ES2366962T3 - ANTIBODIES AGAINST THE ALFA-1 RECEIVER OF IL-13 AND USES OF THE SAME. - Google Patents

Abstract

An antibody that binds to IL-13Rα1 and inhibits IL-13, characterized in that the amino acid sequence of the variable part of the CDR3 heavy chain of said antibody is selected from the group of sequences of the CDR3 heavy chain composed of the Sequences with ID No.: 1, 3, 5 or 7 whereby said antibody is obtained from hybridoma cell lines DSM ACC2709, DSM ACC2710, DSM ACC2711 or DSM ACC2712.

Description

The present invention relates to human antibodies against the alpha-1 receptor of IL-13 (IL-13Rα (alpha) 1), the methods for their production and their uses.

Background of the invention

IL-13 is a monomeric peptide secreted and produced basically by Th2 cells, and can also be produced by mast cells and killer T cells. The biological functions of IL-13 include the regulation of IgE production and the modulation of Th2 development. IL-13 binds to a complex receptor consisting of an IL-13 receptor alpha-1 chain (Il-13Rα1) and an IL-4 receptor alpha chain (IL-4Rα). The binding of IL13 triggers a series of signal transductions mediated primarily by STAT6. IL-13 binds with low affinity to isolated IL-13Rα1 and does not bind to IL-4Rα1. In contrast, IL-4 binds to isolated IL-4Rα and does not bind to isolated IL-13Rα1. Another receptor for IL-13, IL-13Rα2, has been described. IL-13 binds to this receptor with high affinity. This receiver is likely to act as a decoy.

The inducible overexpression of IL-13 in transgenic mice generates a phenotype that shares many characteristics with asthmatic patients. These show mucus metaplasia and inflammation rich in macrophages, lymphocytes and eosinophils, positive protease regulation such as MMP-9, -12, -13, -2 and –14, cathepsin B, H, K and S, also presenting subepithelial fibrosis . Mice with the gene for nullified IL-13 (knockout) show a significant reduction in the production of Th2-type cytokines due to the decrease in Th2 development. These mice do not develop airway hyperreactivity (AHR) despite the presence of inflammation with eosinophils. The AHR is recovered by the administration of IL-13, indicating that IL-13 is necessary and sufficient for the induction of AHR in mice. Other important biological functions of IL-13 in relation to asthma include induction of metaplasia and mucus production of goblet cells. It acts directly on the epithelial cells of the respiratory tract, fibroblasts and smooth muscle cells, inducing different transcriptional programs in each of these cell types. It should be noted that IL-13 decreases the α-adrenergic response in smooth muscle cells, contributing to a narrowing of the airways. The polymorphism of the IL-13 promoter is associated with an increased risk of allergic asthma. Polymorphisms in the IL-13 gene are associated with high levels of serum IgE. Polymorphisms of a single nucleotide in the intergenic sequence between the genes of IL-4 and IL-13 are associated with atopic asthma.

IL-13 antagonists have been used in animal models. For example, it has been shown that a soluble fusion protein IL-13Rα2-IgGFc manages to reverse the ovalbumin-induced AHR in mice and reduce the number of mucus-producing cells. The reversal was obtained even when the treatment is administered after a complete development of the phenotype. In addition, the treatment of mice with a fusion molecule between IL-13 and a cytotoxin resulted in a reduction of all the characteristics of the respiratory tract disease characteristic of a fungal-induced chronic allergic inflammation. In conclusion, IL-13 is a key mediator in triggering the allergic response.

IL-13Rα1 is a member of the hematopoietin receptor superfamily (type 1 cytokine receptor family) and was identified and described by Obiri NI, et al., J. Biol. Chem., 270 (1995) 8797- 8804) and in WO 96/29417. It is a protein of 427 amino acids including the signal sequence. DNA and protein sequences are described in WO 97/15663 and in SwissProt No. P78552. IL-13Rα1 is a glycosylated protein that binds to IL-13 with low affinity, but, when it binds to IL-4Rα forming a heterodimer, it binds to IL-13 with high affinity. This complex is also a receptor for IL-4.

Antibodies against IL-13Rα1 are described in WO 96/29417, WO 97/15663, WO 03/080675, Graber P., et al., Eur. J. Immunol., 28 (1998) 4286-4298; Poudrier J., et al., J. Immunol., 163 (1999) 1153-1161; Poudrier J., et al., Eur. J. Immunol., 30 (2000) 3157-3164; Aikawa M., et al., Cytokine, 13 (2001) 75-84. Antibodies against IL13Rα can be obtained commercially from R&D Systems Inc. USA. EP 449851 refers to rat antibodies that bind to IL-13αl. The problem solved by the present invention is to provide alternative antibodies against IL-13αl with a very high affinity to IL-13αl.

Summary of the Invention

The invention comprises an antibody that binds to IL-13R1 and that inhibits IL-13, characterized in that the amino acid sequence of the variable part of the CDR3 heavy chain is selected from the group of sequences of the CDR3 heavy chain composed of the Sequences with ID No.: 1, 3, 5 or 7 whereby said antibody is obtained from hybridoma cell lines DSM ACC2709, DSM ACC2710, DSM ACC2711 or DSM ACC2712.

The antibody is preferably a human antibody.

The antibody is preferably characterized by an IL-13R1 binding affinity of 10-9 M (KD) or less, preferably 10-9 to 10-13 M.

Preferably, the antibody is characterized in that the CDR3 variable heavy chain amino acid sequence of said antibody is selected from the group consisting of the CDR3 heavy chain sequences of Secondary ED No.: 1, 3, 5, 7 or 9 and comprises as variable region of heavy chain with ED No. of Sec: 1 and as variable region of light chain with ID of Sec: 2, as variable region of heavy chain with ID of Sec: 3, and as variable region of light chain with Sec ID number: 4, as a heavy chain variable region with Sec ID number: 5 and as a light chain variable region with Sec ID number: 6, as a heavy chain variable region with No. Sec ID: 7 and as a light chain variable region with Sec ID number: 8 or as a heavy chain variable region with Sec ID number: 9 and as a light chain variable region with Sec ID number: 10 .

Sequences for CDRs can be determined according to the standard definition of Kabat et al., Sequences of Proteins of Immonological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). On this basis, the complementarity determining regions (CDR) of the sequences with ID No.: 1-8 have the following sequences:

Heavy chain CDRs: CDR1 (aa 31-35) of SEC ID No.: 1, 3, 5, 7, 9, CDR2 (aa 50-66) of SEC ID No.: 1, 3, 5, 7, 9, CDR3 (aa 99-108) of SEQ ID NO: 1, 3, 9, CDR3 (aa 99-107) of SEQ ID NO: 5, CDR3 (aa 99-112) of SEQ ID NO: 7;

Light chain CDRs: CDR1 (aa 24-34) of SEC ID No.: 2, 4, 6, 10, CDR1 (aa 24-35) of SEC ID No.: 8, CDR2 (aa 50-56) of ID No. SEC: 2, 4, 6, 10, CDR2 (aa 51-57) of SEQ ID NO: 8 and CDR3 (aa 89-97) of SEQ ID NO: 2, 4, 6, 10, CDR3 (aa 90-97 ) of SEQ ID NO: 8.

The antibody is preferably characterized by containing as a variable region of the heavy chain the SEC with ID No.: 1 and as a variable region of the light chain the SEC with No. ID: 2, as a variable region of the heavy chain the SEC with ID No.: 3 and as a variable region of the light chain the SEC with ID No.: 4, as a variable region of the heavy chain the SEC with ID No.: 5 and as a variable region of the light chain the SEC with ID No.: 6, as a variable region of the heavy chain the SEC with ID No.: 7 and as a variable region of the light chain the SEC with ID No.: 8 or as a variable region of the heavy chain the SEC with ID No.: 9 and as a variable region of the light chain the SEC with ID No.: 10.

The antibody is preferably characterized by containing

a) as a variable region of the heavy chain the SEC with ID No.: 1, as a variable region of the light chain the SEC with ID No.: 2, as a constant region of the light chain κ the SEC with ID No.: 11 and as a region γ1 heavy chain constant SEC with ID No.: 12, optionally with mutations L234A and L235A or D265A and N297A,

b) as a variable region of the heavy chain the SEC with ID No.: 3, as a variable region of the light chain the SEC with ID No.: 4, as a constant region of the light chain κ the SEC with ID No.: 11 and as a region γ1 heavy chain constant SEC with ID No.: 12 optionally with mutations L234A and L235A or D265A and N297A,

c) as a variable region of the heavy chain the SEC with ID No.: 5, as a variable region of the light chain the SEC with ID No.: 6, as a constant region of the light chain κ the SEC with ID No.: 11 and as a region γ1 heavy chain constant SEC with ID No.: 12 optionally with mutations L234A and L235A or D265A and N297A,

d) as a variable region of the heavy chain the SEC with ID No.: 7, as a variable region of the light chain the SEC with ID No.: 8, as a constant region of the light chain κ the SEC with ID No.: 11 and as a region γ1 heavy chain constant SEC with ID No.: 12 optionally with mutations L234A and L235A or D265A and N297A, or

e) as a variable region of the heavy chain the SEC with ID No.: 9, as a variable region of the light chain the SEC with ID No.: 10, as a constant region of the light chain κ the SEC with ID No.: 11 and as a region γ1 heavy chain constant SEC with ID No.: 12 optionally with mutations L234A and L235A or D265A and N297A,

The antibody is preferably characterized by its binding to IL-13R1 in competition with the LC5002002, LC5002-003, LC5002-005, LC5002-007 and / or LC5002-018 antibodies. The antibody is preferably characterized by including as variable regions the variable regions of LC5002-002, LC5002-003, LC5002-005, LC5002-007 or LC5002-018. The variable regions of these antibodies are those shown in the SEC with ID No.: 1-10. In the state of the art there are very useful and well characterized constant regions. In the SEC with ID Nº: 11-12 some of them are shown.

The antibody is preferably monoclonal or recombinantly produced.

In a preferred embodiment of the invention the antibody is a human antibody with class alteration.

In a preferred embodiment of the present invention the antibody contains a human heavy chain 1 that includes:

a) the amino acid sequence Pro233Val234Ala235 with deletion of the Gly236 and / or the amino acid sequence Gly327Leu328Pro329Ser330Ser331

b) the amino acid sequence Ala234Ala235 or

c) amino acids Ala265 and Ala297.

Preferably, according to the invention, the antibody inhibits the phosphorylation of the IL-13 induced Stat-6 with an IC50 value of 6nM or less, it also inhibits the production of IL-13 induced eotaxin with an IC50 value of 20nM or less and / or inhibits cell proliferation induced by IL-13 or IL-4, preferably TF-1 cells (ATCC CRL 2003) with an IC50 value of 10nM or less (IL-13) and 60nM or less ( IL-4). The phosphorylation of Stat-6, production of eotaxin and induction of cell proliferation are determined according to examples 6 to 8.

The antibody, according to the invention, does not bind to the denatured IL-13R1 (binding KD of 10-6 M or greater). The antibody is preferably characterized by showing no substantial cross reaction with IL-13R2 and IL-4Rα (binding KD of 10-6 M or greater).

The invention also provides hybridoma cell lines that produce antagonistic monoclonal antibodies against IL-13R 1.

Preferred hybridoma cell lines according to the invention (hu-MAB <h-IL-13R alpha> LC.5002-002 (DSM ACC2709), hu-MAB <h IL-13R alpha> LC.5002-003) DSM ACC2710),), hu-MAB <h IL-13R alpha> LC.5002-005) DSM ACC2711), hu-MAB <h IL-13R alpha> LC.5002-007 (DSM ACC2712)) were deposited at 13 -01-2005 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany.

The invention further provides the nucleic acids encoding the polypeptides that make up the antibodies, expression vectors including said nucleic acids and host cells for the recombinant production of said antibodies. The invention also provides methods for the recombinant production of said antibodies.

