Immunoglobulins

FIELD: medicine.

SUBSTANCE: invention represents immunoglobulins, in particular antibodies, which specifically bind to human interleukin 13 (hIL-13). Claimed are recombinant or transfected mammalian host cell, for cloning, as well as for expression of vectors, coding antibodies. Claimed are antibody-containing pharmaceutical composition, set, containing said pharmaceutical composition and pharmaceutical composition, containing monoclonal antibody against IL-4, such as pascolizumab. Claimed is method ob obtaining antibody, and applications of said antibodies.

EFFECT: antibodies can be used for treatment of various diseases to disorders, responsible for modulation of hIL-13 interaction with human IL-13 receptor.

51 cl, 29 dwg, 22 tbl, 7 ex

 

The scope to which the invention relates

The present invention relates to antibodies that specifically bind to the interleukin-13 (IL-13), in particular with human IL-13 (hIL-13). In one of its variants the present invention relates to antibodies that specifically bind to the hIL-13. The present invention also relates to methods of treating diseases or disorders using these antibodies, to pharmaceutical compositions containing these antibodies, and to methods for their preparation. Other aspects of the present invention will be apparent from the following description.

Prior art

Interleukin-13 (IL-13)

IL-13 is a secretory cytokine size of 12 kDa, which was originally described as a T-cell cytokine and which inhibits the production of inflammatory cytokines. Studies of its structure showed that he is chetyrehspalnyh beam, linked by two disulfide bonds. Although IL-13 has four potential glycosylation site, however, analysis of the native IL-13 isolated from the lungs of rats, showed that it is produced in the form of deglycosylation molecules. Expression of human IL-13 in NSO cells and COS-7 confirms this observation (Eisenmesser et al., J. Mol. Biol. 2001, 310(1):231-241; Moy et al., J. Mol. Biol. 200, 310(1):219-230; Cannon-Carlson et al., Protein Expression and Purification, 1998, 12(2):239-248).

IL-13 is a pleiotropic cytokine produced by cells of various types, including activated Th2 cells, mast cells, basophils, dendritic cells, keratinocytes, and NKT cells. It can also be produced Th0 cells, Th1 cells, CD8 cells, and “untrained” CD45RA+-T-cells. IL-13 has immunoregulatory activity, which partially overlaps with the activity of IL-4, and such duplication can be explained by the presence of common components in the receptors of IL-4 and IL-13. IL-13 signals through the IL-4 receptor type II, which is heterodimer consisting of IL4Rα - and IL-13Rα1 chains. IL-13Rα1 binds to IL-13 with low affinity (Kd = 2-10 nm), but mating with IL4Rα it binds to IL-13 with high affinity (Kd = 400 PM) and forms a functional IL-13 receptor (human receptor, hereinafter referred to as hIL-13R”), which transmits signals that lead to activation of pathways JAK/STAT - and IRS-1/IRS-2. Was also characterized and the other chain of the IL-13 receptor (IL-13Rα2), which binds to IL-13 with high affinity (Kd = 250 PM), but does not pass the signal, but instead it probably acts as a receptor-trap. Functional receptors for IL-13 is expressed on cells of a wide variety, including epithelial cells of the respiratory tract, smooth muscle cells, fat cells and, eosinophils, basophils, b cells, fibroblasts, monocytes and macrophages. T-cells do not have functional receptors for IL-13 (Hilton et al., PNAS, 1996, 93(1):497-501; Caput et al., J. Biol. Chem. 1996, 271(28):16921-16926; Hershey G.K., J. Allergy Clin. Immunol. 2003 111(4):677-690).

IL-13 and IL-4 modify the immune and inflammatory responses by stimulation associated with allergic inflammation and suppress the inflammation caused by bacteria, viruses and intracellular pathogens. The main biological effects of IL-13 are inducing the proliferation of b-cells and the regulation of isotype switching to IgE; inducing the expression of MHC II and CD23 on b cells and monocytes; positive regulation of VCAM-1 on endothelial cells; regulation of the production of chemokines; activation of mast cells, eosinophils and neutrophils, and inhibits the expression of Pro-inflammatory genes in populations of monocytes and macrophages. IL-13 has no proliferative actions on T-cells. Thus, IL-13, in contrast to IL-4, probably does not play an important role in the initial differentiation of CD4 T-cells into cells of the Th2 type, but nonetheless, it is clear that it plays an important role in the effector phase of allergic inflammation (McKenzie et al., PNAS 1993 90(8):3735-3739; T.A. Wynn, Annu. Rev. Immunol. 2003 21:425-456).

IL-13 and asthma

Asthma is a chronic lung disease caused by inflammation of the lower respiratory the ways, and is characterized by recurrent breathing difficulties. Airways of patients become sensitized and swollen, or there are some inflammation during the entire period of the disease, even if it is asymptomatic. Inflammation leads to airway narrowing and reduce the intake air flow into the lungs and from the lungs, causing difficulty breathing and cause wheezing, chest tightness and cough. Asthma caused by increased sensitivity to allergens (for example, for the clamp household dust, pollen and mold), irritants (e.g., Smoking, inhalation of smoke and the sharp smell), respiratory infections, and also to physical exercise and dry weather. Such triggers can lead to irritation of the respiratory tract, after which the lining of the Airways swells and becomes even more inflamed, mucus in the Airways forms a clot, the muscles surrounding the Airways, compressed and breathing becomes difficult and more intense, indicating that the appearance of symptoms of asthma.

Studies conducted in animal models, and the results of patient surveys clearly showed that asthmatic inflammation and other diseases caused by disorders of regulation of Th2 responses to aeroallergen and on the other stimuli (Busse et al., Am. J. Resp. Crit. Care Med. 1995, 152(1):388-393). In particular, it is clear that IL-13 is the key effector cytokine that triggers various cellular responses in the lung, including Hyper-reactive Airways, eosinophilia, metaplasia goblet cells and increased secretion of mucous.

Clinical data on the role of IL-13 in the development of asthma

The gene encoding IL-13, is located on chromosome 5q31. This region also contains genes encoding IL-3, IL-4, IL-5, IL-9 and GM-CSF and associated with the development of asthma. Genetic variants of IL-13 associated with the development of asthma and atopy were found in the promoter and coding regions (Vercelli D., Curr. Opin. Allergy Clin. Immunol. 2002 2(5):389-393). Data were obtained functional studies for coding variants Q130 IL-13 (hereinafter referred to as Q130 IL-13”). Polymorphism single nucleotide +2044 G → A (SNP)found in the fourth exon, leads to the substitution of arginine for glutamine at position 130 (Q130 IL-13). In addition, in the sequence of SEQ ID NO:9 this replacement is equivalent to a substitution at position 110, where the first amino acid residue G at the beginning of the Mature amino acid sequence of IL-13 is in position 1. According to the survey of people living in Japan and in Europe, it was found that this variant is associated with asthma, with increased levels of IgE and development topices the CSOs dermatitis. Obviously, Q130 IL-13 has a higher stability compared with IL-13 wild-type. He also has a slightly lower affinity to the receptor-trap IL-13Rα2, and according to these observations, higher mean levels of IL-13 in serum were found in patients who are homozygous for the options Q130 IL-13, compared to non-homozygous patients. These results showed that Q130 IL-13 may have an impact on local and systemic concentrations of IL-13 (Kazuhiko et al., J. Allergy Clin. Immunol. 2002 109(6):980-987).

Elevated levels of IL-13 were detected in patients with atopic and non-atopic asthma. In one study, patients with asthma were measured mean levels of IL-13 in serum, constituting 50 PG/ml, whereas in healthy control individuals such levels was 8 PG/ml (Lee et al., J. Asthma 2001 38(8):665-671). Elevated levels of IL-13 were also detected in plasma, bronchoalveolar lavage fluid, samples of lung biopsy and sputum (Berry et al., J. Allergy Clin. Immunol. 2004 114(5):1106-1109; Kroegel et al., Eur. Respir. J. 1996 9(5):899-904; Huang et al., J. Immunol. 1995 155(5):2668-2694; Humbert et al., J. Allergy Clin. Immunol. 1997 99(5):657-665).

In vivodata supporting the involvement of IL-13 in the development of asthma

Several studies have identified an important effector role of IL-13 in the development of pathology in mice that were used as the models of acute and chronic allergic asthma. In these models to neutralize the biological activity of murine IL-13 was used high-affinity IL-13 receptor (IL-13Rα2) or polyclonal antibodies against IL-13. Blockade of IL-13 during infection allergen completely inhibited OVA-induced Hyper-reactive Airways, eosinophilia and metaplasia goblet cells. In contrast, the introduction of antibodies against IL-4 after sensitization and during infection allergen only partially weakened asthmatic phenotype. Thus, although exogenous IL-4 and IL-13 have the ability to induce a phenotype similar to the phenotype of asthma, however, it is clear that the effector activity of IL-13 exceeds the activity of IL-4. These data suggest that IL-4 plays a major role in the induction of immune response (in particular, the formation of Th2 cells and their recruitment in the Airways, and IgE production, whereas IL-13, probably mainly responsible for the induction of various effector functions, including Hyper-reactivity of the Airways, increased mucus secretion and inflammatory processes in cells (Wills-Karp et al., Science 1998, 282:2258-2261; Grunig et al., Science 1998, 282:2261-2263; Taube et al., J. Immunol. 2002 169:6482-6489; Blease et al., J. Immunol. 2001 166(8):5219-5224).

In additional experiments, the levels of IL-13 in the lungs were increased as a result of overexpression in transgenic mice or is the result of the introduction into the trachea of the protein IL-13 wild-type mice. In both cases, were induced symptoms similar to the symptoms observed in asthma, namely non-specific Hyper-reactivity of the Airways in response to stimulation of cholinergic receptor, pulmonary eosinophilia, hyperplasia of epithelial cells, metaplasia of mucus cells, subepithelial fibrosis, airway stenosis and the formation of crystals, such as crystals Charcot-Leiden. In addition, it was found that IL-13 is a potent stimulator of action of matrix metalloproteinases and Katasonov proteases in the lungs, leading to emphysematous changes and mucous metaplasia. Therefore, IL-13 may be an effector molecule, which plays an important role in the development of asthma phenotypes and phenotypes of the disease COPD (Zhu et al., J. Clin. Invest. 1999 103(6):779-788; Zheng et al., J. Clin. Invest. 2000 106(9):1081-1093).

These data showed that the activity of IL-13 is necessary and sufficient for the development of some of the main clinical and pathological signs of allergic asthma in animal models of what the data indicates, confirmed by clinical studies.

Chronic obstructive pulmonary disease (COPD)

COPD is a General term that encompasses several clinical syndromes, including emphysema and chronic bronchitis. COPD symptoms are similar to symptoms of asthma, and therefore Hellmouth to be treated with the same drugs. COPD is characterized by chronic, progressive and mostly irreversible airway stenosis. The cause of this disease in humans is not yet known, but it is believed that in 90% of cases it is caused by cigarette Smoking. The symptoms of this disease are cough, chronic bronchitis, shortness of breath and respiratory infections. Ultimately, this disease can lead to almost total disability and death. Chronic bronchitis is diagnosed in patients who have a prolonged period, for example at least 3 months to 2 years or more, there is nothing unexplained cough or education sputum. Emphysema is characterized by abnormal permanent enlargement of the volume of the Airways and destruction of the walls of the alveoli.

In the development of COPD may play a role of IL-13. Smokers who develop COPD, the lung inflammatory cells are present many types (neutrophils, macrophages, eosinophils). IL-13 is a Pro-inflammatory Th2-cytokine, and therefore it is used for modeling progressive emphysema; for example, in the work of Zheng et al. discusses the overexpression of IL-13 in the epithelium of the respiratory tract in IL-13 transgenic mice. In these animals it causes inflammation of the parenchyma of the respiratory tract and the easier the x and develops emphysema. These mice developed metaplasia mucosa resembling chronic bronchitis (J. Clin. Invest. 2000 106(9):1081-1093).

It was also reported that a polymorphism in the promoter of IL-13 (-1055 → T), which is associated with allergic asthma is more common in patients with COPD than in healthy control individuals. This indicates a functional role of promoter polymorphism of IL-13 in the increased risk of COPD (Kraan et al., Genes and Immunity 2002 3:436-439). In addition, the increase in the number of IL-13 and IL-4-positive cells was observed in smokers with chronic bronchitis, but was not observed in smokers who have no symptoms of such disease (Miotto et al., Eur. Resp. J. 2003, 22:602-608). However, in recent studies to assess the level of expression of IL-13 in the lungs of patients with severe emphysema did not reveal any relationship between the levels of IL-13 and development of this disease (Boutten et al., Thorax 2004, 59:850-854).

Allergic diseases, including atopic dermatitis and allergic rhinitis

IL-13 is also involved in the development of atopic disorders such as atopic rhinitis and atopic dermatitis. In USA allergic rhinitis is the most common atopic disease, and according to experts this disease affects up to 25% of adults and more than 40% of children. Between allergic rhinitis and asthma, there t is SNA relationship. Both these States have a common immune pathology and pathophysiology, and in these diseases there similar immunological processes in which a role is played by eosinophils and Th2-lymphocytes present in the tissues of the nose and the bronchi. It is believed that excessive production of Th2-cytokines, in particular IL-4 and IL-5 plays a major role in the pathogenesis of allergic diseases. IL-13 and IL-4 have some common properties and effector functions, and this fact, combined with the overlap of their functions during their interaction with the receptors of IL-4 and IL-13 and with components that transmit intracellular signals and genetic organization of the IL-13 give conclusive evidence (albeit indirect) that this cytokine plays a role in stimulating or providingin vivothe immediate type hypersensitivity in humans. These data were confirmed by Li and others (Li et al., J. Immunol. 1998; 161:7007), which demonstrated that individuals with atopic disease, such as seasonal allergic rhinitis, there is a much stronger IL-13 responses to Ag-dependent, but not polyclonal activation.

Atopic dermatitis is a widespread chronic, relapsing, inflammatory skin disease with severe itching. The skin lesions of patients with atopic dermatitis guy who teologichesky characterized by inflammatory T-cell infiltration, and this defeat in its acute phase is associated with the predominant expression of IL-4, IL-5 and IL-13 (Simon et al.,J. Allergy Clin. Immunol.2004; 114:887; Hamid et al.,J. Allergy Clin. Immunol.1996; 98:225). In addition, Tazawa et al. demonstrated that the mRNA of IL-13 but not IL-4) significantly activated in patients suffering from atopic dermatitis with subacute and chronic lesions of the skin (Tazawa et al., Arch. Derm. Res. 2004; 296:459). These patients also significantly increased the level of IL-13-expressing circulating CD4+- and CD8+-T-cells (Aleksza et al., British J. Dermatol. 2002; 147:1135). Such increased activity of IL-13, obviously, leads to the production of elevated levels of serum IgE, which also contributes to the pathogenesis of atopic dermatitis. In addition, increased levels of production of IL-13 neonatal CD4+T-cells are a valuable marker for the identification of newborns with a high risk of developing allergic diseases such as atopic dermatitis (Ohshima et al., Pediatr. Res. 2002; 51:195). Additional evidence of the important role of IL-13 in the etiology of atopic dermatitis is given in Simon et al. (Simon et al.,J. Allergy Clin. Immunol.2004; 114:887); and local treatment ointment tacrolimus (immunosuppressant, inhibiting intracellular transmission signal producing cytokines, leads to significant clinical and Geest is logical weakening of atopic skin lesions, accompanied by a significant decrease in levels of local expression of Th2 cytokines, including IL-13. In addition, it was found that IL-13Rα1 (a protein on the cell surface, which together with IL-4Rα forms a functional IL-13 receptor) sverkhekspressiya on suprabasally the keratinocytes in the skin of patients with atopic dermatitis, and IL-13 is able to stimulate mRNA IL-13Rα1in vitro(Wongpiyabovorn et al., J. Dermatol. Science 2003;33:31).

Overall, these data indicate that IL-13 as a target, including a monoclonal antibody against IL-13, can be an effective method of treating allergic diseases in humans.

Esophageal eosinophilia

Accumulation of eosinophils in the esophagus is a common phenomenon, occurring in patients with various diseases, including gastroesophagal reflux, eosinophilic esophagitis, eosinophilic gastroenteritis and parasitic infections. Esophageal eosinophilia is associated with allergic reactions, and re-infection of mice with aeroallergens allows to establish a link between allergic inflammation of the respiratory tract and esophageal eosinophilia. It is believed that Th2 cells induce associated with eosinophils inflammation through the secretion of several cytokines, including IL-4 and IL-13, which directly and indirectly activitytitle and effector path. It is obvious that such activation of IL-13 plays a particularly important role, because it is produced in large quantities Th2-cells and regulates many of the symptoms of allergic diseases (for example, the production of IgE, overproduction mucus, recruitment and survival of eosinophils and Hyper-reactivity of the Airways). Eosinophils can generate functionally active IL-13 treated with cytokines GM-CSF and/or IL-5 in terms ofin vitro,ex vivoandin vivowhen eosinophilic inflammatory reactions (Schmid-Grendelmeier J. Immunology, 2002, 169:1021-1027). It was found that delivery of IL-13 by insertion through the trachea into the lungs of wild-type mice, mice deficient in STAT-6, eotaxin-1 or IL-5, pneumonia, run IL-13 is associated with the development of esophageal eosinophilia (Mishra et al., Gastroenterol. 2003;125:1419). Overall, these data confirm the role of IL-13 in the development of esophageal eosinophilia.

Indications in Oncology

Another important field of interest of researchers is the delivery of IL-13 receptor, IL-13 in some tumor types for inhibiting their growth. Protection of the host mediated T-cell type 1, obviously, provides optimal tumor rejectionin vivoand switching on the response of the Th2-type can contribute to the blocking rejection of the tumor and/or stimulation of tumour recurrences (M. Kobayashi et al., J. Immunol. 1998;160:5869). And the following, performed on multiple animals using transplantable tumor cell lines, confirmed this view by establishing the fact that Stat-6, IL-4 and IL-13 (produced partially N-cells) have the ability to inhibit tumor rejection (Terabe et al., Nat. Immunol. 2000; 1:515; Kacha et al., J. Immunol. 2000; 165:6024-28; Ostrand-Rosenberg et al., J. Immunol. 2000;165:6015). It is obvious that a strong antitumor activity in the absence of Stat-6 is due to the increased production of tumor-specific IFNg and L activity. In addition, it was shown that the absence of cells NK leads to lower levels of production of IL-13 and simultaneously to increase the risk of tumor recurrence, which indicates that IL-13, produced partly by the cells NK, is important for immune surveillance (Terabe et al., Nat. Immunol. 2000; 1:515). Essentially, these data confirm the fact that inhibitors of IL-13 or new antagonists of IL-13, including mAb against IL-13, can be an effective anticancer immunotherapy drugs, which act by inhibiting negative regulation of cytokine IL-13, which plays a role in the inhibition of immune responses to tumor cells.

In addition to enhancing antitumor protection associated with cell type Th-1, inhibitors of IL-13 can also directly block the growth of cancer cells. For example, Pref-cell chronic lymphocytic leukemia (b-CLL) and Hodgkin's disease IL-13 or blocks apoptosis, either stimulates the proliferation of tumor cells (Chaouchi et al., Blood 1996; 87:1022; Kapp et al., J. Exp. Med. 1999; 189:1939). B-CLL is a clinically heterogeneous disease caused by b-lymphocytes, which are involved in the violation of apoptosis of leukemic cells. It is obvious that IL-13 does not act directly as a growth factor, and only protects tumor cells fromin vitrospontaneous apoptosis (Chaouchi et al., Blood 1996; 87:1022; Lai et al., J. Immunol. 1999; 162:78) and may contribute to the development of b-CLL by preventing the death of neoplastic cells.

Hodgkin's disease is one of the types of lymphoma, which affects mainly young people, and, according to experts, in the U.S. this disease is diagnosed in approximately 7,500 people per year. This type of cancer characterized by the presence of large multinuclear cells of Hodgkin/reed-Sternberg (H/RS). In most cases, this population of malignant cells derived from b cells. Some cell lines, associated with Hodgkin's disease, and lymph node tissue taken from patients with Hodgkin's lymphoma, sverkhekspressiya IL-13 and/or receptors of IL-13. (Kapp et al., J. Exp. Med. 1999;189:1939; Billard et al., Eur. Cytokine Netw. 1997; 8:19; Skinnider et al., Blood 2001; 97:250; Oshima et al., Cell Immunol. 2001; 211:37). It has been shown that neutralizing anti-IL-13-mAb or antagonists of IL-13 dose-dependently inhibit the proliferation of cells in H/RS (Kapp t al., J. Exp. Med. 1999; 189:1939; Oshima et al., Cell Immunol. 2001; 211:37). Similarly, the introduction of soluble receptor-traps IL-13Rα2 mice ND/SCID with implanted cell line associated with Hodgkin's disease, slows down the beginning of the development and tumor growth and increases survival of these mice, suggesting that neutralization of IL-13 can inhibit the growth of jackinsky lymphomain vitroandin vivo(Trieu et al., Cancer Research 2004;64:3271). In General, these studies have shown that IL-13 stimulates autocrine proliferation of cells N/a RS (Kapp et al., J. Exp. Med. 1999;189:1939; Ohshima et al., Histopathology 2001;38:368).

Therefore, neutralization of IL-13 is an attractive and effective way to treat Hodgkin's disease and other associated with b-cells of cancer by inhibiting the growth of tumor cells while increasing antitumor protection.

Inflammatory bowel disease

It is possible that IL-13 plays a role in the pathogenesis of inflammatory bowel disease (IBD). Inflammatory bowel disease includes a number of diseases, clinically classified as ulcerative colitis, Crohn's disease and colitis of unknown etiology. The main manifestations of such diseases is chronic inflammation of the small intestine caused by the amplification of the immune response with simultaneous imbalance activation and Th1 - and Th2-lymphocytes in the mucosa of the small intestine. Such demonstrations have been demonstrated in animal models of Crohn's disease (Bamias et al., Gastroenterol 2005; 128:657) and ulcerative colitis (Heller et al., Immunity 2002; 17:629). Neutralization of IL-13 by injecting IL-13Rα2-Fc leads to prevent the development of colitis in mice with Th2 model of human ulcerative colitis (Heller et al., Immunity 2002; 17:629). In addition, in this model, the production of IL-13 is faster than the production of IL-4, and the production of IL-13 can be induced by stimulation NK-cells, suggesting that the observed tissue destruction may be the result of toxic effects of IL-13 on epithelial cells. When examining some of the patients were obtained some of the data supporting these conclusions, namely the frequency of IL-13 in IL-13-positive biopsies taken from the rectum of patients with ulcerative colitis were much higher than the frequency of IL-13 in individuals with inflammation and the control of individuals, with a higher level of expression of IL-4 and IL-13 was observed in acute ulcerative colitis, but not in normal ulcerative colitis (Inoue et al., Am. J. Gastroenterol. 1999;94:2441). In addition, Akido et al. was characterized by immune activity in the external muscle plate (muscularis externa), isolated from segments of the small intestine of patients with Crohn's disease, and it was found that IL-4 and IL-13 mediate increased the e reduction of smooth muscle cells of the small intestine by STAT-6-ways. The authors concluded that this pathway may contribute to hyperactive muscle contraction of the small bowel in patients with Crohn's disease (Akiho et al., Am. J. Physiol. Gastrointest. Liver. Physiol. 2005;288:619).

Thus, the use of mAb against IL-13, possibly in combination with molecules directed to other cytokines, can be a way of stopping or slowing the progression of IBD.

Psoriasis and psoriatic arthritis

Psoriasis is a chronic skin disease characterized by hyperproliferative keratinocytes and immune cell infiltration, including activation of T cells that produce a variety of cytokines that can influence the phenotype of epidermal keratinocytes. CDw60 is a carbohydrate-bearing molecule that is activated on the surface of psoriatic basal and suprabasal keratinocytes of psoriatic skin. It was shown that IL-4 and IL-13, secreted by T-cells derived from the areas affected by psoriasis, highly stimulate the CDw60 expression on keratinocytes (Skov et al., Am. J. Pathol. 1997;15:675), whereas interferon-gamma blocks the IL-4/IL-13-mediated induction of CDw60 on cultured keratinocytes (Huang et al., J. Invest. Dermatol. 2001;116:305). Thus, it is believed that the CDw60 expression in psoriatic epidermal keratinocytes induced by IU is greater least in part, the cytokine IL-13, secretively activated T-cells in the affected area. In addition, IL-13Rα1 and IL-4Rα, i.e. cell surface proteins, which together form a receptor complex of IL-13, in varying degrees, expressed in skin biopsies taken from patients suffering and not suffering from psoriasis (Cancino-Diaz et al., J. Invest. Dermatol. 2002;119:1114; Wongpiyabovorn et al., J. Dermatol. Science 2003;33:31), while experimentsin vitrodemonstrated that IL-13 but not IL-4) can activate the expression of IL-13Rα1 (Wongpiyabovorn et al., J. Dermatol. Science 2003;33:31). Because IL-13 affects cells of various types, the data from these studies suggest that the IL-13 receptor may play a role in the early stage of the inflammatory process in psoriasis.

Psoriatic arthritis is characterized by synovitis, which is mediated proinflammatory and anti-inflammatory cytokines. Currently, the role of IL-13 in the emergence of various forms of arthritis is increasing interest. Spadaro and colleagues have demonstrated that the levels of IL-13 in synovial fluid of patients with psoriatic arthritis are significantly higher than the levels of IL-13 in patients with osteoarthritis. In addition, levels of IL-13 in synovial fluid was significantly greater than the levels of IL-13 in the serum of patients with psoriatic arthritis, and IL-13 in synovial fluid IL-13 in serum Zn is significantly higher in patients with psoriatic arthritis, than in patients with rheumatoid arthritis, which suggests that in the development of psoriatic arthritis may play a role of IL-13, locally produced in synovial tissues of patients (Spadaro et al., Ann. Rheum. Dis. 2002;61:174).

The possible role of IL-13 in the development of other conditions

Acute graft versus host disease is a major cause of morbidity and mortality after transplantation of stem cells and directly associated with the degree of incompatibility between human leukocyte antigen (HLA) in the donor and recipient. Jordan and other first identified IL-13 as a typical Th2-cytokine, which is produced abundantly in the process reactions mixed culture unrelated incompatible lymphocytes (LR) (reaction mixed culture of lymphocytes;in vitroanalysis for thin selection of the donor after the preliminary LA-typing)(Jordan et al., J. Immunol. Methods; 2002;260:1). Then this group of researchers found that the production of IL-13 donor T-cells after transplantation of donor stem cells is an indicator of acute graft versus host disease (aGVHD) (Jordan et al., Blood 2004;103:717). All patients after stem cell transplantation was observed severe aGVHD of the third degree, were donors who have producirovanie very strong responses to L-13 prior to transplantation, indicating a close relationship between the levels of IL-13 and aGVHD and to increase the likelihood that IL-13 may be directly responsible for some of aGVHD-associated pathologies. Therefore, therapy, based on the specific blocking of IL-13, can be used for the treatment of aGVHD after stem cell transplant.

In Western countries, diabetic nephropathy is one of the main causes of kidney disease end-stage. Although the number of cases of disease nephropathy associated with type 1 diabetes, is reduced, however, at present, diabetes mellitus type 2 is one of the most common causes of kidney failure in the United States, Japan and Europe. In addition, this group of patients has a very poor prognosis regarding the results of maintenance dialysis due to the exceptionally high mortality from bouts of cardio-vascular diseases. Currently, it is becoming increasingly clear that the hemodynamic, metabolic and structural changes are interrelated with each other, and it was found that different enzymes, transcription factors and growth factors play a role in the pathogenesis of these diseases. In particular, GF-β plays an important role in the development of kidney hypertrophy and accumulation of extracellular matrix components, the ri is defined as, this cytokine plays a crucial role in mediating the formation of collagen in the kidney (Cooper. Diabetologia 2001; 44:1957; Wolf. Eur. J. Clin. Invest. 2004; 34(12):785). In diabetic nephropathy in humans and in experimental animals the biological activity of TGF-1 increases, and the introduction of antibodies against GF-β1 mice with diabetes leads to improved renal function and reduced accumulation of extracellular matrix. Recently it was shown that in transgenic murine models of pulmonary fibrosis effect of IL-13 is directed, at least in part, on the regulation of the production and activation GF-β1 and deposition of collagen (Lee et al., J. Exp. Med. 2001; 194:809; Zhu et al., J. Clin. Invest. 1999;103:779), indicating a direct functional relationship between IL-13 and GF-β. Therefore, we can draw a similar conclusion about the role of IL-13 in regulating the activity of GF-β1 in the kidney in patients with diabetes, and therefore the suppression of the action of IL-13 may, likely to have some effect in the treatment of diabetic nephropathy.

