Antibodies with binding specificity for human IL-13

Abstract

The present invention relates to antibody molecules specific for epitopes of human IL-13, the therapeutic use of the antibody molecules, and methods for producing the antibody molecules.

Description

Field of the Invention

[0001] The present invention relates to IL-13 antibodies and fragments thereof, such as binding fragments thereof, compositions comprising the same, and in particular to their use in preventing and / or treating IL-13 related diseases. Background Art

[0002] IL-13 is a short-chain cytokine that shares 25% sequence identity with IL-4. It contains approximately 132 amino acids, forming a secondary structure of four helices spanning residues 10-21 (helix A), 43-52 (helix B), 61-69 (helix C), and 92-110 (helix D), and two beta strands spanning residues 33-36 and 87-90. The solution structure of IL-13 has been solved, revealing a predicted up-up-down-down four-helix bundle conformation, which is also observed in IL-4.

[0003] Human IL-13 is a 17 kDa glycoprotein and is produced by activated T cells of the Th2 lineage, although Th0 and Th1 CD4+ T cells, CD8+ T cells, and several non-T cell populations such as mast cells also produce IL- 13. The functions of IL-13 include switching immunoglobulin isotypes to IgE in human B cells and inhibiting inflammatory cytokine production in humans and mice.

[0004] IL-13 binds to its cell surface receptors IL-13R-α1 and IL-13R-α2. IL-13R-α1 binds to IL-13R-α2 with low affinity (K D ~10 nM) interacts with IL-13 and subsequently recruits IL-4R-α to form a high affinity (K D ~0.4nM) signaling heterodimeric receptor complex.

[0005] The IL-4R / IL-13R-α1 complex is expressed on many cell types, such as B cells, monocytes / macrophages, dendritic cells, eosinophils, basophils, fibroblasts, endothelial cells, airway epithelial cells, and airway smooth muscle cells. Ligation of the IL-13R-α / IL-4R receptor complex leads to the activation of multiple signal transduction pathways, including the signal transducer and activator of transcription 6 (STAT6) and insulin receptor substrate 2 (IRS2) pathways.

[0006] The IL-13R-α2 chain alone has a high affinity for IL-13 (K D ~0.25-0.4 nM). It functions both as a decoy receptor that negatively regulates IL-13 binding and as a signaling receptor that induces TGF-β synthesis and fibrosis through the AP-1 pathway in macrophages and possibly other cell types.

[0007] IL-13 is involved in the pathogenesis of many human diseases, and therapeutic strategies have been designed to inhibit or counteract IL-13 activity. In particular, antibodies that bind and neutralize IL-13 have been sought as a means of inhibiting IL-13 activity. However, there is a need in the art for suitable and / or improved antibodies that can bind to IL-13, especially human IL-13, and in particular antibodies that can neutralize human IL-13. The present invention provides a new family of binding proteins, CDR-grafted antibodies, humanized antibodies and fragments thereof that can bind to human IL-13, bind with high affinity, and bind and neutralize human IL-13. Summary of the invention

[0008] The present invention provides an improved antibody that binds to human IL-13, in particular a neutralizing antibody that inhibits the biological activity of IL-13. The present invention further provides a pharmaceutical composition comprising the antibody and its use in treating IL-13 related diseases.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1. Ab650 humanization alignment.

[0011] Alignment of rat antibody (donor) V region sequences with human germline (acceptor) V region sequences and designed humanized sequences.

[0012] (A) Light chain graft 650:

[0013] 650 = rat variable light chain sequence.

[0014] 650gL8 = variable light chain of a humanized graft of 650 using the IGKV1-39 human germline as acceptor framework.

[0015] CDRs are shown in bold / underlined.

[0016] Donor residues are shown in bold / italic and highlighted: I58 and Y71.

[0017] (B) Heavy chain graft 650:

[0018] 650 = rat variable heavy chain sequence.

[0019] 650gH9 = variable heavy chain of a humanized graft of 650 using the IGHV1-69 human germline as acceptor framework.

[0020] CDRs are shown in bold / underlined.

[0021] Donor residues are shown in bold / italic and highlighted: A67, F69 and V71.

[0022] Figure 2. Amino acid and DNA sequences of anti-IL13.

[0023] Amino acid and DNA sequences encoding the CDRs, heavy and light chain variable regions, scFv and dsscFV forms of antibody 650. DETAILED DESCRIPTION OF THE INVENTION

[0025] Antibody

[0026] Antibodies used in the context of the present disclosure include whole antibodies and functionally active fragments thereof, i.e. molecules comprising an antigen binding domain that specifically binds to IL-13, also referred to as antigen binding fragments. Unless the context dictates otherwise, the features described herein with respect to antibodies also apply to antibody fragments.

[0027] Whole antibodies, also called "immunoglobulins (Ig)", generally refer to complete or full-length antibodies, i.e., elements comprising two heavy chains and two light chains interconnected by disulfide bonds, which assemble to define a characteristic Y-shaped three-dimensional structure. Classical natural whole antibodies are monospecific, in that they bind one antigen type, and are bivalent, in that they have two independent antigen-binding domains. The terms "whole antibody", "full-length antibody" and "whole antibody" are used interchangeably and refer to a monospecific, bivalent antibody having a structure similar to a natural antibody structure, including an Fc region as defined herein.

[0028] Each light chain is composed of a light chain variable region (abbreviated herein as V L ) and the light chain constant region (C L Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H ) and the heavy chain constant region (C H ), the heavy chain constant region (C H ) consists of three constant domains C H1 , C H2 and C H3 or four constant domains C H1 , C H2 , C H3 and C H4 The composition depends on the Ig class. The "class" of Ig or antibody refers to the type of constant region, and includes IgA, IgD, IgE, IgG, and IgM, and several of them can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0029] V of the antibody according to the present invention H and V LThe V domain can be further subdivided into highly variable regions (or "hypervariable regions") that determine antigen recognition, called complementarity determining regions (CDRs), interspersed with regions that are more structurally conserved, called framework regions (FRs). H and V L It consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. CDR and FR together form the variable region. By convention, the CDRs in the heavy chain variable region of an antibody or its antigen-binding fragment are referred to as CDR-H1, CDR-H2 and CDR-H3, and the CDRs in the light chain variable region are referred to as CDR-L1, CDR-L2 and CDR-L3. They are numbered sequentially in the direction from the N-terminus to the C-terminus of each chain.

[0030] CDRs are generally numbered according to the system devised by Kabat et al. This system is described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al. (supra)"). Unless otherwise indicated, this numbering system is used in this specification.

[0031] The Kabat residue designations do not always correspond directly to the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to shortening or insertion of structural components (whether framework or complementarity determining regions) of the basic variable domain structure. The correct Kabat numbering of residues in a given antibody can be determined by aligning residues with homology in the antibody sequence with the "standard" Kabat numbering sequence.

[0032] According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3). However, according to Chothia (Chothia C. and Lesk A. MJ Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Therefore, unless otherwise indicated, 'CDR-H1' as used herein means residues 26 to 35, as described by the combination of the Kabat numbering system and the Chothia topological loop definition.

[0033] According to the Kabat numbering system, the CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2), and residues 89-97 (CDR-L3).

[0034] In addition to the CDR loops, a fourth loop exists between CDR-2 (CDR-L2 or CDR-H2) and CDR-3 (CDR-L3 or CDR-H3), which is formed by framework 3 (FR3). The Kabat numbering system defines framework 3 as positions 66-94 in the heavy chain and positions 57-88 in the light chain.

[0035] Based on alignments of sequences of different members of the immunoglobulin family, numbering schemes have been proposed, as described, for example, in Kabat et al., 1991 and Dondelinger et al., 2018, Frontiers in Immunology, Vol. 9, Article 2278.

[0036] As used herein, the terms "constant domain", "constant region" are used interchangeably and refer to antibody domains located outside the variable region. The constant domain is the same in all antibodies of the same isotype, but is different between one isotype and another. Typically, the constant region of the heavy chain is formed from N to C terminus by CH1-hinge-CH2-CH3-optionally CH4, which contains three or four constant domains.

[0037] The constant domains of the antibody molecules of the present invention (if present) can be selected considering the functions of the proposed antibody molecules, particularly the effector functions that may be required. For example, the constant domains can be human IgA, IgD, IgE, IgG or IgM domains. In particular, when the antibody molecule is intended for therapeutic use and antibody effector functions are required, human IgG constant domains, particularly IgG1 and IgG3 isotypes can be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, IgG2 and IgG4 isotypes can be used. It should be understood that sequence variants of these constant domains can also be used. For example, IgG4 molecules can be used, wherein the serine at position 241 (numbered according to the Kabat numbering system) has been changed to proline, as described by Angal et al. (Angal et al., 1993, A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody as observed during SDS-PAGE analysis, Mol Immunol 30, 105-108) and referred to herein as IgG4P.

[0038] "Fc", "Fc fragment" and "Fc region" are used interchangeably and refer to the C-terminal region of an antibody, which contains the antibody constant region excluding the first constant immunoglobulin domain. Thus, Fc refers to the last two constant domains C of IgA, IgD and IgG. H2 and C H3 , or the last three constant domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. The human IgG1 heavy chain Fc region is defined herein as comprising residue C226 to its carboxyl terminus, wherein numbering is according to the EU index in Kabat. In the context of human IgG1, according to the EU index in Kabat, the lower hinge refers to positions 226-236, the CH2 domain refers to positions 237-340, and the CH3 domain refers to positions 341-447. The corresponding Fc regions of other immunoglobulins can be identified by sequence alignment.

[0039] In the context of the present disclosure, when present, the constant region or Fc region can be native, as defined above, or can be modified in various ways, provided that it contains a functional FcR binding domain, and preferably a functional FcRn binding domain. Preferably, the modified constant region or Fc region leads to improved function and / or pharmacokinetics. The modification may include the deletion of certain parts of the Fc fragment. The modification may further include various amino acid substitutions that can affect the biological properties of the antibody. There may also be mutations for increasing FcRn binding and thus increasing the half-life in vivo. The modification may further include modifications of the glycosylation profile of the antibody. The native Fc fragment is glycosylated in the CH2 domain, wherein there is an N-glycan bound to the asparagine residue (Asn297) at position 297 on each of the two heavy chains. In the context of the present disclosure, the antibody may be glycosylated, i.e., engineered to have a specific glycosylation profile, which, for example, leads to improved properties, such as improved effector function or improved serum half-life.

