Anti-il13 antigen binding proteins

Abstract

To provide an anti-human IL-13 antibody that binds with high affinity not only to human IL-13 but also to cynomolgus monkey IL-13.SOLUTION: The present invention is related to antibodies directed to IL-13 and uses of such antibodies. For example, in accordance with the present invention, human monoclonal antibodies directed to IL-13 are provided. Isolated polynucleotide sequences encoding heavy and light chain immunoglobulin molecules, and amino acid sequences comprising the same, particularly sequences corresponding to contiguous heavy and light chain sequences spanning the framework regions (FR's) and / or complementarity determining regions (CDR's), are provided. Additionally, methods of using these antibodies to treat patients are also provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 879,335, filed July 26, 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference

[0001] This application contains a Sequence Listing in computer-readable format. The Sequence Listing is provided as a text file named A-2421-WO-PCT_SeqList_ST25.txt, created on July 24, 2020, and is 205,493 bytes in size. The information in the electronic format of this Sequence Listing is incorporated herein by reference in its entirety.

[0003] The present invention relates to the field of biopharmaceuticals. In particular, the present invention relates to antibodies and IL-13-binding fragments and derivatives thereof that specifically bind to human IL-13. The present invention also relates to pharmaceutical compositions containing anti-IL-13 antibodies for treating inflammatory diseases, and methods for producing such antibodies. [Background technology]

[0004] IL-13 was the first cytokine recognized for its effects on B cells and monocytes, upregulating class II expression, promoting IgE class switching, and inhibiting the production of proinflammatory cytokines. The IL-13 receptor shares the IL-4 receptor alpha chain with the IL-4 receptor. As a result, IL-13 has many similar biological activities to IL-4.

[0005] IL-13 inhibits inflammatory cytokine release in vivo and has anti-inflammatory effects. IL-13 plays a role in IgE-mediated allergic responses and is a central mediator of allergic asthma (Wills-Karp M., Curr. Opin. Pulm. Med., 2003;9:21-27). In the lung, IL-13 regulates eosinophilic inflammation, mucus secretion, and airway hyperresponsiveness. In addition to asthma, IL-13 is involved in the pathogenesis of numerous diseases (Wynn TA. Annu. Rev. Immunol. 2003.21:425-456).

[0006] The human antibody Ab731 binds to human IL-13 with high affinity. However, this antibody binds to cynomolgus monkeys (macaca fascicularis, also referred to as "cyno" IL-13 ("cyIL-13")) with relatively low affinity. Because cynomolgus monkeys are commonly used to evaluate the preclinical safety of antibodies, it would be desirable to have an anti-human IL-13 antibody that also binds to cynomolgus IL-13 with high affinity. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Wills-Karp M.,Curr.Opin.Pulm.Med.,2003;9:21-27 [Non-patent document 2] Wynn TA.Annu.Rev.Immunol.2003.21:425-456 Summary of the Invention

[0008] It is desirable for therapeutic antibodies to exhibit high affinity binding to both the human and cynomolgus monkey orthologs of the therapeutic target. The affinity gap is required to be within a 10-fold affinity window to enable toxicological testing. The AMGN12 antibody, derived from in vivo immunization in XenoMouse®, demonstrated many favorable properties, including single-digit pM affinity for the human ortholog of the target protein. However, the antibody exhibited 200-fold weaker binding to the ortholog present in cynomolgus monkeys. The goal of this study was to "close" the affinity gap without compromising binding affinity for the human target and to identify mutants with over 100-fold improved affinity for the cynomolgus monkey ortholog and 10-fold improved potency in functional biological assays. Resolving high-resolution crystal structures of the final mutant antibodies in complex with the target demonstrated that the mutations resulting in the improved affinity obtained by the HuTARG platform would be difficult to design a priori in silico.

[0009] The present invention relates to the field of biopharmaceuticals. In particular, the present invention relates to antibodies that specifically bind to human IL-13 and cyno IL-13, as well as IL-13-binding fragments and derivatives thereof. The present invention also relates to pharmaceutical compositions containing anti-IL-13 antibodies for treating inflammatory diseases, and methods for producing such antibodies.

[0010] In embodiments, anti-IL-13 antibodies, antigen (IL-13) binding fragments, and derivatives (collectively referred to as "antigen binding proteins") relate to the field of biopharmaceuticals. The present invention relates to anti-IL-13 antibodies and other IL-13 binding proteins that specifically bind to human IL-13. The present invention also relates to pharmaceutical compositions comprising anti-IL-13 antigen binding proteins for treating inflammatory diseases, and methods for making such antibodies.

[0011] In one embodiment of the present invention, minor amino acid sequence changes to the CDRs of Ab731 increase binding to cynomolgus IL-13.

[0012] The present invention includes the following embodiments: 1. An antigen-binding protein that specifically binds to human IL-13, comprising a light chain immunoglobulin variable region (VL1) and a heavy chain immunoglobulin variable region (VH), VL1 comprises (i) a CDRL1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) a CDRL2 comprising the amino acid sequence of SEQ ID NO: 12, and (iii) a CDRL3 comprising the amino acid sequence of SEQ ID NO: 13; An antigen-binding protein, wherein the VH comprises the amino acid sequences of (i) CDRH1 comprising the amino acid sequence of SEQ ID NO: 8, (ii) CDRH2 comprising the amino acid sequence of SEQ ID NO: 9, and (iii) CDRH3 comprising the amino acid sequence of SEQ ID NO: 10.

[0013] 2. An antigen-binding protein that specifically binds to human IL-13, comprising a light chain immunoglobulin variable region (VL1) and a heavy chain immunoglobulin variable region (VH), VL comprises the CDRs of the antibody expressed by cell 623, An antigen-binding protein in which the VH comprises the CDRs of an antibody expressed by cell 623.

[0014] 3. The antigen binding protein of embodiment 1, further comprising a framework region similar to an antibody expressed by cell 623.

[0015] 4. The antigen-binding protein of any one of embodiments 1 to 3, wherein the antigen-binding protein is an antibody.

[0016] 5. The antigen-binding protein of any one of embodiments 1 to 3, wherein the antigen-binding protein is an antibody fragment.

[0017] 6. The antigen-binding protein of any one of embodiments 1 to 3, wherein the antigen-binding protein is an antibody derivative, including a bispecific antibody, a fusion protein.

[0018] 7. The antigen binding protein of any one of embodiments 1-6, wherein the antigen binding protein has a human sequence.

[0019] 8. The antigen-binding protein of any one of embodiments 1 to 6, wherein the antigen-binding protein is a monoclonal antibody.

[0020] K of 9.2 cM to 50 pM D A human antibody that binds to IL-13.

[0021] K of 10.2 cM to 40 pM D A human antibody that binds to IL-13.

[0022] 11. A human antibody or antigen-binding fragment thereof that binds to human IL-13, having the amino acid sequence: (a) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 11, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 13; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 8, an HCDR2 of SEQ ID NO: 106, and an HCDR3 of SEQ ID NO: 10; (b) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 11, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 13; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 8, an HCDR2 of SEQ ID NO: 83, and an HCDR3 of SEQ ID NO: 10; (c) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 11, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 13; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 8, an HCDR2 of SEQ ID NO: 83, and an HCDR3 of SEQ ID NO: 10; (d) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 74, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 76; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 107, an HCDR2 of SEQ ID NO: 85, and an HCDR3 of SEQ ID NO: 10; (e) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 77, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 76; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 107, an HCDR2 of SEQ ID NO: 85, and an HCDR3 of SEQ ID NO: 10; (f) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 79, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 78; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 107, an HCDR2 of SEQ ID NO: 85, and an HCDR3 of SEQ ID NO: 10; (g) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 79, an LCDR2 of SEQ ID NO: 80, and an LCDR3 of SEQ ID NO: 78; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 107, an HCDR2 of SEQ ID NO: 85, and an HCDR3 of SEQ ID NO: 10; (h) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 81, an LCDR2 of SEQ ID NO: 80, and an LCDR3 of SEQ ID NO: 78; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 107, an HCDR2 of SEQ ID NO: 85, and an HCDR3 of SEQ ID NO: 10; and (i) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 82, an LCDR2 of SEQ ID NO: 80, and an LCDR3 of SEQ ID NO: 78; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 107, an HCDR2 of SEQ ID NO: 85, and an HCDR3 of SEQ ID NO: 10.

[0023] 12. A human antibody or antigen-binding fragment thereof that binds to human IL-13, having the amino acid sequence: (a) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 86 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 87; (b) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 88 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 89; (c) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 90 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 91; (d) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 92 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 93; (e) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 94 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 95; (f) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 96 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 97; (g) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 98 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 99; (h) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 100 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 101; (i) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 102 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 103; and (j) a human antibody or antigen-binding fragment thereof, comprising a variable light chain region and a variable heavy chain region selected from the group comprising: an antibody variable light chain amino acid sequence comprising SEQ ID NO: 104; and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 105.

[0024] 13. A human antibody or antigen-binding fragment thereof that binds to human IL-13, having the amino acid sequence: (a) a light chain selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37; SEQ ID NO:39, SEQ ID NO:41; SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, and SEQ ID NO:73; and (b) a human antibody or antigen-binding fragment thereof comprising a heavy chain selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36; SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:72.

[0025] 14. An antibody comprising a light chain and a heavy chain having an amino acid sequence according to any one of embodiments 1 to 14, 20, 25 to 27.

[0026] 15. A nucleic acid sequence encoding the antibody or antibody fragment thereof according to any one of embodiments 1 to 14.

[0027] 16. A vector comprising a nucleic acid sequence encoding the antibody or antibody fragment thereof according to any one of embodiments 15, 20, 25 to 27.

[0028] 17. A host cell comprising the vector of embodiment 16.

[0029] 18. The host cell according to embodiment 17, wherein the host cell is a CHO cell or an Sp2 / 0 cell.

[0030] 19. The host cell of embodiment 18, wherein the host cell is a CHO cell.

[0031] 20. An antibody or antibody fragment produced by a host cell according to any one of embodiments 18 to 19.

[0032] 21. A pharmaceutical composition comprising an antibody or antibody fragment according to embodiment 20.

[0033] 22. A method for producing an antibody or a fragment thereof by culturing a host cell according to any one of embodiments 17 to 19.

[0034] 23. A method of treating a patient suffering from COPD, emphysema, asthma, or atopic dermatitis by administering to said patient an effective amount of the antibody or fragment thereof of embodiment 20.

[0035] 24. A method of treating a patient suffering from COPD, emphysema, asthma, or atopic dermatitis by administering to said patient an effective amount of the pharmaceutical composition of embodiment 21.

[0036] 25. The antibody of embodiment 20, wherein a half-life extending mutation is present.

[0037] 26. The antibody of embodiment 25, wherein the half-life extending mutations are mutations at Eu positions M252Y, S254T, and T256E within Fc.

[0038] 27. The antibody of embodiment 25, wherein the half-life extending mutation is a complement hexamer disrupting mutation at Eu position S583K in Fc. [Brief explanation of the drawings]

[0039] The present invention will be better understood from the detailed description and accompanying drawings, which are meant to illustrate, not to limit, the invention. [Figure 1] Figure 1 shows a plot of the neutralization data for each well versus relative antibody concentration. The data was used to identify wells with the most potent antibodies. [Figure 2] Figure 2 is not a plot showing the relationship between antigen coating, it is a plot of the ELISA OD for each antibody sample at 31 ng / mL Ag coating against the concentration of antibody. [Figure 3] FIG. 3 is a graph showing the percent inhibition of IL-13-induced eotaxin release by recombinant antibodies 643 and 731 compared to isotype-matched controls. [Figure 4] FIG. 4 is a bar graph comparing the ability of IL-13 or IL-13Q110R to inhibit the binding of 731 or 623 to IL-13 coated ELISA plates. [Figure 5A] Figure 5A is a bar graph comparing cell receptor competition between antibody 643 and an isotype control. [Figure 5B] Figure 5B is a bar graph comparing cell receptor competition between antibody 731 and an isotype control. [Figure 5C] Figure 5C is a schematic diagram showing the protocol of Figure 5A and various predicted results. [Figure 5D]Figure 5D is a schematic diagram showing the protocol of Figure 5B and various predicted results, which we will call 5C. These modifications may facilitate tracking. [Figure 6A] FIG. 6A shows an alignment of the phage display-derived peptide recognized by antibody 693 with a portion of the IL-13 sequence. [Figure 6B] FIG. 6B is a chart showing the secondary structure of IL-13 and indicates which regions of human IL-13 were substituted with mouse IL-13 to construct the chimeric protein. [Figure 7] FIG. 7 is a chart showing the different bins into which different antibodies can be classified. [Figure 8A] 8A and 8B are bar graphs showing that CD4+ T cells from humanized IL-13 mice produce human IL-13 but not mouse IL-13. [Figure 8B] 8A and 8B are bar graphs showing that CD4+ T cells from humanized IL-13 mice produce human IL-13 but not mouse IL-13. [Figure 9] FIG. 9 is a graph showing that anti-IL-13 antibodies 731 and 623 inhibit airway hyperresponsiveness. [Figure 10] FIG. 10 is a bar graph showing that 731 and 623 inhibit mucus production. [Figure 11] FIG. 11 shows the crystal structure of the interaction between an affinity matured anti-IL-13 antibody and IL-13. [Figure 12A] FIG. 12A shows the crystal structure details of the interaction between an affinity matured anti-IL-13 antibody and IL-13. [Figure 12B] FIG. 12B shows the crystal structure details of the interaction between an affinity matured anti-IL-13 antibody and IL-13. [Figure 13] FIG. 13 shows the crystal structure details of the interaction between an affinity matured anti-IL-13 antibody and IL-13. [Figure 14] FIG. 14 is a chart showing the amino acid sequences of high affinity anti-IL13 antibodies with half-life extending mutations. DETAILED DESCRIPTION OF THE INVENTION

[0040] Embodiments of the present invention relate to isolated antibodies that bind to IL-13 and methods of using those antibodies to treat diseases in humans. Preferably, the antibodies are fully human neutralizing monoclonal antibodies that bind to IL-13 with high affinity, high potency, or both. In one embodiment, the antibody or antibody fragment specifically binds to a region of the IL-13 molecule and prevents the molecule from signaling through the IL-13 receptor complex.

[0041] Further, embodiments of the present invention include methods of using these anti-IL-13 antibodies as diagnostic agents or treatments for diseases. For example, the antibodies may be used to treat conditions such as asthma (including both allergic (atopic) and non-allergic (non-atopic)), bronchial asthma, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), hay fever, rhinitis, urticaria, angioedema, allergic dermatitis (including contact dermatitis), Stevens-Johnson syndrome, anaphylactic shock, food allergies, keratitis, conjunctivitis, steroid-resistant nephritic syndrome, mastocytosis, fibrotic diseases (e.g., pulmonary fibrosis, including idiopathic pulmonary fibrosis, cystic fibrosis, bleomycin, erythrocyte sedimentation syndrome, leukemia ... The compounds are useful for treating diseases such as leukemia, leukemia, inflammatory bowel disease, leukemia, leukemia, leukemia-induced fibrosis, hepatic fibrosis, and systemic sclerosis), cancers such as Hodgkin's disease, B-cell proliferative disorders (e.g., B-cell lymphoma, particularly mediastinal large B-cell lymphoma), B-cell leukemia, ovarian cancer, diseases characterized by non-malignant B-cell proliferation such as systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, and cryoglobulinemia, diseases characterized by high levels of autoantibodies such as hemolytic anemia, thrombocytopenia, phospholipid syndrome, and pemphigus, inflammatory bowel disease, and graft-versus-host disease.

