BRPI0706481A2 - antibody or antigen-binding fragment thereof, humanized antibody, antigen-binding fragment, transformed or transfected recombinant host cell, methods of producing an antibody, for treating a human patient afflicted with a disease, to prevent acute asthmatic attacks in a human patient , and to reduce the frequency and / or mitigate the effects of acute asthmatic attacks on a human patient, pharmaceutical composition, kit of parts, and use of an antibody or antigen-binding fragment thereof. - Google Patents

Abstract

ANTIBODY OR ITS ANTIGEN BINDING FRAGMENT, HUMANIZED ANTIBODY, ANTIGEN BINDING FRAGMENT, TRANSFORMED OR TRANSFORMED HOST STUDY, METHODS TO PRODUCE AN ANTANGULAR PATIENT TO A HUMAN PRESSURE , AND TO REDUCE THE FREQUENCY AND / OR MITIGUE THE EFFECTS OF ACUTE ASTHMATIC ATTACKS ON A HUMAN PATIENT, PHARMACEUTICAL COMPOSITION, PARTS KIT, AND USE OF ANTIBODY OR ANTIGEN CONNECTION FRAGMENT, particularly the present invention pertains to the present invention. antibodies specifically binding to human interleukin 13 (hIL-13). Antibodies of the invention may be used in the treatment of a variety of diseases or disorders responsive to modulating the interaction between hIL-13 and the human IL-13 receptor. Such diseases include severe asthma, atopic dermatitis, COPD and various fibrotic diseases. Pharmaceutical compositions comprising said antibodies and methods of manufacture are also presented.

Description

"ANTIBODY OR ITS binding fragment ANTIGEN, ANTIBODY HUMANIZED, CONNECTION SHRED AANTÍGENO, host cell RECOMBINANTETRANSFORMADA or transfected, METHODS PARAPRODUZIR AN ANTIBODY TO TREAT A PACIENTEHUMANO DISTRESSED BY A DISEASE FOR PREVENIRATAQUES ASTHMATICS TREBLE IN A PATIENT MAN, eto REDUCE FREQUENCY AND / OR MITIGATING THE EFFECTS OF ACUTE ASMATIC DOSAGE ATTACKS IN A HUMAN PATIENT, PHARMACEUTICAL COMPOSITION, PARTS KIT, AND USE OF AN ANTIBODY CONNECTION FRAGMENT "

Field of the Invention

The present invention relates to immunoglobulins that specifically bind interleukin 13 (IL-13) and in particular human IL-13 (hIL-13). One embodiment of the invention relates to antibodies that specifically bind hIL-13. The present invention also relates to methods of treating diseases or disorders with said immunoglobulins, pharmaceutical compositions comprising said immunoglobulins and methods of manufacture. Other aspects of this invention will be apparent from the following descriptive report.

Background of the Invention

Interleukin-13 (IL-13)

IL-13 is a 12 kDa secreted cytokine originally described as a T-cell derived cytokine that inhibits inflammatory docytocin production. Structural studies indicate that it has a quadruple helical beam arrangement maintained by two disulfide bonds. Notwithstanding that IL-13 has four potential glycosylation sites, analysis of rat rat's native IL-13 indicated that it is produced as a non-glycosylated molecule. The expression of human IL-13 from NOS and COS-7 cells confirms this observation (Eisenmesser et al., J. Mol. Biol. 2001 310 (1): 231-241; Moy et al., J. Mol. Biol. 2001 310 (1): 219-230; Cannon-Carlson et al., Protein Expression and Purification 1998 12 (2): 239-248).

IL-13 is a pleotropic cytokine produced by a variety of cell types, including activated Th2 cells, osmastocytes, basophils, dendritic cells, keratinocytes, and NKT cells. It can also be produced by ThO, Th1, CD8 cells and TCD45RA + cells. IL-13 has immunoregulatory activities that partially overlap with those of IL4, this redundancy can be explained by the shared components in IL4 and IL-13 receptors. The IL-13 signals through the type II IL4 receptor, which is a heterodimer composed of all IL4Ra and IL-13Ral chains. IL-13Ral binds to low affinity IL-13 (Kd = 2 to 10 nM), but when paired with IL4Ra it binds to a high affinity (Kd = 400 pM) and forms a functional IL-13 receptor (the receptor). (hereinafter referred to as "hIL-13R") which signals, resulting in activation of the JAK / STAT and IRS-1 / IRS-2 pathways. An additional IL-13 receptor chain was also characterized (IL-13Ra2), the high affinity IL-13 qualliga (Kd = 250 pM). IL-13Ra2 is believed to act as a bait receptor modulating IL-13 activity. IL-13 may also signal via the IL13Rot2 chain (Fichter-Feigl 2006 NatureMedicine 12: 99-106) to induce TGFbetal and as such may contribute to fibrosis associated with asthma pathology. ParaIL-13 functional receptors are expressed in a wide range of cells, including an airway epithelium, smooth muscle, mast cells, eosinophils, basophils, B cells, fibroblasts, monocytes, and macrophages. T cells lack functional IL-13 receptors [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].

Both IL-13 and IL-4 act to modify immune and inflammatory responses by promoting inflammation associated with suppressive allergy and inflammation due to intracellular bacteria, viruses, and pathogens. Major biological effects of IL-13 include: induction of B cell proliferation and regulation of isotype switching to IgE; induction of MHC and CD23 expression on B cells and monocytes; VCAM-Overregulation of endothelial cells; regulation of chemokine production; activation of mast cell, eosinophil and neutrophil function, as well as inhibition of proinflammatory gene expression in monocyte and macrophage populations. IL-13 has no proliferative effects on T cells. Thus, unlike IL4, IL-13 does not appear to be important in the initial differentiation of CD4 T cells into Th2-type cells, but instead appears to be important in the effector phase of allergic inflammation (McKenzie et al., PNAS 1993 90 (8). : 3735-3739; Wynn, A., Annu, Rev. Immunol., 200321: 425-456).

IL-13 and Asthma

Asthma is a chronic lung disease caused by inflammation of the lower airways and is characterized by recurrent respiratory problems. Patients' airways are tender and swollen or inflamed to some degree all the time, even when there are no symptoms. Inflammation results in narrowing of the airways and reduces air flow into and out of the lungs, making breathing difficult by raising wheezing, chest constriction and cough. Asthma is triggered by susceptibility to allergens (eg dust mites, pollens, fungus), irritants (eg smoke, gas, strong odors), respiratory infections, exercise and dry weather. The outbreaks irritate the airways and the airway lining swells to become even more inflamed, the mucus then blocks the airways and the muscles around the airways narrow until breathing becomes difficult and stressful, and asthma symptoms appear. There is strong evidence from animal and patient models whose asthmatic inflammation and other pathologies are driven by unregulated Th2 response to aeroallergens and other stimuli (Busse et al., Am. J. Crit. Care Med. 1995 152 (1): 388-393). In particular, IL-13 is believed to be the major effector cytokine driving a variety of cellular responses in the lungs, including airway hyperreactivity, eosinophilia, goblet cell metaplasia, and domino hyper secretion.

Clinical Evidence for the Role of IL-13 in 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 has been linked to asthma. Genetic variants of IL-13 that are associated with asthma and atopy have been observed in both the motor and coding regions (Vercelli, D., Curr. Opin. Allergy Clin. Immunol. 2002 2 (5): 389-393). Data from functional studies are available for the coding variant, Q130 IL-13 (referred to herein as "Q130 IL-13"). Single nucleotide polymorphism (SNP) +2044 GaA Found in the fourth exon results in a substitution of an arginine with a glutamine at position 130 (Q130 IL-13). Also note that in SEQ ID NO: 9, this is equivalent to position 110, where the first amino acid residue 'G' at the beginning of the human-mature IL-13 amino acid sequence is position 1. This variant has been observed to be associated with asthma, increased IgE levels and atopic dermatitis in Japanese and European populations. Q130 IL-13 is believed to have enhanced stability compared to wild-type IL-13. It also has significantly lower affinity for the IL-13Ra2 bait receptor and, consistent with these observations, higher median serum IL-13 levels are found to be homozygous for Q130 variant IL-13 compared to inhomozygous patients. These results indicate that Q130 IL-13 may influence local and systemic IL-13 concentrations (Kazuhiko et al, J. Allergy Clin. Immunol 2002 109 (6): 980-987).

Elevated levels of IL-13 have been measured in both atopic and nonatopic asthmatics. In one study, mean serum IL-13 levels of 50 pg / ml were measured in asthmatic patients compared with 8 pg / ml in normal control patients (Lee et al, J. Asthma 200138 (8): 665-671). . Increased levels of IL-13 were also measured in noplasma, bronchial-alveolar lavage fluid, lung biopsy specimens, and sputum (Berry et al., J Allergy Clin. Immunol 2004 114 (5): 1106-1109; Kroegel et al. , Eur. Respir J. J. 9 (5): 899-904; Huang et al., J. Immunol. 1995 155 (5): 2688-2694; Humbert et al., J. Allergy Clin. ): 657-665).

In vivo evidence for IL-13 involvement in asthma

Several studies have defined a critical effector role for IL-13 in driving both acute and chronic allergic asthma pathology in mouse models. High affinity IL-13 receptor (IL-13Ra2) or polyclonal anti-IL-13 antibodies were used to neutralize mouse IL-13 abioactivity in these models. IL-13 blockade at the time of allergen challenge completely inhibited OVA-induced airway hyperresponsiveness, eosinophilia, and goblet cell metaplasias. In contrast, administration of the IL-4 antibody after sensitization and during the allergen challenge phase only partially reduced the asthma phenotype. Thus, although exogenous IL-4 and EL-13 are both capable of inducing a phenotype such as asthma, the effector activity for IL-13 appears to be superior to that for IL-4. These data suggest a major role for IL-4 in immune induction (particularly for Th2 cell development and recruitment to the airway, and IgE production), while IL-13 is believed to be primarily involved in a number of effector outcomes, including hyperresponsiveness. airways, mucus overproduction, and cellular inflammation (Wills-Karpet 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 complementary experiments, lung IL-13 levels were elevated by overexpression in a transgenic mouse or by instillation of IL-13 protein into the wild type mouse trachea. In both settings, characteristics similar to asthma were induced: hyperresponsiveness to non-specific airway cholinergic stimulation, pulmonary eosinophilia, hyperplastic epithelial cells, mucus cell metaplasia, subepithelial fibrosis, airway obstruction, and Charcot-Leyden-like crystals . In addition, IL-13 has been found to be a potent stimulator of lung metalloproteinases and cathepsin proteases, resulting in emphysematous changes and mucous metaplasia. Therefore, IL-13 may be an important effector molecule in both asthma and COPD disease phenotypes (Zhu et al., J. Clin. Invest. 1999 103 (6): 779-788; Zheng et al., J. Clin.Invest. 2000 106 (9): 1081-1093).

These data indicate that IL-13 activity is both necessary and sufficient to produce many of the major as well as pathological aspects of allergic asthma in well-approved animal models.

Chronic Obstructive Pulmonary Disease (COPD)

COPD is a generic expression that covers several syndromesclinics, including emphysema and chronic bronchitis. The symptoms are similar to asthma and COPD can be treated with the same drugs. ACOPD is characterized by a widely reversible airflow obstruction. The contribution of the individual to the disease is unknown, but cigarette smoke is thought to be the cause of 90% of cases. Symptoms include cough, chronic bronchitis, shortness of breath and respiratory infections. Finally the disease will lead to severe disability and death. Chronic bronchitis is diagnosed in patients with a history of sputum or sputum production most days for at least 3 months for 2 years without further explanation. Pulmonary emphysema is characterized by abnormal permanent dilation of air spaces and destruction of alveolar walls.

IL-13 may play a role in the development of COPD. Smoking humans who develop COPD have many types of inflammatory cells (neutrophils, macrophages, eosinophils) in the lung parenchyma. AIL-13 is a proinflammatory Th2 cytokine, therefore, to model the progression of emphysema. Zheng et al. indicated IL-13 overexpression in airway epithelium in IL-13 mice. These animals developed parenchymal inflammation and airway and lung emphysema. They also developed mucosal metaplasia reminiscent of chronic bronchitis (J. Clin.Invest. 2000 106 (9): 1081-1093).

IL-13 (-1055 CaT) promoter polymorphism associated with allergic asthma has also been reported to have an increased frequency in COPD patients compared to healthy controls. This implies a functional role for IL-13 promoter polymorphism in the increased risk of developing COPD (Kraan et al., Genes and Immunity 2002 3: 436-439). In addition, an increased number of IL-13 and IL-4 positive cells was observed in smokers with chronic bronchitis compared with asymptomatic smokers (Miottoet al., Eur. Resp. J. 2003 22: 602-608). However, a recent study to evaluate the level of IL-13 expression in the lungs of patients with emphysemagrave did not find an association between IL-13 levels and the disease (Boutten et al., Thorax 2004 59: 850-854). including atopic dermatitis and allergic rhinitis

IL-13 has also been implicated in atopic disorders such as atopic rhinitis and atopic dermatitis. Allergic rhinitis is the most common atopic disease in the United States, and is estimated to affect up to 25% of adults and more than 40% of children. There is a close relationship between allergic rhinitis and asthma. Both conditions share common pathology and pathophysiology; they have similar immunological processes in which eosinophils and Th2 lymphocytes in thebronchial nasal tissue play a role. Excessive production of Th2 cytokines, particularly IL-4 and IL-5, is thought to be critical in the pathogenesis of allergic disease. IL-13 shares several effector characteristics and functions with IL-4 and this, combined with functional overlap in IL-4 and IL-13 receptor utilization, intracellular signaling components, and genetic organization, provides compelling (though indirect) evidence. ) for a role of IL-13 in promoting or maintaining immediate human hypersensitivity in vivo. This was corroborated by Li et al. (Li et al. JImmunol 1998; 161: 7007), who demonstrated that atopic patients with seasonal allergic rhinitis had significantly more intense IL-13 responses in response to Ag-dependent but not polyclonal activation.

Atopic dermatitis is a common, chronic, relapsing, highly pruritic inflammatory skin disease. Skin with the atopic dermatitis lesion is histologically characterized by an inflammatory β-cell infiltrate, which, during acute phases, is associated with a predominance of IL-4, IL-5 and IL-13 expression (Simonet 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 IL-13 (but not IL-4) omRNA is significantly up-regulated in subacute and chronic skin lesions of patients with atopic dermatitis (Tazawa et al., Arch Derm Res 2004: 296: 459). The frequency of IL-13 expressing circulating CD4 + and CD8 + T cells is also significantly increased in these patients (Aleksza et al. British JDermatol 2002; 147: 1135). This increased IL-13 activity is thought to result in elevated serum IgE levels, thereby contributing to the apatogenesis of atopic dermatitis. In addition, increased IL-13 production by neonatal CD4 + T cells is a useful marker for identifying newborns at high risk for subsequent development of allergic diseases, especially atopic dermatitis (Ohshima et al. Pediatr Res 2002; 51: 195 ). Further evidence for the importance of IL-13 in the etiology of dermatiteatopy was provided by Simon et al. (Simon et al., J Allergy Clin Immunol 2004; 114: 887); topical treatment with tacrolimus ointment (an immunosuppressive drug that inhibits intracellular signaling pathways for cytokine production) resulted in significant clinical and histological improvement of atopic skin lesions, accompanied by significant reductions in local expression of Th2 cytokines, including IL-13. In addition, IL-13 Ral (a cell surface protein that, together with IL-4Roc forms a functional receptor for IL-13) has been observed to be overexpressed on suprabasal keratinocytes in the skin of dermatiteatopic patients, and IL-13 was able to up-regulate IL-13 Ral mRNA in vitro (Wongpiyabovom et al., JDermatol Science 2003; 33:31).

These data collectively indicate that IL-13 targeted interventions, including an IL-13 monoclonal antibody, may provide an effective approach for the treatment of human allergic disease.

Esophageal Eosinophilia

Eosinophil accumulation in the esophagus is a common medical problem in patients with various diseases, including refluxogastroesophageal disease, eosinophilic esophagitis, eosinophilic gastroenteritis, and parasitic infections. Esophageal eosinophilia is associated with allergic responses, and the repeated challenge of mice with aeroallergens has established a link between allergic airway inflammation and eosinophiliaesophageal. Th2 cells are thought to induce inflammation associated with oseosinophils by secreting a series of cytokines, including IL-4 and aIL-13, which directly and indirectly activate inflammatory and effector pathways. IL-13 appears to be particularly important because it is produced in large quantities by Th2 cells and regulates multiple aspects of allergic disease (eg, IgE production, overproduction, eosinophil recruitment and survival, and airway hyperreactivity). Eosinophils can generate functionally active IL-13 following GM-CSF and / or IL-5 exposure under in vitro, ex vivo and in vivo conditions in eosinophilic inflammatory responses (Schmid-Grendelmeier J Immunology, 2002, 169: 1021-1027) IL-13 released into the lungs of STAT-6, eotaxin-1 or IL-5 deficient wild type mice by intratracheal administration has established that pulmonary inflammation, triggered by IL-13, is associated with the development of eosinophiliaesophageal ( Mishra et al Gastroenterol 2003; 125: 1419) Taken together, these data provide evidence for a role of IL-13 in eosinophiliaesophageal.

Oncology indications

Another important area of interest is in indicating IL-13 or LL-13 receptors to inhibit the growth of certain types of tumors. Type 1 T-cell mediated host defenses are believed to mediate optimal tumor rejection in vivo. , and shift to the Thh2-type response may contribute to block tumor rejection and / or tumor recurrence promotion (Kobayashi, M. et al. J. Immunol. 1998; 160: 5869). Transplantable tumor cell lines support this opinion by demonstrating that Stat6, IL-4 and IL-13 (produced in part by NKT cells) were able to inhibit tumor rejection (Terabe et al. Nat. Immunol. 2000; 1: 515; Kacha et al J. Immunol 2000; 165: 6024-6028; Ostrand-Rosenberg et al J. Immunol 2000,165: 6015).

Potent antitumor activity in the absence of Stat-6 was believed to be due to the intensification of IFNg production and tumor-specific deCTL activity. In addition, a loss of NKT cells was observed to reduce IL-13 production with a concomitant increase in tumor recurrence, indicating that IL-13, produced in part by NKT cells, is important for immunovigilance (Terabe et al. Nat. Immunol. 2000; 1: 515). As such, these findings suggest that IL-13 inhibitors or new IL-13 antagonists, including IL-13 mAbs, may be effective as cancer immunotherapeutic agents by interfering with the negative regulatory role of IL-13 in down-regulating responses. immune to tumor cells.

In addition to enhancing Type 1 Th-associated antitumor defenses, IL-13 inhibitors may also be able to block tumor cell growth more directly. For example, in chronic B-cell leukemialinocytic (B-CLL) and Hodgkin's disease, IL-13 either blocks apoptosis or promotes tumor cell proliferation (Chaouchi et al. Blood 1996; 87: 1022; Kapp et al. J. Exp Med. 1999; 189: 1939). B-CLL is a clinically heterogeneous disease that originates from B-lymphocytes involving the apoptotic defect in leukemic cells. IL-13 is not thought to act as a direct growth factor, but protects tumor cells against spontaneous apoptosis in vitro (Chaouchi et al. Blood1996; 87: 1022; Lai et al. J. Immunol.1999; 162: 78) and may contribute to aB-CLL by preventing neoplastic cell death.

Hodgkin's disease is a type of lymphoma that primarily affects young adults and accounts for about 7,500 cases a year in the United States. Cancer is characterized by the presence of large Hodgkin / Reed-Stemberg (H / RS) multinucleated cells. In a large majority of cases, the malignant cell population arises from B cells. Several cell lines derived from Hodgkin's disease, as well as the lymph node tissue of Hodgkin's lymphoma patients, overexpress IL-13e and / or IL-1 receptors. 13 (Kapp et al. J. Exp Med. 1999: 189: 1939, Billardet al. Eur Cytokine Netw 1997; 8:19; Skinnider et al Blood 2001; 97: 250; Oshima et al., Cell Immunol 2001, 211: 37). Neutralization of anti-IL-13 mAbs or IL-13 antagonists has been observed to inhibit H / RS cell proliferation in a dose-dependent manner (Kapp et al J. ExpMed. 1999; 189: 1939; Oshima et al. , Cell Immunol 2001; 211: 37). Similarly, release of the soluble IL-13Ra2 bait receptor to NOD / SCID mice with an implanted cell line derived from Hodgkin's disease slowed tumor initiation and growth, and increased survival, demonstrating that IL-13 neutralization may suppress IL-13 growth. Hodgkin's lymphoma in vitro and in vivo (Trieu et al. Cancer Research 2004; 64: 3271). Collectively, these studies indicate that IL-13 stimulates H / RS cell proliferation in an autocrine form (Kapp et al. J. Exp Med. 1999; 189: 1939; Ohshima et al. Histopathology 2001; 38: 368).

Neutralization of IL-13 may therefore represent an attractive and effective treatment for Hodgkin's disease and other B cell-associated cancers by inhibiting tumor cell growth, while at the same time strengthening antitumor defenses.

Inflammatory Bowel Diseases

There is a possible role for IL-13 in the pathogenesis of inflammatory bowel disease (IBD). Inflammatory bowel disease comprises several diseases clinically classified as ulcerative colitis, Crohn's disease, and indeterminate colitis. Its main manifestation is chronic intestinal inflammation due to an exaggerated immune response with an imbalance in Th1 and Th2 lymphocyte activation in the intestinal mucosa. This has been demonstrated in animal models of Crohn's disease (Bamias et al. Gastroenterol 2005; 128: 657) and ulcerative colitis (Heller et al, Immunity2002; 17: 629). Neutralization of IL-13 by IL-13Ra2-Fc administration prevented colitis in a murine Th2 model of human ulcerative colitis (Heller et al., Immunity 2002; 17: 629). In addition, IL-13 production rapidly replaces that of IL-4 in this model, and IL-13 production may be induced by stimulation of NKT5 cells suggesting that damage may result from toxic IL-13 activity on epithelial cells. There is some human data to support these findings: the frequency of IL-13 positive rectal biopsy specimens from patients with colitis ulcerative was significantly higher than that from inflammatory or noninflammatory control patients, and a higher IL-4 index expression and IL-13 has been observed in acute ulcerative colitis than acute non-acute colitis (Inoue et al. Am J Gastroenterol 1999; 94: 2441). In addition, Akido et al. characterized the immune activity in the external muscularis of the intestinal segments of Crohn's disease patients, and observed that aIL-4 and IL-13 mediate the hypercontractility of the lysointestinal muscle cells via a STAT-6 pathway. The authors concluded that this pathway may contribute to the hypercontractility of the intestinal muscles in Crohn's disease (Akiho et al., Am J Fysiol Gastrointest Liver Fysiol 2005; 288: 619).

Thus, an IL-13 mAb, possibly in combination with molecules directed at other cytokines, may provide an approach to arrest or slow the progression of IBDs.

Psoriasis and Psoriatic Arthritis

Psoriasis is a chronic skin disease characterized by keratinocyte hyperproliferation and an immune cell infiltrate, including activated T cells, producing various cytokines that may influence the epidermal keratinocyte phenotype. CDw60 is a carbohydrate-carrying molecule that is up-regulated on the surface of the basal and suprabasal psoriatic skin cancers. Secreted T-cell IL-4 and aIL-13 derived from psoriatic lesions have been shown to strongly over-regulate CDwO expression over keratinocytes (Skov et al., Am JPathol 1997; 15: 675), whereas IL-4 / Interferon-gamma-blocked IL-13 mediated induction of CD50s on cultured keratinocytes (Huang et al., JInvest Dermatol 2001; 116: 305). Thus, CDwOO expression on epidermal epidermal keratinocytes is thought to be induced, at least in part, by IL-13 secreted by activated T cells within the lesion. In addition, IL-13Ral and IL-4Ra, the cell surface proteins that together form a receptor complex for IL-13, are differently expressed on skin biopsies of patients with and without psoriasis (Cancino-Diaz et al., JInvest Dermatol 2002 ; 119: 1114; Wongpiyabovorn et al., J Dermatol Science 2003; 33: 31), and in vitro experiments have shown that IL-13 (but not IL-4) can over-regulate IL-13Ral expression (Wongpiyabovorn et al. al., J Dermatol Science 2003.33: 31). Given that IL-13 has an effect on a variety of cell types, these studies suggest that the IL-13 receptor may play a part in the early inflammatory process of psoriasis.

Psoriatic arthritis is characterized by synovitis, which is mediated by both pro-inflammatory and anti-inflammatory cytokines. The role of IL-13 in various forms of arthritis has received increasing interest. Padaro et al. observed significantly higher synovial IL-13 levels in patients with psoriatic arthritis and rheumatoid arthritis than in patients with osteoarthritis. In addition, IL-13 levels of IL-13 were significantly higher than those in psoriatic arthritis-friendly serum, and the IL-13 synovial fluid / serum ratio was markedly higher in the psoriatic arthritis group than in the arthritis group. suggesting a possible role for IL-13 locally produced in the synovial tissues of psoriatic arthritis patients (Spadaro et al., Ann Rheum Dis 2002; 61: 174). Potential role of IL-13 in other conditions

Acute graft versus host disease is a cause of morbidity and mortality following primary cell transplantation and is directly related to the degree of human leukocyte antigen (HLA) mismatch between donor and recipient. Jordan et al. first identified IL-13 as a typical Th2 cytokine that is abundantly produced during unrelated, uncompatible, non-compatible MLRs (mixed lymphocyte reaction; initial in vitro HLA screening assay) (Jordan et al. JImmunol Methods; 2002; 260: 1). The same group subsequently showed that IL-13 production by T Donor cells is a predictor of acute graft versus host disease (aGVHD) following transplantation of unrelated donor primordial cells (Jordan et al. Blood 2004; 103: 717). All patients with severe grade III GVHD following primary cell transplantation had donors who produced very high pre-transplant IL-13 responses, demonstrating a significant link between IL-13 and aGVHD levels and raising the possibility that IL -13 may be directly responsible for some of the conditions associated with GVHD. Consequently, an IL-13 specific block-based therapy may be useful for the treatment of aGVHD following primary cell transplantation.

Diabetic nephropathy is a major cause of end-stage disease in the western world. Although the incidence of type 1 diabetes mellitus is declining, type 2 diabetes mellitus is now the single most common cause of renal failure in the United States, Japan and Europe. In addition, this group of patients has a very poor prognosis for maintenance dialysis due to the extremely high mortality caused by cardiovascular events. It is now increasingly clear that hemodynamic, metabolic and structural changes are intertwined, and that several enzymes, transcription factors and growth factors have been identified that play a role in the pathogenesis of this disease. Particularly, TGF-β is important in the development of renal hypertrophy and accumulation of extracellular matrix components, and is considered the main cytokine in mediating decollagen formation in the kidneys [Cooper. Diabetologia 2001; 44: 1957; Wolf. Eur J ClinInvest 2004; 34 (12): 785]. In experimental and human diabetic nephropathy, TGF-I abioactivity is increased and administration of TGF-β 1a diabetic mice led to improvement in renal and reduced accumulated function of the extracellular matrix. IL-13 has recently been presented in a transgenic mouse pulmonary fibrosis model to mediate its effects, at least in part, by regulating the production and activation of TGF-β 1 and collagen deposition (Lee et al. J. Exp. Med. 2001 ; 194: 809; Zhuet al. J. Clin. Invest. 1999; 103: 779) thereby establishing a direct elofunctional between IL-13 and TGF-β. Consequently, a similar role for IL-13 in regulating TGF-β1 activity in the kidney disease can be considered and indicated IL-13 interventions may potentially have a role in controlling diabetic nephropathy.

Fibrotic Conditions

Pulmonary fibrosis is a condition of improper and harmful lung healing, leading to disability and often death. Expression encompasses a variety of different conditions with different etiologies, pathologies and responses to treatment. In some cases, the cause of fibrosis is identified. Causes include: (1) inhaled pro-fibrotic material, such as asbestos or silicon, or hard metal dust, (2) organinalized material, to which the patient has an idiosyncratic immune response leading to fibrosis (eg, farmer's lungs). ), (3) medicinal products such as nitrofurantoin, amiodarone and methotrexate, (4) in combination with a systemic inflammatory disease such as Systemic Sclerosis or Rheumatoid Arthritis.

However, in many cases no underlying cause or condition is identified. Many of such patients are diagnosed with Idiopathic Pulmonary Fibrosis (PFI). This is a relatively rare condition (prevalence 20/100 000). The diagnosis is based on the absence of a identified cause combined with certain radiological and pathological features, particularly CT blunt or lung biopsy. The disease is usually seen in older patients (> 50) and often follows a relentless course of progressive pulmonary debilitation leading to death, with median survival rated 2 to 5 years. In addition, patients have the most unpleasant experience of the lack of progressing breathing for months or years. This initially restricts physical activity, but in the terminal phase - which can last several months - the patient is breathless even at rest and is also oxygen dependent.

