WO2013103783A1 - Murine il-13 antibodies - Google Patents

Abstract

The invention provides antibodies and fragments thereof that specifically bind human IL-13. The invention also provides methods for treating or ameliorating an IL-13 mediated, Th2-mediated, or allergic disease, disorder, or condition. In one aspect, the invention is an isolated antibody or fragment thereof that specifically binds human IL-13, wherein the antibody or fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 1; a heavy chain variable region comprising SEQ ID NO: 9; a heavy chain variable region comprising SEQ ID NO: 17; or a heavy chain variable region comprising SEQ ID NO: 25. In another aspect, the invention is an isolated antibody or fragment thereof that specifically binds human IL-13, wherein the antibody or fragment thereof comprises: a light chain variable region comprising SEQ ID NO: 2; a light chain variable region comprising SEQ ID NO: 10; a light chain variable region comprising SEQ ID NO: 18; or a light chain variable region comprising SEQ ID NO: 26.

Description

MURINE IL-13 ANTIBODIES

CROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 61/583, 161 filed January 4, 2012, incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to antibodies and fragments thereof that specifically bind human IL-13. The invention also relates to the use of antibodies and fragments thereof that specifically bind human IL-13 for the treatment, amelioration, or prevention of an IL-13 mediated, Th2-mediated, or allergic disease, disorder, or condition.

BACKGROUND OF THE INVENTION

[0003] IL-13 is a (Minty et al, 1993, Nature 362: 248-50; McKenzie et al, 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 3735-39) cytokine of 112 amino acids secreted by the activated T lymphocytes, the B lymphocytes and the mastocytes after activation. By virtue of its numerous biological properties shared with IL-4, IL-13 has been described as an IL-4-like cytokine. Its activities are indeed similar to those of IL-4 on B cells (Defrance et al, 1994, J. Exp. Med. 179: 135-43; Punnonen et al, 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 3730-34; Fior et al, 1994, Eur. Cytokine Network 5: 593-600), the monocytes (Muzio et al, 1994, Blood 83: 1738-43,;De Waal Malefytef al, 1993, J. Immunol. 151 : 6370-81 ; Doyle et al, 1994, Eur. J. Immunol. 24: 1441-45; Montaner et al, 1993, J. Exp. Med. 178: 743-47;

Sozzani et al, 1995, J. Biol. Chem. 270: 5084-88) and other non-haematopoietic cells (Herbert et al, 1993, Febs Lett. 328: 268-70; Derocq et al, 1994, Febs Lett. 343 : 32-36). On the other hand, contrary to IL-4, it does not exert a specific effect on resting or activated T cells (Zurawuki et al, 1994, Immunol. Today 15: 19-26).

[0004] Various biological activities of IL-13 on the monocytes/macrophages, the B lymphocytes and certain haematopoietic are well known in the art. Several data indicate, in addition, that this cytokine has a pleiotropic effect on other cell types. The non- haematopoietic cells that are directly affected by IL-13 are endothelial and microglial cells, keratinocytes and kidney and colon carcinomas.

[0005] The principle components of receptors and receptor complexes that bind IL-13 are IL-13Ral and IL-13Ra2 (see Figure 1). These chains are expressed on the surface of cells as monomers or heterodimers of IL-13Rod. IL-13Ral and IL-13Ra2 monomers bind IL-13, but do not bind IL-4. (Murata, 1999, Int. J. Hematol. 69: 13-20).

[0006] Th2-type immune responses promote antibody production and humoral immunity, and are activated to fight off extracellular pathogens. Th2 cells are mediators of Ig production (humoral immunity) and produce IL-4, IL-5, IL-6, IL- 9, IL-10, and IL-13 (Tanaka et, ah, 1996, Cytokine Regulation of Humoral Immunity 251- 72 (Snapper, ed., John Wiley and Sons, New York). Th2-type immune responses are characterized by the generation of certain cytokines (e.g., IL-13) and specific types of antibodies (IgE, IgG4) and are typical of allergic reactions, which may result in watery eyes and asthmatic symptoms, such as airway inflammation and contraction of airway muscle cells in the lungs.

[0007] IL-13 is a therapeutically important cytokine that plays a critical role in many diseases, including asthma (Ngoc et al, 2005, Curr. Opin. Allergy Clin. Immunol. 5: 161-66). IL-13 has shown the potential to enhance anti-tumor immune responses. Because the cytokine is involved in the pathogenesis of allergic diseases, inhibitors of these cytokines could provide therapeutic benefits. Accordingly, a need exists for improved agents that inhibit IL-13.

SUMMARY OF THE INVENTION

[0008] In one aspect, the invention is an isolated antibody or fragment thereof that specifically binds human IL-13, wherein the antibody or fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 1 ; a heavy chain variable region comprising SEQ ID NO: 9; a heavy chain variable region comprising SEQ ID NO: 17; or a heavy chain variable region comprising SEQ ID NO: 25.

[0009] In another aspect, the invention is an isolated antibody or fragment thereof that specifically binds human IL-13, wherein the antibody or fragment thereof comprises: a light chain variable region comprising SEQ ID NO: 2; a light chain variable region comprising SEQ ID NO: 10; a light chain variable region comprising SEQ ID NO: 18; or a light chain variable region comprising SEQ ID NO: 26.

[0010] In an additional aspect, the invention is an isolated antibody or fragment thereof that specifically binds human IL-13, wherein the antibody or fragment thereof comprise a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 2; a heavy chain variable region comprising SEQ ID NO: 9, and a light chain variable region comprising SEQ ID NO: 10; a heavy chain variable region comprising SEQ ID NO: 17, and a light chain variable region comprising SEQ ID NO: 18; or a heavy chain variable region comprising SEQ ID NO: 25, and a light chain variable region comprising SEQ ID NO: 26.

[0011] In another aspect, the invention is an isolated antibody or fragment thereof that specifically binds human IL-13, wherein the antibody or fragment thereof comprises: a heavy chain variable region comprising CDR1 (SEQ ID NO: 3), CDR2 (SEQ ID NO: 4), and CDR3 (SEQ ID NO: 5) of SEQ ID NO: 1, and a light chain variable region comprising CDR1 (SEQ ID NO: 6), CDR2 (SEQ ID NO: 7), and CDR3 (SEQ ID NO: 8) of SEQ ID 2; a heavy chain variable region comprising CDR1 (SEQ ID NO: 1 1), CDR2 (SEQ ID NO: 12), and CDR3 (SEQ ID NO: 13) of SEQ ID NO: 9, and a light chain variable region comprising CDR1 (SEQ ID NO: 14), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 8) of SEQ ID 16; a heavy chain variable region comprising CDR1 (SEQ ID NO: 19), CDR2 (SEQ ID NO: 20), and CDR3 (SEQ ID NO: 21) of SEQ ID NO: 17, and a light chain variable region comprising CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 23), and CDR3 (SEQ ID NO: 24) of SEQ ID 18; or (d) a heavy chain variable region comprising CDR1 (SEQ ID NO: 27), CDR2 (SEQ ID NO: 28), and CDR3 (SEQ ID NO: 29) of SEQ ID NO: 25, and a light chain variable region comprising CDR1 (SEQ ID NO: 30), CDR2 (SEQ ID NO: 31), and CDR3 (SEQ ID NO: 32) of SEQ ID 26.

[0012] In one aspect, the invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the above-described antibodies or fragments thereof.

[0013] In one aspect, the invention is an isolated nucleic acid molecule that encodes: a heavy chain variable region comprising SEQ ID NO: 1 ; a heavy chain variable region comprising SEQ ID NO: 9; a heavy chain variable region comprising SEQ ID NO: 17; or a heavy chain variable region comprising SEQ ID NO: 25.

[0014] In another aspect, the invention is an isolated nucleic acid molecule that encodes: a light chain variable region comprising SEQ ID NO: 2; a light chain variable region comprising SEQ ID NO: 10; a light chain variable region comprising SEQ ID NO: 18; or a light chain variable region comprising SEQ ID NO: 26.

[0015] In an additional aspect, the invention is a vector comprising either of the above-described nucleic acid molecules. [0016] In another aspect, the invention is an isolated host cell comprising either of the above-described nucleic acid molecules.

[0017] In yet another aspect, the invention is an isolated host cell comprising the above-described vector.

[0018] In a further aspect, the invention is a method for treating or ameliorating an IL-13 mediated, Th2-mediated, or allergic disease, disorder, or condition, comprising administering to a patient in need thereof a pharmaceutically effective amount of any of the above-described antibodies or fragments thereof.

[0019] In one aspect, the invention is the use of a composition comprising any of the above-described antibodies or fragments thereof for the manufacture of a medicament for treating or ameliorating an IL-13 mediated, Th2-mediated, or allergic disease, disorder, or condition.

[0020] In another aspect, the invention is a composition comprising any of the above-described antibodies or fragments thereof for the treatment or amelioration of an IL-13 mediated, Th2-mediated, or allergic disease, disorder, or condition.

[0021 ] Additional features and advantages are described herein, and will be apparent from the following Detailed Description and the Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows a schematic of the JAK/STAT signal transduction pathway mediated by binding of IL-4 or IL-13 to IL-4Ra/IL-13Ral receptor.

[0023] Figure 2 shows FACS analysis of A105. CI 1-10, A101, F232, and mlgG binding to A375 human epithelial cells, which express the high affinity IL-13 receptor, IL-

13Ra2.

[0024] Figure 3 shows IC₅₀ values of (a) A105; (b) CI 1 ; (c) A105; and (d) F232 for inhibition of IL-13 induced reporter gene expression in HEK-Blue™ IL-4/IL-13 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The section headings are used herein for organizational purposes only, and are not to be construed as in any way limiting the subject matter described. The present invention is not limited to the particular methodology, protocols, cell lines, vectors, or reagents described herein because they may vary without departing from the spirit and scope of the invention. [0026] Standard techniques may be used for recombinant DNA molecule, protein, and antibody production, as well as for tissue culture and cell transformation.

Enzymatic reactions and purification techniques are typically performed according to the manufacturer's specifications or as commonly accomplished in the art using conventional procedures known in the art, or as described herein. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0027] Further, the terminology used herein is for the purpose of

exemplifying particular embodiments only and is not intended to limit the scope of the present invention. Any method and material similar or equivalent to those described herein can be used in the practice of the present invention and only exemplary methods, devices, and materials are described herein.

[0028] All patents and publications mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing the proteins, enzymes, vectors, host cells, and methodologies reported therein that might be used with and in the present invention. However, nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

1. Definitions

[0029] As utilized in accordance with the present disclosure, the following terms unless otherwise indicated, shall be understood to have the following meanings:

[0030] "Interleukin-13" (IL-13) refers to naturally occurring or endogenous mammalian IL-13 proteins and to proteins having an amino acid sequence which is the same as that of a naturally occurring or endogenous corresponding mammalian IL-13 protein (e.g., recombinant proteins, synthetic proteins, i.e., produced using the methods of synthetic organic chemistry). Accordingly, as defined herein, the term includes mature IL-13 protein, polymorphic or allelic variants, and other isoforms of IL-13 (e.g., produced by alternative splicing or other cellular processes), and modified or unmodified forms of the foregoing (e.g., lipidated, glycosylated). Naturally occurring or endogenous IL-13 includes wild type proteins such as mature IL-13, polymorphic or allelic variants, and other isoforms and mutant forms that occur naturally in mammals (e.g., humans, non- human primates). Such proteins can be recovered or isolated from a source that naturally produces IL-13, for example. These proteins and proteins having the same amino acid sequence as a naturally occurring or endogenous IL-13 are referred to by the name of the corresponding mammal. For example, where the corresponding mammal is a human, the protein is designated as a human IL-13. Several mutant IL-13 proteins are known in the art, such as those disclosed in International Publication No. WO 03/035847.

[0031] The term "antibody" is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, antibody fragments, or synthetic polypeptides carrying one or more CDR or CDR- derived sequences so long as the polypeptides exhibit the desired biological activity.

Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. Generally, antibodies are considered Igs with a defined or recognized specificity. Thus, while antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. The antibodies of the invention can be of any class (e.g., IgG, IgE, IgM, IgD, IgA and so on), or subclass (e.g., IgGi, IgG₂, IgG_2a, IgG₃, IgG₄, IgAi, IgA₂ and so on) ("type" and "class," and "subtype" and "subclass," are used interchangeably herein). Native or wildtype (obtained from a non-artificially manipulated member of a population) antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (V_H) followed by a number of constant domains. Each light chain has a variable domain at one end (V_L) and a constant domain at the other end. The term "non-artificially manipulated" means not treated to contain or express a foreign antigen binding molecule. Wildtype can refer to the most prevalent allele or species found in a population or to the antibody obtained from a non-manipulated animal, as compared to an allele or polymorphism, or a variant or derivative obtained by a form of manipulation, such as mutagenesis, use of recombinant methods and so on to change an amino acid of the antigen-binding molecule.

[0032] As used herein, "anti-IL-13 antibody" means an antibody or polypeptide derived therefrom (a derivative) which binds specifically to IL- 13 as defined herein, including, but not limited to, molecules which inhibit or substantially reduce the binding of IL-13 to its receptor or inhibit IL-13 activity. [0033] "Agonist" refers to a compound, including a protein, a polypeptide, a peptide, an antibody, an antibody fragment, a conjugate, a large molecule, or a small molecule that activates one or more biological activities of IL-13. Agonists may interact with the binding of a receptor to a ligand and vice versa, by acting as a mitogen of cells activated by a ligand, and/or by interfering with cell inactivation or signal transduction inhibition after ligand binding to a receptor. All such points of intervention by an agonist shall be considered equivalent for purposes of the invention.

[0034] "Antagonist" refers to a molecule capable of inhibiting one or more biological activities of a target molecule, such as signaling by IL-13. Antagonists may interfere with the binding of a receptor to a ligand and vice versa, by incapacitating or killing cells activated by a ligand, and/or by interfering with receptor or ligand activation (e.g., tyrosine kinase activation) or signal transduction after ligand binding to a receptor. The antagonist may completely block receptor-ligand interactions or may substantially reduce such interactions.

[0035] The term "allergic disease" refers to a pathological condition in which a patient is hypersensitized to and mounts an immunologic reaction against a substance that is normally nonimmunogenic. Allergic disease is generally characterized by activation of mast cells by IgE resulting in an inflammatory response (e.g.. local response, systemic response) that can result in symptoms as benign as a runny nose, to life-threatening anaphylactic shock and death. Examples of allergic disease include, but are not limited to, allergic rhinitis (e.g., hay fever), asthma (e.g., allergic asthma), allergic dermatitis (e.g., eczema), contact dermatitis, food allergy and urticaria (hives).

[0036] The term "antigen" as used herein refers to a molecule or a portion of a molecule capable of being bound by the antibodies of the invention. An antigen can have one or more than one epitope. Examples of antigens recognized by the antibodies of the invention include, but are not limited to, serum proteins, e.g., cytokines such as IL-13, bioactive peptides, cell surface molecules, e.g., receptors, transporters, ion-channels, viral and bacterial proteins.

[0037] The term "antigen binding domain," "antigen binding site," or "antigen binding region" refers to that portion of the antibody molecule which contains the specific amino acid residues (or other moieties) that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen. In an antibody, the antigen-binding domain is commonly referred to as the "complementarity-determining region" (or "CDR"). Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is called an epitope. An antigen binding domain may be provided by one or more antibody variable domains. In certain aspects, an antigen binding domain is made of the association of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0038] The term "epitope" refers to that portion of any molecule capable of being recognized by and bound by an antibody, at one or more of the binding agent's antigen binding regions. Epitopes usually consist of chemically active surface groupings of molecules, such as for example, amino acids or carbohydrate side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics.

Epitopes as used herein may be contiguous or non-contiguous. Moreover, epitopes may be mimetic in that they comprise a three-dimensional structure that is identical to the epitope used to generate the antibody, yet comprise none or only some of the amino acid residues found in the IL-13 used to stimulate the antibody immune response.

[0039] The term "antibody fragment" refers to a portion of an intact or a full- length chain or an antibody, generally the target binding or variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab¾ and Fv fragments. A "functional fragment" or "analog of an anti-IL-13 antibody" is one that can prevent or substantially reduce the ability of the receptor to bind to a ligand or to initiate signaling. As used herein, functional fragment generally is synonymous with "antibody fragment," and with respect to antibodies, can refer to fragments such as Fv, Fab, F(ab')2, and so on which can prevent or substantially reduce the ability of the receptor to bind to a ligand or to initiate signaling. An "Fv" fragment consists of a dimer of one heavy and one light chain variable domain in a non-covalent association (VH-VL dimer). In that configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer, as in an intact antibody. Collectively, the six CDRs confer target binding specificity on the intact antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) can have the ability to recognize and to bind target. The Fab fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the CHI domain to include one or more cysteines from the antibody hinge region. Fab' fragments can be produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab¾ pepsin digestion product. Additional enzymatic and chemical treatments of antibodies can yield other functional fragments of interest.

[0040] The term "autoimmune disease" as used herein refers to a non- malignant disease or disorder arising from and directed against an individual's own tissues. Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses such as inflammatory skin diseases including psoriasis and dermatitis; allergic conditions such as eczema and asthma; other conditions involving infiltration of T cells and chronic inflammatory responses; atherosclerosis; diabetes mellitus (e.g., Type I diabetes mellitus or insulin dependent diabetes mellitis); multiple sclerosis and central nervous system (CNS) inflammatory disorder.

[0041] The term "biologically active" when used in relation to IL-13 or an IL-

13 antibody refers to a peptide or polypeptide having at least one activity characteristic of IL- 13 or of an IL-13 antibody. An IL-13 antibody may have agonist, antagonist, or neutralizing or blocking activity with respect to at least one biological activity of IL-13.

[0042] The term "cancer" as used herein refers to or describes the physiological condition in mammals, in particular humans, which is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

[0043] The term, "carrier," refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered. Such physiological carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a suitable carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, depots, and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Such compositions will contain an effective amount of the antibody, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. As known in the art, the formulation will be constructed to suit the mode of administration.

[0044] The terms "cell," "cell line," and "cell culture" include progeny thereof.

It is also understood that all progeny may not be precisely identical, such as in DNA content, due to deliberate or inadvertent mutation. Variant progeny that have the same function or biological property of interest, as screened for in the original cell, are included.

[0045] The term "CDR grafted antibody" refers to an antibody in which the

CDR from one antibody of a particular species or isotype is recombinantly inserted into the framework of another antibody of the same or different species or isotype.

[0046] The term "chimeric antibodies" refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass (type or subtype), with the remainder of the chains identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity of binding to IL-13 or impacting IL-13 activity or metabolism. Monoclonal antibodies herein specifically include chimeric antibodies. Thus, CDRs from one class of antibody can be grafted onto the framework (FR) regions or sequences of an antibody of different class or subclass.

[0047] The invention thus includes administration of one or more antibodies of the invention administered to the same patient in combination with one or more additionally suitable agents, each being administered according to a regimen suitable for that medicament. This includes concurrent administration of an antibody of the invention and one or more suitable agents. As used herein, the terms "concurrently administered" and "concurrent administration" encompass substantially simultaneous administration of one or more antibodies according to the invention and one or more additionally suitable agents.

[0048] "Derivatives" include those antibodies that have been chemically modified in some manner distinct from insertion, deletion, or substitution variants.

[0049] The term "disorder" as used herein refers to any condition that would benefit from treatment with the antibody of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal, and in particular humans, to the disorder in question. Non-limiting examples of disorders to be treated herein include cancers, inflammation, autoimmune diseases, infections, cardiovascular diseases, respiratory diseases, neurological diseases, and metabolic diseases.

[0050] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to the amount of a therapy (e.g., a prophylactic or therapeutic agent), which is sufficient to reduce the severity and/or duration of an IL-13 mediated disease, ameliorate one or more symptoms thereof, prevent the advancement of an IL-13 mediated disease or cause regression of an IL-13 mediated disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of an IL-13 mediated disease or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effects of another therapy (e.g., another therapeutic agent) useful for treating an IL-13 mediated disease.

[0051] The term "expression vector" refers to a plasmid, phage, virus, or vector, for expressing a polypeptide from a DNA (RNA) sequence. An expression vector can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters, or enhancers, (2) a structural or sequence that encodes the binding agent which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.

Alternatively, where a recombinant antibody is expressed without a leader or transport sequence, it may include an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final antibody product.

[0052] The term "fully human" antibody refers to an antibody in which both the CDR and the framework are derived from one or more human DNA molecules.

[0053] Also included within the scope of the invention are functional equivalents of an antibody of interest. The term "functional equivalents" includes antibodies with homologous sequences, antibody homologs, chimeric antibodies, artificial antibodies and modified antibodies, for example, wherein each functional equivalent is defined by the ability to bind to IL-13, inhibiting IL-13 signaling ability or function, or inhibiting binding of IL- 13 to its receptor. The skilled artisan will understand that there is an overlap in the group of molecules termed "antibody fragments" and the group termed "functional equivalents." Methods of producing functional equivalents that retain IL-13 binding ability are known to the person skilled in the art.

[0054] The terms "functional fragment," "variant," "derivative," "analog," and the like, as well as forms thereof, of an antibody or antigen refer to a compound or molecule having qualitative biological activity in common with a full-length antibody or antigen of interest. For example, a functional fragment or analog of an anti-IL-13 antibody is one that can bind to an IL-13 molecule or one which can prevent or substantially reduce the ability of a ligand, or an agonistic or antagonistic antibody, to bind to IL-13.

[0055] The term "heavy chain" when used in reference to an antibody collectively refers to five distinct types, called alpha, delta, epsilon, gamma, and mu, based on the amino acid sequence of the heavy chain constant domain. The combination of heavy and light chains give rise to five known classes of antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, including four known subclasses of IgG, designated as IgGi, IgG₂, IgG₃, and IgG₄.

[0056] The term "hinge" or "hinge region" as used herein refers to the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody.

[0057] "Antibody homolog" or "homolog" refers to any molecule which specifically binds IL-13 as taught herein. Thus, an antibody homolog includes native or recombinant antibody, whether modified or not, portions of antibodies that retain the biological properties of interest, such as binding IL-13, such as an Fab or Fv molecule, a single chain antibody, a polypeptide carrying one or more CDR regions and so on. The amino acid sequence of the homolog need not be identical to that of the naturally occurring antibody but can be altered or modified to carry substitute amino acids, inserted amino acids, deleted amino acids, amino acids other than the twenty normally found in proteins, and so on to obtain a polypeptide with enhanced or other beneficial properties.

[0058] The terms "humanized antibody" or "chimerized antibody" refer to a specific type of CDR-grafted antibody in which the antibody framework region is derived from a human but each CDR is replaced with that derived from another species, such as a murine CDR. The non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Accordingly, such "humanized" antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. [0059] The terms "identity" or "homology" refer to the percentage of nucleotide bases or amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N-terminal or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are available and well known in the art. Sequence identity may be measured using sequence analysis software.

[0060] The term "inhibiting and/or neutralizing epitope" is an epitope which when bound by an antibody, results in the loss of (or at least the decrease in) biological activity of the molecule, cell, or organism containing such epitope, in vivo, in vitro, or in situ. In the context of the present invention, the neutralizing epitope is located on or is associated with a biologically active region of IL-13. Alternatively, the term "activating epitope" is an epitope, which when bound by an antibody of the invention, such as an antibody, results in activation, or at least maintenance of a biologically active conformation, of IL-13.

[0061] The term "isolated" when used in relation to IL-13 or to an IL-13 antibody refers to a compound that is free from at least one contaminating polypeptide or compound that is found in its natural environment, and preferably substantially free from any other contaminating mammalian polypeptides that would interfere with its therapeutic or diagnostic use.