[0042] In connection with such treatments, embodiments of the invention include articles of manufacture comprising the antibodies. One embodiment of the invention is an assay kit comprising an IL-13 antibody used to screen for diseases or disorders associated with IL-13 activity.

[0043] The nucleic acids described herein, and fragments and variants thereof, can be used, by way of non-limiting example, (a) as recombinant or heterologous gene products to direct the biosynthesis of corresponding encoded proteins, polypeptides, fragments, and variants, (b) as probes for the detection and quantification of the nucleic acids disclosed herein, (c) as sequence templates for preparing antisense molecules, etc. Such uses are described more fully below.

[0044] In one aspect, methods for identifying these antibodies are provided, which in one embodiment comprise an eotaxin release assay.

[0045] In one aspect, antibodies that bind to mutants of IL-13 are also provided. Of particular relevance are antibodies that bind to IL-13 mutants that have a glutamine at position 110 of the endogenous IL-13 polypeptide.

[0046] In one embodiment, a mouse humanized for human IL-13 is provided, which is useful for providing a subject for testing airway hyperresponsiveness and inhibition of mucus production.

[0047] Definition: Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0048] Generally, the nomenclature used in connection with, and techniques relating to, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art, as described in various general and more specific references, such as those cited and discussed throughout this specification. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2000, pp. 111-114, 1997.nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), incorporated herein by reference. Standard techniques (e.g., electroporation, lipofection) are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation. Enzymatic reactions and purification procedures are performed according to manufacturer's instructions or as commonly accomplished in the art or as described herein. Standard techniques may also be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0049] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0050] "Polymerase chain reaction" or "PCR" refers to a procedure or technique by which minute amounts of specific pieces of nucleic acid, RNA and / or DNA, are amplified, as described in U.S. Pat. No. 4,683,195, issued July 28, 1987. Generally, sequence information from the ends of the region of interest or beyond must be available so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may correspond to the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987); Erlich, ed., PCR Technology (Stockton Press, NY, 1989). As used herein, PCR is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which involves the use of known nucleic acids as primers and a nucleic acid polymerase to amplify or generate specific pieces of nucleic acid.

[0051] "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. Antibodies exhibit binding specificity to a specific antigen, while immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. The latter type of polypeptides is produced, for example, at low levels by the lymphatic system and at increased levels by myelomas.

[0052] Antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical, substantially full-length light (L) chains and two identical, substantially full-length heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds varies among heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end, with the light chain constant domain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains (Chothia et al. J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:4592 (1985); Chothia et al., Nature 342:877-883 (1989)).

[0053] "Antibody fragment" includes a fragment of an antibody that binds to a target antigen. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments.

[0054] As used herein, "antigen-binding protein" refers to a protein that specifically binds to a particular antigen derived from an antibody. Examples of antigen-binding proteins include, but are not limited to, antibodies, antibody fragments, antibody constructs, fusion proteins, bispecific antibodies, and scFv proteins.

[0055] The antigen-binding protein has a dissociation constant (KD) of ≦10 as measured by surface plasma resonance techniques (e.g., BIACore, GE-Healthcare Uppsala, Sweden) or equilibrium exclusion binding techniques (KinExA, Sapidyne, Boise, Idaho). -7When an antigen-binding protein binds to an antigen at M, it is said to "specifically bind" to that antigen.

[0056] The antigen binding proteins of the invention are capable of neutralizing and inhibiting the binding of IL-13 to signaling receptors such as IL-13 receptor alpha-1 (IL-13Rα1) by at least 60% or 80%, more typically by more than about 85%, as measured in an in vitro competitive binding assay. In one embodiment, the antibody also inhibits binding to the decoy receptor IL-13Rα2, while in other embodiments, the ability of IL-13 to bind to IL-13Rα2 is maintained upon binding of the antibody to IL-13.

[0057] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes." There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0058] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. 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, monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0059] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody is purified to (1) greater than 95% by weight, as determined by the Lowry method and terminal or internal amino acid sequence using a spinning cup sequencer, or (2) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, or preferably silver staining. Isolated antibody includes antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0060] A "neutralizing antibody" is an antibody molecule that can eliminate or significantly reduce the effector function of a target antigen to which it binds. Thus, a "neutralizing" IL-13 antibody can eliminate or significantly reduce an effector function, such as IL-13 signaling activity via the IL-13 receptor. In one embodiment, a neutralizing antibody reduces an effector function by 1-10%, 10-20%, 20-30%, 30-50%, 50-70%, 70-80%, 80-90%, 90-95%, 95-99%, or 99-100%.

[0061] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which nonspecific cytotoxic cells expressing Ig Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on target cells, subsequently causing lysis of the target cell. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay (e.g., that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337) can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1988).

[0062] The term "variable" refers to the fact that certain portions of the variable domains differ significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. In both Ig light and heavy chain variable domains, variability is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions that largely adopt a β-sheet configuration, connected by three CDRs that form loops connecting and occasionally forming part of the β-sheet structure. The CDRs of each chain are held in close proximity together by the FR regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991)). The constant domains are not directly involved in binding an antibody to an antigen but exhibit various effector functions, such as the participation of antibodies in antibody-dependent cellular cytotoxicity.

[0063] Digestion of antibodies with the enzyme papain produces two identical antigen-binding fragments, also known as "Fab" fragments, and an "Fc" fragment, which lacks antigen-binding activity but has the ability to crystallize. Digestion of antibodies with the enzyme pepsin produces an F(ab')2 fragment, in which the two arms of the antibody molecule remain linked and contain two antigen-binding sites. The F(ab')2 fragment has the ability to cross-link antigen.

[0064] "Fv," as used herein, refers to the minimum antibody fragment that retains both the antigen-recognition and antigen-binding sites.

[0065] "Fab," as used herein, refers to a fragment of an antibody containing the constant domain of the light chain and the CH1 domain of the heavy chain.

[0066] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. In two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In single-chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker, such that the light and heavy chains can associate in a "dimeric" structure similar to that in two-chain Fv species. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0067] "Fusion protein" refers to a protein comprising an antibody fragment linked to another protein.

[0068] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally consist of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-62 (L2), and 89-97 (L3) of the light chain variable domain and residues 31-55 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5 thEd. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light-chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy-chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0069] The term "complementarity determining region" or "CDR," as used herein, refers to the portion of an immune receptor that contacts a particular ligand and determines its specificity. The CDRs of an immune receptor are the most variable parts of the receptor protein, conferring diversity to the receptor, and are carried on six loops at the distal end of the receptor's variable domain, with three loops derived from each of the receptor's two variable domains.

[0070] The term "epitope" is used to refer to the binding site of an antibody on a protein antigen. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to bind to an antigen when the dissociation constant is ≦1 μM, preferably ≦100 nM, and most preferably ≦10 nM. An increased or larger equilibrium constant ("K D ") means that the affinity between the epitope and the antibody is less. In other words, the antibody and the epitope are less likely to bind or remain bound together. A decreased or lower equilibrium constant means that the affinity between the epitope and the antibody is greater. In other words, the antibody and the epitope are more likely to bind or remain bound together. A K "less than" or equal to some degree D means that the antibody binds to the epitope with a given affinity or more strongly (or tightly).

[0071] K D describes the epitope and binding properties of an antibody, while "potency" describes the effectiveness of the antibody itself in terms of its function. D However, this does not necessarily mean high potency. Therefore, antibodies with relatively low K D and high potency (e.g., they bind well and potently alter function), relatively high K D and high potency (e.g., they do not bind well but strongly affect function), relatively low K D and low potency (e.g., they bind well but not in a form that is effective in altering a specific function), or a relatively high K D and low potency (e.g., they simply do not bind well to the target). In one embodiment, high potency means that there is a high level of inhibition at a low concentration of antibody. In one embodiment, an antibody has an IC 50 is a small number, for example, 130-110, 110-90, 90-60, 60-30, 30-25, 25-20, 20-15, or less pM, it is potent or has high efficacy.

[0072] "Substantially," unless otherwise specified in conjunction with another term, means that a value may vary within any amount that can be attributed to errors of measurement that may occur in making or practicing an embodiment. "Significantly" means that a value may vary sufficiently to enable the claimed invention to function for its intended use.

[0073] The term "amino acid" or "amino acid residue," as used herein, refers to naturally occurring L-amino acids or D-amino acids, as further described below with respect to variants. Commonly used one-letter and three-letter abbreviations for amino acids are used herein (Bruce Alberts et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York (3rd ed. 1994)).

[0074] The term "mAb" refers to a monoclonal antibody.

[0075] The term "human antibody" refers to an antibody in which the majority (at least 95%) of the antibody sequence is derived from the human genome.

[0076] The term "XENOMOUSE®" refers to a mouse strain engineered to contain 245 kb and 190 kb sized germline fragments of the human heavy chain and kappa light chain loci as described in Green et al. Nature Genetics 7:13-21 (1994), incorporated herein by reference. The XENOMOUSE® strain is available from Abgenix, Inc. (Fremont, CA).

[0077] The term "XENOMAX®" when used with XENOMOUSE® animals refers to the use of the "Selected Lymphocyte Antibody Method" (Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996)).

[0078] The term "SLAM®" refers to the "Selective Lymphocyte Antibody Method" (Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996), and Schrader, U.S. Pat. No. 5,627,052, both of which are incorporated herein by reference in their entireties).

[0079] The terms "disease," "disease state," and "disorder" refer to a physiological condition of a cell or a whole mammal in which a cellular or bodily function, system, or organ is disrupted, stopped, or impaired.

[0080] The term "symptom" means any physical or observable sign of a disorder, whether or not generally characteristic of that disorder. The term "symptom" can refer to all such signs or any subset thereof.

[0081] The term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (attenuate) the onset or spread of an undesirable physiological change or disorder, such as cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of the progression of the disease, improvement or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" also means prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder, or those in whom the condition or disorder is to be prevented. The term "inhibit," when used in conjunction with a disease or symptom, can mean that the antibody may reduce or eliminate the disease or symptom.

[0082] The term "patient" includes human and animal subjects.

[0083] For purposes of treatment, "administering" means delivering to a patient. For example, and without limitation, such delivery may be intravenous, intraperitoneal, by inhalation, intramuscular, subcutaneous, oral, topical, transdermal, or surgical.

[0084] A "therapeutically effective amount" for purposes of treatment means that amount such that an observable change in a patient's condition and / or symptoms can result from its administration, either alone or in combination with other treatments.

[0085] A "pharmaceutically acceptable vehicle" for purposes of treatment is a physical entity that can be administered to a patient. A pharmaceutically acceptable vehicle can be, but is not limited to, a pill, capsule, caplet, tablet, oral fluid, injectable fluid, spray, aerosol, lozenge, dietary supplement, cream, lotion, oil, solution, paste, powder, inhalant, or liquid. One example of a pharmaceutically acceptable vehicle is a buffered isotonic solution such as phosphate-buffered saline (PBS).

[0086] "Neutralize" for purposes of treatment means to partially or completely inhibit chemical and / or biological activity.

[0087] "Downregulation," for purposes of treatment, means decreasing the level of a particular target composition.

[0088] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, sport, or pet animals, such as monkeys, dogs, horses, cats, cows, etc.

[0089] The term "polynucleotide," as referred to herein, means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or a modified form of either type of nucleotide. The term includes single- and double-stranded forms of DNA.

[0090] As used herein, the term "isolated polynucleotide" shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, and by its origin, an "isolated polynucleotide" is one that: (1) is not associated with all or a portion of a polynucleotide with which it is found in nature; (2) is operably linked to a polynucleotide with which it is not linked in nature; or (3) is not found in nature as part of a larger sequence.

[0091] The term "oligonucleotide" as referred to herein includes naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. An oligonucleotide is a portion of a polynucleotide generally comprising a length of 200 bases or less. Preferably, oligonucleotides are 10-60 bases in length, most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 bases in length. Oligonucleotides are usually single-stranded, e.g., for probes, but oligonucleotides may also be double-stranded, e.g., for use in constructing gene mutants. Oligonucleotides may be either sense or antisense oligonucleotides.

[0092] As used herein, the term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides having modified or substituted sugar groups, etc. The term "oligonucleotide linkage" referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoroamidate, etc. See, for example, LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984); Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp.87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990) (the disclosures of which are incorporated herein by reference). Oligonucleotides can optionally contain a label for detection.

[0093] The term "selectively hybridize" as used herein means detectably and specifically bind. Polynucleotides, oligonucleotides, and fragments thereof selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions, as known in the art and discussed herein. Generally, the nucleic acid sequence homology between a polynucleotide, oligonucleotide, or antibody fragment and a nucleic acid sequence of interest is at least 80%, and more typically, the homology is preferably increased to at least 85%, 90%, 95%, 99%, and 100%.

[0094] As used herein, the term "control sequence" refers to polynucleotide sequences necessary to effect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include a promoter, a ribosomal binding site, and a transcription termination sequence; in eukaryotes, such control sequences generally include a promoter and a transcription termination sequence. The term "control sequence" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0095] As used herein, the term "operably linked" refers to the positioning of components described herein in a relationship permitting them to function in their intended manner. For example, a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0096] The term "isolated protein" as referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof; depending on its origin or source, an "isolated protein" is (1) not associated with proteins found in nature, (2) free of other proteins from the same source (e.g., free of murine proteins), (3) expressed by cells from a different species, or (4) non-naturally occurring.

[0097] The term "polypeptide" is used herein as a general term to refer to a naturally occurring protein, fragment, or analog of a polypeptide sequence. Naturally occurring proteins, fragments, and analogs are therefore species of the polypeptide genus. Polypeptides according to the present invention include, for example, human heavy chain immunoglobulin molecules represented in Tables 1 and 21 by SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, and 83-105, and human kappa light chain immunoglobulin molecules represented, for example, by SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, and 106-126, as well as antibody molecules formed by combinations comprising heavy chain immunoglobulin molecules and light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules (and vice versa), and fragments and analogs thereof.

[0098] Unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction in which nascent RNA transcripts are added from 5' to 3' is referred to as the transcription direction. The sequence region on the DNA strand with the same sequence as the RNA and located 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence." The sequence region on the DNA strand with the same sequence as the RNA and located 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0099] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids such as alpha, alpha disubstituted amino acids, N-alkylamino acids, lactic acid, and other unconventional amino acids may also be suitable components of the polypeptides of the invention. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0100] The term "corresponding to" is used herein to mean that a polynucleotide sequence is homologous to all or a portion of a reference polynucleotide sequence (i.e., identical, but not strictly evolutionarily related), or that a polypeptide sequence is identical to a reference polypeptide sequence.

[0101] In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. By way of example, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA."

[0102] The following terms: "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," "substantial identity," and "homology" are terms used to describe the sequence relationships between two or more polynucleotide or amino acid sequences. A "reference sequence" is a predetermined sequence used as a basis for sequence comparison. A reference sequence may be a portion of a larger sequence, for example, a segment of a full-length cDNA or gene sequence set forth in a sequence listing, or may include the entire cDNA or gene sequence. Generally, a reference sequence is at least 18 nucleotides or 6 amino acids in length, often at least 24 nucleotides or 8 amino acids in length, and often at least 48 nucleotides or 16 amino acids in length. Because two polynucleotide or amino acid sequences each (1) contain similar sequences between the two molecules (i.e., a portion of the complete polynucleotide or amino acid sequence), and (2) may further contain sequences that differ between the two polynucleotide or amino acid sequences, sequence comparison between two (or more) molecules is usually performed by comparing the sequences of the two molecules over a "comparison window" to identify and compare local regions of sequence similarity.

