Antibody having binding specificity to human IL-13

Abstract

The present invention relates to antibody molecules having specificity for antigenic determinants of human IL-13, therapeutic uses of said antibody molecules and methods for producing said antibody molecules.

Description

Technical Field

[0001] The present invention relates to IL-13 antibodies and fragments such as binding fragments thereof, compositions containing the same, and particularly to their use in the prevention and / or treatment of IL-13 related diseases.

Background Art

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

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

[0004] IL-13 binds to cell surface receptors IL-13R-α1 and IL-13R-α2. IL-13R-α1 interacts with IL-13 with low affinity (K D ~10 nM), and then IL-4R-α forms a high-affinity (K D ~0.4 nM) signal-transducing heterodimeric receptor complex.

[0005] The IL-4R / IL-13R-α1 complex is expressed in 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 activates various signaling 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 as a decoy receptor that negatively regulates the binding of IL-13 and also as a signaling receptor that induces TGF-β synthesis and fibrosis via 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 devised to inhibit or neutralize the activity of IL-13. 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, particularly human IL-13, and in particular antibodies that can neutralize human IL-13. The present invention provides a novel 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 improved antibodies that bind to human IL-13, particularly neutralizing antibodies that inhibit the biological activity of IL-13. The present invention further provides pharmaceutical compositions comprising the antibodies and their use in the treatment of IL-13-related diseases.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0010] Detailed Description of the Invention Antibody Antibodies for use in the context of the present disclosure include whole antibodies and functionally active fragments thereof, i.e., molecules that contain an antigen-binding domain that specifically binds to IL-13 (also referred to as antigen-binding fragments). The features described herein with respect to antibodies apply to antibody fragments as well, unless otherwise indicated by context.

[0011] Full antibodies are generally also referred to as "immunoglobulins (Ig)" and are intact or full-length antibodies in which two heavy chain and two light chain elements are joined to each other by disulfide bonds and assembled so as to define their characteristic Y-shaped three-dimensional structure. Classical native full antibodies have monovalency, which binds to one antigen type, and bivalency, which has two independent antigen-binding domains. The terms "intact antibody", "full-length antibody" and "full antibody" are used interchangeably herein to refer to monovalent bivalent antibodies having a structure similar to the native antibody structure including the Fc region defined herein.

[0012] Each light chain consists of a variable light chain region (abbreviated herein as V L and a constant light chain region (C L ). Each heavy chain consists of a variable heavy chain region (abbreviated herein as V H and a heavy chain constant region (CH) composed of three constant domains C H1 , C H2 and C H3 , or four constant domains C H1 , C H2 , C H3 and C H4 depending on the Ig class. The "class" of an Ig or antibody refers to the type of constant region and includes IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses such as IgG1, IgG2, IgG3, IgG4, etc. The constant region of an 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 of the classical complement system (Clq).

[0013] The V H and V L regions of the antibodies according to the present invention can be further subdivided into regions of hypervariability (or "hypervariable regions") that determine antigen recognition, called complementarity-determining regions (CDRs), and are interspersed with more structurally conserved regions called framework regions (FRs). Each V H , V LIt is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FRs together form the variable region. Conventionally, the CDRs in the heavy chain variable region of an antibody or its antigen-binding fragment are called CDR-H1, CDR-H2, and CDR-H3, and the CDRs in the light chain variable region are called CDR-L1, CDR-L2, and CDR-L3. These are sequentially numbered in the direction from the N-terminus to the C-terminus of each chain.

[0014] CDRs are conventionally 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)"). In this specification, this numbering system is used unless otherwise indicated.

[0015] The Kabat residue designation does not necessarily directly correspond to the linear numbering of amino acid residues. The actual linear amino acid sequence, whether in the framework or the complementarity-determining region, may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to deletions or insertions of structural components of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody by aligning homologous residues in the antibody sequence with the "standard" Kabat numbering sequence.

[0016] 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. M. J. Mol. Biol., 196, 901-917 (1987)), the loop corresponding to CDR-H1 extends from residue 26 to residue 32. Therefore, as used herein, "CDR-H1", unless otherwise indicated, is intended to refer to residues 26 to 35 as described by a combination of the Kabat numbering system and the topological loop definition of Chothia.

[0017] 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).

[0018] In addition to the CDR loops, there is a fourth loop between CDR-2 (CDR-L2 or CDR-H2) and CDR-3 (CDR-L3 or CDR-H3), which is formed by framework 3 (FR3). In the Kabat numbering system, framework 3 is defined as positions 66-94 of the heavy chain and positions 57-88 of the light chain.

[0019] Based on the alignment of the sequences of different members of the immunoglobulin family, numbering schemes have been proposed, for example, as described by Kabat et al., 1991, and Dondelinger et al., 2018, Frontiers in Immunology, Vol 9, article 2278.

[0020] The terms "constant domain" and "constant region" as used herein are used interchangeably to refer to the domain of an antibody that is outside the variable region. The constant region is identical in all antibodies of the same isotype, but differs from one isotype to another. Typically, the constant region of the heavy chain is formed from CH1 - hinge - CH2 - CH3 - optionally CH4 from the N - terminus to the C - terminus and contains three or four constant domains.

[0021] The constant domain of the antibody molecule of the present invention, if present, can be selected considering the proposed function of the antibody molecule, particularly the effector function that may be required. For example, the constant domain may be a human IgA, IgD, IgE, IgG or IgM domain. In particular, when the antibody molecule is intended for therapeutic use and an antibody effector function is required, particularly the human IgG constant domains of the IgG1 and IgG3 isotypes can be used. Alternatively, when the antibody molecule is for therapeutic purposes and an antibody effector function is not required, the IgG2 and IgG4 isotypes can be used. It will be understood that sequence variants of these constant domains can also be used. For example, serine at position 241 (numbering according to the Kabat numbering system) can be changed to proline as described by 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 an IgG4 molecule called IgG4P herein can be used.

[0022] "Fc", "Fc fragment", and "Fc region" are used interchangeably to refer to the C - terminal region of an antibody that includes the constant region of the antibody excluding the first constant immunoglobulin domain. Thus, Fc is the last two constant domains of IgA, IgD, and IgG, CH2 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 to include residues C226 to its carboxyl terminus, where the numbering is according to the EU index as in Kabat. In the context of human IgG1, 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 according to the EU index in Kabat. The corresponding Fc regions of other immunoglobulins can be identified by sequence alignment.

[0023] In the context of the present disclosure, when present, the constant region or Fc region may be native as defined above, provided that it includes a functional FcR binding domain, preferably a functional FcRn binding domain, or may be variously modified otherwise. Preferably, the modified constant region or Fc region leads to an improvement in functionality and / or pharmacokinetics. The modification may include deletion of a specific portion of the Fc fragment. The modification can further include various amino acid substitutions that can affect the biological properties of the antibody. Mutations for increasing FcRn binding, and thus in vivo half-life, may also be present. The modification can further include modification of the glycosylation profile of the antibody. The native Fc fragment is glycosylated at the CH2 domain, and in each of the two heavy chains, there is an N-glycan attached to the asparagine residue (Asn297) at position 297. In the context of the present disclosure, the antibody may be glycan-modified. That is, it is modified to have a specific glycosylation profile, for example, leading to improved properties such as improved effector function or improved serum half-life.

[0024] The antibodies described herein are isolated. An "isolated" antibody is one that has been separated from the components of its natural environment (e.g., by purification means).

[0025] The term "antibody" encompasses monovalent antibodies, i.e., antibodies containing only one antigen-binding domain (e.g., one-armed antibodies in which a full-length heavy chain and a full-length light chain are linked, also called "half antibodies"), and multivalent antibodies, i.e., antibodies containing two or more antigen-binding domains.

[0026] 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 for use in the present invention include Fab and Fab’ fragments described in international patent applications WO2011 / 117648, WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171.

[0027] Methods for making and producing these antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0028] The term "Fab fragment" as used herein means an antibody fragment comprising a light chain fragment containing the VL (variable light chain) domain and the constant domain (CL) of the light chain, and the VH (variable heavy chain) domain and the first constant domain (CH1) of the heavy chain.