Fibrotic condition

Pulmonary fibrosis is a condition characterized by unwanted and dangerous scarring in the lungs, leading to disability, and often fatal. This term covers a number of conditions of different etiology, pathology and susceptibility to treatment. In some cases, was the determined cause of fibrosis. These reasons are (1) VDI is the W propertiesgo substances, such as asbestos or silica dust, or powder heavy metals, (2) inhalation of organic matter, which causes the patient's idiosyncratic immune response, leading to fibrosis (for example, to the state called “the lungs of a farmer”), (3) the use of drugs such as nitrofurantoin, amiodarone and methotrexate, (4) presence of systemic inflammatory diseases, such as systemic sclerosis or rheumatoid arthritis.

However, in many cases, the cause of the disease or underlying asymptomatic condition remain unknown. In many such patients diagnosed with idiopathic pulmonary fibrosis (IFL). This disease is quite rare (mostly 20 cases per 100,000 people). This diagnosis is usually no obvious cause of the disease in combination with some radiological and pathological features of, in particular, the honeycomb structure of the lungs is determined using computed tomography (CT) or lung biopsy. This disease usually occurs in older people (>50) and is often accompanied by persistent progressive lung damage, leading to death, and, according to experts, this state can last from 2 to 5 years. Moreover, patients experience very unpleasant sensations in the form of shortness of breath, progress is highlighted in several months or years. This condition first leads to restriction of physical activity, but at the final stage of the disease, which can last several months, the patient begins to breath even at rest, and his health is increasingly dependent on oxygen supply.

Currently no satisfactory treatment of this disease. Modern treatment methods typically involve the administration of corticosteroids and immunosuppressants, such as azathioprine. However, for many patients corticosteroids can be effective, but their side effects can even worsen the condition of the patient. There are many possible treatments under study, including interferon-gamma, which, as shown by recent large-scale study can increase life expectancy, and pirfenidone.

There is evidence that IL-13 and cytokines associated with Th2-phenotype, involved in repair of tissue affected by fibrosis (Wynn T.A., Nat. Rev. Immunol. 2004 4:583-594; Jakubzick et al., Am. J. Pathol. 2004 164(6):1989-2001; Jakubzick et al., Immunol. Res. 2004 30(3):339-349; Jakubzick et al., J. Clin. Pathol. 2004, 57:477-486). IL-13 and IL-4 are involved in the development of various fibrotic conditions. Liver fibrosis inducedSchistosomais probably the IL-13-dependent, and there are few data indicating that IL-otsutvuet in the pathogenesis of scleroderma (Hasegawa et al., J. Rheumatol. 1997 24:328-332; Riccieri et al., Clin. Rheumatol. 2003:22:102 to 106).

With regard to pulmonary fibrosis,in vitrostudies have shown that IL-13 stimulates the fibrogenic phenotype. Studies performed on animals have found elevated levels of expression of IL-13 models with artificially induced fibrosis, and also showed that the degree of fibrosis can be reduced by elimination of IL-13.

IL-13 stimulates propertiesi phenotype. At the cellular level there are several mechanisms by which IL-13 can promote fibrosis. Transmission signal and the important role of these different mechanisms are still not precisely defined.

There is evidence that IL-13 acts on fibroblasts and thereby stimulates the production of collagen and inhibits its destruction, thus favouring the development fibroticheski phenotype. Skin fibroblasts are the receptors of IL-13, and processing of cultured skin fibroblasts by the cytokine IL-13 leads to the activation of collagen (Oriente et al., J. Pharmacol. Exp. Ther. 2000 292:988-994). IL-4 has the same, but more short-term action. Cell line the human lung fibroblasts (ICIG7) expresses the IL-4 receptor type II (Jinnin et al., J. Biol. Chem. 2004, 279:41783-41791). Treatment of these cells with the cytokine IL-13 stimulates the secretion of various inflammatory and profibrotic mediators: GM-CSF, G-CSF and multi is on V-beta 1 (Doucet et al., Int. Immunol. 1998 10(10):1421-1433).

IL-13 inhibits IL-1A-induced production of proteins 1 and 3 of matrix metalloproteinases by fibroblasts of the skin, which can reduce the level of destruction of the extracellular (VC) matrix (Oriente et al., J. Pharmacol. Exp. Ther. 2000 292:988-994). IL-13 together with TGF-β act synergistically in human fibroblasts, obtained by biopsy of the Airways in asthma, thereby stimulating the expression of tissue inhibitor of metalloproteinases 1 (TIMP-1). The destruction of the extracellular matrix is carried out under the action of matrix metalloproteinases, which inhibited TIMP-1. Thus, this action of IL-13 should lead to a reduction in the destruction matrix (Zhou et al., Am. J. Physiol. Cell. Physiol. 2005:288:S-S).

Overexpression of IL-13 in transgenic mice leads to subepithelial fibrosis, hypertrophy of epithelial cells, hyperplasia of goblet cells, the deposition of crystals (acidic chitinases mammals), hyperreactivity respiratory tract, interstitial fibrosis, hypertrophy of cells of type 2 and the accumulation of surface-active substances (Zhu et al., J. Clin. Invest. 1999 103(6):779-788).

Various species of mice have different susceptibility to induced by bleomycin fibrosis of the lungs. In susceptible mice 57BL/6J observed rapid activation of IL-13, IL-13Rα and IL-4 (as well as GFβ, TNFRα and IL-1Rs) in response to bleomycin. In BALB/c mice, which are not vos is ioncinema, activation of IL-13 does not occur.

Belpero and others (Am. J. Respir. Cell. Mol. Biol. 2002:27:419-427) conducted studies on the expression and role of IL-13, IL-4 and CC-chemokine C10 in the development of fibrosis in mice with a model of fibrosis induced by bleomycin. The levels of IL-13 and IL-4 in lung tissue was increased in response to bleomycin. Pre-neutralization of IL-13 using polyclonal antibodies against IL-13 resulted in a significant reduction of pulmonary fibrosis induced by bleomycin, as was assessed by the levels of hydroxyproline in the lung. Despite the increased expression of IL-4 in the same model, neutralization of IL-4 did not have any impact on pulmonary fibrosis.

In another model of acute lung fibrosis induced by FITZ in BALB/c mice, the absence of IL-13 (deficient mice), but not IL-4 prevented pulmonary fibrosis. A deficit of IL-4 IL-13-deficient mice did not give any additional protection (Kolodsick et al., J. Immunol. 2004 172:4068-4076). Protective effect of the absence of IL-13 is not associated with differences in recruitment of cells into the lungs, that is all deficient mice and BALB/c mice the total number updated (recruited) cells was the same, and therefore it is obvious that the source of the inflammatory component remained unchanged. The recruitment of eosinophils from IL-4 - and IL-13-deficient mice was lower than in BALB/c mice, but since IL-4-/-mice were not protected is from fibrosis, it cannot serve as an explanation of differences in the degree of fibrosis. Probably, it might seem surprising that IL-13+/+- and IL-13-/--mice there was no difference in the levels of cytokines, including IL-10, MCP-1, IFN-γ, TGF-i. In addition, from the lungs of various animals after they are processed FITZ was allocated the same number of fibroblasts, however, the IL-13-/-mice the level of production of collagen I declined. This indicates that the absence of IL-13 is not simply prevents the inflammatory response, but even plays a more specific antifibrotics role. It has been suggested that IL-13 may have the fibrogenic action by TGF-i (Lee et al., J. Exp. Med. 2001 194:809-821). However, in this WORK models the expression of TGF-i was not decreased in IL-13-deficient mice.

It has been suggested that interleukin-4 has the same effect as IL-13, and they both act through the same receptor. IL-4 significantly activated in the lungs of mice with bleomycin-induced lung fibrosis (Gharaee-Kermani et al., Cytokine 2001 15:138-147). However, compared to bleomycin-induced lung fibrosis in mice 57BL/6J, which is observed overexpression of IL-4, IL-4-deficient mice and wild-type mice (Izbicki et al. Am. J. Physiol. Lung Cell. Mol. Physiol. 2002 283(5):L1110-L1116) was not detected any evidence of the fact, IL-4 is involved in the development of pulmonary fibrosis. The IL-4-deficient mice was not observed decrease the degree of fibrosis, whereas in mice with overexpression of IL-4 was observed an increase in the degree of fibrosis.

Levels of the cytokine IL-13 in bronchoalveolar lavage (BAL) from patients with various forms of pulmonary fibrosis were significantly increased, albeit to varying degrees. The expression of IL-13 were significantly increased in alveolar macrophages obtained from patients with pulmonary fibrosis.

The most convincing clinical data were obtained from studies conducted at the University of Michigan. Jakubzick and colleagues investigated the gene expression of IL-13 and IL-4 and their receptors in lung biopsies obtained after surgery in patients with pulmonary fibrosis. The gene expression of IL-13 in samples taken from the lungs of affected IFL, was significantly higher than in normal lung or lungs affected other fibrotic conditions. Cultured fibroblasts taken from patients with IFL/IPRs were found significantly higher level of expression of the receptor for IL-13 and IL-4 than tissue and fibroblasts, obtained by biopsy tissue of patients with normal light or other forms of pulmonary fibrosis. In particular, fibroblasts, concentrated on areas that were supposedly epicenters of disease activity, gave the most vivid color, indicating when OUTSTA of these receptors (Jakubzick et al., J. Immunol. 2003 171:2684-2693; Jakubzick et al., Am. J. Pathol. 2003 162:1475-1486; Jakubzick et al., Am. J. Pathol. 2004 164(6):1989-2001; Jakubzick et al., Immunol. Res. 2004 30(3):339-349; Jakubzick et al., J. Clin. Pathol. 2004 57:477-486).

There is compelling datain vitrothat indicates that mainly Th2-cytokines, in particular IL-13 stimulate propertiesi phenotype. It was shown that at least in 2 animal models of chemically induced fibrosis can be reduced by elimination of IL-13 (either by gene deletion or by using antibodies against IL-13). Some data indicate that IL-13 plays a more important role in the stimulation of pulmonary fibrosis than IL-4. Clinical data regarding the role of IL-13 in the development of pulmonary fibrosis suggest that in the lungs of patients with IFL there is no regulation of IL-13 and its receptors.

The increasing amount of data suggests that medicines derived from IL-13 are effective for the treatment of a number of fibrotic conditions, including that induced by schistosomiasis liver fibrosis, and various forms of lung fibrosis (e.g., IFL [discussed in the present description] and scleroderma).

Experiments in which independently inhibited IL-4 and IL-13 showed that IL-13 is a dominant effector cytokine of fibrosis in some models (Chiaramonte et al.,J. Clin. Invest.1999; 104:777-785; Blease et al. J. Immunol. 2001; 166:5219;Kumar et al., Clin. Exp. Allergy 2002;32:1104). In the case of schistosomiasis induced in the egg inflammatory response were not affected by blockade of IL-13, however, despite the ongoing and continued production of IL-4, deposition of collagen in animals with chronic infections have declined by more than 85% (Chiaramonte et al. J. Clin. Invest. 1999; 104:777; Chiaramonte et al., Hepatology 2001; 34:273).

Amino acid sequence of hIL-13 represented in SEQ ID NO:9 (This sequence is a sequence of the Mature protein, i.e. the sequence in which no signal sequence).

cDNA encoding hIL-13 presented in SEQ ID NO:10 (This DNA sequence encodes the sequence of the Mature protein, i.e. the sequence in which no signal sequence).

All references to patents and literature cited in this application accurately and in its entirety introduced into the present description by reference (including any patent application, the priority of which is claimed in this application).

Recently received the vaccine produces an immune response against IL-13 designed for the treatment of asthma have been described in the literature (WO 02/070711). Was recently also described the role of IL-13 in the susceptibility of the skin to external allergens (Herrick et al., The Journal of Immunology, 2003, 170:2488-2495).

The present invention, inter alia, on sitsa to the antibody, marked A. As demonstrated below, binding A with hIL-13, obviously, depends on the presence of arginine at position 107 of SEQ ID NO:9. It was reported that arginine at position 107 of SEQ ID NO:9 represents the balance, which plays an important role in the interaction between hIL-13 and hIL-13R. Cm. the publication of Thompson J.P. & Debinski W. (1999) J. Biol. Chem. vol.24, No. 42, pp.29944-29950, entitled “Glutamic Acids at position 13 and 16 in hIL13α-helix A, arginine and serine at positions 66 and 69 in helix C, and arginine at position 109 in helix D were found to be important in inducing biological signalling since their specific mutation resulted in loss and/or gain of function phenomena” (see summary and description). In this work, the arginine at position 109 is the equivalent of the arginine at position 107 of SEQ ID NO:9 of the present application, due to the difference in the numbering systems used by the authors of the present invention and the authors of the work. Thus, the antibody A, binding to hIL-13, includes one of these residues hIL-13, which was previously identified as a residue that plays an important role in the interaction between hIL-13 and hIL-13R, and therefore is involved in the biological signaling by IL-13-path.

Description of the invention

Therefore, the present invention relates to therapeutic antibody or its antigennegative fragment that specifically binds to hIL-13 and neutralizes the activity of hIL-13. See, for example, a table As shown below.

The term “specification is logically associated”, used in the present description with respect to antibodies and their antigennegative fragments according to the invention means that the antibody binds to hIL-13, but does not bind or binds to a small extent with other human proteins, in particular with human IL-4. However, this term does not actually include antibodies according to the invention, which can cross-react with IL-13 cynomolgus monkeys.

In another aspect, the present invention relates to therapeutic antibody or its antigennegative fragment that specifically binds to hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R. This inhibition include, but are not limited to, competitive inhibition. In some embodiments of the invention, the antibodies according to the invention at least inhibit the interaction between hIL-13 and hIL-13R, but can also block the interaction between hIL-13 and hIL-13R and thereby block the transmission signal hIL-13/hIL-13R.

In another aspect the present invention relates to therapeutic antibody or its antigennegative fragment that specifically binds to hIL-13 and contains an area CDR3 having the sequence represented in SEQ ID NO:3.

In another aspect of the present invention apply is to therapeutic antibody or its antigennegative slice which specifically binds to hIL-13 and contains an area CDR3, representing a variant of the sequence presented in SEQ ID NO:3, where one or two of the remainder of the specified area CDR3 this option differ from the residues in the corresponding position of SEQ ID NO:3.

In another aspect the present invention relates to therapeutic antibody or its antigennegative fragment that specifically binds to hIL-13 and consists of the following CDRs:

CDR1: SEQ ID NO:1,

CDR2: SEQ ID NO:2,

CDR3: SEQ ID NO:3,

CDRL1: SEQ ID NO:4,

CDRL2: SEQ ID NO:5,

CDRL3: SEQ ID NO:6.

In the present description amino acid residues in the sequences of the antibodies are numbered according to the Kabat numbering. Similarly, the term “CDR”, “CDRL1”, “CDRL2”, “CDRL3”, “CDR1”, “CDR2”, “CDR3” correspond to the numbering system of Kabat, etc. described in the publication “Sequences of proteins of Immunological Interest, NIH, 1987”. The term “CDR1” means a fragment of a sequence that includes a fragment CDR1 defined by Kabat (residues 31-35V), and the fragment CDR1 defined by Chothia (Chothia et al.(1989); Conformations of immunoglobulins hypervariable regions; Nature 342, R-883), which includes the fragment 26-32 according to Kabat. Therefore, in accordance with the present invention CDR were defined as follows:

CDR: Residues
CDR1:26-35V
CDR2:50-65
CDR3:95-102
CDRL1:24-34
CDRL2:50-56
CDRL3:89-97

In another aspect the present invention relates to therapeutic antibody or its antigennegative fragment comprising VH domain having the sequence represented in SEQ ID NO:7, and a VL domain having the sequence represented in SEQ ID NO:8.

In another aspect the present invention relates to the selected domain VH antibody containing (or essentially consisting of, or consisting of a) SEQ ID NO:7 or 11, 12, 13, 14.

In another aspect the present invention relates to therapeutic antibody or its antigennegative fragment comprising VH domain selected from the group consisting of SEQ ID NO:7 or SEQ ID NO: 11, 12, 13, 14.

In another aspect the present invention relates to therapeutic antibody or its antigennegative fragment, which competitively inhibits the binding of therapeutic antibody containing CDR3 SEQ ID NO:3 with hIL13.

In another aspect the present invention relates to therapeutic antibody or its antigennegative fragment, which competitively inhibits the binding of therapeutic antibody containing CDR SEQ ID NO:1, 2, 3, 4, 5 and 6 with hIL-13.

In another aspect the present invention relates to therapeutic antibody or its antigennegative fragment, which competitively inhibits the binding of therapeutic antibody containing heavy chain SEQ ID NO:18 and a light chain of SEQ ID NO:22, hIL-13.

In accordance with this present invention relates to gumanitarnogo therapeutic antibody containing a VH domain selected from the group consisting of SEQ ID NO: 11, 12, 13, 14, and VL domain selected from the group consisting of SEQ ID NO: 15, 16.

In another aspect the present invention relates to a method of treating a person suffering from a disease or disorder susceptible to modulation of the interaction between hIL-13 and hIL-13R (such as asthma, COPD, allergic rhinitis, atopic dermatitis), where the method includes a stage of introduction of the indicated patient a therapeutically effective amount described herein, therapeutic antibody or antigennegative fragment.

The present invention also relates to the use of the antibodies according to the invention for the manufacture of drug among the STW for treatment of a disease or disorder, susceptible to modulation of the interaction between hIL-13 and hIL-13R.

In another aspect the present invention relates to a therapeutic antibody that specifically binds to human IL-13, where the specified antibody specifically binds to human IL-13 in position residues 97-108 SEQ ID NO:9. Based on the results below, it is clear that the term “residue in position 97-108 SEQ ID NO:9 implies, including residues at positions 97 and 108.

In another aspect the present invention relates to therapeutic antibody that competitively inhibits the binding of therapeutic antibody that has an area CDR3 SEQ ID NO:3, human IL-13 (such as a therapeutic antibody containing heavy chain SEQ ID NO:18 and a light chain of SEQ ID NO:22), where the specified competitive antibody specifically binds to human IL-13 in the positions of residues 97-108 SEQ ID NO:9.

In another aspect the present invention relates to a therapeutic antibody that specifically binds to human IL-13 in the positions of residues 103-107 SEQ ID NO:9, inclusive, and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R.

In one of its variants the present invention relates to pharmaceutical compositions containing a variety of therapeutic monoclonal antibodies (usually human and the and gumanitarnyh), which specifically bind to human IL-13 in the positions of residues 103-107 SEQ ID NO:9 and modulate (e.g. inhibit or block) the interaction between hIL-13 and hIL-13R, and a pharmaceutically acceptable carrier.

In another aspect the present invention relates to a method for production of therapeutic antibodies, which specifically binds to hIL-13 in the positions of residues 103-107 SEQ ID NO:9 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R, where the method includes a stage of cultivation in serum-free culture medium of recombinant host cell containing the first and second vectors, where the first vector contains polynucleotide encoding a heavy chain of the indicated antibodies, and the second vector contains polynucleotide that encodes a light chain of the indicated antibodies. Based on the results below, it is clear that the term “residue in position 103-107 SEQ ID NO:9 includes residues at positions 103 and 107.

In another embodiment, the present invention relates to a method for production of therapeutic antibodies, which specifically binds to hIL-13 in the positions of residues 97-108 SEQ ID NO:9 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R, where the method includes a stage of cultivation in serum-free Kul the Ural environment recombinant host cell, containing the first and second vectors, where the first vector contains polynucleotide encoding a heavy chain of the indicated antibodies, and the second vector contains polynucleotide that encodes a light chain of the indicated antibodies.

In another embodiment, the present invention relates to the intact therapeutic antibody that binds to hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R, where the specified antibody interacts with residue 107 of SEQ ID NO:9.

In another embodiment, the present invention relates to the intact therapeutic antibody that binds to hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R, where the specified binding of therapeutic antibody and hIL-13 depends on the presence of arginine residue (or positively correlated) in position 107 of SEQ ID NO:9.

In another embodiment, the present invention relates to a therapeutic antibody that specifically binds to hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R, and has a dissociation constantkoffin the range from 1.4 × 10-4to 8.22 × 10-5with-1(for example, as was measured in the Biacore analysisTM). Such an antibody may contain CDRH3 SEQ ID NO:3 or its option and in addition to SEQ ID NO:3 or its variant, it can is also contain SEQ ID NO:1, 2, 4, 5 and 6.

In another embodiment, the present invention relates to an antibody that specifically binds to hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R, where the specified antibody contains CDRH3 SEQ ID NO:3 and, optionally, contains each of the regions CDRH1 SEQ ID NO:1, CDRH2 SEQ ID NO:2, CDRL1 SEQ ID NO:4, CDRL2 SEQ ID NO:5 and CDRL3 SEQ ID NO:6, and where the specified antibody also cross-reacts with IL-13 monkeys cynomolgus (cIL-13).

Brief description of drawings

Figure 1:

Sandwich ELISA illustrating the ability of monoclonal antibodies A contact recombinant IL-13 expressed inE. coliwith increasing concentrations.

Figure 2a:

ELISA illustrating the ability of monoclonal antibodies A, at increasing concentrations to inhibit the binding of recombinant human IL-13 expressed inE. coliwith the α1 chain of human IL-13 receptor.

Figure 2b:

ELISA illustrating the ability of monoclonal antibodies A, at increasing concentrations to inhibit the binding of recombinant human IL-13 expressed inE. coliwith the α2 chain of the human IL-13 receptor.

Figure 3:

The neutralization test, illustrating the ability of monoclonal antibodies A, at increasing concentrations to inhibit the biological activity of recombinant human is a mini-IL-13 and IL-13 cynomolgus monkeys, expressed inE. coliin the analysis for cell proliferation, TF-1.

Figure 4:

The neutralization test, illustrating the ability of monoclonal antibodies A, at increasing concentrations to inhibit the biological activity of human IL-13 expressed in mammalian cells (cells SNO), the analysis on the cell proliferation, TF-1.

Figure 5:

The neutralization test, illustrating the ability of monoclonal antibodies A, at increasing concentrations to inhibit the biological activity of recombinant human IL-13 expressed inE. coliin position Q130, in the analysis on cell proliferation, TF-1.

Figure 6:

Sandwich ELISA, which showed that A is not associated with recombinant human IL-4 expressed inE. coli.

Figure 7:

The neutralization test IL5, which demonstrated that A not inhibit the biological activity of recombinant human IL-5 expressed inE. coliin the analysis for cell proliferation, TF-1.

Figure 8:

Sandwich ELISA illustrating the ability of the chimeric mAb A contact recombinant human IL-13 and IL-13 cynomolgus monkeys, expressed inE. coliwith increasing concentrations.

Figure 9:

Sandwich ELISA illustrating the ability of 8 gumanitarnyh mAb against human IL-13 has been Atisa with recombinant human IL-13, expressed inE. coliwith increasing concentrations.

Figure 10A:

Sandwich ELISA illustrating the ability of the chimeric A, L1 + A1 and L2 + A1 contact recombinant human IL-13 expressed inE. coliwith increasing concentrations.

Figure 10b:

Sandwich ELISA illustrating the ability of the chimeric A, L1 + A1 and L2 + A1 contact recombinant IL-13 cynomolgus monkeys, expressed inE. coliwith increasing concentrations.

Figure 11:

Sandwich ELISA illustrating the ability of the chimeric A, L1 + A1 and L2 + A1 contact with native human IL-13 with increasing concentrations.

Figure 12A:

ELISA illustrating the ability of monoclonal antibodies A, chimeric antibodies A, L1 + A1 and L2 + A1 at increasing concentrations to inhibit the binding of recombinant human IL-13 expressed inE. coliwith the α1 chain of human IL-13 receptor.

Figure 12b:

ELISA illustrating the ability of monoclonal antibodies A, chimeric antibodies A, L1 + A1 and L2 + A1 at increasing concentrations to inhibit the binding of recombinant human IL-13 expressed inE. coliwith the α2 chain of the human IL-13 receptor.

Figure 13A

The neutralization test, illustrating the ability of the antibodies A, chimeric antibodies A, L1 + A1 and L2 + A1 with increasing concentrations inhibit is to the biological activity of recombinant human IL-13, expressed inE. coliin the analysis for cell proliferation, TF-1.

Figure 13b

The neutralization test, illustrating the ability of the antibodies A, chimeric antibodies A, L1 + A1 and L2 + A1 at increasing concentrations to inhibit the biological activity of recombinant IL-13 cynomolgus monkeys, expressed inE. coliin the analysis for cell proliferation, TF-1.

Figure 13C

The neutralization test, illustrating the ability of the antibodies A, chimeric antibodies A, L1 + A1 and L2 + A1 at increasing concentrations to inhibit the biological activity of recombinant human IL-13 expressed inE. coliin position Q130, in the analysis on cell proliferation, TF-1.

Figure 13d:

The neutralization test, illustrating the ability of the antibodies A, chimeric antibodies A, L1 + A1 and L2 + A1 at increasing concentrations to inhibit the biological activity of human IL-13 expressed in mammals (in cells SNO), the analysis on the cell proliferation, TF-1.

Figure 14a

Sandwich ELISA, which showed that the antibody A, chimeric antibody A, L1 + A1 and L2 + A1 is not associated with recombinant human IL-4 expressed inE. coli.

Figure 14b

Sandwich ELISA, which showed that the antibody A, chimeric antibody A, L1 + A1 and L2 + A1 is not associated with recombinant human GM-CSF, expre serwerem in E. coli.

Figure 14C:

The neutralization test IL5, which demonstrated that the antibody A, chimeric antibody A, L1 + A1 and L2 + A1 does not inhibit the biological activity of recombinant human IL-5 expressed inE. coliin the analysis for cell proliferation, TF-1.

Figure 15

The epitope mapping using ELISA to determine the epitope to which antibody binds A, human IL-13 or IL-13 cynomolgus monkeys.

Figure 16A

The epitope mapping using ELISA to identify high specificity of antibody binding sites A with human IL-13.

Figure 16b

The epitope mapping using ELISA to identify high specificity of antibody binding sites A with IL-13 cynomolgus monkeys.

Figure 17A

The epitope mapping using ELISA to identify key amino acid residues required for binding A with human IL-13.

Figure 17b

The epitope mapping using ELISA to identify key amino acid residues required for binding L1+A1 with human IL-13.

In figures 17c and 17d presents graphs illustrating the alanine-scanning analysis of parent (mouse) antigen A (Fig. 17c) and gumanitarnogo antibody L1-A1.

