JP2014101372A - Human antibody molecules for il-13 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for using anti-IL antibody molecules in diagnosis or treatment of IL-13 related disorders, e.g., asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease and Hodgkin's lymphoma.SOLUTION: The present invention provides specific binding members, in particular, human anti-IL-13 antibody molecules and especially those which neutralize IL-13 activity.

Description

  The present invention relates to specific binding members, particularly human anti-IL-13 antibody molecules, and particularly those that neutralize IL-13 activity. The invention further relates to the use of anti-IL-13 antibody molecules in the diagnosis or treatment of IL-13 related diseases including asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin lymphoma.

A preferred embodiment of the present invention uses the antibody VH and / or VL domains of the antibody molecule designated herein as BAK502G9 and other antibody molecules as defined herein of BAK502G9 and BAK278D6 strains. . A further preferred embodiment uses the VAK CDR3 in the BAK278D6 strain and preferably the complementarity determining region (CDR) of BAK502G9, especially in other antibody framework regions. A further aspect of the present invention provides a composition containing a specific binding member of the present invention, the use of the composition in a method of inhibiting or neutralizing IL-13, including methods of treating the human or animal body. .
The present invention provides antibody molecules of particular value in binding and neutralizing IL-13 and antibody molecules useful in any of the various therapies described herein and in the supporting technical literature. .

  Interleukin (IL) -13 is a 114 amino acid cytokine with an unmodified molecular weight of approximately 12 kDa [1,2]. IL-13 is most closely related to IL-4 and shares 30% sequence similarity at the amino acid level. The human IL-13 gene is located on chromosome 5q31 adjacent to the IL-4 gene [1] [2]. This region of chromosome 5q contains the gene sequence of cytokines from other Th2 lymphocytes, including GM-CSF and IL-5, and the level is in conjunction with IL-4 in rodent models of asthma and allergic inflammation It has been shown to correlate with disease severity [3] [4] [5] [6] [7] [8].

  Although IL-13 was initially identified as a cytokine derived from Th2 CD4 + lymphocytes, IL-13 is also identified as Th1 CD4 + T cells, CD8 + T lymphocyte NK cells, and non-T cell populations (eg, mast cells, basophils, Eosinophils, macrophages, monocytes, and airway smooth muscle cells).

  IL-13 is an IL-4 receptor alpha chain (IL-4Rα) (which itself binds IL-4 but not IL-13) and at least two other cell surface proteins (IL-13Rα1 and It has been reported to mediate its action through receptor systems including IL-13Rα2) [9] [10]. IL-13Rα1 can bind to IL-13 with low affinity and then recruit IL-4Ra to form a high affinity functional receptor that transmits signals [11] [12]. The GeneBank database describes the amino acid sequence and nucleic acid sequence of IL-13Rα1 as NP_001551 and Y10659, respectively. Studies in mice lacking STAT6 (transcriptional signal transducer and activator 6) have demonstrated that IL-13 uses the JAK-STAT6 pathway to signal in mammals, similar to IL-4 [13]. [14]. IL-13Rα2 shares 37% sequence identity with IL-13Rα1 at the amino acid level and binds IL-13 with high affinity [15] [16]. However, IL-13Rα2 has a shorter cytoplasmic tail that lacks a known signaling motif. Cells expressing IL-13Rα2 do not respond to IL-13 even in the presence of IL-4Rα [17]. Therefore, IL-13Rα2 is thought to act as a decoy receptor that regulates IL-13 function but not IL-4 function. This is supported by studies in IL-13Rα2-deficient mice whose phenotype is consistent with an increased response to IL-13 [18] [19]. The GeneBank database describes the amino acid sequence and nucleic acid sequence of IL-13Rα2 as NP_000631 and Y08768, respectively.

  Signaling IL-13Rα1 / IL-4Rα receptor complex is a human B cell, mast cell, monocyte / macrophage, dendritic cell, eosinophil, basophil, fibroblast, endothelial cell, airway epithelial cell, And expressed on airway smooth muscle cells.

  Bronchial asthma is a common persistent inflammatory disease of the lung and is characterized by high airway responsiveness, mucus overproduction, fibrosis, and high serum IgE levels. Airway hyperresponse (AHR) is excessive contraction of the airway to nonspecific stimuli such as cold air. Both AHR and mucus overproduction are thought to be responsible for airway obstruction that causes shortness of breath (aggravation) that is characteristic of asthma attacks, which is attributed to mortality from this disease (about 2000 deaths annually in the UK). Responsible.

  In recent years, the incidence of asthma has increased significantly with other allergic diseases [20] [21]. For example, about 10% of the UK population is currently diagnosed with asthma.

  The Current British Thoracic Society (BTS) and the Global Initiative for Asthma (GINA) guidelines suggest a phased approach to the treatment of asthma [22,23]. Weak to moderate asthma can usually be controlled by using inhaled corticosteroids in combination with beta agonists or leukotriene inhibitors. However, due to the described side effects of corticosteroids, patients tend to fail to follow treatment, which reduces the effectiveness of the treatment [24-26].

  There is a clear need for new therapies for subjects with more severe illnesses for which the high dose inhalation or oral corticosteroids recommended by the Asthma Guidelines are less effective. Long-term treatment with oral corticosteroids causes osteoporosis, delayed child growth, diabetes, and oral candidiasis [88]. Treatment is a balance between safety and efficacy because the beneficial and adverse effects of corticosteroids are mediated by the same receptors. Hospitalization of these patients as a result of worsening severity (about 6% of the UK asthma population) accounts for the majority of the major economic burden of asthma on health authorities [89].

  The pathology of asthma is thought to be caused by ongoing Th2 lymphocyte-mediated inflammation caused by an inappropriate response of the immune system to innocuous antigens. Evidence has accumulated that IL-13 is involved as a major mediator of the pathogenesis of established airway disease, but not the classic Th2-derived cytokine IL-4.

  Administration of recombinant IL-13 to the airways of unsensitized rodents with no medication experience caused many aspects of asthma symptoms, including airway inflammation, mucus production, and AHR [27] [28 ] [29] [30]. Similar symptoms were observed in transgenic mice that specifically overexpressed IL-13 in the lung. In this model, more chronic exposure to IL-13 also caused fibrosis [31].

  Furthermore, in rodent models of allergic diseases, many aspects of asthma symptoms have been associated with IL-13. Soluble mouse IL-13Rα2 (a potent IL-13 neutralizing agent) has been shown to inhibit AHR, mucus hypersecretion, and inflammatory cell influx, which is characteristic of this rodent model [27] [28] [30]. In a complementary study, mice lacking the IL-13 gene did not develop allergen-induced AHR. In these IL-13 deficient mice, AHR recurs upon administration of recombinant IL-13. In contrast, IL-4 deficient mice caused airway disease in this model [32] [33].

  Using a longer-term allergen-induced pulmonary inflammation model, Taube et al. Demonstrated the efficacy of soluble mouse IL-13Rα2 against established airway disease [34]. Soluble mouse IL-13Rα2 inhibited AHR, mucus hypersecretion, and to a lesser extent airway inflammation. In contrast, soluble IL-4Rα that binds to and antagonizes IL-4 had little effect on AHR or airway inflammation in this system [35]. These findings are supported by Blease et al., Who created a chronic fungal model of asthma where polyclonal antibodies to IL-13 but not IL-4 reduced mucus hypersecretion, AHR, and subepithelial fibrosis [36]

  Many genetic polymorphisms of IL-13 have also been associated with allergic diseases. In particular, a variant of the IL-13 gene in which the arginine residue of amino acid 130 is replaced with glutamine (Q130R) is associated with bronchial asthma, atopic dermatitis, and high serum IgE levels [37] [38] [ 39] [40]. This specific IL-13 variant is also referred to as Q110R (the arginine residue of amino acid 110 is replaced with glutamine) by some groups that exclude the 20 amino acid signal sequence from the number of amino acids. Arima et al. [41] report that this variant is associated with high levels of IL-13 in serum. The IL-13 variant (Q130R) and antibodies to this variant are discussed in WO 01/62933. IL-13 promoter polymorphism, which alters IL-13 production, is also associated with allergic asthma [42].

  High levels of IL-13 have also been measured in asthma, atopic rhinitis (hay fever), allergic dermatitis (eczema), and chronic sinusitis. For example, IL-13 levels were higher in bronchial biopsy, sputum, and bronchoalveolar lavage (BAL) cells of asthmatic patients compared to control subjects [43] [44] [45] [46]. Furthermore, IL-13 levels in BAL samples were increased by allergen challenge with asthmatic patients [47] [48]. Furthermore, the ability of CD4 (+) T cells to produce IL-13 has proven to be a useful marker of the subsequent risk of developing neonatal allergic disease [49].

  Li et al. [11 4] recently reported the effect of neutralizing anti-mouse IL-13 antibody in a chronic mouse model of asthma. Chronic asthma-like responses (eg, AHR, severe airway inflammation, excessive mucus production) were induced in OVA-sensitized mice. Li et al. Report that administration of IL-13 antibody upon stimulation of each OVA antigen suppresses AHR, eosinophil infiltration, serum IgE levels, pro-inflammatory cytokine / chemokine levels, and airway remodeling. [14].

  In summary, these data indicate that IL-13 but not IL-4 is a more attractive target for the treatment of human allergic diseases.

  IL-13 plays a role in the development of inflammatory bowel disease. Heller et al. [116] reported that neutralization of IL-13 by administration of soluble IL-13Rα2 alleviates inflammation of the large intestine in a mouse model of human ulcerative colitis [11 6]. Correspondingly, IL-13 expression was higher in rectal biopsy samples from patients with ulcerative colitis than in controls [117].

  IL-13 is associated with other fibrotic symptoms other than asthma. Increased levels of IL-13 (up to 1000 times higher than IL-4) have been measured in sera from patients with systemic sclerosis [50] and BAL samples from patients with other types of pulmonary fibrosis [51] . Correspondingly, overexpression of IL-13 (not IL-4) in the mouse lung caused significant fibrosis [52] [53]. The contribution of IL-13 to non-lung tissue fibrosis has been extensively studied in mouse models of parasite-induced liver fibrosis. Specific inhibition of IL-13 by administration of soluble IL-13Rα2 or IL-13 gene disruption prevented fibrosis in the liver but did not prevent this by reducing IL-4 production [54] [55 ] [56].

  Chronic obstructive pulmonary disease (COPD) includes a population of patients with varying degrees of chronic bronchitis, small airway disease, and emphysema and is characterized by progressive irreversible pulmonary decline that is less responsive to current asthma-based treatment [90]. In recent years, the incidence of COPD has risen dramatically and has become the fourth leading cause of death worldwide (World Health Organization). Therefore, COPD is a major unmet medical need.

  The cause of COPD is not well understood. The “Dutch hypothesis” proposes that there is a common susceptibility to COPD and asthma, and that similar mechanisms contribute to the pathogenesis of both disorders [57].

  Zheng et al. [58] showed that overexpression of IL-13 in mouse lung caused emphysema, high mucus production, and inflammation, reflecting aspects of human COPD. Furthermore, AHR, an IL-13-dependent response in a mouse model of allergic inflammation, has been shown to be able to predict reduced lung function in smokers [59]. An association between the IL-13 promoter polymorphism and the ease of onset of COPD has also been established [60].

  Thus, there are indications that IL-13 plays an important role in the pathogenesis of COPD, particularly in patients with asthmatic features, including AHR and eosinophilia. It has been demonstrated that IL-13 mRNA levels are higher in autopsy tissue samples from subjects with a history of COPD compared to lung samples from subjects with no reported lung disease (J. Elias , 2002 oral communication at the American Thoracic Society Annual Meeting). In another study, immunohistochemical studies of peripheral lung sections from COPD patients showed high levels of IL-13 [91].

  Hodgkin's disease is a common type of lymphoma, which has approximately 7,500 cases per year in the United States. Neoplastic Reed-Sternberg cells (often obtained from B cells) represent only a small portion of the clinically detected mass, so Hodgkin's disease is rare among malignant tumors . Hodgkin's disease-derived cell lines and primary Reed-Sternberg cells often express IL-13 and its receptor [61]. Since IL-13 promotes cell survival and proliferation in normal B cells, it has been proposed that IL-13 may act as a growth factor for Reed-Sternberg cells. Skinnider et al. Demonstrated that neutralizing antibodies against IL-13 can inhibit the growth of Hodgkin's disease-derived cell lines in vitro [62]. This finding suggested that Reed-Sternberg cells enhance their survival by IL-13 autocrine and paracrine cytokine loops. Consistent with this hypothesis, high levels of IL-13 have been detected in the serum of some Hodgkin's disease patients when compared to normal controls [63]. Thus, IL-13 inhibitors can prevent the spread of disease by inhibiting the growth of malignant Reed-Sternberg cells.

  Many human cancer cells express immunogenic tumor-specific antigens. However, many tumors regress spontaneously, but some slip through the immune system (immune surveillance) by suppressing T cell immunity. Terabe et al. [64] demonstrated a role for IL-13 in immunosuppression in a mouse model in which the tumor spontaneously regressed and then recurred after initial growth. Specific inhibition of IL-13 by soluble IL-13Rα2 protected these mice from tumor recurrence. Terrabe et al. [64] subsequently demonstrated that IL-13 suppresses the differentiation of tumor-specific CD8 + cytotoxic lymphocytes that mediate anti-tumor immune responses.

  Thus, IL-13 inhibitors could be used therapeutically to prevent tumor recurrence or metastasis. Inhibition of IL-13 has been shown to enhance antiviral vaccines in animal models and may be beneficial in the treatment of HIV and other infectious diseases [65].

  When referring generally to interleukin-13 or IL-13 herein, it should be noted that human IL-13 is indicated unless otherwise stated. This is also referred to as “antigen” instead. The present invention provides antibodies against human IL-13, particularly human antibodies, that cross-react with non-human primate IL-13, such as IL-13 of cynomolgus or rhesus monkey. The antibody in some embodiments of the invention recognizes a variant of IL-13 in which the arginine residue at amino acid position 130 is replaced with glutamine. In other aspects and embodiments, the present invention provides specific binding members for murine IL-13, particularly mouse IL-13.

FIG. 1 shows the neutralization titer (% inhibition) of BAK167A11 (black square) and its derivative BAK615E3 (white square) as scFv against 25 ng / ml human IL-13 in a TF-1 cell proliferation assay. Triangles are irrelevant scFv. Data show the average of triplicate measurements within the same experiment, with standard error bars. FIG. 2 shows the neutralization titer (% inhibition) of BAK278D6 (black square) and its derivative BAK502G9 (white square) as scFv against 25 ng / ml human IL-13 in the TF-1 cell proliferation assay. Triangles are irrelevant scFv. Data show the average of triplicate measurements within the same experiment, with standard error bars. FIG. 3 shows the neutralization titer (% inhibition) of BAK209B11 (black squares) as scFv against 25 ng / ml murine IL-13 in the TF-1 cell proliferation assay. Triangles are irrelevant scFv. Data show the average of triplicate measurements within the same experiment, with standard error bars. FIG. 4 shows the neutralization titer (% inhibition) of BAK278D6 (black squares) as scFv against IL-13 in the TF-1 cell proliferation assay. Triangles are irrelevant scFv. Data show the average of triplicate measurements within the same experiment, with standard error bars. FIG. 4A shows the titer against 25 ng / ml human IL-13. FIG. 4B shows the titer against 25 ng / ml human variant IL-13. FIG. 4C shows the titer against 50 ng / ml non-human primate IL-13. FIG. 5 shows a comparison of anti-human IL-13 antibody titers in the TF-1 cell proliferation assay. The data shows the mean neutralization titer of 5-7 experiments against 25 ng / ml human IL-13 with a standard error bar. Performance compared to a commercially available antibody (B-B13) was statistically evaluated by one-sided ANOVA using Dunnett's test. Compared with B-B13, * P <0.05, ** P <0.01. FIG. 6 shows the neutralization titers (% inhibition) of BAK502G9 (black squares), BAK1167F2 (black triangles), and BAK1183H4 (black inverted triangles) as human IgG4 against labeled IL-13 in the F-1 cell proliferation assay. . Open triangles are irrelevant IgG4. Data show the average of three different experiments with standard error bars. FIG. 6A shows the titer against 25 ng / ml human IL-13. FIG. 6B shows the titer against 25 ng / ml human variant IL-13. FIG. 6C shows the titer against 50 ng / ml non-human primate IL-13. FIG. 7 shows BAK502G9 (black squares), BAK1167F2 (black triangles), BAK1183H4 (black inverted triangles) as human IgG4 in a native IL-13-dependent HDLM-2 cell proliferation assay, and commercially available anti-human IL-13. The neutralization titer (% inhibition) of an antibody (B-B13-white square; JES10-5A2-white inverted triangle) is shown. Open triangles are irrelevant IgG4. Data show the average of triplicate measurements within the same experiment, with standard error bars. FIG. 8 shows a comparison of anti-human IL-3 antibody titers in the NHLF assay. The data shows the mean neutralization titer (IC ₅₀ pM) of 4-5 experiments against 10 ng / ml human IL-13 in the NHLF eotaxin release assay with a standard error bar. Performance compared to a commercially available antibody (B-B13) was statistically evaluated by one-sided ANOVA using Dunnett's test. Compared with B-B13, * P <0.05, ** P <0.01. FIG. 9 shows BAK502G9 (black squares), BAK1167F2 (black triangles), and BAK1183H4 (black inverted triangles) as human IgG4 against VCAM-1 upregulation on the surface of HUVEC in response to 10 ng / ml human IL-13. The neutralization titer (% inhibition) is shown. Open triangles are irrelevant IgG4. Data show the average of triplicate measurements within the same experiment, with standard error bars. FIG. 10 shows human IgG4 against eotaxin release from VCAM-1 upregulation on the surface of HUVEC in response to 1 ng / ml human IL-4 (FIG. 10A) or 0.5 ng / ml human IL-1β (FIG. 10B). As neutralization titers (% inhibition) of BAK502G9 (black square), BAK1167F2 (black triangle), and BAK1183H4 (black inverted triangle). Open triangles are irrelevant IgG4. Data show the average of triplicate measurements within the same experiment, with standard error bars. FIG. 11 shows the neutralizing titer (% inhibition) of BAK209B11 (square) as human IgG4 against 1 ng / ml murine IL-13 in a factor-dependent B9 cell proliferation assay. Open triangles are irrelevant IgG4. Data show the average of triplicate measurements within the same experiment, with standard error bars. FIG. 12 shows IL-13 in lung homogenates from sensitized (s) (right bar) and non-sensitized (ns) (left bar) mice after antigen stimulation in a murine model of acute lung allergic inflammation. Indicates the relative level. The effect of sensitization was statistically assessed by Student's t-test using IL-13 content data. * <0.05, ** <0.01 compared to non-sensitized control animals (n = 5-6 mice). Data show mean with standard error bar. FIG. 13 shows the effect of intravenous administration of BAK209B11 as different amounts of human IgG4 compared to isotype matched IgG4 irrelevant control antibody on ovalbumin-induced leukocyte recruitment to the lungs of ovalbumin sensitized mice. The number of white blood cells is indicated (× 104). The effect of antibody treatment was statistically evaluated by one-sided ANOVA with Dunnett's test using white blood cell percentage data. * <0.05, ** <0.01 compared to ovalbumin challenged PBS control animals (= 0% inhibition; n = 5-8 mice). Data show mean with standard error bar. FIG. 14 shows the effect of intravenous administration of BAK209B11 as a different amount of human IgG4 compared to isotype matched IgG4 irrelevant control antibody on ovalbumin-induced eosinophil recruitment to the lung of ovalbumin-sensitized mice. The number of eosinophils is indicated (x104). The effect of antibody treatment was statistically evaluated by one-sided ANOVA with Dunnett's test using white blood cell percentage data. * <0.05, ** <0.01 compared to ovalbumin challenged PBS control animals (= 0% inhibition; n = 5-8 mice). Data show mean with standard error bar. FIG. 15 shows the effect of intravenous administration of BAK209B11 as a different amount of human IgG4 compared to isotype matched IgG4 irrelevant control antibody on ovalbumin-induced neutrophil recruitment to the lungs of ovalbumin-sensitized mice. The number of neutrophils is indicated (× 104). The effect of antibody treatment was statistically evaluated by one-sided ANOVA with Dunnett's test using white blood cell percentage data. * <0.05, ** <0.01 compared to ovalbumin challenged PBS control animals (= 0% inhibition; n = 5-8 mice). Data show mean with standard error bar. FIG. 16 shows the effect of intravenous administration of BAK209B11 as different amounts of human IgG4 compared to isotype matched IgG4 irrelevant control antibody on ovalbumin-induced lymphocyte mobilization to the lung of ovalbumin-sensitized mice. Lymphocyte induction was inhibited by BAK209B11 in a dose-dependent manner, with maximum inhibition obtained with 3 μg / ml BAK209B11. The effect of antibody treatment was statistically evaluated by one-sided ANOVA with Dunnett's test using white blood cell percentage data. * <0.05, ** <0.01 compared to ovalbumin challenged PBS control animals (= 0% inhibition; n = 5-8 mice). Data show mean with standard error bar. FIG. 17 shows the effect of intravenous administration of BAK209B11 as a different amount of human IgG4 compared to isotype matched IgG4 irrelevant control antibody on ovalbumin-induced monocyte / macrophage mobilization to the lungs of ovalbumin-sensitized mice. There was no significant increase in monocyte / macrophage levels of sensitized animals compared to control animals. However, such background levels of these cells were suppressed by ≧ 36 μg / ml BAK209B11 in sensitized animals. The effect of antibody treatment was statistically evaluated by one-sided ANOVA with Dunnett's test using white blood cell percentage data. * <0.05, ** <0.01 compared to ovalbumin challenged PBS control animals (= 0% inhibition; n = 5-8 mice). Data show mean with standard error bar. FIG. 18 shows a commercially available anti-IL-13 neutralizing antibody against the influx of cells (indicating leukocyte count (× 104)) into a murine air pouch induced by administration of bacterial-derived recombinant human IL-13. The effect | action of JES10-5A2 is shown. The effect of antibody treatment was statistically evaluated by one-sided ANOVA with Dunnett's test using white blood cell percentage data. * <0.05, ** <0.01 compared to CMC control animals (= 0% inhibition; n = 11-13 mice). Data show mean with standard error bar. FIG. 19 shows the alignment of the sequence of cynomolgus IL-13 to the human IL-13 amino acid sequence. Seven amino acid residues that are different between human and cynomolgus IL-13 are blackened. Rhesus monkey (rhesus) and cynomolgus IL-13 have the same amino acid sequence. FIG. 20 shows 10 mg / kg vein of BAK502G9 as human IgG4 against the serum IgE levels of 4 allergic but non-antigenic cynomolgus primates (2 males / 2 females) for 29 days. The effect of a single large dose is shown. Serum IgE concentrations are significantly reduced from 100% of control values (before administration) to 66 ± 10% (p <0.05) at 4 and 5 days after administration. This reduction in serum IgE concentration has recovered to 88 ± 8% of control levels by day 22. * = P <0.05, comparison with pre-dose IgE levels, ANOVA followed by Dunnett's multiple comparison test (n = 4 animals). FIG. 20B shows the time course of relative serum IgE levels in male and female cynomolgus primates after a single intravenous dose of 10 mg / kg BAK502G9. Relative serum IgE data is expressed as the arithmetic mean ± SEM% of baseline values. FIG. 21 shows different amounts (H = 237 μg / day, M = 23.7 μg / day, and L = 2.37 μg) compared to isotype-matched IgG1 irrelevant control antibody for lung function in ovalbumin-sensitized and antigen-stimulated mice. / Day) shows the effect of intraperitoneal administration of BAK209B11. In FIG. 21A, pulmonary function is shown by logPC50s (log methacholine concentration required to increase baseline PenH by 50%) before treatment (day 0) and after sensitization, antigen stimulation, and drug treatment (day 25). . FIG. 21A shows the raw data used to calculate the end point (delta log PC50) of the test shown in FIG. 21B. The data shows the average of n = 8 with standard error bars. In FIG. 21B, the changing lung function is shown by the change in log PC50 (delta log PC50) of individual mice. The delta log PC50 is defined as the individual change in the log PC50 for 25 days relative to day 0. Data show group mean delta log PC50 (individual change averaged within treatment group) with standard error bars. The effect of antibody treatment was statistically evaluated by one-sided ANOVA with Dunnett's test using delta log PC50 data. * P <0.01 compared to ovalbumin antigen sensitized and stimulated control animals (n = 8 mice). Figure 22 shows the locality of BAK502G9 as a different amount of human IgG4 compared to isotype matched IgG4 irrelevant control antibody for total leukocyte recruitment (Figure 22A) and eosinophil recruitment (Figure 22B) to BALB / C mouse air pouches. The effects of general (intraperitoneal) and systemic (intravenous) administration are shown. The data shows the average of n = 10 with standard error bars. The effect of antibody treatment was statistically assessed by one-sided ANOVA with Dunnett's test using log transformed data. * P <0.05, ** P <0.01 compared to huIL-13 antigen stimulated mice (n = 10). FIG. 23 shows the effect of intraperitoneal administration of BAK502G9 as human IgG4 compared to isotype matched IgG4 irrelevant control antibody on the development of AHR after intratracheal administration of human IL-13 to the respiratory tract of mice. The effect of antibody treatment was statistically evaluated by one-sided ANOVA using Dunnett's test using PC200 methacholine data. * <0.05, ** <0.01 compared to the human IL-13 positive control group (n = 6-8 mice). Data show mean with standard error bar. FIG. 24 shows the neutralizing titer (% maximal response) of BAK502G9 (black squares) as IgG4 against 30 ng / ml IL-13 in a human B cell IgE production assay. Open squares indicate irrelevant IgG4. The data shows the average of 6 donors from separate experiments with standard error bars. FIG. 25 shows the effect of BAK502G9 on IL-13-induced enhancement of agonist-induced Ca2 + signaling in bronchial smooth muscle cells. For each antibody +/- IL-13 pretreatment condition, the area under the Ca2 + signaling response curve (AUC) was measured. Combined data from three independent experiments are shown for the irrelevant antibodies CAT-001 (a) and BAK502G9 (b) as percent difference over untreated cells of AUC ± SD (ns = not significant (p> 0.05 ), * P <0.05, ** p <0.01). Results were statistically evaluated by one-sided analysis of variance (ANOVA) using the Bonferroni's multiple post-comparison test. FIG. 26 shows the effect of BAK502G9 administered in Phase II. FIG. 26A shows the effect on AHR measured in area under the histamine dose response curve (n = 14). FIG. 26B shows the effect on AHR measured by changes in PC30 (n = 18). FIG. 26C shows the effect on antigen priming (n = 20). FIG. 26D shows the effect on BAL inflammation (n = 21). FIG. 27 shows the effect of BAK502G9 on IL-13 induced CD23 expression. Data are shown as percent response to IL-13 alone (100%) and are expressed as the mean ± SEM% control of 6 different experiments (performed in triplicate) from each of 6 donors. FIG. 28 shows the effect of IgG4 independent of BAK502G9 on IL-13 and / or IL-4-induced PBMC CD23 expression. Data are shown as percent response to IL-4 alone (100%) and are expressed as the mean ± SEM% control of 4 different experiments (performed in triplicate) from each of 4 donors. FIG. 29A shows the effect of BAK502G9 on NHLF eotaxin-1 induced by 48 hour culture using IL-13 / TNF-α / TGF-β1-containing medium. Data are presented as the arithmetic mean ± SEM of triplicate media used in this study to induce changes in leukocyte shape. FIG. 29B shows the effect of BAK502G9 on the morphological changes of human eosinophils induced by 1:16 dilution of conditioned medium. Data points are the mean ± SEM% blank medium morphological change of separate experiments from 4 independent donors. FIG. 30 shows the alignment of human IL-13 to murine IL-13 highlighting the mutations introduced into human IL-13 to create the first panel of IL-13 chimeras. Four alpha helices are highlighted in the box and loop 1 and loop 3 are shown. Five chimeric proteins have been created, with helices B, C and D and loops 1 and 3 replaced with murine sequences. Four more chimeric proteins were made and numbered according to amino acids in the human preprotein (not the multiple alignment numbering above), the arginine at residue 30 (position 34 above) was mutated, residues 33 and 34 (Positions 37 and 38) are mutated, residues 37 and 38 (VH) are mutated (positions 41 and 42), and residues 40 and 41 (TQ) are mutated (positions 44 and 45). ). FIG. 31 shows the alignment of human IL-13 to murine IL-13 highlighting the mutations introduced into human IL-13 to create a second panel of IL-13 chimeras. Six chimeras were created in which human residues were used instead of murine residues (highlighted in the box). Four more chimeric proteins were created (numbering follows the amino acid position in the human preprotein), leucine at residue 58 (position 62 in the figure above) was mutated, and leucine at residue 119 (residue 123 above) was Mutated, the lysine at residue 123 (residue 127 above) is mutated, and the arginine at residue 127 (residue 132 above) is mutated. FIG. 32 shows the mutations made in human IL-13. The dark gray mutation decreased binding to BAK502G9 and the light gray mutation did not change binding. A linear sequence of prehuman IL-13 and mutated residues is shown.