[0029] A typical "Fab’ fragment" comprises a pair of heavy and light chains, where the heavy chain comprises the variable region VH, the constant domain CH1, and a native or modified hinge region, and the light chain comprises the variable region VL and the constant domain CL. A dimer of Fab’ according to the present disclosure makes, for example, F(ab’)2 in which dimerization can occur via the hinge.

[0030] As used herein, the term "single domain antibody" means an antibody fragment comprising a single monomeric variable antibody domain. Examples of single domain antibodies include V H or V L or V H H or V-NAR.

[0031] "Fv" refers to two variable domains, for example, co-variable domains such as a homologous pair or an affinity matured variable domain, i.e., a pair of VH and VL.

[0032] As used herein, "single chain variable fragment" or "scFv" means a single chain variable fragment stabilized by a peptide linker between the V H variable domain and the V L variable domain.

[0033] As used herein, "disulfide stabilized single chain variable fragment" or "dsscFv" means a single chain variable fragment stabilized by a peptide linker between the V H and V L variable domains and comprising an inter-domain disulfide bond between the V H and V L domains (see, for example, Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012, WO2007109254).

[0034] In one embodiment, the variable domains V H and V LThe disulfide bond between V1 or V2 is between two of the residues listed below (except where the context indicates otherwise; Kabat numbering is used in the following list). When referring to Kabat numbering, the relevant reference is Kabat et al., 1991 (5th edition, Bethesda, Md), Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.

[0035] In one embodiment, the disulfide bond is at a position selected from the group consisting of: -V H 37 + V L 95C See, for example, Protein Science 6,781-788 Zhu et al (1997). -V H 44 + V L 100 See, for example, Weatherill et al, Protein Engineering, Design & Selection, 25(321-329), 2012. -V H 44 + V L 105 See, for example, J Biochem. 118,825-831 Luo et al (1995). -V H 45 + V L 87 See, for example, Protein Science 6,781-788 Zhu et al (1997). -V H 55 + V L 101 See, for example, FEBS Letters 377 135-139 Young et al (1995). -V H 100 + V L 50 See, for example, Biochemistry 29 1362-1367 Glockshuber et al (1990). -V H 100b + VL 49; See, for example, Biochemistry 29 1362-1367 Glockshuber et al (1990). -V H 98 + V L 46 See, for example, Protein Science 6,781-788 Zhu et al (1997). -V H 101 + V L 46 See, for example, Protein Science 6,781-788 Zhu et al (1997). -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). -V H 106 + V L 57 See, for example, FEBS Letters 377 135-139 Young et al (1995). and at the corresponding position(s) in the variable region pair(s) located within the molecule. In one embodiment, the disulfide bond is at position V H 44 and V L is formed between 100.

[0036] In one embodiment, the anti-IL13 antibody of the present invention is an antagonistic antibody. As used herein, the term "antagonistic antibody" describes an antibody that can inhibit or neutralize the biological signaling activity of IL-13, for example, by blocking or reducing the binding of IL-13 to its IL-13 receptor, and thus inhibiting receptor activation.

[0037] Antibodies that inhibit the activity of IL-13 can act via several possible mechanisms of action. Bin1 represents an antibody that binds to human IL-13, blocks the binding of IL-13Rα1, and as a result, also blocks the binding of IL-4R. The Bin1 antibody can also inhibit the binding of IL-13 to IL-13Rα2. Bin2 represents an antibody that binds to hIL-13 such that it allows binding to IL-13Rα1 but blocks recruitment to the IL-4R complex. We had selected antibodies that act via Bin1.

[0038] In one embodiment, the anti-IL13 antibody binds to human IL-13 and blocks the binding of IL-13Rα1.

[0039] In one embodiment, the anti-IL13 antibody binds to human IL-13 and blocks the binding of IL-13Rα2.

[0040] In one embodiment, the anti-IL13 antibody binds to human IL-13 and blocks the binding of IL-13Rα1 and IL-13Rα2.

[0041] In one embodiment, the anti-IL13 antibody binds to human IL-13 with a K D of <100 pM.

[0042] The antibodies for use in the present invention may be, but are not limited to, monoclonal antibodies, humanized antibodies, fully human antibodies or chimeric antibodies.

[0043] Monoclonal antibodies can be prepared by any method known to those skilled in the art, such as the hybridoma method (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma method, the human B cell hybridoma method (Kozbor et al, 1983, Immunology Today, 4:72), the EBV-hybridoma method (Cole et al, Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc, 1985), etc.

[0044] Alternatively, antibodies can be generated using the single lymphocyte antibody method by cloning and expressing immunoglobulin variable region cDNAs generated from single lymphocytes selected for the production of a specific antibody, for example, by the methods described in Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-7848l; WO92 / 02551; WO2004 / 051268 and International Patent Application No. WO2004 / 106377.

[0045] Antibody screening can be performed using an assay that measures binding to IL-13 and / or an assay that measures the ability to block binding to one or more receptors of IL-13. Examples of binding assays include, for example, ELISA using a fusion protein of IL-13 immobilized on a plate, which employs a secondary antibody conjugated to detect an anti-IL-13 antibody bound to IL-13. An example of a blocking assay is a flow cytometry-based assay that measures the blocking of the binding of an IL-13 ligand protein to IL-13R. A fluorescently labeled secondary antibody is used to detect the amount of IL-13 ligand protein that binds to IL-13R.

[0046] Humanized antibodies (including CDR-grafted antibodies) are antibody molecules having one or more complementarity-determining regions (CDRs) derived from non-human species and framework regions derived from human immunoglobulin molecules (see, for example, US 5,585,089; WO 91 / 09967). It will be understood that it may be necessary to transplant only the specificity-determining residues of the CDRs, rather than the entire CDRs (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanized antibodies may optionally further include one or more framework residues derived from the non-human species from which the CDRs are derived.

[0047] Chimeric antibodies are composed of elements derived from two different species and are designed to retain the characteristics of the species from which the elements are derived. Generally, chimeric antibodies contain a variable region derived from one species, such as a mouse, rat, rabbit, etc., and a constant region derived from another species, such as a human.

[0048] Antibodies can also be generated using various phage display methods known in the art, including those described by Brinkman et al. (J. Immunol. Methods, 1995, 182:41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic (Gene, 1997 187 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO90 / 02809; WO91 / 10737; WO92 / 01047; WO92 / 18619; WO93 / 11236; WO95 / 15982; WO95 / 20401; and US 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.

[0049] A fully human antibody is an antibody in which both the variable and constant regions (if present) of both the heavy and light chains are of human origin or are substantially identical to human-derived sequences, but not necessarily obtained from the same antibody. Examples of fully human antibodies include, for example, antibodies produced by the phage display method described above, and antibodies produced by mice in which the mouse immunoglobulin variable region gene and optionally the constant region gene have been replaced with human counterparts, for example, antibodies described in general terms in EP0546073, US5,545,806, US5,569,825, US5,625,126, US5,633,425, US5,661,016, US5,770,429, EP0438474 and EP0463151.

[0050] The antibody of the present invention may be a multispecific antibody. As used herein, "multispecific antibody or multispecific antibody" refers to an antibody having at least two binding domains, i.e., two or more binding domains, for example, two or three binding domains, and at least two binding domains independently bind two different antigens or two different epitopes on the same antigen as described herein. Multispecific antibodies are generally monovalent for each specificity (antigen). The multispecific antibodies described herein include monovalent and multivalent, for example, divalent, trivalent, tetravalent multispecific antibodies.

[0051] In one embodiment, the construct is a bispecific antibody. As used herein, "bispecific or bispecific antibody" refers to an antibody having two antigen-binding specificities. In one embodiment, the antibody comprises two antigen-binding domains, one binding domain binding antigen 1 and the other binding domain binding 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, for example, two binding domains binding antigen 1 and the other two binding domains binding antigen 2. In one embodiment, the antibody is a trivalent bispecific antibody.

[0052] In one embodiment, the antibody construct is a trispecific antibody. As used herein, a "trispecific or trispecific antibody" refers to an antibody having three antigen-binding specificities. For example, the antibody is one that independently binds to three different antigens or three different epitopes on the same antigen, i.e., an antibody having three antigen-binding domains (trivalent) where each binding domain is monovalent for each antigen.

[0053] A paratope is the region of an antibody that recognizes and binds to an antigen. The antibodies of the present invention may be multi-paratopic antibodies. As used herein, a "multi-paratopic antibody" refers to an antibody that contains two or more different paratopes that interact with different epitopes from the same antigen or from two different antigens, as described herein. The multi-paratopic antibodies described herein may be biparatopic, triparatopic, or tetraparatopic.