[0103] As used herein, a "comparison window" refers to a conceptual segment of at least about 18 contiguous nucleotide positions or about 6 amino acids, in which the polynucleotide sequence or amino acid sequence is compared to a reference sequence of at least 18 contiguous nucleotide positions or 6 amino acids, and the portion of the polynucleotide sequence in the comparison window may contain no more than 20 percent additions, deletions, substitutions, etc. (i.e., gaps) when compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences over a comparison window can be achieved by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.), GENEWORKS™, or MACVECTOR™ software packages), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) produced by the various methods is selected.

[0104] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., nucleotide-by-nucleotide or residue-by-residue) over a comparison window. The term "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions in both sequences where the nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue is identical to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to determine the percentage of sequence identity. As used herein, the term "substantial identity" refers to a property of a polynucleotide or amino acid sequence that the polynucleotide or amino acid contains a sequence having at least 85 percent sequence identity, preferably at least 90-95 percent sequence identity, and more preferably at least 99 percent sequence identity, when compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions (often a comparison window of at least 24-48 nucleotide (8-16 amino acid) positions), where the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that may contain deletions or additions totaling no more than 20 percent of the reference sequence over the comparison window. The reference sequence may be a portion of a larger sequence.

[0105] Two amino acid or polynucleotide sequences are "homologous" if there is partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum correspondence. Gaps (in either of the two sequences being matched) are allowed in maximizing correspondence, with gap lengths of 5 or less being preferred, and 2 or less being more preferred. Alternatively, and preferably, two protein sequences (or polypeptide sequences derived therefrom that are at least about 30 amino acids in length) are homologous, as the term is used herein, if they have an alignment score of greater than 5 (standard deviation units) using the program ALIGN with a mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, MO, in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10. Two sequences or portions thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.

[0106] When applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned using programs such as GAP or BESTFIT with default gap weighting, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, amino acids with aliphatic side chains include glycine, alanine, valine, leucine, and isoleucine; amino acids with aliphatic-hydroxyl side chains include serine and threonine; amino acids with amide-containing side chains include asparagine and glutamine; amino acids with aromatic side chains include phenylalanine, tyrosine, and tryptophan; amino acids with basic side chains include lysine, arginine, and histidine; and amino acids with sulfur-containing side chains include cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0107] As discussed herein, minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated as encompassed by the present invention, provided that the variations in amino acid sequence are maintained at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids, relative to their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are the aliphatic hydroxy family; asparagine and glutamine are the amide-containing family; alanine, valine, leucine and isoleucine are the aliphatic family; and phenylalanine, tryptophan and tyrosine are the aromatic family.

[0108] For example, it is reasonable to expect that a single substitution of leucine with isoleucine or valine, a single substitution of aspartic acid with glutamic acid, a single substitution of threonine with serine, or a similar substitution of an amino acid with a structurally related amino acid will not significantly affect the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within a framework region. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein.

[0109] Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino or carboxy termini of fragments or analogs occur near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains present in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known (Bowie et al., Science 253:164 (1991)). The foregoing examples demonstrate that those skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the present invention.

[0110] Preferred amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acids should not tend to disrupt helices present in the parent sequence or other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991), each of which is incorporated herein by reference.

[0111] As used herein, the term "polypeptide fragment" refers to a polypeptide having an amino- and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in a naturally occurring sequence as deduced, for example, from a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, preferably at least 14 amino acids in length, and more preferably at least 20 amino acids in length. In other embodiments, polypeptide fragments are at least 25 amino acids in length, more preferably at least 50 amino acids in length, and even more preferably at least 70 amino acids in length.

[0112] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. These types of non-peptide compounds are called "peptide mimetics" or "peptidomimetics." Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al. J. Med. Chem. 30:1229 (1987) (which are incorporated herein by reference). Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to achieve the same therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to paradigm polypeptides (i.e., polypeptides with biochemical properties or pharmacological activity), such as human antibodies, but have one or more peptide bonds optionally replaced by a bond selected from the group consisting of -CHNH-, -CHS-, -CH-CH-, -CH=CH- (cis and trans), -COCH-, -CH(OH)CH-, and -CHSO-, by methods well known in the art. To generate more stable peptides, one or more amino acids of a consensus sequence can be systematically substituted with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine). Furthermore, constrained peptides containing the consensus sequence or substantially identical consensus sequence variations can be generated by methods well known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

[0113] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid or by attachment of a biotinyl moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). In certain circumstances, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0114] As used herein, the term "pharmaceutical product or drug" refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient. Other chemical terms herein are used in accordance with conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)), which is incorporated herein by reference.

[0115] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., the species is more abundant than any other individual species in the composition on a molar basis); preferably, a substantially purified fraction is a composition in which the target species constitutes at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will constitute greater than about 80%, more preferably greater than about 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. Most preferably, the target species is purified to substantial homogeneity (it is impossible to detect contaminating species in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species.

[0116] Antibody structure The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype as IgM, IgD, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 10 or more amino acids. (See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)), incorporated by reference in its entirety for all purposes.) The variable regions of each light / heavy chain pair form the antigen-binding site.

[0117] Thus, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.

[0118] All chains exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, thereby enabling binding to a specific epitope. From the N-terminus to the C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignment for each domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or the definitions in Chothia & Lesk J. Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

[0119] Bispecific or bifunctional antibodies are artificial hybrid antibodies with two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by various methods, including hybridoma fusion or Fab' fragment linkage. (See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al. J. Immunol. 148:1547-1553 (1992)). Compared with conventional antibody production, the production of bispecific antibodies can be a relatively labor-intensive process, and the yield and purity of bispecific antibodies are generally lower. Bispecific antibodies do not exist in the form of fragments with single binding sites (e.g., Fab, Fab', and Fv).

[0120] Human antibodies and antibody humanization Human antibodies avoid some of the problems associated with antibodies that have mouse or rat variable and / or constant regions. The presence of such mouse- or rat-derived proteins can lead to rapid clearance of the antibody or can cause an immune response against the antibody by the patient. To avoid the use of mouse- or rat-derived antibodies, fully human antibodies can be generated by introducing human antibody function into rodents such that the rodents produce fully human antibodies.

[0121] One method for producing fully human antibodies is through the use of the XENOMOUSE® strain of mice engineered to contain 245 kb and 190 kb sized germline fragments of the human heavy chain and kappa light chain loci. See Green et al. Nature Genetics 7:13-21 (1994). The XENOMOUSE® strain is available from Abgenix, Inc. (Fremont, CA).

[0122] The production of XENOMOUSE® is described in U.S. patent application Ser. Nos. 07 / 466,008, filed Jan. 12, 1990; 07 / 610,515, filed Nov. 8, 1990; 07 / 919,297, filed Jul. 24, 1992; 07 / 922,649, filed Jul. 30, 1992; 08 / 031,801, filed Mar. 15, 1993; and 08 / 031,801, filed Aug. 27, 1993. No. 08 / 112,848 filed on April 28, 1994, No. 08 / 234,145 filed on April 28, 1994, No. 08 / 376,279 filed on January 20, 1995, No. 08 / 430,938 filed on April 27, 1995, No. 08 / 464,584 filed on June 5, 1995, No. 08 / 464,582 filed on June 5, 1995, No. 08 / 463,191, filed June 5, 1995; 08 / 462,837, filed June 5, 1995; 08 / 486,853, filed June 5, 1995; 08 / 486,857, filed June 5, 1995; 08 / 486,859, filed June 5, 1995; 08 / 462,513, filed June 5, 1995; 08 / 724,752, filed October 2, 1996 This is further discussed and described in the specification and in U.S. Patent No. 08 / 759,620, filed December 3, 1996, as well as U.S. Patent Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598, and Japanese Patent Nos. 3068180, 3068506, and 3068507. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998).See also European Patent Application No. 0463151B1, published June 12, 1996, International Publication Nos. WO 94 / 02602, published February 3, 1994, WO 96 / 34096, published October 31, 1996, WO 98 / 24893, published June 11, 1998, and WO 00 / 76310, published December 21, 2000. The disclosures of each of the above-cited patents, applications, and references are incorporated herein by reference in their entirety.

[0123] In another approach, other companies, including GenPharm International, Inc., have utilized a "minilocus" approach, in which an exogenous Ig locus is mimicked by including small pieces (individual genes) from the Ig locus. Thus, one or more V H Gene, one or more D H Gene, one or more J HThe gene, the mu constant region, and the second constant region (preferably a gamma constant region) form a construct that is inserted into the animal. This approach is described in U.S. Pat. No. 5,545,807 by Surani et al., U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458, each by Lonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023,010 by Krimpenfort and Berns, and U.S. Pat. Nos. 5,591,669 and 6,023,010 by Berns et al. U.S. Patent Nos. 5,612,205, 5,721,367, and 5,789,215 to Choi and Dunn, U.S. Patent No. 5,643,763 to Choi and Dunn, and U.S. Patent Applications Serial Nos. 07 / 574,748 filed August 29, 1990, 07 / 575,962 filed August 31, 1990, 07 / 810,279 filed December 17, 1991, 07 / 853,408 filed March 18, 1992, 07 / 904,068 filed June 23, 1992, 07 / 990,860 filed December 16, 1992, and 1993 to GenPharm International. Nos. 08 / 053,131, filed April 26, 08 / 096,762, filed July 22, 1993, 08 / 155,301, filed November 18, 1993, 08 / 161,739, filed December 3, 1993, 08 / 165,699, filed December 10, 1993, and 08 / 209,741, filed March 9, 1994, the disclosures of which are incorporated herein by reference.See also EP 0546073B1, WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852, and WO 98 / 24884, and U.S. Pat. No. 5,981,175, the disclosures of which are incorporated herein by reference in their entireties. See also Taylor et al., 1992, Chen et al., 1993, Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild et al., (1996), the disclosures of which are incorporated herein by reference in their entireties.

[0124] Kirin has also demonstrated the production of human antibodies from mice into which large chromosome fragments or entire chromosomes have been introduced by microcell fusion, see European Patent Applications Nos. 773288 and 843961, the disclosures of which are incorporated herein by reference in their entireties.

[0125] Human anti-mouse antibody (HAMA) responses have also led the industry to prepare chimeric or otherwise humanized antibodies. Although chimeric antibodies have human constant regions and mouse variable regions, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly with long-term or multiple-dose use of the antibodies. Therefore, it would be desirable to provide fully human antibodies against multimeric enzymes to reduce the risk and / or impact of HAMA or HACA responses.

[0126] Antibody preparation The antibodies described herein were prepared using XENOMOUSE® technology, as described below. Such mice are capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies. Techniques utilized to accomplish the same are disclosed in the patents, applications, and references cited herein. However, particularly preferred embodiments of the transgenic production of mice and antibodies derived therefrom are disclosed in U.S. patent application Ser. No. 08 / 759,620, filed Dec. 3, 1996, and WO 98 / 24893, published Jun. 11, 1998, and WO 00 / 76310, published Dec. 21, 2000, the disclosures of which are incorporated herein by reference. See also Mendez et al., Nature Genetics 15:146-156 (1997), the disclosure of which is incorporated herein by reference.

[0127] Using such techniques, fully human monoclonal antibodies against IL-13 have been generated, as detailed below. Essentially, the XENOMOUSE® strain of mice was immunized with human IL-13, lymphoid cells (such as B cells) were collected from the mice that expressed the antibodies, and the collected cell lines were fused with myeloid cell lines to prepare immortal hybridoma cell lines. These hybridoma cell lines were screened and selected to identify hybridoma cell lines that produce antibodies specific to IL-13. Further, characterization of the antibodies produced by such cell lines, including nucleotide and amino acid sequence analyses of the heavy and light chains of such antibodies, is provided herein.

[0128] Alternatively, instead of fusing with myeloma cells to generate hybridomas, recovered cells isolated from the immunized XENOMOUSE® strain of mice can be further screened for reactivity to the primary antigen, preferably human IL-13. Such screening includes ELISA using the desired IL-13 protein and functional assays such as IL-13-induced eotaxin-1 production. Single B cells secreting antibodies that specifically bind to IL-13 can then be isolated using a desired IL-13-specific hemolytic plaque assay (Babcook et al., Proc. Natl. Acad. Sci. USA, 1996, 193:7843-7848). Cells targeted for lysis are preferably sheep red blood cells (SRBCs) coated with IL-13. In the presence of a B cell culture secreting the desired immunoglobulin and complement, the formation of plaques indicates specific IL-13-mediated lysis of the target cells.

[0129] A single antigen-specific plasma cell at the center of the plaque can be isolated, and the genetic information encoding the antibody specificity can be isolated from that single plasma cell. Using reverse transcriptase PCR, DNA encoding the variable region of the secreted antibody can be cloned. Such cloned DNA can then be further inserted into an appropriate expression vector, preferably a vector cassette such as pcDNA (Invitrogen, Carlsbad, CA), more preferably such a pcDNA vector containing the constant domains of immunoglobulin heavy and light chains. The generated vector can then be transfected into host cells, preferably CHO cells, and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0130] Described herein is the isolation of a plurality of single plasma cells that produce antibodies specific for IL-13. Additionally, genetic material encoding antibodies that specifically bind to IL-13 has been isolated and placed into a suitable expression vector, which is then transfected into host cells.

[0131] In general, the antibodies produced by the above cell lines had fully human IgG1 or IgG2 heavy chains with human kappa light chains. The antibodies typically had a denatured IgG1 / IgG2 antibody content of about 10, as measured by either solid phase or liquid phase. -9 ~about 10 -13 It had high affinity with a KD of M.

[0132] As mentioned above, anti-IL-13 antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding a particular antibody can be used to transform suitable mammalian host cells, such as CHO cells. Transformation can be by any known method for introducing polynucleotides into host cells, including, for example, packaging the polynucleotide into a virus (or viral vector) and transducing the host cell with the virus (or vector), or by transfection procedures known in the art, such as those exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which are incorporated herein by reference). The transfection procedure used will depend on the host being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus.

[0133] Mammalian cell lines available as hosts for expression are well known in the art and include the many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, Sp2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and many other cell lines. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with IL-13 binding properties.

[0134] Antibody sequence The nucleotide and amino acid sequences of the heavy and light chain variable regions of representative human anti-IL-13 antibodies are shown in the Sequence Listing and summarized in Table 1 below.

[0135] [Table 1]

[0136] Antibody therapy Anti-IL-13 antibodies have therapeutic value for treating symptoms and conditions associated with IL-13 activity. IL-13 has been implicated in a wide variety of diseases and disorders, including inflammatory diseases, cancer, fibrotic diseases, and diseases characterized by non-malignant cell proliferation. In certain embodiments, the anti-IL-13 antibodies disclosed herein are used to treat inflammatory diseases or disorders, such as asthma (including both allergic (atopic) and non-allergic (non-atopic)), bronchial asthma, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), hay fever, rhinitis, urticaria, angioedema, allergic dermatitis (including contact dermatitis), Stevens-Johnson syndrome, anaphylactic shock, food allergies, keratitis, conjunctivitis, and steroid-resistant nephritic syndrome. In other embodiments, the anti-IL-13 antibodies are used to treat mastocytosis. In yet other embodiments, anti-IL-13 antibodies are used to treat fibrotic diseases such as pulmonary fibrosis, including idiopathic pulmonary fibrosis, cystic fibrosis, bleomycin-induced fibrosis, hepatic fibrosis, and systemic sclerosis. In further embodiments, anti-IL-13 antibodies are used to treat cancer, e.g., Hodgkin's disease, B-cell proliferative disorders (e.g., B-cell lymphoma, particularly mediastinal large B-cell lymphoma), B-cell leukemia, and ovarian cancer.

[0137] In yet another embodiment, anti-IL-13 antibodies are used to treat diseases characterized by non-malignant B-cell proliferation, such as systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, and cryoglobulinemia; diseases characterized by high levels of autoantibodies, such as hemolytic anemia, thrombocytopenia, phospholipid syndrome, and pemphigus; inflammatory bowel disease; and graft-versus-host disease.