  In various aspects and embodiments of the present invention, the subject matter of the appended claims is provided.

  The present invention provides specific binding members for IL-13, in particular human and / or primate IL-13 and / or IL-13 variants (Q130R), and mouse IL-13. A preferred embodiment of the present invention is an antibody molecule, which may be a whole antibody (eg, IgG such as IgG4) or an antibody fragment (eg, scFv, Fab, dAb). Like the antibody VH and VL domains, an antigen binding region of the antibody is provided. Within the VH and VL domains, complementarity determining regions (CDRs) are provided, which are provided within different framework regions (FR), optionally forming a VH or VL domain. The antigen binding site may consist of an antibody VH domain and / or a VL domain.

  Antigen binding sites are provided by alignment of CDRs on non-antibody protein backbones such as fibronectin or cytochrome B [115,116]. The framework for creating new binding sites within proteins is reviewed in detail in Nygren et al. [11 6]. The protein backbone of an antibody mimetic is disclosed in WO / 0034784 and the inventors describe a protein (antibody mimetic) comprising a fibronectin type III domain with at least one randomized loop. A suitable scaffold for incorporating one or more CDRs (eg, a set of HCDRs) is provided by any domain member of the immunoglobulin gene superfamily.

  A preferred embodiment of the present invention is referred to herein as the “BAK278D6 line”. This is a set of six CDR sequences of BAK278D6 as follows: HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2), HCDR3 (SEQ ID NO: 3), LCDR1 (SEQ ID NO: 4), LCDR2 (SEQ ID NO: 5), And LCDR3 (SEQ ID NO: 6). In one aspect, the invention relates to a specific binding member for human IL-13, comprising an antibody antigen binding site composed of a human antibody VH domain and a human antibody VL domain and comprising a set of CDRs. Providing a binding member, wherein the VH domain comprises HCDR1, HCDR2, and HCDR3, and the VL domain comprises LCDR1, LCDR2, and LCDR3 (wherein the HCDR1 has the amino acid sequence of SEQ ID NO: 1) The HCDR2 has the amino acid sequence of SEQ ID NO: 2, the HCDR3 has the amino acid sequence of SEQ ID NO: 3, the LCDR1 has the amino acid sequence of SEQ ID NO: 4, and the LCDR2 has the amino acid sequence of SEQ ID NO: 5. Or the LCDR3 has the amino acid sequence of SEQ ID NO: 6); The set is the above set of CDRs (HCDR1 has the amino acid sequence of SEQ ID NO: 1, HCDR2 has the amino acid sequence of SEQ ID NO: 2, HCDR3 has the amino acid sequence of SEQ ID NO: 3, and LCDR1 has the amino acid sequence of SEQ ID NO: 4. Having an amino acid sequence, LCDR2 has the amino acid sequence of SEQ ID NO: 5, and LCDR3 has the amino acid sequence of SEQ ID NO: 6).

  HCDR1 has the amino acid sequence of SEQ ID NO: 1, HCDR2 has the amino acid sequence of SEQ ID NO: 2, HCDR3 has the amino acid sequence of SEQ ID NO: 3, LCDR1 has the amino acid sequence of SEQ ID NO: 4, and LCDR2 has A set of CDRs having the amino acid sequence of SEQ ID NO: 5 and LCDR3 having the amino acid sequence of SEQ ID NO: 6 is referred to herein as a “BAK278D6 CDR set”. HCDR1, HCDR2, and HCDR3 in the CDR set of BAK278D6 are referred to as “HCDR set of BAK278D6”, and LCDR1, LCDR2, and LCDR3 in the CDR set of BAK278D6 are referred to as “LCDR set of BAK278D6”. A BAK278D6 CDR set, a BAK278D6 HCDR set, or a BAK278D6 LCDR set, or a set of CDRs with one or two substitutions therein, is said to be a BAK278D6 strain.

  As described above, in certain embodiments, the present invention provides a specific binding member for human IL-13 comprising an antibody antigen-binding site composed of a human antibody VH domain and a human antibody VL domain and comprising a set of CDRs. Wherein, the CDR set is a CDR set of BAK278D6 or a set of CDRs that includes one or two substitutions compared to the CDR set of BAK278D6.

  In a preferred embodiment, one or two substitutions are made using one or two of the following residues in the CDRs of the VH and / or VL domains using Kabat's standard numbering [107]: It is in. 31, 32, 34 in HCDR1 52, 52A, 53, 54, 56, 58, 60, 61, 62, 64, 65 in HCDR2 96, 97, 98, 99, 101 in LCDR1, 26, 27 in LCDR1 28, 30, 31 95 in LCDR2 95A in 97 LCDR3, 97.

  A preferred embodiment has two substitutions at HCDR3 residue 99 and LCDR1 residue 27 compared to the CDR set of BAK278D6. A preferred embodiment of these embodiments is that N is replaced with S at HCDR3 residue 99 and N is replaced with I at LCDR1 residue 27. Further preferred embodiments include substitution with HCDR3 residue 99 selected from the group consisting of S, A, I, R, P and K, and I, L, M, C, V, K, Y, F, R , Substitution with LCDR1 residue 27 selected from the group consisting of T, S, A, H and G.

In preferred embodiments, one or two substitutions are identified groups of the following possible substitution residues: Substitution positions Substitution residues selected from the group consisting of 31 in HCDR1: Q, D, L , G, E
32 in HCDR1: T
34 in HCDR1: V, I, F
52 in HCDR2: D, N, A, R, G, E
52A in HCDR2: D, G, T, P, N, Y
53 in HCDR2: D, L, A, P, T, S, I, R
54 in HCDR2: S, T, D, G, K, I
56 in HCDR2: T, E, Q, L, Y, N, V, A, M, G
58 in HCDR2: I, L, Q, S, M, H, D, K
60: R in HCDR2
61 in HCDR2: R
62 in HCDR2: K, G
64 in HCDR2: R
65 in HCDR2: K
96 in HCDR3: R, D
97 in HCDR3: N, D, T, P
98 in HCDR3: R
99 in HCDR3: S, A, I, R, P, K
101 in HCDR3: Y
26 in LCDR1: D, S
27 in LCDR1: I, L, M, C, V, K, Y, F, R, T, S, A, H, G
28 in LCDR1: V
30 in LCDR1: G
31 in LCDR1: R
56 in LCDR2: T
95A in LCDR3: N
97 in LCDR3: I
According to one or two of the above residues in the CDR set of BAK278D6:

  A preferred embodiment has a CDR set of BAK278D6 where N is replaced with S at residue 99 of HCDR3 and N is replaced with I at residue 27 of LCDR1. The set of CDRs thus defined is as follows: HCDR1-SEQ ID NO: 7; HCDR2-SEQ ID NO: 8, HCDR3-SEQ ID NO: 9; LCDR1-SEQ ID NO: 10, LCDR2-SEQ ID NO: 11, LCDR3-SEQ ID NO: 12. This set of CDRs is referred to herein as “the set of CDRs of BAK502G9”.

  A further preferred embodiment has a CDR set of BAK278D6 with one or two substitutions in the CDR, but with substitution of N at residue 99 of HCDR3 and N at residue 27 of LCDR1 Replacement pairs are not included.

  Other preferred embodiments are as follows: BAK1166G2: HCDR1-SEQ ID NO: 67, HCDR2-SEQ ID NO: 68, HCDR3-SEQ ID NO: 69, LCDR1-SEQ ID NO: 70, LCDR2-SEQ ID NO: 71, LCDR3-SEQ ID NO: 72 .

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

  BAK1184C8: HCDR1-SEQ ID 73, HCDR2-SEQ ID 74, HCDR3-SEQ ID 75, LCDR1-SEQ ID 76, LCDR2-SEQ ID 77, LCDR3-SEQ ID 78.

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

  BAK1167F4: HCDR1-SEQ ID NO: 85, HCDR2-SEQ ID NO: 86, HCDR3-SEQ ID NO: 87, LCDR1-SEQ ID NO: 88, LCDR2-SEQ ID NO: 89, LCDR3-SEQ ID NO: 90.

  BAK1111D10: HCDR1-SEQ ID 91, HCDR2-SEQ ID 92, HCDR3-SEQ ID 93, LCDR1-SEQ ID 94, LCDR2-SEQ ID 95, LCDR3-SEQ ID 96.

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

  BAK1185F8: HCDR1-SEQ ID NO: 103, HCDR2-SEQ ID NO: 104, HCDR3-SEQ ID NO: 105, LCDR1-SEQ ID NO: 106, LCDR2-SEQ ID NO: 107, LCDR3-SEQ ID NO: 108. All of these are derived from BAK502G9 by randomization of heavy chains CDR1 and CDR2 and are therefore the BAK502G9 strain.

  A VH domain comprising the set of CDRs of any clone (HCDR1, HCDR2, and HCDR3) is shown in Table 1. Alternatively, VL domains comprising the set of clonal CDRs (LCDR1, LCDR2 and LCDR3) shown in Table 1 are also shown in Table 1 according to the present invention. Preferably such VH domains are paired with such VL domains, most preferably the VH and VL domain pairs are the same as the clones shown in Table 1.

  Further provided by the invention is a VH domain comprising a set of CDRs (HCDR1, HCDR2, and HCDR3), wherein the set of CDRs corresponds to that of any clone shown in Table 1 having one or two amino acid substitutions. The

  Further provided by the present invention is a VL domain comprising a set of CDRs (LCDR1, LCDR2, and LCDR3), wherein the set of CDRs corresponds to that of any clone shown in Table 1 having one or two amino acid substitutions. The

  Specific binding members comprising an antigen binding domain of an antibody comprising a VH and / or VL domain are also provided by the present invention.

  The inventor has identified the BAK278D6 strain as a strain that provides the antigen-binding domain of a human antibody against IL-13, which is particularly valuable. Within this system, BAK502G9 is identified as being particularly useful. The BAK278D6 and BAK502G9 CDR sets have already been identified above.

  In accordance with advances in computer chemistry applying multivariate data analysis techniques to structure / property-activity relationships [94], using known mathematical methods such as statistical activity-property relationships, pattern recognition and classification of antibodies, Quantitative activity-property relationships of antibodies can be obtained [95-100]. Antibody properties can be obtained from empirical and theoretical models of antibody sequences (eg, analysis of similar contact residues or analysis of calculated physicochemical properties), functional and three-dimensional structures. Properties can be considered alone and in combination.

  The antigen binding site of an antibody consisting of a VH domain and a VL domain is formed by six loops of the polypeptide (three from the light chain variable domain (VL) and three from the heavy chain variable domain (VH)). Analysis of antibodies of known atomic structure revealed the relationship between antibody binding site sequence and three-dimensional structure [101,102]. These relationships suggest that, with the exception of the third region (loop) in the VH domain, the binding site loop has one of a few backbone conformations (standard structure). The standard structure formed by a particular loop has proven to be determined by its size and the presence of several residues in both the loop and framework regions [101,102].

  This study of sequence-structure correlation can be used to predict antibody residues of known sequence, but not for unknown three-dimensional structures, which maintains the three-dimensional structure of its CDR loops. And therefore important in maintaining binding specificity. These predictions can be supported by a comparison of predictions on the output from the lead optimization experiment.

  In the structural approach, models are available for antibody molecules [103], either freely available or using commercially available packages (eg, WAM [104]). Using a protein visualization and analysis software package, such as Insight II [105] or Deep View [106], to evaluate possible substitutions at each position in the CDR it can. This information is then used to create substitutions that have minimal or beneficial effects on activity.

  The inventor analyzed the sequence data of a panel of clones whose CDR sets are shown in Table 1.

  This analysis tested the hypothesis that a binary combination of the stated amino acid changes in the CDRs from the stated set of scFv variants gives an scFv variant with a starting titer of at least the parent scFv BAK278D6.

  All scFv variants have been screened for improved affinity and confirmed to show higher titers.

  The observed amino acid changes indicate that its effect on the starting titer of 44 nM of scFv BAK278D6 is preferred, unfavorable or neither in the TF-1 assay.

  No association was observed between any two amino acid changes, confirming that there was no “positive” or “negative” synergy between any two selected amino acid changes.

  There are four scenarios where the two combinations satisfy the hypothesis and three scenarios where the hypothesis is not valid. Since no association was observed, synergistic amino acid changes were not considered.

  The hypothesis is as follows: A1: Mutation 1 is preferred and Mutation 2 is preferred A2: Mutation 1 is preferred and Mutation 2 is neither A3: Mutation 1 is neither and Mutation 2 is neither A4: Mutation 1 is effective and mutation 2 is not effective (1 action is stronger than 2 action):

  The hypothesis is as follows: B1: Mutant 1 is not preferred and Mutant 2 is neither B2: Mutant 1 is not preferred and Mutant 2 is not preferred B3: Mutant 1 is preferred and Mutant 2 is not preferred (2 Is not effective).

In order for A4 to be possible, mutation 1 must be highly favorable so that it can offset the negative effect of mutation 2 on titer. Since such highly preferred mutations are not present in the library of changes used for selection, this will be selected for a panel of variants and will therefore often appear there. Since synergies can be eliminated, such mutations will work for any kind of sequence and should therefore reappear in different scFv variants. An example of such a high frequency amino acid change is the light chain CDR1 change Asn27Ile. However, this mutation itself (in clone BAK531E2) has only a 2-fold effect on titer (final IC ₅₀ is 23.2 nM). Since this is not a very favorable mutation by itself, the scenario shown in A4 would not be possible. This is because each clone (Table 1) in the described IL-13 binding clone set with an Asn27Ile change of light chain CDR1 with one or more additional mutations is at least a variant with a single chain light chain CDR1 Asn27Ile mutation. Suggests equivalent titer. Other mutations are neither or positive, but have no negative or harmful effects.

  A further example is Asn99Ser of heavy chain CDR3 (see Table 1). Since no clone with this single amino acid change has been observed, the titer of such a clone is estimated to be about 12.0 nM by the following theory:

  The BAK278D6 titer is 44 nM. The change in VL CDR1 N27I + VH CDR3 N99S gives BAK502G9 with a titer of 8 nM, ie a 5.5 fold improvement.

  The BAK278D6 titer is 44 nM. The change in VL CDR1 N27I gives BAK531E2 with a titer of 23 nM, ie an improvement of 1.9 times.

  The BAK278D6 titer is 44 nM. The change in VH CDR3 N99S gives a possible clone with a titer of 12.2 nM, ie a 2.9-fold improvement (5.5 / 1.9 = 2.9).

  The binary combination of heavy chain CDR3 Asn99Ser and light chain CDR1 Asn27Ile gives scFv BAK0502G9 with a titer of 8 nM. Because synergy is excluded, the contribution of the change of heavy chain CDR3 Asn99Ser in BAK502G9 is additive.

  Thus, all clones in the described set of IL-13 binding clones with heavy chain CDR3 Asn99Ser changes (Table 1) with one or more additional mutations are within a 2.5-fold acceptable assay window for n = 1-2 Will have a titer of at least 12 nM or higher.

  That is, the inventor believes that no highly preferred amino acid changes preferentially selected are observed. As described above, two changes mainly shown in Table 1 of scFv variants were analyzed in detail. The scFv variants of Table 1 having any of these mutations with one or more additional mutations are at least improved as clones containing any one of these two single amino acid changes in the parent BAK278D6. It was. There is therefore no evidence that there is a highly favorable amino acid change in this panel that would allow scenario A4.

  From this observation, the inventor concluded that there are no undesirable mutations in this set of scFv variants. This means that this hypothesis is valid regardless of scenarios A4 and B1-B3.

  Thus, as already described, the present invention relates to a defined set of CDRs, in particular a BAK278D6 CDR set and a BAK278D6 lineage CDR set, with one or two substitutions within the CDR set (eg, BAK502G9 CDR set). A specific binding member comprising is provided.

  A set of related CDRs is provided within the antibody framework region or other protein backbone (eg, fibronectin or cytochrome B) [115,116]. Preferably antibody framework regions are used, and if they are used, these are preferably germline, more preferably the heavy chain antibody framework regions may be DP14 from the VH1 family. A suitable framework region of the light chain may be λ3-3H. In the CDR set of BAK502G9, the antibody framework regions are SEQ ID NO: 27 for VH FR1, SEQ ID NO: 28 for VH FR2, SEQ ID NO: 29 for VH FR3, SEQ ID NO: 30 for light chain FR1, and SEQ ID NO: 30 for light chain FR2. It is preferable that it is sequence number 32 about sequence number 31 and light chain FR3. In a highly preferred embodiment, a VH domain having the amino acid sequence of SEQ ID NO: 15 is provided, which is referred to as a “BAK502G9 VH domain”. In a further highly preferred embodiment, a VL domain having the amino acid sequence of SEQ ID NO: 16 is provided, which is referred to as a “BAK502G9 VL domain”. The antigen-binding site of the highly preferred antibody provided in the present invention is composed of BAK502G9 VH domain SEQ ID NO: 15 and BAK502G9 VL domain SEQ ID NO: 16. The antigen binding site of this antibody can be provided in any desired antibody molecule format, eg, scFv, Fab, IgG, IgG4, dAb, etc., as further discussed elsewhere herein.