[0054] As used herein, an "antigen-binding domain" refers to a part of an antibody that includes part or all of one or more variable domains that specifically interact with a target antigen, for example, part or all of a pair of variable domains VH and VL. The binding domain may include single-domain antibodies. In one embodiment, each binding domain is monovalent. Preferably, each binding domain includes one or fewer VH and one VL.

[0055] A variety of multispecific antibody formats have been generated. Different classifications have been proposed, but multispecific IgG antibody formats generally include bispecific IgG, appended 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 (Alternative molecular formats and therapeutic applications of bispecific antibodies).

[0056] Technologies for making 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), Knobs-in-holes engineering (e.g., WO1996027011, WO1998050431), DuoBody technology (e.g., WO2011131746), Azymetric technology (e.g., WO2012058768), etc. Further technologies for making bispecific antibodies are described, for example, in 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 include, inter alia, CrossMab antibodies, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-lgG, Knobs-in-holes common LC, Knobs-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, kappa lambda body, and orthogonal Fab.

[0057] The appended IgG generally includes full-length IgG modified by adding an additional antigen-binding domain or antigen-binding fragment to the N-terminus and / or C-terminus of the heavy chain and / or light chain of IgG. Examples of such additional antigen-binding fragments include sdAb antibodies (e.g., VH or VL), Fv, scFv, dsscFv, Fab, scFav. Appended IgG antibody formats include, in particular, 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, those described in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106 (Alternative molecular formats and therapeutic applications of bispecific antibodies.).

[0058] Examples of multispecific antibody fragments include, for example, nanobody, nanobody-HAS, BiTE, 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-F C , Diabody-FC, tandem scFv-F C ; and intrabodies.

[0059] Examples of multispecific fusion proteins include Dock and Lock, ImmTAC, HSAbody, scDiabody-HAS, and tandem scFv-toxin.

[0060] Examples of multispecific antibody conjugates include IgG-lgG, Cov-X-Body, scFv1-PEG-scFv2.

[0061] Additional multispecific antibody formats are described, for example, in Brinkmann and Kontermann, The making of bispecific antibodies, mAbs, 9:2, 182-212 (2017), in particular in Figure 2, for example tandem scFv, triple bodies, Fab-VHH, taFv-Fc, scFv4-Ig, scFv2-Fcab, scFv4-IgG. Bibodies, tribodies and methods for their production are disclosed, for example, in WO99 / 37791.

[0062] Preferred antibodies for use in the present invention include additional IgG and additional Fab, wherein the full IgG or Fab fragment is designed, for example, as described in WO2009 / 040562, WO2010 / 035012, WO2011 / 030107, WO2011 / 061492, WO2011 / 061246 and WO2011 / 086091, by adding at least one additional antigen-binding domain (e.g., two, three or four additional antigen-binding domains), such as a single domain antibody (VH or VL, or VHH, etc.), scFv, dsscFv, dsFv, to the N-terminus and / or C-terminus of the heavy chain and / or light chain of said IgG or Fab. In particular, the Fab-Fv format is described in WO2009 / 040562, and its disulfide-stabilized version, Fab-dsFv, is described in WO2010 / 035012. A single linker Fab-dsFv, in which the dsFv is linked to the Fab via a single linker between either the VL or VH domain of the Fv and the C-terminus of the LC or HC of the Fab, is described in WO2014 / 096390. An additional IgG comprising a full-length IgG1 designed by adding dsFv to the C-terminus of the heavy chain or light chain of IgG is described in WO2015 / 197789.

[0063] Another preferred antibody for use in the present invention comprises a Fab linked to two scFvs or dsscFvs, where each scFv or dsscFv binds to the same or different targets (e.g., one scFv or dsscFv that binds a therapeutic target and one scFv or dsscFv that increases the half-life by binding, for example, albumin). Such antibodies are described in WO2015 / 197772. Another preferred antibody for use with the fragments of the present invention comprises a Fab that binds to only one scFv or dsscFv, as described, for example, in WO2013 / 068571 and Dave et al., Mabs, 8(7)1319-1335 (2016).

[0064] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that is specific for human IL-13 and comprises a light chain variable domain comprising at least one CDR having the sequence shown in SEQ ID NO: 1 for CDR-L1, a CDR having the sequence shown in SEQ ID NO: 2 for CDR-L2, or a CDR having the sequence shown in SEQ ID NO: 3 for CDR-L3.

[0065] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that is specific for human IL-13 and comprises a light chain variable domain comprising a CDR having the sequence shown in SEQ ID NO: 1 for CDR-L1, a CDR having the sequence shown in SEQ ID NO: 2 for CDR-L2, and a CDR having the sequence shown in SEQ ID NO: 3 for CDR-L3.

[0066] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that is specific for human IL-13 and comprises a heavy chain variable domain comprising at least one CDR having the sequence shown in SEQ ID NO: 4 for CDR-H1, a CDR having the sequence shown in SEQ ID NO: 5 for CDR-H2, or a CDR having the sequence shown in SEQ ID NO: 6 for CDR-H3.

[0067] In one embodiment, the present invention provides an antibody or an antigen-binding fragment thereof that is specific for human IL-13 and comprises a heavy-chain variable domain including a CDR having the sequence shown in SEQ ID NO: 4 for CDR-H1, a CDR having the sequence shown in SEQ ID NO: 5 for CDR-H2, and a CDR having the sequence shown in SEQ ID NO: 6 for CDR-H3.

[0068] The antibody molecule of the present invention may optionally comprise a complementary light chain or a complementary heavy chain.

[0069] Accordingly, in one embodiment, the present invention provides an antibody or an antigen-binding fragment thereof that binds to human IL-13 and comprises: (a) A light-chain variable region comprising: i. CDR-L1 comprising SEQ ID NO: 1, ii CDR-L2 comprising SEQ ID NO: 2, and iii CDR-L3 comprising SEQ ID NO: 3; and (b) A heavy-chain variable region comprising: i. CDR-H1 comprising SEQ ID NO: 4, ii CDR-H2 comprising SEQ ID NO: 5, and iii CDR-H3 comprising SEQ ID NO: 6; and

[0070] It will be understood that one or more amino acid substitutions, additions and / or deletions can be made to the CDRs provided by the present invention without significantly altering the ability of the antibody to bind to IL-13 and neutralize IL-13 activity. The effect of any amino acid substitution, addition and / or deletion can be readily tested by one skilled in the art, for example, by determining IL-13 binding and inhibition of IL-13 / IL-13 receptor interaction using the methods described herein, particularly the methods exemplified in the Examples.

[0071] Accordingly, the present invention provides an antibody having specificity for human IL-13, which comprises 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), and in these, one or more amino acids in one or more CDRs are substituted with another amino acid, for example, a similar amino acid defined below.

[0072] In one embodiment, the present invention provides an antibody having specificity for human IL-13, which comprises, for example, 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), and in these, one or more amino acids in one or more CDRs are substituted with another amino acid, for example, a similar amino acid defined below.

[0073] As used herein, "identity" indicates that at any given position in the aligned sequences, the amino acid residues are identical between the sequences. As used herein, "similarity" indicates that at any given position in the aligned sequences, the amino acid residues are of a similar type between the sequences. For example, leucine may be replaced with isoleucine or valine. Other amino acids that can often be replaced with each other include, but are not limited to, the following. - phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains), - lysine, arginine and histidine (amino acids having basic side chains), - aspartic acid and glutamic acid (amino acids having acidic side chains), - asparagine and glutamine (amino acids having amide side chains), and - cysteine and methionine (amino acids having sulfur-containing side chains). The degree of identity or similarity can be easily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Inc, Academic Press, 1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, BLAST available from NCBI TM as described in the software (Altschul, S.F. et al, 1990, J. Mol. Biol. 215:403 - 410; Gish, W. & States, D.J. 1993, Nature Genet. 3:266 - 272. Madden, T.L. et al., 1996, Meth. Enzymol. 266:131 - 141; Altschul, S.F. et al., 1997, Nucleic Acids Res. 25:3389 - 3402; Zhang, J. & Madden, T.L. 1997, Genome Res. 7:649 - 656,).