[0062] An "isolated" or "purified" antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source or medium from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. Generally, "purified" will refer to an antibody composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. The language "substantially free of cellular material" includes preparations of an antibody in which the polypeptide is separated from cellular components of the cells from which antibody is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of the antibody having less than about 30%, 20%, 10%, 5%, 2.5%, or 1% (by dry weight) of contaminating protein. Where the term "substantially purified" is used, this designation will refer to an antibody composition in which the antibody forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins in the composition.

[0063] When the antibody is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 5%, 2.5%, or 1% of the volume of the protein preparation. When antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals and reagents, i.e., the antibody of interest is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. Accordingly, such preparations of the antibody have less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or compounds other than antibody of interest. In a preferred embodiment of the invention, antibodies are isolated or purified.

[0064] The antibodies of interest can be screened or can be used in an assay as described herein or as known in the art. Often, such assays require a reagent to be detectable, which is, for example, labeled. The word "label" when used herein refers to a detectable compound or composition that can be conjugated directly or indirectly to a molecule or protein, e.g., an antibody. The label may itself be detectable (e.g., radioisotope labels, particles, or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0065] The term "light chain" when used in reference to an antibody collectively refers to two distinct types, called kappa (k) or lambda (1) based on the amino acid sequence of the constant domains.

[0066] The term "linear Fab" refers to a tetravalent antibody. The "linear Fab" is composed of a tandem of the same CHI-VH domain, paired with the identical light chain at each CHI-VH position.

[0067] The term "linker" as used herein refers to a peptide adapted to connect the variable domains of the antibody constructs of the invention. The peptide linker may contain any amino acids, the amino acids glycine (G) and serine (S) being preferred. The linkers may be equal or differ from each other between and within the heavy chain polypeptide and the light chain polypeptide. Furthermore, the linker may have a length of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. A preferred peptide linker unit for the heavy chain domains as for the light chain domains is GGGGS. The numbers of linker units of the heavy chain and of the light chain may be equal

(symmetrical order) or differ from each other (asymmetrical order). A peptide linker is preferably long enough to provide an adequate degree of flexibility to prevent the antibody moieties from interfering with each other's activity, for example by steric hindrance, to allow for proper protein folding and, if necessary, to allow the antibody molecules to interact with two or more, possibly widely spaced, receptors on the same cell; yet it is preferably short enough to allow the antibody moieties to remain stable in the cell.

[0068] As used herein, the phrase "low to undetectable levels of aggregation" refers to samples containing no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, and often no more than 0.5% aggregation, by weight protein, as measured by, for example, high performance size exclusion chromatography (HPSEC).

[0069] As used herein, the term "low to undetectable levels of fragmentation" refers to samples containing equal to or more than 80%, 85%, 90%, 95%, 98%, or 99%, of the total protein, for example, in a single peak, as determined by HPSEC, or in two (2) peaks (heavy chain and light chain) as determined by, for example, reduced capillary gel electrophoresis (rCGE), and containing no other single peaks having more than 5%, more than 4%, more than 3%, more than 2%, more than 1%, or more than 0.5% of the total protein, each. rCGE as used herein refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in an antibody or antibody-type or derived molecule.

[0070] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo, sports or pet animals, such as dogs, horses, cats, and cows.

[0071] The term "mature" when used in relation to IL-13, anti-IL-13 antibody, or to any other proteinaceous IL- 13 antibody refers to a peptide or a polypeptide lacking a leader or signal sequence. When a binding agent of the invention is expressed, for example, in a prokaryotic host cell, the "mature" peptide or polypeptide may also include additional amino acid residues (but still lack a leader sequence) such as an amino terminal methionine, or one or more methionine and lysine residues. A peptide or polypeptide produced in this manner may be utilized with or without these additional amino acid residues having been removed.

[0072] The term "monoclonal antibodies" refers to a collection of antibodies encoded by the same nucleic acid molecule, which are optionally produced by a single hybridoma or other cell line or by a transgenic mammal such that each monoclonal antibody will typically recognize the same epitope on the antigen. The term "monoclonal" is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse and rat. Monoclonal antibodies are highly specific, being directed against a single target site, epitope, or determinant. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes) of an antigen, each monoclonal antibody is directed against a single determinant on the target. In addition to their specificity, monoclonal antibodies are advantageous being synthesized by a host cell, uncontaminated by other immunoglobulins, which provides for cloning the relevant gene and mRNA encoding the antibody of chains thereof. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies for use with the present invention may be isolated from phage antibody libraries using well-known techniques or can be purified from a polyclonal preparation. The parent monoclonal antibodies to be used in accordance with the invention may be made by classic hybridoma methods or may be made by recombinant techniques.

[0073] The term "multi-specific antibody" refers to an antibody having variable regions that recognize more than one epitope on one or more antigens. A subclass of this type of antibody is a "bi-specific antibody" which recognizes two distinct epitopes on the same or different antigens.

[0074] The term "naturally occurring" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to those which are found in nature and not modified by a human being.

[0075] The term "pharmaceutical composition" as used herein refers to formulations of various preparations. The formulations containing therapeutically effective amounts of the antibodies of the invention are sterile liquid solutions, liquid suspensions, or lyophilized versions, and optionally contain stabilizers or excipients.

[0076] Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein. The term

"physiologically acceptable," "pharmacologically acceptable," and so on mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans.

[0077] "Single-chain Fv," "sFv," or "scAb" antibody fragments comprise the

VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker, often a flexible molecule, between the VH and VL domains, which enables the sFv to form the desired structure for target binding.

[0078] "Specifically binds IL-13" refers to the ability of a specific binding agent (such as an antibody or fragment thereof) of the invention to recognize and bind mature, full-length or partial-length human IL-13 polypeptide, or an ortholog thereof, such that its affinity (as determined by, e.g., affinity ELISA or BIAcore assays as described herein) or its neutralization capability (as determined by, e.g., neutralization ELISA assays described herein, or similar assays) is at least 10 times as great, but optionally 50 times as great, 100, 250, or 500 times as great, or even at least 1000 times as great as the affinity or neutralization capability of the same for any other interleukin or other peptide or polypeptide.

[0079] The phrase "substantially identical" with respect to an antibody chain polypeptide sequence may be construed as an antibody chain exhibiting at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference polypeptide sequence. The term with respect to a nucleic acid sequence may be construed as a sequence of nucleotides exhibiting at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference nucleic acid sequence.

[0080] "Substitutional" variants are those that have at least one amino acid residue in a native sequence removed and replaced with a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule is substituted, or may be multiple, where two or more amino acids are substituted in the same molecule. The plural substitutions may be at consecutive sites. Also, one amino acid can be replaced with plural residues, in which case such a variant comprises both a substitution and an insertion. "Insertional" variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native sequence. Immediately adjacent to an amino acid means connected to either the a-carboxyl or a-amino functional group of the amino acid. "Deletional" variants are those with one or more amino acids in the native amino acid sequence removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule.

[0081] As used herein "Th2-mediated disease" refers to a disease in which pathology is produced (in whole or in part) by an immune response (Th2-type immune response) that is regulated by CD4⁺ Th2 T lymphocytes, which characteristically produce IL-4, IL-5, IL-9, and IL-13. A Th2-type immune response is associated with the production of certain cytokines (e.g., IL-13) and of certain classes of antibodies (e.g., IgE), and is associated with humoral immunity. Th2-mediated diseases are characterized by the presence of elevated levels of Th2 cytokines (e.g., IL-13) and/or certain classes of antibodies (e.g., IgE) and include, for example, allergic disease (e.g., allergic rhinitis, atopic dermatitis, asthma, e.g., atopic asthma, allergic airways disease (AAD), anaphylactic shock, conjunctivitis), autoimmune disorders associated with elevated levels of IL-13 (e.g., rheumatoid arthritis, host-versus-graft disease, renal disease, e.g., nephritic syndrome, lupus nephritis), and infections associated with elevated levels of IL-13 (e.g., viral, parasitic, fungal, e.g., C. albicans, infection). Certain cancers are associated with elevated levels of IL-13 or associated with IL-13 -induced cancer cell proliferation (e.g., B cell lymphoma, T cell lymphoma, multiple myeloma, head and neck cancer, breast cancer, and ovarian cancer). These cancers can be treated, suppressed, or prevented using the antibodies of the invention.

[0082] As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to any agents which can be used in the treatment, management, or amelioration of a disease, disorder, malady, and the like associated with aberrant IL-13 metabolism and activity.

[0083] The term "treatment" as used herein refers to both therapeutic treatment and prophylactic or preventative measures. It refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing, or halting the deleterious effects of a disease state, disease progression, disease causative agent (e.g., bacteria or viruses), or other abnormal condition.

[0084] The term "variants," as used herein, include those polypeptides wherein amino acid residues are inserted into, deleted from, and/or substituted into the naturally occurring (or at least a known) amino acid sequence for the binding agent. Variants of the invention include fusion proteins as described below.

[0085] The term "variable" in the context of a variable domain of antibodies refers to certain portions of the pertinent molecule which differ extensively in sequence between and among antibodies and are used in the specific recognition and binding of a particular antibody for its particular target. However, the variability is not evenly distributed through the variable domains of antibodies. The variability is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) also known as hypervariable regions, both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework (FR) regions or sequences. The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together often in proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the target (epitope or determinant) binding site of antibodies.

[0086] As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et ah, unless otherwise indicated. One CDR can carry the ability to bind specifically to the cognate epitope.

2. Antibody Types and Components

[0087] Antibodies and antibody fragments that specifically bind IL-13 polypeptides are within the scope of the present invention. The antibodies may be polyclonal including mono-specific polyclonal, monoclonal (mAbs), recombinant, chimeric, humanized such as CDR-grafted, human, single chain, catalytic, multi-specific and/or bi-specific, as well as fragments, variants, and/or derivatives thereof.

[0088] Alternatively, transgenic animals (e.g., mice) that are capable of producing a repertoire of antibodies in the absence of endogenous immunoglobulin production can be used to generate such antibodies. This can be accomplished by immunization of the animal with an IL-13 antigen or fragments thereof where the IL-13 fragments have an amino acid sequence that is unique to IL-13. Such immunogens can be optionally conjugated to a carrier. Antibodies may also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.

[0089] The antibodies of the invention may comprise polyclonal antibodies, although because of the modification of antibodies to optimize use in human, as well as to optimize the use of the antibody per se, monoclonal antibodies are preferred because of ease of production and manipulation of particular proteins. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies directed toward an IL-13 polypeptide generally are produced in animals (e.g., rabbits, hamsters, goats, sheep, horses, pigs, rats, gerbils, guinea pigs, mice, or any other suitable mammal, as well as other non- mammal species) by means of multiple subcutaneous or intraperitoneal injections of IL-13 polypeptide or a fragment thereof with or without an adjuvant. Such adjuvants include, but are not limited to, Freund's complete and incomplete, mineral gels such as aluminum hydroxide, and surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants. It may be useful to conjugate an antigen polypeptide to a carrier protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also, aggregating agents such as alum are used to enhance the immune response. After immunization, the animals are bled and the serum is assayed for anti-IL-13 polypeptide antibody. Polyclonal antibodies may be utilized in the sera from which they were detected, or may be purified from the sera, using, for example, antigen affinity chromatography or Protein A or G affinity chromatography.

[0090] Antibodies of the invention may be described or specified in terms of the epitopes or portions of IL-13 that the antibody recognizes or specifically binds. The epitopes or polypeptide portions may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, conformational epitopes and so on.

[0091 ] Antibody fragments that recognize specific epitopes may be generated by known techniques. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies. For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain. However, those fragments can be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from an antibody phage library. Alternatively, F(ab')2-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab¾ fragments. According to another approach, F(ab¾ fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of the invention is a single chain Fv fragment (Fv).