There is currently no satisfactory treatment for this disease. Current treatment usually takes the form of corticosteroids and immunosuppressants such as azathioprine. However, corticosteroids may be ineffective in many patients and their side effects may make the situation worse. There are many potential treatments under research that include interferon-gamma, which presented itself as an attempt for improved survival in a large recent student, and perphenidone.

There is evidence that IL-13 and associated cytokines as a Th2 phenotype are involved in the repair process of fibrosis (Wynn, A., Nat. Rev. Immunol. 2004 4: 583-594; Jakubzick et al. J. PathoL 2004 164 (6): 1989-2001; Jakubzick et al., Immunol. Res. 200430 (3): 339-349; Jakubzick et al. J. Clin. Pathol. 2004 57: 477-486 ). IL-13e and IL-4 have been implicated in a variety of fibrotic conditions. Schistosoma-induced liver fibrosis appears to be dependent on IL-13 and there is limited evidence that IL-13 is involved in the pathogenesis of scleroderma (Hasegawa et al., J. Rheumatol. 1997 24: 328-332; Riccieri eti, Clin. Rheumatol. 2003 22: 102-106).

In terms of pulmonary fibrosis, in vitro studies have shown that IL-13 promotes a fibrogenic phenotype. Animal studies have shown high levels of IL-13 expression in artificially induced fibrosis models, and that fibrosis can be reduced by eliminating IL-13.

IL-13 promotes a pro-fibrotic phenotype. At a cellular level, there are several mechanisms by which IL-13 can promote fibrosis. The signal pathways and the importance of these various mechanisms are not well defined.

There is evidence that IL-13 acts on fibroblast both to promote collagen production and to inhibit its decomposition, thereby favoring a fibrotic phenotype. Skin fibroblasts have IL-13 receptors (= the type II IL-4 receptor) and exposure of cultured skin fibroblasts to IL-13 leads to up-regulation of the collagen generation (Oriente et al., J. Farmacol. Exp. Ther. 2000 292: 988-994). IL-4 also has a similar but more transient effect. A human pulmonary fibroblast cell line (ICIG7) expresses the daIL-4 type II receptor (Jinnin et al., J. Biol. Chem 2004 279: 41783-41791). Exposure of these cells to IL-13 promotes secretion of a variety of inflammatory and pro-fibrotic mediators: GM-CSF, G-CSF, VCAM betai integrin (Doucet et al, Int. Immunol. 1998 10 (10): 1421-1433) .

IL-13 inhibits protein production of IL-1-induced matrix metalloproteinases 1 and 3 by skin fibroblasts, which should tend to reduce EC matrix decomposition (Oriente et al, J. Pharmacol. Exp. Ther 2000 292: 988-994). IL-13 acts synergistically with TGF-β on human fibroblasts obtained from asthma airway biopsy to promote the expression of tissue inhibitor dametaloproteinase 1 (TIMP-1). Extracellular matrix decomposition is effected by matrix metalloproteinases, which are inhibited by TIMP-1. This IL-13 action should thus tend to reduce matrix degradation (Zhouet al., Am. J. Fysiol CellFysiol 2005 288: C435-C442 ).

Overexpression of IL-13 in transgenic mice leads to subepithelial fibrosis, epithelial cell hypertrophy, goblet cell hyperplasia, crystal deposition (mammary acidic chitinase, airway hyperresponsiveness, interstitial fibrosis, type cell hypertrophy). 2 and the accumulation of surfactants (Zhu et al., J. Clin.InvesL 1999 103 (6): 779-788).

The different mouse races have different susceptibilities to bleomycin-induced pulmonary fibrosis. C57B1 / 6J mice, which are susceptible, have rapid up-regulation of IL-13, IL-13Ra and IL-4 (as well as TGFβ, TNFRa and ILlRs) in response to bleomycin. BALB / c mice, which are not susceptible, do not show up-regulation of IL-13.

Belperio et al (Am. J. Respir. Cell Mol. Biol. 2002 27: 419-427) studied the expression and role of IL-13, IL-4 and CC CIO chemokine in a mouse bleomycin fibrosis model. Lung tissue levels of both IL-13 and IL-4 increased in response to bleomycin. Prior to IL-13 neutralization, the use of polyclonal antanth-IL-13 antibodies significantly reduced pulmonary fibrosis in response to bleomycin, as assessed by lung hydroxyproline levels. Despite increased IL-4 expression in the same model, neutralization of IL-4 had no effect on pulmonary fibrosis. In another model of FITC-induced acute pulmonary fibrosis in the BALB / C mouse, the absence of IL-13 (in silenced ), but not IL-4, protected against pulmonary fibrosis. There is no added protection for IL-4 mufflers in IL-13 mufflers (Kolodsicket al., J. Immunol. 2004 172: 4068-4076). The protective effect of the absence of IL-13 is not due to the difference in cellular recruitment within the lungs: in all silent and BALB / c, the total numbers of recruited cells are similar, so that the initial inflammatory component appears to be unaffected. Eosinophilic recruitment is lower in IL-4 and IL-13 silencers compared to BALB / c, but since IL-4 - / - was not protected against fibrosis, this cannot explain the difference in fibrosis. surprisingly, there was no difference in decytocin levels between IL-13 + / + and - / -, including for IL10, MCP-1, interferon-gamma, TGF-1. In addition, the same number of fibroblasts were isolated from the lungs of different animals after FITC, but in IL-13 - / - mice collagen I production is reduced. This indicates that the loss of IL-13 is not simply preventing the inflammatory response but is instead playing a more specific antifibrotic role. It has been suggested that IL-13 may exert its fibrotic effect through TGF-I (Lee et al., J. Exp. Med. 2001194: 809-821). However, in this FITC model, TGF-I expression was not reduced in silent IL-13 mice.

Interleukin-4 can be expected to exert a similar effect to IL-13 as both act through the same receptor. IL-4 is significantly over-regulated in the lungs of bleomycin-induced fibrosepulmonary mice (Gharaee-Kermani et al., Cytokine 200115: 138-147). However, in comparing pelableomycin-induced pulmonary fibrosis in C57BL6 / J mice that overexpress IL-4, ossilent and wild-type IL-4, Izbicki et al. {Am. J. Fysiol. Lung CellMol. Fysiol 2002 283 (5): Ll 110-L1116) found no evidence that aIL-4 was involved in pulmonary fibrosis. Fibrosis was not reduced in our IL-4 cells, and mice overexpressing IL-4 had increased levels of fibrosis.

IL-13 BAL cytokine levels are significantly elevated in patients with a variety of forms of pulmonary fibrosis, despite considerable variability. IL-13 expression is significantly up-regulated in alveolar macrophages obtained from pulmonary fibrosis patients.

The strongest clinical evidence comes from research at the University of Michigan. Jakubzick and colleagues studied the expression of IL-13 and IL-4 genes and their receptors in surgical biopsies of lungs with pulmonary fibrosis. Expression of IL-13 genes is markedly higher in affected IPF lung specimens than in normal lungs or other pulmonary fibrotic conditions. Cultured fibroblasts from patients with IPF / UIP show elevated IL-13 and IL-4 receptor expression compared to tissue and fibroblast biopsies from patients with normal lungs or other forms of pulmonary fibrosis. In particular, fibroblast foci, presumably the epicenter of disease activity, stain in particular with respect to 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 good in vitro evidence that Th2 cytokines in general and IL-13 in particular promote a pro-fibrotic phenotype. In at least two animal models, it has been observed that chemically induced fibrosis can be reduced by eliminating IL-13 (either in silent dogene or by anti-IL-13 antibodies). Some evidence indicates that IL-13 is more important in promoting pulmonary fibrosis than IL-4. Clinical evidence for the role of IL-13 in pulmonary fibrosis suggests that IL-13 and its receptors are dysregulated in the lungs of patients with IPF. A growing body of data suggests an important role for IL-13-based therapies for the treatment of a variety of fibrotic conditions, including schistosomiasis-induced hepatic fibrosis, and various forms of fibrosepulmonary [eg, IPF (examined elsewhere). , scleroderma].

Experiments in which IL-4 and IL-13 were inhibited independently identified IL-13 as the dominant cytokine effector of fibrosis in several models (Chiaramonte et al. J. Clin. Invest. 1999; 104: 777-785; Blease et. J. Immunol 2001; 166: 5219; Kumar et al. Clin Exp.Allergy 2002; 32: 1104). In schistosomiasis, although the egg-induced inflammatory response was not affected by IL-13 blockade, collagen deposition decreased by more than 85% in chronically infected animals (Chiaramonte et al. J. Clin. Invest. 1999; 104: 777; Chiaramonte et al., Hepatology 2001; 34: 273) despite continued unreduced IL-4 production.

The amino acid sequence for hIL is presented as SEQ ID NO: 9 (This is the mature protein sequence, ie no signal sequence is present).

A polynucleotide encoding hIL-13 is presented in SEQID NO: 10 (ie, the DNA sequence for the mature protein sequence, that is, the signal sequence is present).

All patents and literature references filed within this specification (including any patent application for which this application claims priority) are hereby expressly incorporated by reference in their entirety.

Recently, vaccines eliciting IL-13 immune responses for the treatment of asthma have been described (WO 02/070711). A role for IL-13 in skin sensitization to environmental allergens has also been recently described (Herrick et al., The Journal of Immunology, 2003, 170: 2488-2495).

The present invention provides an antibody that binds hIL-13 and inhibits the binding of hIL-13 to both hIL-13R chains, i.e. IL-13Rale IL-13Ra2.

The present invention therefore provides an antibody or antigen binding fragment thereof which specifically binds to hIL-13 and neutralizes hIL-13 activity. The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to hIL-13 and comprises a CDRH3 which is a variant of the sequence set forth in SEQ ID NO: 3 or a variant wherein one or two amino acid residues within said CDHR3. Said variant differ from the amino acid residue at the corresponding position in SEQ ID NO: 3. In one embodiment of the present invention, these differences in amino acid residues are conservative substitutions.

The term "specifically binds" as used throughout this disclosure of antibodies and antigen-binding fragments thereof of the invention means that the antibody selects for hIL-13 without any binding, or with insignificant binding, to other human and human proteins. in particular human IL-4. The expression, however, does not exclude the fact that the antibodies of the invention may be reactivated cross-linked with cinomolgus IL-13.

The term "neutralizes" as used throughout the present disclosure of the antibodies and their antigen-binding fragments of the invention means that the biological activity of IL-13 is reduced in the presence of the antibodies and antigen-binding fragments of the present invention. Comparison with IL-13 activity in the absence of such antibodies and their antigen binding fragments. Neutralization levels can be measured in various ways, for example by using the assays presented in the examples below, for example in a TF-I cell proliferation assay which can be performed, for example, as described in Examples 3.3 to 3.5. IL-13 neutralization in this assay is measured by assessing the reduced proliferation of TF-I cells in the presence of neutralizing antibody.

If an antibody or antigen-binding fragment is capable of neutralization, then this is indicative of inhibition of interaction between hIL-13 and its receptor. Antibodies that are considered to have neutralizing activity against human IL-13 should have an ND 50 of less than 100 micrograms / ml, or less than 80 micrograms / ml in the TF-I cell proliferation assay shown in Examples 3.3, 3.4 or 3.5. .

In an alternative aspect of the present invention antibodies or antigen-binding fragments thereof having neutralization activity equivalent to the antibodies exemplified therein are provided, for example antibodies that retain H2L1 neutralizing activity in the TF-I cell proliferation assay shown in Examples 3.3, 3.4 or 3.5.

As used herein, the term "modulating" means inhibiting IL-13 binding to its receptor, and / or blocking the interaction between IL-13 and its receptor, thereby decoupling the hIL-13 / hIL-signaling pathway. 13R. This may be inhibition and / or blockage of either or both of IL-13Ral and IL-13Rot2. The hIL-13 receptor, as used in the descriptive report, means one or both of these receptors. Inhibition of IL-13 binding to its receptor can be measured in a number of ways, for example by using the assays presented in the examples below, for example an ELISA method such as that described in Examples 6.5 and 6.6.

The antibodies and antigen-binding fragments thereof of the present invention may be therapeutic antibodies and antigen-deleting fragments thereof, that is, suitable for use in therapy.

In one aspect, an antibody or antigen-binding fragment thereof, which specifically binds to hIL-13, is provided and comprises a CDRH3 containing the sequence set forth in SEQ ID NO: 3.

In one embodiment, the antibody or antigen-deleting fragment of the present invention neutralizes human IL-13.

In another embodiment, the antibody or antigen-deleting fragment of the present invention modulates binding of human IL-13 to its receptor.

In another aspect of the present invention there is provided an antigen binding antibody or fragment thereof that specifically binds hIL-13 and modulates the interaction between hIL-13 and hIL-13R.

In certain embodiments, the antibodies of the invention at least inhibit the interaction between hIL-13 and hIL-13R, but may also block the interaction between hIL-13 and hIL-13R, thereby decoupling the hIL-13 / hIL-13R signaling pathway. .

In another embodiment, the present invention provides an antigen binding antibody or fragment wherein CDRH3 comprises the sequence of SEQ ID NO: 3. In another embodiment, the antibody or antigen binding fragment thereof of the present invention further comprises one or more More of the following sequences: CDRH2: SEQ ID NO: 2, CDRH1: SEQ ID NO: 1, CDRL1: SEQ ID NO: 4, CDRL2: SEQ ID NO: 5, and CDRL3: SEQ ID NO: 6. In another embodiment The present invention further comprises these CDR sequences in the context of a human structure, for example as a humanized antibody or fragment thereof.

In another aspect of the present invention, there is provided an antigen binding antibody or fragment thereof that specifically binds hIL-13, and comprises the following CDRs: CDRH1: SEQ. ID NO: 1CDRH2: SEQ. ID NO: 2CDRH3: SEQ. ID NO: 3CDRL1: SEQ. ID NO: 4

CDRL2: SEQ. ID NO: 5CDRL3: SEQ. ID NO: 6

In one embodiment of the present invention, one or more of the CDRs of the antibody or antigen binding fragment may comprise variants of the CDRs shown in the sequences listed above. Each variant CDR will comprise one or two amino acid residues which have been derived from the amino acid residue at the corresponding position in the sequence. string listed above. Such amino acid residue substitutions may be conservative substitutions, for example by replacing a hydrophobic amino acid with an alternative hydrophobic amino acid, for example by replacing Leucine with Valine or Isoleucine.

Throughout this descriptive report, amino acid residues in antibody sequences are numbered according to the Kabat scheme. Similarly, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH 1", "CDRH2", "CDRH3" follow the Kabat numbering system presented in Kabat et cd \ Sequences of proteins ofImmunological Interest NIH, 1987, except that heavy chain position 30 is assumed to be part of a CDR.

As used herein the terms "comprising" and "comprising" incorporate "consisting of" and "consisting of".

In another aspect of the invention, an antibody or antigen-binding fragment thereof comprising a VH domain containing the sequence shown in SEQ ID NO: 7 and a VL domain containing the sequence shown in SEQ ID NO: 8. is provided. An isolated VH domain of an antibody is provided containing the selected group sequence consisting of SEQID NOs: 7, 11, 12, 13, and 14. In one embodiment, the isolated VH domain of an antibody consists of, or consistently of, a VH domain. isolated from an antibody selected from the group consisting of SEQ ID NOs: 7, 11, 12, 13 and 14.

In another aspect of the invention, there is provided an antibody or antigen-binding fragment thereof comprising a VH domain selected from the group consisting of: SEQ ID NOs: 7, 11, 12, 13 and 14.

In another aspect of the invention, an antibody is provided that specifically binds hIL-13 and at least inhibits the interaction between hIL-13 and hIL-13R, whose antibody comprises a heavy chain of SEQ ID NO: 18, a light chain selected from the group. consisting of: SEQ ID NOs: 22, 23 and 24.

In another aspect of the invention, there is provided an antibody that specifically binds to hIL-13 and at least inhibits the interaction between hIL-13 and hIL-13R, whose antibody comprises a heavy chain of SEQ ID NO: 19, a light chain selected from the group. consisting of: SEQ ID NOs: 22, 23 and 24.

In another aspect of the invention, there is provided an antibody that specifically binds hIL-13 and at least inhibits the interaction between hIL-13 and hIL-13R, whose antibody comprises a heavy chain of SEQ ID NO: 20, a light chain selected from the group. consisting of: SEQ ID NOs: 22, 23 and 24.

In another aspect of the invention, an antibody is provided that specifically binds hIL-13 and at least inhibits the interaction between hIL-13 and hIL-13R, whose antibody comprises a heavy chain of SEQ ID NO: 21, a light chain selected from the group. consisting of: SEQ ID NOs: 22, 23 and 24. In another aspect of the invention, there is provided an antibody or antigen-binding fragment that inhibits antibody binding comprising a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO. : 22, at hIL-13.

In accordance with the present invention, a humanized antibody is provided which comprises a VH domain of: SEQ ID NO: 1 and a VL domain selected from the group consisting of: SEQ ID NOs: 15, 16 and 17.

According to the present invention, a humanized antibody is provided which comprises a VH domain of: SEQ ID NO: 12 and a VL domain selected from the group consisting of: SEQ ID NOs: 15, 16 and 17.

In accordance with the present invention, a humanized antibody is provided which comprises a VH domain of: SEQ ID NO: 13 and a VL domain selected from the group consisting of: SEQ ID NOs: 15, 16 and 17.

In accordance with the present invention, a humanized antibody is provided which comprises a VH domain of: SEQ ID NO: 14 and a VL domain selected from the group consisting of: SEQ ID NOs: 15, 16 and 17.

In accordance with the present invention, there is provided an antibody or antigen binding fragment thereof which binds to the peptides shown in SEQ ID NOs: 90, 99, 102, 103, 105, 106, 107, 108, 109, 110, 111, 112 e114, but does not bind the peptides set forth in SEQ ID NOs: 100, 101, 104 and 113, wherein the binding is defined as having binding activity equivalent to the antibodies exemplified herein, for example antibodies which retain binding activity similar to 3G4 binding to peptides. Human IL-13 in the ELISA assay presented in Example 6.4.

In another aspect of the invention, a method is provided for detecting a human patient afflicted with a disease or disorder responsive to modulating the interaction between hIL-13 and hIL13R (such as asthma, COPD, allergic rhinitis, atopic dermatitis), which method comprises the step. administering to said patient a therapeutically effective amount of the antibody or antigen-binding fragment thereof, as described in this report.

The use of an antibody of the invention in the manufacture of a medicament for the treatment of a disease or disorder responsive to modulating the interaction between hIL-13 and hIL-13R is also provided.

In another aspect, the invention provides a method for selecting an anti-IL-13 antibody suitable for use in therapy, which method comprises i) providing an antibody that specifically binds IL-13Ral, ii) determining whether the antibody specifically binds to IL-13Ra2, and select an antibody that binds in step ii) for further development.

Detailed Description Of The Invention

The antibodies of the present invention may be intact antibodies or fragments thereof; human, chimeric or humanized antibodies; and mono- or bispecific.

1. Antibody Structures

1.1 Intact Antibodies

Intact antibodies include heteromimeric glycoproteins comprising at least two heavy and two light chains. With the exception of IgM, intact antibodies are usually approximately 150 Kda heterotetrameric glycoproteins composed of two identical light chains (L) and two identical heavy chains (H). Typically, each light chain is linked to one heavy chain by a covalent disulfide bond, while The number of disulfide joints, between heavy strands of different immunoglobulin isotypes, varies. Each heavy and light chain also has disulfide bridges between chains. Each chain weighs at one end a variable domain (VH) followed by several constant regions. Each light chain has a variable domain (VL) and a constant region at its other end; the light chain constant region is aligned with the first heavy chain constant region and the light chain variable domain is aligned with the heavy chain variable domain. The antibody light chains of most vertebrate species can be attributed to one of two types called of Kapa and Lambda, based on the constant region amino acid sequence. Depending on the amino acid sequence of the constant region of their heavy chains, human antibodies can be assigned to five different classes, IgA, IgD, IgE, IgG and IgM. IgG and IgA may be further subdivided into the IgG1, IgG2, IgG3 and IgG4 subclasses; and IgAl and IgA2. Species variants exist with mice and rats having at least IgG2a, IgG2b. The antibody variable domain confers binding specificity over the antibody, with certain regions showing specific variability called complementarity determining regions (CDRs). The most conserved portions of the variable region are called Structural (FR) regions. The intact heavy and light chain variable domains each comprise four FRs connected by three CDRs. The CDRs in each chain are held together in close proximity by the FR regions, and the CDRs in the other chain contribute to the formation of the antigen binding site of antibodies. Constant regions are not directly involved in antigen-antibody binding, but have several effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), Fcy receptor-binding phagocytosis, half-life / clearance through neonatal Fc receptor (FcRn) and complement dependent acitotoxicity through the complement component Clq dacascata.

In one embodiment, therefore, we provide an intact antibody that specifically binds to hIL-13, which antibody modulates the interaction between hIL-13 and hIL-13R, for example the antibody inhibits interaction between hIL-13 and its receptor. The intact antibody may comprise a constant region of any isotype or subclass thereof described above. In one embodiment, the antibody is of the IgG isotype, particularly IgG1. The antibody may be rat, mouse, rabbit, primate or human. In a typical embodiment, the antibody is primate (such as decynomolgus, Old World monkey or Anthropoid Ape; see for example WO 99/55369, WO 93 / 02108) or human.

In another embodiment, an isolated antipointpoint comprising a CDRH3 of SEQ ID NO: 3 is provided. In another embodiment, an intact antibody comprising a variable region containing CDRs of SEQ ID NOs: 1, 2, 3, 4, 5 is provided. and 6.

In another embodiment, an isolated murine antibody or antigen-binding fragment thereof comprising a VH domain containing the sequence of SEQ ID NO: 7 and a VL domain of the sequence of SEQ ID NO: 8 is provided.

1.2 Human Antibodies

Human antibodies may be made by various methods known to those of skill in the art. Human antibodies can be produced by the hybridoma method using human domieloma or human mouse heteromyeloma cell lines; see Kozbor, J. Immunol 133, 3001, (1984) and Brodeur, Monoclonal Antibody Production Teehniques and Applications, pp 51-63 (Mareei Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both of which use human V region repertoires [see Winter, G., (1994), Annu. Rev. Immunol 12, 433-455, Green, L.L. (1999), J. Immunol. methods 231, 11-23).

Several strains of transgenic mice are now available, in which their mouse immunoglobulin loci have been replaced by human immunoglobulin gene segments [seeTomizuka, K., (2000) PNAS 97, 722-727; Fishwild, D.M. (1996) Nature Biotechnol. 14, 845-851, Mendez, Μ. J., 1997, Nature Genetics, 15, 146-156]. After antigen challenge, such mice are capable of producing a human antibody repertoire from which antibodies of interest may be selected. Of particular mention is the Trimera® system (see Eren, R. etal., (1998) Immunology 93: 154-161) in which human lymphocytes are transplanted into irradiated mice, the Selected Lymphocytic Antibody System (SLAM, see Babcook et al. , PNAS (1996) 93: 7843-7848), wherein human (or other species) lymphocytes are effectively placed by a massive pooled in vitro antibody generation procedure, followed by limiting decomvulated, dedilution and selection procedure and Xenomouse II® (Abgenix Inc.). An alternative approach is available from Morfotek Inc. using Morfodoma® technology.

Phage display technology can be used to produce human antibodies (and fragments thereof). See McCafferty; Nature, 348, 552-553 (1990) and Griffiths, A. D. et al. (1994) EMBO 13: 3245-3260. According to this technique, antibody V domain genes are matrix cloned within either a major or secondary protein degene cover of a filamentous bacteriophage, such as M13 or fd, shown (usually with the aid of an auxiliary phage) as functional antibody fragments on the surface of the phage particle.

Selections based on the antibody's functional properties result in the selection of the gene encoding the antibody that has these properties. The phage display technique can be used to select antigen-specific antibodies from libraries produced from human B cells harvested from individuals afflicted with a disease or disorder described above or, alternatively, from nonimmunized human donors (see Marks; J. Mol. Bio. 222, 581-597, 1991). When an intact human antibody comprising an Fc domain is desired, it is necessary to clone the fragment derived from the fact presented in a mammal, the expression vectors comprising the desired constant regions and establishing stable expression cell lines.

The affinity maturation technique [Marks; Bio / technol 10,779-783 (1992)] can be used to improve binding affinity in which the affinity of primary human antibody is improved by sequential substitution of HeL chain V regions with naturally occurring variants, and selection based on improved binding affinities. . Variants of this technique, such as "epitope printing" are now also available. See WO 93/06213. See also Waterhouse; Nucl.Acids Res 21, 2265-2266 (1993).

Thus, in another embodiment an isolated human anti-point or antigen-binding fragment thereof which specifically binds hIL-13 and modulates the interaction between hIL-13 and hlL-13R is provided, for example when it inhibits the interaction between hIL -13 and its receiver.

In another aspect, an isolated intact human antibody or antigen-binding fragment thereof comprising a CDRH3 of SEQ ID NO: 3 that specifically binds hIL-13 and modulates the interaction between hIL3 and hIL-13R is provided, for example when it inhibits interaction. between hIL-13 and its receiver. In another aspect, an isolated human isolated intact antibody or antigen binding fragment thereof comprising a variable region comprising CDRs of SEQ ID NOs: 1, 2, 3, 4, 5 and 6 as defined above is provided.

1.2 Chimeric and humanized antibodies

The use of non-human intact antibodies in the treatment of human diseases or disorders carries with it the potential for the well-established problems of immunogenicity, that is, the patient's immune system may recognize the human intact antibody as not having a neutralizing response. This is particularly evident after multiple administration of the non-human antibody to a human patient. Several techniques have been developed over the years to overcome these problems, and generally involve reducing the composition of non-human amino acid sequences in the intact antibody, while retaining relative ease in obtaining non-human antibodies from an immunized animal, for example a mouse, rat or rabbit. Two approaches were widely used to achieve this. The first consists of chimeric antibodies, which generally comprise a non-human variable domain (e.g. rodent such as mouse) fused to a human constant region. Since the antigen binding site of an antibody is located within the variable regions, the chimeric antibody retains its antigen binding affinity, but acquires effector functions from the human constant region, and is therefore capable of performing effector functions such as those. described above. Chimeric antibodies are typically produced using recombinant DNA methods. ODNA encoding antibodies (for example cDNA) is isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that are capable of specific binding to genes encoding the H and L chains of the antibody of the invention, for example oDNA encoding SEQ ID NOs: 1, 2, 3, and, 5 and 6 described above). Hybridoma cell cells serve as a typical source of such DNA. Once isolated, the DNA is placed into expression vectors, which are then transfected into host cells such as E. coli, COS cells, CHO cells, or myeloma cells that otherwise do not produce immunoglobulin protein to obtain the synthesis of the antibody. DNA can be modified by replacing the coding sequence for the LeH human chains with the corresponding non-human (e.g., demurino) HeL constant regions. See, for example, Morrison; PNAS 81, 6851 (1984). The second approach involves the generation of humanized antibodies in which the non-human antibody content is reduced by the humanization of the variable regions. Two techniques for humanization gained popularity. The first is humanization by CDR grafting. CDRs construct circuits near the N-terminus of antibodies where they form a surface mounted on a scaffold provided by the framework regions. Antigen binding specificity of the antibody is mainly defined by the topography and chemical characteristics of its CDR surface. These aspects are in turn determined by the formation of the individual CDRs, the relative deposition of the CDRs, and the nature and arrangement of the residue side chains comprising the CDRs. A large reduction in immunogenicity can be achieved by grafting only non-human (e.g. murine) CDRs ("donor" antibodies to human structure ("acceptor structure" and constant regions) [see Jones et al. (1986) Nature 321, 522-525 and Verhoeyen, M. et al (1988) Science 239, 1534-1536) However, CDR porsi grafting may not result in complete retention of antigen binding properties and it is often observed that some structure residues ( sometimes referred to as "back mutations") of the donor antibody need to be preserved in the humanized molecule if significant antigen binding affinities must be recovered (see Queen, C. etal. (1989) PNAS 86, 10.029-10.033, Co, M., et al. (1991) Nature 351, 501-502) In this case, the human V regions showing the greatest homology to the non-human donor antibody are chosen from a database to provide the human structure (FR). . The selection human FRs can be made either from human consensus or from individual human antibodies. Where necessary, key donor antibody residues are replaced in the human acceptor framework to preserve the conformations of the CDR. Antibody computer modeling can be used to help identify such structurally important residues. See WO 99/48523.