  In a further highly preferred embodiment, the present invention provides an IgG4 antibody molecule comprising BAK502G9 VH domain SEQ ID NO: 15 and BAK502G9 VL domain SEQ ID NO: 16. This is referred to herein as “BAK502G9 IgG4”.

  Like other antibody molecules that contain an HCDR set of BAK502G9 (SEQ ID NO: 7, 8, 9) within the antibody VH domain and / or an LCDR set of BAK502G9 (SEQ ID NO: 10, 11, 12) within the antibody VL domain. An IgG4 or other antibody molecule comprising a VH domain (SEQ ID NO: 15) and / or a BAK502G9 VL domain (SEQ ID NO: 16) is provided by the present invention.

  It is pointed out here that “and / or” as used herein is understood to be a specific disclosure of each of the two specific features or components, with or without the other. Is convenient. For example, “A and / or B” is understood to be a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, each as individually described. I want.

  As described above, the present invention provides specific binding members that bind to human IL-13 and comprise a BAK502G9 VH domain (SEQ ID NO: 15) and / or a BAK502G9 VL domain (SEQ ID NO: 16).

  In general, the VH domain is paired with the VL domain to provide the antigen binding site of the antibody, but as described below, the VH domain alone may be used to bind to the antigen. In certain preferred embodiments, the BAK502G9 VH domain (SEQ ID NO: 15) is paired with the BAK502G9 VL domain (SEQ ID NO: 16), thereby forming an antigen binding site for an antibody comprising both the BAK502G9 VH and VL domains. In other embodiments, BAK502G9 VH is paired with a VL domain other than BAK502G9 VL. Promiscuous binding of light chains is established in the art.

  Similarly, any HCDR set of the BAK278D6 strain can be provided in a VH domain that is used alone or in combination with a VL domain as a specific binding member. For example, as shown in Table 1, a VH domain having an HCDR set of BAK278D6 antibodies is provided, and when such a VH domain is paired with a VL domain, for example, a VL domain having an LCDR set of BAK278D6 antibodies shown in Table 1 Is provided. Pairing of the HCDR set with the LCDR set is shown in Table 1 and provides the antigen binding site of the antibody comprising the set of CDRs as shown in Table 1. The framework region of the VH and / or VL domain may be a germline framework. The framework region of the heavy chain domain is selected from the VH-1 family and the preferred VH-1 framework is the DP-14 framework. The framework region of the light chain is selected from the λ3 family, and a preferred such framework is λ3 3H.

  One or more CDRs are taken from the BAK502G9 VH or VL domain and incorporated into the appropriate framework. This is further described herein. BAK502G9 HCDR 1, 2, and 3 are shown in SEQ ID NOs: 7, 8, and 9, respectively. BAK502G9 LCDR's 1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively.

  The same applies to the CDRs of other BAK278D6 lines as shown in Table 1.

  A further embodiment of the invention is a specific binding member comprising a VH and / or VL domain, or an antibody molecule shown as 167A11 (VH: SEQ ID NO: 23, and VL: SEQ ID NO: 24), and derivatives 615E3 (VH: SEQ ID NO: 33 and VL: SEQ ID NO: 34), antigen binding sites comprising CDRs of the VH and / or VL domains of BAK582F7 (VH CDR SEQ ID NO: 141-143) and BAK612B5 (VH CDR SEQ ID NO: 147-149) About. They recognize human IL-13. The derivative of 167A11 from VH CDR3 randomization is a high titer scFv molecule (5-6 nM). The 167A11 strain is used in any aspect and embodiment of the present invention as disclosed herein for other molecules, for example, for antigen binding site mutation and selection methods with improved titers. .

  Variants of the VH and VL domains and CDRs of the present invention, including those whose amino acid sequences are described herein and which can be used as specific binding members for IL-13, can be obtained by sequence modification or mutation methods and screening. Can be obtained. Such a method is also provided by the present invention.

  Any variable domain amino acid sequence variant of the VH and VL domains whose sequences are specifically disclosed herein is utilized in accordance with the present invention as described. Specific variants include one or more amino acid sequence modifications (additions, deletions, substitutions, and / or insertions of amino acid residues), less than about 20 modifications, less than about 15 modifications, less than about 10 modifications, Or less than about 5 modifications, 4, 3, 2, or 1 modification. Modifications are made in one or more framework regions and / or one or more CDRs.

  In a further aspect of the invention, a specific binding member that competes with any specific binding member for binding to an antigen, where both bind to the antigen, the VH and / or VL disclosed herein. A domain, or HCDR3 disclosed herein, or any variant thereof is provided. Competition between binding members is facilitated in vitro, eg, by labeling a reporter molecule specific for one binding member that can be detected using an ELISA and / or in the presence of other unlabeled binding members. Allowing the identification of specific binding members that bind to the same or overlapping epitopes.

  Thus, a further aspect of the invention provides a specific binding member comprising a human antibody antigen binding site that competes for binding to IL-13 with a BAK502G9 antibody molecule, particularly BAK502G9 scFv and / or IgG4. In a further aspect, the present invention provides a specific binding member comprising an antigen binding site of a human antibody that competes with the antigen binding site of an antibody for binding to IL-13, wherein the antigen binding site of the antibody comprises a VH domain and a VL domain. And the VH and VL domains provide said specific binding member comprising the CDR set of the BAK278D6 strain.

  In order to obtain antibodies that are antibodies against IL-13 and that compete with IL-13 for binding to BAK502G9 antibody molecules, antibody molecules having a CDR set of BAK502G9, or antibody molecules having a CDR set of BAK278D6, various methods are available. Available in the field.

  In a further aspect, the present invention provides a method for obtaining one or more specific binding members capable of binding to an antigen, comprising contacting said antigen with a library of specific binding members of the present invention, A method comprising selecting one or more specific binding members of a library capable of binding to an antigen is provided.

  The library is displayed on the surface of bacteriophage particles, each particle containing a nucleic acid encoding an antibody VH variable domain displayed on the surface, and optionally a displayed VL domain.

  After selection of a specific binding member that can bind to the antigen and is displayed on the bacteriophage particle, nucleic acid is collected from the bacteriophage particle that displays the selected specific binding member. Such a nucleic acid is expressed from a nucleic acid having a sequence of nucleic acids taken from a bacteriophage particle that displays the selected specific binding member, by binding to a specific binding member or antibody VH variable domain (optionally antibody VL variable domain). ) For subsequent production.

  An antibody VH variable domain having the amino acid sequence of the selected specific binding member antibody VH variable domain is provided in an isolated form, such as a specific binding member comprising such a VH domain. In addition, the ability to bind to IL-13 and to compete with BAK502G9 (eg, in scFv format and / or IgG format, eg, IgG4) for binding to IL-13 is also tested. As further described below, the ability to neutralize IL-13 is tested.

  A specific binding member of the invention binds to IL-13 with an affinity of a BAK502G9 antibody molecule (eg, scFv or preferably BAK502G9IgG4) or with a stronger affinity.

  A specific binding member of the invention neutralizes IL-13 with a titer of a BAK502G9 antibody molecule (eg, scFv or preferably BAK502G9 IgG4) or better.

  The specific binding member of the present invention neutralizes naturally occurring IL-13 with a titer of BAK502G9 antibody molecule (eg, scFv or preferably BAK502G9 IgG4) or better.

  The binding affinity and neutralization titer of different specific binding members can be compared under appropriate conditions.

  The antibodies of the present invention include existing commercially available rodent anti-human IL-13 antibodies, namely JES10-5A2 (Biosource), B-B13 (Euroclone), and clone 321166 (R & D). There are many advantages over Systems (R & D Systems). The antibody titer of the present invention was compared with commercially available JES10-A2 and B-B13. Clone 321166 was not evaluated because it was found in previous experiments to be significantly less potent than known commercial antibodies.

  Since the rodent commercial IL-13 antibody is likely to induce an immunogenic response and is therefore cleared more rapidly from the body, it appears to have limited efficacy and use in humans. . Kinetic analysis of the antibodies of the invention in non-human primates suggests that these antibodies have a clearance rate comparable to other known human or humanized antibodies.

  The antibodies provided in the various embodiments of the invention recognize non-human primate IL-13 (including rhesus monkey and cynomolgus IL-13). Measuring antibody efficacy and safety profiles in non-human primates is extremely useful because it provides a means to predict antibody safety, pharmacokinetics, and pharmacodynamic profiles in humans.

  Furthermore, the antibodies of the various embodiments of the invention further recognize a human IL-13 variant (Q130R) associated with asthma. Cross-reactivity with variant IL-13 allows the antibodies of the invention and compositions comprising the antibodies of the invention to be used in the treatment of patients with wild type and variant IL-13.

  A preferred embodiment of the present invention has a titer equivalent to or superior to the titer of the IL-13 antigen binding site formed by the BAK502G9 VH domain (SEQ ID NO: 15) and the BAK502G9 VL domain (SEQ ID NO: 16). Including antibodies that neutralize naturally occurring IL-13. For example, the inventors have shown that representative clones such as BAK502G9, 1167F2, and 1183H4 are much more potent against naturally occurring IL-13 than known commercially available antibodies.

  In addition to antibody sequences, specific binding members of the invention include other amino acids, e.g., form peptides or polypeptides (e.g., folding domains) or have other functional characteristics in the molecule other than the ability to bind antigen. Give. Specific binding members of the invention may have a detectable label or may be bound (eg, through a peptide bond or linker) to a toxin or targeting moiety or enzyme.

  In a further aspect, the present invention relates to an isolated nucleic acid comprising a sequence encoding a specific binding member, VH domain and / or VL domain of the present invention and a specific binding member, VH domain and / or VL domain of the present invention. In which the nucleic acid is expressed and recovered under conditions that cause production of the specific binding member, VH domain and / or VL domain.

  The specific binding member of the present invention is a method of treating or diagnosing the human or animal body, eg, treating a disease or disorder in a human patient, comprising administering to the patient an effective amount of the specific binding member of the present invention ( Which may include prophylactic treatment). Symptoms to be treated in the present invention include any symptoms involving IL-13, particularly asthma, atopic dermatitis, allergic rhinitis, fibrosis, chronic obstructive pulmonary disease, scleroderma, inflammatory bowel disease, And there is Hodgkin lymphoma. Furthermore, the antibodies of the invention also inhibit IL-13 mediated immunosuppression [64,65] and are therefore used to treat tumors and viral infections.

  A further aspect of the invention provides a generally isolated nucleic acid encoding a VH variable domain and / or VL variable domain disclosed herein.

  Another aspect of the invention is a VH CDR or VL CDR sequence disclosed herein, in particular a VH CDR selected from SEQ ID NOs: 7, 8 and 9, or a VL CDR selected from SEQ ID NOs: 10, 11 and 12. Most preferably, a generally isolated nucleic acid encoding BAK502G9 VH CDR3 (SEQ ID NO: 9) is provided. Nucleic acids encoding the BAK502G9 CDR set, nucleic acids encoding the BAK502G9 HCDR set, and nucleic acids encoding the BAK502G9 LCDR set are also included in the individual CDR, HCDR, LCDR, and CDR, HCDR, LCDR sets of the BAK278D6 strain. Like the encoding nucleic acid, it is provided by the present invention.

  A further aspect provides a host cell transformed with a nucleic acid of the invention.

  A further aspect provides a method for producing an antibody VH variable domain comprising causing expression from an encoding nucleic acid. Such methods include culturing host cells under conditions for production of the antibody VH variable domain.

  Similar methods for the production of VL variable domains and specific binding members comprising VH and / or VL domains are provided as a further aspect of the invention.

  Production methods may include product isolation and / or purification steps.

  The production method involves formulating the product into a composition comprising at least one additional ingredient, such as a pharmaceutically acceptable excipient.

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

  The term specific binding member "Specific binding member" describes members of a pair of molecules that have binding specificity for each other. The members of the specific binding pair may be obtained in nature or may be wholly or partly synthesized. One member of a molecular pair specifically binds to its surface or cavity to the specific spatial and polar configuration of the other member of the molecular pair and thus has a complementary region thereto. That is, the members of the pair have the property of binding specifically to each other. Examples of specific binding pair types are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The present invention relates to antigen-antibody type reactions.

  Antibody molecule An “antibody molecule” describes a natural or partially or fully synthesized immunoglobulin. The term also encompasses any polypeptide or protein that comprises an antibody binding domain. Antibody fragments that contain the antigen binding domain are molecules such as Fab, scFv, Fv, dAb, Fd, and diabodies.

  It is possible to take monoclonal and other antibodies and use recombinant DNA technology to produce other autoantibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques involve introducing DNA encoding the immunoglobulin variable region or complementarity determining region (CDR) of an antibody into the constant region or constant region + framework region of a different immunoglobulin. See, for example, EP-A-184187, GB2188638A or EP-A-239400, and many subsequent references. Hybridomas or other antibodies that produce antibodies are subject to genetic variation or other changes that may or may not alter the binding specificity of the antibodies produced.

  Since an antibody can be modified in a number of ways, the term “antibody molecule” should be understood to encompass a specific binding member or substance that spans the antigen-binding domain of an antibody with the required specificity. That is, the term encompasses antibody fragments and derivatives, including polypeptides comprising naturally occurring or fully or partially synthesized immunoglobulin binding domains. Thus, chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to other polypeptides are also included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023, and many subsequent references.

  Further methods available in the field of antibody engineering have made it possible to isolate human and humanized antibodies. For example, human hybridomas can be made as described by Kontermann et al. [10 7]. Phage display methods, another established technique for generating specific binding members, are described in detail in many publications such as Kontermann et al. [10 7] and WO 92/01047 (discussed below). Use transgenic mice in which the mouse antibody gene has been inactivated and functionally replaced with human antibodies, leaving other components of the mouse immune system intact, to isolate human antibodies to human antigens [1008].

  For example, as described in Knappik et al., J. Mol. Biol. (2000) 296, 57-86, or Krebs et al., J. Immunol. Meth. 254 2001 67-84. Synthetic antibody molecules may be generated by expression from genes generated using assembled oligonucleotides.

  It has been shown that fragments of whole antibodies can function to bind antigen. Examples of binding fragments are: (i) Fab fragment consisting of VL, VH, CL, and CH1 domains; (ii) Fd fragment consisting of VH and CH1 domains; (iii) Fv consisting of VL and VH domains of single chain antibodies Fragments; (iv) dAb fragments consisting of VH domains (Ward, ES et al., Nature 341, 544-546 (1989), McCafferty et al. (1990) Nature, 348, 552-554); (v) isolated CDR regions (Vi) a F (ab ′) 2 fragment that is a bivalent fragment comprising two linked Fab fragments; (vii) a single chain Fv molecule (scFv), where the VH and VL domains are two domains (Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); viii) bispecific single chain Fv dimer (PCT / US92 / 09965), and (ix) “dia Bodies ", multivalent or multispecific fragments constructed by gene fusion (WO 94/13804; P. Hollinger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993). Fv, scFv, or diabody molecules are stabilized by the incorporation of disulfide bonds that link the VH and VL domains (Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996). Minibodies containing scFv bound to the CH3 domain can also be made (S. Hu et al., Cancer Res., 56, 3055-3061, 1996).

  If bispecific antibodies are used, these may be conventional bispecific antibodies, which can be produced in various ways (Holliger, P. and Winter G., Current Opinion Biotechnol. 4, 446-449 (1993)), for example chemically or prepared from hybrid hybridomas, or any bispecific antibody fragment as described above. An example of a bispecific antibody is that of BiTE ™ technology, which can use the binding domains of two antibodies with different specificities, linked via a short flexible peptide Is done. This combines two antibodies on a short single chain polypeptide. Diabodies and scFvs can be constructed without the Fc region using only variable domains and may reduce the effects of anti-idiotypic responses.

  Bispecific diabodies for bispecific whole antibodies are also particularly useful because they can be easily constructed and expressed in E. coli. Appropriate binding specific diabodies (and many other polypeptides such as antibody fragments) can be readily selected from the libraries using phage display (WO 94/13804). If, for example, the specificity for IL-13 is used to keep one arm of the diabody constant, a library in which the other arm has changed can be generated and antibodies with the appropriate specificity selected. it can. Bispecific whole antibodies may be made by the “knobs-into-holes” method (J.B.B. Ridgeway et al., Protein Eng., 9, 616-621, 1996).

  Antigen-binding domain "Antigen-binding domain" describes a part of an antibody molecule that specifically binds to and is complementary to part or all of an antigen. If the antigen is large, the antibody binds only to a specific part of the antigen, and this part is called an epitope. One or more antibody variable domains provide an antigen binding domain (eg, a so-called Fd antibody fragment consisting of a VH domain). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

  Specific “Specific” is used to refer to a situation in which one member of a specific binding pair does not show significant binding to a molecule other than that specific binding pair. The term also applies when, for example, an antigen binding domain is specific for a particular epitope carried by many antigens, in which case a specific binding member having an antigen binding domain may be a different antigen having an epitope. Can be combined.

  Contain “comprising” is generally used in the sense of including, ie, allowing the presence of one or more features or elements.

  Isolated “Isolated” means that a specific binding member of the invention or a nucleic acid encoding such a binding member is generally in accordance with the invention. Isolated members and isolated nucleic acids may be obtained by recombination DNA technology where such preparation is performed in vitro or in vivo, the material with which they are originally associated, such as the natural environment, or the members and nucleic acids. A situation where there is no or substantially no other polypeptide or nucleic acid found in the environment in which it is prepared (eg, cell culture). Members and nucleic acids are formulated with diluents or adjuvants and are isolated for practical use, eg, if the members are used to coat microtiter plates used in immunoassays, they are usually gelatin Or it may be mixed with other carriers or mixed with a pharmaceutically acceptable carrier or diluent when used in diagnosis or therapy. A specific binding member may be glycosylated in the natural state or by a heterologous eukaryotic cell (eg, CHO cell or NSO (ECACC 85110503) cell) system, or (eg, if produced by expression in a prokaryotic cell). It may not be glycosylated.

  Native IL-13 Native IL-13 generally refers to the presence of an IL-13 protein or fragment thereof. Native IL-13 refers to an IL-13 protein that is naturally produced by a cell without prior introduction of the encoding nucleic acid using recombinant techniques. That is, naturally occurring IL-13 is produced naturally by, for example, CD4 + T cells and / or isolated from mammals (eg, humans, non-human primates, rodents (eg, rats or mice)). Can be done.

  Recombinant IL-13 “Recombinant IL-13” means a condition in which an IL-13 protein or fragment thereof occurs. Recombinant IL-13 refers to an IL-13 protein or fragment thereof produced by recombinant DNA in a heterologous host. Recombinant IL-13 differs from native IL-13 in terms of glycosylation.

  Recombinant proteins expressed by prokaryotic bacterial expression systems are not glycosylated, while those expressed in eukaryotic systems (eg, mammalian or insect cells) are glycosylated. However, proteins expressed in insect cells differ in glycosylation from proteins expressed in mammalian cells.

  “Substantially as described” means that the relevant CDR or VH or VL domain of the present invention is identical or very similar in sequence to a particular region described herein. Means. “Very similar” means that 1-5, preferably 1-4, eg 1-3 or 1 or 2, or 3 or 4 amino acid substitutions are made in the CDR and / or VH or VL domains I think it has been.

  The structure for carrying a CDR or set of CDRs of the invention is generally an antibody heavy or light chain sequence or a sufficient portion thereof, in which the CDR or CDR set is encoded by a reconstituted immunoglobulin gene. Is located at a position corresponding to a CDR or CDR set of a variable domain of a natural VH and / or VL antibody. The structure and location of immunoglobulin variable domains is described in Kabat, EA et al., `` Sequences of Proteins of Immunological Interest '', 4th edition, US Department of Health and Human Services), 1987, and this updated version, now available on the Internet (http://immuno.bme.nwu.edu or can be found by "Kabat" using any search engine ).

  CDRs are also retained by other scaffolds such as fibronectin or cytochrome B [115,116].

  Preferably, a CDR amino acid sequence substantially as described herein is retained as a CDR within a human variable domain or a substantial portion thereof. Substantially the HCDR3 sequences described herein are preferred embodiments of the present invention, each of which is preferably retained as HCDR3 in the human heavy chain variable domain or a substantial portion thereof.

  The variable domains used in the present invention can be derived from any germline or rearranged human variable domain or can be a synthetic variable domain based on a consensus sequence of known human variable domains. The CDR sequences of the present invention (eg, CDR3) may be introduced into a repertoire of variable domains lacking a CDR (eg, CDR3) using recombinant DNA techniques.

For example, Marks et al. (Bio / Technology, 1992, 10: 779-783) is a method for producing a repertoire of antibody variable domains, within or within the variable domain directed to the 5 ′ end of the variable domain region. Is used to provide a repertoire of VH variable domains lacking CDR3 using a consensus primer adjacent to the 3 along with a consensus primer for the third framework region of the human VH gene. Marks et al. Further illustrate how this repertoire is combined with the CDR3 of a particular antibody. A similar method is used to shuffle the CDR3-derived sequences of the present invention with a repertoire of VH or VL domains lacking CDR3, and the shuffled complete VH or VL domain is combined with the homologous VL or VH domain, Specific binding members of the invention are provided. The repertoire is then described in a suitable host system, such as WO 92/01047 or many subsequent references (Kay, BK, Winter, J. and McCafferty, J. (1996), “Peptide and Protein Phage Display: Laboratory Manual ( Phage Display of Peptides and Proteins: A Laboratory Manual), San Diego: Academic Press), resulting in selection of specific binding members. The repertoire consists of 10 ⁴ or more individual members, for example 10 ⁶ to 10 ⁸ or 10 ¹⁰ members. Other suitable host systems are yeast display, bacterial display, T7 display, ribosome display, and the like. For a review of ribosome display, see Lowe D and Jermutus L, 2004, Curr. Pharm. Biotech., 517-27, and WO92 / 01047.

  Similar shuffling or combinatorial methods are also disclosed by Stemmer (Nature, 1994, 370: 389-391), which describes this technology in the context of β-lactamases, but this approach is useful for antibody generation. Observed to be used.

  Another method uses random mutagenesis of one or more selected VH and / or VL genes to create a novel VH or VL region having a CDR-derived sequence of the invention within the entire variable domain. To create mutations. Such a method is described by Gram et al. (1992, Proc. Natl. Acad. Sci. USA 89: 3576-3580) and they used error-prone PCR. In preferred embodiments, one or two amino acid substitutions are made within the set of HCDRs and / or LCDRs.

  Another method used is to induce mutagenesis in the CDR region of the VH or VL gene. Such methods are disclosed by Barbas et al. (1994, Proc. Natl. Acad. Sci. USA 91: 3809-3813) and Schier et al. (1996, J. Mol. Biol. 263: 551-567).

  All the above techniques are known in the art and do not form part of the present invention. One skilled in the art could use such techniques to provide specific binding members of the invention using routine methods in the art.