[0040] The antibodies described herein are isolated. An "isolated" antibody is one that has been separated from a component of its natural environment, such as by purification means.

[0041] The term "antibody" encompasses monovalent antibodies, i.e. antibodies comprising only one antigen binding domain (e.g., one-armed antibodies comprising interconnected full-length heavy and full-length light chains, also called "half antibodies"), and multivalent antibodies, i.e. antibodies comprising more than one antigen binding domain.

[0042] The term "antibody" according to the present invention also encompasses antigen-binding fragments of antibodies. Antigen-binding fragments of antibodies include single-chain antibodies (e.g., scFv and dsscfv), Fab, Fab', F(ab'), 2, Fv, single domain antibodies or nanobodies (e.g. V H or V L , or V HH or V NAR Other antibody fragments useful in the present invention include Fab and Fab' fragments described in international patent applications WO2011 / 117648, WO2005 / 003169, WO2005 / 003170 and WO2005 / 003171.

[0043] Methods for generating and manufacturing these antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0044] The term "Fab fragment" as used herein refers to an antibody fragment comprising a light chain fragment comprising the VL (light chain variable region) domain and the constant domain (CL) of the light chain, and the VH (heavy chain variable region) domain and the first constant domain (CH1) of the heavy chain.

[0045] A typical "Fab' fragment" comprises a heavy chain and a light chain pair, wherein the heavy chain comprises a variable region VH, a constant domain CH1 and a native or modified hinge region, and the light chain comprises a variable region VL and a constant domain CL. A dimer of Fab' according to the present disclosure produces a F(ab') 2 , where dimerization is possible, for example, via the hinge.

[0046] As used herein, the term "single domain antibody" refers to an antibody fragment consisting of a single monomeric variable antibody domain. Examples of single domain antibodies include V H or V L or V H H or V-NAR.

[0047] "Fv" refers to two variable domains, eg, cooperating variable domains, such as a cognate pair or affinity matured variable domains, ie, a VH and VL pair.

[0048] As used herein, "single-chain variable fragment" or "scFv" refers to a single-chain variable fragment stabilized by a peptide linker between the VH and VL variable domains.

[0049] As used herein, "disulfide-stabilized single-chain variable fragment" or "dsscFv" refers to a single-chain variable fragment that is H and V L The single-chain variable fragment is stabilized by a peptide linker between the variable domains and also includes V H and V L(See, e.g., Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012, WO2007109254.

[0050] In one embodiment, the variable domain V H and V L or V 1 or V 2 A disulfide bond is between two residues listed below (Kabat numbering is used in the following list unless the context indicates otherwise). Wherever Kabat numbering is mentioned, the relevant reference is Kabat et al., 1991 (5 th edition, Bethesda, Md.), Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.

[0051] In one embodiment, the disulfide bond is at a position selected from the group consisting of:

[0052] ·V H 37+V L 95C, see e.g. Protein Science 6, 781-788, Zhu et al., (1997);

[0053] ·V H 44+V L I00, see, e.g., Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012;

[0054] ·V H 44+V L 105, see, e.g., J Biochem, 118, 825-831, Luo et al., (1995);

[0055] ·V H 45+V L 87, see for example Protein Science, 6, 781-788, Zhu et al., (1997);

[0056] ·V H 55+V L101, see, e.g., FEBS Letters, 377, 135-139, Young et al., (1995);

[0057] ·V H 100+V L 50, see, e.g., Biochemistry, 29, 1362-1367, Glockshuber et al., (1990);

[0058] ·V H 100b+V L 49; see, e.g., Biochemistry, 29, 1362-1367, Glockshuber et al., (1990);

[0059] ·V H 98+V L 46, see for example Protein Science, 6, 781-788, Zhu et al., (1997);

[0060] ·V H 101+V L 46; see, e.g., Protein Science, 6, 781-788, Zhu et al., (1997);

[0061] ·V H 105+V L 43, see for example; Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 7538-7542, Brinkmann et al., (1993); or Proteins 19, 35-47, Jung et al. (1994),

[0062] ·V H 106+V L 57, see, e.g., FEBS Letters, 377, 135-139, Young et al., (1995) and one or more positions corresponding thereto in the variable region pair in the molecule.

[0063] In one embodiment, the disulfide bond is at position V H 44 and V L Formed between I00.

[0064] In one embodiment, the anti-IL13 antibody of the present invention is an antagonist antibody. As used herein, the term "antagonist antibody" describes an antibody that can inhibit or neutralize the biological signaling activity of IL-13, for example, by blocking the binding of IL-13 to the IL-13 receptor or reducing the binding of IL-13 to the IL-13 receptor, thereby inhibiting the activation of the receptor.

[0065] Antibodies that inhibit IL-13 activity can work through several possible mechanisms of action. Bin 1 represents an antibody that binds human IL-13 and prevents IL-13Rα1 binding and thus also blocks IL-4R binding. Bin 1 antibodies can also prevent IL-13 binding to IL-13Rα2. Bin 2 represents an antibody that binds hIL-13 in a way that allows binding to IL-13Rα1 but prevents IL-4R recruitment into the complex. We selected antibodies that work through Bin 1.

[0066] In one embodiment, the anti-IL13 antibody binds to human IL-13 and prevents the binding of IL-13Rα1.

[0067] In one embodiment, the anti-IL13 antibody binds human IL-13 and prevents the binding of IL-13Rα2.

[0068] In one embodiment, the anti-IL13 antibody binds to human IL-13 and prevents the binding of IL-13Rα1 and IL-13Rα2.

[0069] In one embodiment, the anti-IL13 antibody has a K of <100 pM. D Binds human IL-13.

[0070] The antibody used in the present invention may be, but is not limited to, a monoclonal antibody, a humanized antibody, a fully human antibody or a chimeric antibody.

[0071] Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler & Milstein, 1975, Nature, 256: 495-497), the trioma technique, human B cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4: 72) and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan Rliss, Inc., 1985).

[0072] Antibodies can also be produced using the single lymphocyte antibody method by cloning and expressing immunoglobulin variable region cDNAs produced by a single lymphocyte selected for production of specific antibodies, as described, for example, by Babcook J. et al., 1996, Proc. Natl. Acad. Sci. USA, 93(15):7843-78481; WO92 / 02551; WO2004 / 051268 and International Patent Application No. WO2004 / 106377.

[0073] Screening of antibodies can be performed using assays that measure binding to IL-13 and / or assays that measure the ability to block the binding of IL-13 to one or more of its receptors. An example of a binding assay is an ELISA, for example, using a fusion protein of IL-13 fixed on a plate and using a bound secondary antibody to detect anti-IL-13 antibodies bound to IL-13. An example of a blocking assay is a flow cytometry-based assay that measures the blocking of IL-13 ligand protein binding to IL-13R. A fluorescently labeled secondary antibody is used to detect the amount of IL-13 ligand protein bound to IL-13R.

[0074] Humanized antibodies (including CDR-grafted antibodies) are antibody molecules having one or more complementary determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules (see, e.g., US 5,585,089; WO91 / 09967). It should be understood that only the specificity determining residues of the CDRs may need to be transferred rather than the entire CDRs (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). Humanized antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived.

[0075] Chimeric antibodies are composed of elements derived from two different species such that the elements retain the characteristics of the species from which they were derived. Typically, a chimeric antibody will contain a variable region from one species (e.g., mouse, rat, rabbit, etc.) and a constant region from another species (e.g., human).

[0076] Antibodies can also be produced using various phage display methods known in the art and include Brinkman et al. (in J. Immunol. Methods, 1995, 182:41-50-325), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough et al. (Eur. J. Immunol., 1994, 24:952-958), Persic et al. (Gene, 1997, 187, 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and those methods disclosed in US Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0077] Fully human antibodies are those wherein the variable region and constant region (if present) of heavy and light chains are all human-derived, or are substantially identical to sequences in human-derived, but not necessarily those antibodies from identical antibodies. The example of a fully human antibody can include, for example, antibodies produced by the above-mentioned phage display method and antibodies produced by mice, wherein mouse immunoglobulin variable region genes and optional constant region genes have been replaced by their human counterparts, for example, in EP 0546073, US 5,545,806, US5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, US 5,770,429, EP 0438474 and EP0463151 in general terms.

[0078] The antibody of the present invention can be a multispecific antibody. As used herein, "multispecific or multispecific antibody" refers to an antibody as described herein, which has at least two binding domains, i.e., two or more binding domains, such as two or three binding domains, wherein at least two binding domains independently bind to two different antigens or two different epitopes on the same antigen. Multispecific antibodies are generally monovalent for each specificity (antigen). The multispecific antibodies described herein encompass monovalent and multivalent, such as bivalent, trivalent, tetravalent multispecific antibodies.

[0079] In one embodiment, the construct is a bispecific antibody. As used herein, "bispecific or bispecific antibody" refers to an antibody with two antigen binding specificities. In one embodiment, the antibody comprises two antigen binding domains, one of which binds antigen 1 and the other binds antigen 2, i.e., each binding domain is monovalent for each antigen. In one embodiment, the antibody is a tetravalent bispecific antibody, i.e., the antibody comprises four antigen binding domains, wherein, for example, two binding domains bind antigen 1 and the other two binding domains bind antigen 2. In one embodiment, the antibody is a trivalent bispecific antibody.

[0080] In one embodiment, the antibody construct is a trispecific antibody. As used herein, "trispecific or trispecific antibody" refers to an antibody with three antigen binding specificities. For example, an antibody is an antibody with three antigen binding domains (trivalent) that independently bind to three different antigens or three different epitopes on the same antigen, i.e., each binding domain is monovalent for each antigen.

[0081] A paratope is a region of an antibody that recognizes and binds an antigen. The antibodies of the present invention may be multiparatopic antibodies. As used herein, a "multiparatopic antibody" refers to an antibody as described herein that comprises two or more different paratopes that interact with different epitopes from the same antigen or from two different antigens. The multiparatopic antibodies described herein may be biparatopic, triparatopic, tetraparatopic.