[0092] To determine whether a particular antibody homo log binds to an IL-13 polypeptide, any conventional binding assay may be used. Useful IL-13 binding assays include FACS analysis, ELISA assays, Surface Plasmon Resonance, radioimmunoassays, and the like, which detect binding of antibody, and functions resulting therefrom, to an IL-13 polypeptide. Full-length and soluble forms of IL-13 polypeptides are useful in such assays. The binding of an antibody or homolog to IL-13, or to soluble fragments thereof, may conveniently be detected through the use of a second antibody specific for immunoglobulins of the species from which the antibody or homolog is derived.

[0093] To determine whether a particular antibody or homolog significantly blocks binding to IL-13, any suitable competition assay may be used. Useful assays include, for example, ELISA assays, FACS assays, radioimmunoassays, and the like that quantify the ability of the antibody or homolog to compete with IL-13. Preferably, the ability of ligand to block binding of labeled IL-13 to immobilized antibody or homolog is measured.

[0094] In another embodiment of the invention, the anti-IL-13 antibody need not be labeled, and the presence of the anti-IL-13 antibody can be detected using a labeled antibody that binds to the anti-IL-13 antibody, another form of a second antibody.

[0095] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. Methods for producing chimeric antibodies are known in the art.

[0096] Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR grafting, veneering or resurfacing, and chain shuffling.

[0097] Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other target-binding subsequences of antibodies) which contain sequences derived from non-human immunoglobulin, as compared to a human antibody. In general, the humanized antibody will comprise substantially all of one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin template sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of the human

immunoglobulin template chosen. In general, the goal is to have an antibody molecule that is minimally immunogenic in a human. Thus, it is possible that one or more amino acids in one or more CDRs also can be changed to one that is less immunogenic to a human host, without substantially minimizing the specific binding function of the one or more CDRs to IL-13. Alternatively, the FR can be non-human, but those amino acids most immunogenic are replaced with ones less immunogenic. [0098] The antibodies of the invention may be bispecific antibodies. Bispecific antibodies can be monoclonal, preferably humanized, antibodies that have binding specificities for at least two different antigens. In a preferred embodiment, the bispecific antibody has a binding specificity for IL-13.

[0099] In addition, one can generate single-domain antibodies to IL-13.

Examples of that technology have been described in the art for antibodies derived from Camelidae heavy chain Ig, as well as for the isolation of single domain human antibodies from phage libraries.

3. Target Sites for Antibody Mutagenesis

[00100] Certain strategies can be employed to manipulate inherent properties of an IL-13 -specific antibody, such as the affinity of the antibody for its target. These strategies include the use of site-specific or random mutagenesis of the polynucleotide molecule encoding the antibody to generate antibody variants, followed by a screening step designed to recover antibody variants that exhibit the desired change, e.g., increased or decreased affinity.

[00101] The amino acid residues most commonly targeted in mutagenic strategies are those in the CDRs. As described supra, these regions contain the residues that actually interact with IL-13 and other amino acids that affect the spatial arrangement of these residues. However, amino acids in the framework regions of the variable domains outside the CDR regions have also been shown to make substantial contributions to the antigen-binding properties of the antibody, and can be targeted to manipulate such properties.

[00102] The amino acid sequence of the heavy and/or light chain variable domain may be inspected to identify the sequences of the CDRs by well known methods, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The polynucleotide of interest generated by the combination of the framework regions and one or more CDRs encodes an antibody that specifically binds IL-13, or at least the extracellular domain thereof. For example, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. [00103] By convention, the CDR regions in the heavy chain are typically referred to as HI, H2 and H3 and are numbered sequentially in order counting from the amino terminus to the carboxy terminus. The CDR regions in the light chain are typically referred to as LI, L2 and L3 and are numbered sequentially in order counting from the amino terminus to the carboxy terminus.

[00104] Once the nucleotide sequence and corresponding amino acid sequence of the antibody are determined, the nucleotide sequence of the antibody may be manipulated to obtain the equivalents of interest described herein using methods known in the art for manipulating nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, and PCR, to generate antibodies having a different amino acid sequence, for example, to create amino acid substitutions, deletions and/or insertions.

[00105] The Kabat definition is based on the sequence variability and is the most commonly used definition to predict CDR regions. The Chothia definition is based on the location of the structural loop regions. The AbM definition is a compromise between the Kabat and Chothia definition. AbM is an integral suite of programs for antibody structure modeling produced by Oxford Molecular Group (ABM™, a computer program for modeling variable regions of antibodies, Oxford, UK; Oxford Molecular, Ltd.). The AbM suite models the tertiary structure of an antibody from primary sequencing using a combination of knowledge databases and ab initio methods. An additional definition, known as the contact definition, has been recently introduced. This definition is based on an analysis of the available complex crystal structures.

[00106] Various methods for modifying antibodies have been described in the art. For example, one method is described to produce humanized antibodies wherein the sequence of the humanized immunoglobulin heavy chain variable region framework is 65% to 95% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Each humanized immunoglobulin chain will usually comprise, in addition to the CDRs, amino acids from the donor immunoglobulin framework that are, e.g., capable of interacting with the CDRs to affect binding affinity, such as one or more amino acids which are immediately adjacent to a CDR in the donor immunoglobulin or those within about 3 angstroms as predicted by molecular modeling. The heavy and light chains may each be designed by using any one or all of various position criteria. When combined into an intact antibody, the humanized immunoglobulins of the present invention will be substantially non- immunogenic in humans and retain substantially the same affinity as the donor

immunoglobulin to the antigen, such as a protein or other compound containing an epitope.

[00107] Often, framework residues in the human framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter, and possibly improve, antigen binding. The framework substitutions are identified by methods known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions.

[00108] Certain portions of the constant regions of antibody can be

manipulated and changed to provide antibody homologs, derivatives, fragments and the like with properties different from or better than that observed in the parent antibody. Thus, for example, many IgG4 antibodies form intrachain disulfide bonds near the hinge region. The intrachain bond can destabilize the parent bivalent molecule forming monovalent molecules comprising a heavy chain with the associated light chain. Such molecules can reassociate, but on a random basis.

[00109] It was observed that modifying amino acids in the hinge region of

IgG4 molecules can reduce the likelihood of intrachain bond formation, thereby stabilizing the IgG4 molecule, which will minimize the likelihood of forming bispecific molecules. That modification can be beneficial if a therapeutic antibody is an IgG4 molecule as the enhanced stability will minimize the likelihood of having the molecule dissociate during production and manufacture, as well as in vivo. A monovalent antibody may not have the same effectiveness as the bivalent parent molecule. For example, when bivalent IgG4 is administered to a patient, the percentage of bivalent IgG4 decays to about 30% over a two-week period. An amino acid substitution at position 228 enhances IgG4 stability. The serine that resides at 228 can be replaced with another amino acid, such as one of the remaining 19 amino acids. Such a change can be made particularly with recombinant antibodies wherein the nucleic acid coding sequence can be mutated to yield a replacement amino acid at position 228. For example, the S can be replaced with a proline.

[00110] It is further preferable that humanized antibodies retain high affinity for IL-13, and retain or acquire other favorable biological properties. Thus, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of the displays permits analysis of the likely role of certain residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind IL-13. In that way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is maximized, although it is the CDR residues that directly and most substantially influence IL-13 binding. The CDR regions also can be modified to contain one or more amino acids that vary from that obtained from the parent antibody from which the CDR was obtained, to provide enhanced or different properties of interest, such as binding of greater affinity or greater avidity, for example.

[0011 1] A humanization method of interest is based on the impact of the molecular flexibility of the antibody during and at immune recognition. Protein flexibility is related to the molecular motion of the protein molecule. Protein flexibility is the ability of a whole protein, a part of a protein or a single amino acid residue to adopt an ensemble of conformations which differ significantly from each other.

[00112] Most protein complexes share a relatively large and planar buried surface and it has been shown that flexibility of binding partners provides the origin for their plasticity, enabling them to conformationally adapt to each. As such, examples of "induced fit" have been shown to play a dominant role in protein-protein interfaces. In addition, there is a steadily increasing body of data showing that proteins actually bind ligands of diverse shapes sizes and composition and that the conformational diversity appears to be an essential component of the ability to recognize different partners. Flexible residues are involved in the binding of protein-protein partners.

[00113] Modification of flexible residues such that recognition of B-cell to trigger an immune response is possible, can be achieved by modifying a limited number of residues by: (1) building a homology model of the parent mAb and running an MD simulation; (2) analyzing the flexible residues and identification of the most flexible residues of a non-human antibody molecule, as well as identifying residues or motifs likely to be a source of heterogeneity or of degradation reaction; (3) identifying a human antibody which displays the most similar ensemble of recognition areas as the parent antibody; (4) determining the flexible residues to be mutated, residues or motifs likely to be a source of heterogeneity and degradation are also mutated; and (5) checking for the presence of known T cell or B cell epitopes. The flexible residues can be found using an MD calculation as taught herein using an implicit solvent model, which accounts for the interaction of the water solvent with the protein atoms over the period of time of the simulation. Once the set of flexible residues has been identified within the variable light and heavy chains, a set of human heavy and light chain variable region frameworks that closely resemble that of the antibody of interest are identified. That can be done, for example, using a blast search on the set of flexible residues against a database of antibody human germline sequence. It can also be done by comparing the dynamics of the parent mAb with the dynamics of a library of germline canonical structures. The CDR residues and neighboring residues are excluded from the search to ensure high affinity for the antigen is preserved.

[00114] Flexible residues then are replaced. When several human residues show similar homologies, the selection is driven also by the nature of the residues that are likely to affect the solution behavior of the humanized antibody. For instance, polar residues will be preferred in exposed flexible loops over hydrophobic residues. Residues which are a potential source of instability and heterogeneity are also mutated even if there are found in the CDRs. That will include exposed methionines as sulfoxide formation can result from oxygen radicals, proteolytic cleavage of acid labile bonds such as those of the Asp-Pro dipeptide, deamidation sites found with an exposed asparagine residue followed by a small amino acid, such as Gly, Ser, Ala, His, Asn or Cys and N-glycosylation sites, such as the Asn-X-Ser/Thr site. Typically, exposed methionines will be substituted by a Leu, exposed asparagines will be replaced by a glutamine or by an aspartate, or the subsequent residue will be changed. For the glycosylation site (Asn-X-Ser/Thr), either the Asn or the Ser/Thr residue will be changed.

[00115] The resulting composite sequence is checked for the presence of known B cell or linear T-cell epitopes. A search is performed, for example, with the publicly available IEDB. If a known epitope is found within the composite sequence, another set of human sequences is retrieved and substituted

[00116] Unlike the resurfacing method commonly used in the art, both B-cell- mediated and T-cell-mediated immunogenic responses are addressed by the method. The method also avoids the issue of loss of activity that is sometimes observed with CDR grafting. In addition, stability and solubility issues also are considered in the engineering and selection process, resulting in an antibody that is optimized for low immunogenicity, high antigen affinity and improved biophysical properties.

[00117] Additional strategies and methods for resurfacing antibodies, and other methods for reducing immunogenicity of antibodies within a different host, are known in the art. In one method, (1) position alignments of a pool of antibody heavy and light chain variable regions are generated to yield heavy and light chain variable region framework surface exposed positions, wherein the alignment positions for all variable regions are at least about 98% identical; (2) a set of heavy and light chain variable region framework surface exposed amino acid residues is defined for a non-human, such as a rodent antibody (or fragment thereof); (3) a set of heavy and light chain variable region framework surface exposed amino acid residues that is most closely identical to the set of rodent surface exposed amino acid residues is identified; and (4) the set of heavy and light chain variable region framework surface exposed amino acid residues defined in step (2) is substituted with the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3), except for those amino acid residues that are within 5A of any atom of any residue of a CDR of the rodent antibody, to yield a humanized, such as a rodent antibody retaining binding specificity.

[00118] Methods for identifying the amino acid residues of an antibody variable domain which may be modified without diminishing the native affinity of the antigen binding domain while reducing its immunogenicity with respect to a heterologous species and methods for preparing these modified antibody variable domains which are useful for administration to heterologous species are also known in the art.