Alternatively, humanization may be achieved by a "leafing" process. A statistical analysis of the unique heavy and light decade variable regions of human and murine immunoglobulin revealed that the precise patterns of exposed residues are different in human and murine antibodies, and most surface positions have a strong preference for a small number of different residues [ verPadlan, EA et al; (1991) Mol. Immunol. 28, 489-498 and Pedersen, J.T. et al (1994) J. Mol. Biol 235; 959-973). Therefore, it is possible to reduce the immunogenicity of a non-human Fv by replacing the exposed residues in their framework regions that differ from those usually found in human antibodies. Since protein antigenicity can be correlated with surface accessibility, surface residue substitution may be sufficient to make the mouse variable region "invisible" to the human immune system (see also Mark, GE et al. (1994) in Handbook of Experimental Pharmacology vol.113: The pharmacology of monoclonal Antibodies, Springer-Verlag, pp 105-134). This humanization procedure is referred to as "leafing" because only the surface of the antibody is altered, the support residues remaining undisturbed.

The skilled person will be aware that other antibody humanization methods exist and are available in the literature.

Accordingly, another embodiment of the invention provides a chimeric antibody comprising a non-human (e.g., rodent) variable domain fused to a human constant region (which may be from an IgG isotype, for example IgG1), which specifically binds to hlL -13 and modulates the interaction between hIL-13 and hIL-13R, for example in which it inhibits the interaction between hIL-13 and its receptor.

In another embodiment, a chimeric antibody comprising a non-human variable region (for example scavenger) and a human constant region (which may be from an IgG5 isotype for example IgG1) is provided, which specifically binds hIL-13, whose antibody further comprises a CDRH3 of SEQ ID NO: 3. Such antibodies may further comprise a human constant region of the IgG isotype, for example IgG1.

In another embodiment, a chimeric antibody comprising a non-human variable region (e.g., scavenger) and a human constant region (which may be from an IgG isotype, for example IgG1) that specifically binds hIL-13 is provided. comprising CDRs of SEQID NOs: 1, 2, 3, 4, 5 and 6.

In another embodiment, a chimeric antibody comprising a VH domain of SEQ ID NO: 7 and a VL domain of SEQ ID NO: 8, and a human constant region of an IgG isotype, for example IgG1, that specifically binds is provided. hIL-13 and modulate the interaction between hIL-13 and hIL-13R, for example where it inhibits the interaction between hIL-13 and its receptor.

In another embodiment, a humanized antibody or antigen binding fragment thereof is provided which specifically binds hIL-13 and modulates the interaction between hIL-13 and hIL-13R, for example when it inhibits the interaction between hIL-13 and your receiver.

In another embodiment, a humanized antibody or antigen-binding fragment thereof that specifically binds hIL-13 and comprises a CDRH3 of SEQ ID NO: 3 is provided. Such antibodies may comprise a human constant region of the IgG isotype, for example IgG1. .

In another embodiment, a humanized antibody or antigen binding fragment thereof that specifically binds hIL-13 and comprises CDRs of SEQ ID NOs: 1, 2, 3, 4, 5 and 6 is provided. Such antibodies may comprise a region human constant of the IgG5 isotype for example IgG1.

In accordance with the present invention, there is provided a humanized antibody comprising a VH domain selected from the group: SEQ ID NO: 11, and a VL domain selected from the group of: SEQ IDNOs: 15, 16 and 17. Such antibodies may comprise a region. human constant of the IgG isotype, for example IgG1.

In accordance with the present invention, there is provided a humanized antibody comprising a VH domain selected from the group: SEQ ID NO: 12, and a VL domain selected from the group of: SEQ IDNOs: 15, 16 and 17. Such antibodies may comprise a region. human constant of the IgG isotype, for example IgG1.

In accordance with the present invention, there is provided a humanized antibody comprising a VH domain selected from the group: SEQ ID NO: 13, and a VL domain selected from the group of: SEQ IDNOs: 15, 16 and 17. Such antibodies may comprise a region. human constant of the IgG isotype, for example IgG1.

In accordance with the present invention, there is provided a humanized antibody comprising a VH domain selected from the group: SEQ ID NO: 14, and a VL domain selected from the group of: SEQ IDNOs: 15, 16 and 17. Such antibodies may comprise a region. human constant of the IgG isotype, for example IgG1.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 1 and a VL domain of SEQ ID NO: 15 is provided.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 12 and a VL domain of SEQ ID NO: 15 is provided.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 13 and a VL domain of SEQ ID NO: 15 is provided.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 14 and a VL domain of SEQID NO: 15 is provided.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 11 and a VL domain of SEQ ID NO: 16 is provided.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 12 and a VL domain of SEQ ID NO: 16 is provided.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 13 and a VL domain of SEQ ID NO: 16 is provided.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 14 and a VL domain of SEQ ID NO: 16 is provided.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 1 and a VL domain of SEQ ID NO: 17 is provided.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 12 and a VL domain of SEQ ID NO: 17 is provided.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 13 and a VL domain of SEQ ID NO: 17 is provided.

In another embodiment, a humanized antibody comprising a VH domain of SEQ ID NO: 14 and a VL domain of SEQ ID NO: 17 is provided. In another embodiment, a humanized antibody or antigen binding fragment thereof is provided. specifically select hIL-13 wherein said antibody or fragment thereof comprises CDRH3 of SEQ ID NO: 3, optionally further comprising one or more CDRs of SEQ ID NOs: 1, 2, 4, 5 and 6, wherein one or more of the selected residues of group consisting of positions 10, 30, 67, 69, 71, 73 and 93 of the human acceptor heavy chain structure, and one or both of the residues in the human acceptor light chain structure positions 76 and 98 are replaced by the corresponding residues found in the human structure. donor antibody from which CDRH3 is derived (shown in SEQ IDNO: 7).

In another embodiment, there is provided a humanized antibody or antigen binding fragment thereof that specifically selects hIL-13 wherein said antibody or fragment thereof comprises CDRH3 of SEQ ID NO: 3, optionally further comprising one or more CDRs of SEQ ID NOs. : 1, 2, 4, 5 and 6, wherein the human heavy chain structure comprises one or more (e.g. all) of the following residues (or their conservative substitution):

Residual Position10 D30 I67 A69 L71 A73 K93 T

and a light chain structure comprising either or both of the following residues (or their conservative substitutes): It will be apparent to those skilled in the art that the term "derivative" is intended to define not only the source in the sense of being the physical origin for the subject. material, but also define material that is structurally identical (in terms of the primary amino acid sequence) to the material, but not derived from it, from the reference source. Thus, "residues found in the donor antibody from which CDRH3 is derived" need not necessarily have been purified from the donor antibody.

It is well recognized in the art that certain amino acid substitutions are considered to be "conservative". Amino acids, divided into groups based on common side chain properties and substitutions within groups that maintain all or substantially all of the binding affinity of the antibody of the invention or antigen-binding fragment thereof, are considered conservative substitutions. See a15 Table:

<table> table see original document page 42 </column> </row> <table>

In accordance with the present invention, a humanized antibody comprising a group-selected heavy chain consisting of: SEQ ID NOs: 18, 19, 20, 21 and a group-selected light chain consisting of: SEQID NOs: 22, 23, 24 is provided.

In one embodiment of the invention there is provided a humanized antibody that specifically binds to hIL-13 comprising a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO: 22.

In one embodiment of the invention there is provided a humanized antibody that specifically binds to hIL-13 comprising a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 22.

In one embodiment of the invention there is provided a humanized antibody that specifically binds to hIL-13 comprising a heavy chain of SEQ ID NO: 20 and a light chain of SEQ ID NO: 22.

In one embodiment of the invention, a humanized antibody specifically binding to hIL-13 is provided comprising a heavy chain of SEQ ID NO: 21 and a light chain of SEQ ID NO: 22.

In one embodiment of the invention there is provided a humanized antibody that specifically binds to hIL-13 comprising a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO: 23.

In one embodiment of the invention, a humanized antibody specifically binding to hIL-13 is provided comprising a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 23.

In one embodiment of the invention there is provided a humanized antibody that specifically binds to hIL-13 comprising a heavy chain of SEQID NO: 20 and a light chain of SEQID NO: 23.

In one embodiment of the invention, a humanized antibody specifically binding to hIL-13 is provided comprising a heavy chain of SEQ ID NO: 21 and a light chain of SEQ ID NO: 23.

In one embodiment of the invention there is provided a humanized antibody that specifically binds to hIL-13 comprising a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO: 24.

In one embodiment of the invention there is provided a humanized antibody that specifically binds to hIL-13 comprising a heavy chain of SEQID NO: 19 and a light chain of SEQ ID NO: 24.

In one embodiment of the invention there is provided a humanized antibody that specifically binds to hIL-13 comprising a heavy chain of SEQ ID NO: 20 and a light chain of SEQ ID NO: 24.

In one embodiment of the invention there is provided a humanized antibody that specifically binds to hIL-13 comprising a heavy chain of SEQ ID NO: 21 and a light chain of SEQ ID NO: 24.

In one embodiment of the present invention, there is provided a human or humanized heavy chain variable region comprising one of the CDRs listed in SEQ ID NOs: 1 to 3. In another embodiment of the present invention, a humanized chain-heavy variable region comprising is provided. CDRs listed in SEQ ID NOs: 1 to 3, within the larger sequence of a human heavy chain variable region. In yet another embodiment, the humanized heavy chain variable region comprises the CDRs listed in SEQ ID NOs: 1 to 3 within an acceptor antibody backbone having more than 40% identity in the backbone regions, or more than 50%, or more than 60%, or more than 65%, identity with the donor antibody heavy chain variable region 3G4 murine (SEQ ID NO: 7).

In one aspect of the present invention, the antibodies comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44, further comprising various substitutions at one or more of positions 10, 30, 67, 69, 71, 73, 93 Kabat numbering); wherein each substituted amino acid residue is replaced by the amino acid residue at the equivalent position in SEQ IDNO: 7 (the donor antibody heavy chain variable region 3G4) and the number of substitutions is between 0 and 7. In other embodiments, the number of substitutions is 0, or 1, or 2, or 3, or 4, or 5, or 6, or 7.

In one embodiment of the present invention, there is provided a human or humanized light chain variable region comprising one of the CDRs listed in SEQ ID NOs: 4 to 6. In another embodiment of the present invention, a humanized light chain variable region comprising the CDRs listed in SEQ ID NOs: 4 to 6 within the larger sequence of a human light chain variable region. In yet another embodiment, the humanized light chain variable region comprises the CDRs listed in SEQ ID NOs: 4 to 6 within an acceptor antibody framework having more than 40% identity in the framework regions, or more than 50%, or more than 60%, or more than 65%, identity to the murine donor3G4 antibody heavy chain variable region (SEQ ID NO: 8).

In one aspect of the present invention the antibodies comprise a light chain variable region containing the amino acid sequence of SEQ ID NO: 45, further comprising various substitutions at one or more of positions 76, 98 (Kabat numbering system); wherein each substituted amino acid residue is replaced by the amino acid residue at the equivalent position in SEQ ID NO: 8 (the lightweight variable region of the donor 3G4 antibody) and the number of substitutions is between 0 and 2. In other embodiments, the number of substitutions is 0 or 15 Ior 2.

1.3 Bispecific Antibodies

A bispecific antibody is an antibody having binding specificities for at least two different epitopes. Methods of producing such antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the coexpression of two H-chain L-chain immunoglobulin pairs, wherein the two H-chains have different binding specificities. 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 classification of the HeL chains, a potential mixture of ten different antibody structures is produced, of which only one has the desired binding specificity. An alternative approach involves fusing the variable domains with the desired binding specificities for the heavy chain constant region comprising at least part of the hinge region, the CH2 and CH3 regions. It is preferable to have the CH1 region containing the site necessary for light chain binding present in at least one of the fusions. The DNA encoding these fusions and, if desired, the L chain, are introduced into separate expression vectors and then cotransfected into a suitable host organism. It is possible, however, to introduce coding sequences for two or all three strands into one expression vector. In a preferred approach, the bispecific antibody is composed of an H chain with a first binding specificity on one arm and one HL chain, providing a second binding specificity in the other subject, see WO 94/04690. See also Suresh et al. Methods in Enzyme 121, 210, 1986.

In one embodiment of the invention there is provided a bispecific antibody wherein at least one binding specificity of said antibody is for hIL-13, wherein said antibody modulates the interaction between hIL-13 and IL-13R, for example where it inhibits interaction. entrehIL-13 and its receiver. Such antibodies may further comprise a human constant region of the IgG isotype, for example IgG1. In some embodiments, the bispecific therapeutic antibody has a first binding specificity for hIL-13 and modulates the interaction between hIL-13 and hIL-13R, for example where it inhibits the interaction between hIL-13 and its receptor, and a second specificity of hIL-13. It binds to hIL-4 and modulates the interaction between hIL-4 and a receptor for hIL-4, for example where it inhibits the interaction between hIL-4 and its receptor.

In one embodiment of the invention there is provided a bispecific antibody wherein at least one binding specificity of said antibody is for hIL-13, wherein said antibody comprises a CDRH3 of SEQ ID NO: 3. Such antibodies may further comprise a human constant region of the antibody. IgG isotype, for example IgG1.

In one embodiment of the invention there is provided a bispecific antibody wherein at least one binding specificity of said antibody is for hIL-13, wherein said antibody comprises at least CDRs of SEQ ID NOs: 1, 2, 3, 4, 5 and 6. Such antibodies may further comprise a human constant region of the IgG isotype, for example IgG1.

1.4 Antibody Fragments

In certain embodiments of the invention, antibody fragments that modulate the interaction between hIL-13 and hIL-13R are provided, for example where fragments inhibit the interaction between hIL-13 and its receptor. Such fragments may be intact and / or humanized and / or chimeric antibody functional antigen binding fragments, such as the Fab, Fab ', F (ab') 2, Fv, ScFv fragments of the above described antibodies. Traditionally, such fragments are produced by proteolytic digestion of intact antibodies, for example by papain digestion (see, for example, WO 94/29348), but can be produced directly from recombinantly transformed host cells. For ScFv production, see Bird et al., (1988) Science, 242, 423-426. In addition, antibody fragments can be produced using a variety of engineering techniques as described below.

The fragments appear to have lower interaction energy of their two chains than Fab fragments. To stabilize the association of the VH and VL domains, they have been linked with the peptides (Bird et al., (1988) Science 242, 423-426, Huston et al., PNAS, 85, 5879-5883), with disulfide aspects (Glockshuber et al., (1990) Biochemistry, 29, 1362-1367) and button-in-hole mutations (Zhu et al. (1997) , Protein Sci., 6, 781-788). ScFv fragments can be produced by methods well known to those skilled in the art. See Whitlow et al. (1991) Methods companion Methods Enzymol, 2, 97-105 and Huston et al. (1993) Int.Rev. Immunol 10, 195-217. ScFv can be produced in bacterial cells such as Ε. coli, but more preferably eukaryotic cells are produced. A disadvantage of ScFv is the product monovalence, which prevents increased avidity due to multipurpose binding, and its short half life. Attempts to overcome these problems include divalent (ScFv ') 2 produced from ScFV containing a Cadtional terminal cysteine by chemical coupling (Adams et al. (1993) Can. Res. 534026-4034 and McCartney et al. (1995) Protein Eng. 8, 301-314) or site-specific spontaneous medianmedimerization of ScFv containing an unpaired C-terminal decysteine residue (see Kipriyanov et al. (1995) Cell. Biofys26, 187-204). Alternatively, the ScFv may be forced to form multimeres by shortening the peptide linker to 3 to 12 residues to form "diabodies". See Holliger et al. PNAS (1993), 90, 6444-6448. Further deleterious reduction may result in ScFV trimers ("triacibodies", see Korttet al. (1997) Protein Eng, 10, 423-433) and tetramers ("tetribodies", see LeGall et al. (1999) FEBS Lett, 453, 164-168). Construction of bivalent SCFV molecules can also be obtained by genetic fusion with protein dimerization motifs to form "minianticorpons" (see Pack et al. (1992) Biochemistry 31, 1579-1584) and "minibodies" (see Hu et al. ( 1996), Cancer Res. 56, 3055-3061). ScFv-Sc-Fv [(ScFV) 2] tandens can also be produced by the binding of two ScFv units by a third peptide linker, see Kurucz et al. (1995) J. Immol. 154, 4576-4582. Specific antibodies can be produced by non-covalently associating two single-chain fusion products consisting of the 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 bispecific antibodies may be enhanced by the introduction of disulfide bridges or "buttonhole" mutations as described above, or by the formation of single stranded (ScDb) bodies where two hybrid SCFc fragments are connected via a peptide linker. See Kontermann et al (1999) J. Immunol. Methods 226 179-188. Specific tetravalent molecules are available, for example, by fusing an ScFv fragment to the CH3 domain of an IgG molecule or to a Fab fragment across the hinge region. See Coloma et al. (1997) Nature Biotechnol. 15, 159-163. Alternatively, the bispecific tetravalent molecules were created by the fusion of single-chain bispecific diabodies (see Alt et al, (1999) FEBSLett 454, 90-94.) The smaller tetravalent bispecific molecules can also be formed by the dimerization of any ScFv-ScFv-containing tandens with one ligand. helix-loop-helix motif (DiBi mini-antibodies, see Muller et al. (1998) FEBSLett 432, 45-49) or a single chain molecule comprising four antibody variable domains (VH and VL) in an orientation that prevents intramolecular matching ( tandem, see Kipriyanov et al. (1999) J. Mol. Biol. 293, 41-56) Bispecific F (ab ') 2 fragments can be created by chemical coupling of Fab' fragments or by heterodimerization through leucine (see Shalaby et al., (1992) J.Exp. Med. 175, 217-225 and Kostelny et al. (1992), J. Immunol. 148, 1547-1553.) Isolated VH and VL domains are also available ( Domantis plc) .see the U.S. 6,248,516, U.S. 6,291,158 and U.S. 6,172,197.

In one embodiment, an antibody antibody fragment (e.g. ScFv, Fab, Fab ', F (ab') 2 or an engineered antibody antibody fragment as described above, which specifically binds hIL and modulates the interaction between hIL-13 and hIL is provided). -13R, for example when such fragments inhibit the interaction between hIL-13 and its receptor.The antibody fragment may comprise a CDRH3 comprising the sequence of SEQ ID NO: 3, optionally together with other CDRs comprising one or more of the sequences set forth in SEQ ID NOs: 1, 2, 4, 5 and 6.

An ScFv may be produced comprising the VH and VL regions of the antibodies of the present invention. For example, an ScFv may comprise SEQ ID NOs: 12 and 15, or, for example, may comprise SEQ ID NOs: 13 and 15. This may be done by polynucleotides according to the invention, for example the sequences set forth in SEQ ID NOs. : 93 and 94, or, for example, by the sequences set forth in SEQ ID NOs: 28 and 31. In one embodiment of the invention, an ScFv comprising a protein encoded by the sequences set forth in SEQ ID NOs: 93 and 94 is provided.

1.5 Heteroconjugate Antibodies

Heteroconjugate antibodies also form an embodiment of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies formed using any convenient cross-linking methods. See, for example, U.S. 4,676,980.

1.6 Other Modifications

The interaction between the Fc region of an antibody and various Fc receptors (FcyR) is thought to mediate antibody effector functions, including antibody-dependent cellular cytotoxicity (ADCC), complement fixation, phagocytosis, and half-life. antibody clearance. Various modifications to the Fc region of the antibodies of the invention may be performed depending on the desired property. For example, specific mutations in the Fc region to make an otherwise lytic, non-lytic, antibody are detailed in EP 0629 240B1 and EP 0307 434B2, or a salvage receptor binding epitope can be incorporated into the antibody to increase the life span. serum. See U.S. 5,739,277. There are five currently recognized human FcyRh receptors, the FcyR (I), FcyRIIa, FcyRIIb, FcyRIIIae FcRn neonatal. Shields et al. (2001) J. Biol. Chem 276, 6591-6604 have shown that a common set of IgG1 residues is involved in the binding of all FcyRs, while FcyRII and FcyRIII use distinct sites outside this common set. One group of IgG1 residues reduced binding to all FcyRs when altered in alanine: Pro-238, Asp-265, Asp-270, Asn-297 and Pro-239. All are in the IgG CH2 domain and clusters near the hinge joining CH1 and CH2. While FcyRI uses only the common set of IgG1 residues for binding, FcyRII and FcyRIII interact with distinct residues beyond the common set. Alteration of some residues reduced binding only to FcyRII (eg, Arg-292) or FcyRIII (eg, Glu-293). Some variants showed improved binding to FcyRII or FcyRIII, but did not affect binding to another receptor (for example, enhanced Ser-267Ala binding to FcyRII, but binding to FcyRIII was not affected). Other variants show improved binding to FcyRII or FcyRIII with reduced binding to the other receptor (e.g. Ser-298Ala improved binding to FcyRIII and reduced binding to FcyRII). As for FcyRIIIa, the best binding IgGl variants had combined alanine substitutions in Ser- 298, Glu-333 and Lys-334. The neonatal receptor FcRn is believed to be involved in both antibody clearance and tissue transcytosis (see Junghans, R. P (1997) Immunol. Res 16. 29-57 and Ghetie et al (2000) Annu Rev. Immunol 18, 739-766). Human IgG1 residues determined to interact directly with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435. Exchanges at any of these positions described in this Example may enable increased serum half-life and / or altered effector properties of the antibodies of the invention.

Other modifications include the glycosylation variants of the antibodies of the invention. Glycosylation of antibodies at conserved positions in their constant regions is known to have a profound effect on antibody function, particularly the functioning of the effectors such as those described above. See, for example, Boyd et al. (1996), Mol. Immunol. 32, 1311-1318. Antibody glycosylation variants or antigen-binding fragments thereof of the present invention, wherein one or more carbohydrate components are added, substituted, deleted or modified are considered. The introduction of an asparagine-X-serine or asparagine-X-threonine motif creates a potential site for enzymatic articulation of carbohydrate components and can therefore be used to manipulate the glycosylation of an antibody. In Raju et al. (2001) Biochemistry 40, 8868-8876 The terminal sialylation of a TNFR-IgG immunoadhesin was enhanced by a deregalactosylation and / or resialylation process with the use of beta-1,4-galactosyltranspheres and / or alpha-2,3-sialyltransferase. Increasing sialylation is believed to increase the immunoglobulin half-life. Antibodies, in common with most glycoproteins, are typically produced as a mixture of glycoforms. This mixture is particularly evident when anti-antibodies are produced in eukaryotic cells, particularly mammalian ones. A variety of methods have been developed to manufacture defined glycoforms. 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, the invention takes into account a plurality of (monoclonal) antibodies (which may be of the IgG isotype, for example IgG1) as described herein, comprising a defined number (for example 7 or less, for example5 or least, such as two or one) of glycoforms of said antibodies or antigen-binding fragments thereof.

Other embodiments of the invention include antibodies of the invention or antigen binding fragments thereof coupled to a nonproteinaceous polymer such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene. Protein-PEG conjugation is an established technique for increasing protein half-life as well as reducing protein antigenicity and immunogenicity. The use of PEGylation with different molecular weights and styles (linear or ramified) was investigated with intact antibodies as well as Fab 'fragments. See Koumenis, I. L. et al. (2000) Int. J. Pharmaceut. 198: 83-95.

2. Production Methods

Antibodies of the invention may be produced as a polyclonal population, but are more preferably produced as a monoclonal population (i.e. as a substantially homogeneous population of identical antibodies directed against a specific antigen binding site). It will naturally be apparent to those skilled in the art that a population implies more than one antibody entity. Antibodies of the present invention may be produced in transgenic organisms such as goats (See Pollock et al. (1999), J. Immunol. Methods 231: 147-157), Chickens (see Morrow, KJJ (2000) Genet. Eng.News 20 : 1-55), mice (see Pollock et al.) Or plants (see Doran, PM, (2000) Curr. Opinion Biotechnol. 11, 199-204, Ma J. KC (1998), Nat.Med. 4; 601-606, Baez, J. et al., BioFarm (2000) 13: 50-54, Stoger, E. et al. (2000) Plant Mol. Biol. 42: 583-590). Antibodies may also be produced by chemical synthesis. However, the antibodies of the invention are typically produced using the recombinant cell culture technology well known to those skilled in the art. A polynucleotide encoding the antibody is isolated and introduced into a replicable vector, such as a plasmid, for other cloning (amplification) or expression. A useful expression system is a glutamate synthetase system (such as that sold by Lonza Bilogics), particularly when the host cell is CHO or NSO (see below). The polynucleotide encoding the antibody is easily isolated and sequenced using conventional procedures (e.g. oligonucleotide probes). Vectors that may be used include plasmid, virus, phage, transposons, minichromosomes of which plasmids are a typical embodiment. Generally such vectors further include a signal sequence, origin of replication, one or more marker genes, an enhancer element, a promoter, and transcription termination sequences operably linked to light and / or heavy chain polynucleotide in order to facilitate expression. Opolinucleotide encoding the light and heavy chains may be introduced into separate vectors and transfected into the same host cell or, if desired, both the heavy and light chain may be introduced into the same vector for transfection into the host cell. Accordingly, in accordance with one aspect of the present invention. The invention provides a process of constructing a vector encoding the light and / or heavy chains of an antibody or antigen-binding fragment thereof, which method comprises introducing into a vector a polynucleotide encoding an light chain and / or a heavy chain of an antibody. of the invention.

In another aspect of the invention there is provided a polynucleotide encoding a murine VH domain comprising the sequence shown as SEQ ID NO: 25.

In another aspect of the invention, a polynucleotide encoding a murine VL domain comprising the sequence shown as SEQ ID NO: 26 is provided.

In another embodiment, a polynucleotide encoding a VH domain comprising the selected sequence of the group consisting of SEQ ID NOs: 27, 28, 29, 30 is provided.

In another embodiment, a polynucleotide encoding a VL domain comprising the selected sequence of the group consisting of SEQ ID NOs: 31, 32, 33 is provided.

In accordance with the present invention, there is provided a polynucleotide encoding a heavy chain of the invention, polynucleotide selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37.

In accordance with the present invention, there is provided a polynucleotide encoding the light chain of the invention, polynucleotide selected from the group consisting of SEQ ID NOs: 38, 39, 40.

It will be readily apparent to those skilled in the art that, because of the redundancy of the genetic code, alternative polynucleotides to those presented herein (particularly those optimized for codon for expression in a given host cell) are also available which will encode the polypeptides of the invention.

3.1 Signal Sequences

Antibodies of the present invention may be produced as a fusion protein with a heterologous signal sequence having a specific N-terminal cleavage site of the mature protein. The desinal sequence must be recognized and processed by the host cell. As for eukaryotic host cells, the signal sequence may be a conductive heat stable phosphateal alkaline, penicillinase, or enterotoxin II. As for yeast secretion, the signal sequences may be a conductive yeast invertase, a conducting factor or conductive viral phosphatases. (See, for example, WO 90/13646. In mammalian cellular systems, viral secretory conductors such as herpes simplex gD signal and a native immunoglobulin signal sequence are available. Typically the signal sequence is ligated into the matrix. DNA reading encoding the antibody of the invention.

3.2 Source of Replication

Origins of replication are well known in the compBR322 technique suitable for most Gram-negative bacteria, plasmid2μ for most yeast and various viral origins such as SV40, polyoma, adenovirus, VSV or BPV for most mammalian cells. Generally, the origin of replication component is not required for mammalian expression vectors, but the SV40 can be used as long as it contains the premature promoter.

Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example ampicillin, neomycin, methotrexate or tetracycline, or (b) complementary sauxiotrophic deficiencies or nutrient supply not available in complex media. The selection scheme may involve stopping host cell growth. Cells that have been successfully transformed with the genes encoding the antibody of the present invention survive because of, for example, drug resistance conferred by the selection marker. Another example is the so-called DHFR selection marker in which transformants are grown in the presence of methotrexate. In typical embodiments, cells are cultured in the presence of increasing amounts of methotrexate to amplify the copy number of the exogenous gene of interest. CHO cells are a particularly useful cell line for DHFR selection. Another example is the glutamate synthetase expression system (LonzaBiologics). A suitable selection gene for use in yeast is the trpl gene. See Stinchcomb et al. Nature 282, 38, 1979.

