Multi-specific antibody with binding specificity for human il-13 and il-17

Abstract

This invention relates to a multispecific antibody specific to human IL-13, human IL-17A, and / or human IL-17F. The invention further relates to a method for preparing this multispecific antibody and its therapeutic use in treating atopic dermatitis and other diseases.

Description

[Technical Field]

[0001] This invention relates to multispecific antibodies that are specific to human IL-13, human IL-17A, and / or IL-17F. The invention further relates to a method for preparing multispecific antibodies and their therapeutic use in treating atopic dermatitis and other diseases. [Previous Technology]

[0002] Atopic dermatitis (AD), also known as atopic eczema, is an inflammatory disease that causes intense itching, redness, swelling, oozing, and cracked skin (which usually thickens over time).

[0003] Since the beginning of the 20th century, many inflammatory conditions of the mucous membranes have become more common; atopic dermatitis is a typical example of this disease. It now affects 15% to 30% of children and 2% to 10% of adults in developed countries, and in the United States, this has nearly tripled in the past 30 to 40 years. More than 15 million American adults and children suffer from atopic dermatitis.

[0004] Treatments for Alzheimer's disease (AD) include systemic immunosuppressants such as cyclosporine, methotrexate, interferon-gamma, mycophenolate mofetil, and azathioprine. Antidepressants and naltrexone can be used to control itching. In 2016, crisaborole (a phosphodiesterase-4 inhibitor) was approved for mild to moderate eczema, and in 2017, dupilumab (a monoclonal antibody antagonist of IL-4Rα) was approved for the treatment of moderate to severe eczema.

[0005] Due to the limitations of existing drugs, there is an urgent need for improved treatments for atopic dermatitis.

[0006] WO2013 / 102042A2 (Abbvie) describes a dual-specific binding protein against IL-13 and IL-17 and its potential use in the treatment of a variety of diseases. The binding protein has not progressed to clinical development.

[0007] WO2015 / 127405A2 (Genentech) describes an anti-IL-13 / IL-17 bispecific antibody and its use in the treatment of moderate to severe asthma and / or eosinophilic asthma. In a Phase I clinical trial, BITS7201A was associated with a high incidence of anti-drug antibody (ADA) and clinical development was withdrawn. [Summary of the Invention]

[0008] The present invention provides an improved multispecific antibody capable of binding human IL-13, human IL-17A and / or human IL-17F.

[0009] Compared to currently available antibodies, the antibodies of the present invention possess improved properties, such as lower immunogenicity and / or better pharmacokinetic characteristics. Furthermore, the antibodies of the present invention can be engineered to be purified more efficiently using improved purification methods that involve fewer steps than currently available methods, which is cost- and time-efficient on an industrial scale. The antibodies of the present invention thus possess improved manufacturability.

[0010] The present invention further provides: an isolated polynucleotide encoding a multispecific antibody; an expression vector carrying the polynucleotide; a host cell comprising the vector; a method for preparing a multispecific antibody, the method comprising culturing the host cell and recovering the resulting antibody; a pharmaceutical composition comprising a multispecific antibody; a multispecific antibody or pharmaceutical composition used in a method for therapeutically treating a human or animal; and a method for treating or preventing atopic dermatitis, chronic hand eczema, nasal micropolyps or polyposis, food allergies, or eosinophilic esophagitis, the method comprising administering a therapeutically effective amount of a multispecific antibody or pharmaceutical composition to a patient in need.

Implementation Method

[0011] IL-13

[0012] IL-13 is a short-chain cytokine that shares 25% sequence identity with IL-4. It contains approximately 132 amino acids, forming four helices spanning residues 10 to 21 (helix A), 43 to 52 (helix B), 61 to 69 (helix C), and 92 to 110 (helix D), and two β-strands spanning residues 33 to 36 and 87 to 90. The solution structure of IL-13 has been resolved, showing the predicted up-up-down-down four-helix bundle conformation, which has also been observed in IL-4 (Eisenmesser 2001).

[0013] Human IL-13 is a 17 kDa glycoprotein produced by activated T cells of the Th2 lineage, although Th0 and Th1 CD4+ T cells, CD8+ T cells, and several non-T cell populations (such as mast cells) also produce IL-13. The functions of IL-13 include switching to IgE in human B cells and inhibiting the production of inflammatory cytokines in both humans and mice.

[0014] IL-13 binds to its cell surface receptors IL-13R-α1 and IL-13R-α2. IL-13R-α1 interacts with IL-13 with low affinity (KD approximately 10 nM), and then recruits IL-4R-α to form a high affinity (KD approximately 0.4 nM) signaling heterodimer receptor complex.

[0015] 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. Binding of the IL-13R-α / IL-4R receptor complex leads to the activation of multiple signal transduction pathways, including the signal transducer and activator of transcription 6 (STAT6) and the insulin receptor receptor 2 (IRS2) pathway.

[0016] The IL-13R-α2 chain alone has a high affinity for IL-13 (KD approximately 0.25 to 0.4 nM). It acts as both a decoy receptor that negatively regulates IL-13 binding and a signal transduction receptor that induces TGF-β synthesis and fibrosis via the AP-1 pathway in macrophages and possibly other cell types.

[0017] IL-13 is involved in the pathogenesis of many human diseases, and therapeutic strategies have been designed to inhibit or counteract IL-13 activity. In particular, the search has been for antibodies that bind to and neutralize IL-13 as a means of inhibiting IL-13 activity. However, this technology requires suitable and / or modified antibodies that can bind to IL-13, especially human IL-13, and particularly antibodies that can neutralize human IL-13.

[0018] This invention provides a novel family of binding proteins, CDR-grafted antibodies, humanized antibodies and fragments thereof, which are capable of binding human IL-13 with high affinity and binding to and neutralizing human IL-13.

[0019] Antibodies that inhibit IL-13 activity can act through several possible mechanisms of action. Cage 1 represents an antibody that binds to human IL-13 and prevents the binding of IL-13Rα1, consequently also blocking the binding of IL-4R. Cage 1 antibodies can also prevent IL-13 from binding to IL13Rα2. Cage 2 represents an antibody that binds to hIL-13 in a manner that allows it to bind to IL-13Rα1 but prevents IL-4R recruitment into the complex. We are selecting antibodies that operate via Cage 1.