[00119] As discussed above, modification of an antibody by any of the methods known in the art is typically designed to achieve increased binding affinity for an antigen and/or reduce immunogenicity of the antibody in the recipient. In one approach, humanized antibodies can be modified to eliminate glycosylation sites in order to increase affinity of the antibody for its cognate antigen. Techniques such as "reshaping," "hyperchimerization," and "veneering/resurfacing" have produced humanized antibodies with greater therapeutic potential. While these techniques diminish antibody immunogenicity by reducing the number of foreign residues, they do not prevent anti-idiotypic and anti-allotypic responses following repeated administration of the antibodies.

[00120] In many instances, humanizing antibodies results in a loss of antigen binding capacity. It is therefore preferable to "back mutate" the humanized antibody to include one or more of the amino acid residues found in the original (most often rodent) antibody in an attempt to restore binding affinity of the antibody.

[00121] Once the antibody is identified and isolated, it is often useful to generate a variant antibody or mutant, or mutein, wherein one or more amino acid residues are altered, for example, in one or more of the hypervariable regions of the antibody.

Alternatively, or in addition, one or more alterations (e.g. , substitutions) of framework residues may be introduced in the antibody where these result in an improvement in the binding affinity of the antibody mutant for IL-13. Examples of framework region residues that can be modified include those which non-covalently bind antigen directly; interact with/affect the conformation of a CDR; and/or participate in the VL-VH interface. In certain embodiments, modification of one or more of such framework region residues results in an enhancement of the binding affinity of the antibody for the cognate antigen. For example, from about one to about five framework residues may be altered in this embodiment of the invention. Sometimes, this may be sufficient to yield an antibody mutant suitable for use in preclinical trials, even where none of the hypervariable region residues have been altered. Normally, however, the antibody mutant can comprise one or more hypervariable region alteration(s). The constant regions also can be altered to obtain desirable or more desirable effector properties.

[00122] Ordinarily, the antibody mutant with improved biological properties will have an amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the parent anti-IL-13 antibody, at least 80%, at least 85%, at least 90% and often at least 95% identity. Identity or similarity with respect to parent antibody sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) or similar (i.e., amino acid residue from the same group based on common side- chain properties) with the parent antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[00123] Following production of the antibody mutant, the biological activity of that molecule relative to the parent antibody can be determined as taught herein. As noted above, that may involve determining the binding affinity and/or other biological activities or physical properties of the antibody. In a preferred embodiment of the invention, a panel of antibody mutants is prepared and screened for binding affinity for the antigen. One or more of the antibody mutants selected from the screen are optionally subjected to one or more further biological activity assays to confirm that the antibody mutant(s) have new or improved properties. In preferred embodiments, the antibody mutant retains the ability to bind IL-13 with a binding affinity similar to or better/higher than that of the parent antibody.

[00124] The antibody mutant(s) so selected may be subjected to further modifications, often depending on the intended use of the antibody. Such modifications may involve further alteration of the amino acid sequence, fusion to heterologous polypeptide(s) and/or covalent modifications. For example, a cysteine residue not involved in maintaining the proper conformation of the antibody mutant may be substituted, generally with serine, to improve the oxidative stability of the molecule and to prevent aberrant cross-linking.

Conversely, a cysteine may be added to the antibody to improve stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[00125] Another type of antibody mutant has an altered glycosylation pattern. That may be achieved by deleting one or more carbohydrate moieties found in the antibody and/or by adding one or more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked to Asn or O-linked to Ser or Thr. The tripeptide sequences, asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are common recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. N-acetylgalactosamine, galactose, fucose or xylose, for example, are bonded to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5 -hydroxy lysine also may be used. Addition or substitution of one or more serine or threonine residues to the sequence of the original antibody can enhance the likelihood of O-linked glycosylation.

[00126] The hypervariable region residues which are altered may be changed randomly, especially where the starting binding affinity of the parent antibody is such that randomly-produced antibody mutants can be readily screened for altered binding in an assay as taught herein.

[00127] One procedure for obtaining antibody mutants, such as CDR mutants, is "alanine scanning mutagenesis." One or more of the hypervariable region residue(s) are replaced by alanine or polyalanine residue(s). Those hypervariable region residue(s) demonstrating functional sensitivity to the substitutions then are refined by introducing further or other mutations at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. Similar substitutions can be attempted with other amino acids, depending on the desired property of the scanned residues.

4. Variants of Antibodies

[00128] Variants of antibodies of the invention include insertion, deletion, and/or substitution variants. In one aspect of the invention, insertion variants are provided wherein one or more amino acid residues supplement an antibody amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the antibody amino acid sequence. Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels. Insertion variants include antibody polypeptides wherein one or more amino acid residues are added to an antibody amino acid sequence, or fragment thereof.

[00129] In still another aspect, the invention provides substitution variants of antibodies of the invention. Substitution variants are generally considered to be "similar" to the original polypeptide or to have a certain "percent identity" to the original polypeptide, and include those polypeptides wherein one or more amino acid residues of a polypeptide are removed and replaced with alternative residues. Relating to an IL-13 antibody in particular, percent identity refers to percent identity outside of the complimentary determining regions of the antibody. In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative.

[00130] Antibody fragments include those portions of the antibody that bind to an epitope on the antigen polypeptide. Examples of such fragments include Fab and F(ab')2 fragments generated, for example, by enzymatic or chemical cleavage of full-length antibodies. Other binding fragments include those generated by recombinant DNA techniques, such as the expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions. The invention also embraces polypeptide fragments of an IL-13 antibody wherein the fragments maintain the ability to specifically bind an IL-13 polypeptide. Fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 or more consecutive amino acids of a peptide or polypeptide of the invention are comprehended herein. Preferred polypeptide fragments display immunological properties unique to or specific for the antigen-binding agent so of the invention. Fragments of the invention having the desired immunological properties can be prepared by any of the methods well known and routinely practiced in the art.

[00131] The antibody fragments and functional equivalents of the invention encompass those molecules with a detectable degree of specific binding to IL-13. A detectable degree of binding includes all values in the range of at least 10-100%, preferably at least 50%, 60% or 70%, more preferably at least 75%, 80%, 85%, 90%, 95% or 99% of the binding ability of an antibody of interest. Also included are equivalents with an affinity greater than 100% that of an antibody of interest.

[00132] Alternatively, techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used.

[00133] The CDRs generally are of importance for epitope recognition and antibody binding. However, changes may be made to residues that comprise the CDRs without interfering with the ability of the antibody to recognize and to bind the cognate epitope. For example, changes that do not impact epitope recognition, yet increase the binding affinity of the antibody for the epitope, may be made. Several studies have surveyed the effects of introducing one or more amino acid changes at various positions in the sequence of an antibody, based on the knowledge of the primary antibody sequence, on the properties thereof, such as binding and level of expression.

[00134] Thus, equivalents of an antibody of interest can be generated by changing the sequences of the heavy and light chain genes in the CDR1, CDR2 or CDR3, or framework regions, using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling or mutator-strains of E. coli. The methods of changing the nucleic acid sequence of the primary antibody can result in antibodies with improved.

[00135] Antibodies with homologous sequences are those antibodies with amino acid sequences that have sequence homology with the amino acid sequence of an IL- 13 antibody of the invention. Preferably, homology is with the amino acid sequence of the variable regions of an antibody of the invention. "Sequence homology" as applied to an amino acid sequence herein is defined as a sequence with at least about 90%, 91%, 92%, 93%, 94% or more sequence homology, and more preferably at least about 95%, 96%, 97%, 98% or 99% sequence homology to another amino acid sequence, as determined, for example, by the.

[00136] Preferred methods to determine the relatedness or percent identity of two polypeptides are designed to give the largest match between the sequences tested.

Methods to determine identity are described in publicly available computer programs.

Preferred computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and FASTA. The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources. The well-known Smith Waterman algorithm may also be used to determine identity.

[00137] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage.

[00138] The amino acids may have either L or D stereochemistry (except for Gly, which is neither L nor D) and the polypeptides and compositions of the invention may comprise a combination of stereochemistries. However, the L stereochemistry is preferred. The invention also provides reverse molecules wherein the amino terminal to carboxy terminal sequence of the amino acids is reversed. For example, the reverse of a molecule having the normal sequence X1-X2-X₃ would be X₃-X₂-X₁. The invention also provides retro-reverse molecules wherein, as above, the amino terminal to carboxy terminal sequence of amino acids is reversed and residues that are normally "L" enantiomers are altered to the "D" stereoisomer form.

[00139] Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the invention. Examples of unconventional amino acids include, without limitation: aminoadipic acid, beta-alanine, beta-aminopropionic acid, aminobutyric acid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminoisobutyric acid,

aminopimelic acid, diaminobutyric acid, desmosine, diaminopimelic acid, diaminopropionic acid, N-ethylglycine, N-ethylaspargine, hyroxylysine, allo-hydroxylysine, hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, sarcosine, N-methylisoleucine, N- methylvaline, norvaline, norleucine, orithine, 4-hydroxyproline, γ-carboxyglutamate, ε- Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5 -hydroxy lysine, σ-Ν-methylarginine, and other similar amino acids and amino acids (e.g., 4-hydroxyproline).

[00140] Similarly, unless specified otherwise, the left-hand end of single- stranded polynucleotide sequences is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".

[00141] Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.

[00142] Even more substantial modification in an antibody range and presentation of biological properties can be accomplished by selecting an amino acid that differs more substantially in properties from that normally resident at a site. Thus, such a substitution can be made while maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[00143] Naturally occurring residues may be divided into classes based on common side chain properties:

1) hydrophobic: Met, Ala, Val, Leu, He;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

[00144] Preferred amino acid substitutions include those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity and (4) confer or modify other physico-chemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain (s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence) unless of a change in the bulk or conformation of the R group or side chain.

[00145] For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the antibody that are homologous with antibodies to other orthologs, or into the non-homologous regions of the molecule.

[00146] The antibodies of this invention that are polypeptide or peptide substitution variants may have up to about ten to twelve percent of the original amino acid sequence replaced. For antibody variants, the heavy chain may have 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid replaced, while the light chain may have 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid replaced.

[00147] In making such changes, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);

aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[00148] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included. In certain embodiments, those which are within ±1 are included, and in certain embodiments, those within ±0.5 are included. [00149] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[00150] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those which are within ±1 are included, and in certain embodiments, those within ±0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as "epitopic core regions." Exemplary amino acid substitutions are set forth in Table 1.

[00151] A skilled artisan will be able to determine suitable variants of the polypeptide as set forth herein using well-known techniques. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[00152] In certain embodiments, antibody variants include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of the parent polypeptide. In certain embodiments, protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protein. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional preferred antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence. Cysteine variants may be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[00153] According to certain embodiments, amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (5) confer or modify other functional properties on such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). In certain embodiments, a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).

[00154] It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody. For example, cysteine residue(s) may be introduced in the F_c region, thereby allowing interchain disulfide bond formation in that region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC). Alternatively, an antibody can be engineered which has dual F_c regions and may thereby have enhanced complement lysis and ADCC capabilities.

[00155] The functional equivalents of the present application also include modified antibodies, e.g., antibodies modified by the covalent attachment of any type of molecule to the antibody. For example, modified antibodies include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, deamidation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand, linkage to a toxin or cytotoxic moiety or other protein. The covalent attachment need not yield an antibody that is immune from generating an anti-idiotypic response. The modifications may be achieved by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, or metabolic synthesis. Additionally, the modified antibodies may contain one or more non-classical amino acids. 5. Purification and Refolding of Antibodies

[00156] Once an antibody molecule of the invention has been produced by an animal, chemically synthesized or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule. These techniques involve, at one level, the crude fractionation of the polypeptide and non-polypeptide fractions. Having separated the antibody polypeptide from other proteins, the polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Purification can be achieved, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for IL- 13 after Protein A and size-exclusion chromatography), centrifugation, and differential solubility or by any other standard technique for the purification of proteins. In addition, the antibodies of the invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification. Analytical methods particularly suited to the preparation of a pure antibody peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.

