemmer (Nature, 1994, 370: 389-391), which describes the way in connection with the gene of β-lactamase, but notes that the method can be used to generate antibodies.

The next alternative is the creation of new areas of VH or VL, bearing obtained from the CDR sequences according to the invention, using random mutagenesis of one or more selected genes of the VH and/or VL, in order to create mutations in the complete variable domain. This method is described by Gram et al. (1992, Proc. Natl. Acad. Sci., USA, 89: 3576-3580), who used error-prone PCR. In preferred embodiments, get one or two amino acid substitutions in the set of HCDR and/or LCDR.

Another way that you can use, is directed mutagenesis in the CDR regions of genes of VH or VL. Such methods are described in Barbas et al.,(1994, Proc. Natl. Acad. Sci., USA, 91: 3809-3813) and Schier et al. (1996, J. Mol. Biol. 263: 551-567).

All of the above methods as such are known in this field and of themselves are not part of the present invention. The person skilled in the art will note the thread such ways, to obtain specific binding partners according to the invention, using conventional in this field method.

Another aspect of the invention relates to a method for antigennegative domain antibodies that are specific against the antigen IL-13, the method includes receiving by attaching, deletions, substitutions or insertions of one or several amino acids in the amino acid sequence of the VH domain that is specified in this specification, VH domain, which is a variant amino acid sequence of the VH domain, optionally combining the thus obtained VH domain with one or more VL domains, and testing the VH domain or a combination or combinations of VH/VL to identify specific binding partner or antigennegative domain antibodies, specific for against the antigen IL-13, and optionally having one or more preferred properties, preferably the ability to neutralize the activity of IL-13. Specified VL domain may have an amino acid sequence that is essentially the same as specified in this description.

You can apply a similar method, in which one or more variants of the sequence of the VL domain listed in this description, together with one or more of the VH domains.

In a preferred embodiment, the domain BAK502G9 VH (SEQ ID NO: 15) may be subjected to mutation, to obtain one or more variants of the amino acid sequence of the VH domain and/or the BAK502G9 VL (SEQ ID NO: 16).

Another aspect of the invention relates to a method for obtaining a specific binding partner that is specific to the antigen IL-13, and this method includes:

(a) receiving the original repertoire of nucleic acids encoding the VH domain, which either contains CDR3, which need to be replaced, or does not contain a region that encodes a CDR3;

(b) the Union of the specified repertoire with donor nucleic acid that encodes the amino acid sequence essentially one that is specified in this specification, VH CDR3, so as to embed the specified donor nucleic acid in the CDR3 region in the repertoire, in order to obtain a product repertoire of nucleic acids encoding VH domain;

(c) expression of nucleic acids specified the resulting repertoire;

(d) selecting a specific binding partner that is specific to IL-13; and

(e) removing the indicated specific binding partner or coding him nucleic acids.

In addition, you can use a similar method, in which CDR3 VL according to the invention combined with a repertoire Amu is inovah acids, encoding the VL domain, which either contains CDR3, which must be replaced, or does not contain a region CDR3 encoding.

Similarly one, or several, or all three CDRs may be grafted into a repertoire of VH domains, or VL, which are then subjected to screening for specific binding partner, or specific binding partners, it is specific to IL-13.

In the preferred embodiment, it is possible to use one or more HCDR1 (SEQ ID NO: 7), HCDR2 (SEQ ID NO: 8) and HCDR3 (SEQ ID NO: 9) BAK502G9 or set of HCDR BAK502G9 and/or one or more LCDR1 (SEQ ID NO: 10), LCDR2 (SEQ ID NO: 11) BAK502G9 or set LCDR BAK502G9.

The main part of the variable domain of the immunoglobulin will contain at least three areas CDR together with located between the frame areas. Preferably the part will also contain at least about 50% of one of the two or both the first and fourth frame areas, while 50% are C-terminal 50% of the first frame region and the N-terminal 50% of the fourth frame area. Additional residues at the N-end or C-end of the main part of the variable domain may be the remains, usually not associated with naturally occurring regions of the variable domain. For example, the construction of specific binding partners according to the present invention, carried out by methods based on the e recombinant DNA, may lead to the introduction of N - or C-terminal residues encoded by linkers introduced to facilitate cloning or other processing stage. Other processing stages include the introduction of linkers to join variable domains according to the invention with additional protein sequences, including the heavy chain of the immunoglobulin, other variable domains (for example, when receiving dential) or protein labels, which are discussed more fully in another part of this description.

Although in a preferred aspect of the invention preferred are specific binding partners containing a pair of domains, VH and VL, single binding domains based on sequence domain or VH or VL, form the following aspects of the invention. It is known that the individual domains of immunoglobulin, in particular the VH domains, capable of specific way to bind the antigens of the target.

In case any of the specific binding domains, these domains can be used for screening for complementary domains capable of forming two domains specific binding partner capable of binding IL-13.

This can be done by methods of screening based on the phage display technique using a so-called hierarchical dual is th combinatorial way, which is described in WO 92/01047, in which an individual colony containing the clone of either H-or L-chain, used for infection, a complete library of clones encoding the other chain (L or H), and the resulting double-stranded specific binding partner is selected according to the method based on the phage display technique, such as the methods described in this publication. This method is also described in Marks et al., ibid.

Specific binding partners according to the present invention, in addition, may contain a constant region of the antibodies or portions thereof. For example, domain, VL domain may be linked at its C-end with the constant regions of the light chain of the antibody, including Cκ or Cλ-chain person, preferably Cλ-chain. Similarly, specific binding partner, based on a VH domain may be linked at its C-end full or part (for example, domain (CH1) of the heavy chain of immunoglobulin derived from antibodies of any isotype, e.g. IgG, IgA, IgE and IgM and any subclass isotypes, particularly IgG1 and IgG4. Preferred is IgG4. IgG4 is preferred because it does not bind complement and does not generate effector functions. Any synthetic or other variant constant region that has the specified properties and stabilizes the variable regions, is also preferred for p is imeneniya in embodiments implementing the present invention.

Specific binding partners according to the invention can be marked by registered or functional label. Registered labels include radioactive labels, such as131I or99Tc, which may be associated with the antibody according to the invention using conventional chemistry known in the field of visualization of antibodies. Labels also include enzyme labels, such as horseradish peroxidase. Labels also include chemical residues, such as Biotin, which can be registered by binding with specific related recorded balance, for example labeled Avidya.

Specific binding partners according to the present invention designed for use in methods of diagnosis or treatment of a human or animal, preferably human.

Accordingly, the following aspects of the invention relate to methods of treatment, including the introduction of the proposed specific binding partner, pharmaceutical compositions containing such specific binding partner, and to the use of such a specific binding partner for the manufacture of a medicinal product for administration, for example, in the method of obtaining the drug or pharmaceutical composition, comprising preparing the composition of the JV is citiescape binding partner with a pharmaceutically acceptable excipient.

Clinical indications in which you can apply anti-IL-13 antibody, to obtain a therapeutic effect, include asthma, atopic dermatitis, allergic rhinitis, fibrosis, chronic obstructive pulmonary disease, inflammatory bowel disease, scleroderma, and Hodgkin's lymphoma. As already noted, treatment with anti-IL-13 antibodies is effective in the case of all these diseases.

Anti-IL-13 treatment can be done orally, by injection (e.g. subcutaneously, intravenously, intraperitoneally or intramuscularly), by inhalation, or topically (for example, intraocular introduction, intranasal, rectal, into wounds, on skin). The route of administration can be determined on the basis of physico-chemical parameters of treatment, whereas the disease or requirements optimize effectiveness and minimize side effects.

It is assumed that anti-IL-13 treatment will not be limited to application in the clinic. Therefore, it is also preferred subcutaneous injection using a needleless device.

Combined treatment can be used to obtain significant synergistic effects, in particular the combination of the specific binding partner against IL-13 with one or more other drugs. Specific binding partner solenostoma invention can be used in combination or in addition to the fast-acting or long-acting beta-agonists, corticosteroids, kromoglikatom, leukotriene antagonists (receptor), methylxanthines and their derivatives, inhibitors of IL-4, antagonists of muscarinic receptors, inhibitors of IgE, histamine inhibitors, inhibitors of IL-5, inhibitors eotaxin/CCR3, PDE4 inhibitors, antagonists of TGF-beta, interferon-gamma, pirfenidone, chemotherapeutic drugs and immunotherapy agents.

Combination therapy with one or more fast-acting or long-acting beta-agonists, corticosteroids, kromoglikatom, leukotriene antagonists (receptor), xantina, inhibitors of IgE, inhibitors of IL-4, inhibitors of IL-5, inhibitors eotaxin/CCR3, PDE4 inhibitors can be used to treat asthma. Antibodies according to the present invention can also be used in combination with corticosteroids, antimetabolites, antagonists of TGF-beta and the underlying path of signal transmission for the treatment of fibrosis. Combination therapy of these antibodies with PDE4 inhibitors, xantina and their derivatives, antagonists of muscarinic receptors, fast and long acting beta-antagonists may be applicable for the treatment of chronic obstructive pulmonary disease. Similar reasoning regarding combining acceptable in the case of using anti-IL-13 treatment of atopic dermatitis is, allergic rhinitis, chronic obstructive pulmonary disease, inflammatory bowel disease, scleroderma and Hodgkin's lymphoma.

According to the present invention, the proposed composition can be administered to the people. The introduction is preferably carried out in a "therapeutically effective amount", which is sufficient for the manifestation of the useful life for the patient's actions. This beneficial effect may be at least weakening, at least one symptom. Real input number and speed and the time course of the introduction will depend on the nature and severity of the condition being treated. The purpose of treatment, e.g. decisions regarding dose, etc. is the responsibility of therapists and other doctors. Suitable doses of antibodies are well known in the art; see Ledermann J.A. et al. (1991) Int. J. Cancer 47: 659-664; K.D. Bagshawe et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922.

The exact dose will depend on a number of factors, including the fact whether the antibody for diagnosis or treatment, the size and position of the area being treated, the precise nature of the antibody (e.g., a whole antibody, fragment or dianthicola) and the nature of any registered mark or other molecules associated with the antibody. The typical dose of the antibody is in the range from 100 μg to 1 g for system applications and is 1 μg to 1 mg for local applications. Typically, the antibody will be a whole antibody, preferably IgG4 isotype. Specified is a single dose for the treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other forms of antibodies in proportion to molecular weight. The treatment can be repeated daily, twice weekly, weekly, or monthly intervals according to the prescription of the attending physician. In preferred embodiments of implementing the present invention, the treatment is periodic, and the period between the introduction is about two weeks or more, preferably about three weeks or more, more preferably about four weeks or more, or about once a month.

Specific binding partners according to the present invention, typically, will be introduced in the form of pharmaceutical compositions, which may contain at least one component, in addition to the specific binding partner.

Such pharmaceutical compositions according to the present invention and for use according to the present invention may contain, besides the active ingredient, pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to experts in this field Such substances should be non-toxic and should not hinder the manifestation of the effectiveness of the active ingredient. The specific nature of the media or other substance will depend on the route of administration, which may be oral or by injection, such as intravenous.

Pharmaceutical compositions for oral administration can be in the form of tablets, capsules, powder or liquid. The tablet may contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions typically contain a liquid carrier such as water, oil, animal and vegetable oils, mineral oil or synthetic oil. Can be included physiological saline solution, dextrose or other saccharide, or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol.

For intravenous injection, or injection at the site of lesion active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Professionals related to this field can easily prepare suitable solutions using, for example, isotonic fillers, such as sodium chloride solution for injection, ringer's solution for injection, ringer's solution on the basis of lactate for injection. Can include preservatives, stabilizers, buffers, antioxidants and/or other additives that are required.

The composition may be introduced separately or in combination with other therapeutic means, either simultaneously or sequentially, depending on the condition being treated.

Specific binding partners according to the present invention can be prepared in liquid or solid form, depending on the physicochemical properties of the molecule and the delivery path. The preparations can contain excipients, or a combination of excipients, such as sugars, amino acids and surfactants. Liquid preparations may contain the antibody in a wide range of concentrations and to have a wide range of pH. Solid preparations can be obtained, for example, by lyophilization, spray drying or drying using supercritical fluid technology. Drugs anti-IL-13 will depend upon the intended route of delivery: for example, drugs for pulmonary delivery may consist of particles with physical properties that provide deep penetration into the lungs during inhalation, topical preparations may contain a viscosity modifier agents that prolong the time during which the drug is at the site of action.

The present invention relates to a method of causing or allowing binding of a specific binding p is rtner, proposed in the present invention, with IL-13. As noted, such binding may take place in vivo, for example, after the introduction of a specific binding partner or a nucleic acid that encodes a specific binding partner, or may take place in vitro, for example in ELISA, Western-blotting, immunocytochemistry, thus, affinity chromatography or in cell-based assays, such as the analysis of TF-1.

It is possible to quantify the binding of the specific binding partner with IL-13. Quantification can be related to the amount of antigen in the test sample that may be of interest for diagnostic purposes.

Also offers a collection containing a specific binding partner or antibody molecule according to any aspect or variant implementation of the present invention, as an aspect of the present invention. In the set according to the invention to the specific binding partner or antibody molecule can be labeled, in order to ensure the identification of its reactivity in a sample, for example as described hereinafter. Kit components usually are sterile and are in sealed vials or other containers. Sets can be used in diagnostic assays or other methods where applicable molecule antibodies. Set the can is t to contain instructions for use of the components in the way, for example, in the method according to the present invention. Supporting material that helps or enables the implementation of this method may be included in the set according to the invention.

The reactivity of antibodies in the sample can be determined by suitable methods. One possibility is the radioimmunoassay (RIA). Radioactively labeled antigen is mixed with unlabeled antigen (test sample) and give you the opportunity to contact with the antibody. Associated antigen is physically separated from unbound antigen and determine the amount of radioactive antigen bound to the antibody. The more antigen present in the test sample, the less radioactive antigen will bind with the antibody. Analysis of competitive binding can also be used with non-radioactive antigen, using antigen or similar associated with reporter molecule. Reporter molecule may be fluorochrome, phosphor or laser dye with isolated spectral characteristics of absorption and emission. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas red. Suitable chromogenic dyes include diaminobenzidin.

Other reporters include macromolecular colloidal particles or consisting of particles of material, such as latex beads, to the which are painted, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause the recorded signals observed visually detected electrons ways or recorded otherwise. These molecules can be enzymes that catalyze reactions, which, for example, cause the development or change of color or cause changes in electrical properties. They can be excitable at the level of molecules, so that the electronic transitions between energy States lead to characteristic spectral parameters of absorption or emission. These may include chemical elements used for conjugation with biosensors. Can be used registration system Biotin/avidin or Biotin/streptavidin system and alkaline phosphatase.

The signals generated by the individual conjugates antibody-reporter, can be used to obtain quantitatively measured absolute or relative data linking relevant antibodies in the samples (normal and test).

The present invention also relates to the use of a specific binding partner, as specified above, to measure the levels of antigen in competitor analysis, that is, the method of measuring the level of antigen in the sample using the special is practical binding partner, proposed in the present invention, in the competitive analysis. It is possible in the case when no physical separation of the bound and unbound antigen. One of the features is the binding of the reporter molecule with a specific binding partner, so is physical or optical change upon binding. Reporter molecule can directly or indirectly generate registered and preferably measured signals. The reporter binding molecules may be direct or indirect, covalent, for example via a peptide bond or non-covalent. Linking via a peptide bond may be a result of recombinant expression of the fusion genes encoding the antibody and reporter molecule.