[0020] In one embodiment, the multispecific antibody binds to human IL-13 and prevents the binding of IL-13Rα1.

[0021] In one embodiment, the multispecific antibody binds to human IL-13 and prevents the binding of IL-13Rα2.

[0022] In one embodiment, the multispecific antibody binds to human IL-13 and prevents the binding of IL-13Rα1 and IL-13Rα2.

[0023] In one embodiment, the multispecific antibody binds to human IL-13 and IL-17 at a KD of <100 pM.

[0024] The IL-17 cytokine family consists of six members with molecular weights ranging from 23 to 36 kDa and dimeric structures, based on structural similarity. The founding member, IL-17A (often simply referred to as IL-17 in the literature), shares 16% to 50% amino acid sequence identity with other members: IL-17B, IL-17C, IL-17D, IL-17E (also known as IL-25), and IL-17F. IL-17A and IL-17F share the greatest homology (50%) and bind to the same receptor complex, thus sharing biological activity between these two cytokines. Furthermore, IL-17A and IL-17F exist not only as homodimers but also as IL-17A / F heterodimers. IL-17E (IL-25) has the least similarity to IL-17A. Significant and relevant to the biological activity of IL-17A and IL-17F is the discovery that they share the same IL-17RA / IL-17RC receptor complex. IL-17A exhibits the highest affinity for IL-17RA, while IL-17F binds more strongly to IL-17RC. Another member of the IL-17RA family, IL-17E, is utilized, which signals via the IL-17RA / IL-17RB receptor complex.

[0025] IL-17A and IL-17F are produced by the Th17 subset of CD4+ T cells. In addition, other T cell subsets produce IL-17A and IL-17F, including cytotoxic CD8+ T cells (Tc17), gdT cells, and NK T cells. Other cell populations that have been reported to secrete IL-17A include neutrophils, monocytes, NK cells, lymphoid tissue inducer-like (LTi-like) cells, paneth cells, and even B cells and mast cells. Furthermore, epithelial cells have been reported to secrete IL-17F.

[0026] Cell types that respond to IL-17 cytokines are reflected by the expression of different receptors. IL-17RA is widely expressed, with particularly high levels in hematopoietic tissues, while IL-17RC is expressed at higher levels in non-immune cells of the joints, liver, kidneys, thyroid, and prostate. This differential expression can explain the difference in biological activity between IL-17A and IL-17F, because cells expressing high levels of IL-17RC can respond more strongly to IL-17F, while cells expressing higher levels of IL-17RA than IL-17RC are more likely to respond to IL-17A. Specific cell types that respond to IL-17A and F include fibroblasts, epithelial cells, keratinocytes, synovial cells, and endothelial cells. IL-17A has also been reported to act on T and B cells and macrophages.

[0027] In one embodiment, the multispecific antibody binds to human IL-17A. In one embodiment, the multispecific antibody binds to human IL-17F. In one embodiment, the multispecific antibody binds to both human IL-17A and IL17F.

[0028] In one embodiment, the multispecific antibody binds to human IL-17A with a KD of < 50 pM. In one embodiment, the multispecific antibody binds to human IL-17A with a KD of < 25 pM. In one embodiment, the multispecific antibody binds to human IL-17A with a KD of < 10 pM.

[0029] In one embodiment, the multispecific antibody binds to human IL-17F at a KD of <200 pM. In one embodiment, the multispecific antibody binds to human IL-17F at a KD of <100 pM. Albumin

[0030] The high specificity and affinity of antibodies make them ideal diagnostic and therapeutic agents, especially for regulating protein-protein interactions. However, antibodies can be subject to increased clearance from serum, particularly when they lack the Fc domain that confers long in vivo lifespan (Medasan et al., 1997, J. Immunol. 158:2211-2217).

[0031] Methods for improving antibody half-life are known. One method is to bind the fragment to a polymer molecule. Thus, by binding to polyethylene glycol (PEG; see, for example, WO98 / 25791, WO99 / 64460 and WO98 / 37200), the short circulating half-life of Fab', F(ab')2 fragments in animals is improved. Another method is to modify the antibody fragment by binding to a reagent that interacts with the FcRn receptor (see, for example, WO97 / 34631). Yet another method for prolonging half-life is to use peptides that bind to serum albumin (see, for example, Smith et al., 2001, Bioconjugate Chem. 12:750-756; EP0486525; US6267964; WO04 / 001064; WO02 / 076489; and WO01 / 45746).

[0032] Serum albumin is an abundant protein in blood vessels and extravascular compartments, with a half-life of approximately 19 days in the human body (Peters, 1985, Adv Protein Chem. 37:161-245). This is similar to the half-life of IgG1, which is approximately 21 days (Waldeman and Strober, 1969, Progr. Allergy, 13:1-110).

[0033] The use of anti-serum albumin binding single variable domains and their use as conjugates to increase the half-life of drugs (including NCE (chemical entity) drugs, proteins, and peptides) has been described, see, for example, Holt et al., Protein Engineering, Design & Selection, Vol. 21, 5, pp. 283-288, WO04003019, WO2008 / 096158, WO05118642, WO2006 / 0591056, and WO2011 / 006915. Other anti-serum albumin antibodies and their use in multispecific antibody forms are described in WO2009 / 040562, WO2010 / 035012, and WO2011 / 086091. In particular, we have previously described modified humanized anti-albumin antibodies in WO2013 / 068571.

[0034] The multispecific antibody of the present invention can be engineered to bind to human serum albumin in order to prolong its in vivo serum half-life, thereby resulting in improved pharmacokinetic properties. Antibody

[0035] Antibodies used in the context of this invention include whole antibodies and their functionally active fragments, i.e., molecules (also referred to as antigen-binding fragments), that specifically bind to IL-13, IL-17A, and / or IL-17F. Unless the context otherwise indicates, the characteristics described herein for antibodies also apply to antibody fragments.