Detailed description of the invention

1. The structure of antibodies

1.1. Intact antibodies

Intact antibodies are the two who are heteropolymers glycoproteins, containing at least two heavy and two light chains. Intact antibodies, in addition to IgM, are usually heterotetrameric glycoproteins of approximately 150 kDa, consisting of two identical light chains (L) and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds between the heavy chains of immunoglobulins of various isotypes vary. Each heavy and light chain also have unutilizable disulfide bridges. Each heavy chain has at one end has a variable domain (VH)followed by a number of constant domains. Each light chain is at the other end has a variable domain (VL) and a constant region where the specified constant region light chain corresponds to the first constant region of the heavy chain and the variable domain light chain corresponds to the variable domain of the heavy chain. Light chains of antibodies of most vertebrates, based on their amino acid sequence of the constant region, can be attributed to one of two types of light chains, called Kappa and lambda. Depending on the amino acid sequence of the constant region of the heavy chains of human antibodies may refer to five different classes, IgA, IgD, IgE, IgG and IgM. Antibodies IgG and IgA which may then be subdivided into subclasses IgG1, IgG2, IgG3 and IgG4, as well as Da and Da. In mice and rats there are other types of antibodies, at least IgG2 and IgG2b. Variable domain antibodies tells the antibody ability to specifically bind with some areas that detect particularly high variability and called hypervariable regions (complementarity-determining regions, CDRs). The more conservative parts of the variable regions are called frame regions (FR). The variable domains of each of the heavy and light chains of intact antibodies contain four FR, connected by three CDRs. CDRs in each chain are in close proximity with the FR regions and, with the other chain CDR and participate in education antigennegative site antibodies. Constant region not directly participate in the binding of an antibody to an antigen, but have different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (DCC), phagocytosis via binding to Fcγ receptor, change the time half-life/speed excretion via neonatal Fc receptor (FcRn) and participating in complement-dependent cytotoxicity via component 1q of the complement cascade.

Therefore, in one of its variants the present invention relates to the intact therapeutic antibody, which is specific is associated with hIL-13 and which modulates (e.g., inhibits or blocks) the interaction between hIL-13 and hIL-13R. The intact therapeutic antibody can contain a constant region of any isotype or any subclass described above. In one embodiment of the invention, the specified antibody has an isotype IgG, particularly IgG1. The specified antibody may be a rat antibody, rat antibody, rabbit antibody, antibody primates, or human antibody. In one representative variants of the invention the specified antibody is the antibody of primates, such as cynomolgus monkeys, monkeys or apes, see, for example, WO 99/55369, WO93/02108) or person.

In another embodiment, the present invention relates to the selection of the intact therapeutic antibody containing CDR3 SEQ ID NO:3. In another embodiment, the present invention relates to the selection of the intact therapeutic antibody containing the variable region having CDR SEQ ID NO:1, 2, 3, 4, 5 and 6.

In another embodiment, the present invention relates to the selection of mouse intact therapeutic antibody or its antigennegative fragment containing VH domain having the sequence of SEQ ID NO:7, and a VL domain having the sequence of SEQ ID NO:8.

1.1.2. Human antibodies

Human antibodies can be obtained by various methods known experts who am. Human antibodies can be obtained hybridoma method using human myeloma cell lines or watermelony cell lines “mouse man”, see Kozbor, J. Immunol. 133, 3001 (1984) and Brodeur,Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker Inc., 1987). Alternative methods include the use of phage libraries or transgenic mice with human V-regions (see, Winter G. (1994) Annu. Rev. Immunol. 12, 433-455, Green L.L. (1999), J. Immunol. Methods 231, 11-23).

Currently, there are several strains of transgenic mice in which the murine immunoglobulin loci have been replaced by the gene segments of the human immunoglobulin (see Tomizuka, K. (2000) PNAS 97, 722-727; Fishwild D.M. (1996) Nature Biotechnol. 14, 845-851, Mendez M.J. 1997, Nature Genetics, 15, 146-156). After sensitization with antigen such mice can produce a range of human antibodies, which can be selected interest antibodies. Special attention deserves the Trimera systemTM(see Eren R. et al. (1998) Immunology 93:154-161), in which human cells were transplanted into irradiated mice, that is, the system of the antibodies selected lymphocytes (SLAM, see, Babcook et al. (1996) 93:7843-7848), where human lymphocytes or lymphocytes other species) were subjected effective procedure for transmission through an array of generating antibodies collected byin vitrothe procedure limiting dilution and pried the re-selection and Xenomouse II TM(Abgenix Inc.). Description of the alternative method of using the technology MorphdomaTMcan be found in Morphotek Inc.

For producing human antibodies (and fragments) can be used phage technology view, see McCafferty; Nature, 348, 552-553 (1990) and Griffiths, A.D. et al. (1994) EMBO 13:3245-3260. In accordance with this technology, genes V-domain antibody clone with preservation of the reading frame in the gene is large or small envelope protein of filamentous bacteriophage, such as M13 or fd, and are (usually using helper phage) on the surface ragovoy particles in the form of functional fragments of antibodies. The selection made on the basis of functional properties of the antibody, allows the selection of the gene encoding the antibody possessing these properties. The technology of phage representation can be used for selection of antigen-specific antibodies from a library derived from human b-cells taken from individuals suffering from a disease or disorder described above, or, alternatively, from maimonidean donors (see, Marks, J. Mol.Bio. 222, 581-597, 1991). If it is desirable that the intact human antibody contained Fc-domain, the phage representing the received fragment, you must subclinical in expressing vectors of the mammal containing the desired constant region, and in cell lines with aulnoy expression.

To increase the affinity of binding can be used the technique of affinity maturation (Marks, Bio/Technol. 10, 779-783, 1992)), where the original affinity human antibodies can be increased by replacing the V-regions of H and L natural variants and by selection on the basis of high-affinity binding. Also described other variants of this method, such as methods based on “epitope imprinting”, see, WO 93/06213. Cm. also Waterhouse; Nucl. Acids. Res. 21, 2265-2266 (1993).

Thus, in another embodiment, the present invention relates to selected human intact therapeutic antibody or antigennegative fragment that specifically binds to hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R.

In another aspect the present invention relates to selected human intact therapeutic antibody or antigennegative fragment containing CDR3 SEQ ID NO:3, which specifically binds to hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R. In another aspect the present invention relates to selected human intact therapeutic antibody or antigennegative fragment containing the variable region having CDR SEQ ID NO:1, 2, 3, 4, 5 and 6, defined the above.

1.2. Chimeric and gumanitarnye antibodies

The use of intact non-human antibodies for the treatment of human diseases or disorders may be associated with well-known at the present time, the problems of immunogenicity, which is that the patient's immune system may recognize non-human intact antibody as “foreign” and to develop a neutralizing response. This is particularly evident after repeated administration of human non-human antibodies. To address these problems in recent years, various techniques have been developed, which are mainly aimed at reducing the level of non-human amino acid sequences in intact antibody while maintaining the relative ease of obtaining a non-human antibody from immunising animal, such as a mouse, rat or rabbit. In a broad sense to achieve this goal can be applied two methods. In the first method, get a chimeric antibodies that contain mostly non-human variable domain (e.g. domain, derived from rodent such as a mouse)attached to human constant region. Because antigennegative website antibody localized in the variable regions, the chimeric antibody retains the binding affinity of the antigen, but gets effector function is the human constant region, and so it is able to carry out effector functions described above. Chimeric antibodies are usually obtained by the methods of recombinant DNA. DNA encoding the antibodies (e.g., cDNA), is isolated and is sequenced in accordance with standard procedures (e.g., by using oligonucleotide probes that can specifically bind with the genes encoding the H and L-chains of the antibody according to the invention, for example with DNA that encodes SEQ ID NO: 1, 2, 3, 4, 5 and 6, described above). A typical source of such DNA are hybridoma cells. After extraction of DNA injected into expressing vectors, which are then transferred into the cells of the host, such as cellsE. colicells , COS cells, Cho or myeloma cells that under natural conditions does not produce a protein of the immunoglobulin required for the synthesis of antibodies. Such DNA can be modified by replacing the sequence encoding the constant region of H - and L-chain non-human (e.g. murine) antibodies sequence that encodes the corresponding constant region of a human H - and L-chains, see, for example, Morrison; PNAS 81, 6851 (1984).

The second method involves generating gumanitarnyh antibodies, in which the content of non-human antibodies reduced as a result of humanization of the variable regions. These two methods of humanization become particularly popular is the awn. The first method is the humanization by CDR-grafting. CDRs form loops in close proximity to the N-end of antibodies, where they form a surface that is located on the carrier formed by the frame areas. The binding specificity of the antigen with the antibody is determined mainly by topographic and chemical properties of its CDR-surface. These characteristics, in turn, are determined by the conformation of individual CDR, the relative location of the CDR, as well as the nature and location of the side chains of the residues that comprise the CDR. A significant reduction in immunogenicity can be achieved by attaching only the CDR of non-human (e.g. murine) antibodies (“donor” antibody) to the human frame (“acceptor framework”) and constant regions (see Jones et al. (1986) Nature 321, 522-525 and Verhoeyen, M. et al. (1988) Science 239, 1534-1536). However, the accession CDRper sedoes not completely save antigennegative properties of antibodies, and often it turns out that to maintain the high level of affinity of binding gumanitarnogo antibodies to the antigen, you need to save some frame remains (sometimes called “remnants of reverse mutations”) donor antibodies (see Queen, C. et al. (1989) PNAS 86, 10029-10033, Co, M. et al. (1991) Nature 351, 501-502). In this case, to obtain the human framework region (FR)from the database from eraut human V-region, finding the greatest homology of its sequence with the sequence of non-human donor antibodies. Human FR can be selected either from the FR of a human consensus antibody, or from a separate human antibodies. If it is necessary to save the CDR conformations, the key residues of the donor antibody replace the remnants of the human acceptor framework region. To facilitate the identification of such structurally important residues can be applied computer simulation of antibodies, see, WO 99/48523.

Alternatively, the humanization can be performed by the method of “masking”. Statistical analysis of the unique variable regions of the heavy and light chains of human and mouse immunoglobulin found that accurate profiles of surface residues in human antibodies and mouse antibodies are the most specific provisions of the surface residues have a strong preference towards a small number of other residues (see Padlan E.A. et al. (1991) Mol. Immunol. 28, 489-498 and J.T. Pedersen et al. (1994) J. Mol. Biol. 235;959-973). Therefore, the immunogenicity of non-human Fv can be reduced by replacement of surface residues in the framework regions that differ from the areas normally present in human antibodies. Since the antigenicity of proteins may correlate with access the ability of a surface, the replacement of surface residues may be sufficient to hide the mouse variable region from the supervision of the human immune system (see also Mark G.E. et al. (1994),Handbook of Experimental Pharmacology, vol.113: The pharmacology of monoclonal Antibodies,Springer-Verlag, pp.105-134). This process of humanization is called “masking”, because it allows you to modify only the surface of the antibody, but the core remains unchanged.

Thus, in another embodiment, the present invention relates to chimeric therapeutic antibody containing non-human variable domain (e.g., rodents), joined to human constant region (which may belong to the IgG isotype, e.g. IgG1), which specifically binds to hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R.

In another aspect the present invention relates to chimeric therapeutic antibody containing non-human variable region (e.g., rodents) and human constant region (which may belong to the IgG isotype, e.g. IgG1), which specifically binds to hIL-13, where the specified antibody also contains CDR3 SEQ ID NO:3. Such antibodies may also contain human constant region of the IgG isotype, e.g. IgG1.

In another embodiment, the crust is ASEE the invention relates to chimeric therapeutic antibody containing non-human variable region (e.g., rodents) and human constant region (which may belong to the IgG isotype, e.g. IgG1), which specifically binds to hIL-13, with CDR SEQ ID NO: 1, 2, 3, 4, 5 and 6.

In another embodiment, the present invention relates to chimeric therapeutic antibody containing a VH domain SEQ ID NO:7 and a VL domain SEQ ID NO:8 and a human constant region of the IgG isotype, e.g. IgG1, which specifically binds to hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody or antigennegative fragment that specifically binds to hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody or antigennegative fragment that specifically binds to hIL-13 and contains CDR3 SEQ ID NO:3. Such antibodies may also contain human constant region of the IgG isotype, e.g. IgG1.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody or antigennegative fragment that specifically binds to hIL-13 and contains what it CDR SEQ ID NO: 1, 2, 3, 4, 5 and 6. Such antibodies may also contain human constant region of the IgG isotype, e.g. IgG1.

In accordance with this present invention relates to gumanitarnogo therapeutic antibody containing a VH domain selected from the group consisting of SEQ ID NO:11, 12, 13, 14, and VL domain selected from the group consisting of SEQ ID NO:15, 16. Such antibodies may also contain human constant region of the IgG isotype, e.g. IgG1.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody containing a VH domain SEQ ID NO:11 and a VL domain SEQ ID NO:15.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody containing a VH domain SEQ ID NO:12 and a VL domain SEQ ID NO:15.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody containing a VH domain SEQ ID NO:13 and a VL domain SEQ ID NO:15.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody containing a VH domain SEQ ID NO:14 and a VL domain SEQ ID NO:15.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody containing a VH domain SEQ ID NO:11 and a VL domain SEQ ID NO:16.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody VH-domain of SEQ ID NO:12 and a VL domain SEQ ID NO:16.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody containing a VH domain SEQ ID NO:13 and a VL domain SEQ ID NO:16.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody containing a VH domain SEQ ID NO:14 and a VL domain SEQ ID NO:16.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody or antigennegative fragment that specifically binds to hIL-13, where the aforementioned antibody or its fragment contains CDR3 (SEQ ID NO:3), and optionally contains CDR SEQ ID NO: 1, 2, 4, 5 and 6, where residues selected from the group consisting of residues 19, 38, 73 and 81 of the acceptor framework regions of human heavy chain and the residue at position 85 of the acceptor framework region of human light chain replaced with the corresponding residues present in the frame region of the donor antibody from which CDR3.

For the person skilled in the art it is obvious that the term “place” refers not only to the source, in the sense that the physical source of this material, but also material, which is structurally identical (on the primary amino acid sequence) specified material, but which does not originate from a specified source. Thus, the residues present in the donor ant the body, which is CDR3”, does not need to be isolated from donor antibodies.

In another embodiment, the present invention relates to gumanitarnogo therapeutic antibody or its antigennegative fragment that specifically binds to hIL-13, where the aforementioned antibody or its fragment contains CDR3 SEQ ID NO:3, and optionally contains CDR SEQ ID NO: 1, 2, 4, 5 and 6 where the specified frame region of a human heavy chain contains one or more (e.g. all) of the following residues (or a conservative substitution):

PositionBalance
38I
19R
73T
81R

and the human light chain contains:

PositionBalance
85V

The person skilled in the art it is well known that certain amino acid substitutions are considered to be “conservative”. Amino acids modernization to modernization is considered to groups based on common properties of their side chains and substitutions within groups, which are fully or almost fully retain the binding affinity of the antibodies according to the invention or its antigennegative fragment and which are regarded as conservative substitutions, for example, specified in the following table:

Side chainDenote residues
Hydrophobic residuesMet, Ala, Val, Leu, Ile
Neutral hydrophilic residuesys, Ser, Thr
Acid residuesAsp, Glu
Basic residuesAsn, Gln, His, Lys, Arg
Residues that influence chain orientationGly, Pro
Aromatic residuesTrp, Tyr, Phe

In accordance with this present invention relates to gumanitarnogo therapeutic antibody containing a heavy chain selected from the group consisting of SEQ ID NO:18, 19, 20, 21, and a light chain selected from the group consisting of SEQ ID NO:22, 23.

In one of its variants the present invention relates to gumanitarnogo therapeutic antic the Lu, which specifically binds to hIL-13 and contains a heavy chain SEQ ID NO:18 and a light chain of SEQ ID NO:22.

In one of its variants the present invention relates to gumanitarnogo therapeutic antibody that specifically binds to hIL-13 and contains a heavy chain SEQ ID NO:19 and a light chain of SEQ ID NO:22.

In one of its variants the present invention relates to gumanitarnogo therapeutic antibody that specifically binds to hIL-13 and contains a heavy chain SEQ ID NO:20 and a light chain of SEQ ID NO:22.

In one of its variants the present invention relates to gumanitarnogo therapeutic antibody that specifically binds to hIL-13 and contains a heavy chain SEQ ID NO:21 and a light chain of SEQ ID NO:22.

In one of its variants the present invention relates to gumanitarnogo therapeutic antibody that specifically binds to hIL-13 and contains a heavy chain SEQ ID NO:18 and a light chain of SEQ ID NO:23.

In one of its variants the present invention relates to gumanitarnogo therapeutic antibody that specifically binds to hIL-13 and contains a heavy chain SEQ ID NO:19 and a light chain of SEQ ID NO:23.

In one of its variants the present invention relates to gumanitarnogo therapeutic antibody that specifically binds to hIL-13 and contains a heavy chain SEQ ID NO:20 and a light chain of SEQ ID NO:23./p>

In one of its variants the present invention relates to gumanitarnogo therapeutic antibody that specifically binds to hIL-13 and contains a heavy chain SEQ ID NO:21 and a light chain of SEQ ID NO:23.

1.3. Bespecifically antibodies

Bespecifically antibody is an antibody specifically binding at least two different epitopes. Methods of producing such antibodies known in the art. Typically, the recombinant production of bespecifically antibodies is based on the co-expression of two pairs of H-chain, L-chain immunoglobulin, where the two N-chains have different specificnosti binding, see, Millstein et al., Nature 305:537-539 (1983), WO 93/08829 and Traunecker et al., EMBO, 10, 1991, 3655-3659. Because of the random assortment of H - and L-chains is produced by a potential mixture of ten antibodies with different structures, of which only one antibody possessed desired binding specificity. An alternative method includes attaching variable domains with the desired specificnosti binding to the constant region of the heavy chain that contains at least part of the hinge region, a CH2 region and a CH3 region. While it is preferable that attended the region SN containing the site necessary for binding to the light chain and available at least in one of the hybrids. DNA encoding these hybrids, and e is whether it is necessary L-chain is inserted into the individual expressing the vectors together and then injected into a suitable host organism. In one expressing the vector can be introduced sequence encoding two or all three of these circuits. In one of the preferred methods bespecifically antibody consists of a H-chain having a first binding specificity in one branch, and a pair of N-L-chains having a second binding specificity for another branch, see WO 94/04690. Also see publication Suresh et al., Methods in Enzymology, 121, 210, 1986.

In one of its variants the present invention relates to bespecifically therapeutic antibody that has at least one binding specificity with hIL-13, where the specified antibody modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R. Such antibodies may also contain human constant region of the IgG isotype, e.g. IgG1. In some embodiments of the invention bespecifically therapeutic antibody has a first binding specificity with hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R and a second binding specificity with hIL-4 and modulates (e.g. inhibits or blocks) the interaction between hIL-4 receptor hIL-4.

In one of its variants the present invention relates to bespecifically therapeutic antibody that has the t at least one binding specificity with hIL-13, where the specified antibody contains CDR3 SEQ ID NO:3. Such antibodies may also contain human constant region of the IgG isotype, e.g. IgG1.

In one of its variants the present invention relates to bespecifically therapeutic antibody that has at least one binding specificity with hIL-13, where the specified antibody contains at least CDR SEQ ID NO:1, 2, 3, 4, 5 and 6. Such antibodies may also contain human constant region of the IgG isotype, e.g. IgG1.

1.4. Antibody fragments

In certain embodiments, the present invention relates to fragments of therapeutic antibodies that modulate the interaction between hIL-13 and hIL-13R. Such fragments can be functional antigennegative fragments of intact and/or gumanitarnyh and/or chimeric antibodies, such as Fab, Fab'and F(ab')2-, An Fv, scFv-fragments of the antibodies described above. Typically, such fragments produced by proteolytic hydrolysis of intact antibodies, for example by hydrolysis with papain (see, for example, WO 94/29348), but they can also be obtained directly from recombinante transformed host cells. Description of the method of production of scFv can be found in Bird et al. (1988) Science, 242, 423-426. In addition, antibody fragments can be obtained by genetic engineering methods, described below.

Fv-fragments is s, obviously, they have less interaction energy of its two chains than Fab-fragments. To stabilize the connection between domains VH and VL they are joined by peptide bonds (Bird et al. (1988) Science, 242, 423-426; Huston et al., PNAS, 85, 5879-5883), disulfide bonds (Glockshuber et al. (1990) Biochemistry 29, 1362-1367) and through the introduction of mutations “knob in hole” (Zhu et al. (1997) Protein Sci., 6, 781-788). scFv fragments can be obtained by methods well known in the art, see, Whitlow et al. (1991) Methods companion Methods Enzymol, 2, 97-105 & Huston et al. (1993) Int. Rev. Immunol. 10, 195-217. scFv-fragments can be produced in bacterial cells, such asE. colibut more preferably in eukaryotic cells. One of the drawbacks of scFv is their odnovalentnogo, which does not allow to increase the avidity that can be achieved through multivalent binding, and their short half-life. In an attempt to solve these problems of scFv', containing an additional C-terminal cysteine was produced bivalent (scFv')2by chemical coupling (Adams et al. (1993) Can. Res. 53, 4026-4034 & McCartney et al. (1995) Protein Eng. 8, 301-314) or by spontaneous site-specific dimerization of scFv containing unpaired C-terminal cysteine residue (see Kipriyanov et al. (1995) Cell. Biophys. 26, 187-204). Alternatively, the scFv can be artificially obtained in the form of multimers by shortening the peptide linker to 3-12 residues formed with the eating “di-antibodies”, see Holliger et al. PNAS (1993), 90, 6444-6448. Even greater shortening of the linker may also lead to the formation of scFv-trimers (three antibodies”, see Kortt et al., (1997) Protein Eng. 10, 423-433) and tetramers (“Tetra-antibodies”, see Le Gall et al. (1999) FEBS Lett, 453, 164-168). Construction of bivalent scFv molecules can be carried out also by genetic coupling with protein-dimerize motifs, with the formation of “mini-antibodies” (see, Pack et al. (1992) Biochemistry 31, 1579-1584) and “mini-cells” (see Hu et al. (1996) Cancer Res. 56, 3055-3061). scFv-scFv-tandems ((scFv)2) can also be obtained by attaching two scFv fragments by means of a third peptide linker, see Kurucz et al. (1995) J. Immunol. 154, 4576-4582. Bespecifically di-antibodies can be obtained through non-covalent binding of two single-stranded hybrid products, consisting of a VH domain of one antibody connected by a short linker to the VL domain of another antibody, see, Kipriyanov et al. (1998) Int. J. Can. 77, 763-772. The stability of such bespecifically di-antibodies may be improved by the introduction of disulfide bridges or mutations in the configuration of the type “knob in hole”described above, or through the formation of single-stranded di-antibodies (scDb), where two hybrid scFv-fragment are connected by a peptide linker, see Kontermann et al., (1999) J. Immunol. Methods 226:179-188. Tetravalent bespecifically molecules can be obtained, for example, by attaching scF-slice to the CH3 domain of IgG molecules or Fab fragment via a hinge region, see Coloma et al. (1997) Nature Biotechnol. 15, 159-163. Alternatively, a tetravalent bespecifically molecules can be obtained by attaching bespecifically single-stranded di-antibodies (see Alt et al., (1999) FEBS Lett 454, 90-94). Smaller tetravalent bespecifically molecules can also be obtained by dimerization or scFv-scFv-tandems with a linker containing the motif helix-loop-helix (mini-antibodies DiBi, see, Muller et al. (1998) FEBS Lett. 432, 45-49)or single-stranded molecule that contains four variable domain antibodies (VH and VL), in the orientation that prevents intramolecular pairing (tandem di-antibody, see, Kipriyanov et al. (1999) J. Mol. Biol. 293, 41-56). Bespecifically F(ab')2fragments can be created by chemical joining Fab'fragments or by heterodimerization through latinovich lightning” (see, Shalaby et al. (1992) J. Exp. Med. 175, 217-225 and Kostelny et al. (1992), J. Immunol. 148, 1547-1553). Can be also obtained separate VH and VL domains (Domantis plc), see U.S. patent 6248516, 6291158, 6172197.

In one of its variants the present invention relates to a fragment of therapeutic antibodies (e.g. scFv, Fab, Fab', F(ab')2or designed to fragment antibodies, described above, which specifically binds to hIL-13 and modulates (e.g. inhibits or blocks) the interaction between hIL-13 and hIL-13R. Fragment of therapeutic antibodies usually what keeps CDR3, having the sequence of SEQ ID NO:3, optionally together with a CDR having the sequence presented in SEQ ID NO:1, 2, 4, 5 and 6.

1.5. Heteroconjugate antibodies

One of the variants of the present invention are also heteroconjugate antibodies. Heteroconjugate antibodies consist of two covalently linked antibodies formed by any standard methods of cross-linking. See, for example, U.S. patent 4676980.

1.6. Other modifications

The interaction between the Fc region of antibodies and various Fc receptors (FcγR), it is obvious that mediates effector functions of antibodies, including antibody-dependent cellular cytotoxicity (DCC), fixation of complement, phagocytosis and change the time half-life/speed of removing antibodies. Various modifications in the Fc-region of an antibody according to the invention can be implemented depending on the desired properties. For example, in the Fc-region can be introduced specific mutations, transforming lytic antibody in deletecache antibody, as described in detail in EP V and EP V to increase its half-life in serum, or may be entered epitope binding with the receptor of salvation, see U.S. patent 57399277. Currently, there are five human Fcγ-receptors, namely FcγR(I), FcγRII, FcγRIIb, FcγRIII and neonatal FcRn. In a slave is those Shields et al. (2001) J. Biol. Chem. 276, 6591-6604 demonstrated that binding to all FcγR participates General group IgG1 residues, and binding to FcγRII and FcγRIII are involved other residues that differ from the remnants of this group. One group of residues of IgG1, when replacing residues Pro-238, Asp-265, Asp-270, Asn-297 and Pro-239-alanine reduces the level of binding to all FcγR. All of them are present in the CH2 domain of IgG and form a cluster near the hinge region connecting CN and CH2. In binding to FcγRI involved only the total group of residues of IgG1 and FcγRII and FcγRIII, in addition to this General group of residues, interact with other residues. Modifications in some residues reduce the level of binding only with FcγRII (for example, Arg-292) or FcγRIII (e.g., Glu-293). Some options reveal an increased level of binding to FcγRII or FcγRIII, but does not affect binding to the other receptor (e.g., replacement of Ser-267Ala leads to increased level of binding to FcγRII, but does not affect binding to FcγRIII). Other options detect elevated levels of binding to FcγRII or FcγRIII, but a reduced level of binding to the other receptor (e.g., replacement of Ser-298Ala leads to increased level of binding to FcγRIII and reduce the level of binding to FcγRII). With regard to receptor FcγRIII, the best binding of IgG1 variants occurs when combining alanine substitutions Ser-298, Glu-333 and Lys-334. Neonatal receptor FcRn, Eveno, participates in the clearance of antibodies and transcytosis are activated through the fabric (see Junghans R.P. (1997) Immunol. Res. 16. 29-57 and Ghetie et al. (2000) Annu. Rev. Immunol. 18, 739-766). The remnants of human IgG1, which, as defined, directly interact with human FcRn are Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435. Replacement of any of the provisions described in this section may increase the half-life in serum and/or modify the effector properties of the antibodies according to the invention.

Other modifications are glycosylation variants of antibodies according to the invention. It is known that glycosylation of antibodies in conservative positions in their constant regions has a significant impact on the function of antibodies, in particular effector function, such as function, described above, see for example, Boyd et al., (1996) Mol. Immunol. 32, 1311-1318. Options also discussed glycosylation of therapeutic antibodies or their antigenspecific fragments according to the invention, where have been added, replaced, deleterow or modified one or more carbohydrate parts. The introduction of the motif of “asparagine-X-serine or asparagine-X-threonine” leads to the formation of potential customers enzymatic joining of carbohydrate parts, and therefore, this introduction can be used to change the nature of the glycosylation of antibodies. In the work of Raju et al. (2001) Biohemistry 40, 8868-8876 demonstrated that the level of end sililirovanie immunoadhesin TNFR-IgG is increased in the process of regulationary and/or easilylive under the action of beta-1,4-galactosyltransferase and/or alpha-2,3-sialyltransferase. The higher level end sililirovanie probably leads to increased half-life of immunoglobulin. Antibodies, along with most of glycoproteins, usually produced in the form of a mixture glycoform. This mixture is especially apparent if the antibodies are produced in eukaryotic cells, particularly mammalian cells. For certain glycoform was several methods have been developed, see Zhang et al. Science (2004), 303, 371, Sears et al., Science (2001) 291, 2344, Wacker et al. (2002) Science 298, 1790, Davis et al., (2002) Chem. Rev. 102, 579, Hang et al. (2001) Acc. Chem. Res. 34, 727. Thus, in the present invention discusses a variety of therapeutic (monoclonal) antibodies (which may belong to the IgG isotype, e.g., IgG1), described in this application and containing a certain number (e.g., 7 or less, for example 5 or less, namely, two or one) glycoform these antibodies or their antigenspecific fragments.