  A further aspect of the invention provides a method for obtaining an antigen-binding domain of an antibody specific for IL-13 antigen, which comprises the addition of one or more amino acids to the amino acid sequence of the VH domain described herein. Deletion, substitution or insertion provides a VH domain that is an amino acid sequence variant of the VH domain, optionally combining the VH domain thus provided with one or more VL domains, and a VH domain or VH / VL combination Testing to identify a specific binding member or antigen binding domain of an antibody that is specific for an IL-13 antigen and optionally has one or more suitable properties, preferably the ability to neutralize IL-13 activity including. The VL domain has an amino acid sequence substantially as described herein.

  Similar methods are used that combine one or more sequence variants of the VL domains disclosed herein with one or more VH domains.

  In a preferred embodiment, the BAK502G9 VH domain (SEQ ID NO: 15) is mutated to provide one or more VH domain amino acid sequence variants, and / or BAK502G9 VL (SEQ ID NO: 16).

  A further aspect of the invention is a method of preparing a specific binding member specific for an IL-13 antigen, the method comprising: (a) a region comprising a CDR3 to be substituted or encoding a CDR3 Providing a starting repertoire of nucleic acids encoding the missing VH domain; (b) combining the repertoire with a donor nucleic acid encoding an amino acid sequence substantially as described herein for VH CDR3; Providing a product repertoire of nucleic acids that inserts into the CDR3 region in the repertoire and encodes a VH domain; (c) expresses nucleic acids of the product repertoire; (d) specific for IL-13 Selecting a binding member; and (e) recovering the specific binding member or the nucleic acid encoding it.

  Again, a similar method, wherein the VL CDR3 of the invention is combined with a repertoire of nucleic acids encoding a VL domain comprising a CDR3 to be substituted or lacking a region encoding CDR3. Is used.

  Similarly, one or more or all three CDRs are transplanted into a repertoire of VH or VL domains, which are then screened for specific binding members or specific binding members specific for IL-13. The

  In preferred embodiments, one or more BAK502G9 HCDR1 (SEQ ID NO: 7), HCDR2 (SEQ ID NO: 8), and HCDR3 (SEQ ID NO: 9), or an HCDR set of BAK502G9 is used and / or one or more BAK502G9 LCDR1 An LCDR set of (SEQ ID NO: 10), LCDR2 (SEQ ID NO: 11), or BAK502G9 is used.

  A substantial portion of an immunoglobulin variable domain includes at least three CDR regions along with its intervening framework regions. Preferably, this portion also includes at least about 50% of either or both of the first and fourth framework regions, where 50% is 50% of the C-terminus of the first framework region and the fourth frame. 50% of the N-terminal of the work area. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those that are not normally bound to naturally occurring variable domain regions. For example, the construction of a specific binding member of the invention made by recombinant DNA technology involves the introduction of an N-terminal or C-terminal residue encoded by a linker introduced to facilitate cloning or other manipulation steps. Can bring. Other manipulation steps are described in more detail throughout the various domains of the present invention, additional protein sequences (eg, immunoglobulin heavy chains, other variable domains (eg, in the production of diabodies), or elsewhere herein. In order to link to a protein label), a linker is introduced.

  While specific binding members comprising a pair of VH and VL domains are preferred in a preferred embodiment of the invention, a single binding domain based on either a VH or VL domain sequence constitutes another embodiment of the invention. It is known that a single immunoglobulin domain, in particular a VH domain, can specifically bind to a target antigen.

  In the case of any single specific binding domain, these domains are used to screen for complementary domains that can form a two domain specific binding member capable of binding to IL-13.

  This is done by a phage display screening method using a so-called hierarchical dual combinatorial approach, as disclosed in WO 92/01047. Here, individual colonies containing heavy or light chain clones were used to infect a complete library of clones encoding the strand (L or H) and the resulting double-strand specific binding member was Selected according to the phage display method as described above. This method is also disclosed in Marks et al. (Supra).

  The specific binding member of the present invention further comprises an antibody constant region or part thereof. For example, the VL domain is bound at its C-terminus to an antibody light chain constant domain comprising a human Cκ or Cλ chain, preferably a Cλ chain. Similarly, the specific binding member based on the VH domain is at its C-terminus the immunoglobulin heavy from any antibody isotype (eg, IgG, IgA, IgE, and IgM, and any isotype subclass, especially IgG1 and IgG4). It is bound to all or part of the chain (eg CH1 domain). IgG4 is preferred because it does not bind complement and does not produce effector functions. Synthetic or other constant region variants that have these properties and stabilize variable regions are also suitable for use in embodiments of the present invention.

The specific binding member of the present invention may be labeled with a detectable or functional label. Detectable labels include radiolabels (eg ¹³¹ I or ⁹⁹ Tc) that are coupled to the antibodies of the invention using common chemistry known in the field of antibody imaging. The label also includes an enzyme label such as horseradish peroxidase. The label further comprises a chemical moiety such as biotin, which is detected via binding to a specific homologous detectable moiety (eg, labeled avidin).

  Specific binding members of the present invention are designed for use in the diagnosis or treatment of human or animal subjects (preferably humans).

  Accordingly, a further aspect of the present invention is a therapeutic method comprising the administration of a provided specific binding member, a pharmaceutical composition comprising such a specific binding member, and the manufacture of a medicament to be administered (e.g., a specific binding member and a pharmaceutically acceptable agent). The use of such specific binding members in a method of manufacturing a medicament or pharmaceutical composition comprising blending with an excipient to be prepared.

  Clinical indications where anti-IL-13 antibodies are used to provide therapeutic effects include asthma, atopic dermatitis, allergic rhinitis, fibrosis, chronic obstructive pulmonary disease, inflammatory bowel disease, scleroderma And Hodgkin lymphoma. As already explained, anti-IL-13 treatment is effective for all these diseases.

  Anti-IL-13 treatment can be oral, injection (eg, subcutaneous, intravenous, intraperitoneal, or intramuscular), inhalation, or topically (eg, intraocular, nasal, rectal, intra-wound, on the skin). Be administered. The route of administration can be determined by the physicochemical characteristics of the treatment, special considerations for the disease, or requirements to optimize efficacy and minimize side effects.

  Anti-IL-13 treatment is not limited to use in the clinic. That is, subcutaneous injection using an instrument that does not use a needle is also suitable.

  Combination therapy can be used, particularly in combination with an anti-IL-13 specific binding member, with one or more other agents to achieve great synergy. Specific binding members of the invention include short or long acting beta agonists, corticosteroids, cromoglycic acid, leukotriene (receptor) antagonists, methylxanthine and its derivatives, IL-4 inhibitors, muscarinic receptor antagonists, IgE inhibitors, It may be provided in combination with histamine inhibitors, IL-5 inhibitors, eotaxin / CCR3 inhibitors, PDE4 inhibitors, TGF-beta antagonists, interferon-gamma, perfenidone, chemotherapeutic agents, and immunotherapeutic agents.

  With one or more short or long acting beta agonists, corticosteroids, cromoglycic acid, leukotriene (receptor) antagonists, xanthines, IgE inhibitors, IL-4 inhibitors, IL-5 inhibitors, eotaxin / CCR3 inhibitors, PDE4 inhibitors Combination therapy is used to treat asthma. The antibodies of the invention can also be used in the treatment of fibrosis in combination with corticosteroids, antimetabolites, antagonists of TGF-β and its downstream signaling pathways. Combination therapy of these antibodies with PDE4 inhibitors, xanthines and their derivatives, muscarinic receptor antagonists, short and long beta antagonists is useful for treating chronic obstructive pulmonary disease. Similar combinations apply to the use of anti-IL-13 for the treatment of atopic dermatitis, allergic rhinitis, chronic obstructive pulmonary disease, inflammatory bowel disease, scleroderma, and Hodgkin lymphoma.

  The composition provided in the present invention is administered to an individual. Administration is preferably effected in a “therapeutically effective amount”, which is an amount sufficient to benefit the patient. Such benefit is at least an improvement of at least one symptom. The actual amount administered, rate of administration and time course will depend on the nature and severity of the disease being treated. The treatment prescription (eg, determination of dosage, etc.) is within the responsibility of general practitioners and other physicians. Appropriate doses of antibody are known in the art; see Ledermann JA et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe KD et al. (1991) Antibody, Immunoconjugates and Radipharmaceuticals 4: 915-922. I want.

  The exact dose is, for example, whether the antibody is diagnostic or therapeutic, the size and location of the site to be treated, the exact nature of the antibody (eg, whole antibody, fragment or diabody), and detectable It depends on many factors, such as the nature of the label or other molecule attached to the antibody. Typical antibody doses range from 100 μg to 1 g for systemic application and 1 μg to 1 mg for topical application. Typically the antibody is a whole antibody, preferably the IgG4 isotype. This is a single treatment dose for an adult patient, with children and infants adjusted proportionally, and antibody formats adjusted proportionally with molecular weight. Treatment is repeated daily, twice a week, once a week, or once a month at the discretion of the physician. In a preferred embodiment of the invention, the treatment is periodic and the period between administrations is about 2 weeks or longer, preferably about 3 weeks or longer, more preferably about 4 weeks or longer, or monthly. About once.

  The specific binding member of the present invention is usually administered in the form of a pharmaceutical composition, which comprises at least one component in addition to the specific binding member.

  That is, the pharmaceutical compositions of the present invention and used in the present invention contain, in addition to the active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration, which may be oral or injection (eg, intravenous injection).

  Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form, or liquid form. Tablets contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Saline, dextrose, or other sugar solutions or glycols (eg, ethylene glycol, propylene glycol, or polyethylene glycol) may be included.

  For intravenous injection or injection into the affected area, the active ingredient is in the form of a parenterally acceptable aqueous solution which does not contain pyrogens and has a suitable pH, isotonicity and stability. One skilled in the art can prepare suitable solutions using isotonic solvents such as sodium chloride for injection, Ringer's solution, and lactated Ringer's solution. Optionally, preservatives, stabilizers, buffers, antioxidants, and / or other additives are included.

  The compositions are administered alone or in conjunction with other treatments, either simultaneously or sequentially depending on the condition being treated.

  Specific binding members of the present invention are prepared in liquid or solid form depending on the physicochemical properties of the molecule and the delivery route. The formulation may contain excipients or combinations of excipients such as: sugars, amino acids, and surfactants. Liquid formulations include a wide range of antibody concentrations and pH. Solid formulations may be produced, for example, by freeze drying, spray drying, or drying by supercritical fluid technology. The formulation of anti-IL-13 depends on the intended delivery route: for example, the formulation for pulmonary delivery consists of particles with physical properties that ensure penetration to the deep lung by inhalation; Contains a viscosity modifier that extends the time spent at the site.

  The present invention provides methods that cause or enable binding of a specific binding member to IL-13 as described herein. As described above, such binding can occur in vivo after administration of, for example, a specific binding member or a nucleic acid encoding a specific binding member, or in vitro, for example, ELISA, Western blotting, immunocytochemistry, immunoprecipitation, affinity chromatography. Or in a cell-based assay (eg TF-1 assay).

  The amount of specific binding member binding to IL-13 can be measured. Quantification is related to the amount of antigen in the test sample, which is of diagnostic interest.

  Kits comprising specific binding members or antibody molecules of any aspect or embodiment of the invention are also provided as aspects of the invention. In the kit of the invention, the specific binding member or antibody molecule is labeled to allow measurement of reactivity in the sample (described below). The components of the kit are generally sterile and placed in a sealed vial or other container. The kit is used in diagnostic analysis or other methods where antibody molecules are useful. The kit may contain instructions for use of the components in the method, eg, the method of the invention. Ancillary materials that may assist or perform such a method may be included in the kits of the invention.

  The reactivity of the antibody in the sample is measured by any suitable means. Radioimmunoassay (RIA) is one possibility. Radiolabeled antigen is mixed with unlabeled antigen (test sample) and allowed to bind to the antibody. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the antibody is measured. If more antigen is present in the test sample, less radioactive antigen will bind to the antigen. A competitive binding assay with a non-radioactive antigen may be used, using an antigen or analog bound to a reporter molecule. The reporter molecule may be a fluorescent dye, phosphor or laser dye having spectrally separated absorption or emission properties. Suitable fluorescent dyes include fluorescein, rhodamine, phycoerythrin, and Texas Red. A suitable chromogenic dye is diaminobenzidine.

  Other reporters include macromolecular colloidal particles or particulate materials (e.g., colored, magnetic or paramagnetic latex beads) and visually detectable or electronically detected signals that can be detected, or others There are biologically or chemically active substances that allow direct or indirect recording. Molecules may be enzymes that catalyze reactions that develop or change color or cause changes in electrical properties, for example. These may be those in which the molecules can be excited such that characteristic spectral absorption or emission occurs due to electronic transitions of energy states. These include chemicals used with biosensors. Biotin / avidin or biotin / streptavidin and alkaline phosphatase detection systems may be used.

  Signals generated by individual antibody-reporter conjugates may be used to obtain quantifiable absolute or relative data of relevant antibody binding in a sample (normal sample or test sample).

  The present invention also provides the use of a specific binding member as described above for measuring antigen levels in a competitive assay, i.e. using a specific binding member provided by the present invention in a competitive assay in a sample. A method for measuring antigen levels is provided. This may be the case when it is not necessary to separate bound antigen from unbound antigen. Linking a reporter molecule to a specific binding member so that physical or optical changes upon binding occur is one possibility. The reporter molecule produces a detectable, preferably measurable, signal directly or indirectly. The binding of the reporter molecule may be direct or indirect, covalent (eg, peptide bond) or non-covalent. Linkage via a peptide bond may be the result of recombinant expression of a gene fusion encoding an antibody and a reporter molecule.

  The present invention also provides for directly measuring the level of antigen using, for example, a specific binding member of the present invention in a biosensor system.

  The mode of measuring binding is not a feature of the present invention, and those skilled in the art can select an appropriate mode according to their preference and general knowledge.

  As described above, in various aspects and embodiments, the present invention encompasses specific binding members that compete with a specific binding member as defined herein (eg, BAK502G9 IgG4) for binding to IL-13. Competition between binding members can, for example, label a specific reporter molecule to one binding member, which can be detected in the presence of an unlabeled binding member and identify specific binding members that bind to the same or overlapping epitopes. Can be easily measured in vitro.

  Competition may be measured, for example, using an ELISA in which IL-13 is immobilized on the plate and the first labeled binding member is added to the plate along with one or more unlabeled binding members. The presence of an unlabeled binding member that competes with the labeled binding member is observed by a decrease in the signal emitted by the labeled binding member.

  To test for competition, peptide fragments of the antigen are used, in particular peptides containing the epitope of interest. Peptides having an epitope sequence plus one or more amino acids at either end may be used. Such peptides “consist of” a specific sequence. A specific binding member of the invention is such that its binding to an antigen is inhibited by a peptide having or comprising a given sequence. To test this, a peptide with any sequence plus one or more amino acids may be used.

  Specific binding members that bind to specific peptides are isolated from phage display libraries, for example by panning with peptides.

  The invention further provides an isolated nucleic acid encoding a specific binding member of the invention. Nucleic acids include DNA and / or RNA. In a preferred embodiment, the present invention provides a nucleic acid encoding a CDR or CDR set or VH domain or VL domain of the present invention as described above, or an antigen binding site or antibody molecule of an antibody, such as scFv or IgG4.

  The invention also provides a construct in the form of a plasmid, vector, transcription or expression cassette comprising at least one polynucleotide as described above.

  The invention also provides a recombinant host cell comprising one or more constructs as described above. The provided CDR or CDR set or VH domain or VL domain, or an antigen binding site of an antibody or a nucleic acid itself encoding an antibody molecule, such as scFv or IgG4, is a method of producing the encoded product (this method is (Including expression from a nucleic acid encoding). Expression is conveniently effected by culturing recombinant host cells containing the nucleic acid under suitable conditions. After production by expression, the VH or VL domain or specific binding member is isolated and / or purified using any suitable method and then used as appropriate.

  Specific binding members, VH and / or VL domains, and encoding nucleic acid molecules and vectors of the invention can be isolated and / or purified from, for example, their natural environment, in substantially pure or homogeneous form, Alternatively, in the case of a nucleic acid, it is provided without or substantially free of nucleic acids or genes other than sequences encoding a polypeptide having the necessary function. The nucleic acids of the present invention include DNA or RNA and are fully or partially synthesized. References to nucleotide sequences herein include DNA molecules having a particular sequence, including RNA molecules having a particular sequence (U is used instead of T unless otherwise specified herein) ).

  Systems for cloning and expression of polypeptides in a variety of different host cells are known. Suitable host cells include bacteria, mammalian cells, plant cells, yeast, and baculovirus systems, and transgenic plants and animals. Mammalian cell lines available in the art for expression of heterologous polypeptides include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2 / 0 rat myeloma cells, There are human embryonic kidney cells, human embryonic retinal cells, and many other cells. A common preferred bacterial host is E. coli.

  Expression of antibodies and antibody fragments in prokaryotic cells (eg, E. coli) is established in the art. For a review, see Pluckthun, A. Bio / Technology 9: 545-551 (1991). Expression in cultured eukaryotic cells is also known to those skilled in the art as an option for the production of specific binding members. For example, Chad HE and Chamow SM (2001) 110 Current Opinion in Biotechnology 12: 188-194, Andersen DC and Krummen L (2002) Current Opinion in Biotechnology 13: 117, Larrick JW and Thomas DW (2001) Current Opinion in Biotechnology 12: See 411-418.

  Appropriate vectors containing appropriate control sequences including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as appropriate can be selected and constructed. The vector may be a plasmid, a virus, such as a phage as appropriate, or a phagemid. For further details see, for example, “Molecular Cloning: A Laboratory Manual”, 3rd edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for the manipulation of nucleic acids (e.g., preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins) are described in `` molecular biology. Current Protocols in Molecular Biology, 2nd edition, edited by Ausubel et al., John Wiley and Sons, 1988, “Simple Protocols for Molecular Biology: From Modern Protocols of Molecular Biology” (Short Protocols in Molecular Biology: A compendium of Methods from Current Protocols in Molecular Biology), edited by Ausubel et al., John Wiley and Sons, 4th edition, 1999. . The disclosures of Sambrook et al. And Ausubel et al. (Both) are hereby incorporated by reference.

  Thus, a further aspect of the invention provides host cells containing the nucleic acids disclosed herein. Such host cells may be in vitro or cultured cells. Such host cells may be in vivo. The in vivo presence of the host cell allows intracellular expression as “intrabodies” or intracellular antibodies of the specific binding members of the invention. Intrabodies are used in gene therapy [11 2].

  Yet another aspect provides a method comprising introducing such a nucleic acid into a host cell. This introduction may use any available method. For eukaryotic cells, suitable methods use calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and retroviruses or other viruses (eg vaccinia or baculovirus for insect cells). There is an introduction. The introduction of nucleic acids into host cells, particularly eukaryotic cells, uses viral or plasmid based systems. Plasmid systems are maintained episomally or are incorporated into host cells or into artificial chromosomes [110, 111]. Incorporation is random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophage.

  Following introduction, inclusion of the host cell under conditions for expression of the gene causes or allows expression from the nucleic acid.

  In certain embodiments, the nucleic acids of the invention are integrated into the genome (eg, chromosome) of the host cell. Integration is facilitated by including sequences that facilitate recombination with the genome according to standard techniques.

  The invention also provides a method comprising using the construct described above in an expression system to express the specific binding member or polypeptide described above. Aspects and embodiments of the present invention are illustrated with reference to the following experiments.

Example 1
Anti-IL-13 scFv isolated scFv antibody repertoire A large single chain Fv (scFv) human antibody library obtained from the spleen lymphocytes of 20 donors and cloned into a phagemid vector was used for selection [66 ].

scFv selection scFv recognizing IL-13 from a phage display library by a series of repeated selection cycles for human or murine IL-13 (Peprotech) from recombinant bacteria, essentially as described in [67]. Was isolated. Briefly, after incubation with the library, immobilized antigen and bound phage that had been previously bound to paramagnetic beads were recovered by magnetic separation, and unbound phage was washed away. Bound phage was then recovered as described by Vaughan et al. [67] and the selection process was repeated. Different solid surfaces and capture methods were used in different rounds of selection to reduce non-specific binding. Antigen was covalently bound to beads (Dynabeads M-270 carboxylic acid) or modified by biotinylation according to the manufacturer's (Dynal) protocol followed by streptavidin-coated beads (Dynabeads) Secondary capture by M-280). A representative portion of the clones from the output of the selection round was DNA sequenced as described by Vaughan et al. [67] and Osbourn et al. [70]. Unique clones were evaluated for their ability to neutralize IL-13 as a purified scFv preparation in an IL-13 dependent cell proliferation assay.

  A ribosome display library was generated and screened for scFv specifically recognizing human or murine IL-13 (Peprotech) from recombinant bacteria, essentially as described in Hanes et al. [113]. The BAK278D6 lead clone from the initial selection was first converted to a ribosome display format and this template was used to create a glycosylation library. At the DNA level, a T7 promoter was added at the 5 'end for efficient transcription into mRNA. At the mRNA level, the construct contained a prokaryotic ribosome binding site (Shine-Dalgarno sequence). The stop codon was removed at the 3 'end of the single strand and a portion of gIII (gene III) was added to act as a spacer [11 3].

  Ribosome display libraries were generated by mutagenesis of antibody complementarity determining regions (CDRs), where PCR reactions were performed using non-proofreading Taq polymerase. After affinity-based selection and incubation with the library, biotinylated human IL-13 was captured by streptavidin-coated paramagnetic beads (Dynal M280) and bound ternary complex (mRNA-ribosome-scFv) -IL-13) was recovered by magnetic separation and unbound complex was washed away. MRNA encoding binding scFv is recovered by RT-PCR as described by Hanes et al [11 3] and low concentrations of biotinylated human IL-13 present during selection (100 nM to 100 pM in 5 rounds). The selection process was repeated using.

  Error-prone PCR was also used to further expand the library size. In the selection procedure, using three error frequencies (2.0, 3.5, and 7.2 mutations every 1,000 bp after a standard PCR reaction as described in the manufacturer's (Clontech) protocol) did. The first error prone PCR reaction was performed prior to the first round of selection starting at 100 nM. Subsequent rounds of error-prone PCR were performed with 10 nM biotinylated human IL-13 prior to the third round of selection. As noted above, a representative portion of the clones from the output of the selection round was subjected to DNA sequencing as described by Vaughan et al. [67] and Osbourn et al. [70]. Unique clones were evaluated for their ability to neutralize IL-13 as a purified scFv preparation in an IL-13 dependent cell proliferation assay.