The present invention also relates to the measurement of levels of antigen directly using a specific binding partner according to the invention, for example in the biosensor system.

The method of determining the binding is not a characteristic of the present invention and specialists in this field are able to choose the right way according to your preferences and General knowledge.

As stated in various aspects and embodiments, the present invention applies to a specific binding partner, which con is uriroot for binding to IL-13 with any specific binding partner, defined in this description, such as IgG4 BAK502G9. Competition between binding partners can be easily analyzed in vitro, for example by tagging a specific reporter molecule to one binding partner, which can be detected in the presence of another unlabeled binding partner(ROS), thus making it possible to identify specific binding partners that bind the same epitope or an overlapping epitope.

Competition can be defined, for example, using ELISA, in which IL-13 immobilized on the tablet, the tablet add the first labeled binding partner together with one or more other unlabeled binding partner. The presence of unlabeled binding partner, which competes with the labeled binding partner, see reduction of the signal emitted by the labeled binding partner.

When tested against the competition, you can use a peptide fragment of the antigen, in particular, a peptide containing an epitope. You can use a peptide having the sequence of the epitope plus one or more amino acids at either end. We can say that this peptide "is essentially a" of this order. Specific binding partners according to the present invention may be such that Thu is their binding to the antigen is inhibited by peptide with the sequence or peptide, containing the sequence. At the specified testing you can use the peptide with any sequence plus one or more amino acids.

Specific binding partners that bind specific peptide may be selected, for example, from a library of phage display through panning using peptide(Dov).

The present invention also relates to an isolated nucleic acid encoding a specific binding partner according to the present invention. The nucleic acid may include DNA and/or RNA. In a preferred aspect the present invention relates to a nucleic acid which encodes a CDR or set of CDRs or VH domain or VL domain, or antigennegative plot antibodies, or antibody molecule, e.g. scFv or IgG4, according to the invention, which is defined above.

The present invention also relates to structures in the form of plasmids, vectors, transcription cassettes or expression, which contain at least one polynucleotide specified above.

The present invention also relates to a recombinant cell host, which contains one or more of the structures mentioned above. Nucleic acid encoding any CDR or set of CDRs or VH domain or VL domain, or antigennegative plot antibodies or maul the Kullu antibodies for example scFv or IgG4, which are, as such, forms an aspect of the present invention, as the method of obtaining the encoded product, and this method comprises the expression product encoding nucleic acid. The expression can be easily carried out by cultivation in suitable conditions, recombinant host cells containing the nucleic acid. After production by the expression domain of the VH or VL or specific binding partner can be isolated and/or purified using any suitable method and then applied accordingly.

Specific binding partners, the VH domains and/or VL and molecules encoding nucleic acids and vectors according to the present invention can be isolated and/or purified, e.g. from their natural environment, in substantially purified or homogeneous form, or, in the case of nucleic acid containing no or essentially does not contain nucleic acid or genes of different origin, different from the sequence that encodes a polypeptide with the required function. Nucleic acid according to the present invention may contain DNA or RNA and may be wholly or partially synthetic. Specifying a nucleotide sequence that is described in this description covers m is lukulu DNA with the specified sequence and covers the RNA molecule with the specified sequence, in which T is replaced by U, unless the context requires otherwise.

System cloning and expression of the polypeptide in many different cells of the host are well-known. Appropriate cell hosts include bacteria, mammalian cells, plant cells, yeast and baculovirus systems and transgenic plants and animals. Lines of mammalian cells that are available in this area for expression of a heterologous polypeptide include cells of the Chinese hamster ovary (CHO), HeLa cells, kidney cells baby hamster, human melanoma cells in mice NS0, myeloma cells rat YB2/0 cells, embryonic human kidney cells, human embryonic retina of human rights and many others. Well-known preferred bacterial host is E. coli.

The expression of the antibodies and fragments of antibodies in prokaryotic cells such as E. coli, are well developed in this area. Overview see, e.g., Pluckthun, A. Bio/Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available for professionals in this field as a choice to obtain specific binding partner, such as Chadd HE and Chamow SM (2001) 110 Current Opinion in Biotechnology 12: 188-194, Andersen DC and Krummen L (2002) Current Opinion in Biotechnology 13: 117, Larrick JW and Thomas DW (2001) Current opinion in Biotechnology 12: 411-418.

Can be selected or designed suitable vectors containing appropriate regulatory on what sledovatelnot, including promoter sequences, sequences, terminators, polyadenylation sequence, enhancer sequences, marker genes or other sequences depending on the situation. Vectors can be plasmids, viral vectors such as phage or fahmida depending on the circumstances. A more detailed description see, for example, in Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for the processing of nucleic acids, such as obtaining structures of nucleic acids, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins is described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1988, Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons, 4thedition 1999. Description Sambrook et al. and Ausubel et al. (both) are included in this description by reference.

Thus, another aspect of the present invention relates to a cell host containing a nucleic acid listed in this description. Such a host cell may be in vitro, and may be in culture. Such a host cell may be in vivo. The presence of host cell in vivo may provide an opportunity for intracellular expression of specific binding PA is the partners in accordance with the present invention in the form of "inner antibodies or intracellular antibodies. Internal antibodies can be used for gene therapy [112].

Another aspect relates to a method, which includes the introduction of such nucleic acid in cells of the host. For the introduction, you can use any way. In the case of eukaryotic cells suitable methods may include transfection based on the calcium phosphate method using DEAE-dextran, electroporation-mediated liposomes transfection and transduction using retrovirus or other virus, such as a virus vaccine or, in the case of insect cells, baculovirus. For the introduction of nucleic acid into the cell host, in particular eukaryotic cell, you can use viral or based on plasmid system. Plasmid system can be maintained in the form apicom or may be introduced into the chromosome of the host cell or an artificial chromosome [110, 111]. The implementation can be either random or targeted integration of one or more copies in one or many loci. In the case of bacterial cells suitable methods may include transformation using calcium chloride, electroporation and transfection using bacteriophage.

After the introduction may be caused by the expression or provided the opportunity for the expression of nucleic acid, for example, through the om culturing host cells under conditions of gene expression.

In one embodiment, the nucleic acid according to the invention integrated into the genome (e.g., chromosome) of the host cell. Integration can be stimulated by the introduction of sequences that stimulate recombination with the genome according to standard methods.

The present invention also relates to a method, which involves the use of a design that is described above, in expressing the system to Express a specific binding partner or the polypeptide described above.

Aspects and embodiments of the present invention will now be illustrated in an example with reference to the following experiments.

EXAMPLE 1

Selection of anti-IL-13-scFv

The repertoire of scFv-antibodies

For selection used a large library of human antibodies in single-chain Fv (scFv)derived from spleen lymphocytes from 20 donors and cloned in fahmida vector [66].

Selection of scFv

ScFv that recognize IL-13, were isolated from libraries on the basis of phage display in a series of iterative cycles of selection using recombinant obtained in bacteria IL-13 human or mouse (Peprotech), essentially as described in [67]. Briefly, after incubation with the library immobilized antigen, which was previously linked to paramagnetic beads, and the bound phage were removed pic what edstam magnetic separation, while the unbound phage were removed by washing. Then the bound phage were rescued, as described by Vaughan et al. [67], and the selection process repeated. In different rounds of selection used a variety of hard surfaces and methods of capture, to reduce nonspecific binding. The antigen or covalently linked to beads (Dynabeads M-270 carboxylic acid), or modified by biotinylation with subsequent secondary capture on streptavidin coated beads (Dynabeads M-280) according to the protocols of the manufacturer (Dynal). Part of typical clones obtained by rounds of selection were subjected to DNA sequencing as described in Vaughan et al. [67] and Osbourn et al. [70]. Unique clones were analyzed for their ability to neutralize IL-13 in the form of purified scFv preparations in the analysis dependent on IL-13 cell proliferation.

Create library based on ribosomal display, and were subjected to screening for scFv that have been specifically recognize recombinant obtained in bacteria IL-13 human or mouse (Peprotech), essentially as described in Hanes et al. [113]. First the best clone BAK278D6, resulting in the original selections were made in the form of ribosomal display, and then the resulting matrix was used to create the library. At the level of DNA was added to the T7 promoter to the 5'-end for efficient transcription of mRNA. On the mRNA level design contained the prokaryotic binding site of the ribosome (sequence Shine-Dalgarno). On the 3'-end of one strand was removed the stop codon was added in part gIII (gene III) as a spacer [113].

Libraries in the ribosomal display, obtained from BAK278D6, created by mutagenesis complementarity determining regions of antibodies (CDR), was carried out by PCR reaction with polymerase Tag with no corrective activity. Carried out the selection based on affinity, after incubation with the library biotinylated IL-13 man caught paramagnetic beads coated with streptavidin (Dynal M280) and related ternary complexes (mRNA-ribosome-scFv-IL-13) were removed by magnetic separation, while the unbound complexes were removed by washing. Then mRNA encoding a linked scFv, was recovered by RT-PCR, as described in Hanes et al. [113], and the selection process was repeated with decreasing concentrations (100 nm - 100 PM for 5 rounds) biotinylated IL-13 person present during the selection.

Error-prone PCR were also used to further increase the size of the library. In the scheme of selection used three intensity errors (of 2.0 to 3.5 and 7.2 mutations per 1000 BP after a standard PCR reaction, which is described in the Protocol of the manufacturer (Clontech)). The initial reaction of error-prone PCR took place before the loop from one selection, starting at 100 nm. The next round of error-prone PCR OS is Westley before the cycle of the three selection with 10 nm biotinylated IL-13 human. As indicated above, part of typical clones obtained by rounds of selection were subjected to DNA sequencing as described in Vaughan et al. [67] and Osbourn et al. [70]. Unique clones were analyzed for their ability to neutralize IL-13 in the form of purified scFv preparations in the analysis dependent on IL-13 cell proliferation.

EXAMPLE 2

Neutralizing the effectiveness of anti-IL-13-scFv in the analysis dependent on IL-13 cell proliferation, TF-1

Neutralizing the effectiveness of purified scFv preparations against the bioactivity of IL-13 human or mouse were evaluated using analysis of cell proliferation, TF-1. Purified scFv preparations were obtained as described in example 3 in WO 01/66754. The protein concentration of purified scFv preparations were determined using the BCA method (Pierce). TF-1 is a line premierone human cells obtained from a patient with erythroleucus [68]. The cell line TF-1 is a factor-dependent in relation to survival and proliferation. In this regard, cells TF-1, corresponding to either the human or murine IL-13 [69], were maintained in medium containing GM-CSF man (4 ng/ml, R and D Systems). The inhibition is dependent on IL-13 cell proliferation was determined by measuring the reduction of the inclusion of tritium-labeled thymidine into newly synthesized DNA of dividing cells.

Protocol analysis of cells TF-1

Cells TF-1 was obtained from R and D Systems and support agreement is but the attached protocols. Environment for analysis contained RPMI-1640 with GLUTAMAX I (Invitrogen)containing 5% fetal calf serum (JRH) and 1% sodium pyruvate (Sigma). Before each analysis cell TF-1 was besieged by centrifugation at 300×g for 5 min, the medium was removed by suction and the cells resuspendable environment for analysis. This process was repeated twice and the cells resuspendable at a final concentration of 105cells/ml in the medium for analysis. Test solutions antibodies (three repetitions) were diluted to the desired concentration in the medium for analysis. As a negative control was used irrelevant antibody not directed to IL-13. Recombinant obtained in bacteria IL-13 human or mouse (Peprotech) was added to a final concentration of 50 ng/ml when mixed with the appropriate test antibody in a total volume of 100 µl/well in 96-well tablet for analysis. The concentration of IL-13, used in the analysis were chosen in such a dose that is at final concentrations in the analysis gave approximately 80% of the maximum proliferative response. All samples were incubated for 30 minutes at room temperature. Then each analyzed point was added 100 μl resuspending cells, receiving a total analysis of 200 µl/well. Analyzed the plates were incubated for 72 hours at 37°C in 5% CO2. Then each analyzed point was added 25 is CL, tritium-labeled thymidine (10 mccoury/ml, NEN) and analyzed the tablets were returned to the incubator for another 4 hours. Cells were collected on a filter plate made of fiberglass (Perkin Elmer)using the device to collect the cells. The incorporation of thymidine was determined using a liquid scintillation counter for microplate Packard TopCount. Data were analyzed using the computer program Graphpad Prism.

Results

Despite periodic cycles of selection using antigens of human and mouse, have not received cross-reactive neutralizing antibodies. In the selection received two different neutralizing scFv against IL-13 human and one against IL-13 mouse. BAK278D6 (VH SEQ ID NO: 13; VL SEQ ID NO: 14) and BAK167A11 (VH SEQ ID NO: 23; VL SEQ ID NO: 24) learned of IL-13, whereas the BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26) learned of IL-13 mouse. BAK278D6 (figure 2) and BAK167A11 (figure 1) in the form of scFv neutralized 25 ng/ml IL-13 human with IC50component 44 nm and 111 nm, respectively. BAK209B11 (figure 3) in the form of scFv neutralized 25 ng/ml IL-13 mice with IC50component 185 nm.

EXAMPLE 3

Neutralizing the effectiveness of the best clones, obtained as a result of purposeful optimization CDR3 of the heavy chain of the original clones in the analysis dependent on IL-13 cell proliferation, TF-1

Osbourn et al. [70] showed that targeted mutagenesis of residues in CDR3 of the heavy chain can greatly increase the affinity of antibodies. Selection of Westlake, as described in example 1, the scFv repertoire, in which residues in CDR3 of the heavy chain CDR3 BAK278D6 (SEQ ID NO: 6), BAK167A11 (SEQ ID NO: 57) were randomized by mutagenesis. Unique clones obtained by breeding, identified by DNA sequencing and neutralizing their effectiveness has been evaluated in the form of scFv in the analysis of cell proliferation, TF-1, as described in example 2.

Results

A significant increase in efficiency was achieved in both lines. The most effective clones of the line BAK167A11 were BAK615E3, BAK612B5 and BAK582F7, which in the form of scFv had IC50making 3 nm (figure 1), and 6.6 nm, 6,65 nm, respectively, against 25 ng/ml IL-13 man in the analysis of cell proliferation, TF-1. From line BAK278D6 the most effective was a clone of BAK502G9, which is in the form of scFv had IC50equal to 8 nm, against 25 ng/ml IL-13 man in the analysis of cell proliferation, TF-1 (figure 2).

EXAMPLE 4

Neutralizing efficiency lines BAK167A11 and BAK278D6 against IL-13 primates other than man, and the variant IL-13 associated with asthma in the analysis dependent factor cell proliferation, TF-1

No line BAK167A11 or line BAK278D6, neutralizing IL-13 human, were not cross-reactive with respect to the mouse.

Therefore, the inventors chose the following criteria for lines selected for further optimization and clinical development: it should preferably be cross-reacherous the th with IL-13 Primate, other than man, and must find the variant IL-13, in which arginine at amino acid position 130 is replaced by glutamine (R130Q). This option is genetically linked to asthma and other allergic diseases[37, 39, 41, 71]. Cross reactivity was determined by the ability of purified scFv preparations to contact IL-13 Primate, non-human, and a variant of IL-13 using analysis of surface plasmon resonance (BIAcore). Functional activity was determined using analysis of cell proliferation, TF-1.