Another embodiment of the invention is a nucleic acid encoding an antibody that binds to IL-13Rα1 and that inhibits IL-13 which comprises as a region of the heavy chain to the region with SEQ ID NO: 1 and as a variable region of the light chain to that of SEC No.: 2, as a variable region of the heavy chain to the region with SEQ ID No.: 3 and as a variable region of the light chain to that of SEC No.: 4, as a variable region of the heavy chain to the region with SEQ ID NO: 5 and as a variable region of the light chain to that of SEQ ID NO: 6, as a variable region of the heavy chain to the region with SEQ ID NO: 7 and as variable region of the light chain to that of SEQ ID NO: 8 or as a variable region of the heavy chain to the region with SEQ ID NO: 9 and as a variable region of the light chain to that of SEQ ID NO: 10 .

Antibodies, in accordance with the present invention, have proven beneficial for patients in need of corticosteroid therapy. These antibodies have new and innovative properties that benefit the patient suffering from asthma or allergic disease.

The invention further includes the use of the subject antibody of the invention for the treatment of asthma and for the manufacture of pharmaceutical compounds according to the invention. In addition, the invention includes a method for manufacturing a pharmaceutical compound according to the invention.

The invention also includes a pharmaceutical compound containing the antibody subject of the invention with an effective pharmaceutical dose, together with an optional buffer and / or adjuvant, useful for the formulation of antibodies for pharmaceutical purposes.

The invention also provides pharmaceutical compounds that include said antibodies in a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition may be included in a manufactured article or in a kit.

The invention also includes a vector containing a nucleic acid which is capable of being expressed in a eukaryotic or prokaryotic host cell.

The invention also includes a prokaryotic or eukaryotic host cell containing the vector according to the invention.

The invention also includes a method for the recombinant production of the human antibody subject of the invention, characterized by the expression of a nucleic acid according to the invention in a prokaryotic or eukaryotic host cell and the recovery of said antibody from said cell. The invention also comprises the antibody obtainable by said recombinant method.

The invention also comprises a method for the preparation of a pharmaceutical compound characterized by selecting an antibody against IL-13R1 from a plurality of antibodies against IL-13R1 in relation to such an assay without said antibody, producing said antibody by expression. recombinant, recovering said antibody and combining it with a pharmaceutically acceptable buffer and / or adjuvant. The antibody will preferably have one or more of the properties mentioned above.

Detailed description of the invention

The terms "IL-13R1, murine IL-13R1, IL-13, IL-13R2 and IL-4R" and their domains are well defined in the current state of the art and defined for example in SwissProt P78552, O09030, P35225, Q14627 and P24394. If not indicated otherwise, the terms "IL-13R1, IL-13, IL-13R2 and IL-4R" therefore denote the human polypeptides IL-13R1, IL-13, IL-13R2 and IL-4R.

The term "human antibody", as used herein, includes antibodies that possess constant regions (domains) that can be assigned to immunoglobulin sequences belonging to defined human germ lines due to their high sequence similarity or its identity with such germline sequences. Human antibodies are well known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (eg, mice) that are capable, after being immunized, of producing a complete repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production. The transfer of the immunoglobulin gene series from the human germline to the germline of mice results in the production of human antibodies after exposure to the antigen (see e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 33 -40). Human antibodies can also be produced from the "phage display" libraries (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al., J. Mol. Biol. 222 (1991) 581597). The techniques of Cole et al. and Boerner et al. They are also available for the preparation of monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991 ) 86-95). Different forms are included within the definition of human antibody, preferably that of monoclonal antibodies, comprising but not being limited to complete antibodies, antibody fragments, class altered antibodies and genetically modified antibodies (mutant or variant antibodies) while maintaining the characteristic properties of the invention. Especially preferred are human recombinant antibodies. The term "monoclonal antibody", as used hereafter, refers to a preparation of antibody molecules, all of them with a substantially identical amino acid composition.

The term "human recombinant antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant methods, such as antibodies isolated from host cells such as NSO. or CHO or from animals (eg, mouse) that are transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected in said host cell. Such recombinant human antibodies have constant and variable regions in reorganized arrangement. Human recombinant antibodies according to the invention have been subjected to somatic hypermutation in vivo. Therefore, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that can be assigned to certain VH and VL sequences of human germ lines, but which may not exist naturally within the in vivo repertoire of the human germ lines.

The term "class altered antibody" refers to a monoclonal antibody, preferably a human antibody, comprising a variable region, the binding region, which comes from a germline source and at least a portion of a constant region that It corresponds to a constant region of a different germline source, usually prepared by recombinant DNA techniques. Such class-altered antibodies are not found naturally and therefore cannot be obtained directly from xenoimplanted mice. The forms of class-altered antibodies included in the present invention are those in which the constant region exhibits differences with respect to the wild sequence of the constant region, resulting in an antibody with different properties according to the invention, especially in relation to with the binding to C1q and / or the Fc receptor (FcR), either by change or mutation of the Fc. Class-altered antibodies are the gene expression product for immunoglobulin that include DNA fragments encoding the variable regions of the immunoglobulin and DNA fragments encoding the constant regions of the immunoglobulin. Methods for the production of class-altered antibodies involve conventional techniques of recombinant DNA and gene transfection, well known in the state of the art (see, eg: Morrison, SL, et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Patent Nos. 5,202,238 and 5,204,244).

The "variable region" (variable region of the light chain (VL), variable region of the heavy chain (VH)) as applied hereafter, refers to the part of each pair of light and heavy chains that It is directly involved in the binding of the antibody to the antigen. The variable domains of the human light and heavy chains have the same general structure and each domain is formed by four framework regions (FR) whose sequences are widely conserved, connected to each other by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β sheet conformation and the CDRs can form connection bonds between the beta sheet structure. The CDRs of each chain are maintained in a three-dimensional structure by the framework regions and form, together with the CDRs of the other chain, the antigen binding site. The CDR3 regions of the heavy and light chains of the antibody, preferably the CDR3 of the heavy chain, play a particularly important role in the specificity and binding affinity of the antibodies in accordance with the invention and therefore also constitute an object of the invention. .

The terms "hypervariable region" or "antigen binding portion of an antibody" as used herein, refer to the amino acid residues of an antibody that are responsible for binding to the antigen. The hypervariable region comprises the amino acid residues of the "complementarity determining regions" or "CDR". The "frame" regions or "FR" regions are those regions of variable domain apart from the residues of the hypervariable region as defined. Therefore, the light and heavy chains of an antibody comprise, from the N to C end, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR and FR regions are determined according to the standard definitions given by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

"Constant domains" are not directly involved in the binding of an antibody to an antigen, but have several effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, the antibodies or immunoglobulins are divided into the classes: IgA, IgD, IgE, IgG and IgM, and several of these classes can be subdivided into subclasses (isotypes) ), eg: IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. The constant regions of the heavy chain that correspond to the different classes of immunoglobulins are called,,,, and. The antibodies corresponding to the invention are preferably of the IgG1 type.

The Fc part of an antibody is directly involved in complement activation, C1q binding, C3 activation and Fc receptor binding. The C1q junction is mediated by junction sites defined in part Fc. These places of union are known in the current state of the art and defined by eg. in Lukas, T.J., et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Zebra, J.J., Mol. Immunol 16 (1979) 907-917; Burton, D.R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., Mol. Immunol 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434. Such places of attachment are for example: L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbered according to the Kabat EU index, see below). The antibodies of the IgG1, IgG2 and IgG3 subclasses usually have complement activating action, C1q binding and C3 activation, while IgG4 antibodies do not activate the complement system, do not bind Cq1 and do not activate C3. The term "Fc fragment derived from human origin", as used hereafter, defines an Fc fragment that preferably has an amino acid sequence corresponding to the Fc fragment of a human antibody of the IgG1 subclass, modified in such a way that no c1q binding, C3 activation and / or Fc binding can be detected, or the binding is decreased by at least 50%, preferably 70%, compared to the human IgG1 antibody. The term "Fc part of an antibody" is well known to the specialist formed, being defined based on the papain digestion of the antibodies. The antibodies corresponding to the invention contain an Fc part, preferably with an amino acid sequence of an Fc part derived from a human origin and preferably all other parts of the human constant regions. Preferably the Fc part is a human Fc part mutated from the IgG1 subclass. Of priority preference are the Fc parts that include a constant region 1 of the heavy chain (an example is shown in the SEC with ID No. 11) with mutations L234A and L235A or D265A and N297A (WO99 / 51642).

The constant human chains ex. Heavy chains 1 are described in detail by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), and by Brüggemann, M., et al., J. Exp. Med. 166 (1987) 1351-1361; Love, T.W., et al., Methods Enzymol. 178 (1989) 515-527. Preferred constant domains in the invention have no complement binding. The "variable region" (variable region of the light chain (VL), variable region of the heavy chain (VH)) as used hereafter denotes each of the pair of light and heavy chains that are directly involved in antibody binding to antigen

The term nucleic acid or nucleic acid molecule, as used hereafter, is intended to include all DNA and RNA molecules. A nucleic acid molecule may be single stranded or double stranded, but preferably it is double stranded DNA.

A nucleic acid is "operably linked" when it is placed in functional relationship with another nucleic acid sequence. For example, the DNA for a presequence or secretion signal is operably linked to the DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" refers to the fact that the bound DNA sequences are in cis and, in the case of a secretion signal, contiguously and in reading pattern. However, enhancers do not have to be contiguous or in reading pattern. This operational connection can be achieved by ligation through the appropriate restriction sites. If such places do not exist, the necessary oligonucleotide adapters are used in accordance with common practice.

As used herein, the terms "cell", "cell line", and "cell culture" can be applied interchangeably and all of them include progeny, therefore, the words "transformants" and "cells transformed "include the primary subject cell and the cultures derived therefrom regardless of the number of transfers. It is also understood that the entire progeny must not be identical in their DNA content, due to induced or inadvertent mutations. All the variant progeny that has the same biological function or activity defined for the original transforming cell is included, where different designations are applied, it will be evidenced from the context.

The term "IL-13R1 binding" as will be used herein refers to the binding of the antibody to IL13R1 in an in vitro assay, preferably in a binding assay in which the antibody binds to a surface and IL-13R1 binding is measured by Surface Plasma resonance (SPR) .The binding is defined by a binding affinity (KD) of 10-8 M or less, preferably 10-13 to 10-9 M. "No binding "Implies a KD of 10-6 M or greater. Antibodies related to the invention bind to the extracellular domain of human IL-13R1 and preferably also to mouse IL-13R1.

Binding to IL-13R1 can be determined using the BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden). Binding affinity is defined by the terms ka (constant rate for antibody association from the antigen / antibody complex), kd (dissociation rate), and KD (kd / ka).

The binding of IL-13 to IL-13R1 is inhibited by antibodies corresponding to the invention. Inhibition is measured in terms of IC50 in an IL-13 binding ELISA to the IL-13R1 / IL-4R heterodimer. To carry out said test, IL-13R1 is immobilized and IL-13 and IL-4R are added. The IC50 values of the antibodies according to the invention for the binding of IL-13 to IL-13R1 are not greater than 6nM. The IC50 values are measured as mean or average values of at least three independent measures. Individual IC50 values may be out of range.