[0074] In one embodiment, the antibody of the present invention comprises a light chain variable domain comprising three CDRs, wherein the sequence of CDR-L1 has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence shown in SEQ ID NO: 1, CDR-L2 has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence shown in SEQ ID NO: 2, and / or CDR-L3 has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence shown in SEQ ID NO: 3.

[0075] In one embodiment, the antibody of the present invention comprises a heavy chain variable domain comprising three CDRs, wherein the sequence of CDR-H1 has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence shown in SEQ ID NO: 4, CDR-H2 has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence shown in SEQ ID NO: 5, and / or CDR-H3 has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence shown in SEQ ID NO: 6.

[0076] In one embodiment, the antibody of the present invention comprises a light chain variable region comprising the sequence shown in SEQ ID NO: 13 or SEQ ID NO: 17. In one embodiment, the antibody of the present 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 shown in SEQ ID NO: 13 or SEQ ID NO: 17.

[0077] In one embodiment, the antibody of the present invention comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 14 or SEQ ID NO: 18. In one embodiment, the antibody of the present 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 shown in SEQ ID NO: 14 or SEQ ID NO: 18.

[0078] In one embodiment, the antibody of the present invention comprises a light chain variable region and a heavy chain variable region. The light chain variable region comprises the sequence shown in SEQ ID NO: 13, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 14. In one embodiment, the antibody of the present invention comprises a light chain variable region and a heavy chain variable region. The light chain variable region comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to that shown 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 shown in SEQ ID NO: 14.

[0079] In one embodiment, the antibody of the present invention comprises CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences respectively comprising SEQ ID NO: 1 / 2 / 3 / 4 / 5 / 6, and the remainder of the light and heavy chain variable regions has at least 70%, 80%, 90%, 95% or 98% identity or similarity respectively to SEQ ID NO: 13 and 14 or SEQ ID NO: 17 and 18.

[0080] In one embodiment, the antibody of the present invention is Fab, Fab’, F(ab’)2, Fv, dsFv, scFv, or dsscFv. In one embodiment, the antibody of the present invention is a single domain antibody or nanobody, e.g., V H or V L or V HH or V NAR or the like.

[0081] In one embodiment, the antibody of the present invention is an scFv comprising the sequence shown in SEQ ID NO: 21, or a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence shown in SEQ ID NO: 21.

[0082] In one embodiment, the antibody of the present invention is an scFv comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences respectively shown in SEQ ID NO: 1 / 2 / 3 / 4 / 5 / 6, and the remainder of the scFv has at least 70%, 80%, 90%, 95% or 98% identity and similarity to SEQ ID NO: 21.

[0083] In one embodiment, the antibody of the present invention is a dsscFv comprising the sequence shown in SEQ ID NO: 23, or a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence shown in SEQ ID NO: 23.

[0084] In one embodiment, the antibody of the present invention is a dsscFv comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences shown in SEQ ID NOs: 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.

[0085] In one embodiment, the antibody comprises a heavy chain and a light chain, the heavy chain comprises a CH1 domain, and the light chain comprises a CL domain, either kappa or lambda.

[0086] Biomolecules such as antibodies and their fragments contain acidic and / or basic functional groups, thereby imparting a positive or negative charge to the molecule. The amount of the overall "observed" charge is determined by the absolute amino acid sequence of the entity, the local environment of the charged groups in the three-dimensional 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, the 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 pI of the antibody is 8.

[0087] The IL-13 antibodies and fragments of the present invention can be designed to have an appropriate isoelectric point. This can result in antibodies and / or fragments with more robust properties, particularly appropriate solubility and / or stability profiles. Thus, in one aspect, the present invention provides humanized IL-13 antibodies modified to have an isoelectric point different from that of the originally identified antibody. The antibody may be modified by replacing amino acid residues, for example, replacing one or more acidic amino acid residues with basic amino acid residues. Alternatively, basic amino acid residues may be added or acidic amino acid residues may be removed. Alternatively, if the molecule has an unacceptably high pI value, acidic residues may be introduced as necessary to lower the pH. The pI of the modified antibody or fragment can be, for example, 8 or higher, for example 8.5 or 9. It is important to note that when manipulating the pI, care must be taken to retain the desired activity of the antibody or fragment. Thus, in one embodiment, the modified antibody or fragment has the same or substantially the same activity as the "unmodified" antibody or fragment.

[0088] To predict the isoelectric point of an antibody or fragment, ** programs such as ExPASY http: / / www.expasy.ch / tools / pi_tool.html and http: / / www.iut-arles.up.univ-mrs.fr / w3bb / d_abim / compo-p.html can be used.

[0089] epitope An epitope is the region of an antigen that binds to an antibody. The definition of an epitope can be divided into a structural one and a functional one. A functional epitope is generally a subset of a structural epitope and has residues that directly contribute to the affinity of the interaction. An epitope can also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, an epitope may include a determinant that is a chemically active surface group of a molecule such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0090] Whether a particular antibody binds to the same epitope as a reference antibody or competes with the reference antibody for binding can be readily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as the reference antibody of the present invention, the reference antibody is bound to a protein or peptide under saturation conditions. Next, the ability of the test antibody to bind to the protein or peptide is evaluated. If the test antibody can bind to the protein or peptide after saturation binding with the reference antibody, it can be determined that the test antibody binds to an epitope different from that of the reference antibody. On the other hand, if the test antibody cannot bind to the protein or peptide after saturation binding with the reference antibody, the test antibody may be binding to the same epitope as the epitope to which the reference antibody of the present invention binds.

[0091] To determine whether an antibody competes to bind to a reference antibody, the above binding method is performed in two ways. In the first method, the reference antibody is bound to the protein / peptide under saturation conditions, and then the binding of the test antibody to the protein / peptide molecule is evaluated. In the second method, the test antibody is bound to the protein / peptide under saturation conditions, and then the binding of the reference antibody to the protein / peptide is evaluated. In both methods, if only the first (saturating) antibody can bind to the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As will be understood by those skilled in the art, antibodies that compete for binding to a reference antibody do not necessarily bind to the same epitope as the reference antibody, and may sterically block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0092] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, 75%, 90%, or even 99% as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations of the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody.

[0093] Subsequently, additional routine experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is actually 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. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art.

[0094] The antibody can compete with or bind to the same epitope as that defined above from the perspective of the light chain, heavy chain, light chain variable region (LCVR), heavy chain variable region (HCVR), or CDR sequence, and bind to IL-13. In particular, the antibody can compete with or bind to the same epitope as an antibody comprising a combination of the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences of SEQ ID NO: 1 / 2 / 3 / 4 / 5 / 6 and bind to IL-13. The antibody can compete with or bind to the same epitope as an antibody comprising a combination of the LCVR and HCVR sequences of SEQ ID NO: 13 / 14 or 17 / 18 and bind to IL-13. The antibody can compete with or bind to the same epitope as an scFv comprising the sequence shown in SEQ ID NO: 21, or a dsscFv comprising the sequence shown in SEQ ID NO: 23 and bind to IL-13.

[0095] Effector molecule Optionally, the antibodies for use in the present invention may be conjugated to one or more effector molecules. It will be understood that the effector molecule can comprise a single effector molecule or two or more such molecules linked to form a single site capable of binding to the antibody of the present invention. If an antibody fragment conjugated to an effector molecule is desired, this can be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment binds directly or via a coupling agent to the effector molecule. Techniques for conjugating such effector molecules to antibodies are 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 procedures include, for example, those described in WO93 / 06231, WO92 / 22583, WO89 / 00195, WO89 / 01476 and WO03031581. Alternatively, when the effector molecule is a protein or polypeptide, the linkage can be achieved using recombinant DNA procedures as described, for example, in WO86 / 01533 and EP0392745.

[0096] As used herein, the term effector molecule includes, for example, anti-cancer agents, drugs, toxins, biologically active proteins such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides, particularly radioactive iodides, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy.

[0097] Examples of effector molecules can include cytotoxins or cytotoxic agents that include any agent that is harmful to cells (e.g., kills cells). Examples include combretastatin, dolastatin, epothilone, staurosporine, maytansinoid, spongistatin, lysocine, halichondrin, lohistatin, hemiasterlin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, promycin, and analogs or homologs thereof.