Receiving IL-13 wild-type, variant, and IL-13 Primate, non-human

cDNA of IL-13 human wild-type were obtained from InvivoGen and modified site-directed mutagenesis (set Stratagene Quikchange®)to obtain cDNA encoding a variant IL-13. The coding sequence for IL-13 rhesus and IL-13 macaques-Griboedov was obtained by PCR on the matrix genomic DNA using degenerate primers based on the sequence of IL-13 human. Both sequences of primates other than human (RH and Griboedov)were identical to each other, but differed from IL-13 human family amino acids (Fig). Then expressed recombinant IL-13 wild-type, variant and IL-13 Primate, non-human, using the baculovirus expression system (Invitrogen). For expressing the design, the functions added affinity tag at the carboxyl end, to Express the protein, which provides clean air-conditioned environment of the insect cells almost to homogeneity.

Quantitative analysis of binding using BIAcore

The binding affinity of purified scFv preparations in relation to IL-13 Primate other than humans, variant and IL-13 wild-type was determined by measuring the surface plasmon resonance using a biosensor BIAcore 2000 (BIAcore AB)as described in Karlsson et al. [72]. Briefly, IL-13 was associated with sensor chips SM using the set of binding amine (BIAcore), weighing approximately 200 used, and three concentrations tested scFv (about 350 nm, 175 nm and 88 nm) in NW-EP buffer was passed through the surface of sensor chip. The resulting sensograms was estimated using a computer program for BIA evaluation 3.1, receiving relative data binding.

Protocol analysis of TF-1

The analysis was carried out essentially as described in example 2, with the following modifications: IL-13 Primate other than humans, variant IL-13 person (R130Q) and IL-13 human wild type used at concentrations of 50 ng/ml, 25 ng/ml and 25 ng/ml, respectively.

Results

The results of the BIAcore analysis of binding indicate that the line BAK278D6, but not the line VACA, had the desired profile cross-reactivity is La further developments in therapeutic purposes (table 2). The data obtained was confirmed by the results of bioanalysis, demonstrating that BAK278D6 (figure 4) and BAK502G9 (6) were able to neutralize IL-13 human variant IL-13 person (R130Q) and IL-13 Primate, non-human, in the analysis of cell proliferation, TF-1 with almost equal efficiency. On the contrary, although WAKES (VH SEQ ID NO: 33; VL SEQ ID NO: 34) had a significantly increased effectiveness against IL-13 compared with the original WAKA (VH SEQ ID NO: 23; VL SEQ ID NO: 24) in the analysis of cell proliferation, TF-1 (figure 1), none of the clones were not bound IL-13 Primate, non-human, or a variant of IL-13 in the BIAcore analysis of binding.

Frame region BAK278D6 and BAK502G9 germ line

The obtained amino acid sequence of VH BAK278D6 (SEQ ID NO: 13) and VL (SEQ ID NO: 14) were aligned with known sequences of the germline of the person in the VBASE database [73] and identified the closest germ line sequence similarity. The closest germline sequence for the VH domain BAK278D6 (SEQ ID NO: 14) and its derivatives identified as DP14, member of the VH1 family. VH BAK278D6 has 9 differences from DP14 the germ line in frame areas. The closest germline sequence for VL BAK278D6 identified as Vλ3 3h. VL-domain BAK278D6 (SEQ ID NO: 13) has only 5 changes compared to the germ line in ka is the red areas. Frame region BAK278D6 and its derivatives were returned to the areas of the germ line site-directed mutagenesis (set Stratagene Quikchange) for identical match with native human antibody.

EXAMPLE 5

Neutralizing the effectiveness of the best clones, obtained as a result of purposeful optimization of sequences of the heavy chain CDR1 and CDR2 of the heavy chain BAK502G9 in the analysis dependent on IL-13 human cell proliferation, TF-1

The second phase of the optimization was performed using as template sequence BAK502G9 with frame areas, close to the germ line. The selection was carried out essentially as described in example 1, from the repertoire of scFv in which any residues in the heavy chain CDR1 or CDR2 of the heavy chain BAK502G9 subjected to randomization by mutagenesis. Unique clones obtained by breeding, identified by DNA sequencing and were evaluated by neutralizing their effectiveness in the form of purified scFv preparations in the analysis of cell proliferation, TF-1, as described in example 2. Designed vectors for the most effective scFv clones, to allow re-expression in whole IgG4-human antibodies, as described in Persic et al. (1997, Gene 187; 9-18), with minor modifications. The vectors include a fragment of oriP, in order to facilitate the use of cells HEK-293 EBNA and to provide replication EP is catfish. Variable domain VH was cloned in polylinker between the leader sequence for secretion and a constant region of the gamma 4 people in expressing vector pEU8.1(+). Variable domain VL was cloned in polylinker between the leader sequence for secretion and a constant region of human lambda expressing vector pEU4.1(-).

Whole antibody was purified from the conditioned medium of cells EBNA-293, together transfected with constructs expressing the heavy and light chains, by affinity chromatography using protein A (Amersham Pharmacia). Purified preparations of antibodies sterile filtered and stored at 4°C in phosphate-buffered saline (PBS) prior to the assessment. The protein concentration was determined by absorption measurement at 280 nm using the BCA method (Pierce). Converted entire IgG4 antibodies person was compared with commercially available anti-IL-13-human antibodies in the analysis of the proliferation of TF-1, described in example 2.

Results

As illustrated in figure 5, showed that the commercial antibody B-B13, (mouse IgG1 - Euroclone 5) much more effectively against IL-13 human than commercial antibody JES10-5A2 (IgG1 rat - Biosource) with IC50component 1021 PM and PM 471, respectively. Eight clones, namely BAK1111D10, BAK1166G02, BAK1167F02, BAK1167F04, BAK1183H4, BAK1184C8, BAK1185E1, BAK1185F8 obtained from BAK502G9 (and therefore "line BAK52G9"), in which CDR1 or CDR2 of the heavy chain have been deliberately changed, showed increased efficiency in the form of scFv compared with commercial antibodies. This increase was maintained during the transformation of the whole IgG4-human antibody. Each of these domains VH and VL separately and in appropriate pairs according to these claims is the aspect or implementation of the present invention, as well as specific binding partners for IL-13, which contain one or more of these domains, as well as specific binding partners containing one or more CDRs of clone lines BAK502G9, preferably VH domain containing the set of HCDR line BAK502G9 and/or VL domain containing a set of LCDR line BAK502G9. These domains can be used in any and all aspects of the invention, which are indicated throughout this description. Derivatives BAK502G9 in the form of whole antibodies (IgG4) have IC50in the range from 244 PM until PM 283. BAK502G9 in whole IgG4 antibodies is IC50384 PM. Finally, major improvements in efficiency can be obtained purposeful change CDR1 (SEQ ID NO: 7) or CDR2 (SEQ ID NO: 8) heavy chain BAK502G9. Statistical comparison with B-B13 was performed using ANOVA with subsequent analysis of a posteriori criterion of Dunnet (computer program InStat).

More the feature

Selected anti-human antibodies from line BAK278D6 were subjected to additional characterization to determine their specificity. These antibodies included BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16) and its derivatives BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) and BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38), which are typical examples of clones with modifications in the heavy chain CDR1 and CDR2 of the heavy chain BAK502G9, respectively.

EXAMPLE 6

Neutralizing the effectiveness of the best clones, obtained as a result of purposeful optimization of sequences of the heavy chain CDR1 and CDR2 of the heavy chain BAK502G9 against IL-13 primates other than man, and the variant IL-13 associated with asthma, in the analysis of dependent factors, cell proliferation, TF-1

The cross-reactivity of antibodies against IL-13 human was determined by their ability to inhibit mediated IL-13 Primate, non-human, and variant IL-13 cell proliferation, TF-1, as described in example 4.

Results

Optimized antibodies against IL-13 human BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) and BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38) retained the specificity of its original BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16) (6). Increase effectiveness against IL-13 wild type was reflected in their ability to neutralize IL-13 Primate, non-human, and the variant IL-13 is essentially equal efficiency. IC50in the case of BAK502G9 PR is against IL-13 human variants of IL-13 human IL-13 Primate, non-human, amounted to 1.4 nm to 1.9 nm and 2.0 nm, respectively. IC50BAK1167F2 against IL-13 human variant IL-13 human IL-13 Primate, non-human, was 1.0 nm to 1.1 nm and 1.3 nm, respectively. IC50BAK1183H4 against IL-13 human variant IL-13 human IL-13 Primate, non-human, was 0.9 nm, 1.0 nm and 1.6 nm, respectively. These clones are suitable for therapeutic applications.

EXAMPLE 7

The neutralizing efficiency of the best antibodies against IL-13 human anti-native IL-13 man in the analysis of cell proliferation in HDLM-2

The sequence of IL-13 human has four potential N-glycosylation. The inventors have shown the ability BAK278D6 and its derivatives to neutralize recombinant IL-13 expressed either bacterial or baculovirus expression systems. Although there is evidence that many event processing known in systems of mammals also occur in insects, there are key differences in the glycosylation of proteins, in particular in N-glycosylation [74].

The inventors tested the ability of derivatives BAK278D6 to neutralize native IL-13 released from human cells.

Cells HDLM-2 was isolated by Drexler et al. [75] from a patient with Hodgkin's disease. Skinnider et al. [76] in which Asali, cell proliferation HDLM-2 was partly dependent on autocrine and paracrine release of IL-13. The best antibodies against IL-13 individuals were valued for their ability to inhibit cell proliferation HDLM-2-mediated release of native (or naturally occurring) IL-13.

Protocol analysis of cells HDLM-2

Cells HDLM-2 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and maintained according to the supplied protocols. A framework for analysis consisted of RPI-1640 with Glutamax I (Invitrogen)containing 20% fetal calf serum. Before each analysis, the cells were besieged by centrifugation at 300×g for 5 min, the medium was removed by suction and the cells resuspendable in fresh medium. This process was repeated three times and, finally, the cells resuspendable to a final concentration of 2×105cells/ml in the medium for analysis. 50 μl resuspending cells were added to each point for analysis in 96-well tablet for analysis. Test solutions antibodies (three repetitions) were diluted to the desired concentration in the medium for analysis. As a negative control used the irrelevant antibody isotype, not directed to IL-13. To the cells was added to the appropriate test antibody in a total volume of 50 µl per well at each point for the analysis of the received total volume for analysis, 100 µl/well. The analyzed tablets in what was operovali for 72 hours at 37°C in 5% CO 2. Then each analyzed point was added 25 μl of tritium-labeled thymidine (10 mccoury/ml, NEN) and analyzed the tablets were returned to the incubator for another 4 hours. Cells were collected on a filter plate made of fiberglass (Perkin Elmer)using the device to collect the cells. The incorporation of thymidine was determined using a liquid scintillation counter for microplate Packard TopCount. Data were analyzed using the computer program Graphpad Prism.

Results

As shown in Fig.7, BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16) and its derivatives BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38) and BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) were able to cause a dose-dependent inhibition of cell proliferation with relative efficacies similar to the efficiencies observed in the bioassays. IC50for BAK502G9, BAK1183H4, BAK1167F2 in the form of a human IgG4 was 4.6 nm, about 3.5 nm and 1.1 nm, respectively. IC50commercial antibodies JES10-5A2 and B-B13 amounted to 10.7 nm and 16.7 nm, respectively.

EXAMPLE 8

The neutralizing efficiency of the best antibodies against IL-13 rights in relation to dependent on IL-13 responses in primary cells, related to disease

Secondary bioassays were carried out using primary cells and indicators that have more to do with disease of the respiratory tract. These indicators included the release eotaxin the C normal, the human lung fibroblasts (NHLF) and increased regulation of the adhesion molecules vascular cell 1 (VCAM-1) on endothelial cells of the umbilical vein of a person (HUVEC). Both are dependent on IL-13 response could contribute to the mobilization of eosinophila, a symptom of asthma phenotype [92].

Protocol analysis NHLF

It is shown that IL-13 induced the release eotaxin from lung fibroblasts[77], [78], [79]. Dependent factor release eotaxin from NHLF were determined in ELISA.

NHLF were obtained from Biowhittaker and maintained according to the supplied protocols. Environment for analysis was Wednesday FGM-2 (Biowhittaker). Test solutions antibodies (three repetitions) were diluted to the desired concentration in the medium for analysis. As a negative control was used irrelevant antibody not directed to IL-13. Then added recombinant obtained in bacteria IL-13 person (Peprotech) to a final concentration of 10 ng/ml when mixed with the appropriate test antibody in a total volume of 200 μl. The concentration of IL-13, used in the analysis were chosen in such a dose that gave approximately 80% of the maximum response. All samples were incubated for 30 minutes at room temperature. Then the analyzed sample was added to NHLF, which previously were sown at a density of 1×104cells per well in 96-well plates to analysis. Analyzed the plates were incubated at 37°C for 16-24 hours in 5% CO2. Tablets for analysis was centrifuged at 300×g for 5 minutes to precipitate ukrepivshis cells. Levels eotac is in nadeshiko was determined in ELISA, using the reagents and methods described by the manufacturer (R & D Systems). Data were analyzed using the computer program Graphpad Prism.

Results

The clones line BAK278D6 had the ability to inhibit dependent on IL-13 human release eotaxin from NHLF. Relative efficiency was similar to the efficacy observed in the analysis of cell proliferation, TF-1 (Fig). BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16), BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38), BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) had IC50207 PM, 118 PM and PM 69, respectively, versus 10 ng/ml IL-13 human. Commercial antibodies JES10-5A2 and B-B13 had IC50623 PM and 219 PM, respectively.

Protocol analysis HUVEC

It is shown that IL-13 increases the expression of VCAM-1 on the surface of HUVEC cells [80, 81]. Heavily influenced by the expression of VCAM-1 was determined by registering the increasing regulation of cellular expression of the receptor for VCAM-1, using the data of fluorescence with time resolution.

HUVEC were obtained from Biowhittaker and maintained according to the supplied protocols. Environment for analysis was the medium EGM-2 (Biowhittaker). Test solutions antibodies (three repetitions) were diluted to the desired concentration in the medium for analysis. As a negative control was used irrelevant antibody not directed to IL-13. Recombinant obtained in bacteria IL-13 person (Peprotech) was added to a final concentration of 10 ng/ml in cm is shivani with the appropriate test antibody in a total volume of 200 μl. The concentration of IL-13, used in the analysis were chosen in such a dose that gave approximately 80% of the maximum response. All samples were incubated for 30 minutes at room temperature. Then the analyzed sample were added to HUVEC, which previously were sown at a density of 4×104cells per well in 96-well plates to analysis. Analyzed the plates were incubated at 37°C for 16-20 hours in a 5% CO2. Then Wednesday for analysis were removed by suction and replaced with blocking solution (PBS containing 4% dry milk powder Marvel®). Tablets for analysis were incubated at room temperature for 1 hour. The wells three times washed with PBST twin, then to each well was added 100 ál (dilution 1:500 in PBST/1% Marvel®) biotinylated anti-VCAM-1 antibody (Serotec). Tablets for analysis were incubated at room temperature for 1 hour. The wells three times washed with buffer to flush kits are used (Perkin Elmer), then to each well was added 100 μl labeled with europium streptavidin or antibodies against IgG1 mouse (dilution 1:1000 in buffer for analysis kits are used, Perkin Elmer). Then the tablets for analysis were incubated at room temperature for 1 hour. Wells 7 times washed with buffer to flush kits are used (Perkin Elmer). Finally, each well was added 100 μl of the amplifying solution (Perkin Elmer) and determined the intensity of the fluorescence, use the I the reader tablets Wallac 1420 VICTOR2 (standard Protocol for europium). Data were analyzed using the computer program Graphpad Prism.