[0082] As used herein, "antigen binding domain" refers to a portion of an antibody that contains part or all of one or more variable domains that specifically interact with a target antigen, such as part or all of a pair of variable domains VH and VL. The binding domain may comprise a single domain antibody. In one embodiment, each binding domain is monovalent. Preferably, each binding domain comprises no more than one VH and one VL.

[0083] A variety of multispecific antibody formats have been generated. Different classifications have been proposed, but multispecific IgG antibody formats generally include bispecific IgG, attached IgG, multispecific (e.g., bispecific) antibody fragments, multispecific (e.g., bispecific) fusion proteins, and multispecific (e.g., bispecific) antibody conjugates, as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies, Mol Immunol, 67 (2015): 95-106.

[0084] Technologies for preparing bispecific antibodies include, but are not limited to, CrossMab technology (Klein et al., Engineering therapeutic bispecific antibodies using CrossMab technology, Methods 154 (2019) 21-31), pestle-mortar engineering (e.g., WO1996027011, WO1998050431), DuoBody technology (e.g., WO2011131746), Azymetric technology (e.g., WO2012058768). Other technologies for preparing bispecific antibodies have been described in, for example, Godar et al., 2018, Therapeutic bispecific antibody formats: a patent applications review (1994-2017), Expert Opinion on Therapeutic Patents, 28: 3, 251-276. Bispecific antibodies specifically include CrossMab antibodies, DAF (two-in-one), DAF (four-in-one), Dutamab, DT-IgG, knob-in-hole common LC, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body and orthogonal Fab.

[0085] The additional IgG generally comprises a full-length IgG engineered by attaching an additional antigen binding domain or antigen binding fragment to the N- and / or C-terminus of the heavy and / or light chains of the IgG. Examples of such additional antigen binding fragments include sdAb antibodies (e.g., VH or VL), Fv, scFv, dsscFv, Fab, scFav. Additional IgG antibody formats specifically include DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgC(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody and DVI-IgG (four-in-one), for example as described in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies, Mol Immunol, 67 (2015): 95-106.

[0086] Multispecific antibody fragments include nanobodies, nanobody-HAS, BiTEs, diabody, DART, TandAb, scDiabody, sc-Diabody-CH3, Diabody-CH3, Triple Body, Miniantibody; Minibody, Tri Bi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc; and intrabodies, as described in, for example, Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies, Mol Immunol, 67 (2015): 95-106.

[0087] Multispecific fusion proteins include Dock and Lock, ImmTAC, HSAbody, scDiabody-HSA, and tandem scFv-toxin.

[0088] Multispecific antibody conjugates include IgG-IgG; Cov-X-Body; and scFv1-PEG-scFv2.

[0089] Additional multispecific antibody formats have been described, for example, in Brinkmann and Kontermann, The making of bispecific antibodies, mAbs, 9:2, 182-212 (2017), in particular in FIG. 2 , such as tandem scFv, triplebody, Fab-VHH, TaFv-Fc, scFv 4 -Ig, scFv 2 -Fcab, scFv 4 -IgG. Bibodies, tribodies and methods for preparing them are disclosed in, for example, WO99 / 37791.

[0090] Preferred antibodies for use in the present invention include additional IgG and additional Fab, wherein the complete IgG or Fab fragment is engineered by appending at least one additional antigen binding domain (e.g., two, three or four additional antigen binding domains), such as a single domain antibody (e.g., VH or VL, or VHH), scFv, dsscFv, dsFv to the N- and / or C-terminus of the heavy and / or light chain of the IgG or Fab, respectively, as described, for example, in WO2009 / 040562, WO2010 / 035012, WO2011 / 030107, WO2011 / 061492, WO2011 / 061246 and WO2011 / 086091. In particular, the Fab-Fv format is described in WO2009 / 040562, and its disulfide-stabilized format Fab-dsFv is described in WO2010 / 035012. Single linker Fab-dsFv is described in WO2014 / 096390, wherein the dsFv is linked to the Fab via a single linker between the VL or VH domain of the Fv and the C-terminus of the LC or HC of the Fab. Additional IgGs comprising full-length IgG1 are described in WO2015 / 197789, which are engineered by appending a dsFv to the C-terminus of the heavy or light chain of an IgG.

[0091] Another preferred antibody for use in the present invention comprises a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binds a therapeutic target and one scFv or dsscFv increases half-life by binding to, for example, albumin). Such antibodies are described in WO2015 / 197772. Another preferred antibody for use in the fragments of the present invention comprises a Fab linked to only one scFv or dsscFv, as described, for example, in WO2013 / 068571 and Dave et al., Mabs, 8(7)1319-1335 (2016).

[0092] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof specific for human IL-13, comprising a light chain variable domain comprising at least one CDR having the sequence given in SEQ ID NO: 1 for CDR-L1, a CDR having the sequence given in SEQ ID NO: 2 for CDR-L2, and a CDR having the sequence given in SEQ ID NO: 3 for CDR-L3.

[0093] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof specific for human IL-13, comprising a light chain variable domain comprising a CDR having the sequence given in SEQ ID NO: 1 for CDR-L1, a CDR having the sequence given in SEQ ID NO: 2 for CDR-L2, and a CDR having the sequence given in SEQ ID NO: 3 for CDR-L3.

[0094] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof specific for human IL-13, comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises at least one CDR having the sequence given in SEQ ID NO:4 for CDR-H1, a CDR having the sequence given in SEQ ID NO:5 for CDR-H2, or a CDR having the sequence given in SEQ ID NO:6 for CDR-H3.

[0095] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof specific for human IL-13, comprising a heavy chain variable domain comprising a CDR having a sequence given in SEQ ID NO:4 for CDR-H1, a CDR having a sequence given in SEQ ID NO:5 for CDR-H2, and a CDR having a sequence given in SEQ ID NO:6 for CDR-H3.

[0096] The antibody molecules of the present invention may comprise complementary light chains or complementary heavy chains, respectively.

[0097] Therefore, in one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to human IL-13, comprising:

[0098] (a) a light chain variable region comprising:

[0099] i. CDR-L1 comprising SEQ ID NO: 1,

[0100] ii. a CDR-L2 comprising SEQ ID NO: 2, and

[0101] iii. CDR-L3 comprising SEQ ID NO: 3;

[0102] as well as

[0103] (b) a heavy chain variable region comprising:

[0104] i. CDR-H1 comprising SEQ ID NO: 4,

[0105] ii. CDR-H2 comprising SEQ ID NO: 5, and

[0106] iii. CDR-H3 comprising SEQ ID NO: 6; and

[0107] It should be understood that one or more amino acid substitutions, additions and / or deletions may be made to the CDRs provided herein without significantly changing the ability of the antibody to bind to IL-13 and neutralize IL-13 activity. Those skilled in the art can easily test the effect of any amino acid substitution, addition and / or deletion, for example, by using the methods described herein, particularly those described in the examples, to determine the inhibition of IL-13 binding and IL-13 / IL-13 receptor interactions.

[0108] Thus, the present invention provides an antibody specific for human IL-13, comprising one or more CDRs selected from CDR-L1 (SEQ ID NO: 1), CDR-L2 (SEQ ID NO: 2), CDR-L3 (SEQ ID NO: 3), CDR-H1 (SEQ ID NO: 4), CDR-H2 (SEQ ID NO: 5) and CDR-H3 (SEQ ID NO: 6), wherein one or more amino acids in one or more CDRs have been substituted by another amino acid, e.g., a similar amino acid as defined below.

[0109] In one embodiment, the invention provides an antibody specific for human IL-13, comprising CDR-L1 (SEQ ID NO: 1), CDR-L2 (SEQ ID NO: 2 or SEQ ID NO: 20), CDR-L3 (SEQ ID NO: 3), CDR-H1 (SEQ ID NO: 4), CDR-H2 (SEQ ID NO: 5) and CDR-H3 (SEQ ID NO: 6), e.g., wherein one or more amino acids in one or more CDRs have been substituted with another amino acid, e.g., a similar amino acid as defined below.

[0110] As used herein, "identity" means that at any particular position in the aligned sequences, the amino acid residues are the same between the sequences. As used herein, "similarity" means that at any particular position in the aligned sequences, the amino acid residues are of similar type between the sequences. For example, leucine can be substituted for isoleucine or valine. Other amino acids that can generally be substituted for each other include, but are not limited to:

[0111] - phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

[0112] - lysine, arginine and histidine (amino acids with basic side chains);

[0113] - Aspartic acid and glutamic acid (amino acids with acidic side chains);

[0114] - Asparagine and glutamine (amino acids with amide side chains); and

[0115] - Cysteine ​​and methionine (amino acids with sulfur-containing side chains). The degree of identity and similarity can be easily calculated (Computational Molecular Biology, Lesk AM ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin AM, and Griffin HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje G., Academic Press, 1987, Sequence Analysis Primer, Gribskov M. and Devereux J., eds., M Stockton Press, New York, 1991, BLAST TM The software is available from NCBI (Altschul SF et al., 1990, J. Mol. Biol., 215:403-410; Gish W. & States DJ 1993, Nature Genet., 3:266-272. Madden TL et al., 1996, Meth. Enzymol., 266:131-141; Altschul SF et al., 1997, Nucleic Acids Res., 25:3389-3402; Zhang J. & Madden TL, 1997, Genome Res, 7:649-656,).

[0116] In one embodiment, an antibody of the invention comprises a light chain variable domain comprising three CDRs, wherein the sequence of CDR-L1 is at least 70%, 80%, 90%, 95% or 98% identical or similar to the sequence given in SEQ ID NO:1, CDR-L2 is at least 70%, 80%, 90%, 95% or 98% identical or similar to the sequence given in SEQ ID NO:2, and / or CDR-L3 is at least 70%, 80%, 90%, 95% or 98% identical or similar to the sequence given in SEQ ID NO:3.

[0117] In one embodiment, an antibody of the invention comprises a heavy chain variable domain comprising three CDRs, wherein the sequence of CDR-H1 is at least 70%, 80%, 90%, 95% or 98% identical or similar to the sequence given in SEQ ID NO:4, CDR-H2 is at least 70%, 80%, 90%, 95% or 98% identical or similar to the sequence given in SEQ ID NO:5, and / or CDR-H3 is at least 70%, 80%, 90%, 95% or 98% identical or similar to the sequence given in SEQ ID NO:6.