Other variants of the present invention include therapeutic antibodies according to the invention or their antigennegative fragments associated with a non-protein polymer, such as polyethylene glycol (the EG), polypropylenglycol or polyoxyalkylene. Conjugation of proteins with PEG is a well-known method to increase the half-life of proteins, as well as reduce the antigenicity and immunogenicity of proteins. The use of Paglierani using molecules with different molecular weights and structures (linear or branched) was studied in intact antibodies, and Fab'-fragments, see Koumenis I.L. et al. (2000) Int. J. Pharmaceut. 198:83-95.

2. Competitive antibodies

The present invention also relates to antibodies and their antigennegative fragments that specifically bind to hIL-13 and competitively inhibit the binding of c IL-13 therapeutic antibodies according to the invention or its antigennegative fragment containing CDR3 SEQ ID NO:3, and/or therapeutic antibodies according to the invention or its antigennegative fragment containing CDR SEQ ID NO:1, 2, 3, 4, 5 and 6, hIL-13. In some embodiments of the invention specified therapeutic antibody is a murine antibody containing a VH domain SEQ ID NO:7 and a VL domain SEQ ID NO:8. Such competitive antibody binds to the same epitope or an overlapping epitope hIL-13, or spatially adjacent epitope hIL-13, which is associated with a therapeutic antibody containing CDR SEQ ID NO:1, 2, 3, 4, 5 and 6. Competitive antibody or a fragment of such antibodies, in equivalent olyarnik concentrations provides at least 25%, usually 35%or more, and mostly, at least 50%inhibition.

Thus, in one of its variants the present invention relates to a method of screening for antibodies candidate or selection conducted in order to determine whether antibody-applicant or its fragment competitive antibody described in this application, where the method involves the following stages: (a) incubating the antibody of the applicant or its fragment with a therapeutic antibody containing CDR3 SEQ ID NO:3, and optionally containing CDR SEQ ID NO:1, 2, 4, 5 and 6 (such as a mouse, a therapeutic antibody having VH-domain SEQ ID NO:7 and a VL domain SEQ ID NO:8, or gumanitarnoe therapeutic antibody having a heavy chain SEQ ID NO:18 and a light chain of SEQ ID NO:22, or gumanitarnoe therapeutic antibody having a heavy chain SEQ ID NO:19 and a light chain of SEQ ID NO:23), or antigennegative fragment; (b) determine the fact whether the specified antibody candidate or piece of stage (a) competitive inhibition of the binding of the specified therapeutic antibodies or antigennegative fragment with hIL-13.

The present invention also relates to a competitive antibody or its antigennegative fragment, which competitively inhibits the binding of therapeutic antibody or ant is enswathes fragment, containing region CDR having the sequence presented in SEQ ID NO: 1, 2, 3, 4, 5 and 6.

In another embodiment, the present invention relates to a competitive antibody or its antigennegative fragment, which competitively inhibits the binding of therapeutic antibody according to the invention with hIL-13, where the specified therapeutic antibody contains a heavy chain SEQ ID NO:18 and a light chain of SEQ ID NO:22.

Competitive antibody or antigennegative fragment can be any antibody having the above structure. For example, competitive antibody can be an intact antibody primates or humans or gumanitarnoe antibody, preferably with the IgG isotype, e.g., IgG1 or IgG4. Fragments of competitive antibody may be a Fab, Fab', F(ab')2, scFv, etc. Competitive antibody can be obtained by the methods described in this application.

3. Methods of producing antibodies

Antibodies according to the invention can be produced in the form of a polyclonal population, but more preferably in the form of a monoclonal population (i.e. in the form of a largely homogeneous population of identical antibodies directed against specific antigennegative site). For the person skilled in the art it is evident that the population includes more than one molecule of the antibody. Antibodies which according to the invention can be produced in transgenic organisms such as goats (see Pollock et al. (1999) J. Immunol. Methods 231:147-157), chickens (see Morrow K.J.J. (2000) Genet. Eng. News 20:1-55), mouse (see Pollock et al.) or plants (see Doran P.M. (2000) Curr. Opinion Biotechnol. 11, 199-204, Ma JK-C (1998)Nat. Med. 4; 601-606, J. Baez et al., BioPharm (2000) 13:50-54, Stoger E. et al. (2000) Plant. Mol. Biol. 42:583-590). Antibodies can also be produced by the method of chemical synthesis. However, the antibodies according to the invention are usually obtained with the use of recombinant technology, cell culture, well known to specialists. Polynucleotide encoding the antibody is isolated and inserted into a replicable vector such as a plasmid for further cloning (amplification) or expression. One of the commonly used expression systems is the system glutamate synthetase (such as the system supplied by the company Lonza Biologics), where the host-cell, in particular, the cell is Cho or NSO (see below). Polynucleotide encoding the antibody can be readily isolated and sequenced according to standard procedures (e.g., by using oligonucleotide probes). Used vectors can be plasmid, virus, phage, transposons, minichromosome, of which a typical option are plasmids. Typically, such vectors further include a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and consistently is the termination of transcription, functionally attached to polynucleotide light chain and/or heavy chain to facilitate expression. Polynucleotide encoding light and heavy chains, can be built into separate vectors and transfirieran in the same cell of the owner, or, if necessary, the heavy and light chains can be built into the same vector for transfection into the cell host. Thus, in accordance with one of its aspects the present invention relates to a method of construction of the vector encoding the light and/or heavy chains of therapeutic antibodies and/or its antigennegative fragment according to the invention, where the method includes embedding in vector polynucleotide encoding the light and/or heavy chain of a therapeutic antibody according to the invention.

In another aspect the present invention relates to polynucleotide, codereuse murine VH domain having the sequence represented in SEQ ID NO:24.

In another aspect the present invention relates to polynucleotide, codereuse murine VL domain having the sequence represented in SEQ ID NO:25.

In another embodiment, the present invention relates to polynucleotide, codereuse VH domain having the sequence selected from the group consisting of SEQ ID NO:26, 27, 28, 29.

In another embodiment, the infusion is her invention relates to polynucleotide, codereuse VL domain having the sequence selected from the group consisting of SEQ ID NO:30, 31.

In accordance with this present invention relates to polynucleotide, codereuse heavy chain according to the invention, where the specified polynucleotide selected from the group consisting of SEQ ID nos:32, 33, 34, 35.

In accordance with this present invention relates to polynucleotide, codereuse light chain according to the invention, where the specified polynucleotide selected from the group consisting of SEQ ID nos:36, 37.

As it is obvious to a person skilled in this field, due to the degeneracy of the genetic code can also be obtained polynucleotide, which are the alternative described here polynucleotide coding for the polypeptides according to the invention.

3.1. The signal sequence

Antibodies according to the invention can be produced in the form of a hybrid protein with a heterologous signal sequence, with a specific site of cleavage at the N-Terminus of the Mature protein. The signal sequence should be recognized and processionals the host-cell. For prokaryotic host cells signal sequences can be leader sequence of alkaline phosphatase, penitsillinazy or thermostable enterotoxin II. For secretion in yeast signal PEFC what dovalidate can be the leader sequence of yeast invertase, leader sequence of the α-factor leader or sequence of acid phosphatase, see, for example, WO 90/13646. In cellular systems mammals may be present viral secretory leader sequence, such as a signal gD of herpes simplex virus and signal sequence of native immunoglobulin. Typically, the signal sequence are ligated in the same reading frame with the DNA encoding the antibody according to the invention.

3.2. The origin of replication

Origin replication is well known to specialists, while for most gram-negative bacteria, a suitable origin of replication is R322; for most suitable yeast origin of replication is the 2µ plasmid, and for most mammalian cells are suitable various origin replication of viruses such as SV40, polyomavirus, adenovirus, VSV or BPV. Usually, for expressing vectors mammalian origin of replication is not required, however, these vectors can be used SV40, because it contains the early promoter.

3.3. Selective marker

Typical selective genes encode proteins that (a) inform the resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, or (b) auxotrophic failure included the enta or nutrients, missing in complex environments. Sampling scheme may include the termination of growth of the host cell. Cells that were successfully transformed with genes encoding a therapeutic antibody according to the invention, survive, for example, due to its resistance to drug, reported a selective marker. Another example is the so-called marker DFR-selection, in which transformants are cultivated in the presence of methotrexate. In typical embodiments, the cells are cultured in the presence of increasing amounts of methotrexate to amplify the number of copies of interest exogenous gene. For DFR selection of the most suitable target cells are cells SNO. Another example is the expression system glutamate synthetase (Lonza Biologics). For use in yeast suitable selective gene is a gene oftrp1 see, Stinchcomb et al., Nature 282, 38, 1979.

MOT

Promoters suitable for expression of the antibodies according to the invention, functionally attached to the DNA/polynucleotide, codereuse this antibody. Suitable promoters for prokaryotic hosts include the promoters pho, promoter system, a beta-lactamase and lactose, the promoters of alkaline phosphatase, the tryptophan promoter, and hybrid promoters such as TAC. Promoters suitable for rapid and yeast cells, are the promoters for 3-phosphoglycerate-kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate-dehydrogenase, hexokinase, pyruvate-decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate-mutase and glucokinase. Inducible yeast promoters are the promoters of the alcohol dehydrogenase 2, sociogram C, acid phosphatase, metallothionein and enzymes responsible for the metabolism of nitrogen or utilization of maltose/galactose.

Promoters for expression in cellular systems mammals are viral promoters such as the promoters of polyomavirus, the smallpox virus in poultry and adenovirus (such as adenovirus 2)virus, bovine papilloma virus, sarcoma birds, cytomegalovirus (in particular, the promoter pretannage gene), a retrovirus, hepatitis b virus, actin, rous sarcoma virus (RSV) and early or late simian virus 40. It is obvious that the choice of promoter depends on compatibility with the host-cell used for expression. Therefore, in one of its variants the present invention relates to a first plasmid that contains the promoter of RSV virus and/or SV40 and/or CMV, DNA encoding the V-region light chain (VL) according to the invention, the scope κ together with markers for selection for resistance to neomycin and ampicillin, and the second plasmid, containing the her the promoter of the virus RSV or SV40, DNA encoding the V-region heavy chain (VH) according to the invention, DNA encoding a constant region γ1, DHFR marker and a marker of resistance to ampicillin.

3.5. Enhancer

If it is necessary, for example, for expression in higher eukaryotes, it can be used enhancer element, functionally attached to the promoter element in the vector. Suitable enhancer sequences mammals are enhancer elements derived from genes globin, elastase, albumin, fetoprotein, and insulin. Alternatively, it may be used enhancer element derived from the virus eukaryotic cells, such as the SV40 enhancer (BP 100-270), the enhancer early promoter of cytomegalovirus enhancer of polyomavirus, baculovirus enhancer or enhancer of the murine locus IgG2 (see WO 04/009823). The enhancer is preferably localized on the vector at a site located upstream of the promoter.

3.6. Cell owners

For cloning or expression vectors encoding the antibody according to the invention, suitable cell host is prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotic cells are eubacteria, such as enterobacteria, such asEsherichiafor exampleE. coli(for example, ADS 31446; 31537; 27325),Enterobacter,Erwinia, Klebsiellaproteus, Salmonellafor example,Salmonella typhimurium, Serratia, e.g.Serratia marcescansandShigella, as well as bacilli such asB. subtilisandBacillus licheniformis(see DD 266710),Pseudomonas,such as.aeruginosaandStreptomyces. From yeast host cells are also consideredSaccharomyces cerevisiae,schizosaccharomyces pombe, Kluyveromyces (for example, ADS 16045; 12424; 24178; 56500), yarrowia (EP 402226),Pichia pastoris(EP 183070, see also Peng et al., J. Biotechnol. 108 (2004) 185-192),Candida, Trichoderma reesia(EP244, 234),Penicillin, Tolypocladium andAspergillussuch asA. nidulansandA. niger.

Although the present invention specifically addresses prokaryotic and yeast cells-owners, however, the preferred cell host according to the invention are higher eukaryotic cells. Suitable higher eukaryotic cells hosts are mammalian cells such as COS-1 (ATSS No. RL 1650), COS-7 (ATSS No. RL 1651), cell line human kidney embryonic 293, kidney cells baby hamster (KSS)(ATSS No. RL 1632), VNC ATSS No. RL 10314), 293 (ATSS No. RL 1573)cells Chinese hamster ovary Cho (e.g., Cho-K1, ATSS No. CCL 61, cell line DHFR-Cho, such as DG44 (see Urlaub et al., (1986) Somatic Cell Mol. Genet. 12, 555-556)), in particular Cho cell line adapted to grow in suspension culture, murine cells, Sertoli cells, monkey kidney, the kidney cells of the African green monkey (ATSS No. RL 1587), cells NEA, cells of the kidneys of dogs (ATSS CCL 34), cells of human kidney (ATSS CCL 75), cells ner G2 and myeloma cells or lymphoma, for example NS0 (see U.S. patent No. 5807715), S2/0 and Y0.

Thus, in one of its variants the present invention relates to a stably transformed cell line containing a vector encoding a heavy and/or light chain therapeutic antibodies or antigennegative fragment described in this application. Preferably such cells masters contain the first vector encoding a light chain and a second vector encoding the indicated heavy chain.

Bacterial fermentation

For the expression of fragments of antibodies are particularly suitable bacterial system. Such fragments are localized inside the cells or in periplasm. Insoluble periplasmatic proteins can be extracted and subjected to refolding with the formation of active protein by methods known in the art, see Sanchez et al. (1999) J. Biotechnol. 72, 13-20 and Cupit P.M. et al. (1999) Lett. Appl. Environ. 29, 273-277.

3.7. Methods cell culture

Cell host transformed by a vector encoding a therapeutic antibody according to the invention or their antigennegative fragments, can be cultured by any method known in the art. Cell host can be cultured in centrifuge tubes, in the Oller-vials or systems of the hollow fiber, but for large-scale production it is preferable to use vats reactors for mixing, especially for suspension cultures. While it is preferable that the vats for mixing have been adapted for aeration using, for example, bubblers-spray, partitions or paddle mixers with low shear force. In columns-bubblers and aerolitic reactors can be applied directly by ozonation of air or oxygen. If cells-the hosts are cultivated in serum-free culture medium, preferably in this environment was added agent to protect cells, such as pluronic F-68, in order to prevent cell damage in the process of aeration. Depending on the properties of the host cells can be used microneedle as substrates for the growth substrate-dependent cell lines, or these cells can be adapted to suspension culture (which is usually used for these purposes). For culturing the host cells, in particular cells of invertebrate hosts, can be applied to various surgical methods, such as the cultivation of injection, periodic cultivation (see, Drapeau et al., (1994) Cytotechnology 15:103-109), continuous cultivation or perfusion culture. Although recombinante transformed cell x is saeva mammals can be cultured in an environment containing serum, such as fetal calf serum (FCS), however, it is preferable that these cells are the masters were cultivated in synthetic serum-free environment, such as the environment described by Keen et al. (1995) Cytotechnology 17:153-163, or in a commercially available medium such as Rhone-DM or UltraCHOTM(Cambrex, NJ, USA), which, if necessary, add an energy source, such as glucose, and synthetic growth factors such as recombinant insulin. For culturing the host cells in serum-free environment, it is necessary that these cells were adapted to growth in serum-free conditions. One way to adapt is culturing such host cells in a medium containing serum, followed by periodic replacement of 80% of this culture medium serum-free medium, so that the cells of host-adapted to the conditions of cultivation in serum-free medium (see for example, K. Scharfenberg et al. (1995) Animal Cell Technology: Developments towards the 21st century (E.C. Beuvery et al., eds.), pp.619-623, Kluwer Academic publishers).

Antibodies according to the invention, secreted in the specified environment, can be isolated and purified by various methods, allowing clearance to the desired degree. For example, for the treatment of human mainly used therapeutic antibodies according to the invention having at least 95% purity, and is commonly 98% or 99% purity or more (compared with the untreated culture medium). In the first case, the cellular debris is usually removed from the culture medium by centrifugation followed by the stage of purification of the supernatant, for example by means of microfiltration, ultrafiltration and/or deep filtration. There are also various other cleaning methods, such as dialysis and electrophoresis in the gel, as well as chromatographic methods, such as chromatography on hydroxyappatite (HA), affinity chromatography (optional, including a system of affine tagging, such as polyhistidine) and/or hydrophobic chromatography (GFH, see U.S. patent No. 5429746). In one embodiment of the present invention, the antibodies according to the invention after carrying out various stages of purification immobilized using affinity chromatography on protein a or G-protein, and then spend additional chromatographic stage, such as ion exchange chromatography and/or chromatography on hydroxyappatite (HA), anyone - or cation-exchange chromatography, size-exclusion chromatography and precipitation with ammonium sulfate. In addition, usually carried out various stages of elimination of viruses (for example, by nanofiltration using, for example, filter DV-20). After carrying out these various stages get cleaned (preferably monoclonal) product containing at least 75 mg/ml or more, n is an example of 100 mg/ml or more, the specified antibodies according to the invention or its antigennegative fragment, and this drug is one of the variants of the present invention. Preferably, such preparations essentially do not contain aggregated forms of the antibody according to the invention.

4. The pharmaceutical composition

Purified preparations of antibodies according to the invention (in particular, the preparations of monoclonal antibodies, described above, can be entered in pharmaceutical compositions intended for the treatment of diseases and disorders, such as atopic diseases, such as asthma, allergic rhinitis, COPD. Typically, such compositions contain a known pharmaceutically acceptable carrier used in pharmaceutical practice, see, for example, Remington''s Pharmaceutical Sciences, 16th edition (1980), Mack Publishing. Co. Examples of such media are sterilized media, such as saline, ringer's solution or dextrose, buffered with suitable buffers to a pH of 5-8. Pharmaceutical compositions for injection (e.g. intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular or vnutripartiynyh injection or continuous infusion usually do not contain obvious large particles and may contain from 0.1 ng to 100 mg of the antibody, preferably 5 mg to 25 mg of antibody. Methods received the I of such pharmaceutical compositions is well known to specialists. In one embodiment of the invention the pharmaceutical compositions contain from 0.1 ng to 100 mg therapeutic antibodies according to the invention in a uniform dosage form, optionally together with the attached instructions for their use. The pharmaceutical compositions according to the invention can be liofilizovane (dried by freezing), and then before the introduction divorced by methods well known or obvious to a person skilled in this field. If the variation of the present invention include antibodies of isotype IgG1, the pharmaceutical composition may be added to the agent that forms a chelate with copper, as such agent, as the citrate (e.g. sodium citrate or EDTA or histidine to decrease mediated by copper degradation of the antibodies of this isotype, see EP 0612251. Introduction antibody against hIL-13 may be made orally, by inhalation and local (for example, by intraocular injection, intranasal, rectal administration and the introduction of wounds on the skin).

Effective doses and schemes for the treatment of the antibody according to the invention is usually determined empirically, and these doses and schemes depend on such factors as age, weight and health status of the patient, and the disease or disorder being treated. Such factors should be considered curing the m physician. Guidance on how to determine suitable doses can be found in the publication of Smith et al. (1977) Antibodies in human diagnosis and therapy, Raven Press, New York, and typically, these dose ranges from 1 mg to 1000 mg

Depending on the disease or disorder being treated (particularly asthma), pharmaceutical compositions containing a therapeutically effective amount of the antibody according to the invention, can be introduced simultaneously, separately or sequentially in combination with an effective amount of other drugs, such as anti-inflammatory agents (e.g., corticosteroid or NSPs), anticholinergic agents (particularly antagonists of the receptor M1/M2/M3), agonists β2-adrenergic receptors, anti-infective tools (e.g., antibiotics, antivirals, antihistamines, and a PDE4 inhibitor. Examples of agonists β2-adrenergic receptors are salmeterol, salbutamol, formoterol, salmefamol, fenoterol, terbutaline. Preferred agonists β2-adrenergic receptors prolonged action are agonists described in WO 02/66422A, WO 02/270490, WO 02/076933, WO 03/024439 and WO 03/072539. Suitable corticosteroids include methylprednisolone, prednisolone, dexamethasone, fluticasone propionate, S-formerely ester of 6α,9α-debtor-17α-[(2-fornicator)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-CT is otinovaa acid, S-2-oxo-tetrahydrofuran-3S-silt ester of 6α,9α-debtor-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-karbaminovoi acid, esters of beclomethasone (e.g., ether 17-propionic acid or ether 17,21-dipropionate acid), budesonide, flunisolide, esters mometasone (for example, a complex furoate ester), triamcinolone acetonide, rofleponide, ciclesonide (16α, 17-[[(R)-cyclohexylmethyl]bis(oxy)]-11β,21-dihydroxypregna-1,4-diene-3,20-dione), propionate butixocort, RPR-106541, and ST-126. Preferred corticosteroids include fluticasone propionate, S-formerely ester of 6α,9α-debtor-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-karbaminovoi acid and S-formerely ester of 6α,9α-debtor-17α-[(2-fornicator)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-karbaminovoi acid, and more preferably S-formerely ester of 6α,9α-debtor-17α-[(2-fornicator)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-karbaminovoi acid.

Non-steroidal compounds that are agonists glucocorticoids and have selectivity with respect to transrepression through development and which can be used in combination therapy, were described in the following patents: WO 03/082827, WO 01/10143, WO 98/54159, WO 04/005229, WO 04/009016, WO 04/009017, WO 04/018429, WO 03/104195, WO 03/082787, WO 03/082280, WO 0/059899, WO 03/101932, WO 02/02565, WO 01/16128, WO 00/66590, WO 03/086294, WO 04/026248, WO 03/061651, WO 03/08277.

Suitable anti-inflammatory drugs are non-steroidal anti-inflammatory drugs (NSPs).

Suitable NSPs are cromoglicate sodium, nedocromil sodium, phosphodiesterase inhibitor (PDE)(for example, theophylline, PDE4 inhibitors or mixed inhibitors of PDE3/PDE4), leukotriene antagonists, inhibitors of leukotriene synthesis (for example, montelukast), iNOS inhibitors, inhibitors of tryptase and elastase, antagonists of integrin beta-2 agonists or antagonists of adenosine receptor (e.g., agonists of the adenosine 2A), cytokine antagonists (e.g. chemokine antagonists, such as antagonist R3) or inhibitors of cytokine synthesis or inhibitors of 5-lipoxygenase. Other suitable agonists β2-adrenergic receptors are salmeterol (e.g. as xinafoate), salbutamol (e.g. as the sulphate or free base), formoterol (e.g. as fumarata), fenoterol or terbutaline and salts. For oral administration is preferred iNOS (inducible inhibitor (nitric oxide)-synthase). Suitable iNOS inhibitors are inhibitors described in WO 93/13055, WO 98/30537, WO 02/50021, WO 95/34534 and WO 99/62875. Suitable inhibitors R3 are inhibitors described in WO 02/26722.

Of particular interest is the use of anti-Christ. eat according to the invention in combination with an inhibitor of phosphodiesterase 4 (PDE4). PDE-4-specific inhibitor that is used in this aspect of the present invention may be any compounds which are known to inhibit the PDE4 enzyme or has been found to act as a PDE4 inhibitor, and which are only PDE4 inhibitors, but not compounds that inhibit other members of the PDE family, such as PDE3 and PDE5 and PDE4.

Interest compounds are CIS-4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexane-1-carboxylic acid, 2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-deformational)cyclohexane-1-he and CIS-[4-cyano-4-(3-cyclopropylmethoxy-4-deformational)cyclohexane-1-ol]. In addition, CIS-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylic acid (also known as cilomilast) and its salts, esters, prodrugs and physical form described in U.S. patent 5552438, issued September 3, 1996; this patent and it describes the connection in its entirety introduced into the present description by reference.

AWD-12-281 supplied by the company Elbion (Hofgen, N. et al. 15th EFMC Int. Symp. Med. Chem (Sept. 6-10, Edinburgh) 1998, Abst.P.98; summary CAS No. 247584020-9); derived 9-benzyladenine marked NCS-613 (INSERM); D-4418 supplied by firms Chiroscience and Schering-Plough; inhibitor benzodiazepine PDE4 denoted CI-1018 (PD-168787) and supplied by the company Pfizer; derived benzodioxole described Kyowa Hakko in WO 99/1666; K-34 described in Kyowa Hakko; V-A supplied by the company Napp (Landells, L.J. et al., Eur. Resp. J.[Annu. Cong. Eur. Resp. Soc (Sept 19-23, Geneva), 1998], 12 (Suppl. 28): Abst. P2393); roflumilast (summary CAS No. 162401-32-3) and phthalazine (WO 99/47505, the description of which is introduced in the present invention by reference) are delivered by the company Byk-Gulden; pumafentrine, (-)-p-[(4aR*,10bS*)-9-ethoxy-1,2,3,4,4A,10b-hexahydro-8-methoxy-2-methylbenzo[c][1,6]naphthiridine-6-yl]-N,N-diisopropylbenzamide, which is a mixed inhibitor of PDE3/PDE4, was obtained as described in the manual Byk-Gulden, now Altanta; arofylline is under development at Almirall-Prodesfarma; VM554/UM565 are delivered by the company Vernalis; T-440 described Tanabe Seiyaku; Fuji, K. et al., J. Pharmacol. Exp. Ther, 1998, 284(1):162, as also described T.

Other interest compounds described in published International patent application WO 04/024728 (Glaxo Group Ltd), PCT/EP2003/014867 (Glaxo Group Ltd) and PCT/ER/005494 (Glaxo Group Ltd).

Suitable anticholinergic means are compounds that act as antagonists at the muscarinic receptors, in particular compounds that are antagonists of receptors M1or M3dual receptor antagonists M1/M3or M2/M3or pan-antagonists of the receptors M1/M2/M3. Representative compounds for administration by inhalation are ipratropium (e.g. as bromide, CAS 22254-24-6, keyusage is sold under the trademark Atrovent), oxytrope (for example, as the bromide, CAS 30286-75-0) and Tiotropium (for example, as the bromide, CAS 136310-93-5, commercially available under the trademark Spiriva). Interest compounds are also reatreat (for example, in the form of hydrobromide CAS 262586-79-8) and LAS-34273 described in WO 01/04118. Representative compounds for oral administration are pirenzepine (CAS 28797-61-7), darifenacin (CAS 133099-04-4 or CAS 133099-07-7 in the form of hydrobromide, commercially available under the trademark Enablex), oxybutynin (CAS 5633-20-5, commercially available under the trademark Ditropan), terodiline (CAS 15793-40-5), tolterodine (CAS 124937-51-5 or CAS 124937-52-6 in the form of tartrate, commercially available under the trademark Detrol), uilani (for example, as the bromide, CAS 26095-59-0, commercially available under the trademark Spasmomen), chloride of Tropea (CAS 10405-02-4), solifenacin (CAS 242478-37-1 or CAS 242478-38-2 in succinate, also known as YM-905 and commercially available under the trademark Vesicare).

Other suitable anticholinergic means are the compounds of formula (XXI), described in application for U.S. patent 60/487981:

where the preferred orientation of the alkyl chain attached to tripanosoma ring is endo-orientation;

R31and R32independently selected from the group consisting of straight or branched lower alkyl groups having preferred the compulsory from 1 to 6 carbon atoms, cycloalkyl groups having from 5 to 6 carbon atoms, cycloalkenyl having from 6 to 10 carbon atoms, 2-tanila, 2-pyridyl, phenyl, phenyl substituted alkyl group having not more than 4 carbon atoms, and phenyl, substituted alkoxygroup having not more than 4 carbon atoms;

X-represents an anion associated with the positively charged N atom;

X-may represent, but are not limited to, chloride, bromide, iodide, sulfate, bansilalpet and toluensulfonate, including, for example:

(3-endo)-3-(2,2-di-2-dienylidene)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octabrain;

(3-endo)-3-(2,2-diphenylacetyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octabrain;

(3-endo)-3-(2,2-diphenylacetyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]Octan-4-methylbenzenesulfonate;

(3-endo)-8,8-dimethyl-3-[2-phenyl-2-(2-thienyl)ethynyl]-8-azoniabicyclo[3.2.1]octabrain; and/or

(3-endo)-8,8-dimethyl-3-[2-phenyl-2-(2-pyridinyl)ethynyl]-8-azoniabicyclo[3.2.1]octabrain.