[0036] Holoantibodies, also known as "immunoglobulins (Ig)," generally refer to complete or full-length antibodies, that is, elements consisting of two heavy chains and two light chains linked by disulfide bonds, which can be assembled to define a characteristic Y-shaped three-dimensional structure. Typical natural holoantibody systems are monospecific because they bind to one type of antigen, and bivalent because they have two independent antigen-binding domains. The terms "complete antibody," "full-length antibody," and "holoantibody" are used interchangeably to refer to monospecific bivalent antibodies with a structure similar to that of natural antibodies (including the Fc region as defined herein).

[0037] Each light chain comprises a light chain variable region (abbreviated as VL herein) and a light chain constant region (CL). Each heavy chain comprises a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region (CH) consisting of three constant domains CH1, CH2, and CH3 or four constant domains CH1, CH2, CH3, and CH4, depending on the Ig class. The "class" of Ig or antibody refers to the type of constant region and includes IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses, such as IgG1, IgG2, IgG3, and IgG4. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the typical complement system.

[0038] The VH and VL regions of the antibody according to the present invention can be further subdivided into hypervariable regions (or "hypervariable regions") that determine antigen recognition, called complementarity-determining regions (CDRs), and structurally more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FRs together form the variable region. According to the convention, the CDRs in the heavy chain variable region of the 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. They are numbered sequentially from the N-terminus to the C-terminus of each chain.

[0039] The CDR is conventionally numbered according to the system designed 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."). Unless otherwise specified, this numbering system is used in this specification.

[0040] Kabat residue names do not always directly correspond to the linear numbering of amino acid residues. Actual linear amino acid sequences may contain fewer or additional amino acids compared to strict Kabat numbering, corresponding to shortening or insertion of structural components (whether framework or complementarity-determining regions) of the basic variable domain structure. The correct Kabat number of a given antibody residue can be determined by comparing homologous residues in the antibody sequence with the "standard" Kabat number sequence.

[0041] According to the Kabat numbering system, the CDR of the variable domain of the heavy chain is located at residues 31 to 35 (CDR-H1), residues 50 to 65 (CDR-H2), and residues 95 to 102 (CDR-H3). However, according to Chothia (Chothia, C. and Lesk, AMJ Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Therefore, unless otherwise specified, "CDR-H1" as used herein is intended to refer to residues 26 to 35, as described by the combination of the Kabat numbering system and the Chothia topological ring definition.

[0042] According to the Kabat numbering system, the CDR of the variable domain of the light chain is located at residues 24 to 34 (CDR-L1), residues 50 to 56 (CDR-L2) and residues 89 to 97 (CDR-L3).

[0043] In addition to the CDR ring, the fourth ring exists between CDR-2 (CDR-L2 or CDR-H2) and CDR-3 (CDR-L3 or CDR-H3) formed by frame 3 (FR3). The Kabat numbering system defines frame 3 as positions 66 to 94 in the heavy chain and positions 57 to 88 in the light chain.

[0044] Based on sequence alignment of different members of the immunoglobulin family, a numbering scheme is proposed and described, for example, in Kabat et al., 1991, and Dondelinger et al., 2018, Frontiers in Immunology, Vol. 9, Article 2278.

[0045] The terms "constant domain" and "constant region" are used interchangeably as herein to refer to the domain of an antibody outside the variable region. A constant domain is identical in all antibodies of the same isotype but differs between isotypes. Typically, the constant region of the heavy chain, from the N-terminus to the C-terminus, is formed by CH1-hinge-CH2-CH3-(as needed)CH4 and contains three to four constant domains.

[0046] The constant domains (if present) of the antibody molecules of the present invention can be selected considering the proposed function of the antibody molecule, and in particular, the effector function that may be required. For example, the constant domains may be human IgA, IgD, IgE, IgG, or IgM domains. In particular, when the antibody molecule is intended for therapeutic use and requires antibody effector function, the human IgG constant domain, especially the human IgG constant domains of IgG1 and IgG3 isotypes, can be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and does not require antibody effector function, IgG2 and IgG4 isotypes can be used. It should be understood that sequence variants of these constant domains can also be used. For example, the serine acid (numbered according to the Kabat numbering system) at position 241 can be replaced with proline, as described by Angal et al. (Angal et al., 1993. A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody as observed during SDS-PAGE analysis Mol Immunol 30, 105-108), and referred to herein as IgG4P in the IgG4 molecule.

[0047] The terms "Fc," "Fc fragment," "Fc domain," and "Fc region" are used interchangeably to refer to the C-terminal region of an antibody that contains the constant region of the antibody, excluding the first constant immunoglobulin domain. Therefore, Fc refers to the last two constant domains CH2 and CH3 of IgA, IgD, and IgG, or the last three constant domains of IgE and IgM, and the flexible hinge N-terminus of these domains. The human IgG1 heavy chain Fc region is defined herein as containing residue C226 at its C-terminus, where this numbering is based on the EU index in Kabat. In the context of human IgG1, according to the EU index in Kabat, the lower hinge refers to positions 226 to 236, the CH2 domain to positions 237 to 340, and the CH3 domain to positions 341 to 447. The corresponding Fc regions of other immunoglobulins can be identified by sequence alignment.

[0048] In the context of this invention, when present, the constant region or Fc region may be native as defined above, or may be modified in various ways, limited by the inclusion of a functional FcR-binding domain, and preferably a functional FcRn-binding domain. Preferably, the modified constant region or Fc region results in improved functionality and / or pharmacokinetics. Such modifications may include the deletion of certain portions of the Fc fragment. Modifications may further include various amino acid substitutions capable of affecting the biological properties of the antibody. Mutations for increasing FcRn binding and thus increasing the in vivo half-life may also be present. Such modifications may further include modifications to the glycosylation characteristics of the antibody. The native Fc fragment is glycosylated in the CH2 domain, and each of the two heavy chains contains an N-glycan bound to an aspartic acid residue (Asn297) at position 297. In the context of this invention, antibodies may be glycosylated, that is, engineered to have specific glycosylation characteristics, such as resulting in improved properties, such as improved effector function or improved serum half-life.

[0049] The antibodies described herein are isolated. An "isolated" antibody is an antibody that has been separated from its natural environment components (e.g., by purification methods).