[0098] Effector molecules can also include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiammine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (old daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (old actinomycin), bleomycin, mitramycin, anthramycin (AMC), calicheamicin or duocarmycin), and antimitotic agents (e.g., vincristine and vinblastine), but are not limited thereto.

[0099] Other effector molecules include 111 In 90 Y, Lu 177 , bismuth 213 , californium 252 , iridium 192 , tungsten 188 / rhenium188 Chelating radionuclides such as etc., or drugs including but not limited to alkylphosphocholine, topoisomerase I inhibitors, taxoids, slamine, etc.

[0100] Other effector molecules include proteins, peptides, enzymes, etc. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include toxins such as immunoglobulins, 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 angiogenesis inhibitors such as angiostatin or endostatin, or biological response modifiers or other growth factors and immunoglobulins 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), etc., but are not limited thereto.

[0101] Other effector molecules include, for example, detectable substances useful for diagnosis. Examples of detectable substances include various enzymes, haptens, fluorescent substances, luminescent substances, bioluminescent substances, radionuclides, positron-emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. For metal ions that can be conjugated to an antibody for use as a diagnostic agent, generally refer to U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable haptens include streptavidin, avidin, and biotin; suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin, etc.; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radionuclides include 125 I, 131 I, 111 In, and 99 Tc.

[0102] In another example, the effector molecule can increase the half-life of the antibody in vivo, and / or decrease the immunogenicity of the antibody, and / or enhance the delivery of the antibody to the immune system across the epithelial barrier. Examples of suitable effector molecules of this type include polymers, albumin, albumin-binding proteins, or albumin-binding compounds such as those described in WO05 / 117984.

[0103] When the effector molecule is a polymer, it can generally be a synthetic or naturally-derived polymer, for example, a linear or branched polyalkylene, polyalkenylene, or polyoxyalkylene polymer that is optionally substituted, or a branched or unbranched polysaccharide, for example, a homo- or hetero-polysaccharide.

[0104] Specific optional substituents that may be present on the synthetic polymer include one or more hydroxy groups, methyl groups, or methoxy groups.

[0105] Specific examples of the synthetic polymer include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), or derivatives thereof, particularly optionally substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) or derivatives thereof.

[0106] Specific examples of naturally derived polymers include lactose, amylose, dextran, glycogen, or derivatives thereof.

[0107] As used herein, "derivative" is intended to include reactive derivatives, such as thiol-selective reactive groups such as maleimide. The reactive group may be linked directly to the polymer or via a linker segment. Residues of such groups may, in some cases, form part of the product as a linking group between the antibody fragment and the polymer.

[0108] The size of the polymer can be varied as desired, but generally falls within an average molecular weight range of 500 Da to 50,000 Da, such as 5000 to 40,000 Da, such as 20,000 to 40,000 Da. The polymer size can be selected based on the intended use of the product, particularly, for example, the ability to localize in a specific tissue such as a tumor, or the ability to extend the circulation half-life (see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545 for a review). Thus, for example, if the product is intended to leave the circulation and penetrate into tissues, it may be advantageous to use a low molecular weight polymer having a molecular weight of, for example, about 5000 Da. For applications where the product remains in the circulatory system, it may be advantageous to use a higher molecular weight polymer, such as one having a molecular weight in the range of 20,000 Da to 40,000 Da.

[0109] Suitable polymers include polyalkylene polymers such as poly(ethylene glycol), or in particular methoxypoly(ethylene glycol) or derivatives thereof, particularly those having a molecular weight in the range of about 15,000 Da to about 40,000 Da.

[0110] In one example, the antibody for use in the present invention is conjugated to a poly(ethylene glycol) (PEG) moiety. In certain examples, the antibody is an antibody fragment and the PEG molecule may be conjugated via any available amino acid side chain or terminal amino acid functional group located on the antibody fragment, such as any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids may be naturally present in the antibody fragment or may be engineered into the fragment using recombinant DNA methods (see, e.g., US 5,219,996; US 5,667,425; WO 98 / 25971). As an example, the antibody molecule of the present invention is a modified Fab fragment, and the modification is the addition of one or more amino acids to the C-terminus of its heavy chain to enable binding of an effector molecule. Preferably, the additional amino acids form a modified hinge region containing one or more cysteine residues to which an effector molecule can bind. Multiple sites can be used to conjugate two or more PEG molecules.

[0111] Preferably, the PEG molecule may be covalently bound via the thiol group of at least one cysteine residue located in the antibody fragment. Each polymer molecule bound to the modified antibody fragment may be covalently bound to the sulfur atom of the cysteine residue located in the fragment. The covalent bond will generally be a disulfide bond or in particular a sulfur-carbon bond. When the thiol group is used as a binding point for a suitable activating effector molecule, thiol-selective derivatives such as maleimide and cysteine derivatives may be used. The activated polymer can be used as a starting material in the preparation of polymer-modified antibody fragments as described above. The activated polymer may be any polymer containing a thiol-reactive group such as an α-halocarboxylic acid or ester, for example iodoacetamide, an imide, for example maleimide, vinyl sulfone or disulfide. Such starting materials can be obtained commercially (e.g., from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA), or can be prepared from starting materials that are commercially available using conventional chemical procedures. Specific PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

[0112] In one embodiment, the antibody is PEGylated, i.e., a modified Fab fragment or diFab to which PEG (poly(ethylene glycol)) is covalently bound according to the methods disclosed in, for example, EP0948544 or EP1090037 [see also “Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications”, 1992, J. Milton Harris (ed), Plenum Press, New York, “Poly(ethyleneglycol) Chemistry and Biological Applications”, 1997, J. Milton Harris and S. Zalipsky (eds), 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, the PEG is bound to a cysteine in the hinge region. In one example, the PEG-modified Fab fragment has a maleimide group covalently bound to a single thiol group in the modified hinge region. A lysine residue may be covalently bound to the maleimide group, and a methoxypoly(ethylene glycol) polymer having a molecular weight of about 20,000 Da may be bound to each of the amine groups on the lysine residue. Thus, the total molecular weight of the PEG bound to the Fab fragment may be about 40,000 Da.

[0113] In one embodiment, the present invention provides an antagonistic antibody molecule having specificity for human IL-13, which is a modified Fab' fragment having a modified hinge region containing at least one cysteine residue to which an effector molecule is attached at the C-terminus of its heavy chain. Preferably, the effector molecule is PEG and can be attached using the methods described in WO98 / 25971 and WO2004072116 or WO2007 / 003898. The effector molecule can be attached to the antibody fragment using the methods described in international patent applications WO2005 / 003169, WO2005 / 003170 and WO2005 / 003171.

[0114] In one embodiment, the antibody or fragment does not have an effector molecule attached.

[0115] Production of the antibody The present invention also provides an isolated DNA sequence encoding the heavy chain and / or light chain(s) of the antibody molecule of the present invention. Preferably, the DNA sequence encodes the heavy chain or light chain of the antibody molecule of the present invention. The DNA sequence of the present invention may include synthetic DNA produced, for example, by chemical treatment, cDNA, genomic DNA or any combination thereof.

[0116] 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 antibody heavy and light chains can be synthesized as desired from the determined DNA sequence or based on the corresponding amino acid sequence.

[0117] 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.

[0118] To prepare the DNA sequence encoding the antibody molecule of the present invention, standard techniques of molecular biology can be used. The desired DNA sequence can be synthesized completely or partially using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.

[0119] Examples of suitable sequences are provided herein. Thus, in one embodiment, the present invention provides an isolated polynucleotide encoding an antibody or antigen-binding fragment comprising the sequence shown in SEQ ID NO: 8, 10, 15, 16, 19, 20, 22 or 24.

[0120] Common methods by which vectors can be constructed, 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, F.M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual prepared by Cold Spring Harbor Publishing.

[0121] Also provided is a host cell comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding the antibody of the present invention. For the expression of the DNA sequence encoding the antibody molecule of the present invention, any suitable host cell / vector system can be used. Bacteria, such as Escherichia coli, and other microbial systems may be used, or eukaryotes, such as mammalian host cell expression systems, may be used. Suitable mammalian host cells include CHO cells, myeloma cells or hybridoma cells.

[0122] 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 inducing the expression of the protein from the DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule.