3.4 Promoters

Promoters suitable for expressing antibodies of the invention are operably linked to DNA / polynucleotide encoding the antibody. Promoters for prokaryotic hosts include promotorphoA, beta-lactamase and lactose promoter systems, phosphatealcaline, tryptophan, and hybrid promoters such as Tac. Promoters suitable for expression in yeast cells include 3-phosphoglycerate kinase or other glycolytic enzymes, for example enolase, glyceraldehyde 3 phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose 6 phosphate isomerase, 3-phosphoglycerate mutase eglicoquinase. Inducible yeast promoters include alcoholshydrogenase 2, isocytochrome C, acid phosphatase, metallothionein and the enzymes responsible for nitrogen metabolism or the use of maltose / galactose. Promoters for expression in the mammalian cellular systems include viral promoters such as polyoma, epitheliomacontagious and adenovirus (e.g. adenovirus 2), papillomabovine virus, avian sarcoma virus, cytomegalovirus (in particular immediate preterm gene driver), retroviruses, hepatitis B virus, actin, the Rous sarcoma virus (RSV) promoter, and the premature or late simian virus. Of course, the choice of promoter is based on adequate compatibility with the host cell used for expression. In one embodiment, therefore, a first plasmid comprising an RSV and / or SV40 and / or CMV promoter, DNA encoding the light chain V region (VL) of the invention, the kC region together with neomycin resistance selection markers, is provided. and to ampicillin and a second plasmid comprising an RSV or SV40 promoter, DNA encoding the heavy V chain (VH) region of the invention, DNA encoding the γ1 constant region, DHFR, and ampicillin resistance markers.

3.5 Intensifier Element

Where appropriate, for example for higher eukaryotic expression, an enhancer element operably linked to the promoter element in a vector may be used. Suitable mammalian enhancer sequences include the enhancer elements of globin, elastase, albumin, fetoprotein and insulin. Alternatively, an enhancer element from a eukaryotic cell virus such as the SV40 enhancer (at 100 to 270 base pairs), the enhancer of the premature cytomegalovirus promoter, polyoma enhancer, baculoviral enhancer or IgG2 amino locus (see WO 04/009823). The enhancer is preferably located in the vector at a site upstream of the promoter.3.5.5 Polyadenylation Signals

In eukaryotic systems, polyadenylation signals are operably linked to the DNA / polynucleotide encoding the non-inventive antibody. Such signals are typically placed 3 'from the open reading array. In mammalian systems, non-limiting examples include growth hormone derived signals, elongation factor-1 alpha and viral genes (e.g. SV40) or retroviral long terminal repeats. Our yeast systems, non-limiting examples of terminolydenylation / termination signals include those derived from the genes of daphosphoglycerate kinase (PGK) and alcohol dehydrogenase 1 (ADH). In our prokaryotic systems, polyadenylation signals are typically not required and instead it is usual to employ shorter and more defined terminator sequences. Naturally, the choice of termination / termination sequences is based on proper compatibility with the host cell used for expression.

3.6 Host Cells

Suitable host cells for cloning or expressing antibody-encoding vectors of the invention are the prokaryotic, thin or eukaryotic cells. Suitable prokaryotic cells include eubacteria, e.g. Enterobacteriaceae, such as Escherichia, e.g. E. coli (e.g. ATCC 31.446, 31.537, 27.325), Enterobacter, Erwinia, Klebsiella Proteus, Salmonella, e.g. Salmonella tyfimurium, Serratia, e.g. Serratia marcescans and Shigella, as well as Bacilli, such as B. subtilis and B. Iicheniformis (see DD 266 710), Pseudomonas such as P. aeruginosa and Streptomyees. From yeast host cells, Saccharomyees eerevisiae, sehizosaceharomyces pombe, Kluyveromyces (e.g. ATCC 16.045, 12.424, 24.178, 56.500), yarrowia (EP 402.226), Piehia Pastoris (EP 183.070; see also Peng et al. J. Bioteehnol. ) 185-192), Candida, Triehoderma reesia (EP 244,234), Penicillin, Tolypocladium and Aspergillus \ hosts such as A. nidulans and A. nigers are also considered.

Although prokaryotic and yeast host cells are specifically considered by the invention, preferably, however, the host cells of the present invention are superior cellarokaryotic cells. Suitable higher eukaryotic host cells include mammalian cells such as COS-I (ATCC N2CRL 1650) COS-7 (ATCC CRL 1651), Human Embryonic Kidney Line 293 (BHK) (ATCC CRL 1632), BHK570 (ATCC NO: CRL 10314), 293 (ATCC NO. CRL 1573), Chinese CHO hamster cells (e.g. CHO-K1, ATCC NO: CCL 61, DHFR-CHO cell line, such as DG44 [ see Urlaub et al. (1986) Somatic Cell Mol. Genet. 12, 555-556)], particularly those CHO cell lines adapted for suspension culture, mouse Sertoli cells, monkey kidney cells, macacoverde kidney cells African (ATCC CRL-1587), HELA cells, Canine Kidney Cells (ATCC CCL 34), Human Lung Cells (ATCC CCL 75), HepG2 and Myeloma or Lymphoma cells, for example NSO (see US 5,807,715) , Sp2 / .0, YO.

Thus, in one embodiment of the invention, a stably transformed host cell comprising a vector encoding a heavy chain and / or a light chain of the antibody or antigen-binding fragment thereof is provided as described herein. Preferably, such host cells comprise a first vector encoding the light chain and a second vector encoding said heavy chain.

Bacterial Fermentation

Bacterial systems are particularly suitable for expression of antibody fragments. Such fragments are located intracellularly or within the periplasma. Insoluble periplasmic proteins may be extracted and reduplicated to form active proteins according to methods known to those skilled in the art. See Sanchezet there. (1999) J. Biotechnol. 72, 13-20 and Cupit, Ρ. M. et al. (1999) Lett Appl Microbiol, 29, 273-277.

3.7 Methods of Cell Cultivation

Host cells transformed with the vectors encoding the antibodies of the invention or antigen binding fragments thereof may be cultured by any method known to those skilled in the art. Host cells may be grown in conical flasks, round flasks or hollow fiber systems, but it is preferable for large-scale production that stirred tank reactors are used particularly for suspension cultures. Preferably the agitated tanks are adapted for aeration using, for example, low shear sprayers, dampers or impellers. For bubble columns and pneumatic lifting reactors, direct aeration with air or oxygen bubbles can be used. When host cells are grown in a serum-free culture medium, it is preferable that the medium be supplemented with a cell protection agent, such as pluronic F-68, to help prevent cell damage as a result of the aeration process. Depending on the characteristics of the host cells, or microcarriers may be used as growth substrates for the anchor-dependent cell lines, or ascells may be adapted to the suspension culture (which is typical). Cultured host cells, particularly deinvertebrate host cells, may use a variety of operating modes, such as fed mass, repeated mass processing (see Drapeau et al. (1994) eytotechnology 15: 103-109), mass process prolonged or perfusion culture. Although recombinantly transformed mammalian host cells can be cultured in serum-containing medium, such as fetal calf serum (FCS), it is preferable that such host cells are cultured in serum-free synthetic medium as presented in Keen et al. (1995) Cytotechnology 17: 153-163, or commercially available such as ProCHO-CDM or UltraCHO® (CambrexNJ, USA), supplemented, where necessary, with an energy source such as glucose and synthetic growth factors such as insulinarecombinante. Serum free culture of the host cells may require those cells to be adapted to growth under dehydration free conditions. One adaptation approach is to cultivate such serum-containing host cells and repeatedly exchange 80% of the culture medium for the free serum so that the host cells adapt under the free serum conditions (see, for example, Scharfenberg, K. et al. (1995) in Animal Cell technology: Developments Towards the 21st Century (Beuvery, EC et al. Eds.), Pp 619-623, Kluwer Academic Publishers).

Antibodies of the invention secreted in the medium may be recovered and purified using a variety of techniques to provide a degree of purification suitable for the intended use. For example, the use of the antibodies of the invention for treating humanostypically patients determines at least 95% purity, more typically 98% or 99% or more purity (compared to crude culture medium). In the first case, cell debris from the culture medium is typically removed using centrifugation, followed by a supernatant clarification step using, for example, microfiltration, ultrafiltration and / or depth filtration. A variety of other techniques, such as dialysis and gel electrophoresis, and chromatographic techniques such as hydroxyapatite (HA), affinity chromatography (optionally involving affinity labeling system, such as polyhistidine) and / or chromatography of hydrophobic interaction (ICH, see US 5,429,746), is available. In one embodiment, the antibodies of the invention, following various clarification steps, are captured using Protein A or G affinity chromatography, followed by other chromatography steps, such as ion exchange and / or chromatography. HA, anion or cation exchange, size exclusion chromatography and ammonium sulfate appreciation. Typically, various viral removal steps are also employed (e.g. nanofiltration using, for example, a DV-20 filter). Following these various steps, a purified (preferably monoclonal) preparation comprising at least 75 mg / ml or more, for example 100 mg / ml or more, of the invention antibodies or antigen-binding fragments thereof is provided and thus constitutes a embodiment of the invention. Suitably, such preparations are substantially free of aggregated forms of antibodies of the invention.

4. Pharmaceutical Compositions

The purified antibody preparations of the invention (particularly the monoclonal preparations) as described above may be incorporated into the pharmaceutical compositions for use in the treatment of human diseases and disorders, such as atopic diseases, for example asthma, allergic rhinitis, COPD. Typically such compositions comprise a pharmaceutically acceptable carrier as known and required by acceptable pharmaceutical practice. See, for example, Remingtons Pharmaceutical Sciences, 16th edition, (1980), Mack Publishing Co. Examples of such carriers include sterile carrier such as saline, Ringer's solution or dextrose solution, buffered with suitable buffers within a range of Pharmaceutical compositions for injection (e.g. intravenously, intraperitoneally, intradermally, subcutaneously, intramuscularly or intraportally) or continuous infusion are suitably free of visible particulate matter and may comprise from 0.1 ng to 100 mg of antibody, preferably from 5 mg to 100 mg. mg and 25 mg of the antibody. Methods for preparing such pharmaceutical compositions are well known to those skilled in the art. In one embodiment, the pharmaceutical compositions comprise from 0.1 ng to 100 mg of the antibodies of the invention in unit dosage form, optionally together with instructions for use. The pharmaceutical compositions of the invention may be lyophilized (freeze dried) for reconstitution prior to administration according to methods well known or apparent to those skilled in the art. When embodiments of the invention comprise antibodies of the invention with an IgG1 isotype, a copper chelant such as citrate (e.g. sodium citrate), or EDTA or histidine may be added to the pharmaceutical composition to reduce the degree of antibody degradation of this isotype, mediated by copper. See EP 0612251. Anti-hIL-13 treatment may be administered orally, porally, topically (for example by the intraocular, intranasal, backward pathway of skin wounds).

Effective doses and treatment regimens for administering the antibody of the invention are generally empirically determined and depend on factors such as the age, weight and condition of the patient, and the diseases and disorders to be treated. Such factors are sedentary to the attending physician's competence. Guidance in selecting appropriate dosages can be found, for example, in Smith et al. (1977) Antibodies in human diagnosis and therapy, Raven Press, New York, but will generally be between 1 mg and 1000 mg.

Depending on the disease or disorder being treated (but in particular asthma), pharmaceutical compositions comprising a therapeutically effective amount of the antibody of the invention may be used simultaneously, separately or sequentially, with an effective amount of another medicament such as anti-inflammatory agents (e.g. NSAIDs), anticholinergic agents (particularly M1 / M2 / M3 receptor antagonists), β2 adrenoreceptor agonists, anti-infectious agents (eg antibiotics, antivirals), antihistamines, PDE4 inhibitor. Examples of β2 adrenoreceptor agonists include almeterol, salbutamol, formoterol, salmefamol, fenoterol, terbutaline. Preferred long acting β2 adrenoreceptor agonists include those described in WO 02 / 66422A, WO 02/270490, WO 02/076933, WO03 / 0324439 . Suitable corticosteroids include methylprednisolone, prednisolone, dexamethasone, fluticasone propionate, 6a, 9a-difluoro-17a - [(2-furanylcarbonyl) oxide] -11β-hydroxy-16a-methyl-3-oxo-androsta-1-ester 2,4-diene-17β-carbothioic acid, 6α, 9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy acid S- (2-oxo-tetrahydro-furan-3S-yl) ester -androsta-1,4-diene-17β-carbothioic, beclomethasone esters (eg 17-propionate ester or 17,21-dipropionate ester), budesonide, flunisolide, mometasone esters (eg fluroate ester), triamcinolone acetonide, rofleponide, ciclesonide (16α, 17 - [[(R) -cyclohexylmethylene] bis (oxide)] - 11β, 21-dihydroxy pregna-1,4-diene-3,20-dione), butixocort propionate , RPR-106541 and ST-126. Preferred costicosteroids include fluticasone propionate, 6α, 9α-difluoro-11β-hydroxy-16α-methyl-17α - [[4-methyl-1,3-thiazol-S-carbonyl) oxide] -3- oxo-androsta-1,4-diene-17β-carbiotic, the 6α, 9α-difluoro-17α - [(2-furanylcarbonyl) oxide-11β-hydroxy-16α-methyl-2-oxo-androsta acid S-fluoromethyl ester -1,4-diene-17β-carbothioic, more preferable 6α, 9α-difluoro-17α - [(2-furanylcarbonyl) oxide-11β-hydroxy-16α-methyl-2-acid S-fluoromethyl ester 5-fluoromethyl ester oxo-androsta-1,4-diene-17β-carbiotic.

Non-steroidal compounds having glucocorticoid agonism, which may possess selectivity for transrepression through transactivation, and which may be useful in combination therapy, include those covered in the following patents: WO 03/082827, WO 01/10143, WO 98/54159, WO04 / 005229, WO 04/009016, WO 04/009017, WO 04/018429, WO 03/104195, WO 03/082787, WO 03/082280, WO 03/059899, WO 03/101932, WO02 / 02565, WO 01/16128 WO 00/66590, WO 03/086294, WO 04/026248, WO 03/061651, WO 03/08277.

Suitable anti-inflammatory agents include non-steroidal anti-inflammatory drugs (NSAID's).

Suitable NSAIDs include sodium cromoglycate, nedocromil sodium, phosphodiesterase (PDE) inhibitors (e.g. atherophilin, PDE4 inhibitors or mixed PDE3 / PDE4 inhibitors), leukotriene antagonists, leukotriene synthesis inhibitors (eg montelukast ), iNOS inhibitors, tryptase and daelastase inhibitors, beta-2 integrin antagonists and adenosine doreceptor agonists or antagonists (e.g. adenosine 2a agonists), cytokine antagonists (for example chemokine antagonists such as a CCR3) or cytokine synthesis inhibitors, or 5-lipoxygenase inhibitors. Other β2-adrenoreceptor agonists include osalmeterol (e.g. as xinafosto), salbutamol (e.g. as free base), formoterol (e.g. as fumarate), phenbetol outerbutaline and its salts. An iNOS (inducible nitric oxide synthase inhibitor) is preferably for oral administration. Suitable iNOS inhibitors include those set forth in WO 93/13055, WO 98/30537, WO02 / 50021, WO 95/34534 and WO 99/62875. Suitable CCR3 inhibitors include those disclosed in WO 02/26722.

Of particular interest is the use of the antibodies of the invention in combination with a phosphodiesterase 4 inhibitor (PDE4). The specific PDE-4 inhibitor useful in this aspect of the invention may be any compound that is known as a PDE4 enzyme inhibitor or that it finds to act as a PDE4 inhibitor, and which is only a PDE4 inhibitor, not compounds that inhibit other members of the PDE family. , such as PDE3 and PDE5, as well as PDE4.

Compounds of interest include C 1-4-cyano-4- (3-cyclopentyloxy-4-methoxyphenyl) cyclohexane-1-carboxylic acid, 2-carbomethoxy-4-cyano-4- (3-cyclopropylmethoxy-4 (difluoromethoxyphenyl) cyclohexan-1-one and cis- [4-cyano-4- (3-cyclopropylmethoxy-4-difluoromethoxyphenyl) cyclohexan-1-ol].

Also, cw-4-cyano-4- (3-cyclopentyloxy-4-methoxyphenyl) cyclohexane-1-carboxylic acid (also known as cilomilast) and its salts, esters, prodrugs or physical forms which are described in U.S. Patent 5,552,438 issued September 3, 1996. This patent and the compounds it describes are incorporated herein by reference in their entirety.

AWD-12-281 from Elbion (Hofgen, N. et al. 15th EFMC IntSymp Med Chem (September 6-10, Edinburgh) 1998, Summary P.98; CAS Reference No. 247584020-9); a 9-benzyladenominated derivative named NCS-613 (INSERM); D-4418 from Chiroscience and Schering-Plow; a benzodiazepine PDE4 inhibitor identified as CI-1018 (PD-168787) and assigned to Pfizer; a benzodioxol derivative disclosed by Kyowa Hakko in WO 99/16766; K-34 of Kyowa Hakko; V-11294A daNapp (Landells, L.J. et al. Eur Resp J [Annu Cong Eur Resp Soc (19-23 December, Geneva) 1998], 12 (Suppl 28): Summary P2393); roflumilast (CAS reference No. 162401-32-3) and a ptalazinone (WO 99/47505, the contents of which is incorporated herein by reference) by Byk-Gulden; Pumafentrin, (-) - p - [(4aR *, 10òS *) - 9 -ethoxy-1,2,3,4,4a, 1-Ob-hexahydro-8-methoxy-2-methylbenzo [c] [1,6] naphthyridin-6-yl] -N, N-diisopropylbenza-mide, which is a mixed PDE3 / PDE4 inhibitor which was prepared and published by Byk-Gulden, now Altana; arophylline under development by Almirall-Prodesfarma; VM554 / UM565 by Vernalis; or T-440 (Tanabe Seiyaku; Fuji, K. et al. J. Pharmacol Ther, 1998, 284 (1): 162), and T2585.

Other compounds of interest are disclosed in published international patent application WO 04/024728 (Glaxo Group Ltd), PCT / EP2003 / 014867 (Glaxo Group Ltd.) and PCT / EP 2004/005494 (Glaxo Group Ltd).

Suitable anticholinergic agents are those compounds which act as muscarinic receptor antagonists, in particular those compounds which are M1 or M3 receptor antagonists, dual Mi / M3 or M2 / M3 antagonists, Mi / M2 / M3 receptor receptor pan-antagonists . Exemplary compounds for administration by inhalation include ipratropium (eg as CAS 22254-24-6 sold under the name Atrovent), oxytropium (eg as CAS 30286-75-0 bromide) and tiotropium (eg as bromide CAS 136310-93-5, sold under the name ofSpiriva). Also of interest are revatropate (for example as CAS 262586-79-8 hydrobromide) and LAS-34273, which is disclosed in WO01 / 04118. Exemplary compounds for oral administration include apirenzepine (CAS 28797-61-7), darifenacin (133099-04-4, or CAS133099-07-7 for hydrobromide sold as Enablex), oxybutynin (CAS 5633-20- 5, sold under the name Ditropan), terodiline (CAS 15793-40-5), tolterodine (CAS 124937-51-5, or CAS124937-52-6 for tartrate, sold under the name Detrol), otilonium bromide CAS 26095-59-0 sold under the name Spasmomen), trospium chloride (CAS 10405-02-4) and solifenacin (CAS242478-37-1, or CAS 242478-38-2 for succinate). known as YM-905 and sold under the name Vesicare).

Other suitable anticholinergic agents include the compounds of formula (XXI) which are disclosed in U.S.60 / 487981: <formula> formula see original document page 68 </formula>

wherein the preferred orientation of the articulated alkyl chain to the tropane ring is;

R31 and R32 are independently 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, cycloalkyl alkyl having from 6 to 10 carbon atoms, 2-thienyl, 2-pifidyl, phenyl, phenyl substituted by an alkyl group having no more than 4 carbon atoms, and phenyl substituted by an alkoxy group having no more than 4 carbon atoms;

X "represents an anion associated with the positive charge of atom Ν. X" may be, but is not limited to, chloride, bromide, iodide, sulfate, benzene sulfonate and toluene sulfonate, including, for example:

(3-eO) -3- (2,2-di-2-thienylethyl) -8,8-dimethyl-8-azoniabicyclo- [3.2.1] octane bromide;

(3-e (R)) -3- (2,2-diphenylethenyl) -8,8-dimethyl-8-azoniabicyclo [3.2.1] octane bromide;

(3-era / o) -3- (2,2-diphenylethenyl) -8,8-dimethyl-8-azoniabicyclo [3.2.1] octane 4-methylbenzenesulfonate;

(3-E6) -8,8-dimethyl-3- [2-phenyl-2- (2-thienyl) ethenyl] -8-azoniabicyclo- [3.2. ljoctane; and / or

(3-e / o) -8,8-dimethyl-3- [2-phenyl-2- (2-pyridinyl) ethenyl] -8-azonia-bicyclo [3.2.1] octane bromide.

Other suitable anticholinergic agents include the compounds of formulas (XXII) or (XXIII), which are presented in U.S. Patent Application No. 60/511009: <formula> formula see original document page 69 </formula>

on what

the indicated H atom is in the exo position;

R41- represents an anion associated with the positive charge of the atom of N. RI "may be, but is not limited to, chloride, bromide, iodide, sulfate, benzene sulfonate and toluene sulfonate;

consisting of straight or branched lower alkyl groups (preferably from 1 to 6 carbon atoms), cycloalkyl groups (having from 5 to 6 carbon atoms), cycloalkylalkyl (having from 6 to 10 carbon atoms), heterocycloalkyl (having from 5 to 6 carbon atoms) and N or O as the heteroatom, heterocycloalkylalkyl (having from 6 to 10 carbon atoms) and N or O as the optionally substituted heteroatom, aryl, aryl, heteroaryl and heteroaryl;

C3 -C12 cycloalkyl, C3 -C7 heterocycloalkyl, C1 -C6 alkyl-C3 -C12 cycloalkyl, C1 -C6 alkyl-C3 -C7 heterocycloalkyl, aryl, heteroaryl, C1 -C6 alkyl, C1 -C6 alkyletheriyl, -OR45, - CH2OR45, -CH2OH, -CN, -CF3, -CH2O (CO) R46, -CO2R47, -CH2NH2, -CH2N (R47) SO2R45, -SO2N (R47) 5 -CON (R47) (R48) 5 - CH2N (R48) CO (R46), -CH2N (R48) SO2 (R46) 5CH2N (R48) CO2 (R45) 5-CH2N (R48) CONH (R47);

C 1 -C 6 cycloalkyl C 3 -C 12, C 1 -C 6 alkyl-C 3 -C 7 heterocycloalkyl, C 1 -C 6 alkyl-aryl, C 1 -C 6 alkyl-heteroaryl;

C 3 -C 12 cycloalkyl, C 3 -C 7 heterocycloalkyl, C 1 -C 6 alkyl-C 3 -C 12 cycloalkyl,

R42 and R43 are independently selected from the group.

R44 is selected from the group consisting of C1 -C6 alkyl,

R45 is selected from the group consisting of C1 -C6 alkyl, alkyl

R46 is selected from the group consisting of C1 -C6 alkyl, C1 -C6 alkyl-C3 -C7 heterocycloalkyl, aryl, heteroaryl, C1 -C6 alkyl-aryl, C1 -C6 alkylheteroaryl;

R47 and R48 are independently selected from the group consisting of H, C1 -C6 alkyl, C3 -C12 cycloalkyl, C3 -C7 heterocycloalkyl, C1 -C6 alkylcycloC3 cycloalkyl, C3 -C7 alkyl heterocycloalkyl C6-aryl and C1-C6-alkyl heteroaryl, including, for example:

(Endo) -3- (2-methoxy-2,2-di-thiophen-2-yl-ethyl) -8,8-dimethyl-8-azoniabicyclo [3.2.1] octane iodide; Endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenylpropionitrile;

(Endo) -8-methyl-3- (2,2,2-triphenyl-ethyl) -8-aza-bicyclo [3.2.1] octane;

3 - ((Endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenylpropionamide;

3 - ((Endo) 8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenyl-propionic acid;

(Endo) -3- (2-cyano-2,2-diphenyl-ethyl) -8,8-dimethyl-8-azonia-bicyclo-iodide [3.2. 1] octane;

(Endo) -3- (2-cyano-2,2-diphenyl-ethyl) -8,8-dimethyl-8-azonia-bicyclo- [3.2. 1] octane;

3 - ((Endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenyl-propan-1-ol;

A-benzyl-3 - ((endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenylpropionamide;

(Endo) -3- (2-carbamoyl-2,2-diphenyl-ethyl) -8,8-dimethyl-8-azonia-bicyclo [3.2.1] octane iodide; 1-benzyl-3- [3- ( (endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenyl-propyl] -urea;

1-ethyl-3- [3 - ((endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenyl-propyl] -urea;

7V- [3 ((endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenyl-propyl] acetamide;

N- [3 ((endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenyl-propyl] benzamide;

3 - ((endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-di-thiophen-2-yl-propionitrile;

(Endo) -3- (2-cyano-2,2-di-thiophen-2-yl-ethyl) -8,8-dimethyl-8-azonia-bicyclo [3.2.1] octane iodide;

N- [3 - ((endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenyl-propyl] -benzenesulfonamide;

[3 - ((endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenyl-propyl] -urea;

N-3 - ((endo) -8-methyl-8-aza-bicyclo [3.2.1] oct-3-yl) -2,2-diphenyl-propyl] -methanesulfonamide; and / or

(Endo) -3- {2,2-diphenyl-3 - [(1-phenyl-methanoyl) -amino] -propyl} -8,8] -dimethyl-8-azonia-bicyclo [3.2.1] octane bromide .

More preferred compounds useful in the present invention include:

(Endo) -3- (2-Methoxy-2,2-di-thiophen-2-yl-ethyl) -8,8-dimethyl-8-azoniabicyclic iodide [3.2. 1] octane;

(Endo) -3- (2-cyano-2,2-diphenyl-ethyl) -8,8-dimethyl-8-azonia-bicyclo [3.2.1] octane iodide;

(Endo) -3- (2-cyano-2,2-diphenyl-ethyl) -8,8] dimethyl-8-azonia-bicyclo bromide [3.2. 1] octane;

(Endo) -3- (2-carbamoyl-2,2-diphenyl-ethyl) -8,8-dimethyl-8-azonia-bicyclo [3.2.1] octane iodide;

(Endo) -3- (2-cyano-2,2-di-thiophen-2-yl-ethyl) -8,8-dimethyl-8-azoniabicyclo [3.2.1] octane iodide; and / or

(Endo) -3- {2,2-Diphenyl-3 - [(1-phenyl-methanoyl) -amino] -propyl} -8,8-dimethyl-8-azonia-bicyclo [3.2.1] octane bromide. Suitable antihistamines (also referred to as H1 receptor antagonists) include any or more of the numerous known antagonists that inhibit H1 receptors, and are safe for human use. First generation antagonists include derivatives of ethanolamines, ethylenediamines and alkylamines, for example diphenylhydramine, pyrilamine, clemastine, chloropheniramine. Second generation antagonists, which are non-sedative, include loratidine, desloratidine, terfenadine, astemizole, acrivastine, azelastine, levocetirizine, fexofenadine and cetirizine.

Examples of preferred antihistamines include loratidine, desloratidine, fexofenadine and cetirizine.

Other combinations considered include the use of antibodies of the invention in combination with an anti-IL-4 agent (for example anti-IL-4 antibody such as pascolizumab) and / or anti-IL-5 agent (for example anti-IL-4 antibody). -IL-5, such as mepolizumab) and / or anti-IgE agent (for example, anti-IgE antibody such as omalizumab (Xolair®) outalizumab).

Conveniently, a pharmaceutical composition containing a kit of parts of the antibody of the invention or antigen-binding fragment thereof, together with such other drugs, optionally together with instructions for use, is also contemplated by the present invention.

The invention furthermore contemplates a pharmaceutical composition comprising a therapeutically effective amount of the monoclonal therapeutic antibody or antigen-binding fragment thereof as described herein for use in the treatment of diseases responsive to modulating the interaction between hIL-13 and hIL-13R.

According to the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a humanized monoclonal therapeutic antibody, an antibody comprising a VH domain selected from the group consisting of: SEQID NOs: 11, 12, 13, 14, and a selected VL domain from the group consisting of: SEQ ID NO: 15, 16, 17.

In accordance with the present invention there is provided a pharmaceutical composition comprising a monoclonal antibody comprising a heavy chain selected from the group consisting of: SEQ IDNOs: 18, 19, 20, 21, and a light chain selected from the group consisting of: SEQ ID NOs: 22, 23, 24.

According to the present invention there is provided a pharmaceutical composition comprising a monoclonal antibody comprising a heavy chain of SEQ ID NO: 18 and a light chain of SEQID NO: 22, and a pharmaceutically acceptable carrier.

According to the present invention there is provided a pharmaceutical composition comprising a monoclonal antibody comprising (or consisting essentially of) a heavy chain of SEQ IDNO: 18 and a light chain of SEQ ID NO: 22, and a pharmaceutically acceptable carrier.

According to the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a monoclonal antibody population, which antibody comprises a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO: 22.

According to the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a monoclonal antibody population, which antibody comprises a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO: 23.