Results

Typical data for BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16), BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38), BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) in the form of whole antibodies IgG4 person shown in Fig.9. Relative efficiency was similar to the efficacy observed in the analysis of cell proliferation, TF-1. IC50for BAK502G9, BAK1183H4 and BAK1167F2 was 235 PM 58 55 PM and PM, respectively, versus 10 ng/ml IL-13 human.

EXAMPLE 9

Neutralizing the effectiveness of anti-IL-13 antibodies against dependent on IL-1β and IL-4 enhances the regulation of VCAM-1

The specificity of the clones line BAK278D6 was evaluated in a modified bioanalysis HUVEC. It is shown that along with IL-13 and IL-4 and IL-1β increased the expression of VCAM-1 on the surface of HUVEC cells [80, 81].

Protocol analysis HUVEC

The analysis was carried out essentially as described in example 5 with the following modifications. Recombinant IL-1β human IL-4 (R and D Systems) was used instead of IL-13 person at a concentration of 0.5 ng/ml and 1 ng/ml, respectively, and was administered at a dose, which gave approximately 80% of the maximum response.

Results

None of the evaluated clones line BAK278D6 not neutralized increasing regulation of VCAM-1 in response to IL-1β or IL-4 and thus showed specificity in relation to IL-13 (figure 10). IL-4 is most closely related to IL-13, showing 30% Ident is the durability of the sequences at the amino acid level [82].

EXAMPLE 10

Neutralizing efficiency BAK209B11 in the form of a human IgG4 in the analysis dependent on IL-13 mouse proliferation of murine cells B9

BAK209B11 identified as neutralizing clone against IL-13 mouse in the form of scFv as described in example 1, was transformed into a whole antibody in the form of a human IgG4, as described in example 5 and evaluated its effectiveness in the analysis dependent on IL-13 mouse proliferation of B9 cells. B9 is a cell line of B-cell hybridoma mice [83]. B9 depends on factors in relation to survival and proliferation. In this regard, the B-cells that respond to IL-13 mice, maintained in medium containing IL-6 person (50 PG/ml, R and D Systems). The inhibition is dependent on IL-13 mouse proliferation was determined by measuring the reduction of the inclusion of tritium-labeled thymidine into newly synthesized DNA of dividing cells.

Protocol analysis of cells B9

The B9 cells were obtained from European collection of cell cultures, animals ECACC and maintained according to the supplied protocols. The analysis was carried out essentially as described for the analysis of TF-1 in example 2, but with the following modifications. A framework for analysis consisted of RPMI-1640 with GLUTAMAX I (Invitrogen)containing 5% fetal calf serum (Hyclone) and 50 μm 2-mercaptoethanol (Invitrogen). IL-13 human substituted obtained in recombinant bacteria IL-13 mouse (Peprotech) at a final concentration in analyze ng/ml.

Results

BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26) in the form of a human IgG4 neutralized 1 ng/ml IL-13 mice with IC50equal 776 PM, in the analysis B9 (11). Thus, BAK209B11 is a useful tool to study the role of IL-13 in murine models of the disease. This is clearly shown in example 12, which demonstrates the effectiveness BAK209B11 in murine models of acute inflammation of the lungs.

EXAMPLE 11

Determination of the affinity of anti-IL-13 antibodies in the BIAcore analysis

The affinity of BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16), BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) and BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38) with respect to IL-13 human and BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26) with respect to IL-13 mouse in the form of a human IgG4 were determined by measuring the surface plasmon resonance using a biosensor BIAcore 2000 (BIAcore AB), essentially as described in [72]. Briefly, antibodies were associated with sensor chip CM5 using the set of binding amine (BIAcore), weighing approximately 500 srvc. unit, and a serial dilution of IL-13 (from 50 nm to 0.78 nm) in HBS-EP buffer was passed through the surface of sensor chip. The resulting sensograms was estimated using a computer program for BIA evaluation 3.1, obtaining kinetic data.

Results

IgG4 BAK502G9, BAK1167F2 and BAK1183H4 bound IL-13 human high affinity with Kd PM 178, 136 PM and PM 81, respectively, which corresponds to their otnositel the th efficiency in cell-based assays. BAK209B11 bound IL-13 mice with affinity to 5.1 nm (table 3).

EXAMPLE 12

Efficiency BAK209B11 in a murine model of acute allergic inflammation of the lungs

Murine model of acute allergic inflammation of the lungs

Influence BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), neutralizing IgG4-human antibodies against IL-13 mice, was investigated in mice with acute allergic inflammation of the lungs. This model was carried out essentially as described Riffo-Vasquez et al. [84], and the final result was characterized by increased levels of IL-13 in bronchoalveolar lavage (BAL) (Fig), infiltration of cells into the lung and BAL (Fig), elevated levels of IgE in serum and hypersensitivity of the respiratory tract (AHR).

The Protocol model

Female Balb/C mice (Charles River, UK) were treated with either antibody BAK209B11 against IL-13 mice (at doses of 12, 36, 119 or 357 mg)or matched isotype control antibody (dose 357 µg). At 0 and 7 days, mice in each group were senzibilizirani intraperitoneal injection of 10 μg of ovalbumin (Ova) in 0.2 ml of filler (physiological solution containing 2% Al2O3(Rehydragel) as adjuvant). A separate control group desensibilisation mice received an equal volume of filler. Mice were subjected to provocations with ovalbumin on 14th, 15th and 16th day. Ovalbumin was diluted to 1% (wt./about.) in sterile saline solution prior to spraying. Su is provocation by inhalation held in the chamber for processing of Plexiglas. Ova was respectively in the form of an aerosol using a nebulizer deVilbiss Ultraneb 2000 (Sunrise Medical) in the form of a series of three effects for 20 minutes, separated by 1-hour intervals.

BAK209B11 or irrelevant IgG4 person was injected intravenously 1 day before the first provocation and then for 2 hours before each subsequent provocation (total 4 doses). The model was finished on the 17th day of 24 hours after the last provocation. Collected blood (serum) and BAL. Serum was analyzed for total IgE. BAL was obtained, inhazinue 3 aliquots of saline solution (0.3 ml, 0.3 ml and 0.4 ml) and combining the samples. The calculation of the total leukocyte count and differential count of cells received on the basis of BAL cells.

Results

Provocation by ovalbumin sensitized mice caused a significant (p<0.05) increase in the mobilization of the total cells in BAL compared to desencibiliziruuchee, but subjected to provocations animals. This mobilization is dependent on the dose way inhibited BAK209B11; significant (p<0.05) inhibition was observed at ≥36 µg BAK209B11, but not control antibodies (Fig). Similar effects were also observed in eosinophils (Fig) and neutrophils (Fig) with a significant (p<0,05) inhibition of the influx of cells at the lowest dose BAK209B11 36 mcg. Specified inhibition was not observed in the case of the control antibody. Lymphocytes were also induced by p and provocations in sensitized mice and was not induced in desensibilisation mice. This induction is dependent on the dose way inhibited BAK209B11 with maximum inhibition observed at 36 ág BAK209B11. The control antibody had no effect (Fig). Although monocytes/macrophages was not induced in sensitized animals compared to desencibiliziruuchee animals, source levels were lowered under the action of ≥36 µg BAK209B11, but not lowered under the action of the control antibody (Fig). The levels of IgE in serum were significantly increased in sensitized animals compared to desencibiliziruuchee after provocation (p<0,05). These increases were decreased after treatment 36 µg BAK209B11, but not the control antibody.

In conclusion, systemic introduction BAK209B11, antibodies that neutralize murine IL-13, but not control antibodies, inhibited the influx of inflammatory cells and increase the regulation of IgE levels in the serum-induced sensitization and subsequent provocation by ovalbumin in a mouse model of allergic inflammation.

Examples 13-20 are predictive

EXAMPLE 13

Efficiency BAK209B11 in a murine model of Lloyd acute inflammation of the lungs

Murine model of acute allergic inflammation of the lungs

Influence BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), neutralizing antibodies against IL-13 mice, was investigated in a second mouse model of acute allergic inflammation Le is fir. This model was carried out essentially as described by McMillan et al. [85], and characterized the end result of increased levels of IL-13 in BAL and lung tissue, cellular infiltration into the lungs and BAL, elevated levels of IgE in serum and hypersensitivity of the respiratory tract (AHR).

The Protocol model

Female Balb/C mice (Charles River, UK) were administered different doses of antibodies BAK209B11 against IL-13 mouse or matched isotype control antibodies as follows. 0 and day 12 mice in each group were senzibilizirani (SN) intraperitoneal injection of 10 μg of ovalbumin (Ova) in 0.2 ml of filler (physiological solution containing 2 mg Al(OH)3adjuvant [was calculated as described in example 12]). A separate control group desensibilisation mice (NS) received equal volume of filler. Mice were subjected to provocations with ovalbumin for 20 minutes in 19, 20, 21, 22, 23 and 24 the day. Ovalbumin was diluted to 5% (wt./about.) in physiological solution prior to spraying. All provocations by inhalation held in the chamber for processing of Plexiglas. Ova was respectively in the form of an aerosol using a nebulizer deVilbiss Ultraneb 2000 (Sunrise Medical). On 18, 19, 20, 21, 22, 23 and 24 the day the mice were injected different intraperitoneal dose (237 mg, 23,7 mcg or 2,37 mcg; listed on Fig as H, M or L) antibodies BAK209B11 as muIgG1 against IL-13 mouse or matching ISAT the PU control antibodies (237 mg). The function of the respiratory tract were evaluated at 0 and 25 day through provocation increasing amounts of methacholine and registered using plethysmography animals in consciousness (Buxco). PC50(the concentration of methacholine required to increase the initial level PenH 50%) was estimated for individual mice at day 0 and 25 day based on fitting curves 4 flow parameters for the curves of the response to the dose of methacholine.

The model was finished on the 25th day 24 hours after the last provocation. Collected samples of blood, serum, BAL and lung tissue.

Results

Lung function was assessed for individual animals at day 0 (before treatment) and on the 25th day (after provocation) and quantitatively measured, calculating the value PC50(concentration methacholine required to improve baseline PenH 50%) (figa). Hypersensitivity of the respiratory tract of the individual animals (AHR) was determined from the change log PC50on the 25th day compared to day 0 (log PC5025 day - log PC500 day). This difference log PC50was a primary end result of the study, data PC50transformed as log because of the requirements of ANOVA for the final result. Individual changes were averaged in groups, to obtain an average difference of log PC50(which is shown on figv).

Provocation by ovalbumin sensitized mice caused a significant AHR compared with desencibiliziruuchee and subjected to provocations mice (p<0,01). BAK209B11 caused a clear and dose-dependent decrease in AHR, whereas the control antibody had no effect.

EXAMPLE 14

Efficiency BAK209B11 in a murine model of Gerard acute inflammation of the lungs

Murine model of acute allergic inflammation of the lungs

Influence BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), neutralizing antibodies against IL-13 mouse in the form of a human IgG4, was investigated in a third mouse model of acute allergic inflammation of the lungs. This model was carried out essentially as described Humbles et al. [86], and the final result was characterized by an increased content of IL-13 in BAL and lung tissue, cellular infiltration into the lungs and BAL, elevated levels of IgE in serum and hypersensitivity of the respiratory tract (AHR).

The Protocol model

Female Balb/C mice (Charles River, UK) were administered different doses of antibodies BAK209B11 against IL-13 mouse or matched isotype control antibodies. At 0, 7, and day 14 mice in each group were senzibilizirani (SN) intraperitoneal injection of 10 μg of ovalbumin (Ova) in 0.2 ml of filler (physiological solution containing 1.125 mg Al(OH)3adjuvant (counted as described in example 12). A separate control group of desensi lizirovania mice (NS) received equal volume of filler. Mice were subjected to provocations with ovalbumin for 20 minutes at 21, 22, 23 and 24 the day. Ovalbumin was diluted to 5% (wt./about.) in physiological solution prior to spraying. All provocations by inhalation held in the chamber for processing of Plexiglas. Ova was respectively in the form of an aerosol using a nebulizer deVilbiss Ultraneb 2000 (Sunrise Medical).

The model was finished on the 25th day 24 hours after provocation. Collected samples of blood, serum, BAL and lung tissue.

EXAMPLE 15

Efficiency BAK209B11 in the model of Lloyd chronic inflammation of the lungs

Murine model of acute allergic inflammation of the lungs

Influence BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), neutralizing antibodies against IL-13 mouse in the form of a human IgG4, were investigated in a model of acute allergic inflammation of the lungs. This model was carried out essentially as described Temelkovski et al. [87], and the end result is characterized by cellular infiltration in the lungs and BAL, elevated levels of IgE in serum and hypersensitivity of the respiratory tract (AHR).

The Protocol model

Female Balb/C mice (Charles River, UK) were administered different doses of antibodies BAK209B11 against IL-13 mouse or matched isotype control antibodies. 0 and day 11 mice in each group were senzibilizirani (SN) intraperitoneal injection of 10 μg of ovalbumin (Ova) in 0.2 ml of filler (physiological solution containing 2 mg l(OH) 3adjuvant (counted as described in example 12). A separate control group desensibilisation mice (NS) received equal volume of filler. Mice were subjected to provocations by ovalbumin within 20 minutes 18, 19, 20, 21, 22, 23, 28, 30, 32, 35, 37, 39, 42, 44, 46, 49 and 51 days. Ovalbumin was diluted to 5% (wt./about.) in physiological solution prior to spraying. All provocations by inhalation held in the chamber for processing of Plexiglas. Ova was respectively in the form of an aerosol using a nebulizer deVilbiss Ultraneb 2000 (Sunrise Medical).

The model was finished on day 52 after 24 hours after provocation. Collected samples of blood, serum, BAL and lung tissue.

EXAMPLE 16

The effectiveness of antibodies against IL-13 man against exogenous IL-13 person entered in the model of the air sacs of mice

The effect of antibodies against IL-13 man on the anti-inflammatory effect of IL-13 person were studied in primary murine models. The specified model was performed essentially as described by Edwards et al. [93], and characterized the end result of infiltration of cells into the air bag.

The Protocol model

The air bag was created on the back of a female Balb/C mice by subcutaneous injection of 2.5 ml of sterile air at 0 day. The air bag was again inflated the additional volume of 2.5 ml of sterile air on day 3. 2 µg huIL-13 0.75% CMC inje Aravali directly in the pouch on the 6th day. After 24 hours, mice were killed, the air bag were washed in 1 ml of saline solution with heparin. Treatment with antibodies or together with huIL-13 (in the bag), or conducted in a systematic manner.