[0050] The term "antibody" encompasses monovalent antibodies, which contain only one antigen-binding domain (e.g., a single-arm antibody containing an interconnected full-length heavy chain and a full-length light chain, also known as a "half antibody"), and multivalent antibodies, which contain more than one antigen-binding domain.

[0051] The term "antibody" according to the present invention also covers the antigen-binding fragment of an antibody. Antigen-binding fragments of antibodies include single-chain antibodies (e.g., scFv and dsscfv), Fab, Fab', F(ab')2, Fv, single-domain antibodies, or nano-antibodies (e.g., VH or VL, or VHH or VNAR). Other antibody fragments used in the present invention include the Fab and Fab' fragments described in international patent applications WO2011 / 117648, WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171.

[0052] Methods for generating and manufacturing such antibody fragments are well known in this art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0053] The term "Fab fragment" as used herein refers to an antibody fragment containing a light chain fragment comprising a VL (variable light chain) domain and a constant domain of the light chain (CL), and a VH (variable heavy chain) domain and a first constant domain (CH1) of the heavy chain.

[0054] A typical "Fab' fragment" comprises a pair of heavy and light chains, wherein the heavy chain comprises a variable region VH, a constant region CH1, and a natural or modified hinge region, and the light chain comprises a variable region VL and a constant region CL. The dimer of the Fab' according to the present invention produces F(ab')2, wherein, for example, dimerization can be achieved through the hinge.

[0055] The term "single-domain antibody" as used herein refers to an antibody fragment consisting of a single monomeric variable antibody domain. Examples of single-domain antibodies include VH or VL or VHH or V-NAR.

[0056] The term "Fv" refers to two variable domains, such as a co-operative variable domain, or a homologous pair or an affinity maturation variable domain, i.e., VH and VL pairs.

[0057] "Single-chain variable fragment" or "scFv" as used herein refers to a single-chain variable fragment comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) stabilized by a peptide linker between a VH variable domain and a VL variable domain. The VH and VL variable domains may be in any suitable orientation, for example, the C-terminus of the VH may be linked to the N-terminus of the VL or the C-terminus of the VL may be linked to the N-terminus of the VH.

[0058] “Disulfide-stabilized single-chain variable fragment” or “dsscFv” as used herein refers to a single-chain variable fragment stabilized by a peptide linker between the VH and VL variable domains, and also includes interdomain disulfide bonds between VH and VL. (See, for example, Weatherill et al., Protein Engineering, Design & Selection, 25 (321-329), 2012, WO2007109254.)

[0059] “Disulfide-stabilized variable fragment” or “dsFv” as used herein refers to a single-chain variable fragment that is stabilized by an interdomain disulfide bond between VH and VL, excluding the peptide linker between the VH and VL variable domains.

[0060] In one embodiment, the multispecific antibody of the present invention is an antagonistic antibody. As used herein, the term "antagonistic antibody" describes an antibody capable of inhibiting or neutralizing the biological signaling activity of one or more antigens, for example by blocking or reducing the binding of IL-13, IL17A and / or IL17F to their receptors.

[0061] The antibody used in this invention may be (but is not limited to) a monoclonal antibody, a humanized antibody, a fully human antibody, or a chimeric antibody.

[0062] Monoclonal antibodies can be prepared by any method known in this technology, such as fusion tumor technology (Kohler & Milstein, 1975, Nature, 256:495-497), three-tumor technology, human B-cell fusion tumor technology (Kozbor et al., 1983, Immunology Today, 4:72) and EBV-fusion tumor technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R Liss, Inc., 1985).

[0063] Alternatively, a monolymphocyte antibody method may be used to generate antibodies by selecting and expressing cDNA of immunoglobulin variable regions produced from monolymphocytes, which are selected to generate specific antibodies by, for example, the methods described below: 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.

[0064] Antibody screening can be performed using assays that measure the binding to antigens and / or assays that measure the ability to block the binding of antigens to one or more of their receptors. An example of a binding assay is an ELISA, for example, using an IL-13 fusion protein immobilized on a plate and detecting anti-IL-13 antibodies bound to IL-13 using a binding secondary antibody. An example of a blocking assay is a flow cytometry-based assay that measures the blocking of IL-13 ligand protein binding to IL-13R. A fluorescently labeled secondary antibody system is used to detect the amount of IL-13 ligand protein bound to IL-13R.

[0065] Humanized antibodies (including CDR-grafted antibodies) are antibody molecules having one or more complementarity-determining regions (CDRs) derived from one or more non-human species and a framework region derived from a human immunoglobulin molecule (see, for example, US 5,585,089; WO91 / 09967). It should be understood that it may be necessary to transfer only the specific determining residues of the CDR rather than the entire CDR (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanized antibodies may further include one or more framework residues derived from a non-human species derived from the derived CDR, as needed.

[0066] Chimeric antibodies contain elements derived from two different species, thus retaining the characteristics of the species from which they are derived. Generally, chimeric antibodies will contain a variable region from one species (e.g., mouse, rat, rabbit, or similar) and a constant region from another species (e.g., human).

[0067] Antibodies can also be generated using various phage presentation methods known in this technology, including 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 et al. (Gene, 1997, 187: 9-18), Burton et al. (Advances in Immunology, 1994, 57: 191-280) and WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and US The antibodies revealed by 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.

[0068] A fully human antibody is an antibody in which both the variable and constant regions (if present) of the heavy and light chains are of human origin or substantially identical to human-derived sequences, but not necessarily derived from the same antibody. Examples of fully human antibodies may include, for example, antibodies produced by the phage presentation method described above and antibodies produced by mice, wherein the murine immunoglobulin variable and, as needed, constant region genes have been replaced with their human counterparts, as described in general terms, for example in EP 0546073, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, US 5,770,429, EP 0438474, and EP 0463151. Multispecific antibodies

[0069] The antibody of the present invention is a multispecific antibody. As used herein, "multispecific antibody" refers to an antibody as described herein that has at least two binding domains, i.e., two or more binding domains, such as two or three binding domains, wherein the at least two binding domains independently bind to two different antigens or two different antigenic determinants on the same antigen. Multispecific antibodies are generally monovalent for each specific (antigen). The multispecific antibodies described herein encompass both monovalent and multivalent antibodies, such as bivalent, trivalent, and quadrivalent multispecific antibodies.