Other suitable anticholinergic means are compounds of formula (XXII) or (XXIII), described in application for U.S. patent 60/511009:

where

atom N is in the Exo-position;

R41-represents an anion associated with the positively charged atom n R41-may represent, but is only limited by them, chloride, bromide, iodide, sulfate, bansilalpet and toluensulfonate;

R42and R43independently selected from the group consisting of straight or branched lower alkyl groups (having preferably from 1 to 6 carbon atoms), cycloalkyl groups (having from 5 to 6 carbon atoms), cycloalkenyl (having from 6 to 10 carbon atoms), geterotsiklicheskie (having 5 to 6 carbon atoms) and N or O as the heteroatom, geterotsiklicheskikh (having from 6 to 10 carbon atoms) and N or O as the heteroatom, aryl, optionally substituted aryl, heteroaryl and optionally substituted heteroaryl;

R44selected from the group consisting of (C1-C6)alkyl, (C3-C12)cycloalkyl, (C3-C7)geterotsiklicheskie, (C1-C6)alkyl(C3-C12)cycloalkyl, (C1-C6)alkyl(C3-C7)geterotsiklicheskie, aryl, heteroaryl, (C1-C6)alkylaryl, (C1-C6)alkylglycerol, -or SIG45, -CH2Or SIG45, -CH2HE, -CN, -CF3, -CH2O(CO)R46, -CO2R47, -CH2NH2, -CH2N(R47)SO2R45, -SO2N(R47)(R48), -SOP(R47R48, -CH2N(R48)WITH(R46), -CH2N(R48)SO2(R46), -CH2N(R48)CO2(R45), -CH2 N(R48)N(R47);

R45selected from the group consisting of (C1-C6)alkyl, (C1-C6)alkyl(C3-C12)cycloalkyl, (C1-C6)alkyl(C3-C7)geterotsiklicheskie, (C1-C6)alkylaryl, (C1-C6)alkylglycerol;

R46selected from the group consisting of (C1-C6)alkyl, (C3-C12)cycloalkyl, (C3-C7)geterotsiklicheskie, (C1-C6)alkyl(C3-C12)cycloalkyl, (C1-C6)alkyl(C3-C7)geterotsiklicheskie, aryl, heteroaryl, (C1-C6)alkylaryl, (C1-C6)alkylglycerol;

R47and R48independently selected from the group consisting of H, (C1-C6)alkyl, (C3-C12)cycloalkyl, (C3-C7)geterotsiklicheskie, (C1-C6)alkyl(C3-C12)cycloalkyl, (C1-C6)alkyl(C3-C7)geterotsiklicheskie, (C1-C6)alkylaryl and (C1-C6)alkylglycerol, including, for example,

(endo)-3-(2-methoxy-2,2-dateopen-2-yl-ethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octamide;

3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenyl-propionitrile;

(endo)-8-methyl-3-(2,2,2-triphenylmethyl)-8-Aza-bicyclo[3.2.1]octane;

3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenyl-propionamide;

3-((endo)-8-met the l-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenyl-propionic acid;

(endo)-3-(2-cyano-2,2-diphenylether)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octamide;

(endo)-3-(2-cyano-2,2-diphenylether)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octabrain;

3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenyl-propan-1-ol;

N-benzyl-3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenyl-propionamide;

(endo)-3-(2-carbarnoyl-2,2-diphenylether)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octamide;

1-benzyl-3-[3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenylpropyl]urea;

1-ethyl-3-[3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenylpropyl]urea;

N-[3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenylpropyl]ndimethylacetamide;

N-[3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenylpropyl]benzamide;

3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-di-thiophene-2-yl-propionitrile;

(endo)-3-(2-cyano-2,2-dateopen-2-yl-ethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octamide;

N-[3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenylpropyl]benzosulfimide;

[3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenylpropyl]urea;

N-[3-((endo)-8-methyl-8-Aza-bicyclo[3.2.1]Oct-3-yl)-2,2-diphenylpropyl]methanesulfonamide; and/or

(endo)-3-{2,2-diphenyl-3-[(1-phenylethanol)amino]propyl}-8,8-dimethyl-8-azoniabicyclo[3.2.1]octabrain.

More preferred compounds which can be used in the present invention, are:

(endo)-3-(2-methoxy-2,2-ditive the-2-yl-ethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octamide;

(endo)-3-(2-cyano-2,2-diphenylether)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octamide;

(endo)-3-(2-cyano-2,2-diphenylether)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octabrain;

(endo)-3-(2-carbarnoyl-2,2-diphenylether)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octamide;

(endo)-3-(2-cyano-2,2-dateopen-2-yl-ethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octamide; and/or

(endo)-3-{2,2-diphenyl-3-[(1-phenylethanol)amino]propyl}-8,8-dimethyl-8-azoniabicyclo[3.2.1]octabrain.

Suitable antihistamines (also known antagonists of H1-receptor) are any one or more of various known antagonists that inhibit H1-receptors and are safe for humans. Antagonists of the first row are derivatives of ethanolamines, ethylendiamine and bonds alkylamines, such as diphenylhydramine, pyrilamine, clemastine, chlorpheniramine. Antagonists of the second row that are not sedatives include loratidine, desloratidine, terfenadine, astemizole, acrivastine, azelastine, levocetirizine, Fexofenadine and cetirizine.

Preferred examples of antihistamines are loratadine, desloratidine, Fexofenadine and cetirizine.

Other considered combinations are combinations of antibodies according to the invention with the tool against IL-4 (e.g., an antibody against IL-4, such as pascolizumab) and/or remedy against IL-5 (e.g., ant the body against IL-5, such as mepolizumab) and/or remedy against IgE (e.g., an antibody against IgE, such as omalizumab (XolairTMor talisman).

In the present invention is also considered a pharmaceutical composition comprising a kit including components such as an antibody according to the invention or its antigennegative fragment together with the above-mentioned other drugs, and optional instructions for its use.

In addition, in the present invention is considered a pharmaceutical composition comprising a therapeutically effective amount described here monoclonal therapeutic antibodies or antigennegative fragment and used for the treatment of disorders susceptible to modulation of the interaction between hIL-13 and hIL-13R.

In accordance with this present invention relates to pharmaceutical compositions containing a therapeutically effective amount of a monoclonal gumanitarnogo therapeutic antibodies, where the specified antibody contains a VH domain selected from the group consisting of SEQ ID NO:11, 12, 13, 14, and VL domain selected from the group consisting of SEQ ID NO:15, 16.

In accordance with this present invention relates to pharmaceutical compositions comprising a monoclonal therapeutic antibody containing a heavy chain selected from g is uppy, consisting of SEQ ID NO:18, 19, 20, 21, and a light chain selected from the group consisting of SEQ ID NO:22, 23.

In accordance with this present invention relates to pharmaceutical compositions comprising a monoclonal therapeutic antibody containing heavy chain SEQ ID NO:18 and a light chain of SEQ ID NO:22, and a pharmaceutically acceptable carrier.

In accordance with this present invention relates to pharmaceutical compositions comprising a monoclonal antibody containing heavy chain SEQ ID NO:18 and a light chain of SEQ ID NO:22 (or essentially consisting of them), and a pharmaceutically acceptable carrier.

In accordance with this present invention relates to pharmaceutical compositions containing a pharmaceutically acceptable carrier and a therapeutically effective amount of a population of monoclonal therapeutic antibodies, where the specified therapeutic antibody contains a heavy chain SEQ ID NO:18 and a light chain of SEQ ID NO:22.

5. Clinical use

Antibodies according to the invention can be used to treat atopic diseases/disorders and chronic inflammatory diseases/disorders. Of particular interest is their use for treatment of asthma, such as allergic asthma, particularly severe asthma (i.e. asthma, which is not susceptible to treatment by modern methods, the key systemic administration of corticosteroids; see, Busse W.W. et al., J. Allergy Clin. immunol. 2000, 106:1033-1042), “difficult” asthma (defined as asthma phenotype, characterized by the absence of the effect, despite the introduction of the maximum recommended dosage is prescribed steroids, administered by inhalation, see Barnes P.J. (1998), Eur. Respir. J. 12:1208-1218), unstable asthma (defined as severe unstable asthma in subgroups of patients in whom there is wide variability in the maximum speed of expiration, despite the high doses of steroids, administered by inhalation, see Ayres J.G. et al. (1998) Thorax 58:315-321), “night” asthma, premenstrual asthma, steroid-resistant asthma (see Woodcock A.J. (1993) Eur. Respir. J. 6:743-747), steroid-dependent asthma (defined as asthma that can be treated only with high doses of oral input steroids), aspirin-induced asthma, asthma adults and children with asthma. Antibodies according to the invention can be used to prevent, reduce the frequency or severity of asthma (asthma status). Antibodies according to the invention can also be used to decrease the dose of other medicines (defined as the number or frequency of dose)required for the treatment of asthma. For example, the antibodies according to the invention can be used to reduce the dose of steroids, so the x as corticosteroids, required for the treatment of asthma (“to abstain from the use of steroids). Other diseases or disorders that can be treated by the antibodies according to the invention are atopic dermatitis, allergic rhinitis, Crohn's disease, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis, fibrotic diseases or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis (scleroderma), liver fibrosis, granulomas liver, schistosomiasis, leishmaniasis, and diseases associated with dysregulation of the cell cycle, such as Hodgkin's disease and b-cell chronic lymphocytic leukemia. Other diseases or disorders that can be treated by the antibodies according to the invention, described in detail in the above section “Prior art”.

In one of its variants the present invention relates to a method of treating a patient (human)suffering from asthmatic condition that is not treatable with corticosteroids, where the method includes a step of introducing the indicated patient a therapeutically effective amount of the antibody according to the invention.

In another embodiment, the present invention relates to a method for prevention of acute asthma patient is a (person), where the method involves the step of introducing the indicated patient a therapeutically effective amount of the antibody according to the invention.

In another embodiment, the present invention relates to a method of reducing the frequency and/or severity of asthma in a patient (person), where the method includes a step of introducing the indicated patient a therapeutically effective amount of the antibody according to the invention.

In another embodiment, the present invention relates to method bias T-helper cell response toward the Th1 response-type after inflammatory and/or allergic attack in the human, where the method includes a step of introducing the indicated patient a therapeutically effective amount of the antibody or its antigennegative fragment according to the invention.

In another embodiment, the present invention relates to a method of treating a patient (person)having the option Q130hIL-13 and suffering from asthma, such as severe asthma, where the method includes a step of introducing the indicated patient a therapeutically effective amount of the antibody or its antigennegative fragment according to the invention.

Although the present invention is described mainly with respect to treatment of diseases or disorders in humans, however, the present invention can be t is the train used to treat similar diseases or disorders in mammals, not a person.

The following description of the present invention is given only as an example.

Examples

1. Production of monoclonal antibodies and characterization of mouse monoclonal antibodies A

Monoclonal antibodies (mAb) was obtained from hybridoma cells mainly in accordance with the methodology described in the manual, E. Harlow and D. Lane, Antibodies a Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In accordance with this methodology monoclonal antibodies were obtained by fusion of mouse myeloma cells with lymphocytes obtained from mice, immunogenic target antigen. Hybridoma cell becomes immortalizing thanks myeloma partner in the merger, and the ability to produce antibodies reported In lymphocytes.

Five SJL mice were immunized by intraperitoneal injection to each mouse 2 μg of recombinant human IL-13, derived fromE. coli(Cambridge Bioscience, Cat. No SN-013). For generating an immune response in mice with high titers of antibodies against human IL-13 was developed corresponding schema immunization. After 5 immunizations for 64 days mice were killed and collected cells of the spleen. For producing a hybrid of 3 of these mice took spleen cells and b-lymphocytes were subjected to fusion with mouse myeloma cells obtained from cle is OK RH, using PEG (Boehringer). Individual hybridoma cell lines were cloned by limiting dilution (E. Harlow &D. Lane, supra). Wells containing monoclone identified under the microscope and the supernatant was tested for activity. Cells from most active clones were propagated for cryopreservation for producing antibodies, etc.

First supernatant hybridoma culture was skanirovali on the activity of binding to det-1-labeled recombinant human protein IL-13 expressed inE. coli(obtained under laboratory conditions) in the format of “sandwich”assay. The second screening positive for linking cultures were carried out using the BIAcore methodTMfor detection of binding with det-1-labeled human protein IL-13. Then samples of these hybridomas were tested for their ability to neutralize the biological activity of recombinant human IL-13 expressed inE. coli(Cambridge Bioscience, cat. No SN-013) in bioanalysis cells TF-1.

Six positive clones identified in bioanalysis to neutralize human IL-13 was subclinically by limiting dilution to obtain a stable monoclonal cell lines. Immunoglobulins isolated from these hybridomas cultured in cell “factories” in serum-free medium, was purified using columns with immobilize the data protein A. Then the purified mAb was re skanirovali in the following analytical systems analysis:

Analysis of the binding of recombinant human IL-13 expressed inE. coli(in the format of a sandwich-ELISA)

Analysis of the inhibition of binding of det-1-labeled recombinant human IL-13 expressed inE. coli(in the format of a sandwich-ELISA)

Analysis on the neutralization of recombinant IL-13 human or IL-13 cynomolgus monkeys, expressed inE. coli(in bioanalysis cells TF-1)

Analysis to neutralize Q130-variant recombinant human IL-13 expressed in E.coli (in bioanalysis cells TF-1).

Analysis on the neutralization of recombinant IL-13 human or IL-13 cynomolgus monkeys, expressed inE. coli(in bioanalysis cells TF-1)

Analysis on the specificity of binding to human IL-13, which is determined by assessing the cross-reactivity of the mAb with human IL-4 ELISA and by assessing the cross-reactivity of the mAb with human IL-5 in bioanalysis to neutralize IL-5.

Analysis BIAcoreTMto determine the level of affinity binding to human IL-13.

Monoclonal antibody A was identified as the antibody neutralizes the biological activity of IL-13 human and cynomolgus monkeys. In the following analysis describes the profile of monoclonal antibodies A.

1.1. Binding of the recombinant human IL-13, expressed inE. coli

Linking A with recombinant human IL-13 expressed inE. coli, was evaluated in a sandwich ELISA method essentially described in section 7. Cm. figure 1.

1.2. Inhibition of binding det-1-labeled recombinant human IL-13 expressed inE. coliwith IL-13Rα1 and IL-13Rα2 in the ELISA format

Antibody A inhibited binding det-1-labeled recombinant human IL-13 expressed inE. coliwith the two chains of a receptor for human IL-13. In addition, this antibody inhibited binding more effectively than commercially available polyclonal antibody against human IL-13 monoclonal antibody against human IL-13 (R&D Systems). The value of the IC50that was 0,165 μg/ml, was calculated to assess the inhibition of monoclonal antibody A binding human IL-13 human IL-13Rα1. The value of the IC50that was 0,056 μg/ml, was calculated to assess the inhibition of monoclonal antibody A binding human IL-13 human IL-13Rα2. Cm. figures 2a and 2b. Control IgG with irrelevant specificity did not show detectable activity.

1.3. Neutralization of recombinant IL-13 human IL-13 cynomolgus monkeys, expressed inE. coliin bioanalysis on cell proliferation, TF-1

Cells TF-1 m which may proliferates in response to human IL-13 and IL-13 cynomolgus monkeys. Was developed bioanalysis to assess the ability of mAb against IL-13 neutralizing cell proliferation, TF-1 induced IL-13 human and cynomolgus monkeys. In bioanalysis on cell proliferation, TF-1 antibody A neutralized the biological activity of recombinant IL-13 human and cynomolgus monkeys. In addition, this antibody is neutralized IL-13 human and cynomolgus monkeys is more efficient than commercially available polyclonal antibody against human IL-13 monoclonal antibody against human IL-13 (obtained from R&D Systems). Cm. figure 3.

The average value of ND50that was 0,0783 μg/ml, was calculated to assess the neutralizing monoclonal antibody A biological activity of 5 ng/ml recombinant human IL-13 expressed inE. coliin bioanalysis on cell proliferation, TF-1. The value of ND50that was 0.04 µg/ml, was calculated to assess the neutralizing monoclonal antibody A biological activity of 5 ng/ml recombinant IL-13 cynomolgus monkeys, expressed inE. coliin bioanalysis on cell proliferation, TF-1. [The value of ND50(neutralizing dose) represents the concentration of monoclonal antibody required to reduce cell proliferation in TF-1 by 50%, induced a certain concentration of IL-13].

1.4. Neutralization of human IL-13 expressed in mammalian cells (cells Cho in bioanalysis on cell proliferation, TF-1

In the analysis for cell proliferation, TF-1 was assessed by the ability of monoclonal antibodies A to neutralize human IL-13 expressed in cells SNO. Antibody A neutralizes human IL-13 expressed in mammalian cells, is more effective than commercially available polyclonal antibody against human IL-13, as defined by the values of ND50. The value of ND50that was 0,037 μg/ml, was calculated to assess the neutralizing monoclonal antibody A approximately 50 ng/ml human IL-13 expressed in mammals, in bioanalysis on cell proliferation, TF-1. Cm. figure 4.

1.5. Neutralization Q130-variant recombinant human IL-13 in bioanalysis on cell proliferation, TF-1

In the analysis for cell proliferation, TF-1 was assessed by the ability of monoclonal antibodies A to neutralize Q130-variant recombinant human IL-13 expressed inE. coli(Peprotech, Cat No. 200-13A). Antibody A neutralized Q130-variant of human IL-13 is more effective than commercially available polyclonal antibody against human IL-13. The value of ND50that was 0.11 μg/ml, was calculated to assess the neutralizing monoclonal antibody A biological activity of 60 ng/ml Q130-option the human is ical IL-13. Cm. figure 5.

1.6. The specificity of binding to human IL-13

Because the human IL-4 to its structural and functional properties is the most similar to human IL-13, the binding specificity of monoclonal antibodies A with human IL-13 was assessed in ELISA for binding to human IL-4. Antibody A did not bind to recombinant human IL-4 expressed inE. colithat indicated a high level of specificity of binding of the indicated monoclonal antibodies to human IL-13. In addition, the antibody A did not cross-ability to neutralize the biological activity of recombinant human IL-5 expressed inE. coliin bioanalysis on cell proliferation, TF-1. Cm. figures 6 and 7.

1.7. The BIAcore analysisTM

The binding affinity of A with recombinant IL-13 human and cynomolgus monkeys was evaluated in the BIAcore analysisTM. Cm. table 1.

Table 1
Sample IL-13Ka Association (1/MS)d dissociation (1/s)The affinity constant (D)
det-1-labeled human IL-13of 2.25 × 106 of 7.2 × 10-532 PM
human IL-13 (CA)6,82 × 105of 1.84 × 10-4270 PM
IL-13 cynomolgus monkeys (CA)9,14 × 105of 5.6 × 10-561,2 PM

These data showed that the antibody A has no binding affinity of IL-13 human IL-13 cynomolgus monkeys. [In this analysis used two different sample human IL-13 (both of which were produced inE. coli). IL-13, in essence, is insoluble if it is produced inE. colibut it can be solubilisation, and then subjected to reformingin vitro. The differences in the two samples, IL-13, subjected to refolding may serve to explain differences in offendeth linking each of these samples of human IL-13].

2. Cloning of the variable regions of clone A

Full RNA was extracted from hybridoma cell clone A and cDNA variable domains of the heavy and light chains were produced by the reaction of reverse transcription using primers specific to the mouse leader sequence and constant regions of the anti-Christ. ate, corresponding to a predefined isotype (IgG1/κ). Then cDNA variable domains of the heavy and light chains were cloned into the vector CR2.1 for sequencing.

2.1. Extraction RNA

Full RNA was extracted from the sediment of approximately 106cell hybridoma clone A using the system for the allocation of a full size SV RNA (Promega) according to manufacturer's instructions.

2.2. Reverse transcription

RNA was subjected to reverse transcription to produce cDNA variable domains of the heavy and light chains using primers specific to the mouse leader sequence and a murine constant regions of IgG1/κ. Used a mixture of primers described in the publication Jones ST and Bendig MM Bio/technology 9:88-89 (1991).

Pools direct primers leader sequence of the mouse VNand VLreceived in a concentration of 50 μm. Solutions reverse primers murine constant regions of IgG1 and κ also obtained at a concentration of 50 ám.

2.3. PCR with reverse transcriptase (RT-PCR)

Reverse transcription of RNA that encodes a variable region of the heavy and light chains was performed in duplicates using the Access RT-PCR (Promega) according to manufacturer's instructions. Forward and reverse primers VH and VL described above.

2.4. Gel-purification of the product FROM RT-PCR

Products which you RT-PCR (2 × V Nand 2 × VL) were loaded into the solution for loading the preparative 1% agarose gel containing 0.01% ethidiumbromid, and electrophoresis was performed in TAE buffer at 100 V for 1 hour, and then stripe V-region is cut out. To identify VNand VL-bands in the gel electrophoresis was performed 100 BP DNA ladder.

The DNA fragments were extracted and purified from the gel using a kit for the extraction of the QIAquick gelTM(Qiagen) according to manufacturer's instructions.

2.5. Ligation

Purified RT-PCR-fragments (2 × VNand 2 × VL) cloned in the vector pCR2.1 using the kit TA cloning (Invitrogen) according to manufacturer's instructions.

2.6. Transformation

Legirovannye plasmid was transferred into the cells TOP10F' in accordance with the instructions attached to the kit TA cloning. 50 ál and 200 ál of transformed cells were sown on tablets with L-agar containing 100 μg/ml ampicillin and sensitised 8 μl of 500 mm IPTG solution and 16 μl of 50 mg/ml X-Gal in DMF. The plates were incubated over night at 37°C.

2.7. Sequencing

Colonies were sown and cultivated over night at 37°C in 5 ml of medium IN which was added 100 μg/ml ampicillin. Plasmids R2.1 containing VNand VLdomains A, were extracted and purified using the-W set for cooking mini-drugs Qiagen QIAprep Spin Miniprep in accordance with the manufacturer's instructions. VNand VLdomains sequenced using primers T7, direct primer M13 reverse primer M13.

Amino acid sequence of VN-region antibody A (consensus sequence of 10 clones from 2 RT-PCR-reactions) is SEQ ID NO:7.

Amino acid sequence of VL-region antibody A (consensus sequence of 10 clones from 2 RT-PCR-reactions) is SEQ ID NO:8.

3. Chimeric antibody

Chimeric antibody comprising the parent murine V-regions (see section 2.7)attached to the C-region of human IgG1/κ wild type, was designed to confirm the cloning of the corresponding murine V-regions, as well as for its use as a standard when testing gumanitarnyh structures. Chimeric antibody was expressed in cells SNO, purified and tested for binding to human IL-13 by ELISA.

3.1. PCR amplification

The cloned murine V-region amplified by PCR to introduce restriction sites required for cloning in expressing vectors mammals Rld and Rln. In VNdomain createdHindIII - andSeI-sites in the same reading frame and was carried out by cloning into the modified Rld-vector containing a C-area chain γ1 antibody wild type. In VLdomain createdHindIII - andBsiwI-sites in the same reading frame and was carried out by cloning into the modified Rln-vector containing the C-region of the κ chain of the human antibody to wild-type.

Direct primer for VN:

5'-GAT GAA GCT TGC CAC CAT GAA ATG CAG CTG GGT CAT C-3'

(SEQ ID NO:86)

HindIII restriction site is underlined and the Kozak sequence is in bold font.

Reverse primer for VN:

5'-GAT GGA CTA GTG TTC CTT GAC CCC AGT A-3' (SEQ ID NO:87)

RestrictionSeI-site is underlined.

Direct primer for VL:

5'-GAT GAA GCT TGC CAC CAT GAA GTT GCC TGT TAG GCT G-3'

(SEQ ID NO:88)

HindIII restriction site is underlined and the Kozak sequence is in bold font.

Reverse primer for VL:

5'-GAT GCG TAC GTT TGA TTT CCA GCT TGG TGC C-3' (SEQ ID NO:89)

BsiWI restriction site is underlined.

PCR reaction:Water66 ál
10 × PCR buffer10 ál
dNTP (2 mm)10 ál
primer 1 (5 μm)4 ál
primer 2 (5 μm)4 ál
Polymerase mpliTaq2 ál
Purified plasmid4 ál
Total100 µl

Primer 1: direct VNor VLprimer

Primer 2: return VNor VLprimer

Purified plasmid: VNor VL-containing plasmid R2.1 purified using the kit Qiagen Minipreps (200 breeding x)

The PCR cycle:1-95°C for 4 min
2-95°C for 1 min
3-55°C for 1 min
4-72°C for 1 min
5-72°C for 7 min
stages 2-4 were repeated 30 times.

3.2. The clone expressing the vectors in mammals

PCR products were purified with what ispolzovaniem set for PCR MinElute cleanup from Qiagen according to the manufacturer's instructions.

VNthe PCR product and expressing vector mammals RldhCγ1wt hydrolyzed by enzymes HindIII and SeI:

10x buffer (Nuffer2)5 ál
SA 100x (PSUS)0,5 ál
DNA5 ál
HindIII (Promega)2 ál
SeI (PSUS)2 ál
Waterof 35.5 ál
total50 µl

DNA: purified VNthe PCR product or expressing vector mammals RldhCγ1wt (at 0.25 mg/ml) were incubated for 2 h at 37aboutC.

VLthe PCR product and expressing vector mammals RlnhCκ hydrolyzed by enzymes HindIII and BsiWI:

10x buffer (Nuffer2)5 ál
DNA5 ál
HindIII (Promega)2 ál
Water38 ál
overall the volume 50 µl

DNA: purified VLthe PCR product or the vector Rln hCκ (at 0.25 mg/ml) were incubated for 2 h at 37aboutC. Then was added 2 μl of BsiWI (PSUS) and incubated for 2 hours at 55°C.

The products of hydrolysis restricteduse enzymes were loaded into the solution for download on preparative 1% agarose gel containing 0.01% ethidiumbromid, and electrophoresis was performed in TAE buffer at 100 V for 1 hour, and then strip vectors Rld and Rln and stripes VNand VLRT-PCR fragments were cut out. To identify the bands VNVLand vector in the gel electrophoresis was performed 100 BP DNA ladder. DNA was extracted and purified from the gel using a kit for the extraction of the QIAquick gelTM(Qiagen) according to manufacturer's instructions. VNRT-PCR-fragment, hydrolyzed by enzymes HindIII and SeI, ligated into HindIII-SeI-hydrolyzed vector Rld hCγ1wt. VLRT-PCR-fragment, hydrolyzed by enzymes HindIII and BsiWI, ligated into HindIII-BsiWI-hydrolyzed vector RlnhCκ. Ligation was performed using a rapid ligation DNA LigaFast (Promega) according to manufacturer's instructions, where:

VN: vector: RldhCγ1wt, hydrolyzed by enzymes HindIII and SeI,

insert: VNRT-PCR-fragment, hydrolyzed by enzymes HindIII and SeI

VL: vector: RlnhCκ, obtained the first enzymes HindIII and BsiWI,

insert: VLRT-PCR-fragment, hydrolyzed by enzymes HindIII and BsiWI

Legirovannye products was transferred into competent DH5α cells. 200 μl of vessels DH5α were thawed on ice. 50 μl-aliquots were obtained in test tubes for transformation. Then add 2 µl of the mixture for ligation and the mixture is gently stirred with the pipette tip, and then incubated for 30 minutes on ice. The mixture is incubated with shaking for 45 seconds at 42°C. Then the mixture was transferred to ice for 2 minutes. After this was added 450 μl environment S and the tubes were incubated for 1 hour at 37°C in shaker incubator. 100 µl of the culture were sown on tablets with L-agar, to which was added 100 μg/ml ampicillin, and incubated overnight at 37aboutC.