According to the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a monoclonal antibody population, which antibody comprises a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO: 24.

According to the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a monoclonal antibody population, which antibody comprises a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 22.

According to the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a monoclonal antibody population, which antibody comprises a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 23.

According to the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a monoclonal antibody population, which antibody comprises a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 24.

According to the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a monoclonal antibody population, which antibody comprises a heavy chain of SEQ ID NO: 20 and a light chain of SEQID NO: 22.

According to the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a monoclonal antibody population, which antibody comprises a heavy chain of SEQ ID NO: 20 and a light chain of SEQ ID NO: 24.

According to the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a monoclonal antibody population, which antibody comprises a heavy chain of SEQ ID NO: 21 and a light chain of SEQ ID NO: 22.

According to the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a monoclonal antibody population, which antibody comprises a heavy chain of SEQ ID NO: 21 and a light chain of SEQ ID NO: 23.

According to the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a monoclonal antibody population, which antibody comprises a heavy chain of SEQ ID NO: 21 and a light chain of SEQ ID NO: 24.

5. Clinical Uses

Antibodies of the invention may be used in the treatment of atopic diseases / disorders and chronic inflammatory diseases / disorders. Of particular interest is its use in the treatment of asthma, such as allergic asthma, particularly severe asthma (ie, asthma that is not responsive to current treatment, including systemically administered corticosteroids; see Busse, WW et al., J Allergy Clin. Immunol 2000 , 106: 1033-1042), "difficult" asthma (defined as the asthmatic phenotype characterized by failure to achieve control despite prescribed maximum doses of inhaled steroids; see Barnes, PJ (1998), Eur Respir J 12: 1208-1218 ), "fragile" asthma (defines a subset of severely stable asthma patients who maintain wide peak expiratory flow variability (PEF) despite high doses of inhaled steroids; see Ayres, JG et al. (1998) Thorax 58: 315-321), nocturnal asthma, premenstrual asthma, steroid-resistant asthma (see Woodcock, AJ (1993) Eur Respir J 6: 743-747), steroid-dependent asthma (defined as asthma that can be controlled only with high doses of oral steroids, adult onset asthma, asthma pediatric. Antibodies of the invention may be used to prevent, reduce frequency or mitigate the effects of acute asthmatic episodes (statusasthmaticus). Antibodies of the invention may also be used to reduce the required dosage (or in terms of amount administered or dosage frequency) of other medicaments used in the treatment of asthma. For example, the antibodies of the invention may be used to lower the dosage required for the treatment of asthma steroids, such as corticosteroid treatment ("steroid reduction". Other diseases or disorders that may be treated with the antibodies of the invention include atopic adhesmatitis, 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), hepatic fibrosis, hepatic granulomas, schistosomiasis leishmaniasis, and cell cycle regulatory disorders, for example Hodgkins disease, chronic B-cell lymphocytic leukemia. Other diseases or disorders that can be treated with the antibodies of the invention are detailed in the Example above of the Invention Background.

In one embodiment of the invention, there is provided a method of treating a human patient afflicted with an asthmatic condition that is refractory to corticosteroid treatment, which method comprises administering to said patient a therapeutically effective amount of an antibody of the invention.

In another embodiment, a method of preventing an acute asthmatic attack in a human patient is provided, which method comprises the step of administering to said patient a therapeutically effective amount of an antibody of the invention.

In another embodiment, a method of decreasing the frequency and / or mitigating the effects of an acute asthmatic attack on a human patient is provided, which method comprising the step of administering said patient to a therapeutically effective amount of an anti-antibody of the invention.

In another embodiment of the invention there is provided a method of tending the T helper cell response to a Thl-like response following an inflammatory and / or allergic insult in a human patient, which method comprises administering to said patient a therapeutically effective amount of an antibody or antigen-deleting fragment thereof of the invention.

In another embodiment of the invention, there is provided a method of treating a human patient having variant Q130hIL-13, a patient being afflicted with asthma, such as severe asthma, said method comprising the step of administering to said patient an amount therapeutically effective antibody or antigen-deleting fragment thereof of the invention.

Although the present invention has been described primarily with respect to the treatment of human disease or disorder, the present invention may also have applications in the treatment of similar disease or disorder in nonhuman mammals.

The present invention will now be described by way of example only.

Brief Description Of Drawings

FIGURE 1

Sandwich ELISA illustrating binding of mouse monoclonal 3G4 antibody to human IL-13 expressed in E. colirecombinant at increasing concentrations.

FIGURE 2a

Neutralization assay illustrating the ability of mouse monoclonal 3G4 antibody to increase concentrations to inhibit abioactivity of recombinant human IL-13 expressed in E. coli in a TF-1 cell proliferation assay.

FIGURE 2b

Neutralization assay illustrating the ability of chimeric 3G4 to increase concentrations to inhibit the bioactivity of recombinant human IL-13 expressed in E. coli in a TF-1 cell proliferation assay.

FIGURE 3

Neutralization assay illustrating the ability of mouse monoclonal (and chimeric 3G4) 3G4 antibody to increase concentrations to inhibit the bioactivity of E. coli-expressed cinomolgos IL-13 in a TF-1 cell proliferation assay. The 'Campath' labeled curve is that obtained using the humanized anti-CD52 antibody Germantuz, acting as a relevant antibody control in this experiment. The line labeled 'anti-hIL13 poly' is that obtained using a neutralizing polyclonal anti-IL13 preparation (R & DSystems, catalog number AF-213-NA) as a positive control.

FIGURE 4

Neutralization assay illustrating the ability of mouse monoclonal (and chimeric 3G4) 3G4 antibody at increasing concentrations to inhibit the bioactivity of mammalian expressed human IL-13 (CHO cell) in a TF-1 cell proliferation assay. Campath and anti-hIL13 are control reagents as described above.

FIGURE 5

Neutralization assay illustrating the ability of mouse monoclonal (and chimeric 3G4) 3G4 antibody at increasing concentrations to inhibit the bioactivity of recombinant E. coli expressed human IL-13 Q130 in a TF-1 cell proliferation assay. Campath eanti-hIL13 are reagents as described above.

FIGURE 6

An epitope mapping ELISA to determine the binding epitope for mouse monoclonal 3G4 antibody on human and cynomolgus aIL-13.

FIGURE 7a

An epitope mapping ELISA to identify the fine binding specificity of mouse monoclonal 3G4 antibody over human IL-13.

FIGURE 7b

An epitope mapping ELISA to identify the fine-binding specificity of mouse monoclonal 3G4 antibody over cinomolgus IL-13.

FIGURE 8

An epitope mapping ELISA to determine the key amino acid residues required for binding of mouse monoclonal 3G4 antibody to human IL-13.

FIGURE 9

An epitope mapping ELISA to determine the key amino acid residues required for binding of mouse monoclonal 3G4 antibody to human IL-13.

FIGURE 10

Sandwich ELISA illustrating binding of mouse 3G4, H2L1, H3L1 mouse anti-mouse mAbs antibodies to recombinant human IL-13 expressed in E. coli at increasing concentrations.

FIGURE 11

Neutralization assay illustrating the ability of 3G4, H2L1, H3L1 mouse anti-clonoclonal antibodies to 3G4 humanized chimeric mAbs at increasing concentrations to inhibit the recombinant human E. coli-expressed bioactivity in a TF-1 cell proliferation assay.

FIGURE 12

Neutralization assay illustrating the ability of 3G4, H2L1, H3L1 mouse anti-clonoclonal antibodies to 3G4 humanized chimeric mAbs at increasing concentrations to inhibit the recombinant cinomolg-expressed IL-13 bioactivity in a TF-1 cell deproliferation assay.

FIGURE 13

Neutralization assay illustrating the ability of the 3G4, H2L1, H3L1 mouse anti-mouse monoclonal antibody of chimeric 3G4 humanized mAbs at increasing concentrations to inhibit mammalian expressed human IL-13 (CHO cells) bioactivity in a TF-1 cell proliferation assay.

FIGURE 14

Neutralization assay illustrating the ability of the mouse 3G4, H2L1, H3L1 mouse anti-clonoclonal chimeric 3G4 human mAbs at increasing concentrations to inhibit the bioactivity of human E. coli expressed Q130 in a TF-1 cell proliferation assay.

FIGURE 15

Sandwich ELISA demonstrating that mouse monoclonal antibody 3G4, H2L1, H3L1 from chimeric 3G4 humanized mAbs, do not bind to recombinant human IL-4 expressed in E.coli.

FIGURE 16

Sandwich ELISA demonstrating that mouse monoclonal 3G4 antibody, H2L1, H3L1 from 3G4 human chimeric mAbs, do not bind to recombinant human IL-5 expressed in E.coli.

FIGURE 17

Direct binding ELISA demonstrating that mouse monoclonal antibody 3G4, H2L1, H3L1 from 3G4 humanized chimeric mAbs, do not bind human GM-CSF.

FIGURE 18

Sandwich ELISA illustrating the binding of mouse monoclonal 3G4 antibody, H2L1, H3L1 to humanized chimeric 3G4 mAbs, to native human IL-13 in increasing concentrations.

FIGURE 19

ELISA illustrating the ability of mouse monoclonal antibody 3G4, H2L1, H3L1 to chimeric and humanized 3G4 mAbs, in increasing concentrations to inhibit binding of recombinantly expressed human IL-13 in E. coli to the human IL-13 receptor Î ± 1 chain.

FIGURE 20

ELISA illustrating the ability of mouse monoclonal antibody 3G4, H2L1, H3L1 to chimeric and humanized 3G4 mAbs, in increasing concentrations to inhibit the binding of recombinantly expressed human IL-13 in E. coli to the human IL-13 receptor a2 chain.

Examples

1. Generation of monoclonal antibodies and characterization of mouse monoclonal3G4 antibody

Five SJL mice were immunized by intraperitoneal injection, each with 2 μg of E. coli derived recombinant human IL-13 (Cambridge Bioscience, Cat. No. CH-013). Mouse splenic cells were removed and B lymphocytes were fused with mouse myeloma cells derived from P3X cells using PEG1500 (Boehringer) to generate hybridomas. Individual hybridoma cell lines were cloned by limiting dilution (E. Harlow and D.Lane). Reservoirs containing single colonies were microscopically identified and supernatants tested for activity. Cells from most active clones have been expanded for cryopreservation, antibody production, etc. Initially, the hybridoma supernatants were screened for binding activity against a recombinant det-1 labeled human IL-13 protein expressed in E. coli (produced locally) in a sandwich assay format. A secondary screening of these positive was completed using a BIAcore® method for detecting binding to human IL-13 labeled det-1 protein. Samples of these hybridomas were then tested for the ability to neutralize the abioactivity of recombinant human IL-13 expressed in E. coli (Cambridge Bioscience, cat. No. CH-013) in a TF-1 cell bioassay.

Identified positives from the human IL-13 neutralizing bioassay were subcloned by limiting dilution to generate stable monoclonal cell lines. The immunoglobulins of these hybridomas, cultured in cell factories under serum-free conditions, were purified using immobilized Protein A columns. These mAbs were then re-screened in the same three assay systems.

The monoclonal 3G4 antibody was identified as a potent antibody that neutralized the bioactivity of human IL-13.

The 3G4 antibody has the amino acid sequence of the Vha region shown in SEQ ID NO: 7,

The 3G4 antibody has the Vla region amino acid sequence shown in SEQ ID NO: 8,

A chimeric antibody was constructed by taking the variable regions of the murine monoclonal antibody 3G4 and grafting them into human IgG1 / k wild type C regions. A human signal sequence (as shown in SEQ ID NO: 43) was used in the development of these constructs. This chimeric antibody was named 3G4C.

1.1 Binding to recombinant human IL-13 expressed in E. coli

Recombinant human IL-13 expressed in 3G4-linked E. coli in a sandwich ELISA; The method was performed as described in Example 6.1 (See Figure 1).

1.2 Neutralization of human IL-13 and recombinantly expressed cinomolgus in E. coli in a TF-I cell proliferation bioassay

TF-I cells proliferate in response to human IL-13 and cinomolgus IL-13. A bioassay was developed to evaluate the neutralizing ability of an anti-IL-13 mAb on proliferation of human IL-13 and cinomolgus-induced TF-I cells. The method was performed as described in Example 6.2.

The amino acid sequence and a cinomolgo paraIL-13 cDNA sequence (not including the signal sequence) are presented as SEQ ID NO: 41 and SEQ ID NO: 42, respectively.

3G4 neutralized the bioactivity of recombinant human IL-13 in a TF-I cell bioassay (see Figure 2a).

3G4 neutralized the bioactivity of cinomolgo IL-13 less potentially than human IL-13 (see Figure 3).

An average ND50 value of 0.13 μg / ml was calculated for the neutralization of approximately 10 ng / ml bioactivity of E. coli expressed humanarecombinant IL-13 in a TF-I cell bioassay by the monoclonal 3G4 antibody.

An ND50 value of 29 μg / ml was calculated to neutralize approximately 10 ng / ml bioactivity of recombinant decinomolgous IL-13 expressed in E. coli in a TF-I cell bioassay by the monoclonal 3G4 antibody. [The ND50 (neutralizing dose) value is the concentration of monoclonal antibody needed to reduce TF-I cell proliferation by 50% in response to an established IL-13 concentration].

1.3 Neutralization of human IL-13 (CHO cell) expressed in mammals in a TF-I cell proliferation bioassay

The neutralizing ability of the monoclonal 3G4 antibody to human IL-13 expressed from CHO cells was evaluated in a TF-I cell proliferation assay. The method was performed as described in Example 6.2. Mammalian expressed human IL-13 neutralized 3G4 more potentially than a commercially available anti-human IL-13 polyclonal reagent. An ND 50 value of 0.31 µηη was calculated for neutralization of -20 ng / ml of human IL-13 expressed in mammals without TF-I cell bioassay by monoclonal 3G4 antibody. See Figure 4.

1.4 Neutralization of recombinant human IL-13 Q130 variant in a TF-I cell proliferation bioassay

The neutralizing capacity of the monoclonal 3G4 antibody for recombinant human E. coli expressed Q130 IL-13 (Peprotech, Cat.ns 200-13A) was evaluated in a TF-1 cell proliferation assay. The method was performed as described in Example 6.2. 3G4 neutralized human IL-13 Q130 more potentially than a commercially available anti-human IL-13 polyclonal reagent. An ND50 value of 0.025 μg / ml was calculated for the neutralization of -60 ng / ml bioactivity of human IL-13 Q130 in a TF4 I cell bioassay by 3G4monoclonal antibody. See Figure 5.

1.5 BIAcore® Analysis

The affinity of mouse 3G4 mAb for recombinant human and cinomolgus IL-13 was assessed by BIAcore® (surface plasmon resonance) analysis. See Table 2.

BIAcore® analyzes were performed using anti-mouse IgG capture. An anti-mouse IgG antibody was coupled to a CM5 fragment by primary amine coupling according to the manufacturers' recommendation. Parental mouse mAb3G4 was then captured on this surface and human IL-13 or decinomolgus passed at the defined concentrations. The surface was regenerated back to the anti-mouse IgG surface using mild acid-eluting conditions, and this did not significantly affect the ability to capture antibody for a subsequent IL-13 binding event. This work was performed on the Biacore 3000 and analyzed using the machine's inherent evaluation program, and the data were analyzed using the 1: 1 binding model. Data on the binding of human IL-13 were generated using the labeled recombinant human Det-I-expressed IL-13 in E. coli as well as as a reagent of unlabelled human-recombinant human IL-13 expressed in E. coli ( provided by Peprotech). Recombinant cinomolg AIL-13 expressed in E. coli was generated at GSK. All Biacore developments were performed at 25 degrees C.

Table 2. Biacore 3000 data for the binding of 3G4 parental mouse mAb to human IL-13 and cinomolgus

<table> table see original document page 85 </column> </row> <table>

For human IL-13 labeled Det-1, data were derived from 2 independent experiments with both developments performed in duplicate. Data are presented as the mean and standard deviation (in parentheses) of these results.

For human IL-13 (provided by Peprotech) and IL-13 decinomolgo (produced by GSK), the data are the result of a duplicate experiment.

As for the data described above, duplicates were analyzed together to give value for development. These data indicate that the 3G4 mouse mAb has a very high binding affinity for human IL-13, the deletion affinity of the 3G4 mouse mAb for Cinomolgus IL-13 being less powerful in comparison.

2. 3G4 CLONE HUMANIZATION

2.1 Sequence Analysis

A comparison was made between 3G4 variable region sequences and other murine and human immunoglobulin sequences. This was done using the FASTA and BLAST programs and by inspection.

The following framework residues in 3G4 have been identified as potentially important in the design of a CDR (humanized) grafted version of the antibody:

3G4 VH Position

10 D

30 I

93 T

The position is in accordance with the numbering system of Kabat et al.

3G4 VL Position

98 L

A suitable human acceptor structure for 3G4 Vh has been identified: SEQ ID NO: 44

A suitable human acceptor structure for 3G4 Vl has been identified: SEQ ID NO: 45

In SEQ ID NO: 44, CRDH1 and H2 are present and CDRH3 is represented by XXXXXXXXXX. In SEQ ID NO: 45, CRDL1 and L2 are present and CDRL3 is represented by XXXXXXXXX.

In CDR grafting, it is typical to require one or more donor antibody framework residues to be included in place of their pathologists in the acceptor frameworks in order to achieve satisfactory binding. In addition to those listed above, the following murine framework residues were also considered for possible retention in a humanized antibody project:

<table> table see original document page 87 </column> </row> <table>

4 humanized Vh constructs with different back mutations were designed to obtain a humanized antibody with satisfactory activity. These are numbered from H1 to H4.

H1 consists of a CDR graft of the 3G4 VH CDRs in the specified acceptor sequence. Additionally, H1 contains the murine residue at position 30 (isoleucine). This is outside of the CDT Kabat definition, but within Chothia's CDR Hl definition. In this case, this residue is considered to be part of the CDR, rather than a true backtracking of the structure.

H2 is identical to H1, but with a back mutation where the amino acid at position 93 is threonine, rather than alanine. H3 is identical to H2, but with additional retromutation where the amino acid at position 10 is aspartic acid rather than glutamic acid.Η4 is identical to H3 but contains four additional back mutations at positions 67 (alanine instead of valine) 69 (leucine in place of methionine), 71 (alanine in place of arginine) and 73 (lysine in place of detreonine).

3 humanized V1 constructs with different back mutations were designed to obtain a humanized antibody with satisfactory activity. These are numbered from L1 to L3.

L1 consists of a CDR graft of the 3G4 V1 CDRs at the acceptor sequence specified using the CDR Kabat definition.

L2 is identical to L1, but with a back mutation where amino acid at position 98 is leucine in place of phenylalanine.

L3 is identical to L2, but with an additional back mutation where the amino acid at position 76 is asparagine in place of serine.

Humanized Vh Hl ConstructionSEQ ID NO: 11

Humanized Vh H2 ConstructionSEQ ID NO: 12

Humanized Vh Construction H3SEQ ID NO: 13

Humanized Vh Construction H4SEQ ID NO: 14

Humanized Vh Ll ConstructionSEQ ID NO: 15

Humanized Vl Construction L2SEQ ID NO: 16

Humanized Vl Construction L3SEQ ID NO: 17

2.2 Humanization of 3G4

Humanized Vh and Vl constructs were prepared by de novo formation of overlapping oligonucleotides including restriction sites for cloning into Rld and R1n mammalian expression vectors, as well as a human signal sequence. Hind III and Spe I sites were introduced to form the Vh domain containing the human signal sequence (SEQ ID NO: 43) to clone into Rld containing the human γΐ type constant region. Hind III and BsiW I restriction sites were introduced to form domain V1 containing the human signal sequence for cloning into R1n containing the human kappa constant region. This is essentially as described in WO 2004/014953.

3. Expression and characterization of humanized antibodies

Humanized Vh constructs (H1, H2, H3 and H4) and two humanized V1 constructs (L1 and L2) were prepared in the mammalian expression vectors Rld hCylwt and Rln hCic. The heavy chain-light chain plasmid combinations (H1L1, H1L2, H2L1, H2L2, H3L1, H3L2, H4L1, H4L2) were transiently cotransfected into CHO cells and expressed on a small scale to give eight different humanized antibodies.

Antibodies produced in the CHO cell supernatant and subsequent purified antibody batches were analyzed for activity in human IL-13 binding ELISA, human IL-13 neutralization bioassay, and human IL-13 binding by BIAcore®. the eight humanized mAbs showed binding and / or neutralization of human IL-13 in each of these assays. H2L1 and H3L1 were selected for further analysis because of the better performance in human IL-13 neutralization bioassay, and binding to human IL-13 by BIAcore®, while offering a limited number of retromutations.

3.1 Binding to recombinant human IL-13 expressed in E. coli

Recombinant human IL-13 expressed in 3G4C-linked E. coli, H2L1 and H3L1 in a sandwich ELISA with similar profiles. The method was performed as described in Example 6.1A. Table 3 presents average ED50 values (see also Figure 10). [ED50 (effective dose) is antibody concentration required for maximal half-binding to IL-13 in this ELISA]

Table 3

<table> table see original document page 90 </column> </row> <table>

3G4, 3G4C, H2L1 and H3L1 have also been shown to inhibit binding of human IL-13 to human IL-13 receptor chains (IL13Rα1 and IL13Rα2) by ELISA. This method was performed as descriptors Examples 6.5 and 6.6.

Figure 19 presents data demonstrating inhibition of IL13Ral binding.

Table 4 shows the mean IC 50 values for inhibition of EL13Ra2 binding. [IC50 is the concentration of mAb required to inhibit binding of a fixed amount of human IL-13 to IL13Rα2 by 50%].

Table 4

<table> table see original document page 90 </column> </row> <table>

Figure 19 illustrates that 3G4, 3G4, H2L1 and H3L1 inhibit human IL-13 binding to similarly potent IL13Ral.

Figure 20 illustrates that 3G4, 3G4C, H2L1 and H3L1 inhibit human IL-13 binding to similarly potent IL13Rα2.3.2 Binding to native human IL-13

The HDLM-2 cell line (a Reed-Steinberg-like cell line originally derived from the bone marrow) produces human IL-13 and uses it in an autocrine form for growth. This native human IL-13 is secreted into the supernatant of HDLM-2 cells. This was used to evaluate the binding of 3G4, 3G4C, H2L1 and H3L1 to native human IL-13 using a method described in Example 6.1B. By ELISA, all four antibodies bound to native human IL-13 in HDLM2 cell supernatant with performance very similar to that of parental 3G4 mAb. See Figure 18.

3.3 Neutralization of human IL-13 and recombinantly expressed cinomolgus in E. coli in a TF-I cell proliferation bioassay.

3G4, 3G4C, H2L1 and H3L1 neutralized the bioactivity of human IL-13 and recombinant cinomolgus expressed in E. coli. The method was performed as described in Example 6.2A.

3G4, 3G4C, H2L1 and H3L1 neutralized the bioactivity of recombinant cinomolgus IL-13 expressed in E. coli less potentially than human IL-13 [ND5o (neutralization dose) is mA open concentration to reduce TF-cell proliferation I by 50% in response to an established IL-13 concentration].

See Table 5 below and Figures 11 and 12.

Table 5

<table> table see original document page 91 </column> </row> <table>

The mean ND50 value for 3G4 was 0.049 μ§ / ηι1 calculated for neutralization of approximately 10 ng / ml bioactivity of recombinant human IL-13 expressed in E. coli in the TF-1 assay. Results in Table 5 are the average of four separate repetitions. The value obtained is comparable, although approximately twice less than the previously obtained DNA value (see Example 1.2).

The neutralization level of human IL13 expressed in E. coliobeptide to parental 3G4 mouse mAb is comparable to that obtained by chimeric 3G4. The potencies of H2L1 and H3L1 were reduced compared to both parental 3G4 and chimeric 3G4 mouse mAb.

The average ND5O value for parental 3G4 is 34 μg / ml. It was calculated for the neutralization of approximately 10 ng / ml of the bioactivity of recombinant cinomolg IL-13, expressed in δ. coli. This value was similar to the previously reported value for parental 3G4 (see Example 1.2). H2L1 and H3L1 also showed similar potency against IL13 of cinomolgus as 3G4.

3.4 Neutralization of human IL-13 (CHO cell) expressed in mammalian in a TF-1 cell proliferation bioassay.

The neutralizing capacity of monoclonal antibodies G4, 3G4C, H2L1, and H3L1 to CHO cell expressed human IL-13 was evaluated in a TF-I cell proliferation assay according to the method set forth in Example 6.2A.

3G4, 3G4C, H2L1 and H3L1 neutralized the bioactivity of human IL-13 expressed in recombinant CHO (see Table 6 and Figure 13).

[ND50 (neutralizing dose) is mA open concentration to reduce TF-I cell proliferation by 50% in response to an established IL-13 concentration]

Table 6

<table> table see original document page 92 </column> </row> <table>

The median value of parental ND4 for 3G4 was 0.05 μg / ml. This was calculated for the neutralization of -10 ng / ml of expressed human IL-13 in mammals in a TF-1 cell bioassay. This value differs from that previously obtained (see Example 1.3). However, the amount of human IL-13 used to stimulate TF-I cell proliferation in these experiments was lower than previously used (10 ng / ml).

The neutralization level of human IL13 expressed in CHO obtained by parenteral 3G4 mAb was slightly better than the para4G4C level. The potencies of H2L1 and H3L1 were reduced compared to both parenteral 3G4 and chimeric 3G4 mAb.3.5 neutralization of recombinant human IL-13 variant Q130 in a TF-I cell proliferation assay.

The neutralizing capacity of 3G4, 3G4C, H2L1 and H3L1 for recombinant human E. coli expressed Q130 IL-13 was evaluated in a TF-1 cell proliferation assay. The method was performed as described in Example 6.2A.

An ND50 value of 0.274 μ§ / πι1 was obtained. This differs from the previously obtained ND50 value (see Example 1.4). This assay was repeated several times to confirm these ND50 values. The quality of the commercial source human IL-13 preparation Q130 used in both sets of experiments (performed on different occasions) may explain the differences observed for these 2 data sets.

Table 7 shows the powers of 3G4C, H2L1 and H3L1, which were all similar. See also Figure 14.

Table 7

<table> table see original document page 93 </column> </row> <table>

3.6 Analysis of BIAcore® The affinity of 3G4C, H2L1 and H3L1 for recombinant human IL-13 and decinomolgus was evaluated by BIAcore® analysis. See Tables 8, 9 and 10.

Analyzes were performed using Protein A capture. Briefly, Protein A was coupled to a CM5 fragment by primary amine coupling according to manufacturers' recommendations. The chimeric antibody or humanized antibodies were then captured on this surface and human and cynomolgus IL-13 acted at defined concentrations. The surface was regenerated back to the Protein A surface using mild acid elution conditions, which did not significantly affect the ability to capture antibody for subsequent IL-13 binding events. The work was performed on the Biacore 3000 and Biacore T100 machines, the data were analyzed using the inherent evaluation program on the machines and adjusted to the 1: 1 binding model. Data differed slightly between the two machines, although the observed differences between the kinetics of the binding of chimeric and humanized antibodies to human IL-13 were similar for both machines. Human IL-13 binding data fit well with model 1.1 for all constructs, but the adjustment for cinomolgus IL-13 binding was worse, increasing the likelihood that actual values could be slightly worse (eg , a 2 to 3-fold difference) than reported because of their weaker fit and difficulty in analysis. Data were generated using cinomolgus human IL-13 (produced at GSK). All Biacore developments were made at 25 degrees C.

Table 8. BIACORE 3000 Data for Binding of Humanized Chimeric Antibodies to Human IL-13

<table> table see original document page 94 </column> </row> <table> For chimeric 3G4, data were produced from 4 independent experiments (two of which were performed in duplicate, with each duplicate being analyzed separately). As for the humanized H2A1 and H3L1 mAbs, the data were produced from two independent experiments performed in duplicate (with each duplicate being analyzed separately). Data are presented as the mean and standard deviation (within parentheses) of these results.

Table 9. T100 data for binding of humanized chimeric antibodies to human IL-13

<table> table see original document page 95 </column> </row> <table>

Data were produced from 2 independent experiments and are presented as the mean and standard deviation (within parentheses) of these results.

Chimeric antibody 3G4 and human mAbsh H2L1 and H3L1 bind with high affinity to human IL-13, and these data are comparable to the binding affinity of human 3G-parental mouse mAb.

Table 10. T100 data for binding of the humanized chimeric antibody to cinomolgus IL-13

<table> table see original document page 95 </column> </row> <table>

The data were produced from a unique experiment. The chimeric antibody and humanized mAbs H2L1 and H3L1 bind to cinomolgus IL-13 with a lower affinity compared to their binding affinity for human IL-13.