[0070] The complementary site is the region of the antibody that recognizes and binds to the antigen. The antibody of the present invention may be a multi-complementary site antibody. As used herein, "multi-complementary site antibody" refers to an antibody as described herein that contains two or more different complementary sites that interact with different antigenic determinants from the same antigen or from two different antigens. The multi-complementary site antibody described herein may be a bi-complementary site, a tri-complementary site, or a tetra-complementary site.

[0071] "Antigen-binding domain," as used herein, refers to a portion of an antibody that includes part or all of one or more variable domains, such as part or all of a pair of variable domains VH and VL, which specifically interact with the target antigen. Binding domains may comprise monovalent antibodies. In one embodiment, each binding domain is monovalent. Preferably, each binding domain includes no more than one VH and one VL.

[0072] "Specifically" as used herein means a binding domain that recognizes only antigens that are specific to it, or a binding domain that has a significantly higher binding affinity for antigens that are specific to it compared to the affinity for antigens that are non-specific to it. Binding affinity can be determined by standard tests, such as surface plasma resonance, such as BIAcore.

[0073] Various forms of multispecific antibodies have been developed. Different classifications have been proposed, but multispecific IgG antibody forms generally include bispecific IgG, additional 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.

[0074] Techniques for preparing bispecific antibodies include (but are not limited to) CrossMab technology (Klein et al., Engineering therapeutic bispecific antibodies using CrossMab technology, Methods 154 (2019) 21-31), knobs-in-holes engineering (e.g., WO1996027011, WO1998050431), DuoBody technology (e.g., WO2011131746), and Azymetric technology (e.g., WO 2012058768). Other techniques for preparing 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, in particular, CrossMab antibodies, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, common LC of lenticule-entry-acupoint, lenticule-entry-acupoint assembly, charge pairs, Fab-arm exchange, SEED bodies, Triomab, LUZ-Y, Fcab, κλ-body, and orthogonal Fab.

[0075] Added IgG typically comprises full-length IgG engineered by attaching an additional antigen-binding domain or antigen-binding fragment to the N-terminus and / or C-terminus of the heavy and / or light chains of IgG. Examples of such additional antigen-binding fragments include sdAb antibodies (e.g., VH or VL), Fv, scFv, dsscFv, Fab, and scFav. Additional IgG antibody forms 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, Zy antibodies, and DVI-IgG (quadruple combination), as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106.

[0076] Multispecific antibody fragments include nanobody, nanobody-HAS, BiTE, diabody, DART, TandAb, sc bispecific antibody, sc-bispecific antibody-CH3, bispecific antibody-CH3, triple body, minibody; minibody, Tri Bi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFV3, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc bispecific antibody-Fc, bispecific antibody (-Fc, tandem scFv-Fc; and intrabody, as described, for example, by Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106.

[0077] Multi-specific fusion proteins include docking and locking, ImmTAC, HSA antibody (HSAbody), sc bifunctional antibody-HAS and tandem scFv-toxin.

[0078] Multispecific antibody conjugates include IgG-IgG; Cov-X-Body; and scFv1-PEG-scFv2.

[0079] Other forms of multispecific antibodies have been described, for example, in Brinkmann and Kontermann, The making of bispecific antibodies, mAbs, 9:2, 182-212 (2017), particularly in Figure 2, such as tandem scFv, trifunctional antibodies, Fab-VHH, taFv-Fc, scFv4-Ig, scFv2-Fcab, and scFv4-IgG. Bifunctional antibodies, trifunctional antibodies, and methods for producing them are disclosed, for example, in WO99 / 37791.

[0080] The present invention provides a multispecific antibody that binds to human IL-13, human IL-17A and / or human IL-17F.

[0081] In one embodiment, the multispecific antibody includes an antigen-binding site that binds to human IL-13, wherein the IL-13 binding site contains a light chain variable region comprising the sequence shown in SEQ ID NO:15 for CDR-L1, the sequence shown in SEQ ID NO:16 for CDR-L2, and the sequence shown in SEQ ID NO:17 for CDR-L3.

[0082] In one embodiment, the multispecific antibody includes an antigen-binding site that binds to human IL-13, wherein the IL-13 binding site contains a heavy chain variable region comprising the sequence shown in SEQ ID NO:18 for CDR-H1, the sequence shown in SEQ ID NO:19 for CDR-H2, and the sequence shown in SEQ ID NO:20 for CDR-H3.

[0083] In one embodiment, the IL-13 binding site contains a light chain variable region comprising the sequence shown in SEQ ID NO:27.

[0084] In one embodiment, the IL-13 binding site contains a heavy chain variable region comprising the sequence shown in SEQ ID NO:28.

[0085] In one embodiment, the IL-13 binding site contains a light chain variable region comprising the sequence shown in SEQ ID NO:31.

[0086] In one embodiment, the IL-13 binding site contains a heavy chain variable region comprising the sequence shown in SEQ ID NO:32.

[0087] In one embodiment, the multispecific antibody includes an antigen-binding site that binds to human IL-17A and human IL-17F, comprising: a light chain variable region that includes the sequence shown in SEQ ID NO:1 for CDR-L1, the sequence shown in SEQ ID NO:2 for CDR-L2, and the sequence shown in SEQ ID NO:3 for CDR-L3.

[0088] In one embodiment, the multispecific antibody includes an antigen-binding site that binds to human IL-17A and human IL-17F, comprising: a heavy chain variable region comprising the sequence shown in SEQ ID NO:4 for CDR-H1, the sequence shown in SEQ ID NO:5 for CDR-H2, and the sequence shown in SEQ ID NO:6 for CDR-H3.

[0089] In one embodiment, the antigen binding site to human IL-17A and human IL-17F contains a light chain variable region comprising the sequence shown in SEQ ID NO:7.

[0090] In one embodiment, the antigen binding site to human IL-17A and human IL-17F contains a heavy chain variable region comprising the sequence shown in SEQ ID NO:9.

[0091] In one embodiment, the multispecific antibody lacks an Fc domain and half-life, which is provided by binding to an antigen-binding site on serum albumin.