[0138] If desired, the isotype of an anti-IL-13 antibody can be switched, e.g., to take advantage of the biological properties of a different isotype. For example, in some situations, it may be desirable for a therapeutic antibody against IL-13 to be able to fix complement and participate in complement-dependent cytotoxicity (CDC). Numerous isotypes of antibodies exist that are capable of doing the same, including, but not limited to, mouse IgM, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgM, human IgG1, and human IgG3. It is understood that the antibody produced need not initially have such an isotype; rather, the antibody produced can have any isotype, and the antibody can then be isotype-switched using conventional techniques well known in the art. Such techniques include, among others, the use of direct recombinant techniques (see, e.g., U.S. Pat. No. 4,816,397), cell-cell fusion techniques (see, e.g., U.S. Pat. Nos. 5,916,771 and 6,207,418).

[0139] By way of example, the anti-IL-13 antibodies discussed herein are human antibodies. If an antibody has the desired binding to IL-13, it can be readily isotype switched to create a human IgM, human IgG1, or human IgG3 isotype, while still possessing the same variable regions (which define the antibody's specificity and part of its affinity). Such molecules can then fix complement and participate in CDC.

[0140] In the cell-cell fusion approach, a myeloma or other cell line containing a heavy chain of any desired isotype is prepared, and another myeloma or other cell line containing a light chain is prepared. These cells can then be fused, and a cell line expressing an intact antibody can be isolated.

[0141] Thus, once antibody candidates have been generated that meet the desired "structural" attributes as described above, they can generally be conferred with at least some desired "functional" attributes through isotype switching.

[0142] Biologically active antibodies that bind to IL-13 are preferably used in sterile pharmaceutical preparations or formulations to reduce the activity of IL-13. The anti-IL-13 antibody preferably has suitable affinity to potently inhibit IL-13 activity within the target therapeutic range. Inhibition preferably results from the antibody's ability to interfere with IL-13 binding to a signaling receptor, such as IL-13Ra1 (also known as IL-13Rα1, Rα1, IL-13Ralpha1, IL-13 receptor alpha1, or other similar terms). In other embodiments, the antibody may inhibit IL-13 activity even when IL-13 is able to bind by interfering with IL-13's ability to signal through the receptor. For example, the antibody may prevent the interaction of IL-13Ra1 with a coreceptor required for signaling, such as the IL-4 receptor alpha chain. In some embodiments, the antibody may prevent IL-13 activity through a signaling receptor while allowing IL-13 to bind to a decoy receptor, such as IL-13Ra2. In this case, binding to the decoy receptor may allow clearance of bound IL-13 and enhance the ability of the antibody to inhibit IL-13 activity.

[0143] When used for in vivo administration, antibody formulations are preferably sterile. This is readily accomplished by any method known in the art, for example, filtration through sterile filtration membranes. Antibodies are typically stored in lyophilized form or in a solution. Sterile filtration can be performed before or after lyophilization and reconstitution.

[0144] Therapeutic antibody compositions generally are filled into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter (e.g., a stopper pierceable by a hypodermic injection needle) to permit withdrawal of the formulation.

[0145] The antibody can be administered by known methods, such as by injection or infusion via subcutaneous, intravenous, intraperitoneal, intracerebral, intradermal, intramuscular, intraocular, intraarterial, intrathecal, or intralesional routes, or by inhalation, or by sustained release systems as described below. In some situations, the antibody is preferably administered by infusion or bolus injection. In other situations, a therapeutic composition comprising the antibody can be administered via the nose or lungs, preferably as a liquid or powder aerosol (lyophilized). The composition can also be administered intravenously, parenterally, or subcutaneously, as desired. When administered systemically, the therapeutic composition must be sterile, pyrogen-free, and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those of skill in the art.

[0146] Antibodies for therapeutic use as described herein are typically prepared with appropriate carriers, excipients, and other agents incorporated into the formulation to enhance transport, delivery, tolerability, etc. Briefly, dosage formulations of the antibodies described herein are prepared for storage or administration by mixing an antibody having the desired purity with one or more physiologically acceptable carriers, excipients, or stabilizers. These formulations may include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., Lipofectin™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. The formulation may include buffers such as TRIS HCl, phosphate, citrate, acetate, and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) peptides such as polyarginine, proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium, and / or non-ionic surfactants such as TWEEN, PLURONICS, or polyethylene glycol.

[0147] Other acceptable carriers, excipients, and stabilizers are well known to those skilled in the art. Any of the foregoing mixtures may be suitable in treatments and therapies according to the present invention, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible with the route of administration and is tolerable. For further information, see also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance," Regul. Toxicol. Pharmacol. 32(2):210-8 (2000); Wang W. "Lyophilization and development of solid protein pharmaceuticals," Int. J. Pharm. 203(1-2):1-60 (2000); Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts," J. Pharm. Sci. 89(8):967-78 (2000); Powell et al. "Compendium of excipients for parenteral formulations," PDA J. Pharm. Sci. Technol. 52:238-311 (1998), and references cited therein.

[0148] Sterile compositions for injection are prepared as described in Remington: The Science and Practice of Pharmacy (20 th These compositions can be formulated according to conventional pharmaceutical practice, as described in "Publications of Pharmaceuticals and Medical Devices," ed., Lippincott Williams & Wilkens Publishers (2003). For example, it may be desirable to dissolve or suspend the active compound in a vehicle such as water, a natural vegetable oil such as sesame oil, peanut oil, or cottonseed oil, or a synthetic fatty vehicle such as ethyl oleate. Buffers, preservatives, antioxidants, and the like can be incorporated according to accepted pharmaceutical practice.

[0149] The antibody can also be administered in a sustained-release preparation from which it is released over time. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide. The matrices may be in the form of shaped articles, films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol) as described by Langer et al., J. Biomed Mater. Res., (1981) 15:167-277 and Langer, Chem. Tech., (1982) 12:98-105), polylactides (U.S. Pat. No. 3,773,919; European Patent No. 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid (Sidman et al., Biopolymers, (1983) 22:547-556), non-degradable ethylene-vinyl acetate (Langer et al., supra), degradable lactic acid-glycolic acid copolymers, e.g., LUPRON Depot™ (injectable microspheres composed of lactic acid-co-glycolic acid and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988).

[0150] While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, certain hydrogels release proteins for shorter periods. If encapsulated proteins remain in the body for extended periods, they may denature or aggregate as a result of exposure to moisture at 37°C, potentially resulting in loss of biological activity and altered immunogenicity. Rational strategies for protein stabilization can be devised depending on the mechanism involved. For example, if the aggregation mechanism is determined to be the formation of intermolecular S–S bonds via disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0151] Sustained-release compositions also include preparations of antibody crystals suspended in a suitable formulation capable of maintaining the crystals in suspension. These preparations can produce a sustained-release effect when injected subcutaneously or intraperitoneally. Other compositions include antibodies entrapped in liposomes. Liposomes containing such antibodies can be prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, (1985) 82:3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980) 77:4030-4034; EP Patent Nos. 52,322; 36,676; 88,046; 143,949; 142,641; Japanese Patent Application No. 58-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP Patent No. 102,324.

[0152] The dosage of an antibody formulation for a given patient can be determined by the attending physician. In determining the appropriate dosage, the physician can consider various factors known to alter the action of therapeutic agents, including, for example, the severity and type of disease, body weight, sex, diet, time and route of administration, other drug treatments, and other relevant clinical factors. Therapeutically effective dosages can be determined by either in vitro or in vivo methods.

[0153] The effective amount of an antibody described herein to be used therapeutically will depend, for example, on the therapeutic objectives, the route of administration, and the condition of the patient. Therefore, it is preferred that the therapist titrate the dosage and modify the route of administration as needed to obtain the optimal therapeutic effect. Typical daily dosages can range from about 0.001 mg / kg up to 100 mg / kg or more, depending on the factors mentioned above. Typically, a clinician will administer a therapeutic antibody until a dosage is reached that achieves the desired effect. The progress of this treatment is easily monitored by conventional assays.

[0154] The antibodies described herein are expected to have therapeutic efficacy in treating symptoms and conditions caused by or associated with the activity of IL-13.

[0155] Design and production of other therapeutic agents In accordance with the present invention, and based on the activity of the antibodies generated and characterized herein with respect to IL-13, advanced antibody therapeutics may be used to treat specific diseases. These advanced therapeutics may include bispecific antibodies, immunotoxins, radiolabeled therapeutics, peptide therapeutics, gene therapeutics, particularly intracellular antibodies, antisense therapeutics, and small molecules.

[0156] In the context of generating advanced antibody therapeutics where complement fixation is a desirable attribute, it may be possible to circumvent reliance on complement for cell killing, for example, through the use of bispecifics, immunotoxins, or radiolabels.

[0157] For example, bispecific antibodies can be produced that contain (i) two antibodies, one specific for IL-13 and the other specific for a second molecule, conjugated together; (ii) an antibody having one chain specific for IL-13 and a second chain specific for a second molecule; or (iii) a single-chain antibody specific for both IL-13 and another molecule. Such bispecific antibodies can be produced, for example, using well-known techniques related to (i) and (ii) (see, e.g., Fanger et al., Immunol Methods 4:72-81 (1994) and Wright and Harris, supra) and well-known techniques related to (iii) (see, e.g., Traunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)). In either case, the second specificity can be produced as desired. For example, the second specificity can be generated against a heavy chain activating receptor, including, but not limited to, CD16 or CD64 (see, e.g., Deo et al. 18:127 (1997)), or CD89 (see, e.g., Valerius et al. Blood 90:4485-4492 (1997)).

[0158] In some embodiments, an article of manufacture is provided that includes a container comprising a composition containing an anti-IL-13 antibody and a package insert or label indicating that the composition can be used to treat a disease mediated by IL-13. Preferably, a mammal, more preferably a human, is given the anti-IL-13 antibody. In preferred embodiments, the disease to be treated is asthma (including both allergic (atopic) and non-allergic (non-atopic)), bronchial asthma, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), hay fever, rhinitis, urticaria, angioedema, allergic dermatitis (including contact dermatitis), Stevens-Johnson syndrome, anaphylactic shock, food allergy, keratitis, conjunctivitis, steroid-resistant nephritic syndrome, mastocytosis, fibrotic diseases (e.g., pulmonary fibrosis, including idiopathic pulmonary fibrosis, cystic fibrosis, etc.). , bleomycin-induced fibrosis, hepatic fibrosis, and systemic sclerosis), cancer such as Hodgkin's disease, B-cell proliferative disorders (e.g., B-cell lymphoma, particularly mediastinal large B-cell lymphoma), B-cell leukemia, ovarian cancer, diseases characterized by non-malignant B-cell proliferation such as systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, and cryoglobulinemia, diseases characterized by high levels of autoantibodies such as hemolytic anemia, thrombocytopenia, phospholipid syndrome and pemphigus, inflammatory bowel disease, and graft-versus-host disease.

[0159] In some embodiments, the anti-IL-13 antibody is used to treat asthma. In a specific embodiment, the antibody is the 623 antibody or a variant thereof described herein. In another specific embodiment, the antibody is the 731 antibody or a variant thereof described herein. [Example]

[0160] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the teachings herein.

[0161] Example 1: Antibody production Preparation of IL-13 and IL-13 antigen The following IL-13 peptides were used in the experiments described below.

[0162] Recombinant human IL-13 (R&D 213-IL-005; SEQ ID NO: 1): GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN

[0163] Recombinant human IL-13 (Peprotech 200-13; SEQ ID NO: 2): SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN

[0164] Recombinant human IL-13 (Peprotech 200-13A; SEQ ID NO: 3): MSPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN

[0165] Human IL-13-human Fc fusion protein (including leader sequence; SEQ ID NO: 4): [ka]

[0166] Human IL-13-rabbit Fc fusion protein (including leader sequence; SEQ ID NO: 5): [ka]

[0167] Human IL-13-mouse IL-13 Helix A (underlined; SEQ ID NO: 6): [ka]

[0168] Human IL-13-mouse IL-13 Helix B (underlined; SEQ ID NO: 7): [ka]

[0169] Human IL-13-mouse IL-13 Helix C (underlined; SEQ ID NO: 68): [ka]

[0170] Human IL-13-mouse IL-13 Helix D (underlined; SEQ ID NO: 69): [ka]

[0171] As will be appreciated by those skilled in the art, only a portion of the residues may actually be involved in forming the epitope. For example, in SEQ ID NOs: 66-69 above, the epitope may actually be the helical portion of each peptide (the underlined area).

[0172] Animal immunization Monoclonal antibodies against IL-13 were generated by immunizing XenoMouse® mice (XenoMouse® XMG2L3 and XenoMouse® XMG2, Abgenix, Inc. Fremont, CA). Human IL-13-human Fc fusion protein (SEQ ID NO: 64) or human IL-13-rabbit Fc fusion protein (SEQ ID NO: 65) was used as the immunogen for antibody production. Each mouse was immunized via the footpad route. Animals were immunized on days 0, 4, 7, 11, 14, 18, 21, and 25. The initial immunization used 10 μg of antigen in CpG / alum per mouse. Subsequent boosts used 5 μg of antigen in CpG / alum per mouse. The final boost on day 25 used 5 μg of antigen in PBS without adjuvant per mouse. Animals were bled on day 20 to obtain serum for titration as described below.

[0173] Titer analysis Titers were determined using standard protocols. Briefly, Costar 3368 plates were coated overnight at 4°C with either IL-13 rabbit Fc fusion protein (SEQ ID NO: 65) or full-length rabbit antibody. Plates were washed using Titertek Program ADG9, dried, and blocked with 250 μl of 1% non-fat skim milk / 1×PBS. After blocking, plates were washed again using Titertek Program ADGP and dried. Sera to be tested were serially titrated 1:2 twice, starting with an initial dilution of 1:100. Samples were placed at 50 μl / well in 1% non-fat skim milk / 1×PBS and incubated for 1 hour at room temperature.

[0174] After washing and drying using Titertek Program ADG9, the plates were incubated with a rabbit anti-human Fc secondary antibody conjugated to POD (1:8000 dilution; 50 μL / well) in 1% nonfat skim milk / 1×PBS, which has minimal cross-reactivity to rabbit Fc, for 1 hour at room temperature. The plates were then washed a final time using Titertek Program ADG9 and dried. POD substrate One-Step TMB solution (50 μL / well) was added and developed for 30 minutes at room temperature. The reaction was stopped with 1N HCl (50 μL / well), and the optical density was immediately read on a Titertek Plate reader.

[0175] As shown in Table 2, three animals with high titers to the IL-13 immunogen were selected for harvest.

[0176] [Table 2]

[0177] Primary screening Highly immunized animals were harvested, and CD19+ B cells were isolated for subsequent B cell culture. The cells were induced to proliferate and terminally differentiate into plasma cells. Supernatants from these plasma cells were screened by ELISA to identify primary wells containing anti-IL-13-specific antibodies. Routine cultures were performed with 50–500 CD19+ B cells per well to allow for the identification of monoclonal antigen-specific B cell cultures.

[0178] Briefly, IL-13-RbFc was coated onto Costar 3368 96-well plates at 1 μg / mL overnight. Each plate was washed five times with dH2O, and 40 μL of 1% milk in PBS was added to the plate. Subsequently, 10 μL of B cell supernatant was added to each well. After 1 hour at room temperature, the plate was washed again five times with dH2O. 50 μL of rabbit anti-human Fc-HRP with minimal anti-rabbit cross-reactivity (Jackson Laboratories; 1:8000 dilution) was added to each well. After 1 hour at room temperature, the plate was washed again five times with dH2O, and 50 μL of TMB substrate (Neogen) was added to each well. After 30 minutes, the reaction was stopped by adding 50 μL of 1N hydrochloric acid to each well, and the plate was read at 450 nm.