[00157] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography. The suitability of protein A or protein G as an affinity ligand depends on the species and isotype of any immunoglobulin F_c domain that is present in the antibody variant. Protein A can be used to purify antibodies that are based on human IgGl, IgG2 or IgG4 heavy chains. Protein G can be used for mouse isotypes and for human IgG3. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices, such as controlled pore glass or

poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody variant comprises a CH3 domain, the

Bakerbond ABXTM resin (JT Baker; Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin agarose chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation are also available, depending on the antibody or variant to be recovered. [00158] Following any preliminary purification step(s), the mixture comprising the antibody or variant of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25 M salt).

[00159] As discussed supra, the invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide. Fused or conjugated antibodies of the invention may be used for ease in purification. The marker amino acid sequence can be a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein and the "FLAG" tag.

[00160] Various methods for quantifying the degree of purification of the antibody will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the antibody activity of an active fraction, or assessing the amount of antibodies within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of an antibody fraction is to calculate the binding activity of the fraction, to compare it to the binding activity of the initial extract, and to thus calculate the degree of purification, herein assessed by a "-fold purification number." The actual units used to represent the amount of binding activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed antibody exhibits a detectable binding activity.

[00161] There is no general requirement that the antibody always be provided in its most purified state. Indeed, it is contemplated that less substantially purified antibodies will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "- fold" purification than the same technique utilizing a low-pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of antibody, or in maintaining binding activity of an expressed antibody.

[00162] Various techniques suitable for use in antibody purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies (immunoprecipitation) and the like or by heat denaturation, followed by centrifugation; chromatography steps such as affinity

chromatography (e.g., Protein-A-Sepharose), ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified antibody.

[00163] In some cases, the antibodies produced using procedures described above may need to be "refolded" and oxidized into a proper tertiary structure and generating di-sulfide linkages in order to be biologically active. Refolding can be accomplished using a number of procedures well known in the art. Such methods include, for example, exposing the solubilized polypeptide agent to a pH usually above 7 in the presence of a chaotropic agent. The selection of chaotrope is similar to the choices used for inclusion body solubilization, however a chaotrope is typically used at a lower concentration. An exemplary chaotropic agent is guanidine. In most cases, the refolding/oxidation solution will also contain a reducing agent plus its oxidized form in a specific ratio to generate a particular redox potential which allows for disulfide shuffling to occur for the formation of cysteine bridges. Some commonly used redox couples include cysteine/cystamine,

glutathione/dithiobisGSH, cupric chloride, dithiothreitol DTT/dithiane DTT, and 2- mercaptoethanol (bME)/dithio-bME. In many instances, a co-solvent may be used to increase the efficiency of the refolding. Commonly used cosolvents include glycerol, polyethylene glycol of various molecular weights, and arginine.

[00164] The antibody and fragments thereof of the invention may also be used as affinity purification agents. In that process, the antibodies are immobilized on a solid phase, such as a dextran or agarose resin or filter paper, using methods known in the art. The immobilized antibody is contacted with a sample containing IL-13 or cells carrying same to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the IL-13 or cell to be purified, which is bound to the immobilized antibody of interest. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0 that will release the IL-13 or cell from the antibody of interest. 6. Binding Assays

[00165] The antibodies of the invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.

[00166] Antibodies of the invention also may be described or specified in terms of binding affinity to IL-13. Anti -IL-13 antibodies may bind with a K_D of less than about 10^{" 7} M, less than about 10^"6 M, or less than about 10^"5 M. Higher binding affinities in an antibody of interest can be beneficial, such as those with an equilibrium dissociation constant or K_D of from about 10^"8 to about 10^"15 M, from about 10^"8 to about 10^"12 M, from about 10^"9 to about 10^"11 M, or from about 10^"8 to about 10^"10 M. The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

[00167] As used herein, "non-decorating" antibodies block IL-13 binding to IL- 13Ra2. In certain embodiments, an ELISA readout will be negative in comparison with the positive ELISA readout of a decorating antibody. As used herein, "decorating antibodies" will bind IL-13 and prevent downstream signaling but do not prevent IL-13 binding to the receptor, i.e., cells are decorated with antibodies bound to IL-13.

[00168] Two antibodies of the invention, C 1 1 - 10 and A 101 , are non- decorating, meaning that when they bind to IL-13, the IL-13/C11 and IL-13/A101 complexes cannot bind to the IL-13 receptor (IL-13Ra2) on cell surface because the domain on IL-13 that is needed to bind to the receptor is occupied by the antibodies (A101 or CI 1-10). In contrast, when two decorating antibodies of the invention, A 105 and F232, bind to IL-13, the IL-13/A105 and IL-13/F232 complexes can bind to IL-13 receptor (IL-13Ra2) on cell surface with IL-13 serving as a bridge with one end attaching the receptor on cell surface, while another domain attaches the antibodies (A105 or F232). This distinction between decorating and non-decorating antibodies is due to the fact that A 101 and CI 1 have different binding epitopes than A105 and F232 on the antigen IL-13. This is a factor in determing whether the antigen/antibody complex will bind to the cell surface receptor or not. This selective binding may be an important factor is assessing safety and pharmokinetic properties of the antibody as therapeutics. 7. Diseases

[00169] The antibodies of the invention may be used to treat, suppress or prevent disease, such as an allergic disease, a Th2-mediated disease, or IL- 13 -mediated disease. Examples of such diseases include, Hodgkin's disease, asthma, allergic asthma, atopic dermatitis, atopic allergy, ulcerative colitis, scleroderma, allergic rhinitis, COPD3 idiopathic pulmonary fibrosis, chronic graft rejection, bleomycin-induced pulmonary fibrosis, radiation-induced pulmonary fibrosis, pulmonary granuloma, progressive systemic sclerosis, schistosomiasis, hepatic fibrosis, renal cancer, Burkitt lymphoma, Hodgkins disease, non~Hodgkins disease, Sezary syndrome, asthma, septic arthritis, dermatitis herpetiformis, chronic idiopathic urticaria, ulcerative colitis, scleroderma, hypertrophic scarring, Whipple's Disease, benign prostate hyperplasia, an allergic reaction to a medication, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Grave's disease, pre-eclampsia, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, cystic fibrosis, allergic

bronchopulmonary mycosis, chronic obstructive pulmonary disease, bleornycin-induced pneumopathy and fibrosis, pulmonary alveolar proteinosis, adull respiratory distress syndrome, sarcoidosis, hyper IgE syndrome, idiopathic hypereosinophil syndrome, an autoimmune blistering disease, pemphigus vulgaris, bullous pemphigoid, myasthenia gravis, chronic fatigue syndrome, nephrosis.

8. Diagnostic Assays

[00170] The antibodies or antibody fragments of the invention can be used to detect IL-13, and hence cells expressing IL-13, in a biological sample in vitro or in vivo. In one embodiment, the anti-IL-13 antibody of the invention is used to determine the presence and the level of IL-13 in a tissue or in cells derived from the tissue. The levels of IL-13 in the tissue or biopsy can be determined, for example, in an immunoassay with the antibodies or antibody fragments of the invention. The tissue or biopsy thereof can be frozen or fixed. The same or other methods can be used to determine other properties of IL-13, such as the level thereof, cellular localization, mRNA levels, mutations thereof and so on. In some embodiments, the label is a radiolabel, a fluorophore, a chromophore, an imaging agent or a metal ion. [00171] Diagnostic assays for IL-13 include methods utilizing an antibody and a label to detect IL-13 in human body fluids or extracts of cells or tissues. The antibodies of the invention can be used with or without modification. In a particular diagnostic assay, the antibodies will be labeled by attaching, e.g., a label or a reporter molecule. A wide variety of labels and reporter molecules are known, some of which have been already described herein. In particular, the present invention is useful for diagnosis of human disease.

[00172] A variety of protocols for measuring IL-13 proteins using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non- interfering epitopes on IL-13 is preferred, but a competitive binding assay can be employed.

[00173] In order to provide a basis for diagnosis, normal or standard values for human IL-13 expression are usually established. This determination can be accomplished by combining body fluids or cell extracts from normal subjects, preferably human, with an antibody to IL-13, under conditions suitable for complex formation that are well known in the art. The amount of standard complex formation can be quantified by comparing the binding of the antibodies to known quantities of IL-13 protein, with both control and disease samples. Then, standard values obtained from normal samples can be compared with values obtained from samples from subjects potentially affected by disease. Deviation between standard and subject values suggests a role for IL-13 in the disease state. The assay of interest also can be used to diagnose arthritis or other autoimmune diseases characterized by B cell infiltration and concentration, along with development of differentiated lymphoid tissue.

[00174] For diagnostic applications, in certain embodiments, antibodies typically will be labeled with a detectable moiety. The detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as H, C, P, S, or I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[00175] A method for diagnosis is also provided in which said labeled antibodies or epitope-binding fragments thereof are administered to a subject suspected of having a cancer, arthritis, autoimmune diseases or other IL-13 mediated disease, and the distribution of the label within the body of the subject is measured or monitored. 9. Pharmaceutical Compositions

[00176] Pharmaceutical compositions of IL-13 antibodies are within the scope of the present invention. Pharmaceutical compositions comprising antibodies are described in the art. Such compositions comprise a therapeutically or prophylactically effective amount of an antibody, or a fragment thereof, variant, derivative or fusion thereof as described herein, in admixture with a pharmaceutically acceptable agent. In one aspect, pharmaceutical compositions comprise antagonist antibodies that modulate partially or completely at least one biological activity of IL-13 in admixture with a pharmaceutically acceptable agent.

Typically, the antibodies will be sufficiently purified for administration to an animal.

[00177] Therapeutic formulations of the antibodies may be prepared for storage as lyophilized formulations or aqueous solutions by mixing an antibody having the desired degree of purity with optional "pharmaceutically acceptable" carriers, diluents, excipients or stabilizers typically employed in the art, i.e., buffering agents, stabilizing agents,

preservatives, isotonifiers, non-ionic detergents, antioxidants and other miscellaneous additives. Such additives are generally nontoxic to the recipients at the dosages and concentrations employed, hence, the excipients, diluents, carriers and so on are

pharmaceutically acceptable.

[00178] The formulation herein also may contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely impact each other. For example, it may be desirable to further provide an immunosuppressive agent. Such molecules suitably are present in combination in amounts that are effective for the purpose intended.

[00179] Non-ionic surfactants or detergents (also known as "wetting agents") may be added to help solubilize the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stresses without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (e.g., 20, 80), polyoxamers (e.g., 184, 188), Pluronic^® polyols and polyoxyethylene sorbitan monoethers (e.g., TWEEN-20^®, TWEEN- 80^®). Non-ionic surfactants may be present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.

[00180] The pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counter ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides (preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants.

[00181] The optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antibody. A pharmaceutical composition effective in promoting a diminution of an inflammatory effect, for example, may provide a local therapeutic agent concentration of between about 5 and 20 ng/ml, and, preferably, between about 10 and 20 ng/ml.

[00182] The invention also provides that a liquid formulation of the invention is packaged in a sealed container such as an ampule or sachet indicating the quantity of the product of interest. The liquid formulations of the invention can be in a sealed container indicating the quantity and concentration of the antibody or antibody fragment. The liquid formulation of the invention can be supplied in a sealed container with at least 15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml, or 300 mg/ml of IL-13 antibody in a quantity of 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml or 20 ml, for example.

[00183] In addition, the invention also encompasses stable liquid formulations of the products of interest that have improved half-life in vivo. Thus, the antibody of interest has a half-life in a subject, preferably a human, of greater than 3 days, greater than 7 days, greater than 10 days, greater than 15 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, greater than 5 months or more.