[0118] In one embodiment, an antibody of the invention comprises a light chain variable region comprising the sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 17. In one embodiment, an antibody of the invention comprises a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 17.

[0119] In one embodiment, an antibody of the invention comprises a heavy chain variable region comprising the sequence given in SEQ ID NO: 14 or SEQ ID NO: 18. In one embodiment, an antibody of the invention comprises a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO: 14 or SEQ ID NO: 18.

[0120] In one embodiment, an antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence given in SEQ ID NO: 13 and the heavy chain variable region comprises the sequence given in SEQ ID NO: 14. In one embodiment, an antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to that given in SEQ ID NO: 13 and / or the heavy chain variable region comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to that given in SEQ ID NO: 14.

[0121] In one embodiment, an antibody of the invention comprises CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences comprising SEQ ID NO: 1 / 2 / 3 / 4 / 5 / 6, respectively, and the remainder of the light and heavy chain variable regions are at least 70%, 80%, 90%, 95% or 98% identical or similar to SEQ ID NO: 13 and 14 or SEQ ID NO: 17 and 18, respectively.

[0122] In one embodiment, the antibody of the invention is Fab, Fab', F(ab')2, Fv, dsFv, scFv or dsscFv. In one embodiment, the antibody of the invention is a single domain antibody or a nanobody, such as V H or V L or V HH or V NAR .

[0123] In one embodiment, the antibody of the invention is a scFv comprising the sequence given in SEQ ID NO:21, or a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO:21.

[0124] In one embodiment, the antibody of the present invention is a scFv, which comprises the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences given in SEQ ID NO:1 / 2 / 3 / 4 / 5 / 6, respectively, and the remainder of the scFv has at least 70%, 80%, 90%, 95% or 98% identity or similarity with SEQ ID NO:21.

[0125] In one embodiment, the antibody of the invention is a dsscFv comprising the sequence given in SEQ ID NO:23, or a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO:23.

[0126] In one embodiment, the antibody of the invention is a dsscFv comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences given in SEQ ID NO:1 / 2 / 3 / 4 / 5 / 6, respectively, and the remainder of the dsscFv has at least 70%, 80%, 90%, 95% or 98% identity or similarity to SEQ ID NO:23.

[0127] In one embodiment, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a CH1 domain and the light chain comprises a CL domain, either kappa or lambda.

[0128] Biomolecules, such as antibodies or fragments, contain acidic and / or basic functional groups, thereby giving the molecule a net positive or negative charge. The amount of the total "observed" charge will depend on the absolute amino acid sequence of the entity, the local environment of the charged groups in the 3D structure, and the environmental conditions of the molecule. The isoelectric point (pi) is the pH at which a particular molecule or surface carries no net charge. In one embodiment, an antibody or fragment according to the present disclosure has an isoelectric point (pi) of at least 7. In one embodiment, the antibody or fragment has an isoelectric point of at least 8, such as 8.5, 8.6, 8.7, 8.8 or 9. In one embodiment, the antibody has a pI of 8.

[0129] The IL-13 antibodies and fragments of the present invention can be engineered to have an appropriate isoelectric point. This can result in antibodies and / or fragments having more robust properties, particularly suitable solubility and / or stability characteristics. Therefore, in one aspect, the present invention provides humanized IL-13 antibodies that are engineered to have an isoelectric point different from that of the initially identified antibody. The antibody can be engineered, for example, by replacing amino acid residues, such as replacing acidic amino acid residues with one or more basic amino acid residues. Alternatively, basic amino acid residues can be added or acidic amino acid residues can be removed. Alternatively, if the molecule has an unacceptably high pI value, acidic residues can be introduced as needed to lower the pH. The pI of the engineered antibody or fragment can be, for example, 8 or higher, such as 8.5 or 9. Importantly, when manipulating the pI, care must be taken to maintain the activity of the desired antibody or fragment. Therefore, in one embodiment, the engineered antibody or fragment has the same or substantially the same activity as the "unmodified" antibody or fragment.

[0130] Programs such as **EXPASY http: / / www.expasv.ch / Tools / pi_tool.htmh and http: / / www.iut-arles.up.univ-mrs.fr / w3bb / d_abim / compo-p.htmh Can be used to predict the isoelectric point of an antibody or fragment.

[0131] Epitope

[0132] An epitope is a region of an antigen that is bound by an antibody. An epitope can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and have those residues that contribute directly to the affinity of the interaction. An epitope can also be conformational, i.e., composed of nonlinear amino acids. In some embodiments, an epitope can include determinants that are chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in some embodiments, can have specific three-dimensional structural features and / or specific charge characteristics.

[0133] By using conventional methods known in the art, it is easy to determine whether an antibody binds to the same epitope or competes with a reference antibody. For example, in order to determine whether a test antibody binds to the same epitope as a reference antibody of the present invention, the reference antibody is allowed to bind to a protein or peptide under saturation conditions. Next, the ability of the test antibody to bind to a protein or peptide is assessed. If the test antibody can bind to a protein or peptide after saturation binding of the reference antibody, a conclusion can be drawn that the test antibody binds to different epitopes from the reference antibody. On the other hand, if the test antibody cannot bind to a protein or peptide after saturation binding of the reference antibody, the test antibody can bind to the same epitope as the epitope bound by the reference antibody of the present invention.

[0134] In order to determine whether the antibody competes with the reference antibody for binding, the above-mentioned binding method is carried out in two directions. In the first direction, the reference antibody is allowed to bind to the protein / peptide under saturation conditions. Then the binding of the test antibody to the protein / peptide molecule is evaluated. In the second direction, the test antibody is allowed to bind to the protein / peptide under saturation conditions, and then the binding of the reference antibody to the protein / peptide is evaluated. If in both directions, only the first (saturated) antibody can bind to the protein / peptide, then the conclusion that the test antibody and the reference antibody compete for binding to the protein / peptide is drawn. As will be appreciated by those skilled in the art, the antibody that competes with the reference antibody for binding may not necessarily bind to the same epitope as the reference antibody, but can spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0135] If each antibody competitively inhibits (blocks) the binding of the other antibody to the antigen, the two antibodies bind to the same or overlapping epitopes. That is, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res, 1990: 50: 1495-1502), 1, 5, 10, 20 or 100 times excess of one antibody inhibits at least 50%, 75%, 90% or even 99% binding of another antibody. Alternatively, if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, the two antibodies have overlapping epitopes.

[0136] Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is the cause of the observed lack of binding. Such experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0137] The antibody may compete for binding to IL-13, or bind to the same epitope, as those defined above in terms of light chain, heavy chain, light chain variable region (LCVR), heavy chain variable region (HCVR) or CDR sequences. In particular, the antibody may compete for binding to IL-13, or bind to the same epitope, with an antibody comprising a CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequence combination of SEQ ID NOs: 1 / 2 / 3 / 4 / 5 / 6. The antibody may compete for binding to IL-13, or bind to the same epitope, with an antibody comprising a LCVR and HCVR sequence pair of SEQ ID NOs: 13 / 14 or 17 / 18. The antibody may compete for binding to IL-13, or bind to the same epitope, with an scFv comprising the sequence given in SEQ ID NO: 21 or a dsscFv comprising the sequence given in SEQ ID NO: 23.

[0138] Effector molecules

[0139] If desired, the antibodies used in the present invention can be conjugated to one or more effector molecules. It should be understood that the effector molecule can include a single effector molecule or two or more such molecules, which are connected to form a single part that can be attached to the antibody of the present invention. When it is necessary to obtain an antibody fragment connected to an effector molecule, this can be prepared by standard chemical or recombinant DNA procedures, wherein the antibody fragment is directly or through a coupling agent and connected to the effector molecule. The technology for conjugating such effector molecules to antibodies is well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed, Robinson et al. eds, 1987, pp.623-53; Thorpe et al., 1982, Immunol. Rev., 62: 119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemical methods include, for example, those described in WO 93 / 06231, WO 92 / 22583, WO 89 / 00195, WO 89 / 01476 and WO 03031581. Alternatively, when the effector molecule is a protein or polypeptide, linkage may be achieved using recombinant DNA methods, for example as described in WO 86 / 01533 and EP 0392745.

[0140] The term effector molecule as used herein includes, for example, anti-tumor agents, drugs, toxins, biologically active proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (e.g., DNA, RNA and fragments thereof), radionuclides (particularly radioactive iodine), radioisotopes, chelated metals, nanoparticles, and reporter groups (e.g., fluorescent compounds or compounds detectable by NMR or ESR spectroscopy).

[0141] Examples of effector molecules include cytotoxins or cytotoxic agents, including any agent that is harmful to cells (e.g., kills cells). Examples include combretastatins, dolastatins, epothilones, staurosporine, maytansine, spongestatin, rhizobactin, halichondrin, bacilli, hemipteratin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunomycin, dihydroxyanthraquinone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

[0142] Effector molecules also include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicin or duocarmycin), and antimitotic agents (e.g., vincristine and vinblastine).

[0143] Other effector molecules can include chelated radionuclides, e.g. 111 In and 90 Y, Lu 177 ,bismuth 213 , California 252 ,iridium 192 and tungsten 188 / rhenium 188 ; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxanes, and suramin.

[0144] Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lysates, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins (such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin), proteins (such as insulin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor or tissue plasminogen activator, thrombotic agents or anti-angiogenic agents (such as angiostatin or endostatin)), or biological response modifiers (such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factors and immunoglobulins).

[0145] Other effector molecules may include detectable substances that can be used, for example, for diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron emitting metals (for positron emission tomography), and non-radioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents, see generally U.S. Pat. No. 4,741,900. Suitable enzymes include horseradish enzyme, peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; suitable radionuclides include 125 I. 131 I. 111 In and 99 Tc.

[0146] In another example, the effector molecule can increase the half-life of the antibody in vivo, and / or reduce the immunogenicity of the antibody and / or enhance the delivery of the antibody across the epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins or albumin binding compounds, such as those described in WO 05 / 117984.

[0147] When the effector molecule is a polymer, it may typically be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, such as a homo- or heteropolysaccharide.

[0148] Specific optional substituents which may be present on the above synthetic polymers include one or more hydroxyl, methyl or methoxy groups.

[0149] Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, especially optionally substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) or derivatives thereof.

[0150] Particular naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.