[0179] Representative data from the primary screen are shown in Table 3 below. Positive wells were identified as those found to have signals at least three times higher than those of control wells. A total of 968 positive antigen-specific B cell wells were identified in the primary screen. All of these wells were advanced for screening in the functional assays described below.

[0180] [Table 3]

[0181] IL-13-induced eotaxin-1 production assay All 968 ELISA-positive wells were screened twice in the IL-13-induced eotaxin-1 release assay. The assay was performed so that only wells containing high concentrations of antibody or high affinity antibody were identified as neutralizing. A total of 78 neutralizing antibodies were identified as neutralizing in this assay. Specific data from several wells of interest are also shown in Table 4 for illustrative purposes.

[0182] For the assay, HDFa cells were seeded at 4000 cells / well in 50 μL of Medium 106 supplemented with low serum growth supplement (Cascade) onto half of a 96-well assay plate. The plate was then incubated overnight at 37°C in 5% CO2. In a separate plate, 12.5 μL of sample antibody, negative control, or positive control was aliquoted into a sterile 96-well assay plate. Approximately 600 pM of IL-13 was prepared in Medium 106 (4x the final concentration), and approximately 100 ng / mL of TNF-alpha was prepared in Medium 106 (2x the final concentration).

[0183] To initiate the assay, 12.5 μL of IL-13 or medium alone was added to each well and incubated for 1 hour at 37°C in 5% CO2. After the 1 hour incubation, the medium from the HDFa cells was carefully removed using a multichannel pipette. 25 μL of TNF-alpha was added to each well. 25 μL of sample / IL-13 was transferred to the HDFa / TNF-alpha wells, and the cells were incubated for 48 hours at 37°C in 5% CO2.

[0184] After 48 hours of incubation, the supernatants from the HFDa assay wells were collected into a 96-well V-bottom plate. Samples were centrifuged at 1500 rpm for 5 minutes.

[0185] 30 μL of sample was assayed for eotaxin-1 release with an assay kit (R&D systems) according to standard protocols with the following modifications: (1) 50 μL of capture Ab was coated at 2 μg / mL; (2) 50 μL of sample or standard was used (30 μL sample + 20 μL medium for a final volume of 50 μL); (3) 50 μL of detection Ab was used at 0.1 μg / mL; (4) 50 μL of streptavidin-HRP was used at 0.5 μg / mL; and (5) 50 μL of substrate solution was used.

[0186] [Table 4]

[0187] High antigen (HA) analysis of anti-IL-13 specific B cell culture wells An ELISA method was used to normalize the supernatants for the concentration of antigen-specific antibodies. A standard curve was generated using parallel titrations of known concentrations of anti-target (IL-13) antibodies, and the amount of antigen-specific antibody in the supernatant was compared to the standard to determine its concentration (see Table 5 below).

[0188] [Table 5]

[0189] The amount of antigen-specific antibody in each well was quantified and plotted against the neutralization data for that well to identify the most potent wells (Figure 1). The wells containing the most potent antibodies are those that inhibit best with the lowest concentration of antibody (upper left quadrant of the graph).

[0190] Restricting antigen (LA) analysis of anti-IL-13 specific B cell culture wells Limiting antigen analysis is a method for ranking antigen-specific antibodies prepared in B cell culture supernatant by affinity compared to all other antigen-specific antibodies. When the antigen is coated very lightly, only the antibodies with the highest affinity should be able to bind at any detectable level at equilibrium. (See, for example, WO 03 / 048730 A2, which is incorporated herein by reference.)

[0191] Here, biotinylated IL-13 was bound to streptavidin plates at four concentrations (250 ng / mL; 125 ng / mL; 62 ng / mL; and 31 ng / mL) in a 96-well culture plate for 1 hour at room temperature. Each plate was washed five times with dH2O, and 45 μL of 1% milk in PBS containing 0.05% sodium azide was added to the plate. Subsequently, 5 μL of B cell supernatant was added to each well. After 18 hours on a shaker at room temperature, the plate was washed five times again with dH2O. 50 μL of Gt anti-human (Fc)-HRP at 1 μg / mL was added to each well. After 1 hour at room temperature, the plate was washed five times again with dH2O, and 50 μL of TMB substrate was added to each well. The reaction was stopped by adding 50 μL of 1 M phosphoric acid to each well, and the plate was read at 450 nm.

[0192] However, some wells, including 2388A10 and 2357G11, were clearly superior when measured by OD with the least amount of antigen coating, as shown in Figure 2. The results shown in Figure 2 demonstrate the ability of different antibodies to bind at low concentrations of antigen coating. The antibody giving the highest OD signal has the highest affinity under the conditions of the assay. The remaining clones were further analyzed by combining the high-antigen data, measuring specific antibody concentration and limiting antigen production. In this way, it was possible to compare the affinity of different concentrations of antibody in B cell culture supernatants. The wells containing the highest affinity antibodies are those with the highest ELISA OD at the lowest concentration of Ag-specific antibody.

[0193] Based on all of the screening data, the wells listed in Table 6 were identified for further analysis (plaque assay and micromanipulation, single-cell PCR, and recombinant expression). Five wells were selected based on potency (total inhibition / specific Abs): 2372B8, 2383H5, 2398C5, 2401G12, and 2413G11. Three wells were selected based on affinity and inhibition: 2357G11, 2361G5, and 2384G12. Two wells were selected based on neutralization data alone: 2388A10 and 2407G11.

[0194] [Table 6]

[0195] IL-13-specific hemolytic plaque assay Cells secreting the IL-13-specific antibodies of interest were isolated using an IL-13-specific hemolytic plaque assay as generally described in Babcook et al. (Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996)), which is incorporated herein by reference. The isolated cells are identified in Table 7 below.

[0196] Biotinylation of sheep red blood cells (SRBC) SRBCs were stored in RPMI medium as a 25% stock. 1.0 ml of the stock was aliquoted into an Eppendorf tube, and the cells were spun down (pulse-spin at 8000 rpm (6800 rcf) in a microfuge). The supernatant was removed to yield a 250 μl SRBC-enriched cell pellet. The cells were then washed twice with 1 ml of PBS (pH 8.6). The cell pellet was then resuspended in 4.75 ml of PBS (pH 8.6) in a 15 ml tube. In a separate 50 ml tube, 2.5 mg of sulfo-NHS biotin was added to 45 ml of PBS (pH 8.6). Once the biotin was completely dissolved, 5 ml of SRBCs were added, and the tube was rotated at room temperature for 1 hour. The SRBCs were centrifuged at 3000 g for 5 minutes, the supernatant was removed, and the SRBCs were resuspended in 1 ml of PBS (pH 7.4) in an Eppendorf tube. The SRBCs were washed three times with 1 ml of PBS (pH 7.4). The SRBCs were then resuspended in 4.75 ml of immune cell medium (RPMI 1640 containing 10% FCS) in a 15 ml tube (5% B-SRBC stock). The stock was stored at 4°C until needed.

[0197] Streptavidin (SA) coating of B-SRBCs One ml of the 5% B-SRBC stock was transferred to a new Eppendorf tube. The B-SRBCs were pelleted, the supernatant removed, and the pellet resuspended in 1.0 ml of PBS (pH 7.4), followed by repeated centrifugation. The wash cycle was repeated twice, and then the B-SRBC pellet was resuspended in 1.0 ml of PBS (pH 7.4) to a final concentration of 5% (vol / vol). 10 μl of a 10 mg / ml streptavidin (CalBiochem, San Diego, CA) stock solution was added, and the tube was mixed and rotated at room temperature for 20 minutes. The wash step was repeated, and the SA-SRBCs were resuspended in 1 ml of PBS (pH 7.4) (5% (vol / vol)).

[0198] Human IL-13 coating of SA-SRBCs SA-SRBCs were coated with photobiotinylated human IL-13-RbFc fusion at 100 μg / ml, then mixed and rotated for 20 minutes at room temperature. SRBCs were washed twice with 1.0 ml of PBS (pH 7.4) as described above. IL-13-coated SRBCs were resuspended in RPMI (+10% FCS) to a final concentration of 5% (v / v).

[0199] Quality assessment of IL-13-SRBCs by immunofluorescence (IF) Approximately 10 μl of 5% SA-SRBCs and 10 μl of 5% IL-13-coated SRBCs were added to separate, fresh 1.5 ml Eppendorf tubes containing 40 μl of PBS. A control human anti-IL-13 antibody was added to each SRBC sample at 45 μg / ml. The tubes were rotated at room temperature for 20 minutes, and then the cells were washed three times with 100 μl of PBS. The cells were resuspended in 50 μl of PBS and incubated with 20 μg / ml of Gt anti-human IgG Fc antibody conjugated to Alexa488 (Molecular Probes, Eugene, OR). The tubes were rotated at room temperature for 20 minutes, then washed with 100 μl of PBS, and the cells were resuspended in 10 μl of PBS. Ten μl of the stained cells were spotted onto a clean glass microscope slide, covered with a glass coverslip, and observed under fluorescent light. The cells were scored on an arbitrary scale of 0 to 4.

[0200] Preparation of plasma cells The contents of a single B cell culture well, previously identified by the various assays described above as containing a B cell clone secreting the immunoglobulin of interest, were harvested. Using a pipette, the contents of the well were harvested by adding 100–1000 μL of 37°C RPMI (+10% FCS). The cells were resuspended by pipetting and then transferred to a new 1.5 ml Eppendorf tube (final volume approximately 700–1000 μl). The cells were centrifuged in a microfuge at 2500 rpm for 1 minute at room temperature. The tube was then rotated 180 degrees and spun again at 2500 rpm for 1 minute. The freezing medium was removed, and the immune cells were resuspended in 100 μL RPMI (10% FCS) and then centrifuged. This RPMI (+10% FCS) wash was repeated, and the cells were resuspended in 75 μL RPMI (+10% FCS) and stored on ice until ready for use.

[0201] Plaque assay To 75 μL of the cell sample, IL-13-coated SRBCs (5% (vol / vol) stock, diluted as needed if the SRBC lawn was too dense), 4x guinea pig complement (Sigma, Oakville, ON) stock prepared in RPMI (+10% FCS), and 4x enhancing serum stock (1:900 in RPMI (+10% FCS)) (75 μL each) were added. The mixture (3–5 μL) was spotted onto a TC plate lid (BD Biosciences, San Jose, CA), and the spot was covered with undiluted paraffin oil. The slide was incubated at 37°C for a minimum of 1 h.

[0202] [Table 7]

[0203] Cloning and Expression After isolation of single plasma cells, mRNA was extracted and reverse transcriptase PCR was performed to generate cDNA encoding the variable heavy and variable light chains of the antibodies secreted by each cell. The human variable heavy chain region was cloned into an IgG2 expression vector. This vector was generated by cloning the human IgG2 constant domain into the multiple cloning site of pcDNA3.1+ / Hygro (Invitrogen, Burlington, ON). The human variable light chain region was cloned into an IgK or IgL expression vector. These vectors were generated by cloning the human IgK or human IgL constant domain into the multiple cloning site of pcDNA3.1+ / Neo (Invitrogen, Burlington, ON).

[0204] Next, the heavy and light chain expression vectors were co-transfected into 60 mm dishes of 70% confluent human embryonic kidney (HEK) 293 cells using Lipofectamine. Over a period of 24–72 hours, the transfected cells secreted recombinant antibodies with identical specificity to the original plasma cells. Three milliliters of supernatant was harvested from the HEK 293 cells, and a sandwich ELISA was used to demonstrate the secretion of intact antibodies and specifically detect human IgG. Specificity was confirmed by the binding of the recombinant antibodies to IL-13 using ELISA.

[0205] Secretion ELISA tests were performed as follows. Control plates were coated overnight with 2 mg / mL goat anti-human IgG H+L, similar to the binding plates. IL-13 was coated onto Costar Labcoat Universal Binding Polystyrene 96-well plates and kept overnight at 4°C. Plates were washed five times with dH2O. Recombinant antibodies were titrated 1:2 across seven wells from undiluted lipofection supernatants. Plates were washed five times with dH2O. Goat anti-human IgG Fc-specific HRP-conjugated antibodies were added at a final concentration of 1 μg / mL for 1 hour at room temperature for the secretion assay and the two binding assays. Plates were washed five times with dH2O. Plates were developed by adding TMB for 30 minutes, and the ELISA was stopped by adding 1 M phosphoric acid. Each ELISA plate was analyzed to determine the optical density of each well at 450 nm.

[0206] Purification of recombinant anti-IL-13 antibody For larger-scale production, heavy and light chain expression vectors (2.5 μg of each chain / dish) were lipofected into ten 100 mm dishes of HEK293 cells at 70% confluence. The transfected cells were incubated at 37°C for 4 days, at which point the supernatant (6 mL) was harvested and replaced with 6 mL of fresh medium. On day 7, the supernatant was removed and pooled with the first harvest (120 mL total from 10 plates).

[0207] Each antibody was purified from the supernatant using Protein-A Sepharose (Amersham Biosciences, Piscataway, NJ) affinity chromatography (1 mL). The antibody was eluted from the Protein-A column with 500 mL of 0.1 M glycine (pH 2.5). The eluate was dialyzed into PBS (pH 7.4) and filter-sterilized. The antibody was analyzed by non-reducing SDS-PAGE to assess purity and yield. Concentration was also determined by UV analysis at OD280.

[0208] Example 2: Characterization of recombinant antibodies The recombinant antibodies were analyzed for potency in the eotaxin-1 assay, as described above. The results are presented in Table 8 below. Included are the IC50s measured in this assay for mouse IL-13 receptor α2 / Fc and human IL-13 receptor α2 / Fc. Figure 3 shows the percent inhibition of IL-13-induced eotaxin release by recombinant antibodies 643 and 731 compared to an isotype-matched control, e.g., an irrelevant IgG2 monoclonal antibody.

[0209] [Table 8]

[0210] Biacore affinity Six of the antibodies (602, 623, 643, 693rep1, 693rep2, and 731) were investigated for affinity to human IL-13 (R&D) using a BiaCore assay. First, two high-density goat anti-human antibody surfaces were prepared on a CM5 Biacore chip using conventional amine coupling to capture three mAbs at a time. All mAbs were diluted to approximately 5 μg / mL using HBS-P running buffer containing 100 μg / mL BSA. Using a Biacore2000 instrument, each purified mAb was captured on a different flow cell surface for 1 minute per IL-13 injection cycle.

[0211] Using the KINJECT command, IL-13 (R&D) was injected over all surfaces for 1.5 min, followed by a 20-min dissociation period, at concentrations of 100.9 nM, 50.4 nM, 25.2 nM, 12.6 nM, 6.30 nM, 3.15 nM, 1.58 nM, and 0.788 nM for mAbs 693, 713, and 731, and 25.2 nM, 12.6 nM, 6.30 nM, 3.15 nM, 1.58 nM, 0.788 nM, and 0.394 nM for mAbs 602, 623, and 643. IL-13 samples were prepared in HBS-P running buffer containing 100 μg / ml BSA. All samples were injected in duplicate, randomly selected, with several mAb capture / buffer KINJECT cycles inserted for double referencing.

[0212] After each cycle, the high-density goat anti-human antibody surface was regenerated with a 12-second pulse of 1 / 100 diluted concentrated phosphoric acid (146 mM, pH 1.5). mAb 693 was run twice because there were extra flow cells available on the instrument during the final series of medium-resolution experiments.

[0213] The data were fitted to a 1:1 interaction model including a term for mass transport using CLAMP. Data for six antibodies are shown in Table 9.