3.7 Specificity of 3G4C, H2L1 and H3L1 for binding to human IL-13.

The specificities of 3G4C, H2L1 and H3L1 for human IL-13 were evaluated by analyzing the cross-reactivity potential against human IL-4, human IL-5 and human GM-CSF in deletion ELISAs. These methods were performed as described in sections 6.7, 6.8 and 6.9, respectively. See Figures 15, 16 and 17. These mAbs were found to be specific for binding to IL-13, with no cross reactivity to human IL-4, human IL-5, or human GM-CSF at mAb concentrations up to 30 μ £ / ηι1. 3G4 chimeras appear to have some binding to human IL-5 at 30 µg / ml; This is probably due to a pipetting error, as no observations were made for H2L1 and H3L1 of humanized mAbs at a similar concentration.

4. 3G4 epitope mapping using biotinylated peptides

A series of epitope mapping experiments were performed to determine which amino acid residues in IL-13 were required for binding of the 3G4 mouse mAb.

4.1. Crude mapping of the mouse m3b4 mAb binding epitope on human and cinomolgous IL-13

Sixteen 4-displaced biotinylated peptides were synthesized to map the location of the mouse mAb-identified 3G4 binding epitope on human IL-13 and cynomolgus. An ELISA method, as described in Example 6.3, was used to detect binding of immobilized biotinylated peptide to parent mouse mAb 3G4.

Custom details of the 16-mer designated Peptides: 88 χ16 mere, 4-offset (provided by Mimotopes, Australia).

Format: Peptides 25 and 44 = Biotin-SGSG-PEPTID-acid

Peptides 2 to 24 and 27 to 43 = Biotin-SGSG-PEPTIDE-amide <table> table see original document page 97 </column> </row> <table>

(* indicates a high hydrophobicity value).

Results indicated this 3G4 mouse mAb bound to the two peptides shown below (see also Figure 6).

Peptide 25: DLLLHLKKLFREGRFN (SEQ ID NO: 88)

Peptide 44: DLLVHLKKLFREGQFN (SEQ ID NO: 89)

Peptide 25 is derived from human IL-13.

Peptide 44 is derived from cinomolgus IL-13.Note: Bold indicates residue differences between human IL-13 and cinomolgus IL-13 ortholog.

4.2 Fine mapping of mouse 3G4 mAb binding epitope on human IL-13 and cynomolgus using biotinylated peptides

The minimal binding epitope for mAb decoding 3G4 on human IL-13 was determined using a peptide established around the peptide sequence, QFVKDLLLHLKKLFREGRFN (SEQ ID. NO: 90). The peptides were obtained with 1 amino acid sequentially removed from either the N or C terminus of this parental peptide sequence in order to define the precise epitope of linear binding to mouse mAb 3g4. A similar approach was taken to map the binding to cinomolgus IL-13. An ELISA method (performed as described in Example 6.4) was used to detect immobilized biotinylated dopeptide binding to the parental mouse mAb 3G4 (Figures 7a and 7b).

Results indicate that parental mouse mAb 3G4 binds to the minimal amino acid epitope LLHLKKLFREG (SEQID. NO: 91) in the C-terminal region of human IL-13. However, the 2 amino acids (D and L) located adjacent to the N-terminal end of the above peptide sequence (in the human IL-13 sequence) may also be important for binding, as the binding signal is enhanced when these residues are present. Similarly, the 2 amino acids (R, FeN) located adjacent to the C-terminal end of the above peptide sequence (in the human IL-13 sequence) may also be important for binding, since the binding signal is lost when the R and R residues are present. F are present, but the binding signal is restored when residue N is present.

Similar results were obtained for the binding of mouse mAb 3G4 to the cinomolgus IL-13 peptide pool.

Results indicate that parental mouse mAb 3G4 binds to the minimal amino acid epitope LLVHLKKLFREG (SEQ ID NO: 98) C-terminal region of cinomolgus IL-13. However, the 1 amino acid (D) located adjacent to the N-terminal end of the above peptide sequence (in the cinomolgus IL-13 sequence) may also be important for allocation as the binding signal is intensified when the residue is present. . Similarly, the 3 amino acids (Q, FeN) located adjacent to the C-terminal end of the above peptide sequence (cynomolgus IL-13 sequence) may also be important for binding, since the binding signal is lost when Q residues and F are present, but the binding signal is restored when residue N is present.

4.3 Scanning of alanine from the mAb decamound 3G4 binding epitope using biotinylated peptides.

In order to identify the key residues involved in the interaction of human IL-13 with mouse mAb 3G4, an alanine scanning approach was adopted using the parental depeptide sequence QFVKDLLLHLKKLFREGRFN (SEQ ID NO: 90). For this analysis, the peptides shown in Table 11 were obtained (AnaSpecInc.) When an amino acid was sequentially replaced by an alanine residue at each amino acid position in the LKKLFRE (SEQ IDNO: 92) portion of the parental epitope of LKKLFRE (SEQ ID NO: 92) of the parental epitope QFVKDLLLHLKKLFREGRFN (SEQ. ID. NO: 90).

Table 11

<table> table see original document page 99 </column> </row> <table> An ELISA method was used (similar to that presented in Example 6.4) to detect binding of the immobilized biotinylated peptide to parental mouse mAb 3G4 (see Figure 8 ).

These data confirm that the key amino acid residues involved in the interaction of parental mouse mAb 3G4 with human IL-13 are at least arginine (R) at position 107, lysine (K) at position 103 and lysine (K) at position 104. The numbering of these is as previously described in WO 2006/003407.

As the above analysis had only scanned the LKKLFRE portion (SEQ ID NO: 92) of the minimal binding epitope, another set of peptides as shown in Table 12 was obtained (Mimotopes) to expand the study of alanine scanning to other amino acid residues in the minimal binding epitope.

Table 12

<table> table see original document page 100 </column> </row> <table>

Again, an ELISA method (similar to that used in Example 6.4) was used to detect binding of the immobilized biotinylated peptide to parental mouse mAb 3G4. See Figure 9 (in this experiment the QFVKDLLLHLKKLFREGRFN peptide (SEQ ID NO: 90) is the positive control demonstrating maximum binding, and the QFVKDLLLHLKKLFAEGRFN peptide (SEQ ID NO: 104) is the negative control showing minimal binding).

These data suggest that the phenylalanine (F) residue in 111 is also important for the interaction of parental mouse mAb 3G4 with human IL-13. Numbering of this residue is as previously described in WO 2006/003407.

5. Effectiveness of a humanized anti-IL-13 mAb in the decinomolgus asthma model.

This example is prophetic.

The Ascarissuum (A. suum) -induced pulmonary bronchoconstriction model in cynomolgus monkeys (Macaca fascicularis) is widely recognized as the most similar and relevant non-clinical model to human asmans (Patterson, R. et al. Trans. Assoc. Am. Physicians 1980 93: 317-325; Patterson, R. et al., J. Lab. Clin. Med. 1983 (101: 864-872).

In this model, animals having a pulmonarinata sensitivity to A suum are exposed to nebulized A. suum to induce an asthmatic response. This asthmatic response may be characterized by measurement of airway hyperresponsiveness (AHR), measured cellular infiltration into bronchoalveolar lavage fluid (BAL), and serum IgG levels. The experimental methods are similar to those previously described by Mauser, P. et al. in Am. J. Resp. Crit. Care Med. 1995 204: 467-472 and by Evanoff, H., et al. in Immunologic Investigation 1992 21: 39.

This study uses 30 animals pre-selected for entry that have demonstrated a positive bronchoconstrictor response to a specific dose of A. suum antigen. A. suum is administered at the optimal response dose (ORD) for each animal. It is a predetermined dose of A.suum that produces an increase in Rl (lung resistance) of at least 40% and a decrease in Cdyn (dynamic compliance) of at least 35% by aerosol inhalation (for a single dose administered via 15 breaths with the use of a nebulizer).

The study takes place in two phases. During phase 1, AHR is evaluated in response to the challenge of intravenous (i / v) histamine [ie, a sufficient histamine dose to induce an increase in Rl of at least 30% above the baseline (PC30)] both before (the assessment of baseline lung function on day one) and after (on the eleventh day) administration of A. suum antigen (on the ninth and tenth days when A. suum is administered at a predetermined optimal dose for each animal, by aerosol inhalation).

Phase 2 is identical to phase 1 except that animals receive antibody treatment (see below), each antibody is given as 3 doses of approximately 30 mg / kg administered by i / v infusion on the first, fifth and ninth day.

Group 1 (n = 12): a humanized anti-IL-13 mAb (30 mg / kg)

Group 2 (n = 12): a humanized anti-IL-13 mAb (30 mg / kg) and

Pascolizumab (humanized anti-IL4 mAb, 30 mg / kg)

Group 3 (η = 6): Negative control treatment of vehicle only

Phases 1 and 2 AHR reading outputs are calculated taking into account pressure and air flow readings - lung resistance (R1) and dynamic compliance (Cdyn) - in response to histamine using the mechanical system Buxco lung. The maximum percentage change from baseline compared with the post challenge of A. suum antigen [for pulmonary resistance (Rl) and dynamic compliance (Cdyn) is compared for phases 1 and 2, ie with or without antibody treatment. , and these data are used to evaluate the AHR phenotype.

In addition, BAL samples are collected on the first and eleventh days in phases 1 and 2 to measure cell infiltration and, in particular, eosinophilia. Serum samples are also taken to monitor IgE levels.

6.1 Cinomolgus and human IL-13 binding ELISA

This assay describes an ELISA that detects binding of an antibody to human IL-13 and cinomolgus. It is a scrambled ELISA format.

6.1.1 Materials

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

2. Human IL-13 (expressed in E. coli from CambridgeBiosciences, cat. No. CH1-013).

3. Cinomolgo IL-13 (produced by GlaxoSmithkline)

4. Goat anti-human IL-13 polyclonal antibody (R & DSystems, cat. No. AF-213-NA)

5. Anti-human IgG-HRP (Sigma, Cat. No. A-6029)

6. Anti-mouse IgG-HRP (Sigma, Cat η2 A-9309)

7. Carbonate / Bicarbonate Buffer (Sigma; Cat. No. C-3041)

8. TBST [Tris buffered saline (6.06 g Tris + 8.06 g NaCl + 0.2 g KCl + H2 O to 1 liter) + 0.05% Tween 20]

9. BSA (Sigma A-7030)

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

11. Sulfuric acid6.1.2 Method

l.The blocking solution is 3% BSA + TBST

2. The wash solution is TBST

3. Covered 'Nunc Maxisorp' ELISA plates with 50 µl goat anti-human IL-13 polyclonal antibody 5 µg / ml (R & DSystems, cat. No. AF-213-NA. Compound at a stock concentration of 500 µg / ml according to manufacturers 'instructions, and stored at -20 ° C in aliquots in carbonate / bicarbonate buffer (Sigma; cat. No. C-3041, composed according to manufacturers' instructions), plating with a plate sealer and incubation for at night at 4 ° C.

4. Block with 100 ul of 3% BSA / TBST incubated at room temperature and pressure for 1 hour.

5. Wash X3 in TBST (at least 200 μl of wash solution per well per wash).

6. Add 20 ng per well (in a 50 µl volume) of human IL-13 (Cambridge Bioscience, cat. No. CH1-013. Compound at a stock concentration of 100 ng / ul according to manufacturers' instructions, and stored in aliquots at -20 ° C) or 20 ng per cinomolgo IL-13 reservoir in blocking solution and incubate at room temperature for 1 hour.

7. Wash X3 in TBST.

8. Add 50 μl of antibody sample (titrated out to obtain endpoint titer data, if necessary) in blocking solution, incubate at room temperature and pressure for 1 hour.

9. Wash X3 in TBST.

10. As for the chimeric 3G4 antibody or humanized antibody, detect binding with the use of 50 µl per reservoir of anti-human IgG-HRP (Sigma, Cat No. A-6029) at a dilution to 1/2000 blocking solution by 1 µM. hour at ambient temperature and pressure. As for the 3G4 mouse monoclonal antibody, detect binding using 50 µl by anti-mouse IgG-HRP reservoir (Sigma, Cat No. A-9309) in a 1/1000 dilution in blocking solution for 1 hour at ambient temperature and pressure.

11. Wash X3 in TBST.

12. Develop with 100 μl OPD (Sigma, Cat. On P-9187.Composed according to manufacturers instructions), discontinue with 50 μl 3M H2SO4, reading at an absorbance of 490 nm. The development time is ~ 12 minutes.

6.1A Human IL-13 Binding ELISA

This assay describes an ELISA that detects antibody binding to human IL-13. It is a sandwich ELISA format and slightly different from that described in Example 6.1.

6.1A1 Materials

1. Costar 96-reservoir EIA plate (Corning Costarcat. No. 3369)

2. Human IL-13 (Peprotech cat. No. 200-13)

3. Goat anti-human IL-13 polyclonal antibody (R & DSystems, cat. No. AF-213-NA)

4. Anti-human kappa light chain HRP (Sigma, Cat No. A7164)

5. Carbonate / Bicarbonate Buffer (Sigma; Cat. No. C-3041)

6. TBST [Tris buffered saline (6.06 g Tris + 8.06 g NaCl + 0.2 g KCl + H 2 O to 1 liter) + 0.05% Tween 20]

7. BSA (Sigma A-7030)

8. OPD (Sigma, Cat. N-P-9187)

9. Sulfuric acid

6.1A.2 Method

1. Blocking solution is 3% BSA + TBST

2. The wash solution is TBST

3. Covered 'Costar EIA / RIA' ELISA Plates Covered with 50 µl 5 µg / ml Goat Anti-Human Polyclonal IL-13 Antibody (R & DSystems, Cat. No. AF-213-NA. Compound at Stock Concentration 500 æg / ml according to manufacturers instructions, and stored at -20Â ° C aliquots in carbonate / bicarbonate buffer (Sigma; cat. No. C-3041, compound per manufacturers instructions), covered with a plate sealer and overnight incubation at 4 ° C.

4. Block with 100 ul of 3% BSA / TBST incubated at room temperature and pressure for 1 hour or overnight at 4 ° C.5. Wash X2 in TBST (at least 200 μl of wash solution per well per wash).

6. Add 20 ng per well (in a 50 μl volume) of human IL-13 (Peprotech cat. No. 200-13. Compound at a stock concentration of 100 ng / ul according to manufacturers instructions and stored in aliquots at -20 ° C) dilute in blocking solution and incubate at room temperature for 1 hour.

7. Wash X2 in TBST.

8. Add 50 μl of antibody sample (titrated out to obtain endpoint titer data, if necessary) in blocking solution, incubate at room temperature and pressure for 1 hour.

9. Wash X2 in TBST.

10. As for the 3G4 chimeric antibody or humanized antibody, detect binding with the use of 50 µl per well anti-human kappa lightweight HRP reservoir (Sigma, Cat No A-7164) at a 1/2000 dilution in 1 µl blocking solution. hour at ambient temperature and pressure.

11. Wash X2 in TB ST.

12. Develop with 100 μl OPD (Sigma, Cat. On P-9187.Composed according to manufacturers instructions), discontinue with 50 μl 3M H2SO4, reading at an absorbance of 490 nm.

6.1B Native Human IL-13 Binding ELISA

This assay describes an ELISA that detects binding of an antibody to native human IL-13. It is of a sandwich ELISA format.

6.1B.1 Materials

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

2. NATIVA Human IL-13 (HDLM-2 Cell Supernatant)

3. Anti-human IL13 Antibody (Pharmingen, Cat. No. 554570)

4. Biotinylated anti-human IL13 antibody (Pharmingen, Cat. No. 555054)

5. Streptavidin-HRP (Sigma cat # 2 E2886)

6. Carbonate / Bicarbonate Buffer (Sigma; Cat. No. C-3041)

7. RPMI + 20% FBS + 2mM Glutamine

8. PBST (PBS + 0.05% Tween 20)

9. BSA (Sigma A-7030)

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

11. Sulfuric acid

6.1B.2 Method

1. Blocking solution is 1% BSA in PBST

2. Washing solution is PBST

3. Covered 'Nunc Maxisorp' ELISA plates with 50 µl 5 µg / ml chimeric or humanized 3G4 antibody (Pharmigen, cat. # 2554570) diluted in carbonate / bicarbonate buffer, plaque sealer cover and overnight incubation in 4 ° C.

4. Block with 200 ul of 1% BSA / PBST incubated at room temperature for 1 hour

5. Wash with 200 µl BSA / PBST, incubation at room temperature for 1 hour.

6. Add 50 µl native IL-13 present in HDLM2 release sample (titration separation) dilution in RPMI + 20% FBS + 2 mM glutamine solution, incubation at room temperature and pressure for 1 hour.

7. Wash X3 in PBST.

8. Add 50 µl per well of 1 µg / ml biotinylated anti-human IL-13 (Pharmigen, Cat. No. 555054) dilution in PBST + 1% B SA, incubation at room temperature and pressure for 1 hour.9. Wash X3 in PBST.

10.Add 50 µl per well of the depteptavidin-HRP conjugate in 1/1000 dilution, dilute in PBST + 1% BSA, incubate for 1 hour at room temperature.

11. Wash X3 in TB ST.

12.Develop with 100 μl OPD (Sigma, Cat. No. P-9187.Composed according to manufacturers instructions), discontinue with 50 μl per reservoir of 3M H2SO4, reading at an absorbance of 490 nm.6.2. IL-13 Neutralization Bioassay (TF-1 Cell Proliferation Assay)

This is an IL-13 bioassay that can be used to determine the neutralizing capacity of an anti-IL-13 antibody. The method described below uses recombinant human or cinomolgous IL-13. Human IL-13 expressed in mammals or the variant of human IL-13 Q130 may also be used in this assay as well.

6.2.1 Materials

1. TF-I cell line (the internally obtained TF-I cell line, NB, available from ATCC version)

2. 96-well tissue culture plates (Invitrogen)

3. Human IL-13 (Cambridge Bioscience, Cat. No. CHl-013)

4. CellTiter96 Non-Radioactive Cell Proliferation Assay (Promega, Cat. No. G4000)

6.2.2 Method

1. Method for measuring the ability of an anti-human IL-13 mAb to neutralize the bioactivity of recombinant human or cinomolgous IL-13 in a TF-1 cell bioassay.

2. This assay is performed on 96-well sterile tissue culture dishes (Invitrogen) under sterile conditions. All tests are performed in triplicate. Preincubate 10 ng / ml human IL-13 (CambridgeBioscience, cat. No. CH1-013. Compose at a stock concentration of 100 ng / ul according to manufacturers instructions using sterile technique in a tissue culture hood class 2, store in small aliquots at -20 ° C) or 10 ng / ml cinnomolgus IL-13 (GSK generated) with various dilutions of anti-human IL-13 mAb (diluted 6 µg / ml in 3-fold dilutions). to 0.025 µg / ml) in a total volume of 50 µl for 1 hour at 37 ° C. Positive control reservoirs will also be included with IL-13 present but no anti-human IL-13 mAb. In addition, negative control reservoirs will have no IL-13 and no anti-human IL-13 mAbs present. Use a 96-well, low protein-binding fundoredondo well plate for this preincubation (note that the concentration of IL-13 and anti-human IL-13 mAb will be evenly distributed at a later stage when cells are added). ).

4. Plate 50 µl TF-I cells at 2 x 105 per milliliter in a 96-well sterile reservoir tissue culture dish. After 1 hour preincubation, add IL-13 and anti-human IL-13 mAb sample to cells. The final assay volume of 100 µl, containing various demAb dilutions of anti-human IL-13, recombinant IL-13, and TF-1 cells, is incubated at 37 ° C for -70 hours in a moistened CO2 incubator.

5. In ~ 66 hours, scan the reservoirs to confirm that they are sterile and that no bacterial contamination has occurred.

6. Add 15 μl of sterile per-filter filter substrate (Cat. No. G4000, Promega. Compound per manufacturer's instructions) for the final 4 hours of incubation.

7. Stop the reaction with 100 μl Stop Solution (supplied in the MTT Kit) to solubilize the blue metabolised formazan product. Leave for at least 2 hours, then pipette up and down to help dissolve the crystals. Alternatively, cover with a plate sealer and leave at 40 ° C overnight, then pipette up and down the next day (this is easier in terms of pipetting).

8. Read the absorbance of the solution in each well on a 96 well plate reader at 570 nm wavelength.

9. The ability of the anti-human IL-13 mAb to neutralize the bioactivity of human IL-13 or cynomolgus is expressed as that concentration of the anti-human IL-13 mAb required to neutralize the bioactivity of a defined amount of IL-13. human or decinomolgus (5 ng / ml) by 50% (= ND50). The lower the required concentration, the more potent the neutralizing capacity.

6.2.The IL-13 Neutralization Bioassay (TF-1 Cell Proliferation Assay)

This is an IL-13 bioassay that can be used to determine the neutralizing ability of an anti-IL-13 antibody. The method described below uses recombinant human and cinomolgus IL-13. Mammalian expressed human AIL-13 or human IL-13 variant Q130 can also be used in this assay. Note that this method differs only slightly from that described in Example 6.2.

6.2 .A. 1 Materials

1. TF-I Cell Line (Internally Obtained TF-Iob Cell Line, NB5 available from ATCC version)

2. 96-well tissue culture plates (CorningCostar, Cat. No. 3596).

3. Human IL-13 (Peprotech, cat. No. 200-13)

4. Human IL-13 (expressed in CHO cells) generated in GSK.

5. Human IL-13 variant Q130 (Peprotech, cat. Na 200-13 A) 6. Cinomolgus IL-13 (generated at GSK).

7. Polyclonal Anti-Human IL-13 (R&D Systems AF-213-NA)

8. 96-well tissue culture plates (Corning Costar, cat. No. 3790).

9. CellTiter96 Non-Radioactive Cell Proliferation Assay (Promega, Cat. No. G4000) 6.2.A.2 Method

1. Method for measuring the ability of an anti-human IL-13 mAb to neutralize the bioactivity of recombinant human and cinomolgous IL-13 in a TF-1 cell bioassay.

2. This assay is performed on 96-well sterile reservoir tissue culture plates (Corning Costar, cat. No. 3596) under sterile conditions. All tests are performed in triplicate.

3. Preincubate 10 ng / ml human IL-13 (Peprotech, cat. # 2200-13), or 10 ng / ml CHO-expressed human IL13 (generated in GSK) or 60 ng / ml human IL13 Q130 variant (Peprotech, cat. No. 200-13A), or 10 ng / ml cinnomolgus IL-13 (generated at GSK) to compose commercial Ab at a stock concentration according to manufacturers instructions using sterile technique in a Class 2 fabrics, store in small aliquots at -20 ° C. At various domAb dilutions of anti-human IL-13 (diluted 6 µg / ml or 2 µg / ml or 189 µg / ml at triple dilutions below 0.025 µg / ml or 0.008 µg / ml or 0.74 µg / ml, respectively). ) in a total volume of 50 ul for 1 hour at 37 ° C. Also included will be positive control reservoirs, with IL-13 present but no anti-human IL-13 mAb. In addition, the negative control reservoirs will have no IL-13 and no anti-human IL-13 mAb present. Use a sterile low protein-binding round bottom reservoir plate for this preincubation (Corning Costar, cat. No. 3790). (Note that the concentration of IL-13 and anti-human IL-13 mAb will be divided into equal parts at a later stage when the cells are added).

4. Plaque 50 µl TF-I cells at 2 x 105 per milliliter in a 96-well sterile tissue culture plate (Corning Costar, cat. No. 3596). After 1 hour preincubation, add the IL-13 and mAb sample of anti-human IL-13 to the cells. The final 100 µl assay volume, containing various dilutions of anti-human IL-13 mAb, IL-13 recombinantee TF-1 cells, is incubated at 37 ° C for 3 days in a humidified CO2 incubator.

5. Scan the reservoirs to confirm that they are sterile and that no bacterial contamination has occurred.

6. Add 15 µl MTT substrate per well (Cat. No. G4000, Promega) for the final 4 hours of incubation.

7. Stop the reaction with 100 μl Stop Solution (supplied in MTT Kit) to solubilize the blue metabolised formazan product. Leave for at least 2 hours at room temperature, then shake plates on the plate shaker for ~ 30 minutes.

Alternatively, cover with a plate sealer and leave at 40 ° C for night, then shake the plates on the plate shaker for ~ 30 minutes. Read the absorbance of the solution in each reservoir on a 96-well plate reader at 570 nm wavelength.

8. The ability of the anti-human IL-13 mAb to neutralize the bioactivity of human IL-13 or cynomolgus is expressed as that concentration of the anti-human IL-13 mAb required to neutralize the bioactivity of a defined amount of IL-13. human or decinomolgus by 50% (= ND50). The lower the concentration required, the more powerful the neutralizing ability.

6.3. Crude Epitope Mapping ELISA This assay describes an ELISA that detects the binding of mouse mAb 3G4 to human IL-13 or cinomolg peptides.

6.3.1 Materials

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

Tween 20)

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

3. PBST (phosphate buffered saline + 0,05% of

4. BSA (Sigma A-7030)

5. Human IL-13 and cinomolgus 16-mer peptides, displacement = 4 (Mimotopes' ordering orders)

6. 20-mer Positive and Negative Control Peptides (supplied to order from Mimotopes)

7. 3GA mouse mAb

8. Control Ab (Supplied on request from Mimotopes)

9. Rabbit anti-mouse conjugate Ig HRP (DAKO code η-P0260)

10. OPD (Sigma, Cat. Η2 P-9187)

11. 3M Sulfuric Acid

6.3.2 Method

1. The blocking solution is 3% BSA + PBST.

2. The wash solution is PBST.

3. Cover the 'Nunc Maxisorp' ELISA plates with 100 μg of 5μg / ml Streptavidin ImmunoPure® (Pierce, cat. N and 21125 composed in a stock concentration of 1 mg / ml according to manufacturers instructions and stored in aliquots at + 4 ° C) using PBST as a diluent. Incubate overnight at 37 ° C to allow the solution to dry.

4. Block with 200 μΐ of 3% BSA / PBST. Add plate sealer and incubate at room temperature and pressure for 1 hour.

5. Wash X3 in PBST (at least 200 μΐ of wash solution per reservoir per wash).

6. In duplicate and using PBST as diluent, add 100μl per well (except control reservoirs) of 1,000-fold dilutions of each peptide (dissolved according to manufacturers instructions to 200 μΐ, 40% Acetonitrile, 60% water, then divided). aliquoted in 10-fold dilutions in the same solvent and stored at -20 ° C.

7. In control reservoirs, in duplicate and using PBST as a diluent, add 100 μΐ per 10-fold dilution reservoir of control peptides (dissolved as per manufacturer's instructions in 1 ml, 40% Acetonitrile, 60% water, and stored at -20 ° C). Add plate sealer and incubate at room temperature and pressure for 1 hour on a vibrating table.

8. Wash X3 in PBST (at least 200 μΐ of wash-out per wash reservoir).

9. Add 100 μΐ per well (except control reservoirs) of 1.506 μ £ / ηι1 mouse mAb to PBST.

10. Add 100 μΐ per well to the control-only wells, 4, 16, and 32-fold dilutions of control antibody (used as supplied by the manufacturer and stored at -20 ° C) using PBST as a diluent. Add plate sealer and incubators at room temperature and pressure for 1 hour on a vibrating table.

11. Wash X3 in PBST (at least 200 μΐ of wash flush per wash).

12. Add 100 μΐ per 2,000-fold dilution well of rabbit anti-mouse HRP Ig conjugate (DAKO, code n2P0260 as supplied, stored at + 40 ° C) using PBST as a diluent. Add plate sealer and incubate at room temperature and pressure for 1 hour on a vibrating table.

13. Wash X3 in PBST (at least 200 μΐ washing wash solution).

14. Develop with 100 μΐ OPD (Sigma, Cat. No. P-9187.Composed by manufacturers instructions), interrupt with 50 μΐ 3M H2O4, read at an absorbance of 490 nm. Development time of —10 minutes.

6.4. Epitope Fine Mapping ELISA

This assay describes an ELISA that detects mAb 3G4 binding to human IL-13 and cinomolg peptides.

6.4.1 Materials

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

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

3. PBST (phosphate buffered saline + 0.05% of Tween 20)

4. BSA (Sigma A-7030)

5. Pure partial-window peptides of human and decinomolgous IL-13 (13-mer truncated by one amino acid at a time of both N- and C-terminal end; (Mimotopes' ordering orders)

6. Positive Control 16-Mere Peptides (Supplied by Mimotopes Pre-Order)

7. 3G4 mAb (internally produced)

8. Goat anti-mouse IgG (Fc specific) HRP conjugated antibody (Sigma, Cat. No. P-9187)

9. OPD (Sigma, Cat. At P-9187)

10. 3M Sulfuric Acid

6.4.2 Method

1. The blocking solution is 3% BSA + PBST.2. The wash solution is PBST.

3. Cover 'Nunc Maxisorp' ELISA plates with 100 μΐ of 5μ§ / ιη1 of Streptavidin ImmunoPure® (Pierce, cat. No. 21125 composed in a stock concentration of 1 mg / ml according to manufacturer's instructions and stored in + 4 ° C). Incubate overnight at +37 ° C.