[0151] As used herein, "derivatives" are intended to include reactive derivatives, such as thiol selective reactive groups, such as maleimide, etc. The reactive group can be attached to the polymer directly or through a linker segment. It should be understood that in some cases, the residue of such a group will form part of the product as a linker group between the antibody fragment and the polymer.

[0152] The size of the polymer can be varied as required, but is generally within the range of an average molecular weight of 500Da to 50000Da, such as 5000 to 40000Da, such as 20000 to 40000Da. The polymer size can be selected based on the intended use of the product in particular, such as the ability to locate in certain tissues (such as tumors) or to extend the circulation half-life (reviewed in Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Therefore, for example, in the case where the product is intended to leave the circulation and penetrate tissue, it may be advantageous to use a small molecular weight polymer (e.g., a molecular weight of about 5000Da). For applications where the product is retained in the circulation, it may be advantageous to use a higher molecular weight polymer (e.g., a molecular weight in the range of 20000Da to 40000Da).

[0153] Suitable polymers include polyalkylene polymers such as poly(ethylene glycol) or particularly methoxypoly(ethylene glycol) or derivatives thereof, and particularly having a molecular weight in the range of about 15000 Da to about 40000 Da.

[0154] In one example, the antibody for the present invention is connected with poly (ethylene glycol) (PEG) part.In a specific example, the antibody is an antibody fragment, and the PEG molecule can be connected by any available amino acid side chain or terminal amino acid functional group (such as any free amino group, imino group, thiol group, hydroxyl or carboxyl group) located in the antibody fragment.Such amino acid can be naturally present in the antibody fragment, or can be engineered into the fragment using recombinant DNA methods (see, for example, US 5,219,996, US 5,667,425, WO 98 / 25971).In one example, the antibody molecule of the present invention is a modified Fab fragment, wherein the modification is to add one or more amino acids to the C-terminal of its heavy chain to allow the attachment of effector molecules.Suitably, other amino acids form a modified hinge region, which includes one or more cysteine ​​residues, and effectors can be attached to the cysteine ​​residues.Multiple sites can be used to connect two or more PEG molecules.

[0155] Suitably, the PEG molecule can be covalently linked to the thiol group of at least one cysteine ​​residue located in the antibody fragment. Each polymer molecule connected to the modified antibody fragment can be covalently linked to the sulfur atom of the cysteine ​​residue located in the fragment. The covalent bond is generally a disulfide bond or particularly a sulfur-carbon bond. Wherein the thiol group is used as the connection point of the effector molecule of appropriate activation, for example, thiol selective derivatives such as maleimide and cysteine ​​derivatives can be used. The activated polymer can be used as the starting material for preparing the polymer-modified antibody fragment as described above. The activated polymer can be any polymer containing a thiol-reactive group, such as α-halocarboxylic acid or ester, such as iodoacetamide, imide, such as maleimide, vinyl sulfone or disulfide. Such starting materials can be commercially available (e.g., from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or can be prepared from commercially available starting materials using conventional chemical procedures. Specific PEG molecules include 2OK methoxy-PEG-amine (available from Nektar, formerly Shearwater; Rapp Polymere, and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

[0156] In one embodiment, the antibody is a modified Fab fragment or diFab that is PEGylated, i.e., has PEG (poly(ethylene glycol)) covalently attached thereto, for example according to the methods disclosed in EP 0948544 or EP 1090037 [see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York, "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1997, J. Milton Harris and S. Zalipsky (ed), American Chemical Society, Washington DC and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545]. In one example, PEG is connected to the cysteine ​​in the hinge region. In one example, the Fab fragment modified by PEG has a maleimido group covalently connected to a single thiol group in the hinge region modified. Lysine residues can be covalently connected to maleimido groups, and each amine group on the lysine residues can be connected to a methoxy poly (ethylene glycol) polymer having a molecular weight of about 20,000Da. The total molecular weight of the PEG connected to the Fab fragment can therefore be about 40,000Da.

[0157] In one embodiment, the invention provides an antagonist antibody molecule specific for human IL-13, which is a modified Fab' fragment having a modified hinge region at the C-terminus of its heavy chain, wherein the modified hinge region contains at least one cysteine ​​residue attached to an effector molecule. Suitably, the effector molecule is PEG and is attached using the methods described in (WO98 / 25971 and WO 2004072116 or WO 2007 / 003898. The effector molecule can be attached to the antibody fragment using the methods described in International Patent Applications WO 2005 / 003169, WO 2005 / 003170 and WO 2005 / 003171.

[0158] In one embodiment, the antibody or fragment is not linked to an effector molecule.

[0159] Antibody production

[0160] The present invention also provides the DNA sequence of the separation of the heavy chain and / or light chain of the antibody molecule of the present invention.Suitably, the heavy chain or light chain of the antibody molecule of the present invention of the DNA sequence encoding.The DNA sequence of the present invention can comprise synthetic DNA (e.g., produced by chemical processing), cDNA, genomic DNA or any combination thereof.

[0161] The DNA sequence encoding the antibody molecule of the present invention can be obtained by methods well known to those skilled in the art. For example, the DNA sequence encoding part or all of the heavy and light chains of the antibody can be synthesized from a determined DNA sequence or based on the corresponding amino acid sequence as needed.

[0162] DNA encoding the acceptor framework sequence is widely available to those skilled in the art and can be easily synthesized based on its known amino acid sequence.

[0163] Standard techniques of molecular biology can be used to prepare DNA sequences encoding antibody molecules of the present invention. Oligonucleotide synthesis techniques can be used to synthesize the desired DNA sequence in whole or in part. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques can be appropriately used.

[0164] Examples of suitable sequences are provided herein. Thus, in one embodiment, the invention provides an isolated polynucleotide encoding an antibody or antigen-binding fragment comprising the sequence set forth in SEQ ID NO: 8, 10, 15, 16, 19, 20, 22 or 24.

[0165] The general methods for constructing vectors, transfection methods and culture methods are well known to those skilled in the art. In this regard, reference is made to "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual published by Cold Spring Harbor Publishing.

[0166] Also provided are host cells comprising one or more clones or expression vectors, which contain one or more DNA sequences encoding antibodies of the present invention. Any suitable host cell / vector system can be used to express the DNA sequence encoding the antibody molecule of the present invention. Bacteria (e.g., Escherichia coli) and other microbial systems can be used, or eukaryotic (e.g., mammalian) host cell expression systems can also be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

[0167] The present invention also provides a method for producing an antibody molecule according to the present invention, which comprises culturing a host cell containing the vector of the present invention under conditions suitable for expressing proteins from DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule.

[0168] The antibody molecule may comprise only a heavy chain or a light chain polypeptide, in which case only the heavy chain or light chain polypeptide coding sequence needs to be used to transfect the host cell. In order to produce a product comprising both a heavy chain and a light chain, a cell line may be transfected with two vectors, the first vector encoding a light chain polypeptide and the second vector encoding a heavy chain polypeptide. Alternatively, a single vector may be used, the vector comprising sequences encoding both a light chain and a heavy chain polypeptide.

[0169] The antibodies and fragments according to the present disclosure are expressed at good levels from host cells. Therefore, the properties of the antibodies and / or fragments appear to be optimized and conducive to commercial processing.

[0170] Pharmaceutical compositions, dosages and dosing regimens

[0171] The antibodies of the present invention may be provided in a pharmaceutical composition. The pharmaceutical composition is usually sterile and usually includes a pharmaceutically acceptable carrier and / or adjuvant. The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable adjuvant and / or carrier.

[0172] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. The carrier may be suitable for parenteral, such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, such as by injection or infusion. Alternatively, the carrier may be suitable for non-parenteral administration, such as topical, epidermal or mucosal administration routes. The carrier may be suitable for oral administration. Depending on the route of administration, the modulator may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0173] The pharmaceutical composition of the present invention may contain one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0174] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, buffered water, and saline. Examples of other carriers include ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). In many cases, it is desirable to include isotonic agents, such as sugar, polyols (e.g., mannitol, sorbitol), or sodium chloride in the composition.

[0175] The therapeutic composition typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration.

[0176] The pharmaceutical compositions of the present invention may contain additional active ingredients.

[0177] Kits comprising an antibody or modulator of the invention and instructions for use are also within the scope of the invention.The kit may further comprise one or more additional agents, such as additional therapeutic or prophylactic agents as described above.

[0178] The modulators and / or antibodies of the invention, or formulations or compositions thereof, may be administered for prophylactic and / or therapeutic treatments.

[0179] In therapeutic applications, the compound is administered to a subject already suffering from a disease or condition as described above in an amount sufficient to cure, alleviate or partially prevent the condition or one or more symptoms thereof. Such therapeutic treatment may result in a decrease in the severity of disease symptoms, or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this is defined as a "therapeutically effective amount".

[0180] In preventive applications, the formulation is administered to a subject at risk of a disease or condition as described above in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms. An amount sufficient to achieve this is defined as a "prophylactically effective amount." The effective amount for each purpose will depend on the severity of the disease or injury and the weight and general state of the subject.

[0181] The subject of administration can be a human or a non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Administration to humans is typical.

[0182] The antibody / modulator or pharmaceutical composition of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary according to the desired result. Examples of routes of administration of the compound or pharmaceutical composition of the present invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, typically by injection. Alternatively, the antibody / modulator or pharmaceutical composition of the present invention can be administered by non-parenteral routes, such as topical, epidermal or mucosal routes of administration. The antibody / modulator or pharmaceutical composition of the present invention can be used for oral administration.

[0183] The appropriate dosage of the antibody / modulator or pharmaceutical composition of the present invention can be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be changed to obtain an amount of the active ingredient that effectively achieves the therapeutic response required for a specific patient, composition, and mode of administration and is nontoxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition used in the present invention, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health and previous medical history of the patient treated, and similar factors well known in the medical field.

[0184] Suitable dosages can be, for example, about 0.01 μg / kg to about 1000 mg / kg body weight, typically about 0.1 μg / kg to about 100 mg / kg body weight of the patient to be treated. For example, suitable dosages can be about 1 μg / kg to about 10 mg / kg body weight or about 10 μg / kg to about 5 mg / kg body weight per day.

[0185] The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased according to the urgency of the therapeutic situation. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect and a pharmaceutical carrier required in combination.