[0092] In one embodiment, the multispecific antibody comprises the sequence shown in SEQ ID NO:57 or SEQ ID NO:59.

[0093] In one embodiment, the multispecific antibody comprises the sequence shown in SEQ ID NO:61 or SEQ ID NO:63.

[0094] In one embodiment, the multispecific antibody comprises the sequence shown in SEQ ID NO:59 and the sequence shown in SEQ ID NO:63.

[0095] In one embodiment, the multispecific antibody comprises or is composed of the following: a polypeptide chain of formula (I): VH-CH1-(CH2)s-(CH3)tX-(V1)p; and a polypeptide chain of formula (II): VL-CL-Y-V2; wherein: VH represents a heavy chain variable domain; CH1 represents domain 1 of the heavy chain constant region; CH2 represents domain 2 of the heavy chain constant region; CH3 represents domain 3 of the heavy chain constant region; X represents a bond or linker; V1 represents dsscFv, dsFv, or scFv; VL represents a light chain variable domain; CL represents a domain from the light chain constant region, such as Cκ; Y represents a bond or linker; V2 represents dsscFv, dsFv, or scFv; p represents 0 or 1; s represents 0 or 1; t represents 0 or 1; Wherein, when p is 0, X does not exist and when q is 0, Y does not exist; and the polypeptide chain of formula (I) contains a protein A binding domain; and the polypeptide chain of formula (II) does not bind to protein A.

[0096] In one embodiment, when s is 0 and t is 0, the multispecific antibody according to the present invention is provided as a dimer of the following heavy chain and light chain: respectively, of formula (I) and (II), wherein the VH-CH1 portion and the VL-CL portion together form a functional Fab or Fab' fragment.

[0097] In one embodiment, when s is 1 and t is 1, the multispecific antibody according to the present invention is provided as a dimer of two heavy chains and two light chains, respectively, of formulas (I) and (II), wherein the two heavy chains are linked by interchain interactions, particularly at the CH2-CH3 level, and wherein the VH-CH1 portion of each heavy chain and the VL-CL portion of each light chain together form a functional Fab or Fab' fragment. In this embodiment, the two VH-CH1-CH2-CH3 portions and the two VL-CL portions together form a functional full-length antibody. In this embodiment, the full-length antibody may include a functional Fc region.

[0098] VH represents the heavy chain variable field. In one embodiment, the VH system is humanized. In another embodiment, the VH system is completely human.

[0099] VL represents a light chain variable domain. In one embodiment, the VL system is humanized. In another embodiment, the VL system is completely human.

[0100] Generally, VH and VL together form an antigen-binding domain. In one embodiment, VH and VL form a homologous pair.

[0101] As used herein, "homologous pair" refers to a pair of variable domains derived from a single antibody that are produced in vivo, i.e., naturally occurring pairings of variable domains isolated from the host. A homologous pair is therefore a VH and VL pair. In one instance, the homologous pair cooperatively binds to the antigen.

[0102] "Variable region" or "variable domain" as used herein refers to the region in the antibody chain that contains the CDR and the framework (especially the appropriate framework).

[0103] The variable region used in this invention will generally be derived from an antibody, which can be produced by any method known in this art.

[0104] "Derived from" as used herein means, as in this article, the fact that the sequence used or a sequence highly similar to the sequence used is derived from the original genetic material (such as the light or heavy chain of an antibody).

[0105] “Highly similar” as used herein is intended to mean amino acid sequences that are 95% or more similar in length, such as 96, 97, 98 or 99%.

[0106] The variable region used in this invention, as described above for VH and VL, may be from any suitable source and may be, for example, entirely human or humanized.

[0107] In one embodiment, the binding domain formed by VH and VL is specific to the first antigen.

[0108] In one embodiment, the binding domain of V1 is specific for the second antigen.

[0109] In one embodiment, the binding domain of V2 is specific for the third antigen.

[0110] In one embodiment, each of the present VH-VL, V1 and V2 binds to its respective antigen.

[0111] In one embodiment, the CH1 domain is derived from domain 1, which is naturally present in the antibody heavy chain or a derivative thereof. In one embodiment, the CH2 domain is derived from domain 2, which is naturally present in the antibody heavy chain or a derivative thereof. In one embodiment, the CH3 domain is derived from domain 3, which is naturally present in the antibody heavy chain or a derivative thereof.

[0112] In one embodiment, the CL segment in the light chain is a constant κ sequence or a derivative thereof. In one embodiment, the CL segment in the light chain is a constant λ sequence or a derivative thereof.

[0113] As used herein, a derivative of a naturally occurring domain is intended to refer to a location in a naturally occurring sequence where at least one amino acid has been substituted or omitted, for example, to optimize the properties of the domain, such as by eliminating undesirable properties while retaining the characteristics of the domain. In one embodiment, a derivative of a naturally occurring domain comprises two, three, four, five, six, seven, eight, ten, eleven, or twelve amino acid substitutions or omissions compared to a naturally occurring sequence.

[0114] In one embodiment, one or more natural or engineered interchain (i.e., light chain and heavy chain) disulfide bonds are present in the functional Fab or Fab' fragment.

[0115] In one embodiment, a "natural" disulfide bond exists between CH1 and CL in the polypeptide chains of formulas (I) and (II).

[0116] When the CL domain is derived from κ or λ, the natural position for forming the cysteine ​​bond is 214 in human cκ and cλ (Kabat number, 4th edition, 1987).

[0117] The exact position of the disulfide bond forming cysteine ​​in CH1 depends on the specific domain actually used. Thus, for example, in human γ-1, the native position of the disulfide bond is at position 233 (Kabat number, 4th edition, 1987). The positions of the cysteine ​​bonds forming in other human isoforms (such as γ2, 3, 4, IgM, and IgD) are known, for example, position 127 in human IgM, IgE, IgG2, IgG3, and IgG4, and position 128 in the heavy chain of human IgD and IgA2B.

[0118] As needed, disulfide bonds may exist between VH and VL of the polypeptides of Formula I and II.

[0119] In one embodiment, the multispecific antibody according to the invention has a disulfide bond at a position equivalent to or corresponding to the naturally occurring position between CH1 and CL.