[0214] [Table 9]

[0215] Dynamic analysis Kinetic measurements of several antibodies were evaluated using the KinExA® method, which involves solution-based determination of formal affinity measurements at equilibrium.

[0216] 100 μg of each mAb was coupled to CNBr-activated Sepharose 4B or azlactone beads. Remaining active groups on the beads were blocked as recommended by the manufacturer. The beads were then blocked with 10 mg / ml BSA in 1 M Tris and stored in blocking solution. For some experiments, the mAb was directly absorbed onto PMMA beads as recommended by the manufacturer, blocked with 10 mg / ml BSA in PBS, and stored in blocking solution.

[0217] KinExA experiments were performed using the KinExA3000, an automated flow immunoassay system in which beads conjugated with the relevant mAb served as the solid phase. Briefly, a fixed amount of native human IL-13 or macaque IL-13 (10–650 pM), prepared by purifying and stimulating PBMCs according to standard protocols, was incubated with titrated concentrations of anti-human IL-13 mAb starting at 25 nM in sample buffer (PBS containing 0.1% BSA to reduce nonspecific binding). The antigen / antibody complexes were incubated at room temperature for 48–168 h to reach equilibrium. The mixture was recovered by the corresponding antibody-conjugated beads, allowing unbound antigen to accumulate. The volume and flow rate of the mixture were varied depending on the specific signal obtained in each experiment.

[0218] The captured IL-13 was detected using a solution containing a secondary Ab (either a polyclonal anti-IL-13 Ab or a monoclonal Ab binding to a different epitope) and Cy5-conjugated anti-species Ig against the secondary antibody in sample buffer. In some cases, the bead-bound IL-13 was detected using a mixture of SA-Cy5 and a biotinylated antibody that binds to an epitope other than that bound by the bead-immobilized Ab.

[0219] The concentration, volume, and flow rate of the secondary antibody solution were varied to optimize the signal-to-noise ratio in each experiment. Binding signals were converted to relative values as a percentage of the control in the absence of hIL-13. Three replicates of each sample were measured for every equilibrium experiment. The equilibrium dissociation constant (K D ) was obtained from nonlinear regression analysis of the data using the one-part homotypic binding model included in the KinExA software. This software maps the data points to the theoretical K D By fitting the curve, K D Calculate and determine the 95% confidence interval. D and high K D The affinities for native human IL-13 are summarized in Table 10, and for native macaque IL-13 in Table 11.

[0220] [Table 10]

[0221] [Table 11]

[0222] The association rate constants for two antibodies, 623 and 731, were investigated using KinExA. The same IL-13-conjugated beads were used as probes, and either the "direct" or "injection" method was used. These methods were identical to the KinExA equilibrium assay in terms of antigen capture, antigen concentration, and antigen detection. In the direct method, the antigen and antibody were premixed and then subjected to KinExA. In the injection method, the antibody and titrated antigen were mixed together for a fixed time before reading. Briefly, an amount of mAb that was estimated to bind approximately 80% of the antigen based on equilibrium experiments was mixed with hIL-13. Free antigen present in the sample was repeatedly probed before equilibrium. Because the binding signal is proportional to the concentration of free antigen in solution, the signal decreased over time until the solution reached equilibrium. The volume and flow rate of the antigen-mAb mixture and Cy5-labeled secondary antibody were varied depending on the mAb tested. Data were analyzed using KinExA analysis software. The software graphically depicts the decline in binding signal over time and fits the collected data points to an exact solution of the kinetic differential equation for binding. From this curve, K on The optimal solution was found (Table 12). off is K on and K. D was calculated indirectly from the solution of

[0223] [Table 12]

[0224] Binding to IL-13 mutant proteins The ability of antibodies 623 and 731 to bind to an IL-13 mutant protein in which wild-type arginine 110 was replaced with glutamine (IL-13Q110R) was investigated.

[0225] Briefly, plates were coated with IL-13RbFc (2.5 μg / mL, 50 μl) by overnight incubation at 4° C. in 1× PBS (pH 7.4) and 0.05% azide. Plates were then washed with 1× PBS and blocked with 100 μL of 1% nonfat skim milk in 1× PBS for 30 minutes at room temperature.

[0226] IL-13 or IL-13Q110R was preincubated with anti-IL-13 antibody for 1 hour at room temperature. IL-13 was serially titrated from 2000 ng / ml to a final volume of 30 μl per well. 30 μl of mAb was added per well at 40 ng / ml (sc731, 623) and 80 ng / ml (sc693), resulting in a final IL-13 concentration at the first step of the titration of 1000 ng / ml, a final concentration of antibodies 623 and 731 at the first step of the titration of 20 ng / ml, and a final concentration of antibody 693 at the first step of the titration of 40 ng / ml.

[0227] After preincubation, 50 μl / well of the preincubation solution was transferred to the IL-13RbFc-precoated plate and incubated at room temperature for 30 minutes. The plate was washed, and rabbit anti-Hu IgG Fc HRP was added at a concentration of 200 ng / ml. After an additional 30-minute incubation and subsequent washing, TMB was added and incubated for another 30 minutes. The reaction was stopped with 1N HCl, and the plate was read as quickly as possible on a Powerwave X340 96-well microplate reader (Biotek).

[0228] As can be seen in Figure 4, preincubation with IL-13 inhibited the binding of both antibodies 623 and 731 to IL-13-coated ELISA plates, while preincubation with the IL-13 variant IL-13Q110R inhibited the binding of 731 to a much greater extent than that of 623.

[0229] Receptor chain competition The ability of anti-IL-13 antibodies to block IL-13 binding to the receptors IL-13Rα1 and IL-13Rα2 was investigated. Samples were analyzed using a flow cytometer. The results are presented in Figures 5A and 5B. The data demonstrated the ability of Ab643 (Figure 5A) and Ab731 (Figure 5B) or an isotype control antibody to bind IL-13 and the receptors involved in the binding process. The specific receptor (e.g., IL-13Ra2, IL-13Ra1, or IL-4R) that binds IL-13 and allows the antibody to interact with the cells was determined using neutralizing antibodies against all possible IL-13 receptors expressed on HDFa cells. A summary of the various experimental and predicted results is shown in Figures 5C and 5D (adjust the figure legends if this change is permitted).

[0230] Briefly, HDFa cells were resuspended in FACS buffer to obtain approximately 200,000 cells / well / 100 μL, and 100 μL of cells were aliquoted into a 96-well V-bottom plate. Neutralizing anti-receptor antibodies (anti-human IL-13Ra1 (R&D Systems), anti-human IL-13Ra2 (R&D Systems), or anti-human IL-4R (R&D Systems)) were diluted in FACS buffer to twice the final concentration (10 μg / mL). Anti-IL-13 and control Abs were also diluted in FACS buffer to twice the final concentration (1 μg / mL), as was IL-13 (human R&D; final 10 ng / mL).

[0231] A V-bottom plate of HDFa cells was centrifuged at 180 × g for 7 minutes and the supernatant removed by inversion (Plate #1). Cells were resuspended in 50 μL of FACS buffer, and an additional 50 μL of anti-human IL-13Ra1, anti-human IL-13Ra2, anti-human IL-4R, or FACS buffer (no receptor Ab control) was added to the appropriate wells. Cells and antibodies were then incubated on ice for approximately 1.5 hours.

[0232] A second V-bottom plate was used for Ab / IL-13 preincubation (Plate #2). 60 μL of test antibody was aliquoted into the V-bottom plate. 60 μL of IL-13 was added to the appropriate wells, and the mixture was incubated on ice for approximately 1.5 hours.

[0233] After incubation, the HDFa cells were centrifuged at 180 × g for 7 minutes and the supernatant was removed by inversion. The cells in plate #1 were resuspended in 100 μL of FACS buffer or 100 μL of Ab / IL-13 and incubated for an additional 1.5 hours.

[0234] After the second incubation, cells were centrifuged and washed once with FACS buffer, and 100 μL of FACS buffer, 7AAD, or 2 μg / mL goat anti-Hu IgG-Fc-Cy5 was added to the appropriate wells.

[0235] The cells and secondary antibody were incubated on ice for 20 minutes, followed by washing with FACS buffer. The cells were then resuspended in 100 μL of FACS buffer and aliquoted into pre-labeled FACS tubes containing 300 μL of cold FACS buffer.

[0236] Samples were analyzed using a flow cytometer. The results are shown in Figures 5A and 5B. A summary of the above protocol and predicted results for each antibody is shown in Figures 5C and 5D. As shown by Figure 5A, IL-13 does not bind to HDFa cells in the presence of Ab643. As shown in each panel of Figure 5C, Ab643 appears to prevent IL-13 from binding to its receptor on HDFa cells. This is not the case with Ab731, as seen in Figure 5B. IL-13 allows Ab731 to bind to HDFa cells. As shown in Figure 5D, this binding is not blocked by Abs against IL-13Ralpha1 or IL-4R, but is blocked by Abs against IL-13Ralpha2, indicating that Ab731 prevents IL-13 from binding to IL-13Ralpha1 or IL-4R, but not IL-13Ralpha2.

[0237] The surface expression levels of IL-13Ra1, IL-13Ra2, and IL-4R on HDFa cells were determined by FACS analysis using anti-receptor antibodies. HDFa cells prepared as described above were incubated with anti-receptor antibodies at a concentration of 5 μg / mL on ice for 1 hour. The cells were washed with FACS buffer and incubated with 2 μg / mL of Cy5 secondary (anti-human) antibody on ice for 30 minutes. After washing, the samples were analyzed by flow cytometry. The results are shown in Table 13 below.

[0238] [Table 13]

[0239] Epitope mapping The epitopes of the antibody-IL-13 complexes were analyzed by three methods: 1) SELDI, 2) screening of a random peptide phage display library, and 3) expression of chimeric human / mouse IL-13 molecules. These three approaches, combined with knowledge of the IL-13 structure, provided a consistent view of the relative binding sites and antigenic regions of these mAbs, allowing the identification of functional epitopes, particularly those involved in binding to signaling receptors.

[0240] In initial experiments, dot blot analysis of mAb binding to purified IL-13 protein revealed which antibodies bound to which form of epitope (linear or conformational). mAbs 693 and 785 bound to the reduced, denatured antigen, the linear epitope. mAbs 602, 623, 643, and 713 bound to the nonreduced (conformational epitope) IL-13, but not to the reduced, denatured antigen. mAb 763 showed no binding. Subsequently, the linear epitope was mapped using a random peptide phage display library. After two rounds of panning of mAb 693 against a 12-mer random peptide library expressed on phage, a single specific binder was sequenced and aligned to residues 109–120 (helix D) of IL-13 (Figure 6A). IL-13 antibodies were classified into three different bins, although the bins did not always correlate with epitopes determined by other means. One antibody from each bin was selected for mapping by SELDI. Table 14 shows the binning results for IL-13 antibodies.

[0241] [Table 14]

[0242] Epitope mapping using SELDI The antibody-antigen complex was digested with high concentrations of Lys-C and Asp-N. The epitopes were then determined by SELDI and identified by fragment mass. Table 15 shows the predicted masses of peptides digested with endoproteinase Lys-C.

[0243] [Table 15]

[0244] The masses identified after cleavage were 6842.8 (for peptide fragment 45-108), 7733.7 (for peptide fragment 45-116), and 9461.4 (for peptide fragment 21-108). Therefore, the binding site of mAb713 was determined to be within residues 45-108 of IL-13.

[0245] Peptide arrays for mapping conformational epitopes A peptide array of 101 12-mer peptides spanning residues 21–132 of the IL-13 sequence was generated (SIGMA-Genosys). Each consecutive peptide was offset by one amino acid from the previous one, resulting in a nested, overlapping library. The array was probed with mAb 713, and binding of mAb 713 to the peptides was detected by incubating the PVDF membrane with an HRP-conjugated secondary antibody followed by enhanced chemiluminescence. Two consecutive spots corresponding to amino acids 70–80 of IL-13 and three consecutive spots corresponding to amino acids 83–92 of IL-13 were observed.

[0246] Epitope mapping using mouse IL-13 chimeric molecules The mouse sequences for helix A, helix B, helix C, and helix D were shuffled with the human sequences to generate four new mouse chimeras. A designation of the helix positions is shown in Figure 6B. None of the mAbs bound to mouse IL-13. The four chimeras are as follows: [ka] [ka] [ka] [ka]

[0247] The chimeras were then expressed and the secreted IL-13 chimeric proteins were detected in an ELISA assay. The results are summarized in Table 16. "*" indicates weak binding in the sandwich ELISA.

[0248] [Table 16]

[0249] The results of the above three tests of epitopes of IL-13 are summarized in Table 17.

[0250] [Table 17]

[0251] Thus, several different potential epitope locations appear to be used by the various antibodies disclosed herein.

[0252] Antibody binning analysis Anti-IL-13 antibodies were sorted into three distinct bins by measuring the ability of two antibodies to simultaneously bind to the antigen (one antibody captured the antigen on the beads, the other antibody was used for detection). The signal on the beads in the absence of antigen was subtracted from the signal obtained in the presence of antigen. The signal of each detection antibody was divided by the signal of the capture antibody to determine the fold increase in binding, as shown in Figure 7. Antibodies were then binned based on similar binding patterns of the capture antibody. The data confirmed the existence of three bins of antibody binding for the nine detection antibodies tested (Figure 7).

[0253] Briefly, mouse anti-human IgG1,2,3,4 (BD Pharmingen 555784) conjugated beads were added to the capture antibody in individual dark Eppendorf tubes (353-5 μg / mL). The tubes were rotated overnight in the dark at 4°C. The beads were aliquoted into each well of a filter plate (2500 beads / well) and washed.

[0254] IL-13-RbIg (5 μg / ml) and control (media only) were added to the filter plates at 60 μl / well, which were then incubated in the dark on a shaker at room temperature for 1 hour, followed by two washes.

[0255] Secondary antibodies diluted in medium were added at 60 μl per well (one antibody per well). Antibodies were used at the following concentrations: 353B - 5 μg / ml; 11, 18, 31 - 5 μg / ml; 713 - 0.56 μg / ml; 731 - 1.28 μg / ml; 693 - 2.7 μg / ml; 623 - 5.7 μg / ml; 602 - 11 μg / ml; 643 - 4.3 μg / ml; 785 - 5.5 μg / ml; 763 - 5.7 μg / ml; G2 control - 5 μg / ml). Plates were then incubated at room temperature for 2 hours and washed.

[0256] Biotinylated Mo anti-HuIg G1, 2, 3, 4 (BD Pharmingen #555785) diluted in medium at 5 μg / ml was added to each well (60 μl / well), and the plate was incubated on a shaker at room temperature for 1 hour in the dark. After washing, 60 μl / well of streptavidin-PE (5 μg / mL; Pharm #554061) diluted in medium was added. The plate was incubated on a shaker at room temperature for 20 minutes in the dark and washed twice.

[0257] Each well was carefully resuspended in 80 μl of storage / blocking buffer (PBS, 10 mg / ml BSA, 0.05% wt / vol sodium azide) by pipetting up and down several times to resuspend the beads. Each well was analyzed by reading on a Luminex with a gate set at 8,400–14,500.

[0258] The Luminex platform is a fluorescent bead-based technology that allows multiple assays to be performed at once. The Luminex reader can identify positive signal events on differently coded microspheres. This allows each bead to be coated separately, then the differently coated microspheres are mixed together, and antibodies are then conjugated to each of the different microspheres in a single assay. For antibody isotyping, the microspheres were coated so that each bead could specifically bind to a particular heavy or light chain isotype. The microspheres were then mixed together, and hybridoma supernatants for each antibody were added. After a 20-minute incubation, the microspheres were washed, and bound antibodies were detected using fluorescently labeled secondary antibodies. The microspheres were then read using the Luminex reader.