Example 2
Anti-IL-13 scFv neutralization titer in an IL-13-dependent TF-1 cell proliferation assay The neutralizing titer of purified scFv preparations against human and murine IL-13 biological activity was used using the TF-1 cell proliferation assay And evaluated. The purified scFv preparation was prepared as described in Example 3 of WO01 / 66754. The protein concentration of the purified scFv preparation was measured using the BCA method (Pierce). TF-1 is a human promyelocytic cell line established from patients with erythroleukemia [68]. The TF-1 cell line is factor dependent for survival and proliferation. In this regard, TF-1 cells responded to human or murine IL-13 [69] and were maintained in medium containing human GM-CSF (4 ng / ml, R & D Systems). . Inhibition of IL-13 dependent proliferation was determined by measuring the reduced incorporation of tritiated thymidine into newly synthesized DNA of dividing cells.

TF-1 Cell Assay Protocol TF-1 cells were obtained from R & D Systems and maintained according to the provided protocol. The assay medium is RPMI 1640 with GLUTAMAX I (Invitrogen) containing 5% fetal bovine serum (JRH) and 1% sodium pyruvate (Sigma). Included. Prior to each assay, it was pelleted by centrifugation at 300 xg for 5 minutes, the medium was aspirated and the cells were resuspended in assay medium. This procedure was repeated twice with cells resuspended at a final concentration of 10 ⁵ cells in 1 ml of assay medium. Antibody test solutions (triple measurements) were diluted to the desired concentration in assay medium. An antibody unrelated to IL-13 was used as a negative control. Human or murine IL-13 (Peprotech) from recombinant bacteria was added to a final concentration of 50 ng / ml when mixed with the appropriate test antibody in a total volume of 100 μl / well in a 96-well assay plate. The concentration of IL-13 used in this assay was selected as the dose that gave a maximum proliferative response of approximately 80% at the final assay concentration. All samples were incubated for 30 minutes at room temperature. 100 μl of resuspended cells were then added to each assay point to give a total assay volume of 200 μl / well. The assay plate was incubated for 72 hours at 37 ° C. under 5% CO ₂ . 25 μl of tritiated thymidine (10 μCi / ml, NEN) was then added to each assay point and the assay plate was returned to the incubator for an additional 4 hours. Cells were harvested on glass fiber filter plates (Perkin Elmer) using a cell harvester. Thymidine incorporation was measured using a Packard TopCount microtiter plate liquid scintillation counter. Data was analyzed using Graphpad Prism software.

Results Despite alternating selection cycles of human and murine antigens, no cross-reactive neutralizing antibodies were obtained. Selection resulted in two distinct anti-human and one anti-murine IL-13 neutralizing scFv. BAK278D6 (VH SEQ ID NO: 13; VL SEQ ID NO: 14) and BAK167A11 (VH SEQ ID NO: 23; VL SEQ ID NO: 24) recognize human IL-13 and BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26) is murine IL- 13 was recognized. BAK278D6 (FIG. 2) and BAK167A11 (FIG. 1) as scFv neutralized 25 ng / ml human IL-13 with IC ₅₀ of 44 nM and 111 nM, respectively. BAK209B11 (FIG. 3) as scFv neutralized 25 ng / ml murine IL-13 with an IC ₅₀ of 185 nM.

Example 3
Neutralization titer of lead clones from targeted optimization of heavy chain CDR3 of parent clone in IL-13 dependent TF-1 cell proliferation assay Osbourn et al. [70] suddenly targeted residues in heavy chain CDR3. It has been demonstrated that mutagenesis significantly improves antibody affinity. Selection for the scFv repertoire was performed as described in Example 1, where the residues in the heavy chain CDR3 of BAK278D6 (SEQ ID NO: 6) and BAK167A11 (SEQ ID NO: 57) were randomized by mutagenesis. Unique clones from the selected output were identified by DNA sequencing and neutralization titers measured as scFv in the TF-1 cell proliferation assay as described in Example 2.

Results Significant increases in titers were achieved in both lines. The highest titer clones from the BAK167A11 line are BAK615E3, BAK612B5, and BAK582F7, which are each 3 nM as scFv against 25 ng / ml human IL-13 in the TF-1 cell proliferation assay (FIG. 1), It had an IC ₅₀ of 6.6 nM, 6.65 nM. In the BAK278D6 line, the highest titer clone was BAK502G9, which had an IC ₅₀ of 8 nM against 25 ng / ml human IL-13 as scFv in the TF-1 cell proliferation assay (FIG. 2).

Example 4
Neutralizing titers of BAK167A11 and BAK278D6 strains against non-human primate IL-13 and IL-13 variants associated with asthma in a TF-1 factor-dependent cell proliferation assay Both BAK167A11 and BAK278D6 human IL-13 neutralizing strains There was no murine cross-reactivity. The inventor therefore determined the following criteria for the lines selected for further optimization and clinical progress: preferably cross-reactive with non-human primate IL-13, variants of IL-13 Recognize (in this variant, the arginine at amino acid position 130 is replaced by glutamine (Q130R)). This variant is genetically associated with asthma and other allergic diseases [37,38,41,71]. Cross-reactivity was measured by the ability of purified scFv preparations to bind to non-human primate IL-13 and IL-13 variants by surface plasmon resonance (BIAcore) analysis. Functional activity was measured using a TF-1 cell proliferation assay.

Production of wild-type, variant, and non-human primate IL-13 cDNA of wild-type human IL-13 was obtained from Invitrogen and site-directed mutagenesis (Stratagene Quick Change (Quikchange) (Trademark) kit) to obtain cDNA encoding variant IL-13. The coding sequences for rhesus monkey and cynomolgus monkey IL-13 were obtained by PCR of genomic DNA templates using degenerate primers based on the human IL-13 sequence. Non-human primate (rhesus and cynomolgus monkey) sequences were identical to each other, but differed by seven amino acids from human IL-13 (Figure 19). The baculovirus expression system (Invitrogen) was then used to express recombinant wild type, variant, and non-human primate IL-13. The expression construct imparted a carboxyl terminal affinity tag to the expressed protein, which allowed it to be purified to near homogeneity from insect cell conditioned media.

Qualitative binding assay using Biacore The binding affinity of purified scFv preparations to non-human primates, variants and wild-type IL-13 was measured by the BIAcore 2000 biosensor (Biacore) as described by Karlsson et al. [72]. It was measured by surface plasmon resonance measurement using AB (BIAcore AB). Briefly, at a surface density of about 200 Ru, an amine coupling kit (BIAcore) was used to bind IL-13 to a CM5 sensor chip and 3 concentrations of test scFv (about 350 nM, 175 nM, And 88 nM) was passed through the sensor chip surface. The resulting sensorgram was evaluated using BIA Evaluation 3.1 software to obtain relative binding data.

TF-1 Assay Protocol This assay was performed essentially as described in Example 2 with the following modifications: non-human primate IL-13, human variant IL-13 (Q130R), and wild type human IL-13 was used at concentrations of 50 ng / ml, 25 ng / ml, and 25 ng / ml, respectively.

Results BIAcore binding assay data suggested that BAK278D6 but not the BAK167A11 strain had a cross-reactivity profile required for further therapeutic drug development (Table 2). This finding suggests that BAK278D6 (FIG. 4) and BAK502G9 (FIG. 6) have human IL-13, human IL-13 (Q130R) variants, and non-human primate IL- Supported by bioassay data showing that 13 can be neutralized. In contrast, BAK615E3 (VH SEQ ID NO: 33; VL SEQ ID NO: 34) is significant against human IL-13 relative to its parent BAK167A11 (VH SEQ ID NO: 23; VL SEQ ID NO: 24) in the TF-1 cell proliferation assay. However, none of the clones bound to non-human primate or variant IL-13 in the BIAcore binding assay.

Germline sequencing of BAK278D6 and BAK502G9 framework regions The resulting BAK278D6 VH (SEQ ID NO: 13) and VL (SEQ ID NO: 14) amino acid sequences were compared to known human germline sequences in VBASE data [73]. Sequence comparison was performed and the closest germline was identified by sequence similarity. The germline closest to the VAK domain of BAK278D6 (SEQ ID NO: 14) and its derivatives was identified as DP14, a VH1 family member. BAK278D6 VH has nine changes within the framework region relative to the DP14 germline. The closest germline of the VL of BAK278D6 was identified as V _λ 3 3h. The BAK278D6 VL domain (SEQ ID NO: 14) has 5 changes within the framework region relative to the germline. The framework regions of BAK278D6 and its derivatives were returned to the germline by site-directed mutagenesis (Stratagene quick change kit) to fully match the native human antibody.

Example 5
Neutralization titer of lead clones from targeted optimization of heavy chain CDR1 and heavy chain CDR2 sequences of BAK502G9 in human IL-13 dependent TF-1 cell proliferation assay Germlined frame as template using BAK502G9 sequence The work area was used to optimize the second phase. Selection was basically performed as described in Example 1, wherein residues in the heavy chain CDR1 or heavy chain CDR2 of BAK502G9 were randomized by mutagenesis. Unique clones from selected outputs were identified by DNA sequencing and neutralization titers measured as purified scFv preparations in the TF-1 cell proliferation assay as described in Example 2. Vectors were constructed in the highest titer scFv clones to allow slight modification of the method described by Persic et al. (1997 Gene 187: 9-18) to allow re-expression as a fully human IgG4 antibody. The oriP fragment was included in the vector to facilitate use with HEK-EBNA 293 cells and to allow episomal replication. The VH variable domain was cloned into a polylinker between the secretory leader sequence of the expression vector pEU8.1 (+) and the human gamma 4 constant domain. The VL variable domain was cloned into a polylinker between the secretory leader sequence of the expression vector pEU4.1 (-) and the human lambda constant domain.

  Total antibody was purified from conditioned media from EBNA293 cells co-transfected with constructs expressing heavy and light chains by protein A affinity chromatography (Amersham Pharmacia). The purified antibody fraction was sterile filtered and stored at 4 ° C. in phosphate buffered saline (PBS) before evaluation. Protein concentration was measured by measuring absorbance at 280 nm using the BCA method (Pierce). In the TF-1 proliferation assay described in Example 2, the reformatted human IgG4 whole antibody was compared to a commercially available anti-human IL-13 antibody.

Results As shown in FIG. 5, commercially available antibody B-B13 (mouse IgG1-Euroclone 5 (Euroclone 5)) was obtained from commercially available JES10-5A2 (rat IgG1-Biosource) against human IL-13. Significantly higher titers were shown (IC ₅₀ are 1021 pM and 471 pM, respectively). Eight clones derived from BAK502G9 (hence "BAK502G9 strain"), namely BAK1111D10, BAK1166G02, BAK1167F02, BAK1167F04, BAK1183H4, BAK1184C8, BAK1185E1, BAK1185F8 (where heavy chain CDR1 or CDR2 is targeted) Showed improved titers against the antibodies. These improvements were maintained when converted to whole antibody human IgG4. These VH and VL domains represent aspects or embodiments of the invention, respectively, and in each pair of claims, which are specific binding members for IL-13, BAK502G9, which includes one or more of these. Similar to a specific binding member comprising one or more CDRs from a strain clone, preferably a VH domain comprising an HCDR set of BAK502G9 strain and / or a VL domain comprising an LCDR set of BAK502G9 strain. These can be used in any and all aspects of the invention as disclosed herein. The derivative of BAK502G9 as a whole antibody (IgG4) had an IC ₅₀ in the range of 244 pM to 283 pM. BAK502G9 as total antibody IgG4 had an IC ₅₀ of 384 pM. In summary, targeting the heavy chain CDR1 (SEQ ID NO: 7) or CDR2 (SEQ ID NO: 8) of BAK502G9 will provide a significant improvement in titer. Statistical comparison to B-B13 was performed by ANOVA followed by Dunnett's post-test analysis (InStat software).

Further characterization Selected anti-human antibodies from the BAK278D6 strain were further characterized to determine their specificity. These include BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16) and their derivatives BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) and BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38), Is a representative example of clones having modifications to heavy chain CDR1 and heavy chain CDR2, respectively, of BAK502G9.

Example 6
Neutralizing potency of lead clones from targeted optimization of heavy chain CDR1 and heavy chain CDR2 of BAK502G9 against non-human primate IL-13 and IL-13 variants associated with asthma in a TF-1 factor-dependent cell proliferation assay The cross-reactivity of anti-human IL-13 antibody was measured by its ability to inhibit non-human primate IL-13 and IL-13 variant mediated TF-1 cell proliferation as described in Example 4.

Results Optimized anti-human IL-13 antibodies BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) and BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38) are the parent BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16). ) Was maintained (FIG. 6). The increase in titer against wild type IL-13 was reflected in the ability to neutralize non-human primate IL-13 and IL-13 variants with substantially equivalent titers. The IC ₅₀ of BAK502G9 against human, human variant, and non-human primate IL-13 was 1.4 nM, 1.9 nM, and 2.0 nM, respectively. The IC ₅₀ of BAK1167F2 for human, human variant, and non-human primate IL-13 was 1.0 nM, 1.1 nM, and 1.3 nM, respectively. The IC ₅₀ of BAK1183H4 for human, human variant, and non-human primate IL-13 was 0.9 nM, 1.0 nM, and 1.6 nM, respectively. These clones were suitable for therapeutic use.

Example 7
Neutralizing titer of lead anti-human IL-13 antibody against native human IL-13 in HDLM-2 cell proliferation assay The human IL-13 sequence has four potential N-glycosylation sites. The inventor has demonstrated the ability of BAK278D6 and its derivatives to neutralize recombinant IL-13 expressed in bacterial or baculovirus expression systems. Although there are evidence that many processing events known in mammalian systems also occur in insects, there are significant differences in protein glycosylation, particularly N-glycosylation [74].

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

  Drexler et al. [75] isolated HDLM-2 cells from Hodgkin's disease patients. Skinnider et al. [76] demonstrated that HDLM-2 cell proliferation was partially dependent on autocrine and paracrine release of IL-13. Lead anti-human IL-13 antibodies were evaluated for their ability to inhibit HDLLM-2 cell proliferation mediated by native (or naturally occurring) IL-13 release.

HDLM-2 Cell Assay Protocol HDLM-2 cells were obtained from German microbes and cell culture storage facility (Deutsche Sammlung Von Mikroorganismen und Zellkulturen) (DSMZ) and maintained according to the protocol provided. The assay medium consisted of RPMI 1640 with Glutamax I (Invitrogen) containing 20% fetal bovine serum. Prior to each assay, cells were pelleted by centrifugation at 300 xg for 5 minutes, the media was aspirated off and the cells were resuspended in fresh media. This procedure was repeated three times and the cells were finally resuspended at a final concentration of 2 × 10 ⁵ cells in 1 ml of assay medium. 50 μl of resuspended cells were added to each assay point of a 96 well assay plate. Antibody test solutions (triple measurements) were diluted to the desired concentration in assay medium. An irrelevant isotype antibody that does not react with IL-13 was used as a negative control. A total volume of 50 μl per well of the appropriate test antibody was added to the cells and each assay point gave a total assay volume of 100 μl / well. The assay plate was incubated for 72 hours at 37 ° C. under 5% CO 2. 25 μl of tritiated thymidine (10 μCi / ml, (NEN)) was then added to each assay point and the assay plate was returned to the incubator for an additional 4 hours. Cells were harvested on glass fiber filter plates (Perkin Elmer) using a cell harvester. Thymidine incorporation was measured using a Packard TopCount microtiter plate liquid scintillation counter. Data was analyzed using Graphpad Prism software.

Results As shown in FIG. 7, BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16) and its derivatives BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38) and BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36). ) Was able to cause dose-dependent inhibition of cell proliferation with relative titers similar to those observed in other bioassays. The IC ₅₀ of BAK502G9, BAK1183H4, and BAK1167F2 as human IgG4 was 4.6 nM, 3.5 nM, and 1.1 nM, respectively. The IC ₅₀ of commercially available antibodies JES10-5A2 and B-B13 were 10.7 nM and 16.7 nM, respectively.

Example 8
Neutralization titer of lead anti-human IL-13 antibody to IL-13 dependent response in disease-related primary cells Secondary bioassays were performed using readouts more relevant to primary cells and airway diseases. These were eotaxin release from normal human lung fibroblasts (NHLF) and upregulation of vascular cell adhesion molecule 1 (VCAM-1) on the surface of human umbilical vein endothelial cells (HUVEC). Both IL-13-dependent responses contributed to the recruitment of eosinophils [92], a hallmark of asthma symptoms.

NHLF Assay Protocol IL-13 has been shown to cause eotaxin release from lung fibroblasts [77] [78] [79]. Factor-dependent eotaxin release from NHLF was measured by ELISA.

  NHLF was obtained from Biowhittaker and maintained according to the protocol provided. The assay medium was FGM-2 (Bio Whittaker).

Antibody test solutions (triple measurements) were diluted to the desired concentration in assay medium. An irrelevant antibody that does not react with IL-13 was used as a negative control. Next, recombinant IL-derived human IL-13 (Peprotech) was added to a final concentration of 10 ng / ml when mixed with the appropriate test antibody in a total volume of 200 μl. The concentration of IL-13 used in this assay was selected as the dose that gave a maximum response of about 80%. All samples were incubated for 30 minutes at room temperature. The assay sample was then added to NHLF previously seeded at a density of 1 × 10 ⁴ cells per well in a 96-well assay plate. The assay plate was incubated at 37 ° C. under 5% CO ₂ for 16-24 hours. The assay plate was centrifuged at 300 × g for 5 minutes to pellet the detached cells. Eotaxin levels in the supernatant were measured using an ELISA using the reagents and methods provided by the manufacturer (R & D Systems). Data was analyzed using Graphpad Prism software.

Results The BAK278D6 strain clone was able to inhibit IL-13 dependent eotaxin release from NHLF. Relative titers were similar to those observed in the TF-1 cell proliferation assay (Figure 8). BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16), BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38), BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) are against 10 ng / ml human IL-13 IC ₅₀ were 207 pM, 118 pM and 69 pM, respectively. Commercially available antibodies JES10-5A2 and B-B13 had IC ₅₀ of 623 pM and 219 pM, respectively.

HUVEC Assay Protocol IL-13 has been shown to increase the expression of VCAM-1 on the cell surface of HUVEC [80,81]. Factor-dependent VCAM-1 expression was measured by detecting up-regulation of VCAM-1 receptor cell expression using time-resolved fluorescence readout.

HUVECs were obtained from BioWhittaker and maintained according to the provided protocol. The assay medium was EGM-2 (Bio Whittaker). Antibody test solutions (triple measurements) were diluted to the desired concentration in assay medium. An irrelevant antibody that does not react with IL-13 was used as a negative control. Next, recombinant IL-derived human IL-13 (Peprotech) was added to a final concentration of 10 ng / ml when mixed with the appropriate test antibody in a total volume of 200 μl. The concentration of IL-13 used in this assay was selected as the dose that gave a maximum response of about 80%. All samples were incubated for 30 minutes at room temperature. The assay sample was then added to HUVEC that had been pre-plated at a density of 4 × 10 ⁴ cells per well in a 96-well assay plate. The assay plate was incubated at 37 ° C. under 5% CO ₂ for 16-20 hours. The assay medium was then removed by aspiration and replaced with blocking solution (PBS containing 4% dry Marvel ™ milk powder). The assay plate was incubated for 1 hour at room temperature. After the wells were washed 3 times with PBST • Tween, 100 μl (1: 500 dilution in PBST / 1% Marvel ™) of biotinylated anti-VCAM-1 antibody (Serotec) was added to each well. The assay plate was incubated for 1 hour at room temperature. Wells were washed 3 times with Delfia wash buffer (Perkin Elmer) and then 100 μl Europium labeled streptavidin or anti-murine IgG1 (Delfia wash buffer (diluted 1: 1000 with Perkin Elmer)) The assay plate was then incubated for 1 hour at room temperature, the wells were washed 7 times with Delfia wash buffer (Perkin Elmer) and finally 100 μl of enhancement solution (Perkin). Elmer) was added to each well and the fluorescence intensity was measured using a Wallac 1420 Victor 2 plate reader (standard Europium protocol) Data using Graphpad Prism software Was analyzed.

Results Typical data of BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16), BAK1183H4 (VH SEQ ID NO: 37; VL SEQ ID NO: 38), BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36) as whole antibody human IgG4 Is shown in FIG. Relative titers were similar to those observed in the TF-1 cell proliferation assay. The IC ₅₀ of BAK502G9, BAK1183H4, and BAK1167F2 were 235 pM, 58 pM, and 55 pM for 10 ng / ml human IL-13, respectively.

Example 9
Neutralizing titer of anti-IL-13 antibody against IL-1β and IL-4-dependent VCAM-1 upregulation The specificity of BAK278D6 strain clones was assessed by modifying the HUVEC bioassay. Both IL-4 and IL-1β along with IL-13 have been shown to up-regulate VCAM-1 expression on the cell surface of HUVEC [80,81].

  HUVEC assay protocol The assay was performed essentially as described in Example 5 with the following modifications. Recombinant human IL-1β and IL-4 (R & D Systems) were used in place of human IL-13 at 0.5 ng / ml and 1 ng / ml, respectively, and about 80% of the maximum response The dose to give.

Results None of the evaluated clones from the BAK278D6 line did not neutralize VCAM-1 upregulation in response to human IL-1β and IL-4, and thus showed no specificity for IL-13 (FIG. 10). . IL-4 is most closely related to IL-13 and shares 30% sequence identity at the amino acid level [82].

Example 10
Neutralizing titer of BAK209B11 as human IgG4 in murine IL-13-dependent murine B9 cell proliferation assay

  BAK209B11, identified as an anti-murine IL-13 neutralizing clone as scFv as described in Example 1, was reformatted as whole antibody human IgG4 as described in Example 5, and the titer was determined as murine IL-13. Evaluated in a dependent B9 cell proliferation assay. B9 is a murine B cell hybridoma cell line [83]. B9 is factor dependent for survival and proliferation. In this regard, B9 cells respond to murine IL-13 and are maintained in media containing human IL-6 (50 pg / ml, R & D Systems). Inhibition of murine IL-13 dependent proliferation was measured by measuring the reduction in tritiated thymidine incorporation into newly synthesized DNA of dividing cells.

B9 cell assay protocol B9 cells were obtained from the European Collection of Animal Cell culture (ECACC) and maintained according to the protocol provided. The assay was performed essentially as described for the TF-1 assay in Example 2 with the following modifications. The assay medium consisted of RPMI 1640 with glutamax I (Invitrogen) containing 5% fetal bovine serum (Hyclone) and 50 μM 2-mercaptoethanol. Murine IL-13 (peprotech) from recombinant bacteria replaced human IL-13 at a final concentration of 1 ng / ml.

Results BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26) as human IgG4 neutralized 1 ng / ml murine IL-13 with an IC ₅₀ of 776 pM in the B9 assay (FIG. 11). BAK209B11 is therefore a useful tool to study the role of IL-13 in a murine model of disease. This is clearly demonstrated in Example 12, which demonstrates the efficacy of BAK209B11 in a murine model of acute lung inflammation.