Results

IL-13 human injected into the air pouch (i. po.), caused a significant increase in the total infiltration of leukocytes (p<0.01) and eosinophils (p<0,01) 24 hours after provocation compared with mice treated filler (0,75% carboxymethylcellulose (CMC) in physiological solution i. po.).

Locally entered BAK502G9 (200 mg, 20 mg, or 2 mg inside of the bag) and significantly dependent on the dose way inhibited the infiltration of total leukocytes (p<0.01) and eosinophils (p<0.01) in the air bag caused 2 µg huIL-13 0.75% CMC.

Systematically introduced BAK209B11 (30 mg/kg, 10 mg/kg and 1 mg/kg) also significantly dependent on the dose way inhibited the infiltration of total leukocytes (p<0.01) and eosinophils (p<0.01) in the air bag caused 2 µg huIL-13 0.75% CMC.

EXAMPLE 17

The creation of transgenic mice the introduced gene IL-13 person/knocked out by IL-13 mice in order to assess the effectiveness of antibodies against IL-13 human models of allergic inflammation of the lungs

The authors of the present invention have created mice that Express a human, but not mouse IL-13, through targeted changes in genes. Gene IL-3 mouse was replaced from the start codon to the stop codon of the corresponding part of the gene IL-13 human. The resulting mouse line expresses IL-13 human, but not mouse IL-13 in response to the same stimuli as the mouse wild type, because the endogenous promoter of IL-13 and pA-tail IL-13 remained unchanged. It is shown that IL-13 can be contacted and to transmit a signal through the receptor IL-13 mice, leading to the same physiological effects as the signal transmission caused by IL-13 mouse-related receptors IL-13 mouse. For example, exogenous IL-13 human caused mobilization of inflammatory cells into the air pouch of mice (Fig). Transgenic animals has allowed the inventors to evaluate nematinae cross-reacting antibodies against IL-13 human rights in developed mouse models of the disease.

Received the mouse used in models of acute allergic airway inflammation (which are described in examples 18 and 19) and models of chronic allergic airway inflammation (described in example 20), which provided an assessment of the pharmacological treatment with antibody against IL-13 human allergic airway disease.

EXAMPLE 18

The effectiveness of antibodies against IL-13 human rights in a murine model of Lloyd acute inflammation of the lungs based on transgenic huIL-13 mice

Murine model of acute allergic inflammation of the lungs

The effect of neutralizing the I-13 human antibodies in the form of a human IgG4 investigated in a murine model of acute allergic inflammation of the lungs, using transgenic mice, generated in example 17. This model was carried out essentially as described by McMillan et al. [85] and as described in example 13. The final result of the model is characterized by an increased content of IL-13 in BAL and lung tissue, cellular infiltration into the lungs and BAL, elevated levels of IgE in serum and hypersensitivity of the respiratory tract (AHR).

The Protocol model

The Protocol of this model was the same as described in example 13, except that the injected dose of antibody against IL-13 human instead BAK209B11.

EXAMPLE 19

The effectiveness of antibodies against IL-13 human rights in a murine model of Gerard acute inflammation of the lungs based on transgenic huIL-13 mice

Murine model of acute allergic inflammation of the lungs

The effect of neutralizing IL-13 human antibodies in the form of a human IgG4 investigated in another murine model of acute allergic inflammation of the lungs, using transgenic mice, generated in example 17. This model was carried out essentially as described Humbles et al. [86] and as described in example 14. The final result of the model is characterized by an increased content of IL-13 in BAL and lung tissue, cellular infiltration into the lungs and BAL, elevated levels of IgE in serum and hypersensitivity of the respiratory tract (AHR).

The Protocol model

The Protocol of this model was the m, as described in example 14, except that the injected dose of antibody against IL-13 human instead BAK209B11.

EXAMPLE 20

The effectiveness of antibodies against IL-13 human rights in a murine model of Lloyd chronic inflammation of the lungs based on transgenic huIL-13 mice

The effect of neutralizing IL-13 human antibodies in the form of a human IgG4 investigated in a model of chronic allergic inflammation of the lungs, using transgenic mice, generated in example 17. This model was carried out essentially as described Temelkovski et al. [87] and as described in example 15, and the end result is characterized by cellular infiltration in the lungs and BAL, elevated levels of IgE in serum and hypersensitivity of the respiratory tract (AHR).

The Protocol model

The Protocol of this model was the same as described in example 15, except that the injected dose of antibody against IL-13 human instead BAK209B11.

EXAMPLE 21

Pharmacokinetics and pharmacodynamics of antibodies against IL-13 human in macaques-Griboedov allergic to Ascaris suum

The pharmacokinetics and pharmacodynamics 502G9 was evaluated in 4-allergenic, but not subjected to provocations primates macaques-Griboedov (2 males/2 females) after a single bolus dose of 10 mg/kg/C. the Experiment lasted for 29 days. Pharmacokinetic parameters antibodies determined on the basis of the curve of the compound is the concentration of drug in serum and described in detail below in table 4.

The same study also monitored the concentration of IgE in serum, using a test kit IgE person in ELISA (Bethyl laboratories, USA).

Results

The concentration of IgE in serum was significantly decreased after a single bolus dose of 10 mg/kg/BAK502G9, from 100% in control (prior to dose) to 66±10% from the values in the control (p<0,05) on 4 and 5 day post dose. The aforementioned decrease in the concentration of IgE in the serum was recovered to 88±8% from the levels in the control on day 22 (see Fig). These data also were obtained by normalizing the concentration of IgE in the serum of each animal with respect to the levels before the introduction of the dose, the concentrations to dose was 100%, and then averaged curves for the 4 test animals.

Two male monkeys had a relatively low total IgE levels in serum up to a dose of 60 ng/ml and 67 ng/ml). These IgE levels did not change in a way that shows any tendency after processing BAK502G9 (pigv). Two female monkeys had relatively high levels of total IgE in serum (1209 ng/ml and 449 ng/ml). These IgE levels decreased after treatment BAK502G9 maximum 60% on day 7 and returned approximately to levels before the introduction of dose on the 28th day after administration (pigv). The data obtained show that BAK502G9 reduces the concentration of IgE in the serum of the animal is x with relatively high levels of circulating IgE.

EXAMPLE 22

The effectiveness of antibodies against IL-13 human models skin allergies in macaques-Griboedov

The effect of neutralizing IL-13 human antibodies in the form of a human IgG4 investigated in a model of acute allergic inflammation of the skin of primates. The specified model was carried out by injection of IL-13 on human and antigen A. suum intradermal makaka-rabadam. After another 24-96 hour took a skin biopsy and serum samples. The end result in models characterized by infiltration of cells into the skin.

EXAMPLE 23

The effectiveness of antibodies against IL-13 human model of pulmonary Allergy in macaques-Griboedov

The effectiveness of neutralizing IL-13 human antibodies in the form of a human IgG4 investigated in a model of acute allergic inflammation of the lungs in primates. The specified model was implemented, affecting spray antigen A. suum in macaques-Griboedov allergic to A. suum, thus causing an allergic reaction. Specified Allergy characterized ultimately by infiltration of cells into the lungs and BAL, elevated levels of IgE in serum and hypersensitivity of the respiratory tract.

Additionally evaluated the pharmacodynamics ex vivo, using the method of flow cytometry. CD23 is a high-affinity receptor of IgE and can be expressed in mononuclear cells of peripheral blood. The expression of CD23 may be induzirovanna IL-13 and IL-4 in terms of number of cells, expressing CD23, and for how much CD23 expresses each cell. The response mediated by IL-13, but not IL-4, can be Engibarov antibody against IL-13 human.

Animals pre-selected for presentation in 2-phase study on the basis of pre-induced AHR after the provocation spray antigen (extract of Ascaris suum). In phase I the function of the respiratory tract were evaluated during intravenous provocation with histamine 1 and day 11. PC30the dose of histamine needed to cause a 30% increase in lung resistance (RL) above baseline, was determined on the basis of each curve response from the dose of histamine. 9 and day 10, animals were subjected to provocations individually calculated doses sprayed antigen, which, as was shown previously, causes a 40% increase in RLand a 35% reduction in dynamic plasticity (CDYN). Historically in this model, higher RLobserved after the second provocation given dose of allergen than after the first; this means premirovanii antigen. Two antigen provocation caused AHR, which was measured by the increased area under the curve of the dependence of the response on the dose of histamine and/or falling PC30and BAL, and eosinophilia on day 11 compared to 1 day. Animals exhibiting AHR-phenotype were selected for the in troduction the program phase II.

Phase II sang and danced exactly the same as phase I, except that all animals received an infusion of 30 mg/kg BAK502G9 1, 5 and 9 day. The influence of BAK502G9 was assessed by comparing the changes observed in phase II, with the changes observed in phase I, for a single animal.

Collected samples of blood, serum, BAL and lung tissue. The levels of IgE in serum was checked by ELISA. It is shown that the serum of the treated BAK502G9 macaques-Griboedov inhibits the expression of CD23 on mononuclear cells of peripheral blood induced IL-13, but not IL-4. The value specified inhibition corresponded to the levels of BAK502G9 in serum, calculated using ELISA PK.

Results

BAK502G9 significantly inhibited AHR, which was measured by AUC RL(p<0,05) (figa; table 7). Observed inhibitory effect of BAK502G9 on AHR, as measured by PC30but it did not reach statistical significance (figv; table 7). BAK502G9 also significantly inhibited premirovanii both antigens (p<0,01) (figs; table 7) and inflammation BAL. BAK502G9 significantly inhibited the influx of total cells (p<0.05), eosinophils (p<0,05) but not macrophages, lymphocytes or mast cells in BAL (fig.26D; table 7).

EXAMPLE 24

The effectiveness of antibodies against IL-13 in asthmatic phenotype that develops with the introduction of IL-13 human rights in light of the mouse

Murine model GI is ecostiletto respiratory tract

Investigated the efficacy of neutralizing antibodies BAK502G9 against IL-13 man against the development of hypersensitivity of the respiratory tract (AHR), after administration of IL-13 human rights in the lungs of mice. The specified model was performed essentially as described by Yang et al. [119], except that used IL-13 human instead of mouse IL-13.

The Protocol model

For the development of the phenotype in BALB/c mice acted with two doses of IL-13 human, divided 48-hour intervals. Briefly, mice were anestesiologi intravenous injection of 100 μl of a solution of saffan (dilution 1:4 in water). Mice were intubation catheter needle gauge 22, through which poured recombinant IL-13 person (25 mg dissolved in 20 μl of phosphate-saline buffer (PBS)) or filler as a control (PBS). The function of the respiratory tract was evaluated 24 hours after the last injection of IL-13 with increasing methacholine provocation and registered using plethysmography animals in consciousness (Buxco). PC200(the concentration of methacholine required to increase baseline PenH 200%) was determined on the basis of fitting curves 4 flow parameters for the curves of the response to the dose of methacholine. Treatment antibody was performed by intraperitoneal injection 24 hours before injection of each dose of IL-13.

Result is you

Intratracheal injection of IL-13 human native wild-type mice led to development of significant (p<0,05) hypersensitivity of the respiratory tract compared to the control animals, which were determined based on concentrations methacholine PC200. Systematically introduced BAK502G9 (1 mg/kg) significantly inhibited (p<0.01) development of AHR, whereas antibody zero control had no effect (Fig).

EXAMPLE 25

Neutralizing the effectiveness of BAK502G9 in the form of a human IgG4 against dependent IL-13 human release of IgE from B cells of a person

Protocol analysis switching of B-cells

It is shown that IL-13 induces the synthesis of IgE in B-human cells in vitro [120]. Dependent factor in the release of IgE from B-cells was determined in ELISA. The efficiency of neutralization of BAK502G9 in the form of a human IgG4 were evaluated in relation to dependent on IL-13 human release of IgE from B cells of a person.

Mononuclear cells from peripheral blood (PBMC) were purified from leucocytes film person (Blood Transfusion Service) by centrifugation in a gradient density of 1.077 g/L. B-cells were purified from PBMC using the set for the selection of B-cells II (Miltenyi Biotec)using the reagents and methods described by the manufacturer. A framework for analysis contained environment, Dulbecco, modified by the method of Claims (Life Technologies)containing 10% fetal calf serum and 20 μg/ml transfer the person (Serologicals Proteins Inc). After purification of B-cells resuspendable at a final concentration of 106/ml in the medium for analysis. 50 μl resuspending cells were added to each analyzed point in the 96-well plate for analysis. In the corresponding analyzed well) was added 50 μl of a solution of 4 μg/ml anti-CD40 antibodies EA5 (Biosource). Test solutions of antibodies (six repetitions) was diluted to the desired concentration in the medium for analysis. As a negative control was used irrelevant antibody not directed to IL-13. To the cells was added 50 μl/well of the appropriate test antibody. Then added recombinant obtained in bacteria IL-13 person (Peprotech) to a final concentration of 30 ng/ml, receiving the total amount for the analysis of 200 µl/well. The concentration of IL-13, used for the analysis were chosen to give maximum response. Tablets for analysis were incubated for 14 days at 37°C in 5% CO2. The levels of IgE in nadeshiko was determined in an ELISA using reagents and protocols supplied by the manufacturer (BD Biosciences, Bethyl Laboratories). Data were analyzed using the computer program Graphpad prism.

Results

As shown in Fig, BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16) is able to inhibit dependent on IL-13 human IgE production by B-cells. BAK502G9 in the form of a human IgG4 had IC501.8 nm against 30 ng/ml IL-13 human.

P the EMER 26

The effectiveness of BAK502G9 against mediated IL-13 gain induced by histamine signal Ca2+in primary cells of smooth muscles of bronchial tubes of the person

It is shown that IL-13 directly modulates airway smooth muscles of the respiratory tract [121, 122]. Intracellular mobilization of calcium is a necessary condition for the reduction of smooth muscles. Recent studies have shown that the ability of IL-13 to change the airway smooth muscle is partially mediated by modulating the induced contractile agonist signal Ca2+[123, 124].

The effectiveness of BAK502G9, antibodies against IL-13 human, in the form of IgG4 directed against the mediated IL-13 changes the signal responses of smooth muscle cells of human bronchi (BSMC) on the contractile agonist histamine, investigated in the analysis of the signal Ca2+.

Protocol analysis signal Ca2+in BSMC

Primary human BSMC, the growth medium 2 for smooth muscle cells (SmGM-2) and a basic environment for smooth muscle cells (SmBM) was obtained from Bio Whittaker. BSMC supported in SmGM-2 according to the recommendations of the supplier. BSMC were sown at a density of 2×104cells/well in 96-well plate for micrometrology for tissue culture and allowed to attach for 24 hours, then re-fed and inquire is Ali still within 24 hours. Before the experiment, associated with signal Ca2+, BSMC stimulated IL-13 (Peprotech) at a final concentration of 50 ng/ml in the presence or in the absence of antibody, and incubated for 18-24 hours. BAK502G9 and matched irrelevant isotype control monoclonal antibody, CAT-001, were evaluated at a final concentration of 10 μg/ml of Changes in intracellular concentrations of Ca2+in response to histamine (Calbiochem), titrated from 20 μm, measured using the standard method with sensitive Ca2+dye Fluo-4 (Molecular Probes) and using a card reader to visualize fluorescence 96-hole tablet (FLIPR) (Molecular Devices). The area under the curve (AUC) response signal Ca2+histamine was determined for each condition pre-treatment of cells. Data analysis was performed using GraphPad Prism version 4 for Windows (computer program GraphPad).