4. Block with 200 μΐ of 3% BSA / PBST. Add plate sealer and incubate overnight at +4 ° C.

5. Wash X3 in PBST (at least 200 μΐ of wash solution per reservoir per wash).

6. In duplicate and using 3% BSA in co-diluted PBST, add 100 μΐ per 1,000-fold dilution reservoir of each peptide (dissolved according to manufacturers instructions to 200 μΐ of 40% Acetonitrile, 60% water, and stored at -20 ° C). ° C). Add plate sealer and incubate at room temperature for 1 hour over mesavibration.

7. Wash X3 in PBST (at least 200 μΐ of wash-out reservoir wash solution).

8. Add 100 μΐ per well of 3 μg / ml 3G4 diluted in 3% BSA in PBST.

9. Wash X3 in PBST (at least 200 μΐ of wash flush solution).

10. Add 100 μΐ per 1,000-fold dilution reservoir of goat anti-mouse Ig HRP conjugated antibody (Sigma A-9309 used as supplied, stored at +4 ° C) using 3% BSA in PBST as a diluent. . Add plate sealer and incubate at room temperature for 1 hour on a vibrating table.

11. Wash X3 in PBST (at least 200 μΐ of wash flush per wash).

12. Develop with 100 μΐ OPD (Sigma, Cat. No. P-9187.Composed by manufacturer's instructions), stop with 50 μΐ 3M H2O4, read at 490 nm absorbance. Development time is -10 minutes.

6.5 Human IL-13 binding ELISA to human IL13Ral chain

This ELISA determines whether an antibody can inhibit human IL-13 binding to the human IL13 Ral chain.

6.5.1 Materials

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

39454A)

2. Human IL13Ra1-Fc (R&D Systems, cat. No. 146-IR)

3. Human IL-13 (internally produced)

4. Biotinylated anti-human IL-13 (R&D Systems, cat.

BAF213)

5. Streptavidin-HRP (Sigma, Cat. No. E2886)

6. Carbonate / Bicarbonate Buffer (Sigma, Cat. No. C-3041)

7. TBST [Tris-buffered saline (6.06 g Tris + 8.06 g NaCl + 0.2 g KCl + H 2 O to 1 liter) + 0.05% Tween 20]

8. BSA (Sigma A-7030)

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

10. Sulfuric acid

6.5.2 Method

1. The lock solution is 3% BSA + TBST

2. The wash solution is TBST

3. Cover Nunc Maxisorp ELISA plates with 50 µl of 5ng / µl human IL-13Ra I-Fc in carbonate / bicarbonate buffer. Cover with a plate sealer and incubate overnight at 4 ° C.

4. Block with 100 ul of 3% BSA / TBST and incubate at room temperature and pressure for 1 hour.

5. Wash X3 TBST (at least 200 μl washing wash solution per well).

6. In a total volume of 50 µl, preincubate 0.4 ng / µl human IL-13 with antibody sample (titrated) in blocking solution for 30 minutes. Add the preincubated sample to the recipient coated ELISA plate and incubate at room temperature for 1 hour.

7. Wash x3 in TBST

8. Detect any bound human IL-13 using 50 µl per reservoir of biotinylated anti-human IL-13 diluted 1 µg / ml. Incubate for 1 hour at room temperature.

9. Wash x3 in TBST

10. Add 50 µl per well of deestreptavidin-HRP conjugate at 1/1000 dilution. Incubate for 1 hour at room temperature.

11. Wash x3 in TBST

12. Develop with 100 μl per OPD reservoir (Sigma, Cat. No. P-9187. Compose according to manufacturers instructions), discontinue with 50 μl per 3M H2SO4 reservoir, read at an absorbance of 490 nm.

6.6 ELISA binding of human IL-13 to human IL13Ral chain

This ELISA determines whether an antibody can inhibit human IL-13 binding to the human IL13Rα2 chain.

6.6.1 Materials

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

2. Human IL13Rα2-Fc (R&D Systems, cat. No. 614-IR)

3. Human IL-13 (generated at GSK)

4. Biotinylated anti-human IL-13 (R&D Systems, cat. No. BAF213)

5. Streptavidin-HRP

6. Carbonate / Bicarbonate Buffer (Sigma, cat. No. C-3041) 7.TBST [Tris buffered saline (6.06 g Tris + 8.06 g NaCl + 0.2 g KCl + H2O to 1 liter) + 0.05% Tween 20]

8.BSA (Sigma A-7030)

9.OPD (Sigma, Cat. Η2 P-9187) 10. Sulfuric acid

6.6.2 Method

1.Block solution is 3% BSA + TBST

2.The wash solution is TBST

3. Cover Nunc Maxisorp ELISA plates with 50 µl 5ng / µl human IL-13Roc2-Fc in carbonate / bicarbonate buffer. Cover with a plate sealer and incubate overnight at 4 ° C.

4. Block with 100 ul of 3% BSA / TBST and incubate at room temperature and pressure for 1 hour.

5.Wash X3 TBST (at least 200 μl washing wash solution per well).

6.In a total volume of 50 µl, preincubate 0.01 ng / µl human IL-13 with antibody sample (titrated) in blocker solution for 60 minutes. Add the preincubated sample to the recipient coated ELISA plate and incubate at room temperature for 1 hour.

7.Wash x3 on TBST

8. Detect any bound human IL-13 using 50 µl per reservoir of biotinylated anti-human IL-13 diluted 0.5 µg / ml. Incubate for 1 hour at room temperature.

9.Wash x3 on TBST

10.Add 50 ul per reservoir of deestreptavidin-HRP conjugate at 1/1000 dilution. Incubate for 1 hour at room temperature.

11.Wash x3 on TBST

12.Develop with 100 μl per OPD reservoir (Sigma, Cat. Η- Ρ-9187. Compose according to manufacturers instructions), discontinue with 50 μl per 3M H2SO4 reservoir, read at an absorbance of 490 nm.

Development time is ~ 2 minutes .__>

6.7 Human IL-4 Binding ELISA

This assay describes an ELISA that detects antibody binding to human IL-4. It is of a sandwich ELISA format.

6.7.1 Materials

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

13. Human IL-4 (R&D Systems, cat. No. 204IL)

14. Goat anti-human IL-4 polyclonal antibody (R & DSystems, cat. No. AF204-NA)

15. Biotinylated Rat Anti-Human IL-4 Antibody (R & DSystems, cat. No. BAF204)

16. Anti-mouse IgG-HRP (Dako, Cat. No. P0260)

17. Anti-Human Cover Light Chain HRP (Sigma A7164)

18. Streptavidin-HRP (Sigma cat. No. E2886)

19. Carbonate / Bicarbonate Buffer (Sigma, Cat. No. C-3041)

20. TBST [Tris buffered saline (6.06 g Tris + 8.06 g NaCl + 0.2 g KCl + H 2 O to 1 liter) + 0.05% Tween 20]

21. BSA (Sigma A-7030)

22. OPD (Sigma, Cat. No. P-9187)

23. Sulfuric acid

6.7.2 Method

13. Blocking solution is 3% BSA in TBST

14. Washing solution is TBST

15. Cover Nunc Maxisorp ELISA plates with 50 µl of 5 µg / ml goat anti-human IL-4 polyclonal antibody (R&D Systems, cat. No. AF-204-NA. Compound at a stock concentration of 500 µg / ml according to manufacturers 'instructions, and stored in aliquots at ~ 20 ° C) in carbonate / bicarbonate buffer (Sigma, Cat. No. C-3041, produced as per manufacturers' instructions). Cover with a plate sealer and incubate overnight at 4 ° C.

16. Block with 100 ul of 3% BSA / TBST and incubate at room temperature and pressure (rtp) for 1 hour.

17. Wash X3 in TBST (at least 200 μl wash wash solution per well).

18. Add 1 ng / ml (in a volume of 50 µl) of human IL-4 in blocking solution, and incubate at room temperature for 1 hour.

19. Wash x3 in TBST

20. Add 50 μl of antibody sample (separate portitization to obtain endpoint titration data, if necessary) in blocking solution, incubate at room temperature and pressure for 1 hour. As a positive control for IL-4 binding use a biotinylated anti-human IL-4 anti-human monoclonal antibody (separated by titration).

21. Wash x3 in TBST

22. For the 3G4 mouse monoclonal antibody, detect binding with the use of 50 µl per mouse IgG-HRPanticam reservoir (Dako, cat. Η2 P0260) at a dilution of 1/2000 blocking solution for 1 hour at temperature and pressure. environment. For the chimeric 3G4 antibody or humanized antibody, detect binding using 50 µl per anti-human kappa light chain HRP reservoir (Sigma, Cat.nfi Sigma A7164) at a 1/2000 dilution in blocking solution for 1 hour at temperature and pressure. environment. For positive control biotinylated rat anti-human IL-4 monoclonal antibody, detect with the use of a streptavidin-HRP conjugate antibody (Sigma cat. No. E2886) in 1/1000 dilution in the blocking solution for 1 hour at room temperature and pressure.

23. Wash X3 in TBST.

24.Develop with 100 μl of OPD (Sigma, Cat. No. P-9187.Composed according to manufacturers instructions), discontinue with 50 μl per reservoir of 3M H2SO4, read at an absorbance of 490 nm.

6.8 Human IL-5 Binding ELISA

This assay describes an ELISA that detects antibody binding to human IL-5; It is a sandwich ELISA format.

6.8.1 Materials

24.Nunc Maxisorp F96 Immunoplate 1 (Life Technologies, 4-

39454A)

25.IL-5 human (R&D Systems, cat. No. 205IL)

26. Anti-human IL-5 Polyclonal Antibody (R&D Systems, cat. No. AF-205-NA)

27. Anti-human IL-5 epolizumab (in internal chemistry)

28.IgG-HRP anti-mouse (Dako, Cat. Η2 P0260)

29.HRP Anti-Human Cover Light Chain (Sigma A7164)

30.Carbonate / bicarbonate buffer (Sigma, cat. No. C-3041)

31. TBST [Tris buffered saline (6.06 g Tris + 8.06 g NaCl + 0.2 g KCl + H 2 O to 1 liter) + 0.05% Tween 20]

32.BSA (Sigma A-7030)

33.OPD (Sigma, Cat. No. P-9187)

34. Sulfuric acid

6.8.2 Method

25. Blocking solution is 3% BSA in TBST

26.The wash solution is TPBST

27. Cover Nunc Maxisorp ELISA plates with 50 µl of 5 µg / ml goat anti-human IL-4 polyclonal antibody (R&D Systems, cat. AF-205-NA. Compound at a stock concentration of 500 µg / ml according to manufacturers instructions, and stored in aliquots at ~ 20 ° C) in carbonate / bicarbonate buffer (Sigma, cat. No. C-3041, produced as per manufacturers instructions). Cover with a plate sealer and incubate overnight at 4 ° C.

28. Block with 100 ul of 3% BSA / TBST and incubate at room temperature and pressure (rtp) for 1 hour.

29. Wash X3 in TBST (at least 200 μl washing wash solution per well).

30. Add 100 ng / ml (in a 50 µl volume) of human IL-5 (R&D Systems, cat. No. 205IL) in blocking solution, and incubate at room temperature for 1 hour.

31. Wash X3 in TBST

32. Add 50 μl antibody sample (separate portitization to obtain endpoint titration data, if necessary) in blocking solution, incubate at room temperature and pressure for 1 hour. As a positive control for IL-5 binding use an anti-human IL-5 Epolizumab antibody (titration separated).

33. Wash X3 in TBST.

34. For the 3G4 mouse monoclonal antibody, detect binding with 50 µl use per mouse IgG-HRPanticam reservoir (Dako, cat. No. P0260) at a dilution of 1/2000 blocking solution for 1 hour at temperature and ambient pressure. For the chimeric antibody 3G4 or IL5's humanized antibody and Mepolizumab, detect binding using 50 µl per cape-human light chain HRP reservoir (Sigma, Cat. No. Sigma A7164) at a dilution to 1/2000 blocking solution for 1 hour at room temperature and pressure.

35. Wash X3 in TBST.

36. Develop with 100 μl of OPD (Sigma, Cat. On P-9187.Composed as manufacturers instructions), discontinue with 50 μl of 3M H2SO4, read at an absorbance of 490 nm.

6.9 Human GM-CSF Binding ELISA

This assay describes an ELISA that detects antibody binding to human GM-CSF. It is a direct binding ELISA format.

6.9.1 Materials

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

36. Human GM-CSF (Internal Clinical Graduation)

37. Anti-human GM-CSF Monoclonal Antibody (R & DSystems, cat. Η2 MAB-215)

38. Anti-mouse IgG-HRP (Dako, Cat. No. P0260)

39. Anti-Human Cloak Light Chain HRP (Sigma A7164)

40. Carbonate / Bicarbonate Buffer (Sigma, Cat. No. C-3041)

41. PBST [PBS + 0.05% Tween 20)

42. BSA (Sigma A-7030)

43. OPD (Sigma, Cat. Η2 P-9187)

44. Sulfuric acid6.9.2 Method

37. Blocking solution is 3% BSA in PBST

38. The wash solution is PBST

39. Cover Nunc Maxisorp ELISA plates with 50 µl of 2ug / ml dilution of human GM-CSF in PBS, cover with a plate sealer and incubate overnight at 4 ° C.

40. Block with 200 µl of 3% BSA / PBST and incubate at room temperature and pressure (rtp) for 1 hour.

41. Wash X3 in PBST.

42. Add 50 µl of antibody sample (separate porting to obtain endpoint titer data) in blocking solution, and incubate at room temperature and pressure for 1 hour. As a positive control for binding to human IL-GM-CSF, use anti-human GM-CSF antibody (R&D Systems, cat. No. MAB215) (separated by titration).

43. Wash X3 in PBST.

44. For anti-GMCSF mouse monoclonal antibody, detect binding using 50 µl per mouse IgG-HRPanticam reservoir (Dako, cat. On P0260) at a dilution to 1/2000 blocking solution for 1 hour at room temperature and pressure As for the chimeric 3G4 antibody or humanized antibody, detect binding using 50 µl per well of anti-human kappa light chain HRP reservoir (Sigma, Cat. No. Sigma A7164) at a dilution to 1/2000 blocking solution for 1 hour at ambient temperature and pressure.

45. Wash X3 in PBST.

46. Develop with 100 μl of OPD (Sigma, Cat. No. P-9187.Composed according to manufacturers instructions), discontinue with 50 μl of 3M H2SO4, read at an absorbance of 490 nm.

Table 13

<table> table see original document page 125 </column> </row> <table> <table> table see original document page 126 </column> </row> <table>

Note: The DNA protein or polynucleotide sequence in SEQ ID NOs: 11 to 24 and 27 a40 (inclusive) also includes the signal sequence.SEQ.I.P.NO IDYEIH

SlQ-I.D.HO 2

AIDPETGGTAYNQKFKG

SEQ.I.D.NO 3

I LLYYYiPMD Y

ssq, i.d.no 4

RASQWISDYLH

SEQ.I.D, NO 5

YASQSIS

SEQ-1-D.NO <5

QNGRSFPLT

SEQ.I.D.NO 7

QVQLQQ S GADLVH PGASV7LSCKAS GYT FIDYEI HViM KQT PVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKS S TAYMELRS LTS EDSAVY yctri LlfYYYPMDYWGQGTSVTVS S

SEQ.I.D-NQ 8

Divmtqspatlsvtpgdrvslscrasqnisdylhmyqqkshesprllikyasqsxsgipsrpsgsgsgsdftlsiüsvbpsdvgvyycqnishsfpltlgagtklelk

SEQ I. D.MO:9

GP'VPPSTALRELIEELVNITQNQKAPLCNGSMVWSI RLTAGfiYCAALESL 1NVSGCS AIEKTQRMLSGFCPHKVSAGQF SS MÍVRDTKIEVAQFVKDLLLHLKKLFREGRFff

SEQ.I.D.NOt10GGCCCTGTGCGTCCCTCTACAGCCCTCAC? <3GAGCTCATTGAGGAGCrTGGTCAACATCACCCA

gaaccagaaggctccgctctgcaatggcagcatggtatggagcatcaacctgacagctggcatgtactgtgcagccctggaatccctgatcaacgtgtcaggctgcagtgccatcgagaagacccagaggatgagcgcgcggctcgcgctc

TGTCCGAGACACC ^ U ^ ATCGAGGTGGCCCAGTTTGTAAAGGACCTGCTC-rTAGArTTAAAGAAACTTTTTCGCGAGGGACGGTTCAACTGA

SBQ.I.D.NO 11

mgwsciilflvatatgwisqvqlvqsgaevkkpgasvkvsckasgytfidyeihwvrqapgq

GLEWMGAID PETGGTAYNQKFKGRVTMTTDTSTS TAYMELRSLRS DDT A VY YCARILL YY Y PMDYWGQGTLVTVSS

SBQ-1, P- NO 12

MGW1SC11LPLVATÁTGVHSQVQL VQSQAE VKKPGAS VKVSCXASG YTFIDYBIHWVRQAPGQGl ^ WMGAIDPSTGGTAYNQKFKGRVTMTTDTSTS TAYMEtAS LRSDDTAVYYCTR ILLYYGY!

SEQ.I.D.KO 13

Mgwsciilflvatatgvhsqvqlvqsgadvkkpgasvkvsckasgytfidyeihwwqapgq

GLEWMGAI DPETGGTAYNQKf KGRVTMTTDTSTSTAYMELRS LRSDDTAVY YCTRILLYYYPMDYWGQGfLVTVSS

SEQ.I.D.K0 14

Mgwsciilflvatatgvhsqvqlvqsgadvkkpgasvkvsckasgytfidyeihwvrqapgq

GLEWMGAI D PETGGTAYNQKFKGKATLTADKS TSTAYME LRSLRSDDTAVYYCTRILLYYYPMDYWGQGTLVTVSS

SEQ.I.D.WO 15

MGWSCIΪLFLVATATGVHSEIVLTQS PATLSLS PGERATLSCRASQNISDYLHWYQQKFGQA

PrijjIyyasqsisgiparfsgsgsgtoftltisslepedfawycqmghsfpSEQ.I.D.KO 17

MGWS CI IIrFLVATA TG1THSEI VlLTQS PATLSLS PGERATLS CRAS QNIS DYLHWYQQKPGQftPRLLIYYASQSI SGl PARFSGSGS GTD PTLTIMSLEPEDFAW YCQNSHS FPLTLGGGTKVEIK

SEQ.I.D-.NO 18

MGWSC11LF G LRS VATATGVHSQVQLVQS GAEVKXPÇASVKVSCKASGYTFIDYEIHWVRQAPGQGLEWMGAIDPETGGTAYNQKFKGR VTMTTDTSTS TAYiME LRSDDTAVYYCARILL YY YPM YWGOGTLVTVSSASTKGPSVFPKAPSSKSTSGGTA ^ ^^ ^ I GCLVlODYFPEFTOVSiiNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSSLGTQTYIGNWKKPSOTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTL SRTPEVTCWTO

KPREEQ YNSTYE VVS VLTVIiHQDWLNGKEYKCKVS WKALPÃPIEKTISKA.KGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPBMMYKTTPPVLDSDGSKLKDKSKSLVDQSHLVDKLQDKLVDLQDKLVDQVSLWLQDLW

SBQ.I.P.HQ 19

MGW SCIILF L VATATG VMSQ VQL VQSGAE VKKPGASVKVSCKASG YTFIDYEIHV7VRQAPGQGLE WKGAIDPETGGTAYNQKFKGRVTMTTDTSTS tayme LRS LRSDDTAVTf CTRILLYYYPTOYVIGQGTLVTVSSAST GPSVFPLAPSSKSTSGGTAAL ^ ^ S-VP CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWT S SLGTQT YICMVNIIKPSNTKVDKKVE PKS CDK-THTCPPC PAPELLGGPS VFLFP PKP KDTLM Ϊ E SRTP VTCWVDVSHEDPEVKFNWYVDGVEVHNAKT

Kpreeqymstyrwsvltvijiqdwlhgkeykckvsnkalpapiektiskakgq ^ repqvytl

P PSRDELTKNQVSLTCL VKG FYPSDIA VEW AND SNGQ PENWYKTTPPVLDSDG S FFLYS KLTVDKSRWQQGNVFS C SVMIfEALHNHYTQKSLS L SPGK

SEQ.I.D.NO 20

MGWSCIILFLVATATGVHSQVQLVQSGADVKXPGASVKVSCKASGYTFIDYEIH ^ QAPGQGLEWMGAID-PETGGTAYNQKPKGRVTM ^^^

MDYWGGGTLWVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV ^

Gvhtfpavlqssglyslssvvtvpssslgtqtyicnvmhkpshtkvdkkvepkscdkthtcp

PCPAPELLGGPSVFLFPPKPKDTL ISPvTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHKAKTKPREEQ YN ^ YR ST WS VLTVLHQDWLNG KEY KCKVS NKA G PA PIEKTISKAKGQPRE PQVYTLP PSRDELTXMQVS LTCLVKG FYPS DIAVEW S ^ SMGQPEMÍYKTTPP VLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVI IHEALHNHYTQKSLSLSPGK

SBQ.I.D.NO 21

MGWS CIILFLVATÂTGVHS QVQL VQS-AS GAD VKKPG VKVSCKASG YTFIDYEIHWVRQAPGQGLÉWNGAIDPETGGTAYWQKFKriRATLTÃDKSTSTAYMELRSLRSDDTAVYYCTRI LLYYYPMDYWG TLVTVSSASTKGPSVFPI ^ ^ S PSSKSTSGGTAAIiGCLVKDYFPEPVWSWNSGALTSGVHTPPAVLQS SGL YSLSS WTVPS SLGTQT YICNWHKP SOTKVDKKVE PKS CDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMIS RTPBVTC WVD VSHEDPE VKFlíWíVDGVBVHNAKTKPREEQYNS TYR WS VLTVLHQDWLNGKEYKC KVSNKALPAPIE KTISKAKGQPREPQVYTLPPSSDELTKNOVSLTCLVKGFyPSDIAVEWESNGOPENWYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ.I.D.NO 22

MGWSC11LFLVATATGVKSEIVLTQSpatls ls ls PGERATLSCRASQMlSDYLiiWYQQKPGQAPRLL γyyasqs τSGIPARFSGSGSGTDFTLTISS PEDFAvyYCQNGHSFPLTFGGGTKVEIkrtvaaps VFIFPPSDEQLKSGTAS wcllunfyprea KVQWKVDNALQSGKSQE SVTEQDSKDSTYS lss TLTLSKADYBKHKVYAcevthqglsS pvtks FNRGEC

SEQD.MO 33

MGWSCIILFLVATATGVKSÉlVLTQS PATLSLS PGERATLSCRASQN1SDYLHWYQQKPGQAPRLLIYYASQS ΓSGlFARFSGSGSGTDFTLTIS SLE PED FAVYYCQNGHSFPLTLGGGTKVEIKETVAAPSVFIFPPSDSGLKSGTASWCLLNiiFYPREAKVGWKVDNALOSGNSGESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SBQ.I.D.MO 24

mgws ciilflvatatgvhseivltqs fatlsls pgeratlscrasqnisdylhwyqqkpgqapelliyyasqsisgiparfsgsgsgtd ftlt inslepedfâv ^ ycqnghsfpltixxkírtkveikrtvaapsvpifppsdeijljg ^^ ^^ ^^

SKDST YSLS S T LTLS KADY AND KB KtZY ACEVTHQGL S P VTKS FMRGECSEQ.I.D.NO 25

CAGGTTCAJiCTGCAGCAGTCTGGGGCTGACCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTC

ctgcaaggcttcgggctacacati-tattgactatgaaatacactggatgaagcagacacctg

tgcatcgcctggajíTGGA1TGGAC-CTATTGATCC1'GAAACTGGTGGTACAGCCTATAATCAGaagttcaagggcaaggccattctgactgcagacaaatcctccxgtacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgtacaagaattctcttatattactATCCTATGGACTACTGGGGTCAAGGGACCTCAGTCAC / ÍGTCTCCTCA

seq.i.d.no 26

GACATTGTGATGACTCAGTCTCCAGCCACCCTaTCTGTGACTCCAGGAGATAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAATATTAGCGACTACTTACACTGGTATCAACAAAAATCACATGAGTCTCCWÍGGCI ^ CTCATCAAATATGCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGIXGGATCAGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTGGAGTGTÃTTACTGTClfiAAATGGTCATGGAGCTGAAA

seq <i .d.kio 27

atgggctggagctgcatcatcctgttcctggtggccagcgccaccggcgtgcacagcctggtgcagagcggcgccgaggtga / igaagcccggcgccagcgtgaaggtgagctgca

aggccagcggctacaccttc ^ tdgactacgagatccactg ^ tgcgccaggcccccggccag

ggcctggagtggatgggcgccatcgaccccgagaccggcggcaccgcctagaaccagaagrt

caagggccgcgtgaccatgaccaccgacaccagcaccagcaccgcctacatggagctgcgca

gcctgcgcagcgacgacaccgccgtgtactactgcgcccgcatcctgctgtagtactaccccatggactactggggccagggcacactagtcacagrctcctca

seq, i. b, no 28

atgggctggagctgcatcatcctgttcctggtggccaccgccaccggcgtgcacagcctggtgcagagcggcgccgaggtg.aagaagcccggcgccagcgtgaaggtgagctgca

aggccagcggctacaccttcatcgactacgagatccactgggtgcgccaggcccccggccaggggctggagtggatgggcgccatcgaccccgagaccggcggcaccgcctagaaccagaagttcaagggccgcgtgaccatgaccaccgacaccagcaccagcaccgcctacatggagctgcgcagcctgcgcagcgacgacaccgccgtgtactactgcacccgcatcctgctgtactactaccccatggactactggggccagggcacactagtcacagtctcctca