Example 11
Affinity measurement of anti-IL-13 antibody by Biacore analysis BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16), BAK1167F2 (VH SEQ ID NO: 35; VL SEQ ID NO: 36), and BAK1183H4 (VH SEQ ID NO: 37) as human IgG4 The affinity of VL SEQ ID NO: 38) for human IL-13 and the affinity of BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26) for murine IL-13, basically as described in [72] Measured by surface plasmon resonance measurement using a biosensor (BIAcore AB). Briefly, at a surface density of about 500 Ru, an amine binding kit (Biacore) was used to bind the antibody to the sensor chip and serial dilutions of IL-13 in HBS-EP buffer (50 nM-0. 78 nM) was passed through the sensor chip surface. The resulting sensorgram was evaluated using BIA Evaluation 3.1 software to obtain kinetic data.

Results IgG4 of BAK502G9, BAK1167F2, and BAK1183H4 bound to human IL-13 with a high affinity of Kd of 178 pM, 136 pM and 81 pM, respectively, consistent with relative potency in cell-based assays. BAK209B11 bound to murine IL-13 with an affinity of 5.1 nM (Table 3).

Example 12
Efficacy of BAK209B11 in a murine model of acute allergic pulmonary inflammation A murine model of acute allergic pulmonary inflammation BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), ie, the efficacy of anti-murine IL-13 neutralizing human IgG4 antibody is In pneumonia. This model is performed essentially as described in Riffo-Vasquez et al. [84], at which point bronchoalveolar lavage fluid (BAL) IL-13 elevation (FIG. 12), cell infiltration into the lung and BAL (FIG. 13), analyzed by elevated serum IgE levels and airway hyperresponse (AHR).

Model Protocol Female Balb / c mice (Charles River, UK) were treated with anti-murine IL-13 antibody BAK209B11 (12, 36, 119, or 357 μg dose) or an isotype-matched control antibody (357 μg dose) did. On days 0 and 7, each group of mice was treated with 10 μg of ovalbumin (Ova) in 0.2 ml of solvent (saline containing 2% Al ₂ O ₃ (Rehydragel) as an adjuvant). Sensitization by intraperitoneal injection. Another control group of non-sensitized mice received an equal volume of vehicle. Mice were challenged with ovalbumin on days 14, 15, and 16. Ovalbumin was diluted to 1% (w / v) in sterile saline and then sprayed. All inhaled antigen stimulants were administered in a Plexiglas exposure chamber. Ova was aerosolized using a DeVilbiss Ultraneb 2000 nebulizer (Sunrise Medical) in a series of three exposures of 20 minutes, separated by 1 hour intervals.

  BAK209B11 or irrelevant human IgG4 was administered intravenously 1 day prior to the first antigen challenge and 2 hours prior to each subsequent antigen challenge (4 doses total). The model was terminated on day 17 24 hours after the last antigen challenge. Blood (serum) and BAL were collected. Serum was measured for total IgE. BAL was obtained by injecting a fixed amount of physiological saline three times (0.3 ml, 0.3 ml and 0.4 ml) and the samples were pooled. Total leukocyte and leukocyte percentages were obtained from BAL cells.

Results Ovalbumin challenge in sensitized mice caused a significant (p <0.05) increase in total BAL cell mobilization over non-sensitized but challenged animals. The recruit was inhibited by BAK209B11 in a dose-dependent manner; significant (p <0.05) inhibition was seen with BAK209B11 above 36 μg but not with the control antibody (FIG. 13). Similar effects were seen for eosinophils (FIG. 14) and neutrophils (FIG. 15), with significant (p <0.05) inhibition of cell influx at a minimum dose of 36 μg of BAK209B11. This inhibition was not seen with the control antibody. Lymphocytes were induced in sensitized mice when challenged but not in non-sensitized mice. This induction was inhibited by BAK209B11 in a dose-dependent manner, with maximal inhibition seen by 36 μg BAK209B11. The control antibody had no effect (Figure 16). When compared to non-sensitized animals, monocytes / macrophages were not induced in the sensitized animals, but the background level was suppressed by 36 μg or more of BAK209B11 and not by the control antibody (FIG. 17). Serum IgE levels were significantly elevated in sensitized animals compared to non-sensitized animals after antigen stimulation (p <0.05). This increase was reduced after treatment with 36 μg BAK209B11, but was not reduced by the control antibody.

  In summary, systemic administration of the murine IL-13 neutralizing antibody BAK209B11 inhibits inflammatory cell influx and reduces serum IgE levels caused by sensitization with ovalbumin and subsequent antigen stimulation in a murine model of allergic inflammation. Upregulation was inhibited (control antibody had no inhibitory effect).

  Examples 13-20 are predictions.

Example 13
Efficacy of BAK209B11 in Lloyd murine model of acute lung inflammation A murine model of acute allergic pulmonary inflammation The effect of BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), a murine IL-13 neutralizing antibody, was evaluated as acute allergic. A second murine model of lung inflammation was studied. This model is performed essentially as described by McMillan et al. [85], at which end BAL and lung tissue IL-13 elevation, lung and BAL cell infiltration, elevated serum IgE levels, and airway hyperresponsiveness Analyzed by (AHR).

Model Protocol Female Balb / C mice (Charles River, UK) were administered various doses of anti-murine IL-13 antibody BAK209B11 or an isotype matched control antibody as follows. On days 0 and 12, each group of mice was given 10 μg in 0.2 ml of solvent (saline containing 2 mg Al (OH) ₃ as an adjuvant [calculated as described in Example 12]). Sensitization (SN) by intraperitoneal injection of ovalbumin (Ova). Another control group, non-sensitized mice (NS), received an equal volume of vehicle. On days 19, 20, 21, 22, 23, and 24, mice were challenged with ovalbumin for 20 minutes. Ovalbumin was diluted to 5% (w / v) in physiological saline and then sprayed. All inhaled antigen stimulants were administered in a Plexiglas exposure chamber. Ova was aerosolized using a Devilbis Ultra Neb 2000 Nebulizer (Sunrise Medical). On days 18, 19, 20, 21, 22, 23, and 24, mice were challenged with various intraperitoneal doses (237 μg, 23.7 μg, or 2.37 μg; shown as H, M, and L in FIG. 21). Murine IL-13 antibody BAK209B11 muIgG1 or isotype matched control antibody (237 μg) was administered. Airway function was assessed by increasing methacholine antigen stimulation on days 0 and 25 and following conscious volume fluctuations (Buxco). PC ₅₀ (methacholine concentration required to increase baseline PenH by 50%) was estimated from a 4-parameter non-fixed curve fitting of methacholine dose response curves on day 0 and day 25 for individual mice.

  This model was terminated on day 25, 24 hours after the last antigen challenge. Blood, serum, BAL, and lung tissue were collected.

Results Lung function of individual animals was evaluated on day 0 (before treatment) and day 25 (after antigen stimulation), and PC50 values (concentration of methacholine required to raise baseline PenH by 50%) were calculated and quantified. (FIG. 21A). Individual airway hyperresponsiveness (AHR) was measured by the change in log PC ₅₀ on day 25 from day 0 (log 25 day PC ₅₀ -log 0 day PC ₅₀ ). The amount of change in log PC 50 was the primary endpoint of the test; PC ₅₀ data were log-transformed because of the need of the endpoint ANOVA. Individual changes were averaged within the group to give the group average log PC ₅₀ change (shown in FIG. 21B).

  Ovalbumin challenge in sensitized mice caused significant AHR compared to unsensitized challenge mice (p <0.01). BAK209B11 caused a clear and dose-dependent decrease in AHR, whereas the control antibody had no effect.

Example 14
Efficacy of BAK209B11 in Gerad murine model of acute lung inflammation A murine model of acute allergic lung inflammation The effect of BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), an anti-murine IL-13 neutralizing human IgG4 antibody, A third murine model of acute allergic lung inflammation was examined. This model is performed essentially as described by Humbles et al. [86], at which time BAL and lung tissue IL-13 elevation, lung and BAL cell infiltration, elevated serum IgE levels, and airway hyperresponsiveness Analyzed by (AHR).

Model Protocol Female Balb / C mice (Charles River, UK) were administered various doses of anti-murine IL-13 antibody BAK209B11 or isotype matched control antibody. On days 0, 7, and 14, each group of mice was treated with 0.2 ml of vehicle (saline containing 1.125 mg Al (OH) ₃ as an adjuvant (calculated as described in Example 12). Sensitization (SN) by intraperitoneal injection of 10 μg ovalbumin (Ova) in water). Another control group, non-sensitized mice (NS), received an equal volume of vehicle. On days 21, 22, 23, and 24, mice were challenged with ovalbumin for 20 minutes. Ovalbumin was diluted to 5% (w / v) in physiological saline and then sprayed. All inhaled antigen stimulants were administered in a Plexiglas exposure chamber. Ova was aerosolized using a Devilbis Ultra Neb 2000 Nebulizer (Sunrise Medical).

  This model was terminated on day 25, 24 hours after antigen stimulation. Blood, serum, BAL, and lung tissue were collected.

Example 15
Efficacy of BAK209B11 in a Lloyd Chronic Model of Pulmonary Inflammation Murine Model of Chronic Allergic Pulmonary Inflammation The effect of BAK209B11 (VH SEQ ID NO: 25; VL SEQ ID NO: 26), an anti-murine IL-13 neutralizing human IgG4 antibody, was We investigated in a model of allergic lung inflammation. This model is basically performed as described by Temelkovski et al. [87] and is analyzed at the end by cell infiltration into the lungs and BAL, elevated serum IgE levels, and airway hyperresponse (AHR).

Model Protocol Female Balb / C mice (Charles River, UK) were administered various doses of anti-murine IL-13 antibody BAK209B11 or isotype matched control antibody. On days 0 and 11, each group of mice was treated with 10 μg in 0.2 ml of solvent (saline containing 2 mg Al (OH) ₃ as an adjuvant [calculated as described in Example 12]). Sensitization (SN) by intraperitoneal injection of ovalbumin (Ova). Another control group, non-sensitized mice (NS), received an equal volume of vehicle. On days 18, 19, 20, 21, 22, 23, 28, 30, 32, 35, 37, 39, 42, 44, 46, 49, and 51, mice were challenged with ovalbumin for 20 minutes. Ovalbumin was diluted to 5% (w / v) in physiological saline and then sprayed. All inhaled antigen stimulants were administered in a Plexiglas exposure chamber. Ova was aerosolized using a Devilbis Ultra Neb 2000 Nebulizer (Sunrise Medical).

  This model was terminated on day 52, 24 hours after antigen stimulation. Blood, serum, BAL, and lung tissue were collected.

Example 16
Efficacy of anti-human IL-13 antibody against exogenous human IL-13 administered to a murine air pouch model The effect of anti-human IL-13 antibody on the pro-inflammatory action of human IL-13 is a basic murine model. I examined it. This model was basically performed as described by Edwards et al. [93] and analyzed by cell infiltration into the air sac at the end point.

Model Protocol An air pouch was created on the back of female Balb / c mice by subcutaneous injection of 2.5 ml of sterile air on day 0. On the third day, the air pouch was again inflated with 2.5 ml of sterile air. On day 6, 2 μg huIL-13 in 0.75% CMC was injected directly into the sac. After 24 hours, the mice were sacrificed and the air sac was washed with 1 ml of heparinized saline. Antibody treatment was performed with huIL-13 (in the sac) or systemically.

Results Human IL-13 injected into the air sac (intraperitoneally) 24 hours after antigen challenge in vehicle (0.75% carboxymethylcellulose (CMC) in saline, ip) in mice treated with total leukocytes (p <0.01) and eosinophils (p <0.01) caused a significant increase in infiltration.

  Topically administered BAK502G9 (200 mg, 20 mg, or 2 mg in the sac) induced total leukocytes (p <0.01) and eosinophils (p <0.01) into the air sac induced by 2 μg huIL-13 in 0.75% CMC. p <0.01) invasion was significantly and dose-dependently inhibited.

  Systemically administered BAK209B11 (30 mg / kg, 10 mg / kg, and 1 mg / kg) was also added to total leukocytes (p <0.01) into the air sac generated by 2 μg huIL-13 in 0.75% CMC. And eosinophil (p <0.01) infiltration were significantly and dose-dependently inhibited.

Example 17
Generation of human IL-13 knock-in / murine IL-13 knock-out transgenic mice for assessing the efficacy of anti-human IL-13 antibodies in a pulmonary allergic inflammation model. Mice expressing 13 were generated. The mouse IL-13 gene is replaced with the relevant portion of the human IL-13 gene from the start codon to the stop codon. This mouse species expresses human IL-13 rather than mouse IL-13 in response to the same stimulation as wild-type mice because the endogenous IL-13 promoter and IL-13 pA tail are not altered. Human IL-13 can bind to and signal through the murine IL-13 receptor and produce the same physiological results as signaling caused by murine IL-13 binding to the murine IL-13 receptor. Proven. For example, exogenous human IL-13 caused recruitment of inflammatory cells into the murine air sac (FIG. 18). These transgenic animals make it possible to evaluate non-murine cross-reactive anti-human IL-13 antibodies in a murine model of established disease.

  This mouse is used in an acute allergic airway inflammation model (described in Examples 18 and 19) and a chronic allergic airway inflammation model (described in Example 20), and anti-human IL-13 antibody in allergic airway disease Makes it possible to evaluate the pharmacology of

Example 18
Efficacy of anti-human IL-13 antibody in huIL-13 transgenic loid murine model in acute lung inflammation Effect of anti-human IL-13 neutralizing human IgG4 antibody in trans produced in Example 17 The transgenic mice were used to investigate a murine model of acute allergic lung inflammation. This model was performed essentially as described by McMillan et al. [85] and as described in Example 13. This model is analyzed at the endpoint by elevated BAL and lung tissue IL-13, cellular infiltration into the lung and BAL, elevated serum IgE levels, and airway hyperresponse (AHR).

Model Protocol The protocol for this model is the same as Example 13 except that anti-human IL-13 antibody was administered instead of BAK209B11.

Example 19 Potency of anti-human IL-13 antibody in huIL-13 transgenic Gerad murine model in acute lung inflammation Murine model of acute allergic lung inflammation The effect of anti-human IL-13 neutralizing human IgG4 antibody The transgenic mice created in Example 17 were used to investigate another murine model of acute allergic lung inflammation. This model was performed essentially as described by Humbles et al. [86] and as described in Example 14. This model is analyzed at the endpoint by elevated BAL and lung tissue IL-13, cellular infiltration into the lung and BAL, elevated serum IgE levels, and airway hyperresponse (AHR).

Model Protocol The protocol for this model is the same as Example 14 except that anti-human IL-13 antibody was administered instead of BAK209B11.

Example 20
Efficacy of anti-human IL-13 antibody in huIL-13 Transgenic Lloyd (Lloyd) murine model in pulmonary inflammation The effect of anti-human IL-13 neutralizing human IgG4 antibody using the transgenic mice generated in Example 17 We investigated in a model of chronic allergic lung inflammation. This model was performed essentially as described by Temelkovski et al. [87] and as described in Example 15. This model is analyzed at that endpoint by cell infiltration into the lungs and BAL, elevated serum IgE levels, and airway hyperresponse (AHR).

Model Protocol The protocol for this model is the same as Example 15 except that anti-human IL-13 antibody was administered instead of BAK209B11.

Example 21
Pharmacokinetics and pharmacodynamics of anti-human IL-13 antibody in Ascaris suum allergic cynomolgus monkey 4 after the single intravenous high dose of 10 mg / kg One allergic but non-antigen stimulated cynomolgus primate (2 males / 2 females) was evaluated. The experiment was conducted for 29 days. Antibody pharmacokinetic parameters were determined from the geometric mean of the serum-drug concentration curve and are shown in Table 4 below. In the same study, serum IgE concentrations were also followed using a human IgE ELISA kit (Bethyl Laboratories, USA).

Results Serum IgE concentration significantly decreased from 100% control level (pre-dose) to 66 ± 10% of the control value 4 days and 5 days after administration after a single intravenous high dose of 10 mg / kg of BAK502G9 (p <0.05). The decrease in serum IgE concentration recovered to 88 ± 8% of the control level by day 22 (see FIG. 20). Again, these data were obtained by normalizing the serum IgE concentration of each animal to the pre-dose level (pre-dose concentration being 100%) and then averaging the curves from the 4 animals tested. .

  Two male monkeys had relatively low pre-dose total serum IgE (60 ng / ml and 67 ng / ml). These IgE levels did not change to suggest a trend after treatment with BAK502G9 (FIG. 20B). Two female monkeys had relatively high pre-dose total serum IgE (1209 ng / ml and 449 ng / ml). These IgE levels decreased after treatment with BAK502G9, decreased to a maximum of 60% on day 7 and recovered to near pre-dose levels by day 28 after administration (FIG. 20B). These data indicate that BAK502G9 reduces serum IgE levels in animals with relatively high circulating IgE of IgE.

Example 22
Efficacy of anti-HI IL-13 antibody in cynomolgus monkey model of skin allergy The effect of anti-human IL-13 neutralizing human IgG4 antibody was investigated in a primate model of acute allergic lung inflammation. This model was performed by intradermal injection of human IL-13 and Ascaris suum antigen into cynomolgus monkey. After 24-96 hours, skin biopsy samples and serum samples were taken. This model was analyzed by cell infiltration into the skin at its end point.

Example 23
Efficacy of anti-human IL-13 antibody in cynomolgus model of lung allergy The effect of anti-human IL-13 neutralizing human IgG4 antibody was examined in a primate model of acute allergic lung inflammation. This model was performed by exposing the A. suum allergic cynomolgus primate to a nebulized Ascaris suum antigen, thus producing an allergic reaction. This allergy is analyzed at the end by cell infiltration into the lung and BAL, elevated serum IgE levels, and airway hyperresponsiveness.

  Furthermore, pharmacodynamics were evaluated ex vivo using flow cytometry. CD23 is a high affinity IgE receptor and can be expressed on mononuclear cells of human peripheral blood. CD23 expression can be induced by both IL-13 and IL-4 in terms of the number of cells expressing CD23 and how many CD23 each cell expresses. IL-13 (not IL-4) mediated responses can be inhibited by anti-human IL-13 antibodies.

Animals to participate in this two-phase study were selected based on pre-established AHR after antigen stimulation of nebulized antigen (A. suum extract). Phase I airway function was evaluated on days 1 and 11 during intravenous histamine challenge. PC ₃₀ (histamine dose required to cause a 30% increase in lung resistance (R _L ) above baseline was determined from each histamine dose response curve. Individually adjusted doses of atomized antigen that have been shown to cause animals a 40% increase in R _L and a 35% decrease in dynamic compliance (C _DYN ) on days 9 and 10 Stimulated with antigen. In this model, a larger R _L is conventionally observed than the second allergen administration, which is antigen priming. Two antigenic stimulation, the increase in the area under the histamine dose-response curves on day 11 compared to day 1, as measured by a decrease in PC _30, BAL, and eosinophils, cause AHR It was. Animals exhibiting AHR symptoms were selected and placed in Phase II.

  Phase II was performed exactly as Phase I, except that all animals were injected with 30 mg / kg BAK502G9 on days 1, 5 and 9. The effect of BAK502G9 was evaluated by comparing the changes seen in phase II for individual animals with the changes seen in phase I.

  Blood, serum, BAL, and lung tissue were collected. Serum IgE levels were followed by ELISA. BAK502G9-treated cynomolgus monkey serum has been shown to inhibit the expression of CD23 on human peripheral blood mononuclear cells induced by IL-13 (but not IL-4). The magnitude of this inhibition was consistent with the serum BAK502G9 level predicted by PK ELISA.

Results BAK502G9 significantly inhibited AHR (p <0.05) as measured by _RL AUC (FIG. 26A; Table 7). The inhibitory effect of BAK502G9 against AHR as measured by PC _30, but was observed, did not reach statistical significance (Figure 26B; Table 7). BAK502G9 also significantly inhibited antigen priming (p <0.01) (FIG. 26C; Table 7) and BAL inflammation. BAK502G9 significantly inhibited total cell (p <0.01) and eosinophil (p <0.05) influx into BAL, but not macrophage, lymphocyte or mast cell influx ( FIG. 26D; Table 7).

Example 24
Efficacy of anti-human IL-13 antibody against asthma symptoms that develop when human IL-13 is administered to mouse lungs Murine model of airway hyperresponsiveness Airway hyperresponse (AHR) after administration of human IL-13 to mouse lungs The efficacy of the anti-human IL-13 neutralizing antibody BAK502G9 against the onset of the disease was examined. This model was performed essentially as described by Yang et al. [119] except that human IL-13 was used instead of murine IL-13.

Model Protocol Male Balb / c mice were exposed to two doses of human IL-13 at 48 hour intervals to develop symptoms. Briefly, mice were anesthetized by intravenous injection of 100 μl saffan solution (1: 4 dilution with water). Mice were intubated with 22 gauge catheter needles from which human recombinant IL-13 (25 μg dissolved in 20 μl phosphate buffered saline (PBS)) or solvent control (PBS) was instilled. Airway function was assessed by increasing methacholine challenge 24 hours after the last dose of IL-13 and following conscious plethysmography (Buxco). PC ₂₀₀ (the concentration of methacholine required to increase baseline PenH by 200%) was determined from a four parameter non-fixed curve fit of the methacholine dose response curve. Antibody treatment was performed by intraperitoneal injection 24 hours prior to each IL-13 administration.

Results Intratracheal infusion of human IL-13 into wild-type mice without experience with administration resulted in a significant (p <0.05) airway hyperresponse relative to control animals as measured by PC ₂₀₀ methacholine concentration. It was. BAK502G9 (1 mg / kg) administered systemically significantly (p <0.01) inhibited the appearance of AHR, while the zero control antibody had no effect (FIG. 23).

Example 25
Neutralizing titer of BAK502G9 as human IgG4 against human IL-13 dependent IgE release from human B cells B cell switching assay protocol IL-13 has been demonstrated to induce IgE synthesis in human B cells in vitro [120]. Factor-dependent IgE release from human B cells was measured by ELISA. The neutralizing titer of BAK502G9 as human IgG4 was evaluated against IL-13 dependent IgE release from human B cells.

Peripheral blood mononuclear cells (PBMC) were purified from human buffy coat (Blood Transfusion Service) by density gradient centrifugation at 1.077 g / L. B cells were purified from PBMC using B cell isolation kit II (Miltenyi Biotec) using reagents and methods described by the manufacturer. The assay medium consisted of Iscove's modified Dulbecco medium (Life Technologies) containing 10% fetal bovine serum and 20 μg / ml human transferrin (Serologicals Proteins Inc.). After purification, B cells were resuspended in assay medium at a final concentration of 10 ⁶ / ml. 50 μl of resuspended cells were added to each assay point of a 96 well assay plate. 50 μl of 4 μg / ml anti-CD40 antibody EA5 (Biosource) was added to the assay wells as appropriate. Antibody test solutions (sixfold measurements) were diluted to the desired concentration in assay medium. An irrelevant antibody that does not react with IL-13 was used as a negative control. 50 μl / well of the appropriate test antibody was added to the cells. Next, recombinant IL-derived human IL-13 (Peprotech) was added to a final concentration of 30 ng / ml to give a total assay volume of 200 μl / well. The concentration of IL-13 used in the assay was selected to obtain a maximum response. The assay plate was incubated for 14 days at 37 ° C. with 5% CO ₂ . IgE levels in the supernatant were measured by ELISA using reagents and protocols provided by the manufacturer (BD Biosciences, Bethyl Laboratories). Data was analyzed using Graphpad Prism software.