[0186] Administration can be a single dose or multiple doses. Multiple doses can be administered to the same or different locations by the same or different routes. Alternatively, administration can be by sustained release formulations, in which case less frequent administration is required. Dosage and frequency can vary depending on the half-life of the antagonist in the patient and the desired duration of treatment.

[0187] As noted above, the modulators / antibodies or pharmaceutical compositions of the invention may be co-administered with one or more other therapeutic agents.

[0188] The combined administration of two or more agents can be achieved in many different ways. Both can be administered together in a single composition, or they can be administered in separate compositions as part of a combination therapy. For example, one can be administered before, after, or simultaneously with the other.

[0189] Treatment Indications

[0190] The antibodies of the invention can be used to treat, prevent or ameliorate any disorder associated with IL-13 activity; for example, any disorder caused in whole or in part by signaling through the IL-13 receptor.

[0191] IL-13-associated diseases include primary and metastatic cancers, including breast cancer, colon cancer, rectal cancer, lung cancer, oropharyngeal cancer, hypopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, gallbladder cancer and bile duct cancer, small intestine cancer, urinary tract cancer (including renal cancer, bladder cancer and urothelial cancer), female genital tract cancer (including cervical cancer, uterine cancer and ovarian cancer as well as choriocarcinoma and gestational trophoblastic disease), male genital tract cancer (including prostate cancer, seminal vesicle cancer, testicular cancer and germ cell tumors), endocrine gland cancer (including thyroid cancer, adrenal cancer and pituitary cancer) and skin cancer, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissue as well as Kaposi's sarcoma), tumors of the brain, nerves, eyes and meninges (including astrocytomas, gliomas, solid tumors arising from hematopoietic malignancies (e.g., leukemias) and lymphomas (Hodgkin's and non-Hodgkin's lymphomas), rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme disease arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathies, systemic lupus erythematosus, ulcerative colitis, inflammatory bowel disease, insulin-dependent diabetes mellitus, thyroiditis, allergic diseases, psoriasis, dermatitis scleroderma, graft-versus-host disease, organ transplant rejection, acute or chronic immune diseases associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Grave's disease disease), nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schonlein purpura, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancy, heart failure, Addison's disease, sporadic, polyglandular deficiency type I and polyglandular deficiency type II, Schmidt's syndrome, respiratory distress syndrome (acute) in adults, alopecia, alopecia areata, arthropathy, Reiter's diseasedisease), psoriatic arthropathy, ulcerative colitis arthropathy, enteropathic synovitis, chlamydia, yersinia, and salmonella-associated arthropathy, atherosclerotic disease / arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune hemolytic anemia, Coombs-positive hemolytic anemia, acquired pernicious anemia, juvenile pernicious anemia, myalgic encephalitis / Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiency-related diseases, hepatitis B, hepatitis C, common variable immunodeficiency (common variable hypogammaglobulinemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrosing lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonia, interstitial lung disease associated with connective tissue disease, lung disease associated with mixed connective tissue disease, systemic Interstitial lung disease associated with sclerosis, interstitial lung disease associated with rheumatoid arthritis, lung disease associated with systemic lupus erythematosus, lung disease associated with dermatomyositis / polymyositis, lung disease associated with Sjögren's syndrome, lung disease associated with ankylosing spondylitis, diffuse vasculitic lung disease, lung disease associated with hemosiderosis, drug-induced interstitial lung disease, fibrosis, radiation-induced fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrate lung disease, post-infectious interstitial lung disease, gouty arthritis, autoimmune hepatitis , autoimmune hepatitis type 1 (classic autoimmune or lupus hepatitis), autoimmune hepatitis type 2 (anti-LKM antibody hepatitis), autoimmune hypoglycemia, insulin resistance with acanthosis nigricans type B, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthropathy, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leukopenia, autoimmune neutropenia, nephropathy NOS, glomerulonephritis, renal microscopic vasculitis , Lyme disease, discoid lupus erythematosus, idiopathic male infertility or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestations of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren's syndromesyndrome), Takayasu's disease / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goiter autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxedema, crystalline uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver disease, alcoholic cirrhosis, alcoholic liver damage, cholesteatitis, idiopathic liver disease, drug-induced hepatitis, non Alcoholic steatohepatitis, allergy, group B streptococcus (GBS) infection, mental disorders, depression, schizophrenia, Th2- and Th1-mediated diseases, acute and chronic pain, different forms of pain, cancer, lung cancer, breast cancer, stomach cancer, bladder cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, hematopoietic malignancies, leukemia, lymphoma, abetalipoproteinemia, acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infections, acute pancreatitis, acute renal failure, adenocarcinoma, air Ectopic beats, AIDS dementia syndrome, alcoholic hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis (including seasonal allergic rhinitis), non-allergic rhinitis, allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti-CD3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity, aortic and peripheral artery aneurysms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (persistent or paroxysmal), atrial flutter, atrioventricular block, B-cell lymphoma, bone transplant rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, burns, arrhythmias, cardiac dizziness syndrome, cardiac tumors, cardiomyopathy, inflammatory response to cardiopulmonary bypass, cartilage transplant rejection, cerebellar cortical degeneration, cerebellar dysfunction, chaotic or multifocal atrial tachycardia, chemotherapy-related diseases, chronic myeloid leukemia (CML), chronic alcoholism, chronic inflammatory diseases, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate poisoning, colorectal cancer, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob diseasedisease), culture-negative sepsis, cystic fibrosis, cytokine therapy-associated disorders, dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, skin diseases, diabetes, diabetes mellitus, diabetic atherosclerotic disease, diffuse Lewy body disease, dilated congestive cardiomyopathy, basal ganglia disorders, midlife Down syndrome, drug-induced movement disorders induced by drugs that block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, Epstein-Barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hemophagocytic lymphohistiocytosis, fetal thymic implant rejection, Friedreich's ataxia ataxia), functional peripheral arterial disorder, fungal sepsis, gas gangrene, gastric ulcer, glomerulonephritis, transplant rejection of any organ or tissue, gram-negative sepsis, gram-positive sepsis, granuloma due to intracellular organisms, hairy cell leukemia, Hallervorden-Spatz disease, Hashimoto's thyroiditis, hay fever, cardiac transplant rejection, blood disease, hemodialysis, hemolytic uremic syndrome / thrombolytic thrombocytopenic purpura, bleeding, hepatitis A, His bundle arrhythmia arrythmias), HIV infection / HIV neuropathy, Hodgkin disease, hyperkinetic movement disorders, hypersensitivity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis assessment, idiopathic Addison disease, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, weakness, infantile spinal muscular atrophy, aortic inflammation, influenza A, ionizing radiation exposure, iridocyclitis / uveitis / optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, renal transplant rejection, Legionella, leishmaniasis, leprosy, corticospinal system disease, lipedema, liver transplant rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic / idiopathic migraine, mitochondrial multisystem disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multisystem degeneration (Mencel Dejerine-Thomas Shi-Drager and Machado-Joseph), mycobacterium aviumintracellulare), Mycobacterium tuberculosis, Myelodysplastic syndrome, Myocardial infarction, Myocardial ischemic disease, Nasopharyngeal carcinoma, Chronic lung disease of the newborn, Nephritis, Nephropathy, Neurodegenerative disease, Neurogenic muscular atrophy, Neutropenic fever, Non-Hodgkin lymphoma, Occlusion of the abdominal aorta and its branches, Occlusive arterial disease, OKT3 therapy, Orchitis / epididymitis, Orchitis / vasectomy reversal, Organomegaly, Osteoporosis, Pancreatic transplant rejection, Pancreatic cancer, Paraneoplastic syndrome / Hypercalcemia of malignancy, Parathyroid transplant rejection, Pelvic inflammatory disease, Perennial rhinitis, Pericardial disease, Peripheral atherosclerotic disease, Peripheral vascular disease, Peritonitis, Pernicious anemia, Pneumocystis carinii pneumonia, Pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), postperfusion syndrome, post-pump syndrome, post-MI heart disease, preeclampsia, progressive supranuclear palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynaud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcoma, senile chorea, Alzheimer's disease with Lewy bodies, seronegative arthropathy, shock, sickle cell anemia, skin allograft rejection, skin change syndrome, small bowel transplant rejection, solid tumors, specific arrhythmias, spinal ataxia, spinocerebellar degeneration, streptococcal myositis, structural cerebellar lesions, subacute sclerosing panencephalitis, syncope, cardiovascular syphilis, systemic anaphylaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or Fab ALL telangiectasia, thromboangiitis obliterans, thrombocytopenia, toxicity, transplantation, trauma / hemorrhage, type III hypersensitivity, type IV hypersensitivity, unstable angina, uremia, urosepsis, valvular heart disease, varicose veins, vasculitis, venous disease, venous thrombosis, ventricular fibrillation, viral and fungal infections, fatal encephalitis / aseptic meningitis, vitalassociated hemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's diseasedisease), xenograft rejection of any organ or tissue, acute coronary syndrome, acute idiopathic polyneuritis, acute inflammatory demyelinating polyradiculoneuropathy, acute ischemia, adult-onset Still's disease, allergic reaction, antiphospholipid antibody syndrome, aplastic anemia, atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmune disorders associated with streptococcal infection, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarian failure, blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular disease, catastrophic antiphospholipid syndrome, celiac disease, cervical spondylosis, chronic ischemia, cicatricial pemphigoid, clinically isolated syndrome (cis) with risk of multiple sclerosis, childhood-onset psychiatric illness disorders, dacryocystitis, dermatomyositis, diabetic retinopathy, herniated disc, herniated disc, drug-induced immune hemolytic anemia, endometriosis, endophthalmitis, episcleritis, erythema multiforme, erythema multiforme major, pemphigoid gestationis, Guillain-Barré syndrome (GBS), Hughes syndrome, idiopathic Parkinson's disease, idiopathic interstitial pneumonia, IgE-mediated allergic reaction, immune hemolytic anemia, inclusion body myositis, infectious inflammatory eye disease, inflammatory demyelinating disease, inflammatory heart disease, inflammatory kidney disease, IPF / UIP, iritis, keratitis, keratoconjunctivitis sicca, Kussmaul disease or Kussmaul-Meier disease, Landry's palsy, Langerhans cell histiocytosis, livedo reticularis, macular degeneration, microscopic polyangiitis, morbusbechterev, motor neuron disease, mucous membrane pemphigoid, multiple organ failure, myasthenia gravis, myelodysplastic syndrome, myocarditis, radiculopathy, neuropathy, non-A, non-B hepatitis, optic neuritis, osteolysis, oligoarticular JRA, peripheral arterial occlusive disease (PAOD), peripheral vascular disease (PVD), peripheral arterial disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, poliomyelitis, polyarticular JRA, multiple endocrine deficiency syndrome, polymyositis, polymyalgia rheumatica (PMR), primary Parkinson's syndrome symptoms, prostatitis, pure red cell aplasia, primary adrenal insufficiency, recurrent neuromyelitis optica, restenosis, rheumatic heart disease, sapho (synovitis, acne, pustulosis, osteophyte and osteitis), secondary amyloidosis, shock lung, scleritis, sciatica, secondary adrenal insufficiency, silicone-related connective tissue disease, Sneddon-Wilkinson skin disease, ankylosing spondylitis, Stevens-Johnson syndrome (SJS), temporal arteritis, toxoplasmosis retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumor Tumor necrosis factor receptor, type 1 hypersensitivity reactions, type 2 diabetes, urticaria, usual interstitial pneumonia (UIP), vasculitis, vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration or wound healing, aspirin-sensitive asthma, atopic asthma, chronic hand eczema, allergic bronchopulmonary aspergillosis, celiac disease, Churg-Strauss syndrome (periarteritis nodosa plus atopy), eosinophilic myalgia syndrome, hypereosinophilic syndrome, edema reactions including episodic angioedema , helminthic infections, onchocerciasis dermatitis, eosinophil-associated gastrointestinal diseases, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis, eosinophilic colitis, nasal polyposis and polyposis, food allergies, aspirin intolerance, as well as obstructive sleep apnea, chronic asthma, Crohn's disease and endomyocardial fibrosis, cancer (e.g., glioblastoma (such as glioblastoma multiforme), non-Hodgkin lymphoma (NHL)), fibrosis, inflammatory bowel disease, pulmonary fibrosis (including idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis secondary to cirrhosis), COPD, and liver fibrosis.