SEQ.I.D.MO 29

atgggctggagctgcatcatcctgttcctggtggccaccgccaccggcgtgcacagcctggtgcagagcggogccgacgtgaagaagcccggcgccagcgtgaaggtgâgctgca

aggccagcggctacaccttcatcg / dctacgagatccactgggtgcgccaggcccccgggcagggcctgggcgccatcgaccccgagaccggcggcagcgcacacaaccagaagtt

caagggccgcgtgaccatgaccaccgacaccaggaccagcaccgcctacatggagctgcgcaggctgcgcagcgacgacaccgccgtgtactactgcacccgcatgctgctgtactactaccccatggactactggcacctag

sbq-i ^ d, no 30

31 ^ atgggctggagctgcatcatcgtgttcctggtggccaccgccaccggcgtgcacagccaggtgcaggtggtgcagagcggcgccgacgtgaagaagcccggcgccagcgtgaaggtgagctgcaaggccagcggctacaccttcatcgactacgagatccactgggtgcgccaggccgccggccaggggctggagtgoatgggcggcatcgaccccgagaccggcggcaccgcctacaaccagaagttcaagggccgcgccaccctgaccgccgacm gagcaccagcaccgcctacatggagctgcgcagcctgcgcagcgacgacaccgccgtgtactactggacccgcatcgtgctgtactactaggccatggactactggggccagggcacactagtcacagtctcctcaseq.i.d.no

atgggctggagctgcatcatcctgttcctggtggccaccgccaccggcgtgcacagcgagatcgtgct-gacccagagccccgccaccctgagcctgagccccggcgagcgcgccaccctgagctgccgcgccagcca <macatcagcgactacctgcactggtaccagcagaagcccggccaggccccccgcct-gctgatctactacgccagccagagcatcagcggcatccccgcccgcttcagcgg

cagcggcagcggcaccgacttaicccrgaccatcagcagcctggagcccgaggacttcgccgtgtactactgccagaacggccacagcttcccccrgaccttcggcggcggcaccaaggtggagatcaag

seq.i.d.no 32

atgggctggagctgcatcatcctgttcctc-gtggccaccc-ccaccggcgtgcacagcgagatcgtgctgacccagagccccggcccctgagcctgagccccgcggccaccctgagctgccgcggcgcggcggcgg

ccccgcctgctgatctactacgccagccagagcatcagcggcatccccgcccgccccggcagcggcaccgacttcaccctgaccatcaggagcctggagcccgaggacttcgccgtgtactactgggcggggcgggggg

atcaag

seq.i.d.no 33

atgggctggagctgcatcatcctgttcctggtggccaccgccaccggcgtgcacagcgagatcgtgctgacccagagccccgccaccctgagcctgagccccggcgagcgcgccaccctgagctgccgcgccaggcagaacatcagcgactacctgcactggtaccaggagaagcccggccaggccccccgcctgctgatctactacgccagccagagcatcagcggcatccccgcccgcttcagcggcagcggcagcggcaccgacttgaccctgaccatcíacagcctggagcccgaggacttcgccgtgtactactgccagaacggccacagcttccccctgaccctgggcggcggcaccaaggtggagatcaag

seq.i.d.no 34

atgggctggagctgcatcatcctgttcctggtggccaccgccaccggcgtgcacagccaggtgcagctggtgcagagcc ^^ cgccgaggtgaagaagcccggcgccagcgtgaaggtgagctgcaaggccagcggctacaccttcatcgactacgagatccactgggtgcgccaggcccccggccagggcctggagtggatgggcgccatcgaccccgagaccggcggcaccgcctacaaccagaagttcaagggccgcgtgaccatgaccaccgacaccagcaccagcaccgcctacatggagctgcgcagcctgcgcagcgacgacaccgccgtgtactactccgcccgcatcctgctgtactactaccccatggagtactggggccagggcacactagtcacagtctcctcagcctccaccaagggccgatcggtcttccccctggcaccgtcctccaagagcacctctgggggcácagcggccctgggctgcc

tggtcaaggactacttgcccgaa, ccg <3tgaçggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcgtcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctiicatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaaga- ^ ^ agttgagcccaaatcttgtgac aaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtgagtcttcctcrtcccgccaaaacccaa

ggacaccctcatgatctcccggacccctgaggtc ^ catgcgtggtggtcgacgtgagccacg

aagaccctgaggtcaagrtcaactggtâcgtggacggcgtggaggtgcataatgccaagaca

aagccgcgggaggagcagtacaacagcacgtacggtgtggtcagcgtcctcaccgtcctgca

ccaggactggctgaatggcaaggagtacaagtgcaaggtctggaacaaagccctcccagccc

ccatcgagaaaaccatctccaaagccaaaggggagccccgagaaccacaggtgtacaccctg

cccccatcccgggatgaggtgaccaag / i ^ iccaggigagcctgacctgcctggtcaaaggctt

ctatgccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatggatgaggctctgcacaagcactacacgcagaagagcctctccgtgtctccgggtaaatga

seq.i-d.no 3s

atggggtggagctgcatcatcctgttcctggtggccaccgccaccggcgtgcacagccaggtgcagctggtgcagagcggcgccgaggtgaagaagcccggcgccagcgtgaaggtgagctgcaagggcagcgggtacaccttcatggactacgagarccactgggtgggccaggcccccggccagggcctggagtggatgggcgccatcgaccccgagaccggcggcaccgcctacaaccagaagtt

caagggccgcgtgaccatgaccaccgaçaccsígcagcagcaccgcctacatggagctgcgcagcctgcgcagcgacgacaccgccgtgtactactgcacccgcatcctgctgtactactaccccatggactactggggccagggcacactagtgacagtctcctcagcctccaccaagggcccatcggtcttgcccctggcaccctcctccaagagcaccrctgggggcacagcgggcctgggctgcc

tggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccgtgagcagc

ggcgtgcacaccttcctggctgrcctacagtgctcaggactctactccctcagcagcgtggtgaccgtgccgtccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacacgaaggtggagaagaaagttgagccgíu ^ ítcttgtgacaaaactgacacatggccaccgtgccgagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaa

ggacaccctcatgatctcccggaccgctgaggtcacatgcgtggtggtggacgtgagccacgaagac cctg aggtcaagttcaactggtacg tgga cggcgtggaggtgc ataatgccaagaca

aagccgcgggag'3agcagtacaacagcacgtaccgtgtggtcagggtcgtgcaccaggactggctgaatggcaíiggagtacaagtgcaaggtctccaacaaagcccrcccagcccocatcgcatcaccaccaccaccaccacc

cgcccatcccgggatgagctgacciagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaaga

ccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcacggtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacggctagccccggtagga

seq.i, d.no £ 3-atgggctggagctgcatcatcctgttcctggtggccaccgccaccggcgtgcâcagccaggtgcagctggtgcagagcggcgccgacgtgaagaagcccggcgccagcgtgaaggtgagctgcaaggccagcggctaçaccttcatcgactacgagatccactgggtgcgccaggcccccggccagggcctggagtggatgggcgccatcgaccccgagaccggcggcaccgcctacaaccagaagttcaagggccgcgtgaccatgaccaccgacaccagcaccagcaccgcctacatggagcxgcgcagcctgcgcagcgacgacaccgccgtgtagtactgcacccgcatcctgctgtactactaccccatggactactggggccagggcacactagtcacagtctcctcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaac agcãcctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggt

gaccgtgccctccagcagcttgggcacccagacctacatctgcâacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccajatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaaggcgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgca

ccaggactggctgaatggc ^ ggagtacmtgtgcaaggtctccaacaaagccctcccagccc

ccatcgagaaa ^ ccatctccaaagccaaagggcagccccgagaaccacaggtgtagaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggcrccttcttcctctagagcaagctcaccgtggacaagagcaggrggcagcaggggaacg tctt ctcatgc tccgtgatgcatgaggctctgcacaacgactacacgcagaagagcctctccctgtctccgggtaaatga

seq.i.d.no 37

atgggctggagctgcatcatcctgttcctggtggccaccgccaccggcgtgcacagccaggt

gcagctggtgcagagcggcgcggacgtgaagaagcccggcgccagcgtgaaggtgagctgca

aggccagcggctacaccttcatcgactacgagatccactgggtgcgccaggcccccggccagggcctggagtggatgggcgccatggaccccgagaccggcggcaccgcctacaaccagaagttcaagggccgcgccaccctgaccgccgacaagagcaccagcãccgcctaciatggagctgcgcagcctgcgcagcgacgacaccgccgtgtactactgcacccgcatcctgctgtactactacccc

^ ^ atggactactggggccagggcacactagtcacagtctcctcagccrccaccaagggcccatcggtcttccccctggcaccctcctccajagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccg accggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctgaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacacga ggtggacaagaaagttgagcccaaatcttgtoacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgâtctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagcegcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatogcaaggagtacaagtgcaaggtctccaacaaagccctccolGÇÇCCCATCGAGAAAACCATCTCCAAAGCC / ^ GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT

ctatcccagcgacatcgccgtggagtgggagagcâatgggcagccggagaacaactacaagaccacgcgtcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggac

aagagcaggtggcagcaggggaacgtcttgtcatggrccgxgatgcatgaggctctgcacaa

ccactacacgcagaagagcctctccctgtctccgggtaaatga

seq.i.d.ho 38

atgggctggagctgcatcatcctgttcctggtggccaccgccaccggcgtgcacagcgagatcgtgctgacccagagccccgccaccctgagcctgagccccggggagcgcgccaccctgagct

GCCGCGCCAaCCAGAACATCAGCGACTACCTGCACTGGTACCAGCAGAAGCCCGGCGAGGCCCCCCGCCTGCTGATCTAC-TACGCCAGCCAGAGCATCAGCGGCATCCCCGGCCGCTTCAGCGGCAGGGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGAACGGCCACAGCTTGCCCCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCC1GCTGAAT.AA.CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGACA-ACGCCCTCCJi-ATCGGGTAAC-rCCCAGGAGAGTGTCACAGAGCAGGAC

agcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaâgcagactacgagaa

ACACAAAGIGTAGGCCTGCGA / IGTCACCCATCAC-GGCCTGAGCTCGCCCGTCACAAAGAGCTtcaacaggggacsagtgtmg

SBQ.I.D.NO 39

ATGGGGTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGGACAGCGAGATCGTGCTGACCCAGAGCCCCGCCACGCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCA-GCCAGAACATCAGCGACTACCTGCACTGGTÂCGAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACTACGCCAGCCAGAGCATCAGCGGGATCCCCGCCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACC / ^ TCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGGCAGAACGGCCACAGCTTCCCCCTGACrCTGGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGGTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA

atctggaactgcctcigttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgga ^ ggtggacaacgccctccaatcgggtjíactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgaggaaagcagactacgagaaacacaaagtctaggcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagaggttcaacaggggagagtgttag

seqti ^ dfnq 4.0

atgggctgg ^ vgctgcatcatcctgttcctggtggccaccggcaccggcgtgcacagcgagatcgtgctgacccagagccccgccaccctgagcctgagccccggcgagcgcgccaccgtgagct

gccgcgccagccagaacatcagcgactacctc-cactggtaccagcagaagcccggccaggccCCCCGCCTGCTGATCTACTACGCCAGCCAGAGCATCAGCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGC-CACCGACTTCACCCTGACCATCAACAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGAACGGCCACAGCTTGCCCCTGÃCCCIGGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAAGTGCCTCTGTTGTGTGCCTGCTG ^ T ^ CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTmG

SEQ ■ I .D.UO;: 41

S P VPPSTAL KELl EELVlfI TQNQKAPLiCNGSMVWS I NL> T: AG VYCAALES LINVSGCS Al EKTQRMLNGF C PHKVSAGQFS S LR VRDTKIEVAQ FVKDLLVHLKKLFREGQFN

SEQ.1.D.NO:42

AGCCCTGTGCCTCCCTCTACAGCCCTCAAGGAGCTCATTGAGGAGCTGGTCAACATCACCCA

gaaccagaaggccccgctctgcaatggcagcatggtgtggagcatcaacctgacagctggcg

TGTACTGTGCAGCCCTGGAATCCCTGATCMÍCGTGTCAGGCTGCAGTGCCATCGAGAAGACCCAGAGGATGCTGAACGGArTCTGCCCGCACAíiGGTCTCAGCTGGGCAGrTTTCCAGCTTGCGTGTCCGAGACACCAAAATCGAGGTGGCCCAGTTTGTAAAGGACCTGCTCGTACATTTAAAGAAACTTTTTCGCGÃGGGACAGTTCAACTGA

SEQ.I.D.NO; 4 3MGWSC11 LFLVAmiTGVHS

SEQ ■ I.D.HO: 44

SEQ ♦ X. D, NO; 45

EIVLTQSPATLS hS PGERATLSCftAS QS VS Y YLAVTf QQKPGQAPRLLIYDASNRATGI PiJiFSGSGSGTDFTLTISS LBPEDFAVYYCXXXXXXXXXFQGGTKVEΪ K

SEQ, I.D.NO14 6

SGSGPSTALRSLIEELVNITSEQ.I.D.NO:4-7

SGSGIfRBLI BE LVNITQNQKSBQ * I, D.NO; 48SGSGISELWITQNQKA.PLCSEQ.I.D.NO; 4 9SGSGWI TQMQKAPI1CfiGSMS SQ. 1. D. MO: 5 QS GSGQIIQKAPLCiNG SHVWS1SBQ. NO51SGSGAPLCNGSMVWSINLTASBQ ID, IfD.NO 52SGSGNGSMWSIttfLTAGMYCSBQ i, X i .D .NO 5.3SGSGVWSlULTAGMYCAALESEQ.IDNO: 54SGSGNLTAGMYCAALESLINSBQ.IDNO: 5 5SGSGGMYC & ÃLESLINVSGCSEQ.I, D.NO: 5 € SGSGAALE SLINVS GCSAIESEQ ♦ I.DvNO: 57SGSGSLINVSGCSAIEKTQRSEQ.ID .NO : 58SGSOVSGCSAIEKTGRK0E.SGSEQ. I. D .Μ>; 59SGSGSAIEKTQRMLSGFCPHSEQ.1.D.NO. 60SGSGRfQ-RMLSGFCPHKVSASEQ.I.D.NO:61SGSGMLSGFCPMKVSAGQFSSEQ.I.D.NO:62SGSGFCPHKVSAGQFSSLHVSEQ. I.D.NO:63SGSGKVSAGQFS SLHVRDTKSEQ.I.D.NO:64SGSGGQFSSLHVRDTKrEVASBQ.I.D.NO:65SGiSGSLHVRDTKI EVAQFVKSEQ.I.D.NO:66SGSGRDTKIEVAQFVKD LLLSBQ, I. D.NO:67SGSGIEVAQFVKDLLLHbKKSEQ.I.D.NO:68SGSGQFVKDLLLHLKKLFRS

SEQ> I .D.MOi 69SGSGDLLIiHLKKLFREGRFirSEQ. I.D.KSO: 70SGSGFSTALXELIEELVNITSEQ. I. D.NO:71S GSGLKELIEELVWITQNQKSEQ.I.D.NO:72SGS GMGSWWS INLmGVYCSEQ.I.D.NQ: 73SGSGWS INtTAGVYCAAtESEQ, I.D.NO: 74SGSGNLTAGVYCAALES LIN

SEQ.I.D.NO:7 5SGSGGVYCAALESLINVSGCSEQ, I.D.NO t7 6SGSGVSGCSAIEKrQRMLNGSEQ.I, DjNG: 77SG SGSAIE KTQRMLNGF CPHSgQtI.D.NO:78SGSGKTQRKNG I.D, NQ; 79

SGS GMLNGFCPHKVSAGQPSSÉQ.1.D, M0: gQSGSGFCFHKVSAGQFSSLRVSEQ, I.P> KO: S1SGSGKVSAGQFSSL ^ VRDTXSBQ-I, D.KOt82

SGSGGQFS SLRVRDTKIEVASEQ. 1. D. N0_s S 3SGSGSLRVEDTKIEVAQFVKSEQ.I.D.K0: 84SGSGRDTKIE VAQF VKDLWSBQ.I.D.WOtSS

SGSGiBvaqfvkdllvhlkk

SEQ.I.D, H0: 86SGSGQFVKDLLVHXiKKLFRESBQ.I.D, N0; B7SGSGDLLVHLKtCLFREGQFNSEQ, NO, D.NO: SBDliliLHLKKLFRSGRFNSEQ.I.D, NOi89lllvhlk

140

seq. i. d. this: 90

qfvkpllmlkklfregefn

seq.i.d. wo: sl

llhlkxlfreg

seq.i.d.nq

lkklfre

seq id no, 93

caggtgcagctggxgcagagcggagccgaggtgaagaagcctggcgccagcgtcaaggtgtcctgcaaggccagcggctacaccttcatcgactacgagatccactgggtgcggcaggct

gctggacagggcctggaatggatgggcgccatcgaccccgagacaggcggcaccgcctac

aaccagaagttc ^ iagggccgggtcaccatgaccaccgacaccagcaccagcaccgcctat

atggaactgcggagcctgagaagcgacgacaccgccgtgtactactgcacccggatcctgctgtactactaccccatggactactggggccagggcacactagtcccccttagagcagc

ssq id ko. s4

gagatcgtgctgaccgagagccccgccaccctgagcctgagccctggcgagcgggccaccctgtcctgccgggggagccagaacatcagcgactacctgcactggtatcagcag, oagcccggccaggcgcccaggctgctgatciactacgccagccagtccatctccggcatccccgccaggttc gcggcagcggctccggg ^ ^ ^ ccgacttcacgctgaccatcagctctctggaacgcgaggacttcgccgtgtattattgccaga cggccacagcttccccctgacctttggcggcggaacaaaggtggagatcaag

sbq id mo. ss

atgggatggagctgcatcatcctcrrcctggtggccacggclaccggggrgc ^ tagccaggtgckgctcgtccagtctgg ^^ ec ^

aggccagcggctataccttcatcgactacgagatccattgggtgaggcaggctcccgggcagggcctggagtggatgggcgccatcgacccagagaccggaggcacggcgtacaaccagaagttcaagggacgggtcaccatgacaaccgataccagcacctccaccgcttacatggagctgcgcagcctgagaagcgacgacaccgcggtgtactactgtacgcgcatcctgctctactactacccc

^ c ^ atggattactggggccagggcacactagtcacagtctcctcagcctccaccaagggcccatcggtcttgcccctggcaccctcctccaagagcacttctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctcx aggagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagc accaaggtggacaagaaagttgagccc AAATCTTGTGA caaaa CTC acac atgcccaccgtgcx: cãgavcctgaactcctggggggãccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagaca

aagccgcgggaggagcagtacaacagcaxgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaâgggcâgccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacggctcccgtgctggactccgacggctcgttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcâggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggta ^ jítga

ssq id no, 96atgggatggagctgcatcatcctc ^

gcagçtcgtccagtctggggccgacgtgí-agaívgcccggagcttctgtgaaggtgtcctgcaaggccagcggctataccttcatcgactacgagatccattgggtgaggcaggctcccgggcagggcctggagtogatgggcgccatcgaccca ^ gaccggaggcacggcgmcaaccagaagttcaagggacgggtcaccatgacaaccgataccagcacctccaccgcttacatggagctgcgcagcctgagaagcgacgacaccgcggtgtactactgtacgcgcatcctgctctactactacccc

atggattactggggccagggcacactagtcacagtctcctcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggggtgcacaccttcccggctgtcctacagtcctcaggactctactccctgagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaaggcca

gcaacaccmggtggacaagaaagttgag

ccgtgcccagcacctgaagtcctggggggaccgtcagtcttcctcttccccccaaaacccaa

ggacaccctcatgatcrcccggacccctgaggtcacatgcgtggtggtggacgtgagccacg

aagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagaca

aagccgcgggaggagcagtacaacagcacgtaccgtgtgc-tcagcgtcctcaccgtcctgga

ccaggactggctgaatggcaaggagtacaagtgca / iggtctccaacaaagccctcccaggcc

ccatcgagaa / accatctccaaagccaaagggcagccccgagaagcacaggtgtacaccctgcccccatcccggg atg agct ^ acc aa.g aac c aggtcag cctg acctgcctggtcaaaggcttctatcccagcgggaggagggggggggggggggggggg

ccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacgggtagcctgcctgcccggtag 97

ATGGGATGGTCTTGTATCATCCTGTTCCTGGTGGCGACCGCCACCGGCGTGCACTCCGAGATCGTGCTGACCCAGAGTCCAGCCACCCTCAGCCrGAGCCCTGGGGAACGCGCCACCCTGTCCTGCCGGGCGAGT <Gi ^ ^ ^ CATCTCCGACmCCTGCATTGGTACCAGCAGAAGCCCGGCCAGGCCCCTCGCCTGCTGATCTACTACGCCTCCCAC3AGCATCAGCGGAATCCCCGCGCGGTTCTCCGGAAGTGGGTCCGGAACCGACTTTACCCTGACCATCAGCTCTCTCGAGCCAGAGGACl-rCGCGGTGTACTACTGCCAGZiACGGGCATAGTTTCCCACTGACCTTCGGAGGGGGCAC AAGGTGGAGArCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGG AAGTGCCnrGTGTTGTGTGCCTGCTGAATAACTTCTATCCGAGAGAGGCCAAAGTAC

agtggaaggtggaca ^ cgccctgcaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacggggaggaggaggaggagggggggggaggaggaggaggaggaggaggaggaggagg

SEQ ID KQ; 93

Claims (46)

Antibody or antigen-binding fragment thereof, characterized in that it specifically binds hIL-13 and comprises CDRH3 as defined in SEQ ID NO: 3 or variants thereof, wherein one or two amino acid residues within said CDRH3 differ from amino acid residues at the corresponding position in SEQ ID NO: 3. Antibody or antigen-binding fragment thereof according to claim 1, characterized in that it neutralizes human IL-13. Antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that it modulates the binding of human IL-13 to its receptor. Antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that oCDRH3 comprises the sequence of SEQ ID NO: 3. Antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that it further comprises one or more of the following sequences CDRH2: SEQ IDNO: 2, CDRH1: SEQ ID NO: 1, CDRL1: SEQ ID NO: 4, CDRL2: SEQ ID NO: 5 and CDRL3: SEQ ID NO: 6 or a variant thereof, wherein one or two amino acid residues within said CDR differ from the amino acid residues at the corresponding position in SEQ ID NO. Antibody or antigen-binding fragment thereof according to any one of the preceding claims, characterized in that it comprises the following CDRs: CDRH1: SEQ ID NO: 1CDRH2: SEQ ID NO: 2CDRH3: SEQ ID NO: 3CDRL1: SEQ ID NO: 4CDRL2: SEQ ID NO: 5CDRL3: SEQ ID NO: 6 Antibody or antigen-binding fragment thereof, characterized in that it binds to peptides shown in SEQ IDNOs: 90, 99, 102, 103, 105, 106, 107, 108, 109, 110, 111, 112 and 114 but do not bind to the peptides shown in SEQ ID NOs: 100, 101, 104 and 113. Antibody or antigen-binding fragment thereof according to any preceding claim, characterized in that it specifically binds to the epitope set forth in SEQ ID NO: 91. Antibody or antigen-binding fragment thereof according to any preceding claim, characterized in that the antibody is an intact antibody. Antibody or antigen-binding fragment thereof according to claim 9, characterized in that the antibody is a rat, mouse, primate (for example cynomolgus, Old World monkeys or Anthropoid Ape) antibody or human being. Antibody according to any one of Claims 1 to 9, characterized in that it is a humanized or chimeric antibody. Antibody according to any one of claims 9 to 11, characterized in that it comprises a human constant region. Antibody according to Claims 9 to 12, characterized in that it comprises a constant region of the IgG isotype. Antibody according to Claim 13, characterized in that it is IgG1, IgG2 or IgG4. An antibody according to claim 6, characterized in that it comprises a VH domain of SEQ ID NO: 7 and a VL domain of SEQ ID NO: 8. Humanized antibody according to claim 6, characterized in that it comprises a VH domain of SEQ ID NO: 12 and a VL domain of SEQ ID NO: 15. Humanized antibody according to claim 6, characterized in that it comprises a VH domain of SEQ ID NO: 13 and a VL domain of SEQ ID NO: 15. Humanized antibody according to claim 16 or 17, characterized in that it further comprises a human constant region of an IgG isotype (e.g., IgG1 or IgG4). 19. Humanized antibody, characterized in that it comprises a heavy chain of SEQ ID NO: 19 and a light chain of SEQID NO: 22. 20. Humanized antibody, characterized in that it comprises a heavy chain of SEQ ID NO: 20 and a light chain of SEQID NO: 22. An antigen binding fragment according to any one of claims 1 to 8, characterized in that it is a Fab, Fab ', F (ab') 2, Fv, diabody, triacibody, tetribody, minibody, minibody, VHisolate, VL isolated. Antigen-binding fragment according to claim 21, characterized in that it comprises or consists of an ScFv. Antibody according to any one of claims 20, characterized in that it comprises a mutated Fc region, wherein said antibody has reduced ADCC and / or complement activation. 24. A trans-transformed transformed recombinant host cell, characterized in that it comprises a first and a second vector, said first vector comprising a polynucleotide encoding an antibody heavy chain as defined in any preceding claim, and said second vector comprising a polynucleotide encoding a light chain as defined. in any preceding claim. Host cell according to claim 24, characterized in that it is a eukaryotic cell. Host cell according to claim 25, characterized in that it is a mammalian cell. Host cell according to claim 26, characterized in that it is a CHO cell or an NSO cell. Method for producing an antibody as defined in any one of claims 1 to 20 or claim 23, characterized in that it comprises the step of culturing a host cell as defined in any one of claims 24 to 27 in a free culture medium. of serum. The method of claim 28, characterized in that said antibody is secreted by said host cell into said culture medium. The method of claim 29, characterized in that said antibody is further purified to at least 95% or more (e.g. 98% or more) with respect to said culture medium containing the antibody. Pharmaceutical composition, characterized in that it comprises an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 23, and a pharmaceutically acceptable carrier. 32. Kit of parts, characterized in that it comprises a composition as defined in claim 31, together with instructions for use. A method for treating a human patient afflicted with a disease being asthma, comprising the step of administering a therapeutically effective amount of the antibody or antigen-binding fragment as defined in any one of claims 1 to 23. The method according to claim 33, characterized by the fact that said patient is afflicted by asthma, among allergic asthma, severe asthma, fragile asthma, nighttime asthma, premenstrual asthma, steroid resistant asthma, steroid dependent asthma, induced asthma. by aspirin, adult-onset asthma, pediatric asthma. 35. A method for treating a human patient afflicted with a disease being an asthmatic condition that is refractory to corticosteroid treatment, wherein the step comprises administering to said patient a therapeutically effective amount of the antigen binding antibody or fragment as defined in any one of these. claims 1 to 23. A method for preventing acute asthmatic attacks in a human patient comprising the step of administering to said patient a therapeutically effective amount of an antibody as defined in claims 1 to 23. A method for reducing the frequency and / or mitigating the effects of acute asthmatic attacks in a human patient, characterized in that it comprises the step of administering to said patient a therapeutically effective amount of an antibody as defined in any one of claims 1 to 23. 38. Method for treating a human patient afflicted with a disease being a disease or disorder, a group consisting of atopic dermatitis, allergic rhinitis, Crohn's disease, COPD, fibrotic disorders or disorders such as idiopathic pulmonary fibrosis, progressive sclerosis, hepatic fibrosis , hepatic granulomas, schistosomiasis, leishmaniasis, cell cycle regulatory disorders, such Hodgkins disease, chronic B-cell lymphocytic leukemia, characterized in that it comprises administering to a human patient a therapeutically effective amount of an antibody as defined in any of claims 1 to 23. Use of an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 23, characterized in that it is in the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of: allergic asthma, severe asthma, difficult asthma, fragile asthma, nocturnal asthma, premenstrual asthma, resistant steroid asthma, steroid dependent asthma, aspirin-induced asthma, adult onset asthma, pediatric asthma, dermatiteatopic, allergic rhinitis, Crohn's disease, COPD, fibrotic disorders or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis, hepatic fibrosis, hepatic granulomas, schistosomiasis, leishmaniasis, cell cycle regulation disorders such as Hodgkins disease, chronic B-cell lymphocytic leukemia. 40. Method for treating a human patient afflicted with a disease being a disease or disorder selected from the group consisting of allergic asthma, severe asthma, difficult asthma, fragile asthma, nighttime asthma, premenstrual asthma, steroid resistant asthma, steroid dependent asthma, asthma-induced asthma. aspirin, adult onset asthma, pediatric asthma, dermatiteatopic, allergic rhinitis, Crohn's disease, COPD, fibrotic disorders or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis, liver fibrosis, hepatic granulomas, schistosomiasis, leishmaniasis, cell cycle regulation disorders such as Hodgkins disease, chronic B-cell lymphocytic leukemia, characterized in that it comprises administering a therapeutically effective amount of an antibody as defined in any one of claims 1 to 23, and a therapeutically effective amount of an anti-IL-4 monoclonal antibody. . A method according to claim 40, characterized in that the anti-IL-4 monoclonal antibody is administered simultaneously, sequentially or separately with the antibody as defined in any one of claims 1 to 23. Method according to claim 40 or 41, characterized in that the anti-IL-4 antibody is pascolizumab. Use of an antibody as defined in any one of claims 1 to 23, and an anti-IL-4 monoclonal antibody, such as opascolizumab, characterized in that it is in the manufacture of medicament for the treatment of a selected disease or disorder of the group consisting of of: allergic asthma, severe asthma, difficult asthma, fragile asthma, nocturnal asthma, premenstrual asthma, resistant steroid asthma, steroid dependent asthma, aspirin-induced asthma, adult onset asthma, asthma, atopic dermatitis, allergic rhinitis, Crohn, COPD, fibrotic disorders or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis, hepatic fibrosis, hepatic granulomas, schistosomiasis, leishmaniasis, cell cycle regulatory disorders such as Hodgkins disease, chronic B-cell leukemia. Use of an antibody as defined in any one of claims 1 to 23, and an anti-IL-4 monoclonal antibody, such as opascolizumab, characterized in that it is in the manufacture of a part kit for the treatment of a selected disease or disorder. group consisting of: allergic asthma, severe asthma, difficult asthma, fragile asthma, asthma, premenstrual asthma, resistant steroid asthma, steroid-dependent asthma, aspirin-induced asthma, adult onset asthma, asthma, atopic dermatitis, allergic rhinitis, disease Crohn's disease, COPD, fibrotic disorders or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis, hepatic fibrosis, hepatic granulomas, schistosomiasis, leishmaniasis, cell cycle regulatory disorders such as Hodgkins disease, chronic B-cell leukemia. Part kit, characterized in that it comprises a first pharmaceutical composition containing an antibody as defined in any one of claims 1 to 23, and a pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising an anti-IL-4 monoclonal antibody, such as opascolizumab, and a pharmaceutically acceptable carrier, optionally in conjunction with instructions for use. Pharmaceutical composition, characterized in that it comprises a first antibody as defined in any one of claims 1 to 23, and a second antibody, wherein said second antibody is an anti-IL-4 antibody such as pascolizumab and a pharmaceutically acceptable carrier.