[0123] An antibody molecule may contain only a heavy or light chain polypeptide, in which case it is necessary to transfect a host cell using only the coding sequence of the heavy or light chain polypeptide. To produce a product containing both a heavy chain and a light chain, a cell line can be transfected with two vectors, namely a first vector encoding the light chain polypeptide and a second vector encoding the heavy chain polypeptide. Alternatively, a single vector containing the sequences encoding the light and heavy chain polypeptides may be used.

[0124] The antibodies and fragments according to the present disclosure are expressed at good levels from host cells. Thus, the properties of the antibodies and / or fragments appear to be optimized and suitable for commercial processing.

[0125] Pharmaceutical Compositions, Dosages and Administration Regimens The antibodies of the present invention can be provided in a pharmaceutical composition. The pharmaceutical composition is usually sterile and will typically contain a pharmaceutically acceptable carrier and / or adjuvant. The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable adjuvant and / or carrier.

[0126] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier may be suitable for parenteral administration, such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. Alternatively, the carrier may be suitable for non-parenteral administration, such as topical, epidermal or mucosal routes of administration. The carrier may be suitable for oral administration. Depending on the route of administration, the modulator may be coated with a material that protects the compound from the action of acids and other natural conditions that may inactivate the compound.

[0127] The pharmaceutical composition of the present invention may contain one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refer to salts that retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0128] The pharmaceutically acceptable carrier includes an aqueous carrier or diluent. Examples of suitable aqueous carriers that can be employed in the pharmaceutical composition of the present invention include water, buffered water, and physiological saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and their suitable mixtures, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. In many cases, it may be desirable to include in the composition an isotonic agent, such as saccharides, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride.

[0129] The therapeutic composition is usually sterile and must be stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for a high concentration of drug.

[0130] The pharmaceutical composition of the present invention may further contain additional active ingredients.

[0131] Also within the scope of the present invention is a kit containing the antibody or modulator of the present invention and instructions for use. The kit may further contain one or more additional reagents, such as additional therapeutic or prophylactic agents as described above.

[0132] The modulator and / or antibody of the present invention, or its formulation or composition, can be administered for prophylactic and / or therapeutic treatment.

[0133] For therapeutic use, the compound is administered to a subject already suffering from a disorder or condition as described above, in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Such a therapeutic treatment may result in a reduction in the severity of the disease symptoms, or an increase in the frequency or duration of asymptomatic periods. The amount sufficient to achieve this is defined as a "therapeutically effective amount".

[0134] For prophylactic use, the formulation is administered to a subject at risk of a disorder or condition as described above, in an amount sufficient to prevent or mitigate one or more sequelae of the condition or its symptoms. The amount sufficient to achieve this is defined as a "prophylactically effective amount". The amount effective for each purpose depends on the severity of the disease or injury, as well as the body weight and general condition of the subject.

[0135] The subject for administration may 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.

[0136] The antibody / modulator or pharmaceutical composition of the present invention can be administered via one or more routes of administration using one or more of the various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Examples of routes of administration of the compounds or pharmaceutical compositions 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" means a mode of administration other than enteral and topical administration, and is usually by injection. Alternatively, the antibody / modulator or pharmaceutical composition of the present invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration. The antibody / modulator or pharmaceutical composition of the present invention may also be for oral administration.

[0137] The appropriate dosage of the antibody / modulator or pharmaceutical composition of the present invention can be determined by those skilled in the art. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response without toxicity to the patient, for a particular patient, composition, and mode of administration. The selected dosage level will depend on a variety of pharmacokinetic factors including the activity of the particular composition employed in the present invention, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0138] Suitable dosages may be, for example, in the range of about 0.01 μg / kg to about 1000 mg / kg body weight of the patient being treated, typically in the range of about 0.1 μg / kg to about 100 mg / kg body weight. For example, suitable dosages may be about 1 μg / kg to about 10 mg / kg body weight per day, or about 10 μg / kg to about 5 mg / kg body weight per day.

[0139] The dosing regimen may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, it may be administered as a single dose, divided into several doses over time, or the dosage may be proportionally decreased or increased depending on the urgency of the therapeutic situation. As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0140] Administration may be by a single dose or multiple doses. Multiple doses can be administered by the same or different routes, at the same or different sites. Alternatively, administration can be via a sustained release formulation, in which case fewer doses are required. The dosage and frequency of administration can vary depending on the half-life of the antagonist in the patient and the desired duration of treatment.

[0141] As described above, the modulator / antibody or pharmaceutical composition of the present invention can be co-administered with one or more other therapeutic agents.

[0142] Combined administration of two or more agents can be achieved in many different ways. Both can be administered together in a single composition or separately in different compositions as part of a combination therapy. For example, one can be administered before, after, or simultaneously with the other.

[0143] Therapeutic indications The antibodies of the present invention can be used for the treatment, prevention, or amelioration of any condition associated with IL-13 activity, for example, any condition resulting from all or part of the signal via the IL-13 receptor.