[0192] The antibodies of the invention may be particularly useful for treating or preventing atopic dermatitis, chronic hand eczema, nasal polyposis or polyposis, food allergies or eosinophilic esophagitis.

[0193] Thus, in one embodiment, an antibody or pharmaceutical composition of the invention is provided for use in a method of treating the human or animal body by therapy.

[0194] In one embodiment, an antibody or pharmaceutical composition is provided for use in a method of treating atopic dermatitis, chronic hand eczema, nasal polyposis or polyposis, food allergy, or eosinophilic esophagitis.

[0195] In one embodiment, the invention provides a method for treating or preventing atopic dermatitis, chronic hand eczema, nasal polyposis or polyposis, food allergy or eosinophilic esophagitis, comprising administering a therapeutically effective amount of an antibody or pharmaceutical composition to a patient in need thereof.

[0196] In one embodiment, the invention provides the use of an antibody or pharmaceutical composition in the preparation of a medicament for the treatment or prevention of one or more medical indications as described herein.

[0197] The following examples illustrate the invention. Example

[0198] Example 1: Generation and selection of therapeutic anti-IL-13 antibody CA650

[0199] Rats were immunized with purified human IL-13 (Peprotech) or rat fibroblasts expressing human IL-13 (expressing about 1 μg / ml in the culture supernatant), or in some cases a combination of both. After 3 to 6 injections, animals were sacrificed and PBMC, spleen, bone marrow, and lymph nodes were harvested. The binding of serum to human IL-13 was monitored in ELISA, as well as the ability to neutralize hIL-13 in HEK-293IL-13R-STAT-6 reporter cell assays (HEK-Blue assay, Invivogen).

[0200] B cell cultures were established, and the supernatants were first screened for the ability to bind hIL-13 in a bead-based assay in an Applied Biosystems FMAT assay. This is a homogeneous assay using biotinylated human IL-13 and goat anti-rat Fc-Cy5 conjugates coated on streptavidin beads as revealing agents. The positives from this assay were then carried out into a HEK-293IL-13R-STAT-6 reporter cell assay (HEK-Blue assay, Invivogen) to identify neutralizers. The neutralization supernatant was then analyzed in Biacore to estimate the dissociation rate and characterize the mode of action of neutralization. Neutralization was classified as bin1 or bin2. Bin1 represents an antibody that binds to human IL-13 and prevents the binding of IL-13Rα1 and therefore also blocks IL-4R binding. Bin 1 antibodies can also prevent the binding of IL-13 to IL-13Rα2. Bin 2 represents antibodies that bind hIL-13 in a manner that allows binding to IL-13Rα1 but prevents recruitment of IL-4R into the complex. We selected antibodies that act through Bin 1.

[0201] From a total of 27 × 100 plates of SLAM experiments, approximately 7500 IL-13-specific positives were identified in the primary FMAT screen. 800 wells showed neutralization in the HEK-Blue assay. 170 wells had the expected Biacore profile, i.e., off-rates <5 × 10 -4 s -1 Bin 1 antibody. Attempt to clone the variable region from these 170 wells, 160 of which successfully produced fluorescent foci. After reverse transcription (RT)-PCR, 100 wells produced heavy chain and light chain variable region gene pairs. These V region genes were cloned into mouse IgG1 full-length antibodies and re-expressed in HEK-293 transient expression system. Sequence analysis results show that there are 27 unique anti-human IL-13 antibody families. These recombinant antibodies were then retested in cell-based assays to block the ability of recombinant hIL-13 (E. coli-derived and mammalian Lai-derived), recombinant variant hIL-13 (R130Q) (E. coli-derived), natural wild type and variant hIL-13 (human donor-derived) and cynomolgus monkey IL-13 (mammalian-derived). The ability of recombinant antibodies to bind variant human IL-13 (R130Q) and cynomolgus monkey IL-13 was also tested in Biacore. Following this characterization, antibody families were selected that met our criteria of antibodies below 100 pM with minimal decrease in potency and affinity for all human and cynomolgus IL-13 preparations.

[0202] Based on neutralizing potency, affinity and donor content in humanized grafts (see below), humanized CA650 was selected for further advancement.

[0203] Example 2. Humanization of Antibody CA650

[0204] Humanized antibody 650 is carried out by transplanting the CDR from rat V region to the human germline antibody V region framework. In order to restore the activity of the antibody, many framework residues from rat V region are also retained in the humanized sequence. These residues are selected using the scheme outlined by Adair et al. (1991) (Humanised antibodies, WO 91 / 09967). The comparison of rat antibody (donor) V-region sequence and human germline (acceptor) V-region sequence, as well as the designed humanized sequence, are shown. (Fig. 1 (A) light chain graft 650 and Fig. 1 (B) heavy chain graft 650). The CDR transplanted from the donor to the acceptor sequence is as defined by Kabat (Kabat et al., 1987), except for the CDR-H1 defined by Chothia / Kabat using a combination (see Adair et al., 1991, Humanised antibodies, WO91 / 09967).

[0205] The genes encoding the original V-region sequences were designed and constructed by automated synthesis methods at Entelechon GmbH and modified by oligonucleotide directed mutagenesis to generate the grafted forms gL8 and gH9. The gL8 sequence was subcloned into the UCB Celltech human light chain expression vector pVhCK, which contains DNA encoding the human C-κ constant region (Km3 allotype). The gH9 sequence was subcloned into pVhg1Fab, which contains DNA encoding the human heavy chain γ-1 CH1 constant region.

[0206] Select human V region IGRV1-39 plus JK2J region ( (IMGT), http: / / www.imgt.org ) as the acceptor for the light chain CDRs of antibody 650. The light chain framework residues in graft gL8 are all from the human germline gene, except for residues 58 and 71 (according to Kabat numbering), in which the donor residues isoleucine (I58) and tyrosine (Y71), respectively, are retained. The retention of residues I58 and Y71 is essential for the full efficacy of the humanized antibody.

[0207] Select human V region IGHV1-69 plus JH4 J region (IMGT, http: / / www.imgt.org ) as the acceptor of the heavy chain CDR of antibody 650. The heavy chain framework residues in the graft gH9 are all from human germline genes, except residues 67, 69 and 71 (according to Kabat numbering), in which the donor residues alanine (A67), phenylalanine (F69) and valine (V71) are retained, respectively. Retention of residues A67, F69 and V71 is essential for the full efficacy of the humanized antibody. The glutamine residue at position 1 of the human framework is replaced with glutamic acid (E1) to provide a uniform product for expression and purification: the conversion of glutamine to pyroglutamic acid at the N-terminal of antibodies and antibody fragments is widely reported. The variable graft sequences gL8 and gH9 finally selected are shown in Figures 1 (A) and 1 (B), respectively.

[0208] The DNA sequences and amino acids encoding the CDRs, heavy and light chain variable regions, scFv and dsscFv forms of antibody 650 are shown in FIG2 .