[0259] Example 3: Preclinical in vivo data Humanized IL-13 mice Humanized IL-13 mice, in which the gene encoding mouse IL-13 was disrupted by insertion of a cDNA encoding human IL-13, were generated at Lexicon (The Woodlands, Texas). Mice were backcrossed to the A / J strain to ensure susceptibility to allergen-induced airway hyperresponsiveness, as previously described (Ewert et al., (2000) Am. J. Respir. Cell. Mol. Biol.).

[0260] To demonstrate that humanized IL-13 mice produce only human IL-13 and not mouse IL-13, we analyzed OVA-specific CD4+ cells derived from humanized IL-13 mice (6-8 weeks old). + Cytokine production from T cells was measured using CD4 + T cells were compared. Mice were sensitized (three mice per treatment) by intraperitoneal injection of 50 μg OVA / 1 mg Imject Alum (Pierce, Rockford, IL) in 0.9% sterile saline or PBS. Seven days after sensitization, mice were sacrificed and single-cell suspensions of spleens were prepared. Erythrocytes were lysed, and 5 × 10 washed splenocytes were cultured in complete medium consisting of HL-1 (BioWhittaker, Walkersville, MD) containing 10% heat-inactivated FCS, 2 mM L-glutamine, and 50 mg / L neomycin sulfate. 6 The splenocytes were then resuspended at 200 cells / ml. The splenocytes were then cultured at 37°C for 4 days in the presence of 200 μg / ml OVA, and Ag-reactive CD4 + T cells were generated. CD4 + T cells (5×10 5 cells / well) and then freshly isolated splenocytes (5 × 10 ) treated with mitomycin C (25 μg / ml) from wild-type mice were added. 5 The cells were incubated with 100 μg / ml OVA in complete medium in 96-well plates (250 μl / well) for 96 hours.

[0261] Cell-free culture supernatants were collected and tested for cytokine production. Human and mouse IL-13 (DuoSet, R&D Systems, Minneapolis, MN) concentrations were measured by ELISA according to the manufacturer's protocol. As expected, CD4+ from humanized IL-13 mice after in vitro OVA restimulation was significantly elevated. + T cells produced human IL-13, but not mouse IL-13 (Figure 8, panel A). In contrast, CD4 T cells from wild-type mice produced human IL-13, but not mouse IL-13 (Figure 8, panel A). + T cells produced mouse IL-13 but not human IL-13 (Figure 8, panel B).

[0262] Airway hyperresponsiveness The anti-IL-13 antibodies 731 and 623 were tested in an OVA-induced asthma model using the humanized IL-13 mice described above. A 24-day protocol was used to measure airway responsiveness to intravenous acetylcholine. Briefly, mice were immunized by intraperitoneal injection of OVA (10 μg; crude grade IV; Sigma) in PBS (0.2 ml). PBS alone was used as a control. 14 days after immunization, mice were anesthetized with a mixture of ketamine and xylazine [45 mg and 8 mg per kilogram of body weight (mg / kg)] and challenged intratracheally with 50 μl of a 1.5% OVA solution or an equal volume of PBS as a control.

[0263] Seven days after the initial challenge, mice were challenged intratracheally with either OVA or PBS. The day before each challenge (days 13 and 20), 731 and 623 antibodies were administered intraperitoneally at a dose of 100 μg / mouse. Control mice received PBS or an irrelevant IgG2 as an isotype control. Three days after the final intratracheal challenge, mice were anesthetized with sodium pentobarbital (90 mg / kg), intubated, and mechanically ventilated with a constant tidal volume of air (0.2 ml) at a rate of 120 breaths / min, followed by paralysis with decamethonium bromide (25 mg / kg). After achieving stable airway pressure, acetylcholine was intravenously injected (50 μg / kg), and dynamic airway pressure was measured for 5 minutes. Airway hyperresponsiveness (AHR) to acetylcholine challenge was measured. Airway hyperresponsiveness to acetylcholine challenge was defined by the time-integrated rise in peak airway pressure [airway pressure time index (APTI), HO (cm) × seconds]. *P<0.05 compared with the OVA + IgG2 control group [one-way analysis of variance (ANOVA) followed by Fisher's least significant difference test for multiple comparisons]. Treatment with 731 or 623 resulted in complete reversal of OVA-induced AHR (Figure 9). In this example, complete reversal means that the addition of antibody with OVA resulted in an effect similar to that of adding antibody alone (e.g., IgG2) in the absence of OVA.

[0264] OVA-induced mucus production An 18-day protocol was used to measure OVA-induced mucus production. After subcutaneous priming with ovalbumin (OVA, 25 μg; crude grade IV) (Sigma) in 2 mg of Imject Alum on days 0 and 7, mice were anesthetized with isoflurane and challenged intranasally with 50 μl of a 1.5% OVA solution in PBS on days 14, 15, and 17. Control mice received alum for priming or PBS for challenge.

[0265] On days 13, 15, and 17, the 731 and 623 antibodies were administered intraperitoneally at a dose of 100 μg / mouse. Control mice received PBS. On day 18, mice were sacrificed, and lungs were harvested after perfusion. Lung tissues, including central and peripheral airways, were fixed in 10% formalin, washed in 70% ethanol, dehydrated, embedded in glycol methacrylate, cut into 4 μM sections, mounted on slides, and stained with hematoxylin and eosin and periodic acid-Schiff (PAS). Lung sections (one per animal) were examined at 20x magnification. Five areas were randomly selected, and the number of bronchi within each area was counted for each section. Sections were scored on a scale of 0 to 4 (0: <5% PAS). + Goblet cells: 1: 5-25%; 2: 25-50%; 3: 50-75%; 4: >75%. To obtain the histological goblet cell score (expressed as arbitrary units; U), the sum of the airway scores for each lung was divided by the number of bronchi examined. Five of eight mice died in the OVA-treated group. No mice died in the other groups. Administration of 731 and 623 effectively reversed the OVA-induced increase in mucus-containing cells in the airways (Figure 10). Data are means ± SE. n = 3 for the OVA / OVA / PBS group (originally n = 8); n = 8 for the OVA / OVA / 731 group; n = 4 for the OVA / OVA / 623 group; n = 4 for the OVA / PBS / PBS group; n = 5 for the alum / OVA / PBS and alum / PBS / PBS groups. *p < 0.01 vs. the OVA / OVA / PBS group by unpaired Student's t-test.

[0266] Example 4: Antibody structural analysis The variable heavy and variable light chains of the antibodies shown in Table 1 were sequenced to determine their DNA sequences. Complete sequence information for all anti-IL-13 antibodies, including nucleotide and amino acid sequences, is provided in the Sequence Listing submitted herewith.

[0267] Table 18 shows the amino acid sequences of the heavy chain genes of various IL-13 antibodies described herein. Table 18 also shows the amino acid sequences corresponding to the CDR and framework regions of each antibody, as well as a comparison to their germline sequences.

[0268] Table 19 shows the amino acid sequences of the kappa light chain genes of various IL-13 antibodies described herein. Table 19 also shows the amino acid sequences corresponding to the CDR and framework regions of each antibody, as well as a comparison to their germline sequences.

[0269] Table 20 shows the amino acid sequences of the lambda light chain genes of the various IL-13 antibodies described herein. Table 20 also shows the amino acid sequences corresponding to the CDR and framework regions of each antibody, as well as a comparison to the germline sequence.

[0270] [Table 18]

[0271] [Table 19]

[0272] [Table 20]

[0273] [Table 21]

[0274] [Table 22]

[0275] [Table 23]

[0276] [Table 24]

[0277] Example 5: Generation of affinity matured anti-IL-13 antibodies The novel use of mammalian recombination signal sequence (RSS)-directed recombination for complementarity-determining region (CDR)-targeted protein engineering, based on sequences from Xenomouse®-derived antibodies, closes the species affinity gap of antibody 731 (Ab731).

[0278] Using this non-hypothesis-driven affinity maturation method, we generated multiple antibody variants with improved IL-13 affinity, including an antibody with the highest affinity reported to date for human IL-13 while maintaining high cross-reactivity to cyno IL-13.

[0279] HuTARG technology is a novel RSS recombination-based protein engineering platform coupled to cell surface display in mammalian cell culture. Briefly, DNA encoding the heavy and light chain complementarity-determining regions (CDRs) of Ab731 was engineered to contain RSS sites using standard molecular biology methods. The resulting plasmid pools encoded individual CDRs targeted for RSS integration, and successful integration of the RSS signal into each CDR was confirmed by terminal restriction fragment length polymorphism (T-RFLP). Using the Cre-Lox system, the resulting pool of constructs was stably integrated into the HuTARG cell line. HuTARG cells are recombination-competent mammalian cells in which RAG-1-mediated recombinase activity is induced under tetracycline treatment. Upon induction of recombination, a unique rearrangement occurs in each cell, involving removal of the RSS cassette. In the presence of terminal deoxynucleotidyl transferase (TdT), double-strand break repair occurs, resulting in imperfect ligation of the recombined segments and sequence variation in the human IgG antibody. The antibody is then expressed on the cell surface through directed gene integration into loxP sites, and each cell expresses a unique, monospecific antibody. Because the heavy and light chain sequences were targeted separately, two individual pools of vectors were generated and used to create diversified heavy and light chain antibody cell surface display libraries. Because recombination occurs directly within the cells and does not require a transformation step, the limit of library complexity is determined by the cell number. In this case, we utilized the complexity of 342E6 cells for diversified heavy chains and 320E6 cells for diversified light chains. Mutants with improved affinity (determined by higher binding than that of the parent antibody) were isolated by three rounds of FACS sorting by surface staining with 36 pM recombinant soluble cyno IL-13. Mutant antibody sequences selected for improved affinity were found to contain insertions and substitutions distributed relatively evenly across the enriched light chain, consistent with the frequency of RSS incorporation.However, CDR-H3 was the least variable, which is not surprising since it is the major determinant of epitope recognition in B cells that leave the bone marrow. Insertions and deletions were the dominant mutations observed in affinity-enriched FACS-sorted cells.

[0280] PCR-rescued antibody sequences were cloned and transiently expressed on the surface of HEK293T cells, and their binding to soluble human IL-13 and cyno IL-13 was ranked by FACS analysis. Geometric mean fluorescence values for binding to the target proteins were compared across three gates based on cell surface IgG expression. Two of the heavy chain variants that showed improved binding to cyno IL-13, heavy chain 1 (HC1) and HC2, were confirmed by Kinase Analysis to have higher affinity than their parental antibodies (Figure 1C, Supplementary Table 20(a)). Similar analysis was performed on light chain variants, identifying three light chain sequences (LC1, LC2, and LC3) that exhibited higher binding than the parental antibodies. These light chain variants were then combined with the best heavy chain variants in a checkerboard pattern across all possible permutations. This led to the identification of three antibodies (MMAb3, MMAb5, and MMAb7) that exhibited improved affinity for both cyno IL-13 and human IL-13, demonstrating stronger binding than antibodies with individual mutations in the light or heavy chain. Their biological potency was assessed for neutralization of eotaxin-1 release from normal human dermal fibroblast (NHDF) cells stimulated with human IL-13 or cyno IL-13. All antibodies were found to be potent inhibitors of both ligands, with EC50 values limited by the concentration of IL-13 in the assay. In contrast, the parental Ab731 showed no activity against cyno IL-13.

[0281] Using KinExA-based affinity determination, we determined the formal affinities of the newly generated antibody variants for human and cyno IL-13 targets, demonstrating that MMAb3 had an affinity of 5.1 pM for cyno IL-13 and 34 fM for human IL-13, thus representing a 700-fold and 56-fold improvement, respectively. To our knowledge, the affinity of MMAb7 (and MMAb5 at 142 fM) for human IL-13 protein is among the highest described in the literature. We previously reported an in vivo generated anti-IL8 antibody with sub-picomolar (610 fM) affinity measured by KinExA technology. Other examples of very high affinity antibodies include affinity maturation by site-directed mutagenesis of the murine anti-IL1b antibody XMA005, followed by its humanization, XOMA 052, which yields sub-picomolar antibodies (240 fM and 300 fM, respectively) similarly measured by KinExA technology (Owyang, AM et al. XOMA 052, a potent, high-affinity monoclonal antibody for the treatment of IL-1beta-mediated diseases. mAbs 3, 49-60 (2011)).An engineered anti-fluorescein single-chain antibody was also reported to have a Kd of 270 fM for the small molecule hapten fluorescein, similarly measured by equilibrium binding (Boder, E.T., Midelfort, K.S. & Wittrup, K.D. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proceedings of the National Academy of Sciences of the United States of America 97, 10701-10705 (2000); Midelfort, K.S. et al. Substantial energetic improvement with minimal structural perturbation in a high affinity mutant antibody. Journal of molecular biology 343, 685-701 (2004)).

[0282] [Table 25]

[0283] Example 6: Epitopes and Co-crystallization Co-crystal structures of the novel antibody with cynomolgus monkey IL-13 and human IL-13. To understand the molecular determinants of the very high affinity interaction of the MMAb3 antibody with cyno IL-13 and human IL-13, we generated a crystal structure of cyno IL-13 in complex with the MMAb3 fragment antigen binding (Fab) at 2.1 Å resolution (Figure 2A), followed by homology models of MMAb1 and MMAb2 bound to the same ligand. The crystal structure of cyno IL-13 in complex with MMAb3 Fab revealed that helix C of IL-13 is oriented parallel to the Fab cleft and interposed between the Fab heavy and light chains. The resolution is 1784.8 Å. 2The total buried solvent accessible surface area (SASA) of the antibody is less than that observed at the average antibody-antigen interface (1500-1600 Å). 2 ) 25 The overall shape complementarity score (Sc) of 0.714 indicates that the degree of complementarity of the IL-13-Fab interface is on average (0.64-0.68). 26 This suggests that the solubility of the two molecules is much higher than that of the other two molecules, indicating an extensive and compatible interface between the two molecules.

[0284] The crystal structure demonstrated the high affinity of MMAb3 for cyno IL-13 and human IL-13. MMAb3 differs from the parent Ab731 by three consecutive residues in CDR-H2 (Trp54 / Asp55 / Val56 vs. Ser54 / Gly55 / Gly56) and two consecutive residues on CDR-L1 (Ser32 / Phe33 vs. Thr32 / Cys33) (Figure 11). The first set of residues induces the formation of a π-π stacking channel, in which the engineered residue Trp54 from CDR-H2 elevates π-π stacking interactions with Pro103 of CDR-H2, and further along the channel, similar contacts are made between Tyr104 of CDR-H2 and IL-13 residues Pro72 and His73, which are primarily involved in van der Waals contacts (Figure 13). It is also possible that the presence of Asp55 and Val56 in place of the parent Gly55 and Gly56 may serve to further stabilize the backbone of the CDR-H2 loop, although this has been difficult to assess energetically due to the conformational variability associated with the presence of the two subsequent Gly residues. The central role of Trp54 for the binding interface is also explained by the presence of 170.1A, which constitutes nearly 10% of all SASAs. 2This is also evidenced by the buried surface area of 54. Thus, structural evidence suggests that Trp54 is responsible for stabilization rather than a direct determinant of affinity or specificity, two properties we believe are more likely driven by interactions from CDR-L1 (Figures 12A and 12B). Because the CDR-L1 paratopes of MMAb3 and Ab731 differ with respect to two residues (Ser32 / Phe33 vs. Thr32 / Cys33), we sought to identify altered binding interactions in this region that confer greater cross-reactivity between cyno IL-13 and human IL-13. In the crystal structure, Asn68 from cyno IL-13 is located between Tyr31 and the IL-13 backbone carboxyl derived from residues 73–76. The structure suggests that the tight space created by these contacts locks Asn68 in a conformation that allows binding, albeit via a suboptimal hydrogen bond with Tyr31 (Figure 12A and B). In human IL-13, a Ser residue replaces Asn68 (Figure 13). Given its smaller size and greater distance from the surrounding IL-13 backbone residues, Ser68 is conformationally less restricted and better positioned to establish a stronger hydrogen bond with the hydroxyl group of Tyr31, resulting in tighter binding (consistent with experimental data). In either case, although very difficult to predict a priori, the conformation of Tyr31 may be stabilized by the downstream engineered residue Phe33, whose bulky aromatic ring occupies a cavity and prevents rotamer flipping, in contrast to the parent antibody with Cys33 (Figure 13) and the surrounding cavity. In the context of the parent antibody in complex with human IL-13, the higher affinity of the Ser68:Tyr31 interaction favors an outward conformation of Tyr31, despite the presence of a cavity surrounding Cys33. This contrasts with cyno IL-13, where the lower affinity of the Asn68:Tyr31 interaction forces the tyrosine back to occupy the cavity surrounding Cys33, thereby reaching an internal energy minimum. However, this significantly reduces the affinity of the parent antibody for cyno IL-13.