Results

Pre-incubation BSMC with IL-13 was significantly increased transmission signal Ca2+in response to histamine. Pre-incubation of BAK502G9 (pigv) (but not irrelevant control antibodies specific isotype (figa)) with IL-13 significantly inhibited the increased transmission signal Ca2+in response to histamine (Fig).

EXAMPLE 27

Neutralizing the effectiveness of anti-IL-13 antibodies in the analysis dependent on IL-13 human expre what these CD23 PBMC

Efficiency typical IL-13 antibodies were evaluated in the analysis dependent on IL-13 human CD23 expression in mononuclear cells of peripheral blood (PBMC). PBMC were answered as IL-13 and IL-4 increased the expression of CD23 on the cell surface [120]. CD23 (FceRII) is a low-affinity IgE receptor and is expressed on a variety of inflammatory cells, including monocytes. The inhibition is dependent on IL-13 human improve the regulation of CD23 expression was determined by measuring the decrease of the binding of fluorescently labeled monoclonal antibodies to CD23 from PBMC using flow cytometry.

Protocol analysis

Human blood was obtained from the Blood Transfusion Service and got rid of the red blood cells through a 40-minute deposition in the dextran-T500 (Pharmacia) (final concentration of 0.6%). Then separated fraction enriched in leukocytes and platelets, using a 20-min centrifugation at 1137g in step Percoll gradient 3 ml of 64% and 5 ml of 80% (100% consists of 9 parts Percoll to 1 part 10×PBS). PBMC were collected from the top 64% of the layer, washed and resuspendable buffer for analysis (Invitrogen RPMI 1640, 10% FCS, 200 IU/ml penicillin, 100 μg/ml streptomycin, 2 mm L-glutamine). The analysis was carried out in 24-hole tablets using 2×106cells ±80 PM of recombinant IL-13 person (Peprotech) or 21 PM recombinant IL-4 (R and D Systems), BAK502G9 or irrelevant Ig4 in the final volume of 500 ál. Cells were cultured for 48 h at 37°C, and then reassembled and painted CD23-PE (BD Pharmingen) for 20 minutes at 4°C. Finally the cells were detected in a flow cytometer. The expression of CD23 was determined using the "evaluation" CD23; by the percentage of CD23-positive cells multiplied by the brightness of the color (geometric average fluorescence). The "evaluation" of CD23 without stimulator is not subtracted and the data are presented as percentage of the response to IL-13, added separately (100%). Data were expressed as average ± SEM obtained on the basis of 4-6 separate experiments using cells from 4-6 individual donors, carried out in three repetitions for each point.

Results

Incubation of PBMC with 80 gr IL-13 or 21 PM IL-4 for 48 hours resulted in a marked expression of CD23 (Fig and Fig). BAK502G9 dependent dose-dependent manner inhibited induced IL-13 expression of CD23 with geometric average 120,2 PM (Fig). On the contrary, BAK502G9 did not possess the ability to inhibit the expression of CD23 induced 21,4 PM IL-4 (n=4 individual donor, Fig). Irrelevant IgG4 not inhibited either IL-13 or IL-4-dependent expression of CD23 on PBMC (Fig and Fig). Joint stimulation of PBMC 80 gr IL-13 and 21.4 PM IL-4 was given additive CD23-response. BAK502G9, but not CAT-001, reduced levels of CD23 expression to levels observed during stimulation separately IL-4 (Fig).

EXAMPLE 28

Neutralizing the effectiveness of antibodies against IL-13 che is owaka in the analysis dependent on IL-13 human shape changes of eosinophils

The aim of this study was to evaluate the effect of IL-13 antibodies on the change in the shape of eosinophils induced by mediators released from NHLF after stimulation factors found in the lungs of patients with asthma, such as IL-13 [125, 126], TNF-α [127], TGF-β1 [128]. IL-13 acts synergistically with TNF-α [129] or TGF-β1 [130]inducyruya fibroblasts for the production of eotaxin-1, which can then act as a chemoattractant eosinophils. Changing the shape of leukocytes is mediated by reorganization of the cytoskeleton of cells and is essential for the migration of leukocytes from the microcirculation in the place of inflammation. The dependent inhibition of IL-13 release factors that induce shape change, from NHLF was determined by measuring reducing the secretion eotaxin-1 in ELISA and reduce changes in the shape of eosinophils using flow cytometry.

Protocol analysis

NHLF cells were cultured in medium containing no additives, or in a medium containing stimulants (9,6 nm IL-13, 285,7 PM TNF-α (R & D Systems) and 160 PM TGF-β1 (R & D Systems) in the absence or in the presence of BAK502G9 (concentration range 875 nm - 6,84 nm). Then cells were cultured for another 48 h at 37°C, then the resulting conditioned medium was aspirated and stored at -80°C. the Concentration eotaxin-1 in the conditioned medium was evaluated using the ELISA system R ad D systems Duoset (R & D Systems).

Human blood was obtained from the Blood Transfusion Service and got rid of the red blood cells through a 40-minute deposition in the dextran-T500 (Pharmacia) (final concentration of 0.6%). Then separated fraction enriched in leukocytes and platelets, using a 20-min centrifugation at 1137g in step Percoll gradient 3 ml of 64% and 5 ml of 80% (100% consists of 9 parts Percoll to 1 part 10×PBS). Granulocytes were collected at the interface 64%:80%, washed and resuspendable buffer for analysis (Sigma PBS, 1 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, 10 mm D-glucose, 0.1% BSA, Sigma, pH 7.3). The analysis was performed in FACS tubes containing 5×105cells ±3 nm recombinant eotaxin-1 person (R & D Systems) or air-conditioned environment in the final volume of 400 ál. Cells were incubated for 8.5 minutes at 37°C, then transferred to 4°C and fixed with a fixative (CellFix, BD Biosciences) and finally recorded in a flow cytometer. Eosinophils were identified by their FL-2-autofluorescence and registered option direct scattering (FSC). Parameter FSC eosinophils were changed in response as eotaxin-1 and air-conditioned environment, providing the ability to measure changes form. The contents of the tubes were analyzed at high speed and the data collection was stopped after 1000 events the occurrence of eosinophils or after 1 minute, regardless of what it was faster. Change fo what we expected in the form of a percentage of the FSC, called only one buffer to change the shape (100% shape change in control). Data were expressed as mean % change forms control ± SEM derived from 4 separate experiments. In each experiment used cells from individual leukocyte films (and hence, from a single donor) and the experiment was performed in two iterations for each point.

Results

NHLF cells, costimulatory 9,6 nm IL-13, 285,7 PM TNF-α and 160 PM TGF-β1 and cultured for 48 h, secretively 9,6 nm eotaxin-1 into the culture medium. On the contrary, NHLF cells, cultured only supportive growth environment, secretively 0.1 nm eotaxin-1 into the culture medium. The specified production eotaxin-1 was dependent on IL-13, as BAK502G9 dependent dose-dependent manner inhibited the production of eotaxin-1 cells NHLF, costimulatory IL-13/TNF-α/TGF-β1, with IC5032,4 nm (figa).

The main aim of this part of the study was to determine the changes in the shape of eosinophils. The change in shape of eosinophils in response to 3 nm eotaxin (positive control) was 122,2±2,1% (n=4). Induced eotaxin-1 change form completely inhibited 100 nm antibody against eotaxin CAT-213, an average shape change 101,0±1,0% (n=4).

Environment from NHLF cells, costimulatory 9,6 nm IL-13, 285,7 PM TNF-α and 160 PM TGF-β1 and cultured in t the value of 48 hour (air-conditioned environment), induced a clear change in the shape of eosinophils (pigv). On the contrary, from NHLF cells, cultured for 48 hours only in supporting the growth of cells NHLF environment, not induced shape changes of eosinophils (pigv).

Adding anti-IL-13 antibodies BAK502G9 to the environment to costimulatory before cultivation NHLF led to a dependent dose-dependent inhibition of changing the shape of eosinophils with geometric mean IC5016,8 nm when assayed in a dilution of 1:16 (pigv).

Also investigated the ability of stimulants (IL-13, TNF-α and TGF-β1), does not enter into the culture of NHLF cells, to induce a change in the shape of eosinophils and neutrophils. 9,6 nm IL-13, 285,7 PM TNF-α and 160 PM TGF-β1 did not induce visible changes in the shape of eosinophils. This suggests that the ability of the air-conditioned environment to change the form of eosinophils, which develops during the cultivation of cells NHLF with stimulants, arises not because of any stimulator separately or in combination (pigv).

EXAMPLE 29

Mapping of anti-IL-13 antibodies to IL-13 human

Epitope mapping typical IL-13 antibodies BAK502G9 was carried out using the molecular method and standard cutting peptides.

Molecular method

Designed chimeras IL-13, in which parts of the sequence of IL-13 man replaced the murine sequence. The resulting Chimera is used in studies linking with typical IL-13-antibodies to identify specific epitope.

Received two panels of chimeras IL-13. The first panel consisted of nine chimeras (Fig) and was used to locate the General position of the epitope. The second panel contains ten chimeras (Fig) and was used for fine mapping of the epitope.

Chimeric sequences of IL-13 were collected using PCR, and cloned in the vector for the introduction of Gateways®, which is then recombinable with the target vector pDEST8 (modified so that it encodes registered and affinity tag at the C-end of the recombinant protein). These expressing vectors were used to transform chemically competent E. coli DH10Bac™, providing the opportunity for site-specific transposition labeled chimeric IL-13 in a baculovirus Shuttle vector (bacmid). Recombinant backmenu DNA was isolated for each Chimera IL-13 and transfusional in insect cells Sf9 (Spodoptera frugiperda)using Cellfectin reagent®. Recombinant baculoviruses were harvested 72 hours after transfection and were passively in insect cells Sf9 two more times.

Adosados culture volume 2000-500 ml purified on affinity column and suirvey material was concentrated from 16 to 1 ml and was applied on the column for exclusion chromatography size Superdex 200 HR10/300GL for final processing and replacement of the buffer.

Bit is a Protocol homogeneous competitive analysis, using biotinylated IL-13 human streptavidin-Anafiotika and labeled with europium BAK502G9. The analysis was carried out as follows: Eu-BAK502G9 binds biotinylated IL-13 human, then the complex is recognized by the conjugate streptavidin-APC, and in the case of the flash light is energy transfer from APC-labels to europium proximity, and can be measured fluorescence with time resolution. Competition specified create binding by unlabeled IL-13 (quality control) and chimeric constructs. The specified competition assess quantitatively to calculate the relative affinity of the mutant IL-13 in relation to IL-13 antibodies, which makes possible the identification of mutations that alter the binding.

Results

Found that chimeric design IL13-helix D (table 5) is the weakest competitor for biotinylated IL-13 person for binding BAK502G9, indicating that helix D in the molecule IL-13 is involved in epitope binding BAK502G9 (table 5). Reduced activity was also observed in the case of mutants 4041 and 3334, in which residues 40, 41 and 33, 34 of the original sequence, respectively, were changed, indicating a possible involvement of helix A in recognition of BAK502G9. Reduced activity loop 3 has not been taken into account, as specified loop has IU lsee the number of amino acids in the mutant compared with the molecule man, and is likely to change the overall structure of the protein. Other decrease in the ability of chimeric molecules IL-13 competes for binding BAK502G9 not considered significant in the case of such amino acid changes.

Then tested a set of more targeted mutations in helix D (Fig). The results obtained are shown in table 6 and are as follows.

The results show that chimeric constructs 116117TK (in which lysine at position 116 is replaced by threonine and aspartate at position 117 is replaced with lysine), 123KA (which is replaced by lysine at position 123) and 127RA (which is replaced by arginine at position 127) have the least ability to compete for binding with BAK502G9 (123KA and 127RA not compete with 1 μm). Other residues involved in the binding of BAK502G9 on the basis of reduced efficiencies in competitive analysis, include the remains of a spiral D 124Q (where lysine is replaced by glutamine) and 120121SY (pair leucine-histidine is replaced by a pair of serine-tyrosine). Mutation of leucine in position 58L also reduces the binding, and analysis of 3D structures showed that this residue is Packed against helix D and can either directly contact the BAK502G9, or may affect the alignment of helix D.

The experiments show that the residues in helix D is important for the binding of BAK502G9 with IL-13. In particular, the lysine at position 123 and arginine in point is the situation 127 important for the specified binding, since the mutation in any of them ceases to be binding BAK502G9.

Cut epitope

Epitope mapping of BAK502G9 also carried out using the standard method of cutting peptide. In this case IgG was immobilized on the solid phase and gave the opportunity to catch the ligand IL-13. Then the complex formed is subjected to specific proteolytic cleavage, which was split available peptide bonds, ties, secure the contact area of IgG:the ligand remained intact. Thus, the peptide containing the epitope remained associated with IgG. Then it can be decarbonate, collect and identify mass spectrometry (MS).

Used two additional ways, used in the first mass spectrometer MALDI-TOF to read protein chips Ciphergen, this could covalently bind IgG with chip for mass spectrometer and then to perform the splitting and extraction in situ. The second method used biotinylated BAK502G9 associated with the streptavidin coated beads, and provide the capability of collecting a sufficient amount of the peptide to confirm the sequence of tandem mass spectrometry (MS/MS).

Two ways though differed in real detail and scale, essentially consisted of the same stage, the IgG binding, blocking neproreagirovavshimi the x binding sites, flushing, the capture ligand, removing unbound ligand cleavage and final stage of washing.

In the method MALDI-TOF-MS used a proprietary MS-chips, activated carbonyl diimidazol, which covalently binds with the free primary amino groups, which were bound IgG at a concentration of 1-2 mg/ml in PBS overnight at 4°C. Then the chip was blocked with a solution of ethanolamine at room temperature for 1 hour and then thoroughly washed with PBS or HBS plus suitable detergent. Then the chip was applied to the aliquot containing one picomole IL-13, either in PBS or HBS, and gave the opportunity to contact with chemically immobilized IgG for 2 hours at room temperature. Then spent an additional wash in PBS or HBS with detergent or without it, to remove any not specific associated IL-13. Then was added a solution of trypsin in the range from 200 to 3.1 μg/ml in PBS or HBS for IgG complex:ligand and provide the capability of splitting for 30 minutes at room temperature, after which the chip was rinsed in PBS or HBS plus detergent, PBS or HBS and finally with water. Then, after applying a suitable matrix for MALDI-TOF MS, the chip was placed directly into the mass spectrometer and analyzed.

Based on balls way started with biotinidase IgG using the connection NHS-Biotin in the molar ratio of the attachment 1 IgG at 4 molecules of Biotin. After this was followed by removal of unbound Biotin and side reaction products using gel filtration. Then gave the opportunity biotinylating IgG to contact covered neutravidin agarose beads, while trying to maximize the capture of IgG. Then aliquots coated with IgG beads were distributed on concentrating centrifuge the column and washed with PBS, Dulbecco + 0.05% tween-20, then resuspendable in PBS, Dulbecco + 0.05% tween-20. Then to resuspending IgG-beads pulse was added to IL-13 and provided the opportunity for the occurrence of binding for 10 minutes, then the liquid phase was removed by centrifugation and the beads were washed in PBS, Dulbecco + 0.05% tween-20, followed by resuspending in PBS, Dulbecco + 0.05% tween-20.