3.3. Sequencing

VNand VLthe clones were cultured overnight at 37°C in 5 ml of medium IN which was added 100 μg/ml ampicillin. Plasmids Rld and Rln containing VNand VLdomains respectively, were extracted and purified using a kit for the preparation of mini-drugs Qiagen QIAprep Spin Miniprep in accordance with the manufacturer's instructions. VNthe region sequenced using forward primers in the vector Rld and using the signal sequence and the reverse primer in the field of human γ1.

VL-area sequeiro the Ali using forward primers in the vector Rln and using the signal sequence and the reverse primer in the field of human κ. Clones with the corresponding sequences of the VNand VLidentified and obtained plasmids for expression in cells SNO.

3.4. Expression of chimeric antibodies in cells SNO

Plasmids Rld and Rln containing VNand VLdomains antibodies A respectively, were subjected to temporary cotransfection cells SNO and expressed. The obtained chimeric antibody was extracted from cell culture supernatant using affinity chromatography on protein a - sepharose.

3.4.1. Purification of plasmids

Cells DH5α containing Rld-6A1VNand Rln-6A1VL, was cultured in 5 ml of LB medium to which was added 100 μg/ml ampicillin, for 8 hours at 37°C in shaker incubator. 200 ml of LB medium to which was added 100 μg/ml ampicillin, was inoculable 1 milliliter day culture and incubated over night at 37°C in shaker incubator. Plasmids were extracted and purified using a kit for the preparation of mini-drugs Qiagen QIAfilter Plasmid Miniprep in accordance with the manufacturer's instructions. Ethanol precipitate resuspendable 200 μl of THE buffer and the concentration of the plasmid was determined by measuring optical density at 260 nm after 100-fold dilution of the mother liquor.

3.4.2. Transfection

Cells SNO cultivated to confluently modified in ways which Dulbecco environment MEM c glutamax-1 (DMEM), which has been added fetal bovine serum very low concentrations (Ultra low) 1% penicillin-streptomycin, 4 × 175 cm2-flasks for culturing tissue BD Falcon at 37°C.

In each flask, namely in a 50 ml Falcon tube, was added, intensively mixing the following reagents:

8 ml of Optimem 1 with glutamax-1

20 µg of purified plasmid Rld-6A1VN

20 µg of purified plasmid Rln-6A1VL

240 μl of reagent TransFast transfection.

The resulting mixture was incubated for 10-15 minutes at room temperature. Then, the DMEM was removed from the flask, the mixture was intensively mixed and added into the flask. After the mixture is incubated at 37°C for 1 hour. To the flask was added 32 ml of Optimem and the mixture is incubated at 37°C for 48-72 hours.

3.4.3. Purification of chimeric antibodies

Environment, taken from the flask 175 cm2were combined and centrifuged at 1500 rpm for 3 min on the device SE Mistral 2000, and the supernatant was passed through a filter system of 0.22 μm CA a volume of 500 ml Antibody was isolated from the clarified supernatant on the device Amersham Biosciences Akta Explorer using computer programs Unicorn. On the used column was applied to 1 ml of recombinant protein a HiTrap with separate FF. The flow rate was 1 ml/min

The column was balanced by 10 column volumes of PBS, Dulbecco, and then the column was loaded on velenyi supernatant using a pump A. The column washed with 20 column volumes of PBS, Dulbecco, after which the pump And washed to remove debris, and then passed through the column 10 column volumes of PBS, Dulbecco to ensure complete clearance of the supernatant. The antibody was suirable 10 column volumes of buffer for elution of ImmunoPure IgG (Pierce) and collected in 1 ml fractions containing 100 μl of neutralizing buffer 1M Trizma-HCl, pH 8.0. Then the column was again balanced by 5 column volumes of PBS, Dulbecco.

The antibody in the fractions of the eluate were quantitatively evaluated by reading the optical density at 280 nm compared with a control solution containing 10 volumes of buffer for elution of ImmunoPure IgG + 1 volume of 1M Trizma-HCl, pH 8.0, and the fractions containing sufficient amounts of pure antibodies were combined and stored in 100 μl-aliquot at -20°C.

3.4.4. Analysis of chimeric antibodies

Supernatant and purified chimeric antibody A (as) were analyzed for binding to IL-13 human and cynomolgus monkeys using ELISA. Supernatant from cells SNO, temporarily transfetsirovannyh chimeric monoclonal antibody A, was associated with recombinant IL-13 human IL-13 cynomolgus monkeys, expressed inE. coliin sandwich ELISA. The purified antibody was also associated with recombinant human IL-13 and IL-13 cynomolgus monkeys, expressed inE. coliin sandwich ELISA (data not shown). See figure 8.

Amino acid sequence and cDNA sequence of IL-13 cynomolgus monkeys (including the signal sequence) is presented in SEQ ID NO:90 and 91, respectively.

These results confirmed that the desired variable regions A were successfully cloned and produced antigennegative chimeric antibody able to bind to IL-13 human and cynomolgus monkeys.

4. Humanization clone A

4.1. Strategy humanization

4.1.1. Search in the database of mouse sequences

12 murine sequences with the highest homology with the amino acid sequence of the VNA and 11 murine sequences with the highest homology with the amino acid sequence of the VLidentified by searching a database of peptide sequences (Genbank).

Amino acid sequence of VNA compared with all 12 mouse sequences by searching in the database and identified the following frame remains:

PositionVH AmouseThe occurrence
19RTo 12/12
38ITo12/12
81RQ12/12

Provisions are in accordance with the numbering system according to Kabat and other

Amino acid sequence of VlA compared with 11 mouse sequences by searching the database and identifying the frame remains.

4.1.2. Search in the database of human sequences

The human frame sequence with the highest homology with frame sequences VNand VLA identified by searching a database of peptide sequences using the program EasyBlast.

Was identified one series of human sequences for VNA from which to humanize was selected frame sequence SEQ ID NO:92.

The following skeleton remains were identified as the remains that have potentially important for the recovery of affinity, and these residues may require reverse mutations:

Position (Kabat#)Human V NVNA
19ToR
38RI
73ET
81ER

Were designed 4 gumanitarnye VN-design with different reverse mutations, one of which was a direct mutation, and the other was a different reverse mutation (A2, A3, A4). Namely:

A2 was an A1 plus R381

A3 represented A2 plus IT

A4 was a A3 plus 19R plus 81R

Was identified one series of human sequences for VLA from which to humanize was selected frame sequence SEQ ID NO:93.

The following residues were identified as the remains that have potentially important for the recovery of affinity, and these residues may require reverse mutations:

Position (Kabat#)Mouse VLAHuman VL
85VI

Received two constructions, one of which had a direct mutation (L1)and the other had a reverse mutation (L2)(i.e., L1 plus 185V).

Humanitarna VNdesign A1:

SEQ ID NO:11

Humanitarna VNdesign A2:

SEQ ID NO:12

Humanitarna VN-design A3:

SEQ ID NO:13

Humanitarna VN-design A4:

SEQ ID NO:14

Humanitarna VL-design L1:

SEQ ID NO:15

Humanitarna VL-design L2:

SEQ ID NO:16

4.2. Humanization A:

Gumanitarnye VNand VLconstructs were obtainedde novoby constructing overlapping oligonucleotides including restriction sites for cloning in the expression sites Rld and Rln mammals, as well as human signal sequence. RestrictionHindIII - andSeI sites were introduced with the preservation of the reading frame in the VNdomain containing human signal sequence for cloning into the vector Rld containing human constant region γ1 wild type. RestrictionHindIII - andBsiWIsites were introduced with the preservation of the reading frame in the VLdomain containing human signal is ing sequence, for cloning in the vector Rln containing human constant region Kappa.

Human signal sequence is a sequence of SEQ ID NO:17.

Were designed 4 gumanitarnye VN-design and two gumanitarnye VL-design. As a result of this design received 8 different combinations of heavy and light chains.

To create structures were obtained approximately 10 oligonucleotides with a length of about 60 bases, interleaved about 18 bases.

4.2.1. Design of oligonucleotides

Solutions of oligonucleotide pools were obtained from 5 μl of each oligonucleotide mother solutions at a concentration of 100 μm. Synthesis gumanitarnyh VNand VLgenes by constructing overlapping oligonucleotides was performed mainly as described in the publication W.P. Stemmer et al. (1995) Gene 164(1):49-53, using a computer program described Ertl P.F. et al. (2003) Methods 31:199-206.

4.2.1.1. Representative PCR reaction Assembly:

Water
10x PCR buffer ProofStart
dNTP (10 mm)
Oligonucleotide pool
DNA polymerase ProofStart
Total
of 41.5 ál
5 ál
1,5 ál
1 ál
1 ál
50 µl

Cycle PCR Assembly:

1-94°C, 2 minutes

3-40°C, 2 minutes

4-72°C, 10 seconds

5-94°C, 15 seconds

6-40°C, 30 seconds

7-72°C, 20 seconds + 3 seconds/cycle; stage 4-7 repeated 25 times.

4.2.1.2. Representative reaction PCR recovery:

Primers 1 and 2 represent the first top and bottom oligonucleotides used in the PCR Assembly. Reaction PCR recovery allows you to amplify the full-size gene V.

Reaction PCR recovery:
Water
10x PCR buffer ProofStart
dNTP (10 mm)
Primer 1 (100 μm)
Primer 2 (100 µm)
Reagent for PCR Assembly
DNA polymerase ProofStart
Total
42 ál
4 ál
1,5 ál
0,5 ál
0,5 ál
1 ál
0,5 ál
50 µl

Cycle PCR recovery:

1-94°C, 2 minutes

2-94°C, 45 seconds

3-60°C, 30 seconds

4-72°C, 2 minutes

5-72°C, 4 minutes

stages 2-4 were repeated 25 times.

The products of PCR recovery was purified using a kit PCR purification MinElute (Qiagen) according to manufacturer's instructions.

4.2.2. Hydrolysis restricteduse enzymes

Gumanitarnye VN-structures A1, A2, A3 and A4 antibodies A hydrolyzed by enzymes HindIII-SpeI, and gumanitarnye VL-design L1, L2 antibodies A hydrolyzed by enzymes HindIII-BsiWI, as is written in section 3.

4.2.3. Gel-purification

The products of hydrolysis restricteduse the enzyme was purified in a manner analogous to the method described in section 3.

4.2.4. Ligation

Gumanitarnye VN-fragments of antibodies A, hydrolyzed by enzymes HindIII-SpeI, and ligated into HindIII-SpeI-hydrolyzed vector Rld hCγ1wt.

Gumanitarnye VL-fragments of antibodies A, hydrolyzed by enzymes HindIII-BsiWI, ligated into HindIII-BsiWI-hydrolyzed vector Rln hCκ.

Ligation was performed using the rapid ligation DNA LigaFast (Promega) according to manufacturer's instructions.

4.2.5. Transformation

The transformation was performed in a manner analogous to the method described in section 3.

4.2.6. Representative sequencing method

Colonies from each reaction tablet were cultured over night at 37°C in 5 ml of LB medium to which was added 100 μg/ml ampicillin. Then the plasmids were extracted, purified using the kit to get minipreparation QIAprep Spin Miniprep (Qiagen) according to manufacturer's instructions and sequenced using the primers described in section 3. Clones containing the appropriate gumanitarnye sequence VNand VLidentified and obtained plasmids for expression in cells SNO.

5. Expression and characterize the Oia gumanitarnyh antibodies

Four gumanitarnye VN-design (A1, A2, A3, A4) and two gumanitarnye VL-design (L1 and L2) were received expressing vectors Rld hCγ1wt and Rln hCκ mammals. Eight plasmid combinations of heavy chain-light chain (1L1, A1L2, A2L1, A2L2, A3L1, A3L2, A4L1, A4L2) was subjected to a temporary co-transfection into cells SNO and expressed in laboratory scale with getting 8 different gumanitarnyh antibodies. Antibodies produced in the supernatant of cells SNO, were analyzed in ELISA for binding to human IL-13.

5.1. A representative method of purification of plasmids

Cells DH5α containing one of the plasmids described above were cultured in 5 ml of LB medium to which was added 100 μg/ml ampicillin, for 8 hours at 37°C in shaker incubator. 200 ml of LB medium to which was added 100 μg/ml ampicillin, was inoculable 1 milliliter day culture and incubated over night at 37°C in shaker incubator. Plasmids were extracted and purified using the kit QIAfilter Plasmid Maxi (Qiagen) according to manufacturer's instructions. Ethanol precipitate resuspendable 200 μl of THE buffer and the concentration of the plasmid was measured by optical density at 260 nm after 100-fold dilution of the mother liquor.

5.2. A representative method of transfection

9 holes 96-hole tablet Corning Costar 3506 were sown 10 6cells SNO and were cultured overnight in modified according to the method of Dulbecco environment MEME with glutamax-1 (DMEM), which were added fetal bovine serum very low concentrations (Ultra low) and 1% penicillin-streptomycin at 37°C.

Then to each well in 5 ml of medium Bijou was added, with shaking, the following reagents to each mixture for transfection contained various combinations of light and heavy chains, namely:

1 ml of Optimem 1 with glutamax-1;

5 μg of the plasmid containing gumanitarnuyu VN;

5 μg of the plasmid containing gumanitarnuyu VL;

30 µg of reagent TransFast transfection.

Incubation was carried out for 10-15 minutes at room temperature. Then from the wells was removed DMEM, and the mixture was shaken and added to the corresponding well. After this incubation was performed at 37°C for 1 h and Then was added 2 ml of Optimem per well and the mixture incubated at 37°C for 48-72 hours.

5.3. Analysis gumanitarnyh antibodies

The medium from each well was recovered and centrifuged at 13,000 rpm for 1 min on a laboratory table top Eppendorf centrifuge 5415R, and the supernatant was passed through 0.2 μm filter-syringe Pall Acrodisc (25 mm). The cell supernatant were analyzed by ELISA for binding to human IL-13. ELISA analysis showed that all 8 gumanitarnyh the antibodies is contacted with a human IL-13 with a profile similar to the profile of the binding of chimeric antibodies A with human IL-13. Cm. figure 9.

Gumanitarnye antibodies L1+A1 and L2+A1 were selected for large-scale expression, purification and further analysis.

6. Analysis gumanitarnyh antibodies L1+A1 and L2+A1 against human IL-13

6.1. The activity of binding IL-13 human and cynomolgus monkeys in ELISA

Antibodies L1+A1 and L2+A1 were successfully produced by large-scale expression and assessed in ELISA analysis on the binding of IL-13 human and cynomolgus monkeys, expressed inE. coli. Cm. figures 10A and 10b and table C.

Table
ELISAmAbEC50(ág/ml)
Binding to human IL-13The parent mAb 6A10,049
Chimeric A0,015
L1+A10,018
L2+A10,024
Binding of IL-13 cynomolgus monkeysThe parent mAb 6A10,39
Chimeric A0,018
L1+A10,021
L2+A10,028

Profiles of antibody binding to L1+A1 and L2+A1 expressed inE. coliwith human IL-13 and IL-13 cynomolgus monkeys, expressed inE. coliwere similar. Size EU50(obtained by constructing a curve using the computer program Excel Robosage”) showed that the level of activity of the binding of such antibodies to similar level of activity of the chimeric binding standard mAb 6A1.

6.2. Analysis of the binding of the antibody L1+A1 and L2+A1 with native human IL-13 (isolated from MCPC)

To analyze the binding of the chimeric antibody mAb antibody 6A1, L1+A1 and L2 antibody+A1 with native human IL-13 (isolated from MCPC) used supernatant CD4+-Th2-cells (generated from cultures of human MCPC), stimulated by antibodies against CD23 and CD28. ELISA analysis showed that all 3 antibodies was associated with native human IL-13 in supernatants Th2-cells with efficiency, almost similar to the effectiveness of the parental mAb 6A1. Cm. figure 11.

In addition, to determine the level of native human IL-13 present in the supernatant Th2-cells, built with undertow curve using commercially available reagents. All 3 antibodies and commercially available mAb against human IL-13 was found equivalent amounts of IL-13 in the sample supernatant Th2 cells. Cm. the following table 2.

Table 2
MAbNative IL-13 (ng/ml)
The parent mAb 6A122,5
Chimeric A19,6
L1+A125,1
L2+A122,7
+ control mAb28,0

6.3. The activity of inhibiting the binding of L1+A1 and L2+A1 with human IL-13 ELISA-assays for binding to the IL-13 receptor

The parent murine mAb 6A1, chimeric A, antibody L1+A1 and L2 antibody+A1 analyzed for their ability to inhibit the binding of human IL-13 c chains of IL-13Rα1 and IL-13Rα2 in ELISA analysis on competitive binding. Cm. figures 12A and 12b, as well as table 3, below.

Table 3
ELISAmAbEC50
Competitive binding to human IL-13Rα1The parent mAb 6A10,039
Chimeric A0,034
L1+A10,044
L2+A10,056
Competitive binding to human IL-13Rα2The parent mAb 6A10,020
Chimeric A0,040
L1+A10,113
L2+A10,117

All antibodies inhibited binding of det1-labeled human IL-13 expressed inE. colihuman IL-13Rα1 with a similar profile. Similarly, all of the antibodies inhibited the binding of det1-labeled human IL-13 expressed inE. coliwith human IL-13Rα2, although, as shown by this analysis, antibodies L1+A1 and L2+A1 had a few low activity (size EU50were obtained by curve a functional dependency using the computer program Excel is Robosage”).

6.4. Evaluation of the affinity of antibody binding to L1+A1 and L2+A1 with human IL-13

The binding kinetics L1+A1 and L2+A1 with human IL-13 was assessed using a BIAcore systemTM. Description of the methods used is given below in section 7.

Analysis 1:

Conducted analysis on the binding of IL-13 and IL-13 cynomolgus monkeys (protein expressed inE. coli). In table KD values mean average of the 5 curves for various concentrations of IL-13 (data obtained from three replicates). It should be noted that this analysis was observed products of mass transfer and that the modified Protocol of the experiment (adjusted for this product) was carried out in analysis 4 (not attended by the products of mass transfer). Cm. table 4.

Table 4
Sample IL-13mAbKa Association (MS-1)Kd dissociation (-1)The affinity constant KD (PM)
human IL-13the parent mAb 6A11,96×106of 6.78×10-535
Chimera is th A with 4.64×1052×10-543
L1+A15,07×1051,C-4300
L2+A15,07×105of 1.56×10-4310
IL-13 cynomolgus monkeysthe parent mAb 6A19,14×105of 5.6×10-561
chimeric Aof 5.92×105of 3.27×10-555
L1+A1of 4.46×105of 1.55×10-535
L2+A15,77×105to 5.58×10-597

Analysis 2:

Conducted analysis of the binding of human IL-13 (protein expressed inE. coli) with the antibody L1+A1. Cm. table 5.

Table 5
Sample IL-13mAbKa Association (MS-1)Kd dissociation (-1)The affinity constant KD (PM)
Human IL-13L1+A1of 4.66 × 105of 6.95 × 10-5149

Analysis 3:

Conducted analysis on the binding of 16-dimensional biotinylated human peptide of IL-13 No. 24 (identified as a linear epitope to bind to the parent mAb 6A1, see section 6.7). It should be noted that the absolute values of KD obtained for the binding of peptide ligands, often quite different from the values obtained for binding to the full-size protein target. However, it is obvious that these data coincide with the data obtained for full-length protein, and with data on the neutralization of IL-13 (in bioanalysis TF-1), which indicate approximately 3-fold decrease in the affinity of binding of chimeric antibodies A and the affinity of binding antibodies L1+A1. Cm. table 6.

Table 6
Sample IL-13mAbKa Association MS -1)Kd dissociation (-1)The affinity constant KD (nm)
Peptide 24The parent mAb 6A12,95 × 105to 9.15 × 10-43,11
Chimeric Ato 2.57 × 1059,19 × 10-4to 3.58
L1+A1of 1.95 × 1051,7 × 10-39,03
L2+A1to 1.79 × 1051,67 × 10-39,35

Analysis 4:

Conducted analysis on the binding of IL-13 and IL-13 cynomolgus monkeys (with protein expressed inE. coli). In table KD values mean average of the 5 curves for various concentrations of IL-13 (data obtained from three replicates). It should be noted that in this dataset, there were no products of mass transfer.

Table 7
Sample IL-13MAbKa Association (MS-1)Kd dissociation (-1)The affinity constant KD (PM)
Human IL-13Chimeric 6A1of 1.05 × 1064 × 10-538
L1+A18,24 × 105of 1.4 × 10-4170
L2+A19,07 × 105of 1.39 × 10-4153
IL-13 cynomolgus monkeysChimeric Acent to 8.85 × 105to 2.65 × 10-530
L1+A17,3 × 1055,86 × 10-580
L2+A17,72 × 1054,25 × 10-555

The results showed that any meaningful RA is the difference between gumanitarnye structures L1+A1 and L2+A1 none.

The binding affinity of L1+A1 with human IL-13 is approximately 168 gr. Such reaction kinetics was dominant due to the exceptionally low rate of dissociation, as was predicted on the basis of significant neutralizing activity of antibodies. The values of Association constantskonwere constant and is approximately 6 × 105M-1with-1. The values of dissociation constantskoffwere more variable and ranged from 1.4 × 10-4to 8.22 × 10-5with-1that due to some technical problems, not allowing you to accurately calculate the values for the low rate of dissociation.

6.5. Activity L1+A1 and L2+A1 in biological analyses to neutralize IL-13

The parent murine mAb 6A1, chimeric antibody A, antibody L1+A1 and L2 antibody+A1 was evaluated on the activity of neutralizing IL-13 in a biological analysis of cells TF-1in vitro(this biological analysis is an industry standard analysis to assess the biological activity of IL-13 and to assess the neutralizing activity of commercially available anti-IL-13 antibody). In this analysis evaluated several variants of IL-13, including human IL-13 expressed inE. coliIL-13 human-like apes, expressed inE. coliQ130-variant of human IL-13 expressed inE. coli(option associated with Razvitie asthma) and human IL-13, expressed in cells of SSC mammals (note: native human IL-13 in the sample supernatant Th2 cells cannot be used in bioanalysis, because this supernatant also contains other cytokines capable of inducing cell proliferation, TF-1). Cm. figures 13A, 13b, 13c and 13d.

All antibodies tested in this bioassay system neutralized the biological activity of all variants of IL-13; it was determined the ability of each antibody to neutralize each of the variants of IL-13 and this ability was expressed as the amount of ND50. Cm. table 8.

Table 8
A variant IL-13MAbAverage ND502 analyses (ág/ml)
Expressed inE. colihuman IL-13Chimeric 6A10,119
L1+A10,428
L2+A10,608
The parent mAb 6A10,193
Expressed inE. coliIL-13 monkeys cyomolgus Chimeric A0,059
L1+A10,078
L2+A10,120
The parent mAb 6A10,078
Expressed inE. coliQ130-variant of human IL-13Chimeric A0,128
L1+A10,438
L2+A10,705
The parent mAb A0,213
Expressed in Cho human IL-13Chimeric A0,285
L1+A10,975
L2+A11,200
The parent mAb 6A10,440

Note: because this bioanalysis for cell proliferation, TF-1 to the same extent required different to the number of each of the variants of IL-13, it is not desirable to carry out a comparison of ND50obtained for one specific antibodies directed against each of the variants of IL-13. However, it is necessary to conduct a comparison of ND50obtained for each of the antibodies directed against one variant IL-13.

Essentially, the level of neutralization achieved the parent mAb 6A1 and chimeric A was identical, indicating the absence of any detectable loss of activity of the parent mAb and chimeric antibodies. However, the activity of L1+A1 and L2+A1 for each individual tested variants of IL-13 was significantly lower on average about 3-4 times in comparison with the activity of the parent mAb 6A1 and chimeric 6A1. These data are very closely correlated with the data obtained in the analysis BIAcoreTM.

6.6. The binding specificity of antibodies L1+A1 and L2+A1 with human IL-13

The binding specificity of antibodies L1+A1 and L2+A1 with human IL-13 was assessed by using analysis of potential cross-reactivity with human IL-4 and human GM-CSF in ELISA analysis on the binding. Cm. figures 14a and 14b.

It was found that mAb have binding specificity to IL-13 and do not have cross-reactivity with human IL-4 or human GM-CSF at concentrations of mAb to 30 μg/ml. in Addition, these mAb does not have the ability of the Yu cross-neutralize the biological activity of human IL-5 in the bioanalysis of IL-5. Cm. figure 14C.

6.7. Mapping of epitopes for A using biotinylated peptides

Proteins, namely human IL-13 and IL-13 cynomolgus monkeys were subjected to electrophoresis in denaturing SDS page with LTOs. Western blot analysis using mouse mAb 6A1 revealed bands of expected size for the human protein of IL-13 (expressed inE. coliin laboratory scale), and for protein IL-13 cynomolgus monkeys (expressed inE. coliin laboratory scale). Antibody A not detected hIL-13 (expressed inE. coliCambridge Bioscience), which is probably due to technical problems. This analysis gave reason to assume that mAb 6A1 recognize linear peptide epitope sequences of IL-13 human and cynomolgus monkeys (data not shown).

Biotinylated 16-dimensional peptides shifted by 4 residue were synthesized to create a map of localization of b-cell epitope recognized mAb A, IL-13 human IL-13 cynomolgus monkeys. For detection of binding of the immobilized biotinylated peptide with the parent mAb 6A1 was used ELISA method.

16-gauge specially designed peptides have the following options: 88 × 16-measures with a shift of 4 residue (such peptides are delivered by the company Mimotopes, Australia).

Ormat: Peptides 25 and 44 = Biotin-SGSG-peptide-acid;
Peptides 2-24 and 27-43 = Biotin-SGSG-peptide-amide.
No.HydroMolecular weightN-endSequenceWith the end
20,422311,66Biotin-SEQ ID NO:38-NH2
30,272453,82Biotin-SEQ ID NO:39-NH2
40,382326,70Biotin-SEQ ID NO:40-NH2
50,312231,58Biotin-SEQ ID NO:41-NH2
60,432289,66Biotin-SEQ ID NO:42-NH2
70,592190,57Biotin-SEQ ID NO:43-NH2
80,572260,64Biotin-SEQ ID NO:44-NH2
90,62*2255,64Biotin-SEQ ID NO:45-NH2
100,512197,56Biotin-SEQ ID NO:46-NH2
110,562144,52Biotin-SEQ ID NO:47-NH2
120,46 2090,38Biotin-SEQ ID NO:48-NH2
130,292219,54Biotin-SEQ ID NO:49-NH2
140,292180,53Biotin-SEQ ID NO:50-NH2
150,362318,70Biotin-SEQ ID NO:51-NH2
160,322303,73Biotin-SEQ ID NO:52-NH2
170,472209,57Biotin-SEQ ID NO:53-NH2
180,482257,60Biotin- SEQ ID NO:54-NH2
190,172273,57Biotin-SEQ ID NO:55-NH2
200,272300,60Biotin-SEQ ID NO:56-NH2
210,292383,77Biotin-SEQ ID NO:57-NH2
220,352401,83Biotin-SEQ ID NO:58-NH2
230,452407,92Biotin-SEQ ID NO:59-NH2
240,422541,08Biotin-SEQ ID NO:60-NH2
250,332513,97Biotin-SEQ ID NO:61-NH2
270,422283,64Biotin-SEQ ID NO:62-NH2
280,272425,81Biotin-SEQ ID NO:63-NH2
290,572228,57Biotin-SEQ ID NO:64-NH2
300,62*2223,57Biotin-SEQ ID NO:65-NH2
310,512165,49Biotin-SEQ ID NO:66-NH2
320,56 2112,45Biotin-SEQ ID NO:67-NH2
330,272207,56Biotin-SEQ ID NO:68-NH2
340,332345,73Biotin-SEQ ID NO:69-NH2
350,292330,76Biotin-SEQ ID NO:70-NH2
360,452236,60Biotin-SEQ ID NO:71-NH2
370,432276,64Biotin-SEQ ID NO:72-NH2
380,122292,62Biotin- SEQ ID NO:73-NH2
390,222319,64Biotin-SEQ ID NO:74-NH2
400,242402,82Biotin-SEQ ID NO:75-NH2
410,332387,80Biotin-SEQ ID NO:76-NH2
420,432393,90Biotin-SEQ ID NO:77-NH2
430,392527,05Biotin-SEQ ID NO:78-NH2
440,352471,88Biotin-SEQ ID NO:79-NH2
(* means high hydrophobic number)

Example: a typical 96-well plate used for this analysis.