[0144] IL-13-related diseases include breast cancer, colon cancer, rectal cancer, lung cancer, pharyngeal cancer, hypopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, gallbladder cancer, bile duct cancer, small intestine cancer, urinary tract cancer (kidney, bladder, urothelium, etc.), female reproductive system (cervix, uterus, ovary, choriocarcinoma, gestational trophoblastic disease, etc.), male reproductive system (prostate, seminal vesicle, testis, germ cell tumor, etc.), endocrine glands (thyroid, adrenal gland, pituitary gland, etc.), and skin, as well as primary and metastatic cancers such as hemangioma, melanoma, sarcoma (including those arising from bone and soft tissues and Kaposi's sarcoma), tumors of the brain, nerves, eyes, and meninges (astrocytoma, glioma, glioblastoma, retinoblastoma, neuroma, neuroblastoma, schwannoma, meningioma, etc.), solid cancers caused by hematopoietic malignancies such as leukemia, lymphoma (Hodgkin lymphoma and non-Hodgkin lymphoma), rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthritis, systemic lupus erythematosus, ulcerative colitis, inflammatory bowel disease, insulin-dependent diabetes, thyroiditis, allergic diseases, psoriasis, scleroderma, graft-versus-host disease, organ transplant rejection, acute or chronic immune diseases associated with organ transplantation. Sarcoidosis, arteriosclerosis, disseminated intravascular coagulation syndrome, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schönlein purpura, microscopic polyangiitis, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute rhabdomyolysis, Huntington's disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignant tumor, heart failure, Addison's disease, sporadic, polyendocrine deficiency type I - polyendocrine deficiency type II, Schmidt syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, arthritis, Reiter's disease, psoriatic arthritis, ulcerative colitis-associated arthritis, enteric synovitis, Chlamydia - Escherichia coli - Salmonella-related arthritis, 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 encephalomyelitis / Chronic Fatigue Syndrome, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing cholangitis, idiopathic autoimmune hepatitis,Acquired immunodeficiency-related diseases, hepatitis B, hepatitis C, common variable immunodeficiency (common variable hypogammaglobulinemia), dilated cardiomyopathy, female infertility, ovarian insufficiency, premature ovarian insufficiency, fibrotic lung diseases, idiopathic pulmonary fibrosis, post-inflammatory interstitial lung disease, interstitial pneumonia, connective tissue disease-related interstitial lung disease, mixed connective tissue disease-related lung disease, systemic sclerosis-related interstitial lung disease, rheumatoid arthritis-related interstitial lung disease, systemic lupus erythematosus-related lung disease, dermatomyositis / polymyositis-related lung disease, Sjogren's disease-related lung disease, ankylosing spondylitis-related lung disease, vasculitic diffuse lung disease, hemosiderin-related lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, obliterative bronchiolitis, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, post-infectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (classical autoimmune hepatitis or lupoid hepatitis), type 2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune-mediated hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune diseases associated with organ transplantation, chronic immune diseases associated with organ transplantation, osteoarthritis, primary sclerosing cholangitis, psoriasis vulgaris type 1, psoriasis vulgaris type 2, idiopathic leukopenia, autoimmune neutropenia, renal disease NOS, glomerulonephritis, microscopic polyangiitis of the kidney, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, secondary pulmonary hypertension due to connective tissue disease, Goodpasture syndrome, pulmonary manifestations of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjogren's syndrome, Takayasu disease / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid diseases, hyperthyroidism, goiter autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxedema, lens-induced uveitis, primary vasculitis, vitiligo, acute liver diseases, chronic liver diseases, alcoholic cirrhosis, alcoholic liver injury, cholestasis, idiopathic liver diseases, drug-induced hepatitis, non-alcoholic fatty hepatitis, allergies, group B streptococcus (GBS) infections, mental diseases, depression, schizophrenia, Th2- and Th1-mediated diseases, acute and chronic pain, different forms of pain, cancer, lung cancer, breast cancer, gastric cancer, bladder cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, hematopoietic malignancies, leukemia, lymphoma,Abetalipoprotemia, 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, atrial ectopic beats, AIDS dementia complex, alcoholic hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis (including seasonal allergic rhinitis), non-allergic rhinitis, allograft rejection, alpha-I-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti-CD3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aortic and peripheral aneurysms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (persistent or paroxysmal), atrial flutter, atrioventricular block, B-cell lymphoma, bone graft rejection, bone marrow transplantation (BMT) rejection, bundle branch block, Burkitt lymphoma, burns, cardiac arrhythmias, cardiac standstill syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammatory reaction, cartilage graft rejection, cerebellar cortical degeneration, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy-related disorders, chronic myeloid leukemia (CML), chronic alcoholism, chronic inflammatory conditions, 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 disease, culture-negative sepsis, cystic fibrosis, cytokine therapy-related diseases, boxer dementia, demyelinating diseases, dengue hemorrhagic fever, dermatitis, skin diseases, diabetes, diabetes mellitus, diabetic arteriosclerosis, diffuse Lewy body disease, dilated congestive cardiomyopathy, basal ganglia disorders, midlife Down symptoms, drug-induced movement disorders by drugs that block central nervous system dopamine receptors, drug hypersensitivity, eczema, encephalomyelitis, endocarditis, endocrine disorders, laryngotracheitis, Epstein-Barr virus infection, erythromelalgia, extrapyramidal-cerebellar disorders, familial hemophagocytic lymphohistiocytosis, fetal thymus graft rejection, Friedreich ataxia, functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcers, glomerulonephritis, graft rejection of any organ or tissue, gram-negative sepsis, gram-positive sepsis, granulomas by intracellular bacteria, hairy cell leukemia, Hallervorden-Spatz disease, Hashimoto's disease (thyroiditis), hay fever, heart transplant rejection, hemochromatosis,Hemodialysis, hemolytic uremic syndrome / thrombotic thrombocytopenic purpura, bleeding, hepatitis A, His bundle arrhythmia, HIV infection / HIV neuropathy, Hodgkin's disease, hyperkinesia, hypersensitivity reaction, hypersensitivity pneumonia, hypertension, hypokinesia, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, myasthenia, spinal muscular atrophy in children, aortic inflammation, influenza A, exposure to ionizing radiation, iridocyclitis / uveitis / optic neuritis, ischemia-reperfusion injury, ischemic cerebral infarction, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, renal transplant rejection, Legionnaires' disease, leishmaniasis, Hansen's disease, corticospinal tract lesions, lipedema, liver transplant rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcal bacteremia, 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 avium intracellulare, Mycobacterium tuberculosis, Melodyplastic syndrome, myocardial infarction, myocardial ischemia disorder, nasopharyngeal cancer, chronic lung disease in newborns, nephritis, nephrosis, neurodegenerative disease, neurogenic muscular atrophy, neutropenic fever, non-Hodgkin lymphoma, occlusion of the abdominal aorta and its branches, obstructive arterial disease, okt3 therapy, orchitis / epididymitis, orchitis / vasectomy reversal surgery, organomegaly, osteoporosis, pancreatic transplant rejection, pancreatic cancer, tumor-associated syndrome / malignant tumor hypercalcemia, parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, peripheral atherosclerotic disease, peripheral vascular disorder, peritonitis, pernicious anemia, pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrine disorder, monoclonal gamma disease, skin change syndrome), post-perfusion syndrome, post-pump syndrome, post-Ml open heart surgery syndrome, pregnancy-induced hypertension nephropathy (preeclampsia), progressive supranuclear palsy, primary pulmonary hypertension, radiotherapy, Raynaud's phenomenon and Raynaud's disease, Raynaud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcoma, senile tremor, senile dementia with Lewy body dementiaSeronegative arthritis, shock, sickle cell anemia, skin allograft rejection, cutaneous change syndrome, small intestine transplant rejection, solid tumor, specific arrhythmia, spinal ataxia, spinocerebellar degeneration, streptococcal myositis, cerebellar structural lesion, subacute sclerosing panencephalitis, absence, cardiovascular syphilis, general anesthesia, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL capillary angioma, thrombotic vasculitis obliterans, thrombocytopenia, toxicity, transplantation, trauma / bleeding, type III hypersensitivity reaction, type IV hypersensitivity reaction, unstable angina, uremia, uremic sepsis, valvular heart disease, aneurysm, vasculitis, venous disease, venous thrombosis, ventricular fibrillation, viral and fungal infections, viral encephalitis / aseptic meningitis, virus-related hemophagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, allograft rejection of all organs / tissues, acute coronary syndrome, acute idiopathic polyneuritis, acute inflammatory demyelinating polyradiculopathy, acute ischemia, adult Still's disease, anaphylaxis, antiphospholipid antibody syndrome, aplastic anemia, atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmune disease associated with streptococcal infection, autoimmune enteropathy, autoimmune deafness, autoimmune lymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarian insufficiency, blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular disease, catastrophic antiphospholipid antibody syndrome, celiac disease, cervical spondylosis, chronic ischemia, cicatricial pemphigoid, clinical isolated syndrome (cis) associated with the risk of multiple sclerosis, childhood-onset mental disorder, dacryocystitis, dermatomyositis, diabetic retinopathy, intervertebral disc herniation, intervertebral disc prolapse, 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 allergy, immune hemolytic anemia, inclusion body myositis, infectious ophthalmic inflammatory disease, inflammatory demyelinating disease, inflammatory heart disease, inflammatory kidney disease, IPF / UIP, iritis, keratitis, keratoconjunctivitis sicca, Kussmaul disease or Kussmaul-Mayer disease. Landry paralysis, Langerhans cell histiocytosis, reticular skin patch, macular degeneration, microscopic polyangiitis, Morbus Bekteref, motor neuron disorder, mucous membrane pemphigoid, multiple organ failure, myasthenia gravis, myelodysplastic syndrome, myocarditis, radiculopathy, neuropathy, non-A non-B hepatitis, optic neuritis, osteolysis, non-articular JRA, peripheral arterial occlusive disease (PAOD),Peripheral vascular disease (PVD), peripheral arterial disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, vitiligo, polyarticular JRA, polyendocrine deficiency syndrome, polymyositis, rheumatoid polymyalgia (PMR), idiopathic Parkinsonism, prostatitis, pure red cell aplasia, primary adrenal insufficiency, relapsing neuromyelitis optica, restenosis, rheumatoid, Heart disease, SAHO (synovitis, acne, pustulosis, hyperostosis, osteitis), secondary amyloidosis, shock lung, scleritis, sciatica, secondary adrenal insufficiency, silicon-related connective tissue disease, Sneddon-Wilkinson skin disease, ankylosing spondylitis, Stevens-Johnson syndrome (SJS), temporal arteritis, toxoplasma retinitis, toxic epidermal necrolysis, rhabdomyositis, TRAPS (tumor necrosis factor receptor, type 1 allergic reaction, 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 + atopy), eosinophilic myalgia syndrome, hypereosinophilic syndrome, edematous reactions including episodic angioedema, helminth infections, onchocercal dermatitis, eosinophil-related gastrointestinal disorders, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis, eosinophilic colitis, nasal micropolyposis and polyposis, food allergy, aspirin intolerance and obstructive sleep apnea syndrome, 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 (idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis secondary to sclerosis, etc.), COPD, liver fibrosis, etc.

[0145] The antibody of the present invention is considered to be particularly useful for the treatment or prevention of atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergy, or eosinophilic esophagitis.

[0146] Accordingly, in one embodiment, the antibody or pharmaceutical composition of the present invention is provided for use in a method of treating the human or animal body by therapy.

[0147] In one embodiment, the antibody or pharmaceutical composition is provided for use in a method of treating atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergy, or eosinophilic esophagitis.