[0120] In one embodiment, the constant region comprising CH1 and a constant region such as CL have disulfide bonds in non-naturally occurring positions. This can be engineered into the molecule by introducing cysteine ​​at one or more desired positions into the amino acid chain. This non-natural disulfide bond is a complement or replacement of the natural disulfide bond present between CH1 and CL. The cysteine ​​in the natural position can be replaced by an amino acid that cannot form a disulfide bridge (such as serine).

[0121] The introduction of engineered cysteine ​​can be performed using any method known in this art. These methods include (but are not limited to) PCR extension overlap mutagenesis, site-directed mutagenesis, or cassette mutagenesis (generally, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, 1989; Ausbel et al., Current Protocols in Molecular Biology, Greene Publishing & Wiley-Interscience, NY, 1993). Site-directed mutagenesis kits are commercially available, such as the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA). Cassette mutagenesis can be performed based on Wells et al., 1985, Gene, 34:315-323. Alternatively, mutants can be prepared by whole-genome synthesis via annealing, conjugation, and PCR amplification and selection of overlapping oligonucleotides.

[0122] In one embodiment, the disulfide bond between CH1 and CL is completely absent, for example, the interchain cysteine ​​may be replaced by another amino acid (such as serine). Therefore, in one embodiment, interchain disulfide bonds are absent in the functional Fab segment of the molecule. Disclosures such as WO2005 / 003170, incorporated herein by reference, describe how to provide Fab segments without interchain disulfide bonds.

[0123] Examples of antibody forms used in this invention include attached IgG and attached Fab, wherein the complete IgG or Fab fragment is engineered by attaching at least one additional antigen-binding domain (e.g., one, two, three or four additional antigen-binding domains) (e.g., single-domain antibodies (such as VH or VL, or VHH), scFv, dsscFv, dsFv) to the N-terminus and / or C-terminus of the light chain of the IgG or Fab and, as needed, to the heavy chain of the IgG or Fab, as described in, for example, WO2009 / 040562, WO2010035012, WO2011 / 030107, WO2011 / 061492, WO2011 / 061246 and WO2011 / 086091, all of which are incorporated herein by reference. The addition of IgG, which comprises full-length IgG engineered by adding dsFv to the C-terminus of the light chain (and, if necessary, to the heavy chain), was first disclosed in WO2015 / 197789, which is incorporated herein by reference.

[0124] A preferred antibody form used in this invention comprises a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binding to a therapeutic target and another scFv or dsscFv increasing its half-life by binding, such as albumin). Such antibody fragments are described in International Patent Application Publication No. WO2015 / 197772, which is incorporated herein by reference in its entirety and, in particular, the discussion of antibody fragments.

[0125] V1 represents dsscFv, dsFv or scFv.

[0126] V2 represents dsscFv, dsFv or scFv.

[0127] In one embodiment, when V1 and / or V2 are dsFv or dsscFv, the disulfide bond between the variable domains VH and VL of V1 and / or V2 is between two residues listed below (unless the context otherwise indicates, Kabat numbers are used in the following list). Wherever Kabat numbers are mentioned, 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.

[0128] In one embodiment, the disulfide bond is located at a position selected from the group consisting of: ● VH37 + VL95C, see, for example, Protein Science 6, 781-788, Zhu et al. (1997); ● VH44 + VL100, see, for example, Weatherill et al., Protein Engineering, Design & Selection, 25 (321-329), 2012); ● VH44 + VL105, see, for example, J Biochem. 118, 825-831, Luo et al. (1995); ● VH45 + VL87, see, for example, Protein Science 6, 781-788, Zhu et al. (1997); ● VH55 + VL101, see, for example, FEBS Letters 377, 135-139, Young et al. (1995); ● VH100 + VL50, see, for example, Biochemistry 29 1362-1367 Glockshuber et al. (1990); ● VH100b + VL49; see, for example, Biochemistry 29 1362-1367 Glockshuber et al. (1990); ● VH98 + VL46; see, for example, Protein Science 6, 781-788 Zhu et al. (1997); ● VH101 + VL46; see, for example, Protein Science 6, 781-788 Zhu et al. (1997); ● VH105 + VL43, 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); ● VH106 + VL57, see, for example, FEBS Letters 377 135-139 Young et al. (1995) and the corresponding positions in the variable regions of the molecule.

[0129] In one embodiment, a disulfide bond is formed between positions VH44 and VL100.

[0130] The amino acid pairs listed above are located in positions that facilitate the formation of disulfide bonds by cysteine ​​substitution. Cysteine ​​can be engineered to these desired positions using known techniques. Therefore, in one embodiment, the engineered cysteine ​​according to the invention refers to the location where a naturally occurring residue at a given amino acid position has been substituted by a cysteine ​​residue.

[0131] The introduction of engineered cysteine ​​can be performed using any method known in this art. These methods include (but are not limited to) PCR extension overlap mutagenesis, site-directed mutagenesis, or cassette mutagenesis (generally, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, 1989; Ausbel et al., Current Protocols in Molecular Biology, Greene Publishing & Wiley-Interscience, NY, 1993). Site-directed mutagenesis kits are commercially available, such as the QuikChange® site-directed mutagenesis kit (Stratagen, La Jolla, CA). Cassette mutagenesis can be performed based on Wells et al., 1985, Gene, 34:315-323. Alternatively, mutants can be prepared by whole-genome synthesis via annealing, conjugation, and PCR amplification and selection of overlapping oligonucleotides.

[0132] Therefore, in one embodiment, when V1 and / or V2 is dsFv or dsscFv, the variable domains VH and VL of V1 and / or the variable domains VH and VL of V2 can be connected by a disulfide bond between two cysteine ​​residues, wherein the positions of the pair of cysteine ​​residues are selected from the group consisting of: VH37 and VL95, VH44 and VL100, VH44 and VL105, VH45 and VL87, VH100 and VL50, VH100b and VL49, VH98 and VL46, VH101 and VL46, VH105 and VL43 and VH106 and VL57.