The subject antibodies of the invention preferably show a binding to the same epitopes of IL-13R1 as the antibodies selected from the group consisting of antibodies LC5002-002, LC5002-003, LC5002-005, LC5002-007 or LC5002-018 or their binding to IL-13R1 is inhibited by steric impediments by the binding of these antibodies. Binding inhibition can be detected by an SPR assay using an immobilized antibody selected from the group that includes antibodies LC5002-002, LC5002-003, LC5002-005, LC5002-007 or LC5002-018 and IL-13R1 a a concentration of 20-50nM, the antibody being detected at a concentration of 100nM. A 50% or greater signal reduction demonstrates competition from the antibody against an antibody selected from the group of antibodies that includes LC5002-002, LC5002-003, LC5002-005, LC5002-007 or LC5002-018. The term "epitope" refers to a protein determinant capable of specific binding against an antibody. An epitope usually consists of groups of chemically active molecules, side chains of amino acids or sugars and usually have specific three-dimensional structure characteristics, as well as a defined load. The conformational and non-conformational epitopes are distinguished in that the union to the former but not to the latter is lost in the presence of denaturing agents. The invention also includes a human antibody that binds to IL-13R1 by inhibiting the bioactivity of IL-13, characterized by an affinity of 10-9 M (KD) or less, preferably 10-9 to 10-13 M for binding. to IL-13R1 and for an affinity of 10-7 M (KD) or less, preferably 10-8 to 10-9 M for binding to mouse IL-13R1.

In a preferred embodiment of the invention, the antibodies according to the invention are further characterized by having one or more of the characteristics selected from the group of binding parameters ka, kd and KD, joining the same epitope to which binds an antibody selected from the group consisting of the LC5002-002, LC5002-003, LC5002-005, LC5002-007 or LC5002-018 antibodies.

The antibodies according to the invention are preferably produced by recombinant methods. Such methods are widely known in the state of the art and include expression of proteins in eukaryotic and prokaryotic cells with subsequent isolation of the antibody polypeptide and usually a purification until a pharmacologically acceptable purity is obtained. For protein expression, nucleic acids encoding the light and heavy chains or fragments thereof, are inserted into expression vectors by standard methods. Expression is carried out in suitable, eukaryotic or prokaryotic host cells, such as CHO, NSO, SP / 2, HEK293, COS, yeast or E.coli cells, the antibody being recovered from the cells (supernatant or after a cell lysis).

Recombinant antibody production is well known in the current state of the art, being described for example in review articles such as Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol 16 (2000) 151-161; Werner, R.G., Drug Res. 48 (1998) 870-880.

The antibodies may be present in whole cells, in a cell lysate or in partially or substantially purified form. Purification is carried out to remove other cellular components or other contaminants, eg, other nucleic acids or cellular proteins, by standard techniques, including treatment with alkali and SDS, banding with CsCl, column chromatography gel electrophoresis of agarose and other methods well known in the state of the art. See Ausubel, F., et al., Ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

The expression in NSO cells is described by Barnes, L.M., et al., Cytotechnology 32 (2000) 109-123; and Barnes, L.M., et al., Biotech. Bioeng 73 (2001) 261-270. The transient expression is described among others by Durocher, Y., et al., Nucl. Acids Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R., et al., Proc. Natl Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl Acad. Sci. USA 89 (1992) 4285-4289; and by Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is that described by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol Methods 194 (1996) 191-199.

Suitable control sequences for prokaryotes typically include a promoter, optionally an operator sequence and a ribosome binding site. Eukaryotic cells are known to use promoters, enhancers and polyadenylation signals.

Monoclonal antibodies are suitably separated from the culture medium by conventional immunoglobulin purification protocols, such as, for example, sepharose-bound protein A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. The DNA or RNA encoding the monoclonal antibodies can be isolated and sequenced effectively using conventional methods. Hybridoma cells can serve as a source of said DNA and RNA. Once isolated, the DNA can be inserted into expression vectors, which are then transfected into host cells such as CHO, HEK293, or myeloma that would not otherwise produce immunoglobulins, to thereby obtain the synthesis of recombinant monoclonal antibodies. .

Antibodies according to the invention also include those that exhibit "conservative sequence modifications", amino acid or nucleotide modifications that do not affect or alter the characteristics of the antibody according to the invention, mentioned above. Modifications can be made by standard techniques known in the state of the art, such as directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which the amino acid residue is replaced by another amino acid residue with a similar side chain. Amino acid families with similar side chains have been defined in the state of the art. These families include amino acids with basic side chains (for example lysine, arginine, histidine), acidic side chains (for example aspartic acid, glutamic acid), uncharged polar side chains (for example glycine, asparagine, glutamine, serine, threonine, tyrosine , ciseine, tryptophan), non-polar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), side chains with beta branches (for example, threonine, valine, isoleucine) and aromatic side chains (tyrosine , phenylalanine, tryptophan, hisitidine). Thus, an amino acid residue with prediction of not being essential in a human anti-IL-13R1 antibody can be preferably substituted by another amino acid residue of the same side chain family.

Amino acid substitutions can be carried out by mutagenesis based on molecular models as described by Riechmann, L., et al., Nature 332 (1988) 323-327 and Queen, C., et al., Proc. Natl Acad. Sci. USA 86 (1989) 10029-10033.

Variant amino acid sequences of human antibodies against IL-13R1 are prepared by introducing the appropriate nucleotide change in the DNA encoding the antibody, or by peptide synthesis. Such modifications can be carried out, however, only within a very limited range as described above. For example, the modifications should not alter the antibody characteristics mentioned above, such as the IgG isotype and epitope binding, but may improve the yield of recombinant production, protein stability or facilitate its purification.

Any cysteine residue that is not involved in maintaining the proper conformation of the anti-IL13R1 antibody can also be substituted, generally with a serine to improve the oxidative stability of the molecule and prevent the formation of aberrant sulfur bridges. In addition, cysteine-mediated sulfur bridges could be added to the antibody to improve its stability (especially when the antibody is an antibody fragment such as the Fv fragment).

Another type of amino acid variant in the antibody would consist of the alteration of the original glycosylation pattern. By alteration is meant the deletion of one or more carbohydrate groups present in the antibody, and / or the addition of one or more glycosylation sites that were not originally present in the antibody. The glycosylation of antibodies is usually done through the binding of the carbohydrate group to the amino group of the asparagine side chain. The asparagine-X-serine and asparagine-X-threonine tripeptides, where X is any amino acid except proline, result in recognition sequences for the enzymatic binding of the carbohydrate group to the asparagine side chain. Therefore, the presence of any of those tripeptides in a polypeptide creates a potential glycosylation site. The addition of glycosylation targets to an antibody can be achieved by altering the amino acid sequence so as to contain one or more of the above-mentioned tripeptide sequences (for N-glycosylation sites).

Nucleic acid molecules encoding anti-IL-13R1 antibodies with varying amino acid sequences are prepared by a variety of methods known in the state of the art. These methods include, but are not limited to, extraction from the natural source (in the case of naturally occurring amino acid variants) or preparation by oligonucleotide-directed or mediated mutagenesis, PCR or mutagenesis. Cassette mutagenesis of a variant or non-variant version of the anti-13R1 antibody prepared above.

Another type of covalent modification is that which involves the binding to the antibody by chemical or enzymatic route of glycosidic groups. These procedures have the advantage of not requiring the production of the antibody in a host cell that has the ability to glycosylate either by N-or O- route. Depending on the type of coupling used, the sugar / es may be attached (a) to a residue of arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) hydroxyl groups free such as serine, threonine

or hydroxyproline, (e) aromatic residues such as tryptophan, phenylalanine or tyrosine, or (f) the glutamine amide group. These methods are described in WO 87/05330, and in Aplin, J.D., and Wriston, J.C. Jr., CRC Crit. Rev. Biochem. (1981) 259-306.

The elimination of any of the carbohydrate groups present in the antibody can be achieved by enzymatic or chemical methods. Chemical deglycosylation can be achieved by exposure of the antibody to trifluoromethanesulfonic acid, or equivalent compounds. This treatment results in the separation of most of the sugars except the binding ones (N-acetylglucosamine or N-acetylgalactosamine), leaving the antibody intact, chemical deglycosylation is described by Sojahr, HT, and Bahl, OP, Arch. Biochem Biophys 259 (1987) 52-57 and by Edge, A.S., et al. Anal. Biochem 118 (1981) 131-137. The enzymatic separation of the carbohydrate groups from the antibodies can be achieved through the use of a wide variety of endo- and exoglycosylases, as described by Thotakura, N.R., and Bahl, O.P., Meth. Enzymol 138 (1987) 350-359.

Another type of covalent modification of the antibody consists in binding the antibody to any of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner established in US patents under 4,640. 835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

In another different aspect, the invention provides B cells isolated from transgenic animals, for example a transgenic mouse, which expresses the human anti-IL-13R1 antibodies according to the invention. Preferably, isolated B cells are obtained from non-human transgenic animals, for example the mouse, which have been immunized with a purified or enriched preparation of IL-13R1 antigen and / or cells expressing IL-13R1. Preferably, the non-human transgenic animal, for example the mouse, has a genome that includes the transgene encoding the human heavy chain and the transgene for the human light chain, encoding all or part of an antibody of the invention. The isolated B cells are then immortalized to provide a source (a hybridoma) of human anti-IL-13R1 antibodies. Accordingly, the present invention also provides a hybridoma with the ability to produce human monoclonal antibodies according to the invention. In one embodiment, the hybridoma includes a B cell obtained from a non-human transgenic animal, for example a transgenic mouse, which possesses a genome that includes a transgene encoding the human heavy chain and the transgene for the human light chain. , encoding all or part of an antibody of the invention, fused with an immortalized cell.

In a particular embodiment, the non-human transgenic animal is a transgenic mouse that possesses a genome with a transgene encoding the human heavy chain and transgene for the human light chain, encoding all or part of an antibody of the invention. The non-human transgenic animal can be immunized with a purified or enriched preparation of IL-13R1 antigen and / or cells expressing IL-13R1. Preferably, the non-human transgenic animal, for example the transgenic mouse, will be able to produce human monoclonal antibodies of the IgG1 isotype against IL-13R1.

The human monoclonal antibodies according to the invention can be obtained by immunizing a non-human transgenic animal, for example a transgenic mouse, that possesses a genome with a transgene encoding the human heavy chain and the transgene for the human light chain, encoding all or part of an antibody of the invention, with a purified or enriched preparation of IL-13R1 antigen and / or cells expressing IL-13R1. The B cells of the animal (for example spleen B cells) are then obtained and fused with myeloma cells to achieve immortal hybridoma cells that secrete human monoclonal antibodies against IL-13R1.

In a preferred embodiment, human monoclonal antibodies directed against IL-13R1 can be generated, using transgenic mice carrying parts of the human immune system instead of the mouse itself. These transgenic mice, hereinafter referred to as "huMab mice," contain miniloci for non-rearranged human immunoglobulin genes, including genes for constant regions of the heavy (µ and γ) and light κ chains, along with targeted mutations. which inactivate the loci for the endogenous µ and κ chains (Lonberg, N., et al., Nature 368 (1994) 856-859). Correspondingly, these mice will exhibit reduced expression of mouse IgM or K, and in response to immunization, transgenes introduced for human heavy and light chains will undergo class exchange and somatic mutations to generate human IgG monoclonal antibodies. High affinity (Lonberg, N., et al., Nature 368 (1994) 856-859; reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113 (1994) 49-101; Lonberg, N., and Huszar, D ., Intern. Rev. Immunol. 25 (1995) 65-93; and Harding, F., and Lonberg, N., Ann. N. Acad. Sci 764 (1995) 536546). The preparation of HuMab in mice is described by Taylor, L., et al., Nucleic Acids Research 20 (1992) 6287-6295; Chen, J., et al., International Immunology 5 (1993) 647-656; Tuaillon, N., et al., Proc. Natl Acad. Sci USA 90 (1993) 3720-3724; Choi, T.K., et al., Nature Genetics 4 (1993) 117-123; Chen, J., et al., EMBO J. 12 (1993) 821830; Tuaillon, N., et al., Immunol. 152 (1994) 2912-2920; Lonberg, N., et al., Nature 368 (1994) 856-859; Lonberg, N., Handbook of Experimental Pharmacology 113 (1994) 49-101; Taylor, L., et al., Int. Immunol. 6 (1994) 579-591; Lonberg, N., and Huszar, D., Intern. Rev. Immunol. 25 (1995) 65-93; Harding, F., and Lonberg, N., Ann. N. Acad. Sci 764 (1995) 536-546; Fishwild, D.M., et al., Nat. Biotechnol. 14 (1996) 845-851. For more information, see US patents number 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,545,807; 5,770,429; WO 98/24884; WO 94/25585; WO 93/1227; WO 92/22645; and WO 92/03918.