[0148] In one embodiment, the present invention provides a method of treating or preventing atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergy, or eosinophilic esophagitis, the method comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or pharmaceutical composition.

[0149] In one embodiment, the present invention provides the use of an antibody or pharmaceutical composition in the manufacture of a medicament for the treatment or prevention of one or more of the medical indications described herein.

Example

[0150] The present invention will be described by the following examples.

[0151] Example 1. Generation and selection of the therapeutic anti-IL-13 antibody CA650 Rats were immunized with purified human IL-13 (Peprotech) or rat fibroblasts expressing human IL-13 (expressing approximately 1 μg / ml in the culture supernatant), or in some cases, a combination of the two. After 3 to 6 injections, the animals were sacrificed and PBMC, spleen, bone marrow, and lymph nodes were collected. Serum was monitored for binding to human IL-13 by ELISA and for the ability to neutralize hIL-13 in a HEK-293 IL-13R-STAT-6 reporter cell assay (HEK-Blue assay, Invivogen).

[0152] B cell cultures were set up and the supernatants were first screened for binding ability to hIL-13 in a bead-based assay using the Applied Biosystems FMAT assay. This was a homogeneous assay using biotinylated human IL-13 coated on streptavidin beads and goat anti-rat Fc-Cy5 conjugate as the reveal agent. Next, the positives from this assay were progressed to the HEK-293 IL-13R-STAT-6 reporter cell assay (HEK-Blue assay, Invivogen) to identify neutralizing agents. The neutralizing supernatants were then profiled on Biacore to estimate the off-rate and to clarify the mechanism of action of the neutralization. The neutralizations were classified into bin 1 or bin 2. Bin 1 represents antibodies that bind to human IL-13 and inhibit the binding of IL-13Rα1, and as a result, also inhibit the binding of IL-4R. Bin 1 antibodies may also inhibit the binding of IL-13 to IL-13Rα2. Bin 2 represents antibodies that can bind to IL-13Rα1 but bind to hIL-13 in such a way as to prevent recruitment to the IL-4R complex. We had selected antibodies that act via bin 1.

[0153] From a total of 27 x 100 plate SLAM experiments, approximately 7500 IL-13 specific positives were identified in the primary FMAT screen. 800 wells demonstrated neutralization in the HEK-blue assay. 170 wells had the desired Biacore profile, i.e., off-rate < 5 x 10 -4 s -1It had the bin1 antibody. When attempting to clone the variable regions from these 170 wells, fluorescent spots were successfully obtained in 160 wells. Reverse transcription (RT)-PCR was performed on 100 wells to generate heavy and light chain variable region gene pairs. These V region genes were cloned as full-length mouse IgG1 antibodies and re-expressed in the HEK-293 transient expression system. As a result of sequence analysis, it was found that there were 27 unique families of anti-human IL-13 antibodies. These recombinant antibodies were then retested for their ability to block recombinant hIL-13 (from E. coli and mammals), recombinant mutant hIL-13 (R130Q) (from E. coli), native wild-type and mutant hIL-13 (from human donors), and cynomolgus monkey IL-13 (from mammals) in a cell-based assay. The recombinant antibodies were also tested for their binding ability to mutant human IL-13 (R130Q) and cynomolgus monkey IL-13 using Biacore. After this characterization, antibody families that met our criteria, i.e., antibodies with a titer and affinity decrease of less than 100 pM with little decrease against all human and cynomolgus monkey IL-13 preparations, were selected.

[0154] Based on neutralizing power, affinity, and donor content (see below) in humanized grafts, humanized CA650 was selected for further progression.

[0155] Example 2. Humanization of antibody CA650 Antibody 650 was humanized by transplanting the CDRs from the rat V region into the human germline antibody V region framework. To restore antibody activity, a number of framework residues from the rat V region were also retained in the humanized sequence. These residues were selected using the protocol outlined by Adair et al. (1991) (Humanized antibodies, WO91 / 09967). An alignment of the rat antibody (donor) V region sequence with the human germline (acceptor) V region sequence is shown with the designed humanized sequence. (Figure 1(A) light chain graft 650 and Figure 1(B) heavy chain graft 650). The CDRs grafted from the donor to the acceptor sequence are as defined by Kabat (Kabat et al., 1987) when using a combination of the Chothia / Kabat definitions, except for CDR-H1 (see Adair et al., 1991 Humanized antibodies, WO91 / 09967).

[0156] The gene encoding the initial sequence of the V region was designed and constructed by the automated synthesis method of Entelechon GmbH and modified by oligonucleotide-directed mutagenesis to generate the grafted versions gL8 and gH9. The gL8 sequence was subcloned into the UCB Celltech human light chain expression vector pVhCK containing DNA encoding the human C-kappa constant region (Km3 allotype). The gH9 sequence was subcloned into pVhg1Fab containing DNA encoding the human heavy chain gamma-1 CH1 constant region.

[0157] The human V region IGKV1-39+JK2 J region (International Immunogenetics Information System® (IMGT), http: / / www.imgt.org) was selected as the acceptor for the antibody 650 light chain CDRs. The light chain framework residues of the grafted gL8 are all from human germline genes except for residues 58 and 71 (numbering according to Kabat), and the donor residues isoleucine (I58) and tyrosine (Y71) were retained, respectively. The retention of the residues I58 and Y71 was essential for the full potency of the humanized antibody.

[0158] The human V-region IGHV1-69+JH4 J-region (IMGT, http: / / www.imgt.org) was selected as the acceptor for the heavy-chain CDR of antibody 650. The heavy-chain framework residues of graft gH9 are all from human germline genes except for residues 67, 69, and 71 (numbering according to Kabat), and the donor residues alanine (A67), phenylalanine (F69), and valine (V71) were retained, respectively. The retention of the A67, F69, and V71 residues was essential for the full potency of the humanized antibody. The glutamine residue at position 1 of the human framework was substituted with glutamic acid (E1) to enable the expression and purification of a homogeneous product. The conversion of glutamine to pyroglutamic acid at the N-terminus of antibodies and antibody fragments has been widely reported. The finally selected variable graft sequences gL8 and gH9 are shown in Figure 1(A) and Figure 1(B), respectively.

[0159] The amino acid sequences and DNA sequences encoding the CDR, heavy and light variable regions, scFv, and dsscFV formats of antibody 650 are shown in Figure 2.

[0160] Example 3. Biological activity of the anti-IL13 antibody Data showing the confirmation of antigen binding and neutralization of IL-13 are not shown.

Sequence Listing Free-Text

[0161] SEQ ID NOs: 1-24 <223> Recombinant sequences

Claims

1. An antibody or antigen-binding fragment thereof that binds to human IL-13, comprising the following: an antibody or antigen-binding fragment thereof. (a) A light chain variable region comprising the sequence shown in SEQ ID NO: 13 or SEQ ID NO: 17 and (b) A heavy chain variable region comprising the sequence shown in SEQ ID NO: 14 or SEQ ID NO:

18.

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

14.

3. The antibody or antigen-binding fragment according to claim 1, wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 17 and the heavy chain variable region comprises the sequence shown in SEQ ID NO:

18.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein the antibody is a full-length antibody.

5. The antigen-binding fragment is Fab, Fab', F(ab'), ２ , Fv, dsFv, scFv, or dsscFv, the antibody or antigen-binding fragment according to any one of claims 1 to 3.

6. The antibody or antigen-binding fragment according to claim 5, wherein the antigen-binding fragment is an scFv comprising the sequence shown in SEQ ID NO: 21 or a dsscFv comprising the sequence shown in SEQ ID NO:

23.

7. An isolated polynucleotide encoding the antibody or antigen-binding fragment according to any one of claims 1 to 6.

8. An expression vector having the polynucleotide according to claim 7.

9. A host cell comprising the vector according to claim 8.

10. A method for producing the antibody or antigen-binding fragment according to any one of claims 1 to 6, comprising culturing the host cell according to claim 9 under conditions permitting the production of the antibody or antigen-binding fragment and recovering the produced antibody or antigen-binding fragment.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 6 and a pharmaceutically acceptable adjuvant or carrier.

12. The pharmaceutical composition according to claim 11 for treating the body of a human or animal by treatment.

13. The pharmaceutical composition according to claim 12 for the treatment of atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergy, or eosinophilic esophagitis.

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