Results As shown in FIG. 24, BAK502G9 (VH SEQ ID NO: 15; VL SEQ ID NO: 16) was able to inhibit human IL-13-dependent IgE production by human B cells. BAK502G9 as human IgG4 had an IC ₅₀ of 1.8 nM against 30 ng / ml human IL-13.

Example 26
Efficacy of BAK502G9 on IL-13-mediated enhancement of histamine-induced Ca2 ⁺ signaling in primary human bronchial smooth muscle cells IL-13 has been shown to directly regulate airway smooth muscle contraction [121,122]. Intracellular calcium mobilization is an essential requirement for smooth muscle contraction. Recent studies have demonstrated that IL-13's ability to alter smooth muscle contractility is mediated in part by modulation of contractile agonist-induced Ca ²⁺ signaling [123, 124].

The potency of BAK502G9, an anti-human IL-13 antibody formatted as IgG4, against the IL-13 mediated modification of primary human bronchial smooth muscle cell (BSMC) signaling response to the contractile agonist histamine, Ca ²⁺ signaling Investigated by assay.

BSMC Ca ²⁺ Signaling Assay Protocol Primary human BSMC, smooth muscle growth medium-2 (SmGM-2) and smooth muscle basal medium (SmBM) were obtained from BioWhittaker. BSMC was maintained in SmGM-2 according to vendor recommendations. BSMCs were seeded in 96-well microtiter cell culture plates at 2 × 10 ⁴ cells / well, allowed to attach for 24 hours, then re-fed and incubated for an additional 24 hours. Prior to Ca ²⁺ signaling experiments, BSMCs were stimulated with IL-13 (Peprotech) at a final concentration of 50 ng / ml and incubated for 18-24 hours. An irrelevant control monoclonal antibody CAT-0001 isotype matched to BAK502G9 was evaluated at a final concentration of 10 μg / ml. Cells that responded to histamine (Calbiochem) were tested for Ca ²⁺ concentration (titer measurement from 20 μM), Ca ²⁺ sensitive dye Fluo-4 (Molecular Probes) and 96-well fluorescence imaging plate reader ( Measurements were made using standard methods using FLIPR (Molecular Devices). The area under the curve of the Ca ²⁺ signaling response in response to histamine (AUC) was measured for each pretreatment conjugate. Data analysis was performed using GraphPad Prism Windows (registered trademark) Version 4 (GraphPad Software).

Results Preincubation of BSMC with IL-13 significantly enhanced Ca ²⁺ signaling in response to histamine. Preincubation of BAK502G9 with IL-13 significantly inhibited enhancement of Ca ²⁺ signaling in response to histamine (but not an irrelevant control antibody using IL-13 (FIG. 25A)) (FIG. 25) .

Example 27
Anti-IL-13 antibody neutralization titers in human IL-13-dependent PBMC CD23 expression assay. Representative IL-13 antibody titers were assessed by human IL-13-dependent peripheral blood mononuclear cell (PBMC) CD23 expression assay. did. By increasing the cell surface expression of CD23, PBMC responds to both IL-13 and IL-4 [120]. CD23 (FceRII) is a low affinity receptor for IgE and is expressed on a variety of inflammatory cells (including mononuclear cells). Inhibition of human IL-13-dependent CD23 expression upregulation was measured by measuring the decrease in binding of fluorescently labeled CD23 monoclonal antibody to PBMC by flow cytometry.

Assay Protocol Human blood was obtained from Blood Transfusion Service and red blood cells were removed by dextran-T500 (Pharmacia) sedimentation (final concentration 0.6%) for 40 minutes. The leukocyte and platelet rich fractions are then collected on a discontinuous percoll gradient of 3 ml 64% and 5 ml 80% (100% is 9 parts Percoll and 1 part 10 × PBS) for 20 minutes. Separated by centrifugation at 1137 g. PBMCs were collected from the top of the 64% layer and resuspended in assay buffer (Invitrogen RPMI 1640, 10% FCS, 200 IU / ml penicillin, 100 μg / ml streptomycin, 2 mM L-glutamine). The assay contains 2 × 10 ⁶ cells, ± 80 pM recombinant human IL-13 (Peprotech) or 21 pM recombinant human IL-4 (R & D Systems), ± BAK502G9 or irrelevant IgG4 in a final volume of 500 mcl. Performed in well plates. The cells were harvested after culturing at 37 ° C. for 48 hours and stained with CD23-PE (BD Farmingen) for 20 minutes at 4 ° C. Finally, the cells were read on a flow cytometer. CD23 expression was measured by CD23 “score”; the percentage of CD23 positive cells was multiplied by the “brightness” of staining (geometric mean fluorescence). CD23 “score” without stimulant was subtracted and data presented as percent response (100%) to IL-13 alone. Data were expressed as mean ± SEM obtained from 4-6 different experiments, with each point performed in triplicate using cells from 4-6 individual donors.

Results Incubation of PBMC with 80 pM IL-13 or 21 pM IL-4 for 48 hours clearly expressed CD23 (FIGS. 27 and 28). BAK502G9 inhibited IL-13-induced CD23 expression in a dose-dependent manner with a geometric mean of 120.2 pM (FIG. 27). In contrast, BAK502G9 failed to inhibit CD23 expression induced by 21.4 pM IL-4 (n = 4 from individual donors, FIG. 28). Irrelevant IgG4 did not inhibit IL-13 or IL-4-dependent CD23 expression on PBMC (FIGS. 27 and 28). Co-stimulation of PBMC with 80 pM IL-13 and 21.4 pM IL-4 caused additional CD23 expression. BAK502G9 (but not CAT-001) reduced CD23 expression levels to that seen with IL-4 stimulation alone (FIG. 28).

Example 28
Neutralizing titer of human IL-13 antibody in human IL-13-dependent eosinophil shape change assay The purpose of this study was to determine whether IL-13 [125,126], TNF-α [127], TGF-β1 [128] It was to evaluate the effect of IL-13 antibody on eosinophil shape change induced by mediators released from NHLF after stimulation with factors present in the lungs of such asthmatic patients. IL-13 synergizes with TNF-α [129] or TGF-β1 [130] to induce fibroblasts to produce eotaxin-1, which in turn chemoattracts eosinophils directly Can act as follows. The leukocyte shape change response is mediated by cytoskeletal rearrangement and is essential for the leukocyte migration process from the microcirculation to the site of inflammation. Inhibition of IL-13-dependent morphogenic inducer release by NHLF was measured by measuring the decrease in eotaxin-1 secretion by ELISA and the decrease in eosinophil morphological changes by flow cytometry.

Assay Protocol NHLF cells were cultured on medium alone or stimulator [9.6 nM IL-13, 285.7 pM TNF-α (R & D Systems) and 160 pM TGF-β1 (R & D Systems). (R & D Systems))] was co-cultured in the absence or presence of BAK502G9 (concentration range 875 nM to 6.84 nM). The cells were then cultured at 37 ° C. for a further 48 hours, and the resulting conditioned medium was aspirated and stored at −80 ° C. The concentration of eotaxin-1 in the conditioned medium was evaluated using the R & D Systems Duoset ELISA system (R & D Systems).

Human blood was obtained from a Blood Transfusion Service and red blood cells were depleted by dextran-T500 (Pharmacia) sedimentation (final concentration 0.6%) for 40 minutes. The leukocyte and platelet rich fractions are then collected for 20 minutes on a discontinuous percoll gradient of 3 ml of 64 & and 5 ml of 80% (100% is 9 parts Percoll and 1 part 10 × PBS). Separated by centrifugation at 1137 g. 64%: Granulocytes are collected from the 80% interface, washed, assay buffer (Sigma PBS, 1 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 10 mM D-glucose, 0.1% Sigma BSA, Resuspended in pH 7.3). The assay was performed in FACS tubes using 5 × 10 ⁵ cells, ± 3 nM recombinant human eotaxin-1 (R & D Systems) or conditioned media in a final volume of 400 μl. Cells were incubated at 37 ° C. for 8.5 minutes, then transferred to 4 ° C., fixed with fixatives (CellFix, BD Bioscience), and finally read on a flow cytometer. Eosinophils were identified by their FL-2 autofluorescence and forward scatter (FSC) parameters were read. Eosinophil FSC changed in response to both eotaxin-1 and conditioned medium, and morphological changes were measured. The tube was sampled at a high flow rate and collection was stopped after 1000 eosinophil events or 1 minute (whichever comes first). Morphological change was calculated as the percent of FSC (100% blank morphological change) caused by the morphological change buffer alone. Data were expressed as mean% blank shape change ± SEM from 4 different experiments. Each experiment was performed in duplicate for each point using cells from individual buffy coats (and thus individual donors).

Results When NHLF cells were co-stimulated with 9.6 nM IL-13, 285.7 pM TNF-α and 160 pM TGF-β1 and cultured for 48 hours, 9.6 nM eotaxin-1 was secreted into the medium. In contrast, NHLF cells cultured only in the maintenance medium only secreted 0.1 nM eotaxin-1 into the medium. NHLF cell eotaxin-1 production co-stimulated with IL-13 / TNF-α / TGF-β1 was inhibited by BAK502G9 in a dose-dependent manner with an IC ₅₀ of 32.4 nM (FIG. 29A), so this eotaxin-1 production Was IL-13 dependent.

  The main purpose of this part of the study was to examine eosinophil shape change. The magnitude of eosinophil shape change in response to 3 nM eosinophils (positive control) was 122.2 ± 2.1% (n = 4). Eotaxin-1 induced morphological change was completely inhibited by 100 nM anti-eosinophil antibody CAT-213, with an average morphological change of 101.0 ± 1.0% (n = 4).

  The medium (conditioned medium) obtained from NHLF cells co-stimulated with 9.6 nM IL-13, 285.7 pM TNF-α and 160 pM TGF-β1 and cultured for 48 hours showed obvious eosinophil shape change. Induced (Figure 29B). In contrast, medium from NHLF cultured for 48 hours in NHLF maintenance medium alone did not induce eosinophil shape change (FIG. 29B).

Addition of anti-IL-13 antibody BAK502G9 to costimulation medium prior to NHLF culture caused a dose-dependent inhibition of eosinophil shape change with a geometric mean IC ₅₀ of 16.8 nM measured at 1:16 dilution ( FIG. 29B).

  The ability of stimulants (IL-13, TNF-α and TGF-β1) not cultured with NHLF cells to induce eosinophil and neutrophil shape changes was also examined. 9.6 nM IL-13, 285.7 pM TNF-α and 160 pM TGF-β1 did not induce obvious eosinophil shape change. This suggests that the eosinophil shape-changing ability of the conditioned medium that occurs during NHLF cell culture with stimulants is not due to either stimulant alone or in combination (FIG. 29B).

Example 29
Mapping of anti-IL-13 antibodies on human IL-13 Epitope mapping of the representative IL-13 antibody BAK502G9 was performed using a molecular approach and standard peptide cleavage.

Molecular Approach An IL-13 chimera was created in which part of the human IL-13 sequence was replaced with a murine sequence. These chimeras were used in binding studies with representative IL-13 antibodies to help identify specific epitopes.

  Two panels of IL-13 chimera were created. The first panel contained 9 chimeras (Figure 30) and was used to locate the overall location of the epitope. The second panel contained 10 chimeras (Figure 31) and was used to finely map the epitope.

  The chimeric IL-13 sequence is assembled using PCR and cloned into the Gateway ™ entry vector, which is then cloned into the destination vector pDEST8 (detection and affinity tags at the C-terminus of the recombinant protein. And recombination was performed. These expression vectors were used to transform DH10Bac ™ competent E. coli by chemical treatment to site-specificate the tagged chimeric IL-13 into a baculovirus shuttle vector (bacmid). Dislocation was made possible. Recombinant bacmid DNA was isolated for each IL-13 chimera and transfected into Sf9 (Spodoptera frugiperda) insect cells using the Cellfectin ™ reagent. Recombinant baculovirus was harvested 72 hours after transfection and passaged two more times with Sf9 insect cells.

  Insect 2000-500 ml culture supernatant is purified on an affinity column, the eluted material is concentrated from 16 ml to 1 ml and applied to a size exclusion Superdex 200HR10 / 300GL column for final finishing and buffer exchange. Loaded.

  A homogeneous competition assay was developed using biotinylated human IL-13, streptavidin-anthophyocyanate and europium labeled BAK502G9. The assay is as follows: Eu-BAK502G9 binds to biotinylated human IL-13, then the complex is recognized by the streptavidin APC conjugate, and when flash is applied, energy is transferred from the APC label. Transfer to nearby europium and time-resolved fluorescence can be measured. This binding competition is introduced by unlabeled human IL-13 (as a control) and the chimeric construct. By quantifying this competition and calculating the relative affinity of the IL-13 variant for the IL-13 antibody, it becomes possible to identify mutations that alter binding.

Results For binding to BAK502G9, the chimeric construct IL13-helix D (Table 5) is the weakest competitor to biotinylated human IL-13, and helix D within the IL-13 molecule is involved in BAK502G9 epitope binding. (Table 5). A decrease in activity was seen for the 40, 41 and 33, 34 variants in which residues 40, 41, and 33, 34 of the parent sequence were changed, indicating the possible involvement of helix A in the recognition of BAK502G9. Yes. Since loop 3 has a reduced number of amino acids in this mutant compared to the human molecule and may change the overall structure of the protein, this reduced activity of the loop was ignored. Other reductions in the ability of chimeric IL-13 molecules to compete for BAK502G9 binding were not considered important for such amino acid changes.

  Next, a more targeted set of mutations in helix D (FIG. 26) was tested. The results obtained are shown in Table 6 and are as follows:

  The results show that the chimeric construct 116117TK (the lysine at position 116 was replaced with threonine and the aspartic acid at position 117 was replaced with lysine), 123KA (the lysine at position 123 was replaced), and 127RA (position 127) Arginine was substituted) indicates that it can compete at least for binding to BAK502G9 (123KA and 127RA do not compete at 1 μM). Other residues that are supposed to bind to BAK502G9 due to reduced efficacy in competition assays include helix D residue 124Q (lysine substituted with glutamine) and 120121SY (leucine histidine pair is serine tyrosine Is changing in pairs). Mutation of leucine at position 58L also reduces binding, and analysis of the 3D structure reveals that this residue assembles into helix D and is directly contacted by BAK502G9 or affects helix D alignment did.

  These experiments demonstrate that residues in helix D are critical for BAK502G9 binding to IL-13. In particular, lysine at position 123 and arginine at position 127 are critical for this binding because any of these mutations abolish binding to BAK502G9.

Epitope cleavage Epitope mapping of BAK502G9 was also performed using standard peptide cleavage methods. Here, IgG is immobilized on a solid phase to capture the IL-13 ligand. The complex formed then undergoes specific proteolytic digestion during which the accessible peptide bonds are cleaved, while those protected by the IgG: ligand interface remain intact. That is, the peptide containing the epitope binds to IgG. This is then desorbed, collected and identified by mass spectrum (ms).

  Two complementary techniques are used, the first method uses a Ciphergen ProteinChip Reader MALDI-TOF mass spectrometer, where IgG can be covalently attached to the mass spectrometer chip, Digestion and extraction can be performed in situ. The second technique used biotinylated BAK502G9 coupled to streptavidin-coated beads, which allowed the collection of sufficient peptides for sequence confirmation by tandem mass spectrometry (ms / ms).

  The two methods differ in absolute detail and scale, but are essentially the same steps (IgG binding, blocking of unreacted binding sites, washing, ligand capture, removal of unbound ligand, digestion, and final wash steps). Included.

  The MALD-TOF ms approach uses a dedicated ms chip activated with carbonyldiimidazole covalently attached to free primary amine groups conjugated with 1-2 mg / ml IgG in PBS at 4 ° C. overnight. The chip was then blocked with an ethanolamine solution for 1 hour at room temperature and then washed thoroughly with PBS or HBS + suitable surfactant. A 1 picomolar aliquot of IL-13 was then applied to the chip in PBS or HBS and allowed to bind to chemically immobilized IgG for 2 hours at room temperature. Next, it was further washed with PBS with or without surfactant or HBS to remove non-specifically bound IL-13. A trypsin solution in the range of 200-3.1 μg / ml in PBS or HBS is applied to the IgG: ligand complex and digestion is allowed to proceed for 30 minutes at room temperature, followed by PBS or HBS + surfactant, PBS or HBS, Finally, the chip was washed with water. After applying the appropriate MALD-TOF ms matrix, the chip was placed directly into the mass spectrometer for analysis.

  The bead-based approach started with the biotinylation of IgG (1 · IgG to 4 · biotin molecule molar ratio) using NHS biotin compounds. Gel filtration was then used to remove unbound biotin and reaction byproducts. Biotinylated IgG was then bound to neutravidin-coated agarose beads, where IgG capture was maximized. Next, an aliquot of IgG-coated beads was placed in a concentrated spin column, washed with Dulbecco PBS + 0.05% Tween 20, and then resuspended in Dulbecco PBS + 0.05% Tween 20. A pulse of IL-13 is then applied to the resuspended IgG beads and binding is allowed to proceed for an additional 10 minutes, then the liquid phase is removed by centrifugation, the beads are washed with Dulbecco's PBS + 0.05% Tween 20, and then Were resuspended in Dulbecco's PBS + 0.05% Teen20.

  The beads: IgG: ligand complex was then incubated with trypsin or chymotrypsin at room temperature or 37 ° C. for proteolysis. The beads were then washed again with Dulbecco PBS + 0.05% Tween 20, and then further with Dulbecco PBS without surfactant. The beads were then resuspended in a mixture of water, acetonitrile and trifluoroacetic acid and the supernatant was collected. This was then analyzed variously by MALDI-TOF ms or reverse phase HPLC mass spectra (eg tandem (ms / ms fragmentation using a ThermoQuest LCQ ESI ion trap mass spectrometer)). The resulting fragment pattern was matched to the human IL-13 sequence and the other heavy and light chain sequences of BAK502G9 IgG.

  During the experimental process, using many controls (mainly blank surface, IgG only, isotype control), the identified peptide was obtained specifically from IgG capture IL-13 and the product of BAK502G9 Or proved not to be the product of non-specifically bound IL-13 digestion.

Results This experimental series consistently gave a single IL-13 specific peptide for each digestion. Data from the LCQ ion trap revealed that the trypsin fragment has a monoisotopic mass of 3258 Da (MH +) and the chymotrypsin fragment has a monoisotopic fragment of 3937 Da (MH +).

  These mass searches for proper in silico digestion of human IL-13 gave close agreement with related peptides in the C-terminal part of the molecule.

  Tryptic peptide mass match: 3258 Da With a tolerance of 1000 ppm, 3258 Da matches the sequence from aspartic acid at position 106 to the C-terminal asparagine at position 132. There is no other match in this tolerance. This region is highlighted in bold on the sequence of the precursor form of human IL-13 below.

  Chymotrypsin peptide mass match: 3937 Da With a tolerance of 1000 ppm, 3937 Da matches the sequence from serine at position 99 to the C-terminal asparagine at position 132. This region is highlighted in bold on the sequence of the precursor form of human IL-13 below.

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

  The body of both peptides was successfully confirmed by ms / ms and none showed significant sequence analogs with BAK502G9. The ms / ms fragment map, adjusted to identify Y or B ions, corresponds to 26 out of 104 possible ions in one charge state of a trypsin peptide and 19 out of 128 possible ions in a chymotrypsin peptide. Matched. Examination of all charge states shows the identification of 23 of the 27 amino acid residues of the trypsin fragment and 29 of the 33 residues of the chymotrypsin fragment. This is sufficient to confirm identity.

  The overall experimental sequence identified that a portion of the BAK502G9 epitope on human IL-13 is present within 27 C-terminal amino acid residues. These findings confirm the findings of the molecular approach detailed above.

Twenty-one animals showing AHR (PC ₃₀ ) in Phase I and additional animals with antigen-priming symptoms were advanced to Phase II trials (22 in total). As measured by AUC and PC _30, not the necessarily all animals have the AHR. Only animals that showed AHR in Phase I and whose AHR was evaluated in Phase I and Phase II were included in the AHR results. Statistical tests were performed using InStat. The test was a two-tailed Student's t-test with the null hypothesis that the endpoint included the number 0 (ie, there was no change in Phase II compared to Phase I); * p <0.05, ** p <0.01. Data are shown as arithmetic mean ± SEM (n = 14-21). ^a Five animals were excluded from AUC analysis because they did not show AHR (AUC increase) in Phase I. Three more animals were excluded due to technical failure in collecting Phase II airway function data. ^b Three animals were excluded from the PC ₃₀ analysis (same animals as a) due to technical failure in collecting phase II airway function data. Additional animals with antigen priming symptoms were excluded because they did not show PC ₃₀ AHR in Phase I. ^c Two animals were excluded from antigen priming analysis due to technical failure in collecting Phase I airway function data. ^d One animal was excluded from the BAL analysis because of significant BAL inflammation at the start of the study.