To generate fully human monoclonal antibodies against IL-13R1, HuMab mice can be immunized with a purified or enriched IL-13R1 antigen preparation and / or cells expressing IL-13R1 according to the general method, as described. by Lonberg, N., et al., Nature 368 (1994) 856-859; Fishwild, D.M., et al., Nat. Biotechnol. 14 (1996) 845-851 and WO 98/24884. Preferably, the mice will be 6 to 16 weeks of age at the time of the first immunization. For example, a purified or enriched preparation of the IL-13R1 antigen (for example, purified from cells expressing IL-13R1) can be used to immunize HuMab mice intraperitoneally. If it turns out that immunizations using a purified or enriched preparation of the IL-13R1 antigen do not result in antibody production, the mice can also be immunized with cells expressing IL-13R1, for example a tumor cell line, which could induce response. immune The accumulated experience using different antigens has shown that HuMab transgenic mice respond best when the initial immunization is done intraperitoneally using Freund's complete adjuvant antigen, followed by weekly immunizations alternating the intraperitoneal route with the subcutaneous with Freund's incomplete adjuvant antigen . The immune response can be monitored throughout the immunization protocol using plasma samples obtained by retroorbital bleeding. Plasma can be analyzed by ELISA, and mice with sufficient titers of human anti-IL-13R1 immunoglobulins can be used for immortalization of the corresponding B cells. Mice can be realmized with intravenous antigen 3 to 4 days before slaughter and removal of the spleen and lymph nodes. It is expected that it will be necessary to carry out 2 to 3 fusions for each antigen. Several mice will be immunized for each antigen. For example, a total of 5 to 12 HuMab mice of strains HCo7 and HCo12 can be immunized.

HCo7 mice have a JKD disruption in the genes for their endogenous light chain (kappa) (as described in Chen, J., et al., EMBO J. 12 (1993) 821-830), a CMD disruption in their endogenous genes for the heavy chain (as described in example 1 of WO 01/14424), a human KCo5 transgene for the kappa light chain (as described in Fishwild, DM, et al., Nat. Biotechnol. 14 (1996) 845-851), and an HCo7 transgene for the human heavy chain (as described in US Patent No. 5,770,429).

HCo12 mice have a JKD disruption in the genes for their endogenous light chain (kappa) (as described in Chen, J., et al., EMBO J. 12 (1993) 821-830), a CMD disruption in their endogenous genes for the heavy chain (as described in example 1 of WO 01/14424), a human KCo5 transgene for the kappa light chain (as described in Fishwild, DM, et al., Nat. Biotechnol. 14 (1996) 845-851), and an HCo12 transgene for the human heavy chain (as described in Example 2 of WO 01/14424).

Mouse lymphocytes can be isolated and fused with myeloma cell lines to produce hybridomas using standard protocols based on the use of PEG. The resulting hybridomas are screened to check the production of antigen specific antibodies. For example, cell suspensions of lymphocytes obtained from a spleen or lymph node from immunized mice are fused with one sixth of non-secretory mouse myeloma cells SP 2/0 (ATCC, CRL 1581) with 50% PEG. The cells are plated at an approximate density of 2 x 105 in flat bottom microplates, incubating for about two weeks in selective medium.

Individual wells are screened by ELISA to detect IgM and IgG antibodies against IL-13R1. Once sufficient hybridoma growth has occurred, the medium is analyzed, usually after 10 to 14 days. Hybridomas that secrete antibodies are plated again and characterized again, if they remain positive for human monoclonal antibodies of the IgG type against IL-13R1, they can be subcloned at least twice by limiting dilution. Stable subclones are grown in vitro to obtain medium with antibody production for characterization.

Because the CDR sequences are responsible for the interactions between the antigen and the antibody, it is possible to express recombinant antibodies according to the invention by constructing expression vectors that include the CDR sequences according to the invention within framework sequences. for a different human antibody. (see for example, Riechmann, L., et al., Nature 332 (1998) 323-327; Jones, P., et al., Nature 321 (1986) 522-525; and Queen, C., et al. , Proc. Natl. Acad. See. USA 86 (1989) 10029-10033). Such framework sequences can be obtained from public DNA databases that include the germline gene sequences encoding human antibodies . These germline sequences will differ from mature gene sequences because they will not include reorganized variable regions, which are formed by the V (D) J junction during maturation of B cells. Germline gene sequences also have specific differences. with respect to the sequences of the secondary repertoire of high affinity antibodies distributed uniformly throughout the variable region.

The invention preferably includes a nucleic acid fragment encoding a polypeptide capable of binding to IL-13R1, wherein said polypeptide inhibits the binding of IL-13 to IL-13R1, selected from the group that includes

a) an antibody heavy chain that includes one of the heavy chain CDRs with SEQ ID NO: 1, 3.5, 7 or 9;

b) an antibody light chain that includes one of the light chain CDRs with SEQ ID NO: 2, 4, 6, 8 or 10.

The variable regions of the heavy and light chains thus reconstructed are combined with the sequences of a promoter, translation initiation, constant region, 3 'untranslated region, polyadenylation and transcription termination to form the construct in the vector of expression. The expression constructs for the heavy and light chains can be combined into a single expression vector, transfected together, consecutively or separately to the host cells that are then fused to achieve a single host cell that expresses both chains.

As a result, the invention provides a method for the production of a recombinant human antibody according to the invention, including the expression of a nucleic acid encoding for

a) an antibody heavy chain that includes one of the heavy chain CDRs with SEQ ID NO: 1, 3.5, 7 or 9;

b) an antibody light chain that includes one of the light chain CDRs with SEQ ID NO: 2, 4, 6, 8 or 10.

The invention also includes the use of an antibody according to the invention for the detection of IL13R1 in vitro, preferably by means of an immunological assay that determines the binding between IL-13R1 of a sample and the antibody according to the invention.

On the other hand, the present invention provides a composition, for example a pharmaceutical composition, including one or a combination of human monoclonal antibodies, or the corresponding antigen binding part, of the present invention, formulated together with a pharmaceutically acceptable carrier.

As used herein, the term "acceptable pharmaceutical carrier" includes all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and retarding agents and the like that are physiologically compatible. Preferably, the vehicle will be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (by injection or infusion).

The term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the antibody without causing any unwanted toxicological effects (see for example Berge, SM, et al., J. Pharm. Sci. 66 (1977) 1- 19). Such salts are included in the invention. Examples of such salts include those of acidic nature and those of basic nature. Acids include those derived from non-toxic inorganic salts, such as hydrochloride salts.

A compound of the present invention can be administered by a variety of methods known in the state of the art. As will be apparent to the specialist technician, the route and / or mode of administration will vary depending on the expected results.

In order to administer a compound of the invention through certain routes, it may be necessary to coat the compound with, or administer in conjunction with, a material that prevents its inactivation. For example, the compound can be administered to a subject by a suitable carrier, such as liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions. The use of such media and agents with pharmaceutically active substances is known in the state of the art.

The terms "parenteral administration" and "perentially administered", as used herein refer to forms of administration other than topical or enteral administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial routes. , intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intrarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal.

These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers.

or dispersants. To avoid the presence of microorganisms, some sterilization process can be applied in the preparation and include antibacterial and antifungal agents, for example: paraben, chlorobutanol, phenol, sorbic acid and the like. It may also be convenient to include isotonic agents, such as sugars, sodium chloride and the like, to the compounds. In addition, to ensure prolonged absorption of injectable pharmaceutical compound, retarders for absorption such as aluminum monostearate and gelatin can be included.

Regardless of the route of administration selected, the compounds of the present invention, which can be employed in hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated with a pharmacologically acceptable dosage by conventional methods known to those with knowledge about the matter.

Actual dosage levels for the active ingredients within the pharmaceutical compositions of the present invention can be varied in order to have an amount of active ingredient that is sufficient to produce the desired therapeutic response in each particular patient, composition or mode of administration. , without it becoming toxic to the patient. The selected dose will depend on a number of pharmacokinetic factors, including the activity of each compound of the present invention, the ester, salt or amide, the route of administration in particular, the time of administration, the rate of excretion. of one of the specific compounds used, of the duration of treatment, of other drugs, compounds and / or materials administered in combination with the specific compositions employed, of age, sex, condition, general state of health and medical history previous patient in treatment, and style factors, well known to medical specialists.

The compound must be sterile and fluid, to the extent that it can be administered by a syringe. In addition to water, the vehicle can be a buffered and isotonic saline solution, ethanol, polyalcohol (for example glycerol, propylene glycol, liquid polyethylene glycol and the like), and their appropriate mixtures.

Adequate fluidity can be maintained, for example, by the use of lecithin-type coatings, by maintaining the desired particle size in the case of a dispersion and by the use of surfactants. In many cases, it is preferable to include isotonic agents in the composition, for example, sugars, polyalcohols, such as mannitol or sorbitol, and sodium chloride. The long-term absorption of the injectable compounds can be achieved by the inclusion in the composition of an agent that delays the absorption, for example aluminum monostearate or gelatin.

Sequence Listing

Description of the Sequence Listing

SEC ID NO: 1 variable domain of the HuMab LC5002-002 heavy chain

SEC ID NO: 2 variable domain of the HuMab LC5002-002 light chain

SEC ID NO: 3 HuMab LC5002-003 heavy chain variable domain

SEC ID NO: 4 variable domain of the HuMab LC5002-003 light chain

SEC ID NO: 5 variable domain of the HuMab LC5002-005 heavy chain

SEC ID NO: 6 HuMab LC5002-005 light chain variable domain

SEC ID NO: 7 variable domain of the HuMab LC5002-007 heavy chain

SEC ID NO: 8 HuMab LC5002-007 light chain variable domain

SEC ID NO: 9 HuMab LC5002-018 heavy chain variable domain SEC ID NO: 10 variable domain of the HuMab LC5002-018 light chain SEQ ID NO: 11 constant region κ of the light chain SEC ID NO: 12 constant region 1 of the heavy chain

Description of the figures

Figure 1 shows the binding of anti-IL-13Rα1 antibodies to the previously immobilized recombinant human IL-13R1 polypeptide. The human anti-IL-13R1 rabbit polyclonal antibody AF152 (R&D systems) and an anti-KLH as human Monoclonal for negative control are included.

Figure 2 shows the inhibition of IL-13 binding by anti-IL-13α1 antibodies, using an immobilized IL-13R1 / IL-4Rα receptor.

Figure 3 shows the blockade of the binding of IL-13 to CHO cells (expressing IL-13Ralpha1 and IL4R2) by anti-IL-13Rα1 antibodies. A commercial anti-IL-13Rα1 antibody (AF152, R&D Systems, Minneapolis, MN) is used as a positive control.

Figure 4 shows the binding of anti-IL-13Rα1 antibodies to hIL-13R1 and functionally related receptor binding properties such as hIL-13R2 and hIL-4R.

Figure 5 shows the ability of anti-IL-13Rα1 antibodies to bind to the murine IL-13R1 recombinant polypeptide, previously immobilized. Goat polyclonal anti-IL13R1 human antibody AF152 (R&D systems) and an anti-KLH as human Monoclonal for negative control are included.