[0209] Example 3. Biological activity of anti-IL13 antibodies

[0210] Antigen binding and IL-13 neutralization were confirmed, data not shown. Sequence Listing <110> UCB Biopharma SRL <120> Antibodies with binding specificity for human IL-13 <130> PF0209-WO-PCT <150> GB 1919062.8 <151> 2019-12-20 <160> twenty four <170> PatentIn Version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 1 Lys Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 2 Tyr Thr Asp Ile Leu Gln Thr 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 3 Tyr Gln Tyr Tyr Ser Gly Tyr Thr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 4 Gly Tyr Ser Phe Thr Ser Tyr Tyr Ile His 1 5 10 <210> 5 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 5 Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 6 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 6 Phe His Tyr Asp Gly Ala Asp 1 5 <210> 7 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 7 Asp Ile Gln Met Thr Gln Ser Pro Pro Val Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Leu Ser Cys Lys Ala Ser Gln Asn Ile Asn Glu Asn 20 25 30 Leu Asp Trp Tyr His Gln Lys His Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Asp Ile Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr 85 90 95 Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 8 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 8 gacatccaga tgacccagtc tcctccagtc ctgtctgcat ctgtgggaga cagagtcact 60 ctcagttgca aagcaagtca gaatattaat gagaacttag actggtatca tcaaaagcat 120 ggcgaagctc caaaactcct gatatattat acagacattt tgcaaacggg catcccatca 180 aggttcagtg gcagtggatc tggtacagat tacacactca ccatcagcag cctgcagcct 240 gaagatgttg ccacatatta ctgctatcag tattacagtg ggtacacgtt tggacctggg 300 accaagctgg aaataaaa 318 <210> 9 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 9 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Val Asp Lys Tyr Phe Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ser Pro Glu Asp Thr Ala Val Phe Tyr Cys 85 90 95 Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 10 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 10 caggtacaac tgcagcagtc tggagctgag ttggtgaagc ctgggtcttc agtgaagatg 60 tcctgcaagg cttctggcta cagtttcacc agctactaca tacactggat aaagcagagg 120 tcctgcaagg cttctggcta cagtttcacc agctactaca tacactggat aaagcagagg 120 cctggacagg gccttgagtg gattgggcgt attggtcctg gaagtggaga tattaattac 180 cctggacagg gccttgagtg gattgggcgt attggtcctg gaagtggaga tattaattac 180 aatgagaagt tcaagggcaa ggccacattt actgtggaca aatatttcag cacagcctac 240 aatgagaagt tcaagggcaa ggccacattt actgtggaca aatatttcag cacagcctac 240 atgcaactca gcagcctgtc acctgaggac actgcggtct tttactgtgc aagatttcac 300 atgcaactca gcagcctgtc acctgaggac actgcggtct tttactgtgc aagatttcac 300 tatgatgggg ctgactgggg ccaaggcact ctggtcacag tctcgagc 348 tatgatgggg ctgactgggg ccaaggcact ctggtcacag tctcgagc 348 <210> 11<210> 11 <211> 107<211> 107 <212> PRT<212> PRT <213> 人工序列<213> Artificial sequence <220><220> <223> 重组序列<223> Recombinant sequence <400> 11<400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 12 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 13 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Glu Asn 20 25 30 Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Asp Ile Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 14 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Recombinant sequence <400> 14 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 15 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 15 gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga cagggtgacc 60 gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga cagggtgacc 60 atcacctgca aggcctccca gaacatcaac gagaacctgg actggtacca gcagaagccc 120 atcacctgca aggcctccca gaacatcaac gagaacctgg actggtacca gcagaagccc 120 ggcaaggccc ccaagctgct gatctactac accgacatcc tgcagaccgg catcccctcc 180 ggcaaggccc ccaagctgct gatctactac accgacatcc tgcagaccgg catcccctcc 180 aggttctccg gctccggctc cggcaccgac tacaccctga ccatctcctc cctgcagccc 240 aggttctccg gctccggctc cggcaccgac tacaccctga ccatctcctc cctgcagccc 240 gaggacttcg ccacctacta ctgctaccag tactactccg gctacacctt cggccagggc 300 gaggacttcg ccacctacta ctgctaccag tactactccg gctacacctt cggccagggc 300 accaagctgg agatcaag 318 accaagctgg agatcaag 318 <210> 16<210> 16 <211> 348<211> 348 <212> DNA<212> DNA <213> Artificial sequence<213> Artificial sequence <220> <220> <223> Recombinant sequence <223> Recombinant sequence <400> 16 <400> 16 gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60 gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60 tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120 tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120 cccggccagg gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180 cccggccagg gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180 aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240 aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240 atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300 atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300 tacgacggcg ccgactgggg ccagggcacc ctggtgaccg tctcgagc 348 <210> 17 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Glu Asn 20 25 30 Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Asp Ile Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr 85 90 95 Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 18 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 18 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Met 35 40 45 Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 19 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 19 gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga cagggtgacc 60 atcacctgca aggcctccca gaacatcaac gagaacctgg actggtacca gcagaagccc 120 ggcaaggccc ccaagctgct gatctactac accgacatcc tgcagaccgg catcccctcc 180 aggttctccg gctccggctc cggcaccgac tacaccctga ccatctcctc cctgcagccc 240 gaggacttcg ccacctacta ctgctaccag tactactccg gctacacctt cggctgcggc 300 accaagctgg agatcaag 318 <210> 20 <211> 348 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 20 gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60 tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120 cccggccagt gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180 aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240 atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300 tacgacggcg ccgactgggg ccagggcacc ctggtgaccg tgtcctcc 348 <210> 21 <211> 242 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 21 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 130 135 140 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys 145 150 155 160 Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asp Ile Leu Gln Thr 180 185 190 Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 210 215 220 Tyr Gln Tyr Tyr Ser Gly Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys <210> 22 <211> 726 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 22 gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60 tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120 cccggccagg gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180 aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240 atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300 tacgacggcg ccgactgggg ccagggcacc ctggtgaccg tgtcctccgg aggtggcggt 360 tctggcggtg gcggttccgg tggcggtgga tcgggaggtg gcggttctga catccagatg 420 acccagtccc cctcctccct gtccgcctcc gtgggcgaca gggtgaccat cacctgcaag 480 gcctcccaga acatcaacga gaacctggac tggtaccagc agaagcccgg caaggccccc 540 aagctgctga tctactacac cgacatcctg cagaccggca tcccctccag gttctccggc 600 tccggctccg gcaccgacta caccctgacc atctcctccc tgcagcccga ggacttcgcc 660 acctactact gctaccagta ctactccggc tacaccttcg gccagggcac caagctggag 720 atcaag 726 <210> 23 <211> 242 <212> PRT <213> Synthetic Sequence <220> <223> Recombinant Sequence <400> 23 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Met 35 40 45 Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 130 135 140 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys 145 150 155 160 Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asp Ile Leu Gln Thr 180 185 190 Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 210 215 220 Tyr Gln Tyr Tyr Ser Gly Tyr Thr Phe Gly Cys Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys <210> 24 <211> 726 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 24 gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60 tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120 cccggccagt gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180 aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240 atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300 tacgacggcg ccgactgggg ccagggcacc ctggtgaccg tgtcctccgg aggtggcggt 360 tctggcggtg gcggttccgg tggcggtgga tcgggaggtg gcggttctga catccagatg 420 acccagtccc cctctccct gtccgcctcc gtgggcgaca gggtgaccat cacctgcaag 480 gcctcccaga acatcaacga gaacctggac tggtaccagc agaagcccgg caaggccccc 540 aagctgctga tctactacac cgacatcctg cagaccggca tcccctccag gttctccggc 600 tccggctccg gcaccgacta caccctgacc atctctccc tgcagcccga ggacttcgcc 660 acctactact gctaccagta ctactccggc tacaccttcg gctgcggcac caagctgggag 720 atcaag 726

Claims

1. An antibody or antigen-binding fragment thereof that binds to human IL-13, comprising: (a) A light chain variable region comprising: i. CDR-L1 consisting of SEQ ID NO:1, ii. CDR-L2 consisting of SEQ ID NO:2, and iii. CDR-L3 consisting of SEQ ID NO:3; And (b) A heavy chain variable region comprising: i. CDR-H1 consisting of SEQ ID NO:4, ii. CDR-H2 consisting of SEQ ID NO:5, and iii. CDR-H3 consisting of SEQ ID NO:

6.

2. The antibody or antigen-binding fragment according to claim 1, wherein the light chain variable region comprises the sequence given in SEQ ID NO:13 or SEQ ID NO:

17.

3. The antibody or antigen-binding fragment according to claim 1 or claim 2, wherein the heavy chain variable region comprises the sequence given in SEQ ID NO:14 or SEQ ID NO:

18.

4. The antibody or antigen-binding fragment according to claim 1 or claim 2, wherein the light chain variable region consists of the sequence given in SEQ ID NO:13 or a sequence that is at least 95% identical thereto, and the heavy chain variable region consists of the sequence given in SEQ ID NO:14 or a sequence that is at least 95% identical thereto.

5. The antibody or antigen-binding fragment according to claim 1 or claim 2, wherein the light chain variable region consists of the sequence given in SEQ ID NO:17 or a sequence that is at least 95% identical thereto, and the heavy chain variable region consists of the sequence given in SEQ ID NO:18 or a sequence that is at least 95% identical thereto.

6. The antibody or antigen-binding fragment according to claim 1 or claim 2, wherein the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences consist of SEQ ID NO:1 / 2 / 3 / 4 / 5 / 6, respectively, and the remaining portions of the light and heavy chain variable regions have at least 95% identity with SEQ ID NO:13 and 14 or SEQ ID NO:17 and 18, respectively.

7. The antibody or antigen-binding fragment according to claim 1 or claim 2, wherein the antibody is a chimeric, humanized or fully human antibody.

8. The antibody or antigen-binding fragment according to claim 1 or claim 2, wherein the antibody is a full-length antibody.

9. The antibody or antigen-binding fragment according to claim 1 or claim 2, wherein the antigen-binding fragment is Fab, Fab', F(ab') 2 , Fv, dsFv, scFv or dsscFv.

10. The antibody or antigen-binding fragment according to claim 9, wherein the antigen-binding fragment is a scFv comprising the sequence given in SEQ ID NO:21 or a dsscFv comprising the sequence given in SEQ ID NO:

23.

11. An isolated polynucleotide encoding the antibody or antigen-binding fragment according to any one of claims 1 to 10.

12. An expression vector carrying the polynucleotide according to claim 11.

13. A host cell, said host cell comprising the vector according to claim 12.

14. A method for producing an antibody or antigen-binding fragment according to any one of claims 1 to 10, said method comprising culturing the host cell according to claim 13 under conditions permitting production of said antibody or antigen-binding fragment, and recovering the produced antibody or antigen-binding fragment.

15. A pharmaceutical composition, said pharmaceutical composition comprising an antibody or antigen-binding fragment as defined in any one of claims 1 to 10 and a pharmaceutically acceptable adjuvant.

16. A pharmaceutical composition, said pharmaceutical composition comprising an antibody or antigen-binding fragment as defined in any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

17. An antibody or antigen-binding fragment according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 15, for use in a method of treating a human or animal body by therapy.

18. An antibody, antigen-binding fragment or pharmaceutical composition according to claim 17, for use in the treatment of atopic dermatitis, chronic hand eczema, nasal polyposis, food allergy or eosinophilic esophagitis.

19. An antibody, antigen-binding fragment or pharmaceutical composition according to claim 17, for use in the treatment of polyposis.

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