NB: the numbers correspond to the peptide in each well; numbers in parentheses indicate the degree of dilution of the control antibody.

Optical density at 490 nm in a 96 wells

123456789101112
A0,0570,0670,0790,0630,0720,0610,0840,0610,0750,0640,075of 0.066
B0,0680,0700,1050,065 0,0750,0720,0710,0700,0640,0610,0620,063
C0,119of 0.0810,0990,0640,0730,077to 0.0600,0610,0900,1442,1092,200
D0,1150,1290,141to 0.0600,0900,0630.104 g0,0780,076is 0.1352,1482,210
Eto 0.0600,0740,0980,0620,0640,0710,0880,082 0,0890,0730,0680,067
F0,0820,0780,0710,0620,0560,0570,0840,0670,0900,0740,0630,056
G0,0570,055to 0.060to 0.0600,0580,0580.104 gto 0.1082,2362,2372,2292,229
H1,4991,1970,7391,5481,2090,9760,0770,0800,0720,0720,0820,103

This result (one of several attempts) correlates with a positive result for peptides 24, 25, 43 and 44, the following (as well as a positive control peptides). Cm. figure 15. All attempts have demonstrated that peptides 24, 25, 43 and 44 were positive.

Peptide 24:QFVKDLLLHLKKLFRE(SEQ ID NO:80);
Peptide 25:DLLLHLKKLFREGRFN(SEQ ID NO:81);
Peptide 43:QFVKDLLVHLKKLFRE(SEQ ID NO:82);
Peptide 44:DLLVHLKKLFREGQFN(SEQ ID NO:83);

Peptides 24 and 25 were from hIL-13. Peptides 43 and 44 were from IL-13 cynomolgus monkeys.

In addition, all antibodies, namely chimeric antibody A, antibody L1+A1 and L2 antibody+A1, associated with the same linear epitope in the C-terminal region of IL-13 human and cynomolgus monkeys (data for mAb, such as chimeric A, L1+A1 and L2+A1 not given).

In the end, ELISA results showed that all antibodies, namely the parent murine mAb 6A1, chimeric A, L1+A1 and L2+A1, are connected in the following sequence occurring on the human protein IL-13, such as:

DLLLHLKKLFRE (SEQ ID NO:84)

and in the following sequence derived from the protein IL-13 cynomolgus monkeys, such as:

DLLVHLKKLFRE (SEQ ID NO:85).

NB: bold indicates that in this position the rest of the human IL-13 is different from the rest of the ortholog of IL-13 cynomolgus monkeys.

Accordingly, it was found that parental murine mAb 6A1, chimeric A, L1+A1 and L2+A1 immunospecificity contact with human IL-13 in the positions of residues 97-108 SEQ ID NO:9.

6.8. Fine mapping of epitopes for A using biotinylated peptides

The epitope binding with mAb 6A1, was determined by the analysis of the binding of peptides having the approximate sequence KDLLLHLKKLFREG, with human IL-13, and peptides having the approximate sequence KDLLVHLKKLFREG, IL-13 cynomolgus monkeys. For accurate determination of the linear epitope binding to the mAb 6A1, the peptides were added 1-amino acid, which was then removed with either N-or C-end of the parent peptide sequences (i.e. KDLLLHLKKLFREG or KDLLVHLKKLFREG).

For detection of binding of the immobilized biotinylated peptide with the parent mAb 6A1 used ELISA method.

Identification number of peptides (413-447) and their corresponding sequences are given below.

Peptide sequence:

Peptide No.N-endSequenceWith the end
413Biotin-SEQ ID NO:94-NH2
414Biotin-SEQ ID NO:95-NH2
415Biotin-SEQ ID NO:96-NH2
416Biotin-SEQ ID NO:97-NH2
417Biotin-SEQ ID NO:98-NH2
418Biotin-SEQ ID NO:99-NH2
419Biotin-SEQ ID NO:100-NH2
420Biotin-SEQ ID NO:101-NH2
421Biotin- SEQ ID NO:102-NH2
422Biotin-SEQ ID NO:103-NH2
423Biotin-SEQ ID NO:104-NH2
424Biotin-SEQ ID NO:105-NH2
425Biotin-SEQ ID NO:106-NH2
426Biotin-SEQ ID NO:107-NH2
427Biotin-SEQ ID NO:108-NH2
428Biotin-SEQ ID NO:109-NH2
429Biotin-SEQ ID NO:110-NH2
430Biotin-SEQ ID NO:111-NH2
431 Biotin-SEQ ID NO:112-NH2
432Biotin-SEQ ID NO:113-NH2
433Biotin-SEQ ID NO:114-NH2
434Biotin-SEQ ID NO:115-NH2
435Biotin-SEQ ID NO:116-NH2
436Biotin-SEQ ID NO:117-NH2
437Biotin-SEQ ID NO:118-NH2
438Biotin-SEQ.ID.NO:119-NH2
439Biotin-SEQ ID NO:120-NH2
440Biotin-SEQ ID NO:121-NH2
441Biotin-SEQ ID NO:122-NH2
442Biotin-SEQ ID NO:123-NH2
443Biotin-SEQ ID NO:124-NH2
444Biotin-SEQ ID NO:125-NH2
445Biotin-SEQ ID NO:126-NH2
446Biotin-SEQ ID NO:127-NH2
447Biotin-SEQ ID NO:128-NH2
44 (Control)Biotin-SEQ ID NO:79-OH

Example: a 96-well plate used for this analysis.

NB: the Number represents the peptide in each well.

Optical density at 490 nm in a 96 wells

123456789101112
A2,4562,5012,4342,4192,7462,6612,2242,4070,0590,0520,0522,527
B2,4082,4522,4442,6242,6393,1062,1882,4730,0590,0550,0522,568
C2,4720,0990,065 0,0590,0700,0580,0530,054rate £ 0.1622,4792,3892,883
D2,3990,1000,0670,0530,0490,0510,0520,0470,4852,8382,7832,640
E2,5822,3592,5852,5120,0960,0520,0540,0480,0490,1830,0512,424
F2,4312,8722,5222,2430,0970,0590,052 0,0490,0570,0470,0502,342
G0,0560,0510,0580,0650,0560,0670,0490,0470,0530,0570,0520,056
H0,0470,0520,0500,0700,0540,0470,0560,0530,0490,0500,0520,049

Cm. figures 16A and 16b. These results showed that the parent mab 6A1 binds to a linear epitope having the amino acid sequence KKLFR, in the C-terminal region of human IL-13 and the ortholog of IL-13 cynomolgus monkeys.

In addition, all antibodies, namely chimeric antibody mAb 6A1, antibody L1+A1 and L2 antibody+A1, associated with the same linear epitope (i.e., KKLFR) in the C-end is howling human IL-13 (data for chimeric mAb 6A1, L1+A1 and L2+A1 not shown). In addition, it was found that parental mAb 6A1 binds to the same epitope of IL-13 cynomolgus monkeys.

In the end, ELISA results showed that all antibodies, namely the parent murine mAb 6A1, chimeric A, L1+A1 and L2+A1, are connected in sequence, derived from the human protein IL-13, namelyKKLFR.

6.9. Alanine scanning of epitope binding to A, using biotinylated peptides

To identify some of the key residues involved in the interaction of IL-13 mAb 6A1, a method was developed alanine scanning is conducted using the parent peptide sequence containing the binding epitope KKLFR (i.e. QFVKDLLLHLKKLFREGRFN). To implement this analysis was obtained peptides (supplied by the company AnaSpec Inc.), where one amino acid was sequentially replaced with alanine residue at each position the amino acids in the epitope KKLFR (and each of the amino acids, directly limiting the epitope).

For detection of binding of the immobilized biotinylated peptide with the parent mAb 6A1 and L1+A1 was used ELISA method.

Peptides derived for this analysis and their respective identification numbers are listed below:

Peptide No.Sequence
1BiotinSEQ ID NO:129
62BiotinSEQ ID NO:130
63BiotinSEQ ID NO:131
64BiotinSEQ ID NO:132
65BiotinSEQ ID NO:133
66BiotinSEQ ID NO:134
67BiotinSEQ ID NO:135
68BiotinSEQ ID NO:136

Results: the optical density at 490 nm.

The average test results (n=2).

Data for the parent (mouse) mAb 6A1:

Rooms peptides16263646566 6768
Average And4903,5433,4893,27951,4683,84953,59950,5953,581

Data for L1+A1:

Rooms peptides162636465666768
Average And4902,85352,8322,65351,81753,01652,840,8162,8085

Cm. figures 17A and 17b.

These data suggest that the key amino acid residues involved in the interaction of mAb 6A1 or L1+A1 with human IL-13, are arginine (R) at position 107, and lysine (K) at position 103.

To confirm this effect in this interval mAb breeding this analysis was repeated, but with the use of what Finance mAb 6A1 and L1+A1 at various concentrations.

The parent murine mAb 6A1 (figure 17c) and gumanitarnye candidate L1+A1 (figure 17d) were analyzed for binding to the peptides used for alanine scanning (SEQ ID NO:129, 131-135) at various concentrations. Since the peptides were divided into two 96-well tablets, the parent peptide sequence not containing alanine substitutions (SEQ ID NO:129), were analyzed on both tablets, and so I got two results on the graph. This was done in order to determine whether there are any major differences between the tablets, and in both cases it was not observed any visible differences.

It was found that peptides containing substitutions CA, L105A and F106A (SEQ ID NO:131, 133 and 134, respectively; numbering of residues is presented in SEQ ID NO:9), had almost the same profile of binding to mAb as the parent peptide (SEQ ID NO:129), and therefore, these residues do not play a decisive role in linking A/L1+A1 and IL-13. However, it was found that peptides containing substitutions CA and R107A (SEQ ID NO:132 and 135, respectively; numbering of residues is presented in SEQ ID NO:9), had a reduced ability to associate with A/L1+A1 than the parent peptide (SEQ ID NO:129), in particular, at lower concentrations, indicating that these residues do not play a decisive role in the optimal binding A/L1+A1 and IL-13.

Cm. figures 17c and 17d.

These data showed that the key amino acid residues, involved in the interaction between the parent (i.e. mouse) 6A1 or L1+A1 with human IL-13, are arginine (R) at position 107, and lysine (K) at position 103 of SEQ ID NO:9.

Section 7. Materials and methods

In this section, where appropriate, are used the following materials and methods. These materials and methods are representative. In repeated experiments in these materials and methods can be made small changes.

Materials:

The allocation system full-sized RNA SV: Promega Z3100;

System RT-PCR with extension chain: Promega A1250;

Set for extraction from the gel (QIAquick: Qiagen 28704;

The solution is to download gel: Sigma G7654;

Agarose: Invitrogen 15510-019;

Ethidiumbromid: Sigma E1510;

Buffer TAE: received in the laboratory;

DNA ladder 100 BP: New England BioLabs N3231S;

Kit TA cloning: Invitrogen 45-0046;

Cells TOP10F': Invitrogen 44-0300;

L-agar + 100 μg/ml ampicillin: get in the lab;

X-Gal, 50 mg/ml in DMF): Promega V394A;

DNA polymerase AmpliTaq: Applied Biosystems;

10x PCR buffer: Applied Biosystems;

E-gel, a 1.2% agarose: Invitrogen G501801;

Wednesday LB + 100 μg/ml ampicillin: get in the lab;

Set to get mini-drugs QIAprep Spin Miniprep: Qiagen 27106;

Set for PCR MinElute cleanup: Qiagen 28004;

NE buffer 2, 10x conc.: New England Biolabs B7002S;

Purified BSA 100x conc.: New England Biolabs 9001S;

BsiWI: New Englan Biolabs R0553L;

Hind III: Promega R604A;

Spe I: New England Biolabs R0133S;

Rapid ligation DNA LigaFast: Promega M8225;

Chemically competent DH5α cells with maximum efficiency: Invitrogen 18258-012;

Environment SOC: received in the laboratory;

Kit QIAfilter Plasmid Maxi: Qiagen 12263;

Wednesday MEME, Dulbecco with glutamax-1: Invitrogen 31966-021;

Medium Optimem 1 with glutamax-1: Invitrogen 51985-026;

Reagent TransFast transfection: Promega E2431;

1 ml of recombinant protein a - Sepharose FF HiTrap: Amersham Biosciences 17-5079-01;

PBS, Dulbecco: Sigma D8537;

Buffer for elution of ImmunoPure IgG: Pierce 21009;

1M Trizma-HCl, pH 8.0: Sigma T2694;

DNA polymerase ProofStart: Qiagen 1016816;

PCR buffer ProofStart: Qiagen 1016961.

7.1. ELISA analysis of the binding of IL-13 human or IL-13 cynomolgus monkeys

This analysis represents an ELISA, allowing to detect binding of an antibody to IL-13 human or cynomolgus monkeys. This analysis was performed in the format of the “sandwich”ELISA.

7.1.1. Materials:

1. Immunoplate Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A);

2. Human IL-13 (Cambridge Biosciences, cat no. SN-013);

3. IL-13 cynomolgus monkeys (manufactured by the firm GlaxoSmithkline);

4. Goat polyclonal antibody against human IL-13 (R+D Systems, cat. No. AF-213-NA);

5. HRP-conjugated antibody against human IgG (Sigma, Cat. No. a-6029);

6. HRP-conjugated antibody against mouse IgG (Sigma, Cat. No. a-9309);

7. Carbonate/bicarbonate buffer (Sigma; cat. Number C-341);

8. TBST [buffered by tricom saline solution (the 6.06 g Tris + 8.06 g Nl + 0.2 g KCl + H2O to a volume of 1 liter) + 0.05% tween-20];

9. BSA (Sigma A-7030);

10. OPD (Sigma, Cat no. P-9187);

11. Sulphuric acid.

7.1.2. Method

1. A blocking solution of 3% BSA+TBST.

2. Wash solution is TBST.

3. ELISA-tablets “Nunc Maxisorp was senzibilizirani 50 μl of 5 μg/ml goat polyclonal antibodies against human IL-13 (R+D Systems, cat no. AF-213-NA. The solution was used when the initial concentration of 500 µg/ml according to manufacturer's instructions and stored in aliquot at -20°C) in carbonate/bicarbonate buffer (Sigma; cat no. C-3041, prepared in accordance with the manufacturer's instructions), and then the tablets were closed using a device for sealing tablets and incubated over night at 4°C.

4. Blocked with 100 μl of 3% BSA/TBST and incubated at room temperature for 1 hour.

5. Washed 3 times in TBST (at least 200 ál of wash solution/well/wash).

6. Added 20 ng per well (in a volume of 50 μl) of human IL-13 (Cambridge Biosciences, cat. No. SN-013. The solution is prepared at initial concentration of 100 ng/ml according to manufacturer's instructions, and stored in aliquot at -20°C) or 20 ng per well of IL-13 cynomolgus monkeys in blocking solution, and incubated at room temperature for 1H.

7. Washed 3 times in TBST.

8. Added 50 μl of the sample antibodies (if necessary, titration was performed to obtain data titer endpoint) in blocking solution and incubated at room temperature for 1 hour.

9. Washed 3 times in TBST.

10. For chimeric antibodies or gumanitarnogo antibodies A binding was detected using 50 μl per well of HRP-conjugated antibodies against human IgG (Sigma, Cat. No. a-6029) at a dilution of 1/2000 in blocking solution for 1 hour at room temperature. For mouse monoclonal antibodies A binding was detected using 50 μl per well of HRP-conjugated antibodies against mouse IgG (Sigma, Cat. No. a-9309) at a dilution of 1/1000 in blocking solution for 1 hour at room temperature.

11. Washed 3 times in TBST.

12. Showed 100 Microlitre OPD (Sigma, Cat. No. p-9187, prepared in accordance with the manufacturer's instructions), the reaction was stopped by adding 50 μl of 3M H2SO4and the tablets were read at an optical density of 490 nm. The developing time was approximately 12 minutes.

7.2. ELISA analysis of binding human IL-13 human IL-13Rα1-chain

This ELISA analysis allows you to determine whether the antibody to inhibit the binding of human IL-13 human IL-13Rα1-chain.

7.2.1. Matera is Aly:

1. Immunoplate Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A);

2. Human IL-13Rα1-Fc (R&D Systems, cat no. 146-IR);

3. Det1-labeled human IL-13 (prepared in the laboratory);

4. Biotinylated antibody against human IL-13 (R&D Systems, cat no. BAF213);

5. Streptavidin-HRP;

6. Carbonate/bicarbonate buffer (Sigma; cat. # C-3041);

7. TBST [buffered by tricom saline solution (the 6.06 g Tris + 8.06 g Nl + 0.2 g KCl + H2O to a volume of 1 liter) + 0.05% tween-20];

8. BSA (Sigma A-7030);

9. OPD (Sigma, Cat no. P-9187);

10. Sulphuric acid.

7.2.2. Method

1. A blocking solution of 3% BSA+TBST.

2. Wash solution is TBST.

3. ELISA-tablets “Nunc Maxisorp was senzibilizirani 50 ál of 5 ng/ml human IL-13Rα1-Fc in carbonate/bicarbonate buffer. The tablets covered with devices for sealing tablets and incubated over night at 4°C.

4. Blocked 100 Microlitre 3% BSA/TBST and incubated at room temperature for 1 hour.

5. Washed 3 times in TBST (at least 200 ál of wash solution/well/wash).

6. In total volume of 50 μl of pre-incubated 0.04 ng/ál det-1-labeled human IL-13 with the sample antibody (titrated) in blocking solution for 30 minutes. Then pre-incubated sample was added to the sensitized receptor ELISA-Lancet and incubated at room temperature for 1 hour

7. Washed 3 times in TBST.

8. Were detected binding of human IL-13 using 50 μl per well of biotinylated antibodies against human IL-13, diluted at 1 μg/ml and Then incubated for 1 hour at room temperature.

9. Washed 3 times in TBST.

10. Added 50 μl per well of conjugate “streptavidin-HRP” at a dilution of 1/1000. Then incubated for 1 hour at room temperature.

11. Washed 3 times in TBST.

12. Showed 100 Microlitre/well OPD (Sigma, Cat. No. p-9187, prepared in accordance with the manufacturer's instructions), the reaction was stopped by adding 50 μl per well of 3M H2SO4and the tablets were read at an optical density of 490 nm. The developing time was approximately 2 minutes.

7.3. ELISA analysis of binding human IL-13 human IL-13Rα2-chain

This ELISA analysis allows you to determine whether the antibody to inhibit the binding of human IL-13 human IL-13Rα2-chain.

7.3.1. Materials:

1. Immunoplate Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A);

2. Antibody against human IgG (Sigma, cat no. I-3382);

3. Human IL-13Rα2-Fc (R&D Systems, cat no. 614-IR);

4. Det1-labeled human IL-13 (prepared in the laboratory);

5. Biotinylated antibody against human IL-13 (R&D Systems, cat no. BAF213);

6. Streptavidin-HRP

7. To bonaty/bicarbonate buffer (Sigma; cat. # C-3041);

8. TBST [buffered by tricom saline solution (the 6.06 g Tris + 8.06 g Nl + 0.2 g KCl + H2O to a volume of 1 liter) + 0.05% tween-20];

9. BSA (Sigma A-7030);

10. OPD (Sigma, Cat no. P-9187);

11. Sulphuric acid.

7.3.2. Method

1. A blocking solution of 3% BSA+TBST.

2. Wash solution is TBST.

3. ELISA-tablets “Nunc Maxisorp was senzibilizirani 50 ál of antibody against human IgG, diluted to 1/1000 in carbonate/bicarbonate buffer. The tablets covered with devices for sealing tablets and incubated over night at 4°C.

4. Blocked 100 Microlitre 3% BSA/TBST and incubated at room temperature for 1 hour.

5. Washed 3 times in TBST (at least 200 ál of wash solution/well/wash).

6. Added 50 μl per well of 1 μg/ml human IL-13Rα2-Fc in blocking solution. The tablets covered with devices for sealing tablets and incubated at room temperature for 1 hour

7. Washed 3 times in TBST.

8. In total volume of 50 μl of pre-incubated 0.004 ng/ál det1-labeled human IL-13 with the sample antibody (titrated) in blocking solution for 30 minutes. Then pre-incubated sample was added to the sensitized receptor ELISA tablet, and incubated at room temperature during the 1 o'clock

9. Washed 3 times in TBST.

10. Were detected binding of human IL-13 using 50 μl per well of biotinylated antibodies against human IL-13, diluted at 1 μg/ml and Then incubated for 1 hour at room temperature.

11. Washed 3 times in TBST.

12. Added 50 μl per well of conjugate “streptavidin-HRP” at a dilution of 1/1000. Then incubated for 1 hour at room temperature.

13. Washed 3 times in TBST.

14. Showed 100 Microlitre/well OPD (Sigma, Cat. No. p-9187, prepared in accordance with the manufacturer's instructions), the reaction was stopped by adding 50 μl per well of 3M H2SO4and the tablets were read at an optical density of 490 nm. The developing time was approximately 2 minutes.

7.4. The bioanalysis to neutralize IL-13(analysis on cell proliferation, TF-1)

This analysis represents the bioanalysis IL-13, which can be used to determine the neutralizing capacity of antibodies against IL-13. The following method is used recombinant human IL-13 or IL-13 cynomolgus monkeys. This analysis can also be used expressed in mammalian human IL-13 or Q130-variant of human IL-13. (Cells TF-1 also proliferate in response to human IL-5. This analysis can also be applied to assess the ability of antibodies A1 to neutralize the biological activity of human IL-5).

7.4.1. Materials:

1. Cell line TF-1 (obtained in the laboratory).

2. 96-well tablets for culturing tissue (Invitrogen).

3. Human IL-13 (Cambridge Bioscience, cat. No. SN-013).

4. Analysis on the proliferation of non-radioactive cell CellTiter 96 (Promega, Cat. No. G4000).

7.4.2. Method

1. Implemented method for determining the ability of mAb against human IL-13 neutralizing the biological activity of recombinant IL-13 human or cynomolgus monkeys in bioanalysis cells TF-1 (cell line TF-1 was obtained in the laboratory and does not have the registration number of ATSS).

2. This analysis was performed in sterile 96-well tablets for the cultivation of tissues (Invitrogen) under sterile conditions. All tests were performed with three replicates.

3. Pre-incubated 10 ng/ml human IL-13 (Cambridge Bioscience, cat no. SN-013. The mother solution was used at a concentration of 100 ng/μl in accordance with manufacturers ' instructions in sterile conditions in a closet for culturing tissue class 2 and kept in a small aliquot at -20°C) or 10 ng/ml IL-13 cynomolgus monkeys (obtained in the laboratory from SA) with different dilutions of mAb against human IL-13 (in 3-fold dilutions from 6 μg/ml to 0.025 µg/ml) in a total volume of 50 μl for 1 hour at 37°C. In the experiment also included a positive control wells, containing the E. IL-13, but not mAb against human IL-13. In addition, the experiment included a negative control wells containing no IL-13 mAb against human IL-13. This pre-incubation was performed in a sterile round-bottom 96-well tablet with a low level of binding protein. (It should be noted that when added to cells at a later stage, IL-13 mAb against human IL-13 were used at half the concentration).

4. 50 μl of cells TF-1 were sown at a density of 2 × 105/ml in sterile 96-well plate for culturing tissues. After pre-incubation for 1 hour the cells were added to the sample IL-13 mAb against human IL-13. Received the product in the final analytical volume of 100 µl, containing various breeding mAb against human IL-13, recombinant IL-13 cells and TF-1, incubated at 37°C. for about 70 hours in CO2-incubator with high humidity.

5. Approximately 66 hours, the wells were scanned to confirm their sterility and absence of bacterial infection.

6. In the last 4 hours of incubation was added 15 μl/well sterilized in the filter substrate MTT (Cat no. G4000, Promega, prepared in accordance with the manufacturer's instructions).

7. The reaction was stopped by adding 100 μl of the solution to terminate the reaction (as is being provided in the kit MTT) to solubilize the metabolized product formisano blue. The product is then left for at least 2 hours, after which the pipette with the product was shaken to facilitate dissolution of the crystals. Alternatively, the tablet was closed using a device for sealing tablets and left over night at 4°C, and then, the next day, the pipette was all shook up (this procedure simplifies the pipetting).

8. Read the optical density of the solution in each well of 96-well plate reader at a wavelength of 570 nm.

9. The ability of mAb against human IL-13 neutralizing the bioactivity of IL-13 human or cynomolgus monkeys were expressed as the concentration of mAb against human IL-13, is required to neutralize the biological activity of a certain number of IL-13 human or cynomolgus monkeys (5 ng/ml)/50% (=ND50). The smaller the required concentration, the better the ability of neutralization.

Example: a 96-well plate used in this analysis

/tr>
sample 1antibody-positive
123456 789101112
A3 µg/ml mAb against hIL-13 + IL-13 +
TF-1
Sample 2 mAb
sample 3 mAb
3 μg/ml of polyclonal antibody against hIL-13 + IL-13 + TF-1
B1 μg/ml mAb against hIL-13 + IL-13 + TF-11 μg/ml of polyclonal antibody against hIL-13 + IL-13 + TF-1
Cof 0.33 μg/ml mAb against hIL-13 + IL-13 + TF-1of 0.33 μg/ml of polyclonal antibody against hIL-13 + IL-13 + TF-1
Dof 0.11 μg/ml mAb against hIL-13 + IL-13 + TF-1of 0.11 μg/ml of polyclonal antibody against hIL-13 + IL-13 + TF-1
E0,037 μg/ml mAb against hIL-13 + IL-13 + TF-10,037 μg/ml of polyclonal antibody against hIL-13 + IL-13 + TF-1
F0,0123 μg/ml mAb against hIL-13 + IL-13 + TF-10,0123 μg/ml of polyclonal antibody against hIL-13 + IL-13 + TF-1
GPositive control for cell proliferation, TF-1 = cells TF-1 + IL-13 (mAb missing, 12 holes)
HControl to determine background = present only cells TF-1 (IL-13 is not present, the sample mAb missing, 12 holes)

7.5. ELISA analysis of the binding of human IL-4

This analysis represents an ELISA, allowing to detect binding of an antibody to human IL-4. This analysis was performed in the format of the “sandwich”ELISA.

7.5.1. Materials:

1. Immunoplate Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A).

2. Human IL-4 (R+D Systems, cat no.).

3. Goat polyclonal antibody against human body, control the IL-4 (R+D Systems, Cat no. AF-204-NA).

4. Biotinylated rat monoclonal antibody against human IL-4 (BD/Pharmingen, Cat no.).

5. HRP-conjugated antibody against mouse IgG (Dako, Cat. No. P0260).

6. HRP-conjugated antibody against mouse IgG (Sigma, Cat. No. a-9309).

7. Carbonate/bicarbonate buffer (Sigma; cat. # C-3041);

8. PBST (PBS + 0.05% tween-20).

9. BSA (Sigma A-7030).

10. OPD (Sigma, Cat no. P-9187).

11. Sulphuric acid.

7.5.2. Method

1. A blocking solution of 3% BSA+BST.

2. Wash solution is BST.

3. ELISA-tablets “Nunc Maxisorp was senzibilizirani 50 μl of 5 μg/ml goat polyclonal antibodies against human IL-4 (R+D Systems, cat no. AF-204-NA. The mother solution was used at a concentration of 500 µg/ml according to manufacturer's instructions and stored in aliquot at -20°C) in carbonate/bicarbonate buffer (Sigma; cat no. C-3041, prepared in accordance with the manufacturer's instructions), and then the tablets were closed using a device for sealing tablets and incubated over night at 4°C.