EXAMPLES

Example 1 Hybridoma Generation

The human monoclonal antibodies according to the invention can be produced by immunizing transgenic animals, such as a transgenic mouse that has a genome that includes transgenes for the heavy and light chains encoding all or part of an antibody of the present. invention, with cells expressing human IL-13Rα. After that, B cells from the animal's spleen are obtained and fused with myeloma cells to achieve immortal hybridoma lines that can secrete human monoclonal antibodies against IL-13R1. Human monoclonal antibodies against IL13R1 can be generated using human mice carrying parts of the human immune system instead of the human body itself. These transgenic mice will hereinafter be referred to as "HuMab" mice, these contain a minilocus for human immunoglobulin that includes non-rearranged genes from the constant regions of human immunoglobulins, both for the heavy chain (µ e) and light chain ĸ (kappa), together with targeted mutations that inactivate endogenous loci for the µ and kappa chains (Lonberg N., et al., Nature 368 (1994) 856859). Thanks to this, mice show reduced expression of mouse IgM or ĸ, and, in response to immunization, transgenes introduced for human light and heavy chains undergo class switching and somatic mutations to generate human IgG monoclonal antibodies from High affinity to produce fully human monoclonal antibodies against IL-13R1, HuMab mice can be immunized with cells expressing human IL-13R1 according to the general method, as described by Lonberg, N., et al., Nature 368 (1994) 856-859; Fishwild, D.M., et al., Nat. Biotechnol. 14 (1996) 845-851 and WO 98/24884. Preferably, the mice should be 6 to 16 weeks of age upon receiving the first immunization. For example, cells transfected with IL-13R1 can be used to immunize HuMab mice intraperitoneally. The immune response can be monitored throughout the immunization protocol by extracting plasma samples obtained by periorbital bleeds. Plasma can be tracked by ELISA and / or FACS. Mice with sufficient titers of human anti-human IL-13R1 immunoglobulin can be used for immortalization of the corresponding B cells. Mice can be reinvented intravenously with the antigen about 3 or 4 days before slaughter and removal of the spleen and lymph nodes. For example, HuMab mice of the HCo7 or HCo12 strains can be immunized. HCo7 mice have a JKD disruption in their endogenous genes for the light chain (kappa) (described in Chen et al. (1993) EMBO J. 12: 821-830), a CMD disruption in their endogenous genes for the heavy chain ( described in WO 01/14424), a transgene for the human kappa KCo5 light chain (described in Fishwild, DM, et al. (1996) Nature Biotechnology 14: 845-851), and a transgene for the human heavy chain HCo7 (described in US 5,770,429). HCo12 mice have a JKD disruption in their endogenous genes for the light chain (kappa) (described in Chen, J., et al., EMBO J. 12 (1993) 821-830), a CMD disruption in their endogenous genes for the heavy chain (as described in example 1 of WO 01/14424)) a KCo5 transgene for the human kappa light chain (described in Fishwild, DM, et al., Nat. Biotechnol. 14 (1996) 845-851, and an HCo12 transgene for the human heavy chain (described in example 2 of WO 01/14424). Mouse lymphocytes can be isolated and fused with a mouse myeloma line using PEG according to standard protocols for generating hybridomas. The resulting hybridomas are screened to determine the production of antigen-specific antibodies, for example, cell suspensions of spleen-derived lymphocytes and nodules obtained from immunized mice are fused with non-secretory mouse SP / 2 myeloma cells ( ATCC, CRL 1581) with 50% PEG. The cells are cultured at a density of 0.75 x 107 in flat bottom microplates, followed by about two weeks of incubation in selective medium. Individual wells are screened by ELISA and / or FACS for the presence of human monoclonal antibodies IgM and IgG anti-IL-13R1. Once sufficient growth of the hybridomas has been obtained, those that secrete antibodies are plated again, and if they remain positive for anti-IL-13R1 human IgG monoclonal antibodies, they can be subcloned at least twice by limiting dilution. Stable subclones are grown in vitro to produce antibodies in culture medium for characterization.

Immunization procedure of transgenic mice

Three HCo7 mice (3 males), strain GG2201 (Medarex, San Jose, CA, USA) and 2 HCo12 mice (1 male and one female), strain GG2198 (Medarex, San Jose, CA, USA) were immunized with 1 x 106 HEK293 cells, previously transfected with an expression vector for IL-13R1. In total, eight immunizations were performed, alternating the intraperitoneal route (i.p.) with the subcutaneous (s.c.) at the base of the tail. For the first immunization, 100 µl of HEK293: IL-13R1 cells at 1 x 106 were mixed with 100 µl of Freund's complete adjuvant (CFA; Difco Laboratories, Detroit, USA). For all other immunizations, 100 µl of cells in PBS were mixed with 100 µl of Freund's incomplete adjuvant (ICFA; Difco).

Mouse Kingdomculation

When the serum titers of anti-IL-13R1 were demonstrated sufficient, the mice were again reallocated twice with HEK293: IL-13R1 cells at 1x106, in 200 µl of PBS (iv) 4 and 3 days before fusion .

Example 2 ELISA check of binding of HuMab to immobilized IL-13Rα1

To determine the ability of the antibodies of the invention to bind recombinant IL-13R1, the extracellular domain of IL-13R1 (R&D Systems, UK) was dissolved in PBS (1 µg / ml) and adsorbed to microplates (NUNC Maxisorb) by an incubation at 4 ° C overnight. After washing the plates with wash buffer (TL = 0.9% NaCl; 0.1% Tween® 20) the non-specific binding sites were blocked by adding 100 µl incubation buffer (TI = PBS with 1% of protein C and 0.1% Tween® 20) and incubated for 30 minutes at room temperature (RT). After this, serial dilutions of the HuMab and control antibodies (100 µl / well; dilutions in TI) were made, these were added and incubated for 1 h at RT. The plates were washed again and bound human antibodies were detected by incubation with peroxidase-conjugated rabbit anti human kappa (DAKO, Denmark) at a final dilution of 1: 500 in TI. Goat anti-hIL-13R1 polyclonal antibodies were detected with a goat anti-IgG polyclonal produced in donkey and conjugated with peroxidase (Santa Cruz; dilution 1: 1000 in TI). After incubating for 1h at RT and after a new washing step, the plates were revealed with ABTS® solution ready for use (Roche Diagnostics GmbH) at RT in the dark. The absorbance at 405nM was measured at the time when the absorbance of the highest concentration reached a sufficient value.

All antibodies against IL-13Ralpha1 that were tested were able to bind to the immobilized extracellular domain of human IL-13R1. The EC50 values that were determined were in the range of 0.5-2nM for the various LC antibodies tested. The HuMab anti-KLH antibody that was used as a negative control did not bind to the immobilized extracellular domain of IL-13R1. The positive control, goat anti-human IL-13R1 antibody, also efficiently bound to the immobilized extracellular domain of IL-13R1 (Fig. 1).

Example 3 Inhibition of IL-13 binding to the IL-13Rα1 / IL-4Rα hetrodimer (ELISA)

Microplates were upholstered with 100 µl of the hIL-13R1 chimeric protein: hFc (R&D Systems, UK) dissolved in PBS at 3 µg / ml overnight at 4 ° C, under stirring. After washing the plates with TL, serial dilutions of HuMab antibodies and controls (100 µl / well; dilutions in TI) were added and incubated for 30 minutes at RT. The plates were washed again and after that a mixture of IL-13 (R&D Systems, UK; at 0.5 µg / ml; diluted with TI) with IL-4Rα (R&D Systems, UK; 0.75 µg / ml; diluted with TI), incubating for 1h at RT. After washing the plates, 100 µl of biotinylated anti-IL-13 antibody (BAF213; R&D Systems, UK) was added at a concentration of 0.4 µg / ml and incubated for 1 h at RT. After washing the plates, bound IL-13 was detected by peroxidase-bound streptavidin (Roche Diagnostics GmbH, DE) at a dilution of 1: 5000 in TI (1h at RT). Finally, the plates were washed and developed with ABTS® solution ready for use (Roche Diagnostics GmbH, DE) at RT in the dark. The absorbance at 405 measured after 45-60 minutes.

The LC5002-002, LC5002-003, LC5002-005, LC5002-007 and LC5002-018 antibodies were able to inhibit the binding of IL-13 to the heterodimeric receptor with maximum inhibition values of 50 to 80-85% . The positive control was AF152 (rabbit polyclonal antibody). As expected, the negative control, anti-KLH, did not inhibit the binding of IL-13 to the heterodimeric receptor. The IC50 values obtained were between 1.5nM and 10.1nM for LC5002-002, LC5002-003, LC5002-005, LC5002-007 and LC5002-018 (Fig. 2).

Example 4 Radioligand binding assay

The 125I IL-13 binding assay was carried out on CHO cells expressing human IL-13Rα1 and IL-4Rα, dissolved in binding buffer (25mM HEPES, 150mM NaCl, 1mM CaCl2, 5mM MgCl2, and bovine serum albumin 0.5%, adjusted to pH 7.2). 1x105 cells were mixed per well with the antibodies and pre-incubated for 15 minutes to 1 hour. After this, 0.1nM 125I-IL-13 was added and the mixture was incubated at 4 ° C for 4 hours. The concentration of 125IIL-13 used in the assay was determined from analysis of binding saturation, competition and by determining the amount of 125I IL-13 necessary to reach the balance of binding with the cell line. Samples were collected on a plate with GF / C filter pretreated with 1% PEI / 0.5% BSA and measured on a Packard TopCount scintillation counter. The data analysis was carried out with the PRISM software using the non-linear adjustment to a regression curve (GraphPad Software, San Diego, CA).

All antibodies against IL-13Ralpha1 tested blocked the binding of labeled IL-13 to the IL13Rα1 / IL-4Rα complex. The IC50 values calculated for the LC5002-002, LC5002-003, LC5002-005, LC5002007 and LC5002-018 antibodies were between 0.09nM and 0.32nM, with 84.8nM for AF152 (Fig. 3).

Example 5 Inhibition by HuMab antibodies of positive regulation of CD23 induced by IL-13 in human and monocyte B cells.

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Hypaque density gradient. After washing the cells with RPMI, they were resuspended in RPMI / 10% FCS and distributed at a density of 3x105 PBMC / well (50 µl volume) in flat-bottom 96-well microplates (Corning Incorporated Costar). After this, 25 µl of human anti-CD40 antibody (Immunotech) was added to a final concentration of 0.5 µg / ml in RPMI / 10% FCS and 25 µl of human anti IgA + IgG + IgM antibody (Immunoresearch) at a concentration 10 µg / ml final in RPMI / 10% FCS. After this, serial dilutions of the HuMab and control antibodies (50 µl / well; dilutions in RPMI / 10% FCS) were added and incubated for 30 min in the oven (37 ° C; 5% CO2). Recombinant human IL-13 (R&D Systems) was then added to a

final concentration of 0.67 ng / ml (50 µl / well), the cells being incubated for 72h at 37 ° C / 5% CO2. After this

incubation the plates were centrifuged and the medium aspirated. To disengage the cells, 200 ul of Accutase (PAA) was added and the cells were incubated for approximately 5 minutes at 37 ° C; 5% CO2 The cells are detached by repeated rinsing and transferred to a round bottom plate. After centrifuging and aspirating the

Supernatants cells were incubated with 200 ul of a mixture of anti-CD23-PE, anti-CD20-FITC and antiCD14-APC (all of them from BD Biosciences Pharmingen, San Diego, CA). The cells were incubated for 30 minutes at 4 ° C, centrifuged and the supernatants aspirated. This washing step was repeated again and the cells were resuspended in 200 µl of PBS / 0.1% human serum albumin and analyzed by FACS using a Calibur flow cytometer (BD Biosciences Pharmingen, San Diego, CA) using the CellQuest software. In most cases, 10,000 events were acquired, being selected and grouped according to light scattering, including only viable monocytes and lymphocytes. The cells were pre-grouped into B lymphocytes, positive for CD19, or monocytes, positive for CD14, and were analyzed for CD23 expression.

The observed IC50 values for inhibition of positive regulation of CD 23 in B lymphocytes were between 0.5nM and> 70nM for antibodies LC5002-002, LC5002-003, LC5002-005, LC5002-007 and LC5002-018 and 13 , 6nM for AF152. For the inhibition of positive regulation of CD23 induced by IL-13 a similar pattern was observed in human monocytes. In the case of monocytes, the IC50 values were between 0.1nM and 62.8nM for the LC5002-002, LC5002-003, LC5002-005, LC5002-007 and LC5002-018 and 62.9nM antibodies for AF152.