Then the complex ball:IgG:the ligand was subjected to proteolysis by either trypsin or chymotrypsin, incubare at room temperature or at 37°C. then the beads were again washed in PBS, Dulbecco + 0.05% tween-20, followed by additional washes in PBS, Dulbecco with detergent. Then the balls resuspendable in a mixture of water, acetonitrile, triperoxonane acid and extracted adosados. Then adosados analyzed in various ways, either by MALDI-TOF MS, or HPLC with reversed phase and mass spectrometry, including tandem (MS/MS) fragmentation using mass when astrometry with ion trap ThermoQuest LCQ ESI. Then attempt to match the resulting fragmentation pattern with a sequence of IL-13 human and a separate sequence heavy and light chain IgG BAK502G9.

During the series of experiments used a number of controls are initially empty surfaces, IgG separately and isotype controls to show that the identified peptides have received specific IgG-captured IL-13, and they are not the product of cleavage of BAK502G9 or not specific associated IL-13.

Results

In a series of experiments in the same way received a single specific peptides of IL-13 for each splitting. The data obtained with the help of the device with ion trap LCQ, showed that obtained by treatment with trypsin fragment had monoisotopic mass 3258 E (MH+), and the fragment obtained by treatment with chymotrypsin - monoisotopic mass 3937 D (MH+).

The search for the specified mass compared with a suitable splitting of the in silico IL-13 man gave a close match with related peptides in the C-terminal part of the molecule.

The coincidence of the mass of the peptide obtained by treatment with trypsin:3258 D

When tolerance 1000 ppm 3258 D coincides with the sequence from aspartic acid at position 106 to C-terminal asparagine at position 132. This tolerance of other matches there. The area in bold is the sequence form of precursor IL-13 human below.

The coincidence of the mass of the peptide obtained by treatment with chymotrypsin:3937 D

When tolerance 1000 ppm 3937 D coincides with the sequence from serine at position 99 to C-terminal asparagine at position 132. The area highlighted in bold in the sequence form of precursor IL-13 human below.

Both of these matches show that IgG BAK502G9 retains the C-terminal part of the molecule IL-13 during proteolysis of the complex antibody:ligand.

The identity of both peptides was successfully confirmed by MS/MS, for none of them did not observe any substantial parallel sequences with BAK502G9. On the map fragment in MS/MS, is designed to identify either the Y-or B-ions, coincided 26 of 104 possible ions charged in one area in case of a peptide obtained by treatment with trypsin, and 19 out of 128 possible ions in the case of a peptide obtained by treatment with chymotrypsin. Browse all charged sites demonstrates the identification of 23 out of 27 amino acid residues in the case trypsinogen fragment and 29 of 33 residues in the case chymotrypsinogen fragment. This is enough to confirm identity.

In a series of experiments in General has identified this part of the epitope BAK502G9 in IL-13 human, which is located within the far East is dcate seven C-terminal amino acid residues. The obtained results confirm the results of the molecular method, described in detail above.

IgG
Table 2
The binding specificity of antibodies against IL-13 human
IL-13 humanA variant IL-13 humanIL-13 Primate, non-human
BAK278D6+++
BAK502G9+++
BAK615E3+--
Table 3a
Kinetic analysis of antibodies against IL-13 human
The dissociation rate (sec-1)The rate of Association (M-1s-1)KD (PM)
BAK278D67,41e-35,49E513500
BAK502G94,09e-42,49e6178
BAK1167F24,05e-42,99e6136
BAK1183H43,00e-43,7e681
Table 3b
Kinetic analysis of antibodies against IL-13 mouse
IgGThe dissociation rate (sec-1)The rate of Association (M-1s-1)KD (PM)
BAK209B111,98e-23,9e65100

Table 4
Pharmacokinetics BAK502G9 4 allergic, but not subjected to provocations primates macaques-Griboedov (2 males/2 females) after a single bolus/dose 10 mg/kg for 29 days. Levels of BAK502G9 in serum were measured by ELISA (mean values)
Cmax(t=0.25 h) (μg/ml)349,04
Vdinf(ml·kg-1)75,03<80 ml/kg, means no fabric-binding
Clinf(ml·h-1·kg-1)0,23
AUCinf(mg·h·ml-1)42,99
AUCext(%)17,34<30%, so the clearance and volume of distribution must be accurate
T0,5(h)223,55
Vdinf= volume of distribution during the time 0 to infinity, calculated on the basis of the extrapolated AUC.
Clinf= clearance over time 0-beskonechnost is, calculated on the basis of the extrapolated AUC.
AUCinf= area under the curve (a measurement of the total exposure to drug) during time 0 to infinity, including the extrapolated period based on the rate constant of elimination (k) and the last detectable concentration of the drug in serum.
AUCext= the percentage of the AUC of the total AUC, which is extrapolated.
T0,5= the half-life of the drug in the final phase of elimination.

Table 5
The first set of chimeric constructs
Chimeric constructsIC50nm
BAK502G90,17±0,07
Loop 10,71±0,35
hum-flag1,30±0,18
30R1,76±0,45
3738VN1,89±1,9
Helix B2,49±0,88
Spiral C4,11±0,70
Loop 3the 5.45±3,96
404112,02±1,3
333412,17±1,2
Helix D110,07±9,9
Table 6
The second set of chimeric constructs
Chimeric constructsIC50nm
BAK502G90,11±0,04
113H1,6±0,5
128H1,6±1,0
119LA1,96±1,0
130P2,22±0,8
120121SYto 4.73±1,5
58LA5,2±2,0
124Q18,7±15,9
116117T 82±11,3
123KANo
127RANo

Table 7
The influence of BAK502G9 on different pre-defined end-results
The change in the phase INThe change in the phase IINThe end result
AHR (RLAUC)0,020±0,00314a0,004+0,00614a-0,016±0,006*
AHR (PC30)-1,343±0,31818b1,061±0,24418b0,282+0,179
Premirovanii antigen (RLAUC)strength of 0.159+0,03320with0,033+0,02520with0,126+0,043**
Total cells in BAL20,623±3,16021d14,597+1,95121d-6,026+2,194*
Eosinophils in BAL18,453+3,00921d13,412+1,73721d-5,041+2,090*
Mononuclear cells in BAL2,050+0,43821d1,176±0,48121d-0,874+0,506
21 animal expressing AHR (PC30in phase I, and additional animal with the phenotype of premirovany antigen translated for testing in phase II (22). Not every animal had AHR, which was measured as AUC, and PC30. Only those animals that showed AHR in phase I and AHR which has been evaluated in phase I and phase II, were included in the analysis of the results of AHR. A statistical test was carried out using InStat. The test was a test of the null hypothesis using two-sided t-student criterion that the final result did not include the number 0 (i.e. no change in the phase II is compared with the phase I); *p<0,05, **p<0,01. Data are shown as arithmetic average ± SEM (n=14-21).
a5 animals were excluded from the analysis of the AUC, as they did not show AHR (increased AUC) in phase I.
3 the following animals were excluded due to technical failure during data collection on the functioning of the respiratory tract in phase II.
b3 animals were excluded from the analysis PC30due to technical problems during data collection on the functioning of the respiratory tract in phase II (same animals as in case a). Additional animal with the phenotype of premirovany antigen was excluded because it was not observed PC30AHR in phase I.
c2 animals were excluded from the analysis premirovany antigen due to technical problems during data collection on the functioning of the respiratory tract in phase I.
d1, the animal was excluded from the analysis of BAL due noticeable inflammation BAL in the beginning of the study.

1. An isolated specific binding partner for IL-13 human containing antigennegative plot antibodies, which consists of a VH domain of the antibody is human and domain VL of human antibodies and which contains a set of CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the domain VH contains HCDR1, HCDR2 and HCDR3, and VL domain contains LCDR1, LCDR2 and LCDR3, and set the CDR consists of a set of CDRs selected from the group consisting of
set CDR BAK278D6, which, by definition,
HCDR1 has amino acid sequence SEQ ID NO: 1,
HCDR2 has amino acid sequence SEQ ID NO: 2,
HCDR3 has amino acid sequence SEQ ID NO: 3,
LCDR1 has amino acid sequence SEQ ID NO: 4,
LCDR2 has amino acid sequence SEQ ID NO: 5 and LCDR3 has amino acid sequence SEQ ID NO: 6,
set CDR from table 1 with an additional one or two amino acid substitutions as compared to CDR set BAK278D6, and
each set of CDR, which is shown for individual clones in table 1.

2. An isolated specific binding partner according to claim 1, where one or two substitutions affect one or two of the following residues in the CDR, when used in the research Institute of standard numbering according to Kabat:
31, 32, 34 in HCDR1
52, 52A, 53, 54, 56, 58, 60, 61, 62, 64, 65 in HCDR2
96, 97, 98, 99, 101 in HCDR3
26, 27, 28, 30, 31 in LCDR1
56 in LCDR2
95A, 97 in LCDR3.

3. An isolated specific binding partner according to claim 2, where one or two substitutions carried out in the following provisions from among the identified groups of possible residues of alternates for each position:

The position of substitutionThe residue substituent selected from the group consisting of
31in HCDR1:Q, D, L, G and E
32in HCDR1:T
34in HCDR1:V, I and F
52in HCDR2:D, N, A, R, G, and E
52Ain HCDR2:D, G, T, P, N and Y
53in HCDR2:D, L, A, R, T, S, I and R
54in HCDR2:S, T, D, G, K and I
56 T, E, Q, L, Y, N, V, A, M and G
58in HCDR2:I, L, Q, S, M, H, D and K
60in HCDR2:R
61in HCDR2:R
62in HCDR2:K and G
64in HCDR2:R

65in HCDR2:To
96in HCDR3:R and D
97in HCDR3:N, D, T and R
98in HCDR3:R
99in HCDR3:S, A, I, R, P and K
101in HCDR3:Y
26in LCDR1D and S
27in LCDR1: I, L, M, C, V, K, Y, F, R, T, S, A, H, and G
28in LCDR1:V
30in LCDR1:G
31in LCDR1:R
56in LCDR2:T
95Ain LCDR3:N
97in LCDR3:I

4. An isolated specific binding partner according to claim 3, which has two substitutions compared with a set of CDR BAK278D6, HCDR3 replacement residue at position 99 and LCDR1 replacement of the residue in position 27.

5. An isolated specific binding partner according to claim 4, containing a set of CDR BAK278D6 with the substitution of the residue at position 99 in HCDR3 selected from the group consisting of S, A, I, R, P and K and/or a substitution at position residue 27 in LCDR1 selected from the group consisting of I, L, M, S, V, K, Y, F, R, T, S, A, H, and G.

6. An isolated specific binding partner according to claim 4, containing a set of CDR BAK278D6 with replacement of N by S at position 99 in HCDR3 and/or replacement of residue N at I at position 27 in LCDR1.

7. An isolated specific binding partner according to claim 2, in which the HCDR1, HCDR2 and HCDR3 of the VH domain nah who are within the frame of the germ line and/or LCDR1, LCDR2 and LCDR3 domain VL are in the frame of the germ line.

8. An isolated specific binding partner according to claim 7, in which the HCDR1, HCDR2 and HCDR3 of the VH domain are within the framework germline VH1 DP14.

9. An isolated specific binding partner according to claim 7, in which the HCDR1, HCDR2 and HCDR3 of the VH domain are within the framework germline VL Vλ3 3h.

10. An isolated specific binding partner according to claim 2, which additionally binds the variant IL-13 human, in which arginine at position 130 is replaced by glutamine.

11. An isolated specific binding partner according to any one of claims 1 to 10, which additionally binds IL-13 non-human Primate.

12. An isolated specific binding partner according to claim 11, in which IL-13 is non-human Primate is IL-13 macaque-rhesus or macaques of having.

13. An isolated specific binding partner of claim 8, containing a VH domain BAK502G9 (SEQ ID NO: 15).

14. An isolated specific binding partner of claim 8, containing a VL domain BAK502G9 (SEQ ID NO: 16).

15. An isolated specific binding partner for 13 containing a VL domain BAK502G9 (SEQ ID NO: 16).

16. An isolated specific binding partner according to any one of claims 1 to 10, 13 or 15, which binds IL-13 with an affinity equal to or greater than the affinity of antigennegative plot for IL-13, formed VH domain BAK502G9 (SEQ D NO: 15) and the VL domain of BAK502G9 (SEQ ID NO: 16), moreover, the affinity of the specific binding partner and the affinity antigennegative area is determined in the same conditions.

17. An isolated specific binding partner according to any one of claims 1 to 10, 13 or 15, the binding with IL-13 human leads to neutralization of IL-13 human.

18. An isolated specific binding partner for 17, which neutralizes IL-13 human with efficiency equal to or greater than the efficiency antigennegative plot for IL-13, formed VH domain BAK502G9 (SEQ ID NO: 15) and the VL domain of BAK502G9 (SEQ ID NO: 16), and the effectiveness of the specific binding partner and efficiency antigennegative area is determined in the same conditions.

19. An isolated specific binding partner according to any one of claims 1 to 10, 13 or 15, which contains a molecule of antibody scFv or constant region of the antibody, or whole antibody, where the whole antibody is not necessarily IgG4.

20. Isolated VH domain of an antibody comprising a specific binding partner according to any one of claims 1 to 19.

21. Isolated VL domain of an antibody comprising a specific binding partner according to any one of claims 1 to 19.

22. Composition for treatment associated with IL-13 disorders containing a specific binding partner, VH domain or VL of the antibody according to any one of claims 1 to 21 and at least one additional the component.

23. The composition according to item 22, containing pharmaceutically acceptable excipient, filler or carrier.

24. An isolated nucleic acid encoding a specific binding partner or VH domain or VL of the antibody comprising a specific binding partner according to any one of claims 1 to 21.

25. A host cell transformed in vitro nucleic acid according to paragraph 24, to obtain specific binding partner, or VH domain, or VL of the antibody according to any one of claims 1 to 21.

26. A method of obtaining a specific binding partner, or VH domain, or VL of the antibody according to any one of claims 1 to 21 by culturing host cells under A.25.

27. The method according to p, further comprising isolation and/or purification of the specified specific binding partner or variable domain of the VH or VL of the antibody.

28. The method of obtaining antigennegative domain antibodies that are specific against IL-13 human, the method includes
getting through additional substitution in accordance with table 1 of one or two amino acids in the amino acid sequence of CDR of the original VH domain containing the HCDR1, HCDR2 and HCDR3, where HCDR1, HCDR2 and HCDR3 of the original VH domain is a set of HCDR BAK278D6, which, by definition, HCDR1 has amino acid sequence SEQ ID NO: 1, HCDR2 has amino acid sequence SEQ ID NO: 2, and HCDR3 kelamangalam sequence of SEQ ID NO: 3, or a set of HCDR BAK502G9, which, by definition, HCDR1 has amino acid sequence SEQ ID NO: 7, HCDR2 has amino acid sequence SEQ ID NO: 8, HCDR3 has amino acid sequence SEQ ID NO: 9, VH domain, which is a variant amino acid sequence of the original VH domain, and optionally combining the thus obtained VH domain with one or more VL domains, obtaining one or more combinations of VH/VL;
and testing of the specified domain VH, which is a variant of the amino acid sequence of the original VH domain, or a combination or combinations of VH/VL, with the identification of antigennegative domain antibodies that are specific against IL-13 human.