[0285] Example 7: High affinity anti-IL-13 antibodies For stability and viscosity, several mutations were made to the Mmab7 light and heavy chains, and their high affinity anti-IL13 amino acid sequences are listed below in Tables 21-22.

[0286] [Table 26]

[0287] [Table 27]

[0288] [Table 28]

[0289] [Table 29]

[0290] [Table 30]

[0291] [Table 31]

[0292] [Table 32]

[0293] [Table 33]

[0294] [Table 34]

[0295] Table 35

[0296] Table 36

[0297] Table 37

[0298] Table 38

[0299] Table 39

[0300] Table 40

[0301] Table 41

[0302] Table 42

[0303] Table 43

[0304] Table 44

[0305] Example 8: Use of anti-IL-13 antibodies as diagnostic agents for detecting IL-13 in a sample An enzyme-linked immunosorbent assay (ELISA) can be developed to detect IL-13 in a sample. In this assay, a first fully human monoclonal antibody directed against IL-13 is adsorbed to several wells of a microtiter plate, e.g., a 96-well or 384-well microtiter plate, for several hours. The immobilized antibody serves as a capture antibody for IL-13 that may be present in the test sample. The wells are rinsed and treated with a blocking agent, such as milk protein or albumin, to prevent nonspecific adsorption of the analyte.

[0306] The wells are then treated with a test sample suspected of containing IL-13, or with a solution containing a standard amount of antigen.

[0307] After rinsing away the test sample or standard, the wells are treated with a second fully human monoclonal anti-IL-13 antibody labeled by conjugation with biotin. The labeled anti-IL-13 antibody serves as the detection antibody. After rinsing away excess second antibody, the wells are treated with avidin-conjugated horseradish peroxidase (HRP) and an appropriate chromogenic substrate. The concentration of antigen in the test sample is determined by comparison with a standard curve developed from the standard samples.

[0308] Example 9: Treatment of COPD in humans A patient suffering from COPD is identified. The patient is given an effective amount of the anti-IL-13 antibody disclosed above, administered intravenously or subcutaneously to the patient. A booster dose is administered three weeks later and every three weeks thereafter. The anti-IL-13 antibody inhibits mucus production, the development of bronchial epithelial hyperplasia, and bronchial smooth muscle spasm. This inhibition of mucus production and smooth muscle contraction reduces airway obstruction and improves ventilation.

[0309] Example 10: Treatment of chronic bronchitis in humans A patient suffering from chronic obstructive pulmonary disease ("COPD"), characterized by chronic bronchitis, is identified. The patient is administered an effective amount of an anti-IL-13 antibody disclosed herein by intravenous or subcutaneous injection. Treatment can be repeated weekly, or every two weeks, or every three weeks, or every four weeks, or every month, or every other month. The anti-IL-13 antibody causes partial or complete inhibition of mucus production in inflamed respiratory tissue and bronchial smooth muscle contraction. This inhibition of mucus production and smooth muscle contraction reduces airway obstruction and improves ventilation.

[0310] Example 11: Treatment of Emphysema in Humans A patient suffering from emphysema is identified. An effective amount of an IL-13 antibody is administered to the patient via intravenous or subcutaneous injection. Treatment can be repeated weekly, every two weeks, every three weeks, every four weeks, every month, or every other month. The IL-13 antibody causes partial or complete inhibition of neutrophil chemotaxis in inflamed respiratory tissue. Inhibition of neutrophil chemotaxis reduces the severity of tissue damage to the lungs and airways caused by the patient's immune response. IL-13 has a direct effect (at least it is hypothesized) on the induction of proteases that lead to the destruction of lung tissue in emphysema.

[0311] Example 12: Treatment of asthma in humans A patient suffering from asthma is identified. The patient is administered an effective amount of an IL-13 antibody by intravenous or subcutaneous injection into the patient.

[0312] Treatment can be repeated weekly, or every two weeks, or every three weeks, or every four weeks, or every month, or every other month. The anti-IL-13 antibody reduces the severity of tissue damage to the lungs and airways caused by the patient's immune response.

[0313] Example 13: Treatment of atopic dermatitis in humans A patient suffering from atopic dermatitis is identified. The patient is administered an effective amount of an IL-13 antibody by intravenous or subcutaneous injection. Treatment can be repeated weekly, every two weeks, every three weeks, every four weeks, every month, or every other month.

[0314] Example 14: Optimized sequences for high affinity anti-IL-13 antibodies Engineering of the MmAb5 / MmAb7 hotspot began with an in silico scan using internal software for chemical propensity and consensus deviations from the germline sequence. Results revealed four potential isomerization sites in the CDRs (Asp Ser and Asp Gly), one consensus deviation, and one potential Trp oxidation site in the CDR. To evaluate potential mutations and mitigate risk, internally derived co-crystal structures of MmAb7 and IL-13 were analyzed for hotspot residue exposure and interactions. From this structure, it was observed that the Asp residue directly interacts with a positively charged residue on the antigen in LC:DS 67-68. To preserve this interaction, adjustments were limited to changes in the Ser residue, and the Ala substitution was chosen based on internal experience. In LC:DS 110-111, it was observed that Asp interacts with a positively charged residue on the adjacent CDR, providing structure. To preserve its function in the molecule, the Asp was left intact and the DS was changed to DA. In HC:DS 72-73, the isomerization site was found to be exposed on the surface of the molecule and non-interacting. DS was changed to DA to improve homogeneity. The HC:DG 109-110 site was observed to be buried within the structure and not subject to isomerization. G110A mutants were tested only individually and found to lose activity. In HC:Q108, the residue was changed to germline. Hotspot tuning at various sites was tested individually and in combination using rational design. Lead mutant selection was based on production yield, Tm, and functional activity using TARC and eotaxin assays.

[0315] Engineering the viscosity of MmAb5 and MmAb7 began with in silico surface analysis of the MmAb7 / IL-13 cocrystal structure using BioLuminate Schrodinger software, which identified charge patches and contributing residues. Key contributing residues from the most prominent charge patches were analyzed for potential antigen binding and structural impact. The LC:D3 residue was replaced with Val derived from an alternative germline. The LC:D87 residue was replaced with Asn derived from an alternative germline. For optimal patch disruption, residues L:D26, L:D33, and L:D110 were replaced with Lys. Tuning viscosity at various sites was tested individually and in combination using rational design. Lead mutant selection was based on production yield, viscosity measured by the cone-plate method, Tm, and functional activity using the TARC assay.

[0316] The TARC assay measures inhibitory TARC produced by IL-13-sensitive progenitor cells in the presence of IL-13. The anti-IL-13 mAb to be measured for TARC inhibition is serially diluted and then added to a set amount of IL-13 (3 ng / mL) and incubated at room temperature for 20 minutes. After incubation, the mAb and IL-13 solution is added to 2e5 cells in a 96-well tissue culture plate and incubated at 37°C and 5% CO2 for 48 hours. After incubation, samples are taken and TARC is measured using an anti-TARC mAb from MSD's detection kit. The plate is read using an MSD 6000. Dose-response data is analyzed, dose-response curves are generated, and IC50 levels are calculated using Graph Pad Prism software.

[0317] Variants of anti-IL-13 monoclonal antibodies also contain half-life-extending mutations at EU positions M252Y, S254T, and T256E within the Fc, commonly known as YTE mutations. These modifications improve FcRn binding by allowing the antibody to recirculate in the circulation after endocytosis by effector function cells. These modifications are intended to prolong PK and reduce the required dose and / or frequency of administration. See Figure 14.

[0318] The anti-IL-13 monoclonal antibody variant also contains a complement hexamer-disrupting mutation at Eu position S583K within the Fc. By preventing mAb hexamer formation as part of the combined effector function mechanism, the modification has been observed to reduce antibody viscosity at a given formulation and concentration.

[0319] Incorporation by Reference All references cited herein, including patents, patent applications, articles, textbooks, etc., and references cited therein, are incorporated by reference in their entirety, unless already incorporated.

[0320] equivalent The foregoing specification is considered sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and set forth the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears, it will be understood that the invention can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

Claims

1. 1. An antigen-binding protein that specifically binds to human IL-13, comprising a light chain immunoglobulin variable region (VL1) and a heavy chain immunoglobulin variable region (VH), the VL1 comprises (i) a CDRL1 comprising the amino acid sequence of SEQ ID NO: 11, (ii) a CDRL2 comprising the amino acid sequence of SEQ ID NO: 12, and (iii) a CDRL3 comprising the amino acid sequence of SEQ ID NO: 13; An antigen binding protein, wherein the VH comprises: (i) a CDRH1 comprising the amino acid sequence of SEQ ID NO: 8; (ii) a CDRH2 comprising the amino acid sequence of SEQ ID NO: 9; and (iii) a CDRH3 comprising the amino acid sequence of SEQ ID NO:

10.

2. 1. An antigen-binding protein that specifically binds to human IL-13, comprising a light chain immunoglobulin variable region (VL1) and a heavy chain immunoglobulin variable region (VH), the VL1 comprises the CDRs of the antibody expressed by cell 623; An antigen-binding protein, wherein the VH comprises the CDRs of an antibody expressed by cell 623.

3. 2. The antigen binding protein of claim 1, further comprising a framework region similar to that of an antibody expressed by cell 623.

4. The antigen-binding protein of any one of claims 1 to 3, wherein the antigen-binding protein is an antibody.

5. The antigen-binding protein of any one of claims 1 to 3, wherein the antigen-binding protein is an antibody fragment.

6. 4. The antigen-binding protein of any one of claims 1 to 3, wherein the antigen-binding protein is an antibody derivative, including a bispecific antibody, a fusion protein.

7. 7. The antigen binding protein of any one of claims 1 to 6, wherein the antigen binding protein has a human sequence.

8. The antigen-binding protein of any one of claims 1 to 6, wherein the antigen-binding protein is a monoclonal antibody.

9. K between 2 cM and 50 pM Ｄ A human antibody that binds to IL-13, wherein the antibody binds to IL-13 at the

10. K between 2 cM and 40 pM Ｄ A human antibody that binds to IL-13, wherein the antibody binds to IL-13 at the

11. A human antibody or antigen-binding fragment thereof that binds to human IL-13, the antibody having the amino acid sequence: (a) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 11, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 13; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 8, an HCDR2 of SEQ ID NO: 106, and an HCDR3 of SEQ ID NO: 10; (b) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 11, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 13; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 8, an HCDR2 of SEQ ID NO: 83, and an HCDR3 of SEQ ID NO: 10; (c) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 11, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 13; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 8, an HCDR2 of SEQ ID NO: 83, and an HCDR3 of SEQ ID NO: 10; (d) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 74, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 76; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 107, an HCDR2 of SEQ ID NO: 85, and an HCDR3 of SEQ ID NO: 10; (e) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 77, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 76; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 107, an HCDR2 of SEQ ID NO: 85, and an HCDR3 of SEQ ID NO: 10; (f) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 79, an LCDR2 of SEQ ID NO: 12, and an LCDR3 of SEQ ID NO: 78; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 107, an HCDR2 of SEQ ID NO: 85, and an HCDR3 of SEQ ID NO: 10; (g) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO:79, an LCDR2 of SEQ ID NO:80, and an LCDR3 of SEQ ID NO:78; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO:107, an HCDR2 of SEQ ID NO:85, and an HCDR3 of SEQ ID NO:10; (h) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 81, an LCDR2 of SEQ ID NO: 80, and an LCDR3 of SEQ ID NO: 78; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 107, an HCDR2 of SEQ ID NO: 85, and an HCDR3 of SEQ ID NO: 10; and (i) an antibody variable light chain amino acid sequence comprising an LCDR1 of SEQ ID NO: 82, an LCDR2 of SEQ ID NO: 80, and an LCDR3 of SEQ ID NO: 78; and an antibody variable heavy chain amino acid sequence comprising an HCDR1 of SEQ ID NO: 107, an HCDR2 of SEQ ID NO: 85, and an HCDR3 of SEQ ID NO:

10.

12. A human antibody or antigen-binding fragment thereof that binds to human IL-13, the antibody having the amino acid sequence: (a) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 86 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 87; (b) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 88 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 89; (c) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 90 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 91; (d) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 92 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 93; (e) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 94 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 95; (f) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 96 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 97; (g) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 98 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 99; (h) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 100 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 101; (i) an antibody variable light chain amino acid sequence comprising SEQ ID NO: 102 and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO: 103; and (j) a human antibody or antigen-binding fragment thereof, comprising a variable light chain region and a variable heavy chain region selected from the group comprising: an antibody variable light chain amino acid sequence comprising SEQ ID NO: 104; and an antibody variable heavy chain amino acid sequence comprising SEQ ID NO:

105.

13. A human antibody or antigen-binding fragment thereof that binds to human IL-13, the antibody having the amino acid sequence: (a) a light chain selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37; SEQ ID NO:39, SEQ ID NO:41; SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, and SEQ ID NO:73; and (b) a human antibody or antigen-binding fragment thereof comprising a heavy chain selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36; SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:

72.

14. An antibody comprising a light chain and a heavy chain having the amino acid sequence according to any one of claims 1 to 14, 20, and 25 to 27.

15. A nucleic acid sequence encoding the antibody or antibody fragment thereof according to any one of claims 1 to 14.

16. A vector comprising a nucleic acid sequence encoding the antibody or antibody fragment thereof according to any one of claims 15, 20, 25 to 27.

17. A host cell comprising the vector of claim 16.

18. 18. The host cell of claim 17, wherein the host cell is a CHO cell or an Sp2 / 0 cell.

19. 19. The host cell of claim 18, wherein the host cell is a CHO cell.

20. An antibody or antibody fragment produced by a host cell according to any one of claims 18 to 19.

21. A pharmaceutical composition comprising the antibody or antibody fragment of claim 20.

22. A method for producing an antibody or a fragment thereof by culturing the host cell of any one of claims 17 to 19.

23. 21. A method of treating a patient suffering from COPD, emphysema, asthma, or atopic dermatitis by administering to said patient an effective amount of the antibody or fragment thereof of claim 20.

24. 22. A method of treating a patient suffering from COPD, emphysema, asthma, or atopic dermatitis by administering to said patient an effective amount of the pharmaceutical composition of claim 21.

25. 21. The antibody of claim 20, wherein a half-life extending mutation is present.

26. 26. The antibody of claim 25, wherein the half-life extending mutations are mutations at Eu positions M252Y, S254T, and T256E within Fc.

27. 26. The antibody of claim 25, wherein the half-life extending mutation is a complement hexamer disrupting mutation at Eu position S583K in Fc.

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