Example 6 TF-1 proliferation assay in response to stimulation by IL-13 or IL-4

TF-1 cells (ATCC # CRL 2003) were cultured in culture medium containing RPMI modified by ATCC, 10% FBS, 1X Pencilin / Streptomycin, GM-CST at 2ng / ml. One day before the test the cells were transferred to a medium without GM-CSF. 5 x 103 cells were incubated per well against the appropriate concentrations of anti-IL-13Rα1 antibodies at 37 ° C for 1 hour. The cells were then stimulated with human IL-13 at 2ng / ml (R&D Systems, Minneapolis, MN) or human IL-4 at 0.1ng / ml (R&D Systems, Minneapolis, MN) and incubated at 37oC for 48 hours. The cells were exposed to Thymidine-Ci 3H at 0.5 µM and incubated at 37oC for 16 to 18 hours. Samples were collected on GFC plates that had been treated with 1% PEI + 0.25% BSA, using the "Perkin Elmer Filtermate 96 harvester" 96-well collector. GFC plates were counted using a "Perkin Elmer Top count Scintillation counter" scintillation counter. The data analysis was carried out with the PRISM software using the non-linear adjustment to a regression curve (GraphPad Software, San Diego, CA).

Anti-KLH antibodies did not produce any type of inhibition in this assay. The same statement is valid for LC5002-007. All other antibodies inhibited the response, even LC5002-007 inhibited the response with higher IC values than other antibodies. The IC50 values observed for the different antibodies were: 13.50nM for AF152, 9.21nM for LC5002-002, 3.07nM for LC5002-003 and 0.39nM for LC5002

005. For the proliferation of TF-1 induced by IL-4 a similar profile was obtained, however the potency of the antibodies was lower compared to the responses induced by IL-13. The IC50 values for IL-4 induced proliferation were 0.02nM for the anti-IL4R antibody, 74.37nM for AF152 and for the LC5002-002, LC5002-003, LC5002-005, LC5002-007 and LC5002-018 between 4.68nM and 60nM.

Example 7 Inhibition of eotaxin production in response to IL-13 in human lung fibroblasts.

The assay was carried out using HFL-1 cells (Human Lung Fibroblast, ATCC # CCL-153). The cells were seeded at a density of about 100,000 cells per well in a 12-well plate and incubated at 37 ° C for 72 hours until confluence was reached. The cells were subsequently kept in serum-free medium for 24 hours and treated with anti-IL-13Rα1 antibodies at 37 ° C for 1 hour. Following this treatment the cells were stimulated with IL-13 at 10ng / ml (R&D Systems, Minneapolis, MN) at 37oC for 48 hours. Supernatants were collected and the eotaxin content was determined using the commercial R&D Systems kit (Cat. No. DTX00). The absorbance reading was done with a Spectromax plate reader and the data was analyzed with the PRISM program (GraphPad Software, San Diego, CA).

The antibodies tested showed different abilities to inhibit eotaxin secretion. With the exception of LC5002-007 all other antibodies tested showed some inhibition. The calculation of the average IC50 from 3 to 4 experiments was 11.5nM for AF152 and between 2.45nM and 19.8nM for the LC5002-002, LC5002-003, LC5002-005, LC5002-007 and LC5002 antibodies -018.

Example 8 Inhibition of IL-13-induced Stat-6 phosphorylation in human bronchus smooth muscle cells

Smooth muscle cells of human bronchus (BSMC; Clonetics, Cat. No CC-2576) were cultured according to the supplier's instructions. The cells were grown in 12-well plates until they reached confluence. At that time they left 24 hours in serum free medium and various amounts of antibody were added. Plates were incubated for 1 hour and then stimulated with IL-13 at 2.5ng / ml (R&D System). The supernatants were recovered after a 15 minute incubation, the cells were washed with phosphate buffer, finally adding 100 µl of lysis buffer. The mixture was briefly sonicated on ice and centrifuged. The lysate was used for Western Blot detection of phosphorylated Stat-6. An SDS gel was loaded with equivalent amounts of protein and transferred to a membrane. The anti-Stat-6 antibody was obtained from Santa Cruz Biotechnology (Cat. No. SC-11762R) and a secondary antibody conjugated to peroxidase was used. Detection was carried out using the Amersham ECL Plus System (Cat No RPN 2132). The quantification was done with the Typhoon 9400 Imager system. The two HuMabs analyzed in this assay (LC50002-003 and LC5002-005) inhibited Stat-6 phosphorylation induced by IL-13. The power observed in this trial was similar to that of other functional tests. The IC50 values obtained were 18.64nM for AF152, 5.98nM for LC5002-003 and 1.18nM for LC5002

005

Example 9 Cloning and analysis of the coding sequence for the variable domains (light chains- image 1 and heavy- image2 1) HuMab anti-hIL-13Rα1.

Nucleotide sequences encoding the variable region of the VL light chain and the VH heavy chain of the HuMabs anti hIL-13R1 antibodies were isolated by standard cDNA synthesis, followed by PCR. Total RNA was obtained from hybridoma cells at 1x106-1x107 using the GeneRacer ™ kit (Invitrogen). The RNA thus obtained was used as a template for the synthesis of the first cDNA chain and ligation of the oligo dT GeneRacer ™ primer. The synthesis of the second cDNA chain and subsequent PCR amplification of the fragments encoding the VL and the VH was completed with antisense primers for the light and heavy chain, complementary to the nucleotide sequences encoding the constant sequence of the light-ĸ and heavy-1 chain and with specific 5 'GeneRacer ™ primers, respectively. The PCR products were cloned using the TOPO ™ TA cloning kit from InvitrogenTM Life Technologies and the pCR4TOPO ™ cloning vector. Cloned PCR products were identified by restriction of the appropriate plasmids using EcoRI for digestion, with expected fragments with sizes of about 740 and 790 base pairs for VL and VH, respectively. The DNA sequence of the cloned PCR fragments was determined by double chain sequencing. The GCG software package (Genetics Computer Group, Madison, Wisconsin) in version 10.2 and Vector-NTI 8 (InforMax, Inc) were used to process the DNA sequences. The DNA and protein sequences were aligned using the GCUST CLUSTALW module. Sequence alignments were carried out using the GENEDOC program (version 2.1).

Example 10 Construction of expression plasmids for an HuMab anti-hIL-13Rα1 IgG1 antibody

The genes encoding the light and heavy chains of the HuMab anti-hIL-13R1 were assembled separately into expression vectors for mammalian cells. Next the segments coding for the variable region of the light chain (VL) of the HuMab anti-hIL-13R1 and for the constant region κ of the chain (CL, SEQ ID NO: 11) were joined in the same way as was done for the segments for the variable region of the human heavy chain (VH) and for the γ1 constant region of the human heavy chain of the HuMab anti-hIL-13R 1 (CH1bisagra-CH2-CH3, SEQ ID NO: 12). General information regarding the nucleotide sequences of heavy and light human chains, and their codon use is published by: Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman,

K. S., and Foeller, C., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 91-3242.

The κ light chain transcription unit of the HuMab anti-hIL-13R1 is composed of the following 5 elements:

•

The immediate and early enhancer and the human cytomegalovirus (HCMV) promoter,

•

A synthetic 5’-UT including a Kozak sequence,

• A signal sequence for the mouse immunoglobulin heavy chain including the intron of the signal sequence,

10 • The cloned cDNA for the variable part of the HuMab anti-hIL-13R1 light chain, arranged with a single restriction target for BsmI at its 5 'end, a donor place for splicing and a single splice restriction target for NotI at the 3 'end,

• The gene for the human κ constant region, including the mouse 2 intronic enhancer for its Ig-κ (Picard, D., and Schaffner, W., Nature 307 (1984) 80-82) and 15 • The signal sequence for polyadenylation ("poly-A") of the κ region of human immunoglobulin.

The γ1 heavy chain transcription unit of the HuMab anti-hIL-13R1 is composed of the following elements:

•

The immediate and early enhancer and the human cytomegalovirus (HCMV) promoter,

•

A synthetic 5’-UT including a Kozak sequence,

20 • A signal sequence for the mouse immunoglobulin heavy chain including the intron of the signal sequence,

• The cloned cDNA for the variable part of the heavy chain of the HuMab anti-hIL-13R1, arranged with a single restriction target for BsmI at its 5 'end, a donor site for splicing and a single restriction target for NotI on the 3 'end,

25 • The gene for the human γ1 constant region, including the mouse enhancer for its Ig-µ (Neuberger, M.S., EMBO J. 2 (1983) 1373-1378)

• The signal sequence for polyadenylation ("poly-A") of the γ1 region of human immunoglobulin.

The functional elements for expression plasmids of the κ light chain and heavy chain 1 of the 30 HuMab anti-hIL-13R1:

In addition to the expression cassette described above for the κ light chain and heavy chain 1 of the HuMab anti-hIL13R1, these plasmids contain:

• A hygromycin resistance gene 35 • An origin of replication, oriP, of the Epstein-Barr virus (EBV)

• An origin of replication from the pUC18 vector that allows plasmid replication in E.coli, and

• A ß-lactamase gene that provides resistance to ampicillin in E.coli.

Example 11 Construction and expression of plasmids for the mutant antibody (variant) anti-hIL-13Rα1 IgG1 Expression plasmids encoding heavy chains 1 mutants of the anti-hIL-13R1 antibodies can be generated by mutagenesis directed on expression plasmids wild using the kit

45 QuickChangeTM Site-Directed (Stratagene) mutagenesis and are described in Table 1. Amino acids are numbered according to EU numbering (Edelman, GM, et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78 -85; Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS, and Foeller, C., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 91-3242).

50 Table 1 Example 12

Mutation

Description

PVA-236; GLPSS331 as specified by E233P; L234V; L235A; Delta G236;

The amino acid sequence Glu233Leu234Leu235Gly236 of the human heavy chain 1 is replaced by the amino acid sequence Pro233Val234Ala235 of the human heavy chain 2.

A327G; A330S; P331S

The amino acid sequence Ala327Leu328Pro329Ala330Pro331 of the heavy chain

human 1 is replaced by the amino acid sequence Gly327Leu328Pro329Ser330Ser331 of the human heavy chain 4.

L234A; L235A

The amino acid sequence Leu234Leu235 of the human heavy chain 1 is replaced by the amino acid sequence Ala234Ala235

Production of recombinant anti-hIL-13Rα1 HuMabs

Recombinant HuMabs antibodies were generated by transient transfection of adherent cells

5 HEK293-EBNA (ATTC CRL-10852) grown in DMEM medium (Gibco) supplemented with 10% FCS with a very low IgG (Gibco) content, 2 mM Glutamine (Gibco), 1% v / v amino acids non-essential (Gibco) and 250 µg / ml of G418 (Roche Diagnostics GmbH, DE). For transfection, the Fugene ™ 6 transfection reagent (Roche Diagnostics GmbH, DE) was used in a ratio of reagent (µl) to DNA (µg) between 3: 1 and 6: 1. Heavy and light immunoglobulin chains were expressed from two different plasmids, using a

10 mole ratio of plasmids encoding heavy and light chains from 1: 2 to 2: 1. Culture supernatants containing human monoclonal antibodies (HuMab) were recovered between days 4 and 11 after transfection.

General information regarding the recombinant expression of human antibodies can be found in lines 15 such as HEK293 in: Meissner, P., et al., Biotechnol Bioeng 75 (2001) 197-203.