[00184] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefore. In one embodiment of the invention, antibody compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the antibody product may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[00185] The invention encompasses liquid formulations having stability at temperatures found in a commercial refrigerator and freezer found in the office of a physician or laboratory, such as from about -20° C to about 5° C, said stability assessed, for example, by high performance size exclusion chromatography (HPSEC), for storage purposes, such as for about 60 days, for about 120 days, for about 180 days, for about a year, for about 2 years or more. The liquid formulations of the invention also exhibit stability, as assessed, for example, by HSPEC, at room temperatures, for a at least a few hours, such as one hour, two hours or about three hours prior to use.

[00186] The pharmaceutical compositions can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for enteral delivery such as orally, aurally, opthalmically, rectally, or vaginally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. [00187] The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at slightly lower pH, typically within a pH range of from about 5 to about 8.

[00188] The route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, intralesional routes, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal means, by sustained release systems or by implantation devices. Where desired, the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.

[00189] When parenteral administration is contemplated, the therapeutic compositions for use in this invention may be in the form of a pyrogen- free, parenterally acceptable aqueous solution comprising the desired antibody in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a binding agent is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes that provides for the controlled or sustained release of the product which may then be delivered via a depot injection.

Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

[00190] In a preferred embodiment, an aqueous solution of therapeutic polypeptide, antibody or fragment thereof can be administered by subcutaneous injection. Each dose may range from about 0.5 mg to about 50 mg per kilogram of body weight, or more preferably, from about 3 mg to about 30 mg per kilogram body weight. The dosage can be ascertained empirically for the particular disease, patient population, mode of

administration and so on, practicing pharmaceutical methods known in the art.

[00191] The pharmaceutical composition to be used for in vivo administration typically must be sterile. This may be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[00192] In some cases, it may be desirable to use pharmaceutical compositions in an ex vivo manner. In such instances, cells, tissues, or organs that have been removed from the patient are exposed to the pharmaceutical compositions after which the cells, tissues and/or organs are subsequently implanted back into the patient.

[00193] In other cases, an antibody which is a polypeptide can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide. Such cells may be animal or human cells, and may be autologous, heterologous, or xenogeneic. Optionally, the cells may be immortalized. In order to decrease the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues. The encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.

[00194] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[00195] The exact dosage will be determined in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.

[00196] The frequency of dosing will depend upon the pharmacokinetic parameters of the binding agent molecule in the formulation used. Typically, a composition is administered until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as multiple doses (at the same or different concentrations/dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely made. Appropriate dosages may be ascertained through use of appropriate dose-response data.

[00197] To prolong the serum circulation of an antibody in vivo, various techniques can be used. For example, inert polymer molecules, such as high molecular weight polyethylene glycol (PEG), can be attached to an antibody with or without a multifunctional linker either through site-specific conjugation of the PEG to the N-terminus or to the C-terminus of the antibody or via ε amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity can be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by size-exclusion or by ion exchange chromatography. PEG-derivatized antibodies can be tested for binding activity as well as for in vivo efficacy using methods known to those of skilled in the art, for example, by immunoassays described herein.

[00198] An antibody having an increased half-life in vivo can also be generated by introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) into an IgG constant domain, or F_CR binding fragment thereof (such as an F_e or hinge F_e domain fragment).

[00199] Further, an antibody can be conjugated to albumin to make an antibody more stable in vivo or have a longer half life in vivo. The techniques are known in the art. The antibody also can be modified, for example, by glycosylation, acetylation,

phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein and so on.

10. Combination Therapy

[00200] An article of manufacture containing materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for diagnosing, preventing or treating an IL- 13 mediated condition or disease and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label on or associated with the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes and package inserts with instructions for use.

EXAMPLES

[00201] The invention now will be exemplified for the benefit of the artisan by the following non-limiting examples that depict some of the embodiments by and in which the invention can be practiced.

EXAMPLE 1: Identification of IL-13 antibodies

[00202] Recombinant IL-13 in the forms of human IL-13 and cynomolgus monkey ("cyno") IL-13 was used to immunize mice to generate antibodies that bind IL-13. Either human or a mixture of human and cyno IL-13 recombinant proteins were used to immunize the mice. In some cases human IL-13 was used as a primary immunogen followed by the additional injection of cyno IL-13 before the fusion (A 105 was generated from mice immunized this way, the others as previously discussed).

[00203 ] A 1 : 1 mixture of IL- 13 with MPL-TDM (monophosphoryl lipid A, synthetic trehalose dicorynomycolate) as an adjuvant was used to induce the production of sera containing polyclonal antibodies specific for IL-13. Administration of the IL-13 immunogen consisted of 2-3 intraperitoneal injections of the above mixture. The injections were followed by measurement of mouse serum titers for specific binding to IL-13 by completion ELISA. This assay was used to identify antibodies that bind to IL-13 and block it's binding to IL-13Ra2. Plates were coated with IL-13Ra2 and blocked with 1% BSA /PBS. IL-13 was incubated either with the receptor coated on the plate or with the antibodies in solution. Mixtures of antibodies and IL-13 or antibodies only were applied to the plates coated with the receptor and developed with polyclonal secondary anti-mouse HRP conjugated antibodies followed by ABTS substrate and absorbance was measured at OD405. Antibodies that showed binding to IL-13 but not IL-13Ra2 were considered as non- decorating, and antibodies that showed binding to both targets were considered to be decorating (Figure 2). The splenocytes from the mouse with the highest specific anti-IL-13 antibody titer in serum were used for generation of hybridomas.

Example 2: Creation of hybridomas and generation of monoclonal IL-13 antibodies

[00204] Monoclonal antibodies were made using hybridoma technology, as described above. Hybridoma Production Medium (IMDM) for use in creation of hybridomas was created by combining: Iscove's Modified Dulbecco's Medium 500 ml - HyClone SH30259.01 ; Fetal Bovine Serum 50ml - HyClone SH30070.03; L-glutamine 5 ml- Gibco Invitrogen cat # 25030; Non-essential amino acids 5 ml - Gibco Invitrogen cat # 1 1 140050; Sodium pyruvate 5 ml - Gibco Invitrogen cat # 1 1360070; and Penicillin-streptomycin 0.1% 5 ml - Gibco Invitrogen cat # 15140148. The IMDM was then filtered before use.

[00205] Expansion medium for use in creation of hybridomas was created by combining: Serum Free Medium - Gibco Hybridoma SFM # 12045 1000ml; 10% (100ml) HyClone SuperLow IgG Defined FBS # SH30898.03; and 10ml penicillin/streptomycin. HAT used was from Sigma-Aldrich # H0262 (50X), Hybridoma Fusion and Cloning Supplement used was from Roche Diagnostics - 11 363 735 001 (50X), the Trypan Blue Stain 0.4% used was from Invitrogen - cat # 15250-061 or T10282, the PEG 1500 in 75 mM Hepes 50% w/v used was from Roche - cat # 783641 (10783641001), and the sterile filtered Freezing medium, 45ml heat inactivated FBS+ 5ml DMSO, was from HyClone - SH30070.03.

[00206] Three or four days before the fusion, the mouse was boosted with IL- 13. This can be done either intraperitonealy or intravenously. On the day of the fusion, the mouse was sacrificed in CO₂ chamber, blood was collected through cardiac puncture and the spleenwas taken out and placed into 10 ml of serum free IMDM in a Petri dish. All reagents except HAT, Hybridoma Fusion and cloning supplement were then heated to 37°C in a water bath. Fusion partner cells (F0) were then grown at a log phase, and split one day before the fusion (1 :2 and 1 :5). The cells were then collected into 20 ml centrifuge tubes and spun while preparing the spleen. All centrifugations were performed at 1570 rpm for 5 min. Cell pellets were then resuspend in 10ml IMDM and washed two times with serum free IMDM medium. Connective tissue was the dissected from the harvested spleen, and the spleen was injected with 1 ml of serum free IMDM preheated to 37° C using a 1 ml syringe and 25-gauge needle. Splenocytes were then squeezed out of the fibroelastic coat using forceps and then washed two times with 10 ml of serum free IMDM (including initial spin) before finally resuspending in 10ml serum free IMDM. Splenocytes were then diluted 1 : 100 (160ul PBS, 20ul Trypan Blue and 20ul splenocytes) for counting and F0 were counted in undiluted form (lOul + lOul Trypan Blue Cells before filling the counting chamber). The splenocytes were then again diluted 1 : 10. All cells were counted in a Countess Automated Cell Counter.

[00207] For the next step, fusion partner cells and splenocytes were combined in one 50ml tube at ratio of 1 :2 to 1 : 10 (by cell number) and spun down at 970 rpm for 10 min (slow spin) to form a loose pellet. All splenocytes exceeding 150 million were then discarded. The final counts were 30 million F0 + 150 million splenocytes. All cells were utilized for the fusion.

[00208] After the "slow" spin, supernatant was taken out with the precaution not to disturb the pellet, but minimize the amount of liquid over the cells in order not to dilute the concentration of PEG 1500 as discussed below. While tilting the tube, the medium was removed with a 10ml pipette. The last remaining medium was removed with 200ul pipettman and reserved for later use (addition back after PEG was added; below). 1ml preheated PEG 1500 (at 37°C) was added dropwise to the cell pellet over 1 minute period of time and cells were mixed after all drops of PEG were added. The pellet was then incubated with PEG for another 1 minute followed by addition of 10 ml of serum- free IMDM medium over 1 minute, such that the first 1 ml out of 10 was added over 30 sec. The cells were then spun slowly at 970 rpm for 10 min and the supernatant poured off. Two 100ml troughs containing 70ml IMDM with 10% FBS, 2ml HAT and 2ml Hybridoma and Fusion Cloning Supplement were then prepared. Cells were then resuspend in 10ml IMDM with 10% FBS and then split into two 50ml tubes, 5ml cells/tube and 25ml IMDM added with 10%> FBS. All 30ml were then transferred to troughs containing 70ml HBSS/HAT/cloning supplement solution. 200ul cells/well were then pipetted into ten 96-well plates.

[00209] About 10 to 14 days later, or when medium in the wells turns yellow, the fusions were ready for screening using a 50ul ELISA. After the primary screening, positive clones were selected, numbered and moved to a 24-well plate with 500 ul per well of IMDM with 10% FBSHI. The next day, positive clones were rescreened against positive and negative antigens. The clones that showed specific reactivity were cloned in 96-well plates by limiting dilutions: two-fold dilutions horizontally (from 1-12) followed by two-fold dilutions vertically (from A-H). Parental hybridomas can be frozen for back up cloning if necessary. Clones from the positive hybridomas appeared 10 -14 days later. One to two positive clones from first limiting dilution cloning were moved to 24-well plates in 0.5ml IMDM/10% FBS, then at most one was re-cloned by limiting dilution for a second time. 10-14 days later clones from single cells were re-checked for activity. At least 2 clones can be frozen on two different dates. If there no positives are found after the primary screen, there is an option to return to the original 96-well plate and transfer cells to a new 24-well plate well and recloned.

[00210] In one specific experiment, Fusion S clone # 3 from 96-well fusion plate was determined to be positive using ELISA. It was cloned by limiting dilution a assaying of single clones. Positive clones were moved into a 24-well plate and re-cloned by limiting dilution. These clones were re-tested and re-cloned by limiting dilution in 96-well plates. Two positive clones from the second limiting dilution cloning were expanded and frozen for cryopreservation and sequencing and only one of these clones was cultured for purification.

[0021 1] To perform the above-referenced cryopreservation, 5-10 million cells were spun from a confluent T75 flask at 1570 RPM/5 minutes. The pellet was then brought up in 1ml freezing medium, transferred to a freezing vial, and stored at -80°C (short term) or in liquid nitrogen (long term). In order to freeze the cell pellet for sequencing, about 3-5 million cells were spun at 1570 RPM for 5 minutes. The cells were then washed once in 10ml PBS. Pellets were then resuspend in 300ul Qiagen RLT Lysis Buffer # 79216.

[00212] The monoclonal antibodies secreted by the subclones were isolated or purified from the culture medium by protein A/G-sepharose affinity column.