29. The method according to p, in which the amino acid sequence of the original VH domain selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 15.

30. The method according to p, wherein said one or more VL domains get by means of additional substitution in accordance with table 1 of one or two amino acids in the amino acid sequence of CDR of the original VL domain containing LCDR1, LCDR2 and LCDR3, where LCDR1, LCDR2 and LCDR3 of the source domain VL is a set of LCDR BAK278D6, which, by definition, LCDR1 has amino acid sequence SEQ ID NO: 4, LCDR2 has amino acid sequence SEQ ID NO: 5, LCDR3 has and is inoculate sequence of SEQ ID NO: 6, or set LCDR BAK502G9, which, by definition, LCDR1 has amino acid sequence SEQ ID NO: 10, LCDR2 has amino acid sequence SEQ ID NO: 11, LCDR3 has amino acid sequence SEQ ID NO: 12, one or more VL domains, each of which is a variant of the amino acid sequence of the source domain VL.

31. The method according to item 30, in which the amino acid sequence of the original VL domain selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 16.

32. The method according to any of p-31, further comprising receiving antigennegative site of the antibody molecule IgG antibodies, scFv or Fab.

33. A method of obtaining a specific binding partner that binds IL-13 human, the method includes
receipt of the initial nucleic acid that encodes a VH domain, or starting repertoire of nucleic acids, each of which encodes a VH domain, VH domain or the VH domains or contain HCDR1, HCDR2 and/or HCDR3 that need replacement or deprived region HCDR1, HCDR2 and/or HCDR3;
merge the specified source nucleic acid or starting repertoire with donor nucleic acid or donor nucleic acids encoding the amino acid sequence of HCDR1 (SEQ ID NO: 1) or HCDR1 (SEQ ID NO: 7), HCDR2 (SEQ ID NO: 2) or HCDR2 (SEQ ID NO: 8) and/or HCDR3 (SEQ ID NO: 3) or HCDR3 (SEQ ID NO: ) or obtained through additional substitution in accordance with table 1 of one or two amino acids in the above amino acid sequences,
so that this donor nucleic acid, or specified donor nucleic acid has been integrated into the region CDR1, CDR2 and/or CDR3 in the original nucleic acid or starting repertoire, so as to obtain a product repertoire of nucleic acids encoding VH domains;
the expression of the nucleic acids of the obtained product in the form of repertoire, with receipt of the products in the form of VH domains;
not necessarily the Union of the specified product in the form of VH domains with one or more VL domains;
the selection of the specific binding partner specific IL-13 human containing the obtained product in the form of a VH domain and, optionally, a VL domain; and
removing the indicated specific binding partner or coding him nucleic acids.

34. The method according to p, according to which donor nucleic acid is obtained mutations specified HCDR1 and/or HCDR2.

35. The method according to p, according to which donor nucleic acid is obtained by mutation HCDR3.

36. The method according to p, including the receipt of donor nucleic acid by mutation of the nucleic acid that encodes the amino acid sequence HCDR3 (SEQ ID NO: 3) or HCDR3 (SEQ ID NO: 9).

37. The method according to any of PP-36, further comprising linking the obtained VH domain, which finds the camping in the extracted specific binding partner, with the constant region of the antibody.

38. The method according to any of PP-36, which includes obtaining molecules of IgG antibodies, scFv or Fab, containing the obtained VH domain and VL domain.

39. The method according to any of p or 33, further comprising testing antigennegative domain antibody or a specific binding partner that binds IL-13 human, in terms of their ability to neutralize IL-13 human.

40. The method according to p, further comprising testing antigennegative domain antibody or a specific binding partner that binds IL-13 human, in terms of their ability to neutralize IL-13 human.

41. The method according to clause 37, further comprising testing antigennegative domain antibody or a specific binding partner that binds IL-13 human, in terms of their ability to neutralize IL-13 human.

42. The method according to § 38, further comprising testing antigennegative domain antibody or a specific binding partner that binds IL-13 human, in terms of their ability to neutralize IL-13 human.

43. The method according to § 39, according to which receive specific binding partner, which contains a fragment of the antibody that binds and neutralizes IL-13 human.

44. The method according to item 43, according to the which the antibody fragment is a molecule of antibody scFv or antibody molecule Fab, where optional is specified, the method further includes obtaining a domain VH and/or VL domain of the antibody fragment in the whole antibody.

45. The use of a specific binding partner or variable domain of the VH or VL of the antibody obtained by the method according to item 27, or a specific binding partner that binds IL-13, antigennegative plot antibodies or variable domain of the VH or VL of the antibody, which contains a specific binding partner, or antigennegative plot antibody that binds IL-13, obtained by the method according to any of PP, 32, 33, 37 and 38, to obtain a composition for neutralizing IL-13, containing at least one additional component.

46. The method according to any of p or 33, further comprising binding a specific binding partner that binds IL-13 human IL-13 or a fragment of IL-13, where the specified binding occurs in vitro.

47. The method according to p, further comprising binding a specific binding partner that binds IL-13 human IL-13 or a fragment of IL-13, where the specified binding occurs in vitro.

48. The method according to clause 37, further comprising binding a specific binding partner that binds IL-13 human IL-13 or a fragment of IL-13, where the specified binding occurs in vitro.

9. The method according to § 38, further comprising binding a specific binding partner that binds IL-13 human IL-13 or a fragment of IL-13, where the specified binding occurs in vitro.

50. The use of a specific binding partner that binds IL-13 according to any one of claims 1 to 21, in the method for determining the number of IL-13 in the tested sample in vitro, under which create conditions for the specified binding to the specific binding partner with IL-13 or a fragment of IL-13 human rights and determine the amount of bound peroxidase specific binding partner with IL-13 or a fragment of IL-13.

51. The use of a specific binding partner, obtained by the method according to any of PP, 32, 33, 37 and 38, for the manufacture of a medicinal product for the treatment of diseases or disorders selected from the group consisting of asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease and Hodgkin's lymphoma.

52. The use of a specific binding partner according to any one of claims 1 to 19 in the manufacture of drugs for the treatment of diseases or disorders selected from the group consisting of asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease and Hodgkin's lymphoma.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. Described are versions of the humanised antibody CD45RO/RB which carry a light and a heavy strand. Versions of the following are disclosed: isolated polynucleotide, coding antibody, expression vector containing a polynucleotide and host cells containing the expression vector. Described also is use of the antibody to treat and/or prevent various diseases, including as a component of a pharmaceutical composition.

EFFECT: invention provides antibodies identified as CD45RO and CD45RB, which can find use in medicine.

9 cl, 14 dwg, 2 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.

EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.

57 cl, 45 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: genetic makers of siSTRIKE-neo vector producing interfering RNA (siRNA), are inhibitors of reproduction of human immunodeficiency virus type 1. Invention allows producing effective anti-HIV preparations of siRNA produced in cells by the administered genetic makers containing palindrome intended for formation of siRNA production and selected with using non-virus and virus models.

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12 cl, 3 dwg, 3 tbl, 3 ex

Product and method // 2385931

FIELD: medicine.

SUBSTANCE: invention concerns biotechnology and represents a new alpha galactosidase, a DNA molecule coding it. Besides the invention concerns an expression vector containing such DNA molecule, and also a cell transformed by the vector. The invention also concerns the method for making alpha-galaktobiose disaccharides with using new alpha galactosidase.

EFFECT: invention allows for high-efficient alpha-galaktobiose disaccharides.

15 cl, 5 dwg, 1 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: there is described an immunogen for making an immunogenic cancer composition free of DNA-binding function and all domains of a zinc finger, on the basis of polynucleotide coding a nonfunctional mutant form of a related molecule ("brother") of regulator of imprint sites (BORIS) of protein, polypeptide or peptide, containing amino acid sequence presented in the description. The immunogenic cancer composition contains aforementioned immunogen and an adjuvant chosen particularly from cytokine, chemokin, a costimulating molecule. There is described an expression vector containing polynucleotide, coding above-stated protein, e.g., in bacterial systems, mammal systems, in yeast or viral systems. The cancer vaccine under the invention contains polynucleotide (immunogen), additionally the adjuvant and, if necessary, a pharmaceutically acceptable carrier. The invention describes the method for of cancer immunisation of a mammal with using said immunogen on the basis of polynucleotide.

EFFECT: invention allows improving effectiveness of cancer prevention.

28 cl, 7 dwg, 2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.

EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.

14 cl, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there is offered molecule of nucleic acid inducing CEA immune response, containing a nucleotide sequence that codes a fused protein on a basis of carcinoembryonal antigen (CEA) or its functional version fused with a subunit B of thermolabile enterotoxin E coli. There are described versions thereof, as well as the related purified protein. There is disclosed an expression vector containing said molecule of nucleic acid, and a host-cell containing specified vector. There are described adenoviral vaccinal vector for inducing the immune response and a vaccinal plasmid on the basis of the specified molecule.

EFFECT: application of the invention allows to inducing the immune response in a mammal which is stronger, than that induced with natural CEA that can find application in medicine for cancer treatment.

20 cl, 62 dwg, 20 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to the field of biotechnology, specifically, to separation and identification of new genes of spiramycins biosynthesis track and to new polypeptides, which participate in this biosynthesis, and may be used to produce acyltransferase, which is responsible for modification of platenolid in position 3. Polynucleotide coding acyltransferase, which is responsible for modification of platenolid in position 3, cells of bacterium Streptomyces type are transformed, and strain-producer of end polypeptide is made.

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26 cl, 41 dwg, 44 tbl, 31 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to the field of biotechnology and immunology. Separated and cleaned DNA is presented, which codes receptor CTLA-4 (CD 152) of cat. The following is also suggested - diagnostic oligonucleotide, cloning vector, vaccine, methods of induction, strengthening and suppression of immune response in cat.

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24 cl, 10 dwg, 6 tbl, 8 ex

FIELD: pharmacology.

SUBSTANCE: present invention refers to immunology and biotechnology. There are antibody-antagonist to CD40 with their variable areas derived from an antibody produced of hybridoma 4D11 (FERM BP-7758). The constant areas of antibodies are derived from human IgG4 with mutations S228P and L235E. There are described related coding polynucleotides and the based expression vector. There is disclosed host-cell containing said vector. There is described method for preparing monoclonal antibody and application thereof in the pharmaceutical composition.

EFFECT: application of the invention provides reduced ADCC and CDC activity that can find application in therapy of autoimmune diseases and graft rejection.

10 cl, 26 dwg, 2 tbl, 22 ex

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. Described are versions of the humanised antibody CD45RO/RB which carry a light and a heavy strand. Versions of the following are disclosed: isolated polynucleotide, coding antibody, expression vector containing a polynucleotide and host cells containing the expression vector. Described also is use of the antibody to treat and/or prevent various diseases, including as a component of a pharmaceutical composition.

EFFECT: invention provides antibodies identified as CD45RO and CD45RB, which can find use in medicine.

9 cl, 14 dwg, 2 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.

EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.

57 cl, 45 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.

EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.

14 cl, 1 tbl, 6 ex

FIELD: pharmacology.

SUBSTANCE: present invention refers to immunology and biotechnology. There are antibody-antagonist to CD40 with their variable areas derived from an antibody produced of hybridoma 4D11 (FERM BP-7758). The constant areas of antibodies are derived from human IgG4 with mutations S228P and L235E. There are described related coding polynucleotides and the based expression vector. There is disclosed host-cell containing said vector. There is described method for preparing monoclonal antibody and application thereof in the pharmaceutical composition.

EFFECT: application of the invention provides reduced ADCC and CDC activity that can find application in therapy of autoimmune diseases and graft rejection.

10 cl, 26 dwg, 2 tbl, 22 ex

FIELD: pharmacology.

SUBSTANCE: invention concerns immunology and biotechnology. There is offered human monoclonal antibody specific to TNF-alpha containing light and heavy chain with appropriate CDR3 sites. There are described versions thereof including those based on heavy and light chains and coded by human genes VH3-33 and A30VK1 or VH3-53 and L2VK3 respectively. There are disclosed: the method for estimating the TNF-alpha content in the patient's sample with using specified antibodies, and application of antibodies for preparing a medical product. There are described: compositions for diagnostics and treatment of the conditions associated with TNF-alpha activity on the basis of antibodies. There is disclosed coding nucleic acid, a cell for making said antibodies and the method for making said antibodies.

EFFECT: application of the invention ensured high-affinity neutralizing monoclonal antibodies with improved Kd and IC50 in comparison with Infliximab, Adalimumab or Etanercept that can find application in medicine for treatment and diagnostics of the diseases associated with TNF-alpha hyperactivity.

35 cl, 13 dwg, 36 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: human antibodies or their antigen-binding fragments are fully described, which specifically bind to human 4-1BB, and which provide for binding of human 4-1BB to human 4-1BB ligand. Antibody includes variable areas of light and heavy chains with amino-acid sequence, given in formula, and may in one of aspects represent IgG4 antibody. Invention presents polynucleotides, which code amino-acid sequences of heavy chain and light chain of monoclonal antibody. Pharmaceutical compositions are described for cancer treatment on the basis of monoclonal antibody or its fragment and method for treatment of disease in subject, which consists in introduction of therapeutically efficient amount of antibody to this subject.

EFFECT: antibodies of invention have agonistic activity and may be used for treatment or prevention of human diseases, such as cancer, infectious and autoimmune diseases.

10 cl, 24 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.

EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.

10 cl, 21 dwg, 11 ex

FIELD: chemistry, medicine.

SUBSTANCE: novel antibodies and fragments of human antibodies are bound with GDF-8 in a specific way and inhibit its activity in vitro and/or in vivo. On the basis of said invention pharmaceutical composition is created, which can be used for diagnostics, prevention or treatment of degenerative dysfunctions of muscle or bone or disorders of insulin metabolism.

EFFECT: extending range of arsenal of technical means used in treatment of diseases related to muscular, bone tissue or insulin metabolism.

FIELD: medicine.

SUBSTANCE: polypeptides include single-domain antibody against vWF, A1 domain of vWF, A1 domain of activated vWF, A3 domain of vWF, gp1b and/or collagen. Invention claims methods of obtaining indicated polypeptides, methods of coating devices applied in medical practice (e.g. in X-ray structural analysis, endoprosthetics) with indicated polypeptides.

EFFECT: obtainment of polypeptides for treatment of diseases requiring modulation of thrombocyte-mediated aggregation.

40 cl, 69 ex, 30 dwg, 32 tbl

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology and immunology. Proposed here is a polynucleotide, encoding a cyclic single-stranded tri-specific antibody. The antibody is directed against human ovarian carcinoma in vitro, has mass of approximately 84 kD and consists of three components: an antibody against human ovarian carcinoma cells, anti-CD3 antibody and anti-CD28 antibody, which are joined together by peptide interlinks such that, they form a cyclic antibody. Invented is an expression vector, containing a coding polynucleotide and versions of E.coli host cell based on the polynucleotide and expression vector.

EFFECT: use of the invention provides for a stable antibody molecule, optimum for activation of T-cells, which can be used in curing human ovarian carcinoma.

8 cl, 12 dwg

FIELD: medicine.

SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.

EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.

14 cl, 1 tbl, 6 ex

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