Example 13

a) Affinity analysis of HuMabs LC5002-003, -005, and -007 against hIL-13Rα1: chimeric hFc

For interaction analysis, the Biacore 3000 was used as an instrument. As a career and

20 HBS-P reaction (10mM HEPES, 150mM NaCl, 0.005% P polysurfactant, pH 7.4) was used at 25 ° C. The capture molecules (goat antibodies against human IgG, specific for Fc) were linked by their amine at a concentration of 20 µg / ml with a flow of 5 µl / min for 20 minutes. The HuMabs were injected at a concentration of 1 µg / ml with a flow of 10 µl / min for 1 minute. The blocking of goat antibodies against human IgG-F, Fcγ that were free was done by injecting 500nM human gammaglobulin at a flow of

25 30µl / min for 3 minutes. The analyte (the hIL-13R1 chimeric protein: hFc) was injected for two minutes at five different concentrations, between 5.63nM and 90nM, washing with the HBS-P buffer for five minutes. Surface regeneration was obtained by two injections of 100mM HCl HCl for 1 minute. The chip, test format and sequence of injections, as well as the kinetic data are those described in the table

2. For the correction of the intrinsic drift of the system baseline the negative control data (curves with the

30 buffer) were subtracted from the curves generated by the samples. For the analysis of the sensograms and the calculation of the affinity data, the software BiaEvaluation version 4.01 was used. The kinetics data were obtained by adjusting the data to a Langmuir 1: 1 binding model (Table 2).

Table 2:

35 Analysis of HuMab affinities on hIL-13Rα1: hFc. Data analysis based on a Langmuir 1: 1 union model

Chip

Screenshot Flirting Analyte ka (1 / Ms) kd (1 / s) KD (M)

CM5

AntihFcγ LC5002003 hIL-13 R1: hFc 2.1x105 1.4x10-6 <6.5x10-12

CM5

AntihFcγ LC5002005 hIL-13 R1: hFc 1.73x105 3.12x10-6 1.8x10-11

CM5

AntihFcγ LC5002007 hIL-13 R1: hFc 1.19x105 1x10-6 <8.4x10-12

b) Affinity analysis of the HuMabs LC5002-003, -005 and -007 on the extracellular domain of hIL-13Rα1

40 obtained from the chimeric protein hIL-13Rα1: hFc For interaction analysis, the Biacore 3000 was used as an instrument. HBS-P (10 mM HEPES, 150 mM NaCl, polysurfactant P al. 0.005%, pH 7.4) at 25 ° C. The capture molecules (anti-hFc) were linked by their amine at a concentration of 100 µg / ml with a flow of 5 µl / min for 20 minutes. The HuMabs were injected at a concentration of 10 µg / ml with a flow of 10 µl / min

45 for 30 seconds. Processed hIL-13R1 molecules (MW40kDa) were injected for 200 seconds at seven different concentrations, between 1.5mm and 100nM, washing with the HBS-P buffer for five minutes. The surface regeneration was obtained by two injections of 100 mM HCl HCl for 1 minute each with a flow of 10 µl / min. The chip, test format and the sequence of injections, as well as the kinetic data are those described in Table 3. The kinetic data were obtained by adjusting the data to a model.

50 of 1: 1 union of Langmuir.

Table 3:

Chip

Capture or Flirting Analyte ka (1 / Ms) kd (1 / s) KD (M)

C1

Anti-hFcγ LC5002002 hIL-13R1 1.1x106 6.5x10-4 6.2x10-10

C1

Anti-hFcγ LC5002003 hIL-13R1 1.3x106 5.1x10-4 3.9x10-10

C1

Anti-hFcγ LC5002005 hIL-13R1 1.4x106 3.0x10-4 2.2x10-10

C1

Anti-hFcγ LC5002007 hIL-13R1 1.9x105 8.3x10-4 4.4x10-9

C1

Anti-hFcγ LC5002005, mutant L234A; L235A hIL-13R1 1.4x106 2.9x10-4 2.1x10-10

c) Comparative analysis of the affinity of the recombinant variants of LC5002-005, using the extracellular domain of hIL-13Rα1 processed from the chimeric protein hIL-13Rα1: hFc.

5 In these experiments, the affinity of the original IgG1 derived from the hybridoma was compared with the affinities of the recombinant variant IgG1-Ala-Ala. For interaction analysis, Biacore 3000 was used. HBS-P (10mM HEPES, 150mM NaCl, 0.005% P polysurfactant, pH 7.4) was used as a run and reaction buffer, at 25 ° C. The capture antibody molecules (anti-hFc) were linked by their amine at a concentration of 20 µg / ml with a flow of 5 µl / min for 20 minutes. The HuMabs were injected at a concentration of 10 µg / ml with a flow

10 of 10µl / min for 1 minute. The processed hIL-13R1 molecules (analyte) were injected for 5 minutes at eight different concentrations, between 1.5M and 200nM, washing with the HBS-P buffer for five minutes. The surface regeneration was obtained by two injections of 100mM HCl HCl for 1 minute each. The chip, test format and the sequence of injections, as well as the kinetic data are those described in Table 4. The kinetic data were obtained by adjusting the data to an analyte binding model.

15 bivalent.

Table 4:

Example 14

Chip

Captured r Flirting Analyte ka1 (1 / Ms) kd1 (1 / s) ka2 (1 / RUs) kd2 (1 / s) KD (M)

CM5

Anti-hFcγ LC5002005 hIL-13 R1 1.33x105 3.6x10-4 5.8x10-3 0.06 2.7x10-9

CM5

Anti-hFcγ IgG1 wing-wing LC5002005 hIL-13 R1 1.53x105 4.2x10-4 4.1x10-3 0.04 2.8x10-9

ELISA check of the HuMab cross reaction against hIL-13Rα2 and hIL-4Rα

The chimeric proteins hIL-13R2: hFc and hIL-4R: hFc (R&D Systems, UK) were dissolved in PBS (1 µg / ml) and adsorbed to microplates (NUNC Maxisorb) by overnight incubation at 4 ° C. After washing the plates with wash buffer (TL = 0.9% NaCl; 0.1% Tween® 20) the non-specific binding sites were blocked by adding 100 µl incubation buffer (TI = PBS with 1 % protein C and 0.1% Tween® 20) incubating for 30 minutes at room temperature (TA). After this serial dilutions of the

25 HuMab and of the control antibodies (100 µl / well; dilutions in TI), these were added and incubated for 1 h at RT. The plates were washed again and bound human antibodies were detected by incubation with peroxidase-conjugated rabbit anti human kappa (DAKO, Denmark) at a final dilution of 1: 500 in TI. After incubating for 1h at RT and after a new washing step, the plates were revealed with ABTS® solution ready for use (Roche Diagnostics GmbH) at RT in the dark. The absorbance at 405nM was measured at the time

30 that the absorbance of the highest concentration reached a sufficient value.

All antibodies against IL-13Ralpha1 tested demonstrated binding to the immobilized extracellular domain of human IL-13R1, but not hIL-13R2 or hIL-4Rα (Fig. 4).

Example 15 Cross-reaction of them with murine IL-13Rα1 Murine chimeric protein IL-13R1: hFc (R&D Systems, UK) was dissolved in PBS (1 µg / ml) by incubating in

microplates for adsorption (NUNC Maxisorb) by incubation at 4 ° C overnight. After washing the plates with wash buffer (TL = 0.9% NaCl; 0.1% Tween® 20) the non-specific binding sites were blocked by adding 100 µl incubation buffer (TI = PBS with 1 % protein C and 0.1% Tween® 20) and incubated for 30 minutes at room temperature (TA). After this, serial dilutions of the 5 HuMab and control antibodies (HuMab anti-KLH and goat polyclonal anti-hIL-13Rα1 (R&D Systems)) were made, added to the wells (100 µl / well; dilutions in TI), these were added and incubated for 1h at RT. The plates were washed again and bound human antibodies were detected by incubation with peroxidase-conjugated rabbit anti human kappa (DAKO, Denmark) at a final dilution of 1: 500 in TI. Goat anti-hIL-13R1 polyclonal antibodies were detected with a goat anti-IgG polyclonal produced in

10 donkey and peroxidase conjugate (Santa Cruz; dilution 1: 1000 in TI). After incubating for 1h at RT and after a new washing step, the plates were revealed with ABTS® solution ready for use (Roche Diagnostics GmbH) at RT in the dark. The absorbance at 405nM was measured at the moment when the absorbance of the highest concentration reached a sufficient value (Fig. 5).

Example 16 Cross-reaction of HuMab against Cynomolgus IL-13Rα1 The gene coding for IL-13R1 was isolated by RT-PCR from Cynomolgus tissue and was transfected into the murine Ba / F3 cell line. To check whether HuMabs antibodies had cross-reaction or not with Cynomolgus IL-13R1, both transfected and stabilized Ba / F3 cells and parental Ba / F3 were incubated against

20 to HuMab at 10 µg / ml and to control antibodies. As a positive control, a goat polyclonal anti-hIL-13R1 (R&D Systems) was used. The negative controls included were: a human myeloma IgG1 protein (Nordic) and a normal goat serum. Bound antibodies were detected by FACS analysis using an antibody against human IgG conjugated to FITC to detect HuMabs and an antibody specific for goat IgG conjugated to FITC for the detection of goat antibodies. Intensities of

Mean fluorescence (IFM) for the different antibodies tested on the transfected line Ba / F3 against those obtained with the parental line.

All HuMabs of the invention were able to bind to Cynomolgus IL-13R1 expressed by transfected Ba / F3 cells. As expected due to the great homology between the human IL-13R1 and the

30 of Cynomolgus, also the polyclonal antibody AF152 showed binding to Cynomolgus IL-13R1. The negative control antibodies showed only a marginal increase in whether IFM when tested with the transfected Ba / F3 cell line (Table 5).

Table 5:

Antibody

IFM Ba / F3 IFM Ba / F3_Cyno_IL13Rα1 Rate of increase of the MFI in the presence of CynoIL13Rα1

HuMabs

LC5002-002 3.9 83.7 79.8

LC5002-003

3.4 82.4 79.0

LC5002-005

14.8 101.5 86.7

LC5002-007

4.1 19 14.9

AF152

3.2 21.2 18.0

Controls

Normal goat IgG 3.3 5.7 2.4

Normal human IgG1 (Nordic)

3.5 10.2 6.7

Only anti-human IgG-FITC

3.3 5.5 2.2

35

Example 17

Binding of HuMab anti-IL-13Rα1 to Fcγ receptors (binding to FcγRIIIa in killer T cells)

To determine the ability of the antibodies of the invention to bind FcRIIIa (CD16) in killer T cells

(NK), Peripheral Blood Mononucleated Cells (PBMCs) were first isolated and incubated with 40 HuMab antibody at 20 µg / ml and with control antibodies in the presence or absence of blocking mouse antibody

for FcRIIIa (anti-CD16, clone 3G8, RDI, Flanders, NJ) at 20 µg / ml, thus checking the binding via FcRIIIa.

As negative controls were used IgG2 and human IgG4 (The Binding Site), which do not bind FcRIIIa. IgG1 and

Human IgG3 (The Binding Site) were used as positive controls for binding to FcRIIIa. Antibodies

bound to killer T cells were detected by FACS analysis using a murine human anti-CD56 antibody 45 (surface marker of NK cells), labeled with phycoerythrin (PE) (BD Biosciences Pharmingen, San

Diego, CA) in combination with a FITC-labeled goat anti-human IgG (Fc) F (ab) 2 (Protos immunoresearch, Burlingame, CA). Determining the maximum binding at 20 µg / ml (Umax: IFM ± dev. Est.) Of the analyzed HuMab.

The LC5002-005 was able to bind FcRIIIa efficiently (comparable to the control IgG1 antibody) as indicated by a Umax value (IFM) of 580.6 ± 245.8. The addition of a blocking antibody against FcRIIIa greatly reduced the binding of LC5002-005 to NK cells (Umax (IFM) of 260.4 ± 95.90) indicating a specific binding to FcRIIIa.