Claims (91)

An isolated specific binding member for human IL-13, comprising an antibody VH domain and a human antibody VL domain and comprising a set of CDRs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 Comprising an antigen binding site, wherein the VH domain comprises HCDR1, HCDR2, and HCDR3, the VL domain comprises LCDR1, LCDR2, and LCDR3, and the set of CDRs is as follows:
A set of CDRs of BAK278D6, where HCDR1 has the amino acid sequence of SEQ ID NO: 1, HCDR2 has the amino acid sequence of SEQ ID NO: 2, HCDR3 has the amino acid sequence of SEQ ID NO: 3, and LCDR1 has the amino acid sequence of SEQ ID NO: 4 LCDR2 has the amino acid sequence of SEQ ID NO: 5 and LCDR3 has the amino acid sequence of SEQ ID NO: 6;
A set of CDRs comprising 1 or 2 amino acid substitutions compared to the set of CDRs of BAK278D6, and each set of CDRs shown for individual clones in Table 1.
Said member consisting of a CDR set selected from the group consisting of: Said 1 or 2 substitutions are the following residues in CDRs using Kabat's standard numbering:
31, 32, 34 in HCDR1
52, 52A, 53, 54, 56, 58, 60, 61, 62, 64, 65 in HCDR2
96, 97, 98, 99, 101 in HCDR3
26, 27, 28, 30, 31 in LCDR1
56 in LCDR2
95A, 97 in LCDR3
2. The isolated specific binding member of claim 1 in position 1 or 2. Said 1 or 2 substitution is at the following position:
The position of the substitution A substituted residue selected from the group consisting of 31 in HCDR1: Q, D, L, G, and E
32 in HCDR1: T
34 in HCDR1: V, I, and F
52 in HCDR2: D, N, A, R, G, and E
52A D, G, T, P, N, and Y in HCDR2
53 in HCDR2: D, L, A, P, T, S, I, and R
54 in HCDR2: S, T, D, G, K, and I
56 in HCDR2: T, E, Q, L, Y, N, V, A, M, and G
58 in HCDR2: I, L, Q, S, M, H, D, and K
60: R in HCDR2
61 in HCDR2: R
62 in HCDR2: K and G
64 in HCDR2: R
65 in HCDR2: K
96 in HCDR3: R and D
97 in HCDR3: N, D, T, and P
98 in HCDR3: R
99 in HCDR3: S, A, I, R, P, and K
101 in HCDR3: Y
26 in LCDR1: D, S
27 in LCDR1: I, L, M, C, V, K, Y, F, R, T, S, A, H, and G
28 in LCDR1: V
30 in LCDR1: G
31 in LCDR1: R
56 in LCDR2: T
95A in LCDR3: N
97 in LCDR3: I
3. An isolated specific binding member according to claim 2 made from among the identified group of possible substituted residues for each position.   4. The isolated specific binding member of claim 3, wherein there are 2 substitutions at residue 99 of HCDR3 and residue 27 of LCDR1 compared to the CDR set of BAK278D6.   Substitution at residue 99 of HCDR3 selected from the group consisting of S, A, I, R, P and K, and / or I, L, M, C, V, K, Y, F, R, T 5. The isolated specific binding member of claim 4, having a substitution at LCDR1 residue 27 selected from the group consisting of, S, A, H and G.   5. The isolated specific of claim 4, comprising the CDR set of BAK278D6 wherein N is replaced with S at residue 99 of HCDR3 and / or N is replaced with I at residue 27 of LCDR1. Join member.   The HCDR1, HCDR2, and HCDR3 of the VH domain are in a germline framework and / or the LCDR1, LCDR2, and LCDR3 of the VL domain are in a germline framework. An isolated specific binding member according to paragraph.   8. The isolated specific binding member of claim 7, wherein the VH domains HCDR1, HCDR2, and HCDR3 are within the germline framework VH1 DP14.   9. The isolated specific binding member of claim 7 or claim 8, wherein the VH domains HCDR1, HCDR2 and HCDR3 are within the germline framework VL vλ3h.   10. An isolated specific binding member according to any one of claims 1 to 9, which binds to a human IL-13 variant in which position 130 arginine is replaced by glutamine.   11. The isolated specific binding member of any one of claims 1-10 that binds to non-human primate IL-13.   12. The isolated specific binding member of claim 11, wherein the non-human primate IL-13 is a rhesus monkey or cynomolgus.   13. An isolated specific binding member according to any one of claims 8 to 12, comprising a BAK502G9 VH domain (SEQ ID NO: 15).   14. An isolated specific binding member according to any one of claims 8 to 13, comprising a BAK502G9 VL domain (SEQ ID NO: 16).   The antibody binds to IL-13 with an affinity equivalent to or better than the affinity of the IL-13 antigen-binding site formed by the BAK502G9 VH domain (SEQ ID NO: 15) and the BAK502G9 VL domain (SEQ ID NO: 16). The specific binding member according to any one of 1 to 14, wherein the affinity of the specific binding member and the affinity of the antigen binding site are measured under the same conditions, member.   16. A specific binding member according to any one of claims 1 to 15, which neutralizes human IL-13.   Neutralizes human IL-13 with a titer equivalent to or better than the titer of the IL-13 antigen binding site formed by the BAK502G9 VH domain (SEQ ID NO: 15) and the BAK502G9 VL domain (SEQ ID NO: 16), 17. A specific binding member according to claim 16, wherein the titer of the specific binding member and the titer of the antigen binding site are measured under the same conditions.   18. A specific binding member according to any one of claims 1 to 17 comprising an scFv antibody molecule.   18. A specific binding member according to any one of claims 1 to 17 comprising an antibody constant region.   20. The specific binding member of claim 19, comprising a whole antibody.   21. The specific binding member of claim 20, wherein the whole antibody is IgG4.   22. An isolated antibody VH domain of the specific binding member of any one of claims 1-21.   22. An isolated antibody VL domain of the specific binding member of any one of claims 1-21.   24. A composition comprising a specific binding member according to any one of claims 1 to 23, an antibody VH domain or VL domain and at least one additional component.   25. The composition of claim 24, comprising a pharmaceutically acceptable excipient, solvent, or carrier.   24. An isolated nucleic acid comprising a nucleotide sequence encoding a specific binding member according to any one of claims 1 to 23 or an antibody VH domain or VL domain of a specific binding member.   27. A host cell transformed in vitro with the nucleic acid of claim 26.   28. A method of producing a specific binding member or antibody VH or VL domain comprising culturing the host cell of claim 27 under conditions for production of the specific binding member or antibody VH or VL domain.   29. The method of claim 28, further comprising isolating and / or purifying the specific binding member or antibody VH or VL variable domain.   30. The method of claim 28 or claim 29, further comprising preparing a specific binding member or antibody VH or VL variable domain in a composition comprising at least one additional component.   A method for producing an antigen binding domain of an antibody specific for human IL-13, comprising a parent VH domain comprising HCDR1, HCDR2, and HCDR3, wherein the parent VH domains HCDR1, HCDR2, and HCDR3 are HCDR of BAK278D6 Set (where HCDR1 has the amino acid sequence of SEQ ID NO: 1, HCDR2 has the amino acid sequence of SEQ ID NO: 2, and HCDR3 has the amino acid sequence of SEQ ID NO: 3) or a set of HCDRs of BAK502G9 (Wherein HCDR1 has the amino acid sequence of SEQ ID NO: 7, HCDR2 has the amino acid sequence of SEQ ID NO: 8, and HCDR3 has the amino acid sequence of SEQ ID NO: 9)] Amino acid sequence variants of the parent VH domain by addition, deletion, substitution or insertion of amino acids Providing a VH domain and optionally combining the provided VH domain with one or more VL domains to obtain one or more VH / VL combinations; and the VH domain being an amino acid sequence variant of the parent VH domain Or testing the VH / VL combination to identify an antigen binding domain of an antibody specific for human IL-13.   32. The method of claim 31, wherein the parent VH domain amino acid sequence is selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 15.   The one or more VL domains include a parent VL domain including LCDR1, LCDR2, and LCDR3, where the parent VL domains LCDR1, LCDR2, and LCDR3 are a set of LCDRs of BAK278D6 (where LCDR1 is the amino acid sequence of SEQ ID NO: 4). LCDR2 has the amino acid sequence of SEQ ID NO: 5 and LCDR3 has the amino acid sequence of SEQ ID NO: 6) or a set of BKR502G9 LCDRs, where LCDR1 is the amino acid sequence of SEQ ID NO: 10 LCDR2 has the amino acid sequence of SEQ ID NO: 11 and LCDR3 has the amino acid sequence of SEQ ID NO: 12)], by addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence One or more VL domains, each of which is an amino acid sequence variant of the parent VL domain It is provided by producing method according to claim 31 or claim 32.   34. The method of claim 33, wherein the parental VL domain amino acid sequence is selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 16. A method for producing an antigen binding domain of an antibody specific for human IL-13, comprising a parent VH domain comprising HCDR1, HCDR2, and HCDR3, wherein the parent VH domains HCDR1, HCDR2, and HCDR3 are the HCDR of BAK167A11 (Where HCDR1 has the amino acid sequence of SEQ ID NO: 55, HCDR2 has the amino acid sequence of SEQ ID NO: 56, and HCDR3 has the amino acid sequence of SEQ ID NO: 57) or the set of HCDR of BAK615E3 (Where HCDR1 has the amino acid sequence of SEQ ID NO: 153, HCDR2 has the amino acid sequence of SEQ ID NO: 154, and HCDR3 has the amino acid sequence of SEQ ID NO: 155) or a set of HCDRs of BAK582F7 (here HCDR1 is the amino acid sequence of SEQ ID NO: 141 HCDR2 has the amino acid sequence of SEQ ID NO: 142 and HCDR3 has the amino acid sequence of SEQ ID NO: 143), or a set of BAK612B5 HCDR (where HCDR1 represents the amino acid sequence of SEQ ID NO: 147) HCDR2 has the amino acid sequence of SEQ ID NO: 148, and HCDR3 has the amino acid sequence of SEQ ID NO: 149)], by addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of Providing a VH domain that is an amino acid sequence variant of the parent VH domain, and optionally combining the provided VH domain with one or more VL domains to obtain one or more VH / VL combinations; and The VH domain or VH / VL combination, which is an amino acid sequence variant, is tested to determine whether it is specific for human IL-13. Identifying the antigen binding domain comprises the method.   36. The method of claim 35, wherein the parent VH domain amino acid sequence is selected from the group consisting of SEQ ID NO: 55 and SEQ ID NO: 33.   The one or more VL domains include a parent VL domain that includes LCDR1, LCDR2, and LCDR3, where the parent VL domains LCDR1, LCDR2, and LCDR3 are a set of LCDRs of BAK167A11 (where LCDR1 is SEQ ID NO: 58 LCDR2 has the amino acid sequence of SEQ ID NO: 59 and LCDR3 has the amino acid sequence of SEQ ID NO: 60) or a set of LCDRs of BAK615E3 (where LCDR1 is the amino acid sequence of SEQ ID NO: 156) LCDR2 has the amino acid sequence of SEQ ID NO: 157 and LCDR3 has the amino acid sequence of SEQ ID NO: 158) or a set of LCDRs of BAK582F7 (where LCDR1 has the amino acid sequence of SEQ ID NO: 144) LCDR2 is the sequence number 145 Or a set of LCDRs of BAK612B5 (wherein LCDR1 has an amino acid sequence of SEQ ID NO: 150 and LCDR2 has an amino acid sequence of SEQ ID NO: 151). An amino acid sequence, and LCDR3 has the amino acid sequence of SEQ ID NO: 152)], by addition, deletion, substitution or insertion of one or more amino acids in each amino acid sequence 37. The method of claim 35 or claim 36, provided by producing one or more VL domains that are variants.   38. The method of claim 37, wherein the parental VL domain amino acid sequence is selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 34.   35. The method of any one of claims 31 to 34, wherein the VH domain amino acid sequence that is an amino acid sequence variant of the parent VH domain is provided by CDR mutagenesis.   39. The method of any one of claims 35 to 38, wherein the VH domain amino acid sequence that is an amino acid sequence variant of a parent VH domain is provided by CDR mutagenesis.   41. The method of any one of claims 31 to 40, further comprising providing an antigen binding site of the antibody within an IgG, scFv, or Fab antibody molecule. A method for producing a specific binding member that binds human IL-13, comprising:
A starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH domain, where the VH domain includes a substituted HCDR1, HCDR2, and / or HCDR3, or HCDR1, HCDR2, and / or HCDR3 The coding region is deleted);
The starting nucleic acid or starting repertoire is HCDR1 (SEQ ID NO: 1) or HCDR1 (SEQ ID NO: 7), HCDR2 (SEQ ID NO: 2) or HCDR2 (SEQ ID NO: 8), and / or HCDR3 (SEQ ID NO: 3) or HCDR3 (SEQ ID NO: 9) or in combination with a donor nucleic acid produced by this mutation, the donor nucleic acid is inserted into a CDR1, CDR2, and / or CDR3 region in the starting nucleic acid or starting repertoire and encodes a VH domain Providing a product repertoire of nucleic acids;
Expressing the nucleic acid of the product repertoire to produce a product VH domain;
Optionally combining the product VH domain with one or more VL domains;
Selecting a specific binding member specific for human IL-13, said specific binding member comprising a product VH domain and optionally a VL domain; and said specific binding member or nucleic acid encoding it to recover,
Said method comprising:   43. The method of claim 42, wherein the donor nucleic acid is produced by the HCDR1 and / or HCDR2 mutation.   43. The method of claim 42, wherein the donor nucleic acid is produced by mutation of HCDR3.   45. The method of claim 44, comprising providing a donor nucleic acid by mutation of a nucleic acid encoding the amino acid sequence of HCDR3 (SEQ ID NO: 3) or HCDR3 (SEQ ID NO: 9).   43. The method of claim 42, comprising providing the donor nucleic acid by random mutation of nucleic acid.   47. The method of any one of claims 42 to 46, comprising binding a product VH domain contained within the recovered specific binding member to an antibody constant region.   47. A method according to any one of claims 42 to 46 comprising providing an IgG, scFv or Fab antibody comprising a product VH domain and a VL domain.   49. The method of any one of claims 31 to 48, further comprising testing an antigen binding domain or specific binding member of an antibody that binds human IL-13 for the ability to neutralize human IL-13. .   50. The method of claim 49, wherein a specific binding member is obtained comprising an antibody fragment that binds and neutralizes human IL-13.   51. The method of claim 50, wherein the antibody fragment is a scFv antibody molecule.   51. The method of claim 50, wherein the antibody fragment is a Fab antibody molecule.   53. The method of claim 51 or claim 52, further comprising providing a VH domain and / or VL domain of the antibody fragment in the whole antibody.   A specific binding member that binds to IL-13, an antigen binding site of an antibody, or an antigen binding site of an antibody that binds to IL-13 or the VH or VL variable domain of an antibody of a specific binding member 54. The method of any one of claims 31-53, further comprising preparing a composition comprising:   55. The method of any one of claims 31-54, further comprising binding a specific binding member that binds human IL-13 to IL-13 or a fragment of IL-13.   22. A method comprising binding a specific binding member that binds to IL-13 according to any one of claims 1-21 to human IL-13 or a fragment of human IL-13.   57. The method of claim 55 or claim 56, wherein the binding occurs in vitro.   58. The method of any one of claims 55 to 57, comprising measuring the amount of specific binding member binding to IL-13 or a fragment of IL-13.   Further use of the specific binding member in the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin lymphoma 59. The method of any one of claims 31-58, comprising.   Any of claims 1-21 in the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin lymphoma Use of a specific binding member according to paragraph 1.   A method for treating a disease or disorder selected from the group consisting of asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin lymphoma, according to any one of claims 1 to 21. Administering said specific binding member to a patient having or at risk of developing the disease or disorder. An isolated human IL-13 consisting of a human antibody VH domain and a human antibody VL domain and comprising the antigen binding domain site of an antibody comprising a set of CDRs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 A specific binding member, wherein said VH domain comprises HCDR1, HCDR2, and HCDR3, said VL domain comprises LCDR1, LCDR2, and LCDR3;
Where HCDR1 is:
HX ₁ HX ₂ G HX ₃ S
(Where
HX ₁ is selected from the group consisting of N, Q, D, L, G, and E;
HX ₂ is selected from the group consisting of Y and T;
HX ₃ is selected from the group consisting of V, I, F, and L)
An amino acid sequence having
HCDR2 has the following formula:
WI HX ₄ HX ₅ HX ₆ HX ₇ G HX ₈ T HX ₉ Y HX ₁₀ HX ₁₁ HX ₁₂ F HX ₁₃ HX ₁₄
(Where
HX ₄ is selected from the group consisting of S, D, N, A, R, G, and E;
HX ₅ is selected from the group consisting of A, D, G, T, P, N, and Y;
HX ₆ is selected from the group consisting of N, D, L, A, P, T, S, I, and R;
HX ₇ is selected from the group consisting of N, S, T, D, G, K, and I;
HX ₈ is selected from the group consisting of D, T, E, Q, L, Y, N, V, A, M, and G;
HX ₉ is selected from the group consisting of N, I, L, Q, S, M, H, D, and K;
HX ₁₀ is selected from the group consisting of G and R;
HX ₁₁ is selected from the group consisting of Q and R;
HX ₁₂ is selected from the group consisting of E, K, and G;
HX ₁₃ is selected from the group consisting of Q and R;
HX ₁₄ is selected from the group consisting of G and K)
An amino acid sequence having
HCDR3 has the following formula:
D HX ₁₅ HX ₁₆ HX ₁₇ HX ₁₈ W A R W HX ₁₉ F HX ₂₀ L
(Where
HX ₁₅ is selected from the group consisting of S, R, and D;
HX ₁₆ is selected from the group consisting of S, N, D, T, and P;
HX ₁₇ is selected from the group consisting of S and R;
HX ₁₈ is selected from the group consisting of S, N, A, I, R, P, and K;
HX ₁₉ is selected from the group consisting of F and Y;
HX ₂₀ is selected from the group consisting of D and Y)
An amino acid sequence having
LCDR1 has the following formula:
G G LX ₁ LX ₂ LX ₃ G LX ₄ LX ₅ L V H
(Where
LX ₁ is selected from the group consisting of N, D, and S;
LX ₂ is selected from the group consisting of N, I, L, M, C, V, K, Y, F, R, T, S, A, H, and G;
LX ₃ is selected from the group consisting of I and V;
LX ₄ is selected from the group consisting of S and G;
LX ₅ is selected from the group consisting of K and R)
An amino acid sequence having
LCDR2 has the following formula:
D D G D R P LX ₆
(Where
LX ₆ is selected from the group consisting of S and T)
An amino acid sequence having
LCDR3 has the following formula:
Q V WD TG S LX ₇ P V LX ₈
(Where
LX ₇ is selected from the group consisting of D and N;
LX ₈ is selected from the group consisting of V and I)
Said member which is an amino acid sequence having HX ₁ is selected from the group consisting of D and N;
HX ₂ is Y,
HX ₃ is L;
HX ₄ is selected from the group consisting of S and G;
HX ₅ is selected from the group consisting of T and A;
HX ₆ is N;
HX ₇ is selected from the group consisting of N and I;
HX ₈ is D and
HX ₉ is selected from the group consisting of N, D, and K;
HX ₁₀ is G,
HX ₁₂ is selected from the group consisting of E and G;
HX ₁₃ is Q,
HX ₁₉ is F,
LX ₁ is selected from the group consisting of N and S;
LX ₂ is selected from the group consisting of N, Y, T, S, and I;
LX ₆ is S,
LX ₇ is D, isolated specific binding member according to claim 62. HX ₁ is selected from the group consisting of N and D;
HX ₂ is Y,
HX ₃ is L;
HX ₄ is selected from the group consisting of S and G;
HX ₅ is selected from the group consisting of A and T;
HX ₆ is N;
HX ₇ is N;
HX ₈ is selected from the group consisting of D and G;
HX ₉ is selected from the group consisting of I, S, N, and D;
HX ₁₁ is Q,
HX ₁₂ is E and K;
HX ₁₄ is G,
HX ₁₅ is S,
HX ₁₆ is selected from the group consisting of S and N;
HX ₁₇ is S,
HX ₁₈ is selected from the group consisting of S and N;
HX ₁₉ is F,
HX ₂₀ is D,
LX ₁ is selected from the group consisting of N and D;
LX ₂ is I,
LX ₈ is V.
64. The isolated specific binding member of claim 62. HX ₇ is selected from the group consisting of N, S, T, D, G, and K;
HX ₈ is selected from the group consisting of D, T, E, Q, L, Y, N, V, A, M;
HX ₉ is selected from the group consisting of N, I, L, Q, S, M, and H;
HX ₁₀ is G,
HX ₁₁ is Q,
HX ₁₂ is F;
HX ₁₃ is Q,
HX ₁₄ is G,
HX ₁₅ is S,
HX ₁₆ is selected from the group consisting of N and S;
HX ₁₇ is S,
HX ₁₈ is selected from the group consisting of N and S;
HX ₁₉ is F,
HX ₂₀ is D,
LX ₁ is N and LX ₂ is selected from the group consisting of N and I;
LX ₃ is I,
LX ₄ is S,
LX ₅ is K,
LX ₆ is S,
LX ₇ is D and LX ₈ is V.
63. An isolated specific binding member according to claim 62. HX ₁ is selected from the group consisting of N, Q, and D;
HX ₃ is selected from the group consisting of L, V, and I;
HX ₄ is selected from the group consisting of S, N, A, and R;
HX ₅ is selected from the group consisting of A, D, T, G, N, and Y;
HX ₆ is selected from the group consisting of N, A, P, S, D, and I;
HX ₇ is selected from the group consisting of N, T, D, and G;
HX ₈ is selected from the group consisting of D, Q, Y, and N;
HX ₉ is, N, Q, S, and is selected from the group consisting of I, isolated specific binding member according to claim 65.   67. A specific binding member according to any one of claims 62 to 66, which neutralizes human IL-13.   Neutralizes human IL-13 with a titer equivalent to or better than the titer of the IL-13 antigen binding site formed by the BAK502G9 VH domain (SEQ ID NO: 15) and the BAK502G9 VL domain (SEQ ID NO: 16), 68. The specific binding member of claim 67, wherein the specific binding member titer and the antigen binding site titer are measured under the same conditions.   69. A specific binding member according to any one of claims 62 to 68 comprising an scFv antibody molecule.   69. A specific binding member according to any one of claims 62 to 68 comprising an antibody constant region.   71. A specific binding member according to claim 70 comprising a whole antibody.   72. A specific binding member according to claim 71, wherein the whole antibody is IgG4.   73. The isolated specific binding member of any of claims 62-72, which binds to a human IL-13 variant, wherein arginine at position 130 is substituted with glutamine.   73. The isolated specific binding member of any one of claims 62-72, which binds to non-human primate IL-13.   75. The isolated specific binding member of claim 74, wherein the non-human primate IL-13 is a rhesus monkey or cynomolgus.   76. An isolated antibody VH domain of the specific binding member of any one of claims 62-75.   76. An isolated antibody VL domain of the specific binding member of any one of claims 62-75.   78. A composition comprising the specific binding member of any one of claims 62-77, an antibody VH domain or VL domain, and at least one additional component.   79. The composition of claim 78, comprising a pharmaceutically acceptable excipient, solvent, or carrier.   78. An isolated nucleic acid comprising a nucleotide sequence encoding a specific binding member according to any one of claims 62 to 77 or an antibody VH domain or VL domain of a specific binding member.   81. A host cell transformed in vitro with the nucleic acid of claim 80.   82. A method of producing a specific binding member or antibody VH or VL domain, comprising culturing the host cell of claim 81 under conditions for production of said specific binding member or antibody VH or VL domain. .   83. The method of claim 82, further comprising isolating and / or purifying the specific binding member or antibody VH or VL variable domain.   84. The method of claim 82 or claim 83, further comprising formulating the specific binding member or antibody VH or VL variable domain into a composition comprising at least one additional component.   85. The method of any one of claims 82 to 84, further comprising binding a specific binding member that binds human IL-13 to IL-13 or a fragment of IL-13.   76. A method comprising binding to the IL-13 specific binding member of any one of claims 62-75 to human IL-13 or a fragment of human IL-13.   88. The method of claim 85 or claim 86, wherein the binding occurs in vitro.   88. The method of any one of claims 85-87, comprising measuring the amount of specific binding member binding to IL-13 or a fragment of IL-13.   Further comprising the use of a specific binding member in the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin lymphoma The method according to any one of claims 82 to 84.   76. In the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease, and Hodgkin lymphoma Use of a specific binding member according to paragraph 1.   76. A method of treating a disease or disorder selected from the group consisting of asthma, atopic dermatitis, allergic rhinitis, fibrosis, and Hodgkin lymphoma, the specific binding according to any one of claims 62 to 75 Administering the member to a patient having or at risk of developing the disease or disorder.

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