4. Blocked 100 Microlitre 3% BSA/BST and incubated at room temperature and at normal pressure for 1 hour.

5. Washed 3 times in BST (at least 200 ál of wash solution/well/wash).

6. Added 1 ng/ml (50 μl) of human IL-4 in blocking solution and incubated PR the room temperature within 1 hour

7. Washed 3 times in BST.

8. Added 50 μl of the sample antibodies (if necessary, titration was performed to obtain title at the end point) in blocking solution and incubated at room temperature and at normal pressure for 1 hour. In the analysis for binding of human IL-4 as a positive control was used biotinylated monoclonal antibody against human IL-4 (m).

9. Washed 3 times in BST.

10. For mouse monoclonal antibodies A binding was detected using 50 μl per well of HRP-conjugated antibodies against mouse IgG (Sigma, Cat. No. a-9309) at a dilution of 1/1000 in blocking solution for 1 hour at room temperature and under normal pressure. For chimeric antibodies or gumanitarnogo antibodies A binding was detected using 50 μl per well of HRP-conjugated antibodies against human IgG (Sigma, Cat. No. a-6029) at a dilution of 1/2000 in blocking solution for 1 hour at room temperature and under normal pressure. For biotinylated rat monoclonal antibodies against human IL-4, used as a positive control, detection was performed using antibodies conjugated with streptavidin-HRP. (Alternatively, using HRP-conjugated-antibodies is against mouse IgG, P0260 can be detected antibody A and biotinylated rat monoclonal antibody against human IL-4).

11. Washed 3 times in BST.

12. Showed 100 Microlitre OPD (Sigma, Cat. No. p-9187, prepared in accordance with the manufacturer's instructions), the reaction was stopped by adding 50 μl of 3M H2SO4and the tablets were read at an optical density of 490 nm.

7.6. ELISA analysis for mapping epitopes

This analysis is an ELISA that allows detection of binding murine mAb 6A1 with peptides of IL-13 human or cynomolgus monkeys.

7.6.1. Materials:

1. Immunoplate Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A).

2. Streptavidin ImmunoPure® (Pierce, cat. No. 21125).

3. PBST (phosphate buffered saline + 0.05% tween-20).

4. BSA (Sigma A-7030).

5. 16-dimensional peptides of IL-13 human and cynomolgus monkeys, shift = 4 (specially made by the company Mimotopes)

6. 20-dimensional peptides used as positive and negative control (specially made by the company Mimotopes).

7. Mab 6A1.

8. Control Ab (specially made by the company Mimotopes).

9. HRP-conjugated rabbit antibody against mouse Ig (DAKO, code number R).

10. OPD (Sigma, Cat no. P-9187).

11. 3M sulphuric acid.

7.6.2. Method

1. A blocking solution of 3% BSA+BST.

2. Wash solution is BST.

3. ELISA-p is anxiety “Nunc Maxisorp was senzibilizirani 100 Microlitre 5 μg/ml streptavidin ImmunoPure® (Pierce, cat. No. 21125; the drug was used when the initial concentration of 1 mg/ml according to manufacturer's instructions and stored in aliquot at 4°C) using PBST as a diluent. Then the plates were incubated over night at 37°C for drying solution.

4. Blocked 200 Microlitre 3% BSA/BST. After that the tablet was closed using a device for sealing tablets and incubated at room temperature and under normal pressure for 1 hour

5. Washed 3 times in PBST (at least 200 ál of wash solution/well/wash).

6. In wells, taken in duplicate, in which the diluent used PBST, was added 100 μl per well (except control wells) 1000-fold dilutions of each peptide (dissolved in accordance with the manufacturers ' instructions in 200 μl of 40% acetonitrile/60% water, and then divided into aliquots of 10-fold dilutions in the same solvent and kept at -20°C).

7. In control wells, taken in duplicate, in which the diluent used PBST, was added 100 μl per well of 10-fold dilutions of control peptide (dissolved in accordance with the manufacturer's instructions in 1 ml of 40% acetonitrile/60% water, and then kept at -20°C). The tablets covered with devices for sealing tablets and incubated p. and room temperature and under normal pressure for 1 hour to Shuttle machine with a horizontal platform.

8. Washed 3 times in BST (at least 200 ál of wash solution/well/wash).

9. Added 100 μl per well (except control wells) 1,506 μg/ml mouse mAb in PBST.

10. Added 100 μl per well (only in the control wells) 4-, 16 - and 32-fold dilutions of the control antibody (which is used in accordance with manufacturer's instructions and stored at -20°C) using PBST as a diluent. The tablet was closed using a device for sealing tablets and incubated at room temperature and at normal pressure for 1 hour to Shuttle machine with a horizontal platform.

11. Washed 3 times in PBST (at least 200 ál of wash solution/well/wash).

12. Added 100 μl per well of 2000-fold dilutions of HRP-conjugated rabbit antibodies against mouse Ig (DAKO, code number R used in accordance with manufacturer's instructions and stored at +4°C) using PBST as a diluent. The tablet was closed using a device for sealing tablets and incubated at room temperature and at normal pressure for 1 hour to Shuttle machine with a horizontal platform.

13. Washed 3 times in PBST (at least 200 ál of wash solution/well/wash).

14. Showed 100 Microlitre OPD (igma, Cat. No. p-9187, prepared in accordance with the manufacturer's instructions), the reaction was stopped by adding 50 μl of 3M H2SO4and the tablets were read at an optical density of 490 nm. The developing time was approximately 10 minutes.

7.7. ELISA analysis for fine mapping of epitopes

This analysis is an ELISA that allows detection of binding of mAb 6A1 with peptides of IL-13 human or cynomolgus monkeys.

7.7.1. Materials:

1. Immunoplate Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A).

2. Streptavidin ImmunoPure® (Pierce, cat. No. 21125).

3. PBST (phosphate buffered saline + 0.05% tween-20).

4. BSA (Sigma A-7030).

5. The peptides of human IL-13 and IL-13 cynomolgus monkeys, synthesized by the method of “network partial window (“partial window net) (14-dimensional peptide, truncated by one amino acid at the N - and C-end at the same time; this peptide was specially manufactured by the company Mimotopes).

6. 16-dimensional peptide, used as a positive control (pre-custom firm Mimotopes).

7. Mab 6A1 (made in the laboratory).

8. HRP-conjugated goat antibody against mouse IgG (Fc specific)(Sigma A-9309).

9. OPD (Sigma, Cat no. P-9187).

10. 3M sulphuric acid.

7.7.2. Method

1. A blocking solution of 3% BSA+BST.

2. Wash solution is BST.

3. ELISA-tablets “Nunc Maxisorp” sensibiliser the Wali 100 μl of 5 μg/ml streptavidin ImmunoPure® ultrapure water (Pierce, cat. No. 21125; the drug was used when the initial concentration of 1 mg/ml according to manufacturer's instructions and stored at +4°C). Then the plates were incubated over night at 37°C.

4. Blocked 200 Microlitre 3% BSA/BST. After that the tablet was closed using a device for sealing tablets and incubated over night at +4°C.

5. Washed 3 times in BST (at least 200 ál of wash solution/well/wash).

6. In wells, taken in duplicate, in which the diluent used PBST, was added 100 μl per well 1000-fold dilutions of each peptide (dissolved in accordance with the manufacturers ' instructions in 200 μl of 40% acetonitrile/60% water, and then kept at -20°C). The tablet was closed using a device for sealing tablets and incubated at room temperature for 1 hour Shuttle machine with a horizontal platform.

7. Washed 3 times in BST (at least 200 ál of wash solution/well/wash).

8. Added 100 μl per well of 3 µg/ml A, diluted in 3% BSA in PBST. The tablet was closed using a device for sealing tablets and incubated at room temperature for 1 hour Shuttle machine with a horizontal platform.

9. Washed 3 times in BST (at least 200 ál of wash solution/well/wash).

<> 10. Added 100 μl/well 1000-fold dilution of HRP-conjugated goat antibodies against mouse IgG (Sigma A-9309, which is used in accordance with manufacturer's instructions and stored at +4°C) using 3% BSA in PBST as a diluent. The tablet was closed using a device for sealing tablets and incubated at room temperature for 1 hour Shuttle machine with a horizontal platform.

11. Washed 3 times in PBST (at least 200 ál of wash solution/well/wash).

12. Showed 100 Microlitre OPD (Sigma, Cat. No. p-9187, prepared in accordance with the manufacturer's instructions), the reaction was stopped by adding 50 μl of 3M H2SO4and the tablets were read at an optical density of 490 nm. The developing time was approximately 10 minutes.

7.8. Method BiacoreTMfor analysis gumanitarnyh structures on the binding of anti-IL-13 antibodies with full-IL-13

Kinetic analysis was performed on the apparatus Biacore 3000 using the method of immobilization of antibodies. Briefly, chimeric constructs A and gumanitarnogo antibodies used method of capture on protein a, And for the parent murine antibody I used the method of capture antibody against Fc mouse IgG supplied by the company Biacore. Briefly, this method was as follows: the Ligand to capture Immobilise the Wali on the biosensor chip SM by attaching a primary amine in accordance with standard protocols Biacore and using reagents supplied in the kit to attach the primary amine Biacore. This method involves the activation of the touch surface SM by passing through a surface of a solution of 50 mm N-hydroxysuccinimide (NHS) and 200 mm N-ethyl-N'-dimethylaminopropylamine (EDC). Then the ligand to capture (dissolved in acetate buffer, pH 5 or pH 4.5) was added to the activated sensor surface, then any still activated esters were blocked by injecting 1 M ethanolamine hydrochloride, pH 8.5. Then the antibody candidate was passed through the surface, which were immobilized either protein a or antibody against the Fc fragment of mouse immunoglobulin, depending on whether it is human or mouse, and then it was immobilized. After registering a stable signal of binding IL-13 was passed through the surface on which has been immobilized antibody at various certain concentrations. Then binding curves were analyzed using analytical computer programs Biacore, BIAeval v.4.1, to determine reaction kinetics. Experiments were carried out using HBS-EP buffer, Biacore.

7.8.1. Method BiacoreTMfor analysis of the binding of anti-IL-13 antibodies with peptide

Kinetic analysis was performed on the apparatus Biacore 3000 using the method of direct binding of antibodies immobilizovannym peptide of IL-13. Briefly, biotinylated peptide of IL-13 was immobilized using a biosensor Biacore chip SA (streptavidin). After this antibody was passed through the touch surface at various concentrations. Then binding curves were analyzed using analytical computer programs Biacore, BIAeval v.4.1, to determine reaction kinetics. Experiments were carried out using HBS-EP buffer, Biacore.

8. Efficiency gumanitarnogo anti-IL-13-mAb L1+A1 y cynomolgus monkey model of asthma

This section describes the predictive method.

Model of pulmonary bronchoconstriction in cynomolgus monkeys (Macaca fascicularis)induced by Ascaris suum (A.suum), known as the non-clinical model of asthma or model of the disease associated with asthma in humans (R. Patterson et al., Trans. Assoc. Am. Physicians 1980 93:317-325; R. Patterson et al., J. Lab. Clin. Med. 1983, 101:864-872).

In this model, animals with congenital susceptibility of the lung to infection caused A.suum, infected antigen A.suum by inhalation to induce these animals asthmatic response. This asthmatic response can be characterized by measuring the level of hypersensitivity of the respiratory tract (AHR), infiltration of cells defined in the liquid after bronchoalveolar lavage (BAL) and IgE levels in serum. Experimental methods similar to the methods described previously in publications Mauser . et al., Am. J. Resp. Crit. Care Med. 1995 204:467-472 and Evanoff, H. et al., Immunologic Investigation 1992:21:39.

In this study involved 30 animals, pre-selected and included in the research group, which saw a positive bronchospasm in response to the introduction of specific doses of antigen A.suum. Antigen A.suum was administered to each animal in a dose that causes optimal response dose-response (ORD). This dose represents a predefined dose of A.suum, which induces the increase of RL(lung resistance)of at least 40% and a decrease of CDYN(dynamic lung volume changes with pressure variation), at least 35%, at its introduction by aerosol inhalation (for a single dose via nebulizer took more than 15 breaths).

This study was conducted in 2 phases. In phase 1 were evaluated AHR in response to intravenous (i.v.) injection of histamine (doses of histamine, sufficient to induce the increase of RLat least 30% compared with the background level (PC30)) before injection (baseline assessment of lung function on day 1) and after injection (on day 11) antigen A.suum (on days 9 and 10, where A.suum was administered to each animal in optimal pre-defined dose by aerosol inhalation).

Phase 2 is identical to phase 1, except that animals were injected with the antibody (see below), where each of the antibody was administered by intravenous (i.v.) in the form of 3 doses of about 30 mg/kg on days 1, 5 and 9.

Group 1 (n=12): L1+A1 (gumanitarnoe mAb against IL-13, SEQ ID NO:18 and SEQ ID NO:22).

Group 2 (n=12): L1+A1 (gumanitarnoe mAb against IL-13, 30 mg/kg) and pascolizumab (gumanitarnoe mAb against IL-4, 30 mg/kg).

Group 3 (n=6): introduced only media that is used as a negative control.

AHR-data phases 1 and 2 was calculated by measuring the pressure and reception state parameters of the respiratory tract, namely pulmonary resistance (RL) and dynamic lung volume changes with pressure variation (CDYNin response to histamine, using mechanical breathing system Buxco. The maximum percentage change background values after injection of antigen A.suum [for pulmonary resistance (RL) and dynamic lung volume changes with pressure variation (CDYN)] were compared in phases 1 and 2, i.e. with or without treatment with the antibody, and these data were used to assess the phenotype of AHR.

In addition, BAL samples were taken on days 1 and 11 in phases 1 and 2 to determine the level of infiltration of cells, in particular of eosinophilia. To monitor levels of IgE also took serum samples.

Table And
Description of proteins or
polynucleotide (PN)
Identification number of placentas the activity
(SEQ ID NO:)
A, CDRH11
6A1, CDRH22
6A1, CDRH33
6A1, CDRL14
6A1, CDRL25
6A1, CDRL36
6A1, VH (mouse)7
A, VL (mouse)8
hIL-139
hIL-13 (PN)10
6A1, VH, humanitarna design A111
A, VH, humanitarna design A212
A, VH, humanitarna design A313
A, VH, humanitarna design A414
A, VL, humanitarna design L115
A, VL, humanitarna design L216
6A1, heavy chain, humanitarna design A118
A, heavy chain, humanitarna design A219
A, heavy chain, humanitarna design A320
A, heavy chain, humanitarna design A421
A, light chain, humanitarna design L122
A, light chain, humanitarna design L223
A, PN, encoding SEQ ID NO:724
A, PN, encoding SEQ ID NO:825
A, PN, encoding SEQ ID NO:1126
A, PN, encoding SEQ ID NO:1227
A, PN, encoding SEQ ID NO:1328
A, PN, encoding SEQ ID NO:1429
A, PN, encoding SEQ ID NO:1530
A, PN, encoding SEQ ID NO:1631
A, PN, encoding SEQ ID NO:1832
A, PN, encoding SEQ ID NO:1933
A, PN, encoding SEQ ID NO:2034
A, PN, encoding SEQ ID NO:2135
A, PN, encoding SEQ ID NO:2236
A, PN, encoding SEQ ID NO:2337

1. Therapeutic antibody or antigennegative fragment that specifically binds to hIL-13 and inhibits the interaction between hIL-13 and hIL-13R and contains the following CDR's:
CDRH1: SEQ ID NO:1
CDRH2: SEQ ID NO:2
CDRH3: SEQ ID NO:3
CDRL1: SEQ ID NO:4
CDRL2: SEQ ID NO:5
CDRL3: SEQ ID NO:6.

2. Therapeutic antibody or antigennegative fragment according to claim 1, which specifically binds to the epitope represented in the sequence SEQ ID NO:84, contained in SEQ ID NO:9, and inhibits the interaction between hIL-13 and hIL-13R.

3. Therapeutic antibody or antigennegative fragment according to claim 2, where the binding of the indicated antibodies takes place between the residues in positions 103-107, inclusive, in the sequence of SEQ ID NO:9.

4. Therapeutic antibody or antigennegative fragment according to claim 3, where the binding of an antibody to hIL-13 depends on the presence and ginekologa residue at position 107 of the sequence SEQ ID NO:9.

5. Therapeutic antibody or antigennegative fragment according to claim 4, where the replacement of the arginine residue at position 107 of the sequence SEQ ID NO:9 alanine residue leads to loss of binding of the indicated antibodies with hIL-13 in comparison with the binding of the indicated antibodies and antigennegative fragment with the sequence SEQ ID NO:9 in the absence of the substitution at position 107 of the sequence SEQ ID NO:9.

6. Therapeutic antibody or antigennegative fragment according to claim 1, which is an intact antibody.

7. Therapeutic antibody or antigennegative fragment according to claim 6, which is a mouse antibody, rat antibody, antibody primates (e.g., cynomolgus monkeys, monkeys or apes) or human antibody.

8. Therapeutic antibody according to claim 1, which is a humanized or chimeric antibody.

9. The antibody of claim 8, containing human constant region.

10. The antibody according to claim 9, containing the constant region of the IgG isotype.

11. The antibody of claim 10, which is an IgG1 or IgG4.

12. Therapeutic antibody according to claim 7, which is a murine antibody containing a VH domain sequence SEQ ID NO:7 and a VL domain sequence SEQ ID NO:8.

13. therapeutic antibody of claim 8, which is a humanized antibody contains Asim VH domain sequence SEQ ID NO:11 and a VL domain sequence SEQ ID NO:15.

14. therapeutic antibody of claim 8, which is a humanized antibody containing the VH domain sequence SEQ ID NO:12 and a VL domain sequence SEQ ID NO:15.

15. therapeutic antibody of claim 8, which is a humanized antibody containing the VH domain sequence SEQ ID NO:13 and a VL domain sequence SEQ ID NO:15.

16. therapeutic antibody of claim 8, which is a humanized antibody containing the VH domain sequence SEQ ID NO:14 and a VL domain sequence SRQ ID NO:15.

17. therapeutic antibody of claim 8, which is a humanized antibody containing the VH domain sequence SEQ ID NO:11 and a VL domain sequence SEQ ID NO:16.

18. therapeutic antibody of claim 8, which is a humanized antibody containing the VH domain sequence SEQ ID NO:12 and a VL domain sequence SEQ ID NO:16.

19. therapeutic antibody of claim 8, which is a humanized antibody containing the VH domain sequence SEQ ID NO:13 and a VL domain sequence SEQ ID NO:16.

20. therapeutic antibody of claim 8, which is a humanized antibody containing the VH domain sequence SEQ ID NO:14 and a VL domain sequence SEQ ID NO:16.

21. Therapeutic antibody according to any one of PP-20, optionally containing human constant region of the IgG isotype (for example, the R, IgG1 or IgG4).

22. Therapeutic antibody according to claim 1, which is a humanized antibody containing heavy chain SEQ ID NO:18 and a light chain of SEQ ID NO:22.

23. Therapeutic antibody according to claim 1, which is a humanized antibody containing heavy chain SEQ ID NO:19 and a light chain of SEQ ID NO:22.

24. Therapeutic antibody according to claim 1, which is a humanized antibody containing heavy chain SEQ ID NO:20 and a light chain of SEQ ID NO:22.

25. Therapeutic antibody according to claim 1, which is a humanized antibody containing heavy chain SEQ ID NO:21 and a light chain of SEQ ID NO:22.

26. Therapeutic antibody according to claim 1, which is a humanized antibody containing heavy chain SEQ ID NO:18 and a light chain of SEQ ID NO:23.

27. Therapeutic antibody according to claim 1, which is a humanized antibody containing heavy chain SEQ ID NO:19 and a light chain of SEQ ID NO:23.

28. Therapeutic antibody according to claim 1, which is a humanized antibody containing heavy chain SEQ ID NO:20 and a light chain of SEQ ID NO:23.

29. Therapeutic antibody according to claim 1, which is a humanized antibody containing heavy chain SEQ ID NO:21 and a light chain of SEQ ID NO:23.

30. Therapeutic antibody or antigennegative fragment according to claim 1, where the aforementioned antibody is humanized and where residues selected from the group consisting of residues 19, 38, 73 and 81 and capturei frame human heavy chain and residue at position 85 of the acceptor framework region of human light chain, replaced with the corresponding residues present in the frame region of the donor antibody from which there is a CDRH3.

31. Therapeutic antibody or antigennegative fragment according to claim 1, where the aforementioned antibody is humanized and where the frame region of a human heavy chain contains one or more (e.g. all) of the following residues (or a conservative substitution):

PositionBalance
38I
19R
73T
81R

and the human light chain contains
PositionBalance
85V

32. Antigennegative fragment according to any one of claims 1 to 7, where the specified fragment is a Fab, Fab', F(ab')2, Fv, di-antibody, three-antibody, Tetra-antibody, mini-antibody, mini body, selected VH or dedicated VL.

33. The antibody according to claim 9, containing a mutated Fc region, such that the antibody has a reduced D and/or is ctively complement.

34. Therapeutic antibody or antigennegative fragment according to any one of claims 1 to 20, 22-31 or 33, which competitively inhibits the binding of hIL-13 and hIL-13R.

35. Therapeutic antibody or antigennegative fragment by clause 34, where the antibody blocks the binding of hIL-13 and hIL-13R.

36. Therapeutic antibody according to claim 1, which specifically binds to hIL-13 and inhibits the interaction between hIL-13 and hIL-13R and has a dissociation constant koff1.4 · 10-4is 8.22 · 10-5with-1(for example, as was measured in the analysis Biacore™).

37. Therapeutic antibody for p, where the aforementioned antibody is gumanitarnym.

38. Recombinant transformed or transfusiona a host cell of the mammal cloning vectors encoding the antibody according to the invention, containing the first and second vectors, where the first vector contains polynucleotide encoding a heavy chain of the antibody according to any one of the preceding paragraphs, and the second vector contains polynucleotide that encodes a light chain according to any one of the preceding paragraphs.

39. Recombinant transformed or transfusiona a host cell of a mammal for expression vectors encoding the antibody according to the invention, containing the first and second vectors, where the first vector contains polynucleotide encoding thee is Eloy chain antibody according to any one of the preceding paragraphs, and the second vector contains polynucleotide that encodes a light chain according to any one of the preceding paragraphs.

40. A host cell according to § 38 or 39, where the first vector contains polynucleotide selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and the second vector contains polynucleotide selected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36 and SEQ ID NO:37.

41. A host cell according p, where the specified cell is a Cho or NSO.

42. A method of obtaining a therapeutic antibody according to any one of claims 1 to 20, 22-31, 33 or 36 and 37, where the method includes a stage of culturing the host cell according to any one of p-41 in serum-free culture medium.

43. The method according to § 42, where the aforementioned antibody is secreted specified the host-cell in the specified culture medium.

44. The method according to item 43, where the aforementioned antibody is optionally purified to a purity constituting at least 95% or more (for example, 98% or more) relative to the specified containing antibody culture medium.

45. Pharmaceutical composition for use in the treatment of disorders susceptible to modulation of the interaction between hIL-13 and hIL-13R containing a therapeutically effective amount of a therapeutic antibody or antigennegative fragment according to any one of claims 1 to 20, 22-31, 33 or 3 and 37 and a pharmaceutically acceptable carrier.

46. Set for use in the treatment of disorders susceptible to modulation of the interaction between hIL-13 and hIL-13R, part of which is the composition according to item 45, together with instructions for use.

47. The application of therapeutic antibodies or antigennegative fragment according to any one of claims 1 to 20, 22-31, 33 or 36 and 37 for the production of drugs for the treatment of diseases or disorders selected from the group consisting of allergic asthma, severe asthma, asthma, difficult to treat, unstable asthma, "night" asthma, premenstrual asthma, steroid-resistant asthma, steroid-dependent asthma, aspirin-induced asthma, asthma in adults, childhood asthma, atopic dermatitis, allergic rhinitis, Crohn's disease, COPD, fibrotic diseases or disorders, such as idiopathic pulmonary fibrosis, progressive systemic sclerosis, fibrosis, granuloma of the liver, schistosomiasis and leishmaniasis, and diseases associated with dysregulation of the cell cycle, such as Hodgkin's disease, b-cell chronic lymphocytic leukemia.

48. The use of antibodies according to any one of claims 1 to 20, 22-31, 33 or 36 and 37 and monoclonal antibodies against IL-4, such as pascolizumab, in order to manufacture a medicinal product for the treatment of diseases or disorders selected from the group SOS is oasa of allergic asthma, severe forms of asthma, asthma, difficult to treat, unstable asthma, "night" asthma, premenstrual asthma, steroid-resistant asthma, steroid-dependent asthma, aspirin-induced asthma, asthma in adults, childhood asthma, atopic dermatitis, allergic rhinitis, Crohn's disease, COPD, fibrotic diseases or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis, fibrosis, granuloma of the liver, schistosomiasis and leishmaniasis, and diseases associated with dysregulation of the cell cycle, such as Hodgkin's disease and b-cell chronic lymphocytic leukemia.

49. The use of antibodies according to any one of claims 1 to 20, 22-31, 33 or 36 and 37 and monoclonal antibodies against IL-4, such as pascolizumab, in order to prepare a set of several parts for the treatment of diseases or disorders selected from the group consisting of allergic asthma, severe asthma, asthma, difficult to treat, unstable asthma, "night" asthma, premenstrual asthma, steroid-resistant asthma, steroid-dependent asthma, aspirin-induced asthma, asthma in adults, childhood asthma, atopic dermatitis, allergic rhinitis, Crohn's disease, COPD, fibrotic diseases or disorders such as idiopathic pulmonary fibrosis, progressive systemic SK is Eros, liver fibrosis, granuloma of the liver, schistosomiasis and leishmaniasis, and diseases associated with dysregulation of the cell cycle, such as Hodgkin's disease and b-cell chronic lymphocytic leukemia.

50. A set of multiple parts for use in the treatment of disorders susceptible to modulation of the interaction between hIL-13 and hIL-13R, comprising a first pharmaceutical composition comprising a therapeutically effective amount of the antibody according to any one of claims 1 to 20, 22-31, 33 or 36 and 37 and a pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising a monoclonal antibody against IL-4, such as pascolizumab, and a pharmaceutically acceptable carrier, optionally together with the accompanying instructions for use.

51. Pharmaceutical composition for use in the treatment of disorders susceptible to modulation of the interaction between hIL-13 and hIL-13R containing a therapeutically effective amount of a first antibody according to any one of claims 1 to 20, 22-31, 33 or 36 and 37, and a therapeutically effective amount of a second antibody, where the specified second antibody is an anti-IL-4 antibody, such as pascolizumab, and a pharmaceutically acceptable carrier.



 

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16 cl, 18 dwg, 17 tbl, 11 ex

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24 cl, 21 tbl, 32 ex

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18 cl, 5 tbl, 1 dwg, 55 ex

FIELD: medicine, immunobiology, pharmacy.

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EFFECT: valuable medicinal properties of antibody.

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17 cl, 11 dwg, 17 tbl, 4 ex

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15 cl, 11 dwg, 17 tbl

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3 cl, 6 dwg, 6 ex

FIELD: biotechnology, immunology, molecular biology, pharmacy.

SUBSTANCE: invention describes variants of MCP-1-binding molecules. One of MCP-1-binding molecule comprises at least one variable region of immunoglobulin (VH) heavy chain comprising of hypervariable sites CDR1, CDR2 and CDR3 while other molecules comprises both light and heavy chains. Invention proposes DNA constructs encoding indicated MCP-1-binding molecules and expressing vector carrying at least one of these DNA constructs. Invention describes a method for preparing MCP-1-binding molecule. Invention discloses a method for treatment of disease or disorder mediated by MCP-1 or eotaxine-1 based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode. Invention describes a pharmaceutical composition based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode and used in treatment of disease or disorder mediated by MCP-1 or eotaxine-1 in a patient. MCP-1-binding molecules inhibit binding MCP-1 with its receptor. The full immobilized antibody is highly specific as far as it binds human recombinant MCP-1 with value KD = (43 ± 2.9) x 1012 and can be used in medicine.

EFFECT: valuable medicinal properties of antibodies, improved method of treatment.

13 cl, 5 dwg, 4 tbl, 2 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: biotechnology, immunology.

SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.

EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.

26 cl, 16 dwg, 1 tbl, 8 ex

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