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SEQUENCE LIST

<110> F. Hoffmann-La Roche AG

<120> Antibodies against the alpha-1 receptor of IL-13 and their uses

<130> 22922 EP

<150> EP 05002229

<151> 2005-02-03

<160> 12

<170> PatentIn version 3.2

<210> 1

<211> 119

<212> PRT

<213> Artificial

<220>

<223> LC5002-002 VH gamma / heavy chain variable domain

<400> 1 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 15 10 15 Be Leu Arg Leu Be Cys Wing Be Gly Phe Thr Phe Asn Ile Tyr

20 25 30 22

Wing Met Asn Trp Val Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Val Ile Ser Gly Arg Gly Ile Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Wing Asp Asp Thr Wing Val Tyr Tyr Cys 85 90 95

Ala Lys Gly Ser Ser Ser Trp Thr Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115

<210> 2

<211> 107

<212> PRT

<213> Artificial

<220>

<223> LC5002-002 VL kappa chain / variable domain of the chain

<400> 2 Asp Ile Gln Met Thr Gln Be Pro Be Be Leu Be Wing Be Val Gly 15 10 15 Asp Arg Val Thr Ile Thr Cys Arg Wing Be Gln Gly Ile Be Arg Trp

20 25 30 Val Wing Trp Tyr Gln Gln Lys Pro Glu Lys Wing Pro Lys Ser Leu Ile 35 40 45 Tyr Wing Wing Be Being Leu Gln Be Gly Val Pro Be Arg Phe Be Gly

50 55 60

Be Gly Be Gly Thr Asp Phe Thr Leu Thr Ile Be Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp

85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

<210> 3

<211> 119

<212> PRT

<213> Artificial

<220>

<223> LC5002-003 VH gamma / heavy chain variable domain

<400> 3 Glu Val Gln Leu Leu Glu Ser Gly Gly Asp Leu Ile Gln Pro Gly Gly 15 10 15 Be Leu Arg Leu Be Cys Wing Be Gly Phe Thr Phe Asn Ile Tyr

20 25 30 Wing Met Asn Trp Val Arg Gln Wing Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Arg Gly Ile Thr Thr Tyr Tyr Wing Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys

85 90 95 Wing Lys Gly Ser Ser Tyr Trp Thr Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115

<210> 4

<211> 107

<212> PRT

<213> Artificial

<220>

<223> LC5002-003 VL kappa / light chain variable domain

<400> 4 Asp Ile Gln Met Thr Gln Be Pro Be Be Leu Be Ala Be Val Gly 15 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Be Gln Gly Ile Be Be Trp

20 25 30 Leu Wing Trp Tyr Gln Gln Lys Pro Glu Lys Wing Pro Lys Ser Leu Ile 35 40 45 Tyr Wing Wing Be Being Leu Gln Be Gly Val Pro Be Arg Phe Be Gly

50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp

85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

<210> 5

<211> 118

<212> PRT

<213> Artificial

<220>

<223> LC5002-005 VH gamma / heavy chain variable domain

<400> 5 Glu Val Gln Val Leu Asp Be Gly Gly Gly Leu Val Gln Pro Gly Gly 15 10 15 Be Leu Arg Leu Be Cys Thr Wing Be Gly Phe Thr Phe Arg Leu Tyr

20 25 30 Thr Met Ser Trp Val Arg Gln Thr Pro Gly Arg Gly Leu Glu Trp Val 35 40 45

Be Gly Ile Be Gly Be Gly Leu Ser Thr Tyr Phe Ala Asp Ser Val

50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys

85 90 95 Wing Lys Glu Gly Asp Trp Ile Tyr Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Ile Val Ser Ser 115

<210> 6

<211> 107

<212> PRT

<213> Artificial

<220>

<223> LC5002-005 VL kappa / light chain variable domain

<400> 6 Asp Ile Gln Met Thr Gln Be Pro Be Be Leu Be Ala Be Val Gly 15 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Be Gln Gly Ile Be Be Trp

20 25 30 Leu Wing Trp Tyr Gln Gln Lys Pro Glu Lys Wing Pro Lys Ser Leu Ile 35 40 45 Tyr Wing Wing Be Being Leu Gln Be Gly Val Pro Be Arg Phe Be Gly

50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser His Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105

<210> 7

<211> 123

<212> PRT

<213> Artificial

<220>

<223> LC5002-007 VH gamma / heavy chain variable domain

<400> 7 Gln Val Gln Leu Val Gln Be Gly Wing Glu Val Lys Lys Pro Gly Ser 15 10 15 Be Val Lys Val Be Cys Lys Val Be Gly Gly Thr Phe Be Be Tyr

20 25 30 Wing Phe Ser Trp Val Arg Gln Wing Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Arg Thr Asn Tyr Wing Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95 Wing Arg Glu Gly Glu Thr Leu Asp Tyr Phe Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120

<210> 8

<211> 107

<212> PRT

<213> Artificial

<220>

<223> LC5002-007 VL kappa / light chain variable domain

<400> 8 Glu Ile Val Leu Thr Gln Be Pro Gly Thr Leu Be Leu Be Pro Gly 15 10 15 Glu Arg Ala Thr Leu Be Cys Arg Ala Ser Gln Ser Val Be Be Be

20 25 30 Tyr Leu Wing Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Wing Being Being Arg Wing Ile Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Ser Ser Leu

85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105

<210> 9

<211> 119

<212> PRT

<213> Artificial

<220>

<223> LC5002-018 VH gamma / heavy chain variable domain

<400> 9

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

15 10 15

Be Leu Arg Leu Be Cys Wing Be Ser Gly Phe Thr Phe Asn Ile Tyr 20 25 30 Met Wing Asn Trp Val Arg Gln Wing Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Be Gly Ser Gly Val Thr Thr Tyr Tyr Wing Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys

85 90 95 Wing Lys Gly Ser Ser Trp Tyr Val Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115

<210> 10

<211> 107

<212> PRT

<213> Artificial

<220>

<223> LC5002-018 VL kappa / light chain variable domain

<400> 10 Asp Ile Gln Met Thr Gln Be Pro Be Be Leu Be Wing Be Val Gly 15 10 15 Asp Arg Val Thr Ile Thr Cys Arg Wing Be Gln Gly Ile Be Be Trp

20 25 30

Leu Wing Trp Tyr Gln Gln Lys Pro Glu Lys Wing Pro Lys Ser Leu Ile 35 40 45

Tyr Wing Wing Be Ser Leu Gln Be Gly Val Pro Be Arg Phe Be Gly 50 55 60

Be Gly Be Gly Thr Asp Phe Thr Leu Thr Ile Be Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp

85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

<210> 11

<211> 107

<212> PRT

<213> Homo sapiens

<400> 11 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 15 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

<210> 12

<211> 330

<212> PRT

<213> Homo sapiens

<400> 12 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Be Gly Leu Tyr Ser

50 55 60

Leu Be Be Val Val Thr Val Pro Be Be Be Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Wing Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 5 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 10 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 15 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 20 305 310 315 320 Gln Lys Ser Leu Be Leu Be Pro Gly Lys 325 330

Claims (14)

one.

An antibody that binds to IL-13R1 and inhibits IL-13, characterized in that the amino acid sequence of the variable part of the CDR3 heavy chain of said antibody is selected from the group of sequences of the CDR3 heavy chain composed of the Sequences with ID No.: 1, 3, 5 or 7 whereby said antibody is obtained from hybridoma cell lines DSM ACC2709, DSM ACC2710, DSM ACC2711 or DSM ACC2712.

2.

An antibody that binds to IL-13Rα1 and that inhibits IL-13, characterized in that the CDR3 variable heavy chain amino acid sequence of said antibody is chosen from the group consisting of heavy chain CDR3 sequences with SEQ ID NO: 1 , 3, 5, 7 or 9 and includes as a variable region of the heavy chain to the region with SEQ ID NO: 1 and as a variable region of the light chain to that of SEC NO: 2, as a variable region of the heavy chain to the region with SEQ ID NO: 3 and as a variable region of the light chain to that of SEC NO: 4, as a variable region of the heavy chain to the region with SEQ ID NO: 5 and as a variable region of the light chain to that of SEQ ID NO: 6, as a variable region of the heavy chain to the region with SEQ ID NO: 7 and as a variable region of the light chain to that of SEQ ID NO: 8 or as a variable region from the heavy chain to the region with SEQ ID NO: 9 and as a variable region from the light chain to that of SEQ ID NO: 10.

3.

An antibody according to claim 2, characterized in that it comprises

a) as a variable region of the heavy chain the SEQ ID NO: 1, as a variable region of the light chain the SEQ ID NO: 5, as a constant region of the light chain κ the SEQ ID NO: 11 and as a region heavy chain constant γ1 SEQ ID NO: 12, b) as a variable region of the heavy chain the SEQ ID NO: 3, as a variable region of the light chain the SEC ID NO: 4, as a constant region of the light chain κ the SEQ ID NO: 11 and as constant region of the heavy chain γ1 SEQ ID NO: 12, c) as a variable region of the heavy chain the SEQ ID NO: 5, as a variable region of the light chain the SEQ ID NO: 6, as a constant region of the light chain κ the SEQ ID NO: 11 and as constant region of the heavy chain γ1 the SEQ ID NO: 12, d) as a variable region of the heavy chain the SEQ ID NO: 7, as a variable region of the light chain the SEQ ID NO: 8, as a constant region of the light chain κ the SEQ ID NO: 11 and as constant region of the heavy chain γ1 SEQ ID NO: 12, e) as a variable region of the heavy chain the SEQ ID NO: 9, as a variable region of the light chain the SEQ ID NO: 10, as a constant region of the light chain κ the SEQ ID NO: 11 and as constant region of the heavy chain γ1 SEQ ID NO: 12.

Four.

An antibody according to claim 3, characterized in that it additionally comprises mutations L234A and L235A or D265A and N297A.

5.

The use of an antibody according to claims 1 to 4 for the manufacture of a pharmaceutical compound.

6.

A pharmaceutical compound that includes an antibody that is in accordance with claims 1 to 4.

7.

A recombinant host cell capable of producing a recombinant antibody according to claims 1 to 4.

8.

A method for the production of a pharmaceutical compound that includes antibody that is in accordance with claims 1 to 4.

9.

A nucleic acid encoding an antibody that binds to IL-13Rα1 and that inhibits IL-13 which comprises as a region of the heavy chain variable to the region with SEQ ID NO: 1 and as a variable region of the light chain to the of No. of SEC: 2, as a variable region of the heavy chain to the region with No. of SEQ ID: 3 and as a variable region of the light chain to that of No. of SEC: 4, as a variable region of the heavy chain to the region with SEQ ID NO: 5 and as a light chain variable region to that of SEQ ID NO: 6, as a variable region from the heavy chain to the region with SEQ ID NO: 7 and as a variable region of the chain light to that of SEQ ID NO: 8 or as a variable region of the heavy chain to the region with SEQ ID NO: 9 and as a variable region of the light chain to that of SEQ ID NO: 10.

10.

An expression vector that includes a nucleic acid according to claim 9, capable of expressing said nucleic acid in a eukaryotic or prokaryotic host cell.

eleven.

A prokaryotic or eukaryotic host cell including the vector according to claim 10.

12.

A method for the production of a polypeptide capable of binding to IL-13Rα1 and inhibiting the binding of IL

13 to IL-13Rα1, characterized by expressing a nucleic acid encoding a heavy chain and a nucleic acid coding for a light chain according to claim 9 in a prokaryotic or eukaryotic host cell with the recovery of said polypeptide from the cited cell. 13. Antibody according to claims 1 to 4 for use in the treatment of a patient requiring asthma or antiallergic therapy. A method for the preparation of the pharmaceutical compound characterized by producing an antibody according to claims 1 to 4 by recombinant expression, recovery of said antibody and combination of said antibody with a pharmaceutically acceptable buffer and / or adjuvant. 15. The DSM ACC2709, DSM ACC2710, DSM ACC2711 or DSM ACC2712 hybridoma cell lines. fifteen