Four hybridoma clones, A101, CI 1-10, A105, and F232 were identified as having high affinity to bind to human IL-13. The binding affinity of antibodies selected for specifically binding both cyno and human IL-13 antibodies of was determined by Biacore^® Surface Plasmon Resonance (Table 3). These antibodies also inhibited the biological activity of human IL-13 in a cell-based functional assay: IL-13 (5 ng/ml) induced reporter gene expression in HEK-Blue™ IL-4/IL-13 cells. HEK-Blue IL-4/IL-13 Cells (Invivogen) are designed to monitor the activation of the STAT6 pathway induced by IL-4 and IL-13. These cells have been engineered to express a fully active STAT6 signalling pathway.

Furthermore, HEK-Blue IL-4/IL-13 cells stably express the reporter gene secreted embryonic alkaline phosphatase (SEAP) under the control of the IFN-β minimal promoter fused to four STAT6 binding sites. Activation of the STAT6 pathway in HEK-Blue IL-4/IL-13 cells by human IL4 and/or human or mouse IL13 induces the expression of the reporter gene SEAP which is secreted in the supernatant and is detectable using QUANTI-Blue (Invivogen) in a colorimetric assay.

[00213] HEK-Blue IL-4/IL-13 Cells were cultured according to manufacture's protocol. 50,000 cells per well were cultured in a 96 well flat bottom plate and stimulated with 5 or 2ng/ml human IL13 (Chemicon). STAT6 signalling was inhibited by adding different concentrations of an anti-IL13 antibody. After 24 hours incubation at 37°C in a CO₂ incubator 20 ml of induced HEK-Blue IL-4/IL-13 cells supernatant were added to 160μ1 Quanti-Blue Regent (Invivogen, hkb-stat6-kit). The mix was incubated 2 hours at 37°C. SEAP levels were measured by determining the absorbance at 620nm determined using a spectrophotometer. Measurements were performed in triplicates (Table 3 and Figure 3). The amino acid sequences of the antibodies were then mapped out by sequencing cDNAs derived from the hybridoma cells as shown below (CDR or variable region sequences are underined) and in Table 2.

Claims

WHAT IS CLAIMED:

Claim 1. An isolated antibody or fragment thereof that specifically binds human IL-13, wherein the antibody or fragment thereof comprises:

(a) a heavy chain variable region comprising SEQ ID NO: 1;

(b) a heavy chain variable region comprising SEQ ID NO: 9;

(c) a heavy chain variable region comprising SEQ ID NO: 17; or

(d) a heavy chain variable region comprising SEQ ID NO: 25.

Claim 2. An isolated antibody or fragment thereof that specifically binds human IL-13, wherein the antibody or fragment thereof comprises:

(a) a light chain variable region comprising SEQ ID NO: 2;

(b) a light chain variable region comprising SEQ ID NO: 10;

(c) a light chain variable region comprising SEQ ID NO: 18; or

(d) a light chain variable region comprising SEQ ID NO: 26.

Claim 3. An isolated antibody or fragment thereof that specifically binds human IL-13, wherein the antibody or fragment thereof comprises:

(a) a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 2;

(b) a heavy chain variable region comprising SEQ ID NO: 9, and a light chain variable region comprising SEQ ID NO: 10;

(c) a heavy chain variable region comprising SEQ ID NO: 17, and a light chain variable region comprising SEQ ID NO: 18; or

(d) a heavy chain variable region comprising SEQ ID NO: 25, and a light chain variable region comprising SEQ ID NO: 26.

Claim 4. An isolated antibody or fragment thereof that specifically binds human IL-13, wherein the antibody or fragment thereof comprises:

(a) a heavy chain variable region comprising CDRl (SEQ ID NO: 3), CDR2 (SEQ ID NO: 4), and CDR3 (SEQ ID NO: 5) of SEQ ID NO: 1, and a light chain variable region comprising CDRl (SEQ ID NO: 6), CDR2 (SEQ ID NO: 7), and CDR3 (SEQ ID NO: 8) of SEQ ID 2; (b) a heavy chain variable region comprising CDR1 (SEQ ID NO: 1 1), CDR2 (SEQ ID NO: 12), and CDR3 (SEQ ID NO: 13) of SEQ ID NO: 9, and a light chain variable region comprising CDR1 (SEQ ID NO: 14), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 8) of SEQ ID 16;

(c) a heavy chain variable region comprising CDR1 (SEQ ID NO: 19), CDR2 (SEQ ID NO: 20), and CDR3 (SEQ ID NO: 21) of SEQ ID NO: 17, and a light chain variable region comprising CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 23), and CDR3 (SEQ ID NO: 24) of SEQ ID 18; or

(d) a heavy chain variable region comprising CDR1 (SEQ ID NO: 27), CDR2 (SEQ ID NO: 28), and CDR3 (SEQ ID NO: 29) of SEQ ID NO: 25, and a light chain variable region comprising CDR1 (SEQ ID NO: 30), CDR2 (SEQ ID NO: 31), and CDR3 (SEQ ID NO: 32) of SEQ ID 26.

Claim 5. The antibody or fragment thereof of any one of claims 1-4, wherein the heavy chain and light chain variable regions are connected by a flexible linker to form a single- chain antibody.

Claim 6. The antibody or fragment thereof of any one of claims 1-4, wherein the antibody or fragment thereof is a single-chain Fv antibody.

Claim 7. The antibody or fragment thereof of any one of claims 1-4, wherein the antibody or fragment thereof is a Fab antibody.

Claim 8. The antibody or fragment thereof of any one of claims 1-4, wherein the antibody or fragment thereof is a Fab' antibody.

Claim 9. The antibody or fragment thereof of any one of claims 1-4, wherein the antibody or fragment thereof is a (Fab¾ antibody.

Claim 10. The antibody or fragment thereof of any one of claims 1-4, wherein the antibody or fragment thereof inhibits IL-13 activity.

Claim 1 1. The antibody or fragment thereof of any one of claims 1 -4, wherein the antibody or fragment thereof dissociates from human IL-13 with a ¾ of about 9 x 10^~10 or less and neutralizes human IL-13 bioactivity in a standard in vitro assay with an IC₅₀ of about 6 x 10^-9 M or less.

Claim 12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the antibody or fragment thereof of any one of claims 1-4.

Claim 13. An isolated nucleic acid molecule that encodes:

(a) a heavy chain variable region comprising SEQ ID NO: 1;

(b) a heavy chain variable region comprising SEQ ID NO: 9;

(c) a heavy chain variable region comprising SEQ ID NO: 17; or

(d) a heavy chain variable region comprising SEQ ID NO: 25.

Claim 14. An isolated isolated nucleic acid molecule that encodes:

(a) a light chain variable region comprising SEQ ID NO: 2;

(b) a light chain variable region comprising SEQ ID NO: 10;

(c) a light chain variable region comprising SEQ ID NO: 18; or

(d) a light chain variable region comprising SEQ ID NO: 26.

Claim 15. A vector comprising the nucleic acid molecule of claim 13 or 14.

Claim 16. An isolated host cell comprising the nucleic acid molecule of claim 13 or 14.

Claim 17. An isolated host cell comprising the vector of claim 15.

Claim 18. A method for treating or ameliorating an IL-13 mediated, Th2-mediated, or allergic disease, disorder, or condition, comprising administering to a patient in need thereof a pharmaceutically effective amount of the antibody or fragment thereof of any one of claims 1-4.

Claim 19. The method of claim 18, wherein the IL-13 mediated, Th2-mediated, or allergic disease, disorder, or condition is Hodgkin's disease, asthma, allergic asthma, aopic dermatitis, atopic allergy, ulcerative colitis, scleroderma, allergic rhinitis, COPD3 idiopathic pulmonary fibrosis, chronic graft rejection, bleomycin-induced pulmonary fibrosis, radiation- induced pulmonary fibrosis, pulmonary granuloma, progressive systemic sclerosis, schistosomiasis, hepatic fibrosis, renal cancer, Burkitt lymphoma, Hodgkins disease, non~Hodgkins disease, Sezary syndrome, asthma, septic arthritis, dermatitis herpetiformis, chronic idiopathic urticaria, ulcerative colitis, scleroderma, hypertrophic scarring, Whipple's Disease, benign prostate hyperplasia, an allergic reaction to a medication, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Grave's disease, pre-eclampsia, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, cystic fibrosis, allergic

bronchopulmonary mycosis, chronic obstructive pulmonary disease, bleornycin-induced pneumopathy and fibrosis, pulmonary alveolar proteinosis, adull respiratory distress syndrome, sarcoidosis, hyper IgE syndrome, idiopathic hypereosinophil syndrome, an autoimmune blistering disease, pemphigus vulgaris, bullous pemphigoid, myasthenia gravis, chronic fatigue syndrome, or nephrosis.

Claim 20. The use of a composition comprising the antibody or fragment thereof of any one of claims 1 -4 for the manufacture of a medicament for treating or ameliorating an IL- 13 mediated, Th2-mediated, or allergic disease, disorder, or condition.

Claim 21. The use of claim 20, wherein the IL- 13 mediated, Th2-mediated, or allergic disease, disorder, or condition is Hodgkin's disease, asthma, allergic asthma, aopic dermatitis, atopic allergy, ulcerative colitis, scleroderma, allergic rhinitis, COPD3 idiopathic pulmonary fibrosis, chronic graft rejection, bleomycin-induced pulmonary fibrosis, radiation-induced pulmonary fibrosis, pulmonary granuloma, progressive systemic sclerosis, schistosomiasis, hepatic fibrosis, renal cancer, Burkitt lymphoma, Hodgkins disease, non~Hodgkins disease, Sezary syndrome, asthma, septic arthritis, dermatitis herpetiformis, chronic idiopathic urticaria, ulcerative colitis, scleroderma, hypertrophic scarring, Whipple's Disease, benign prostate hyperplasia, an allergic reaction to a medication, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Grave's disease, pre-eclampsia, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, cystic fibrosis, allergic bronchopulmonary mycosis, chronic obstructive pulmonary disease, bleornycin-induced pneumopathy and fibrosis, pulmonary alveolar proteinosis, adull respiratory distress syndrome, sarcoidosis, hyper IgE syndrome, idiopathic hypereosinophil syndrome, an autoimmune blistering disease, pemphigus vulgaris, bullous pemphigoid, myasthenia gravis, chronic fatigue syndrome, or nephrosis.

Claim 22. A composition comprising the antibody or fragment thereof of any one of claims 1 -4 for the treatment or amelioration of an IL- 13 mediated, Th2-mediated, or allergic disease, disorder, or condition.

Claim 23. The composition of claim 22, wherein the IL-13 mediated, Th2-mediated, or allergic disease, disorder, or condition is Hodgkin's disease, asthma, allergic asthma, aopic dermatitis, atopic allergy, ulcerative colitis, scleroderma, allergic rhinitis, COPD3 idiopathic pulmonary fibrosis, chronic graft rejection, bleomycin-induced pulmonary fibrosis, radiation- induced pulmonary fibrosis, pulmonary granuloma, progressive systemic sclerosis, schistosomiasis, hepatic fibrosis, renal cancer, Burkitt lymphoma, Hodgkins disease, non~Hodgkins disease, Sezary syndrome, asthma, septic arthritis, dermatitis herpetiformis, chronic idiopathic urticaria, ulcerative colitis, scleroderma, hypertrophic scarring, Whipple's Disease, benign prostate hyperplasia, an allergic reaction to a medication, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Grave's disease, pre-eclampsia, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, cystic fibrosis, allergic

bronchopulmonary mycosis, chronic obstructive pulmonary disease, bleornycin-induced pneumopathy and fibrosis, pulmonary alveolar proteinosis, adull respiratory distress syndrome, sarcoidosis, hyper IgE syndrome, idiopathic hypereosinophil syndrome, an autoimmune blistering disease, pemphigus vulgaris, bullous pemphigoid, myasthenia gravis, chronic fatigue syndrome, or nephrosis.