[0133] In one embodiment, when V1 and / or V2 are dsFv or dsscFv, the variable domains VH and VL of V1 and / or the variable domains VH and VL of V2 can be linked by a disulfide bond between two cysteine ​​residues (one in VH and one in VL), which are outside the CDR, wherein the positions of the pair of cysteine ​​residues are selected from the group consisting of: VH37 and VL95, VH44 and VL100, VH44 and VL105, VH45 and VL87, VH100 and VL50, VH98 and VL46, VH105 and VL43 and VH106 and VL57.

[0134] In one embodiment, when V1 is dsFv or dsscFv, the variable domains VH and VL of V1 are connected by a disulfide bond between two engineered cysteine ​​residues (one at position VH44 and the other at VL100). In one embodiment, when V2 is dsFv or dsscFv, the variable domains VH and VL of V2 are connected by a disulfide bond between two engineered cysteine ​​residues (one at position VH44 and the other at VL100).

[0135] In one embodiment, when V1 is dsscFv, dsFv or scFv, the VH domain of V1 is connected to X.

[0136] In one embodiment, when V1 is dsscFv, dsFv or scFv, the VL domain of V1 is connected to X.

[0137] In one embodiment, when V2 is dsscFv, dsFv or scFv, the VH domain of V2 is connected to Y.

[0138] In one embodiment, when V2 is dsscFv, dsFv or scFv, the VL domain of V2 is connected to Y.

[0139] Skilled practitioners will understand that when V1 and / or V2 represent dsFv, the multispecific antibody will contain a third polypeptide encoding the corresponding free VH or VL domain not linked to X or Y. When V1 and V2 are dsFv, the "free variable domain" (i.e., the domain linked to the rest of the polypeptide via disulfide bonds) is the same for both chains. Therefore, although the actual variable domains fused to or linked to the polypeptide via X or Y in each polypeptide chain may differ, the paired free variable domains are generally the same.

[0140] In some embodiments, p is 1. In some embodiments, p is 0.

[0141] In some embodiments, s is 1. In some embodiments, s is 0.

[0142] In some embodiments, t is 1. In some embodiments, t is 0.

[0143] In some embodiments, s is 1 and t is 1. In some embodiments, s is 0 and t is 0.

[0144] In one embodiment, p is 1, q is 1, r is 0, s is 0 and t is 0, and V1 and V2 both represent dsscFv. Therefore, in one state, a multispecific antibody is provided that binds to human IL-13, human IL-17A, and / or human IL-17F, comprising or consisting of the following: a) a polypeptide chain of formula (Ia): VH-CH1-X-V1; and b) a polypeptide chain of formula (IIa): VL-CL-Y-V2; wherein: VH represents a heavy chain variable domain; CH1 represents domain 1 of the heavy chain constant region; X represents a bond or linker; Y represents a bond or linker; V1 represents scFv, dsscFv, or dsFv; VL represents a light chain variable domain; CL represents a domain from the light chain constant region, such as Cκ; V2 represents scFv, dsscFv, or dsFv; wherein at least one of V1 or V2 is dsscFv or dsFv; wherein the polypeptide chain of formula (Ia) contains a protein A binding domain; and wherein the polypeptide chain of formula (IIa) does not bind protein A.

[0145] In this embodiment, V2 does not bind protein A, that is, the scFv, dsscFv, or dsFv of V2 does not contain a protein A binding domain. In one embodiment, V2, that is, the scFv, dsscFv, or dsFv of V2, contains a VH1 domain. In another embodiment, V2, that is, the scFv, dsscFv, or dsFv of V2, contains a VH3 domain that does not bind protein A. In one embodiment, V2, that is, the scFv, dsscFv, or dsFv of V2, contains a VH2 domain. In one embodiment, V2, that is, the scFv, dsscFv, or dsFv of V2, contains a VH4 domain. In one embodiment, V2, that is, the scFv, dsscFv, or dsFv of V2, contains a VH5 domain. In one embodiment, V2, that is, the scFv, dsscFv, or dsFv of V2, contains a VH6 domain. In one embodiment, the polypeptide chain of formula (Ia) contains only one protein A binding domain present in VH or V1. In another embodiment, the polypeptide chain of formula (Ia) contains only one protein A binding domain present in V1. In yet another embodiment, the polypeptide chain of formula (Ia) contains two protein A binding domains present in VH and V1, respectively.

[0146] In another embodiment, p is 0, q is 1, r is 0, s is 1, t is 1, and V2 is dsscFv. Thus, in one state, a multispecific antibody is provided that binds to human IL-13, human IL-17A, and / or human IL-17F, and comprises or consists of the following: a) a polypeptide chain of formula (Ib): VH-CH1-CH2-CH3; and b) a polypeptide chain of formula (IIb): VL-CL-Y-V2; wherein: VH represents a heavy chain variable domain; CH1 represents domain 1 of the heavy chain constant region; CH2 represents domain 2 of the heavy chain constant region; CH3 represents domain 3 of the heavy chain constant region; Y represents a bond or linker; VL represents a light chain variable domain; CL represents a domain from the light chain constant region, such as Cκ; V2 represents dsscFv; wherein the polypeptide chain of formula (Ib) contains a protein A binding domain; and wherein the polypeptide chain of formula (IIb) does not bind protein A.

[0147] In this embodiment, V2 does not bind protein A, that is, the dsscFv of V2 does not contain a protein A binding domain. In one embodiment, V2, that is, the dsscFv of V2, contains a VH1 domain. In another embodiment, V2, that is, the dsscFv of V2, contains a VH3 domain that does not bind protein A. In one embodiment, the polypeptide chain of formula (Ib) contains only one protein A binding domain present in VH or CH2-CH3. In another embodiment, the polypeptide chain of formula (Ib) contains two protein A binding domains present in VH and CH2-CH3, respectively.

[0148] In another embodiment, p is 0, q is 1, r is 0, s is 1, t is 1, and V2 is dsFv. Thus, in one state, a multispecific antibody is provided that binds to human IL-13, human IL-17A, and / or human IL-17F, comprising or consisting of the following: a) a polypeptide chain of formula (Ic): VH-CH1-CH2-CH3; and b) a polypeptide chain of formula (IIc): VL-CL-Y-V2; wherein: VH represents a heavy chain variable domain; CH1 represents domain 1 of the heavy chain constant region; CH2 represents domain 2 of the heavy chain constant region; CH3 represents domain 3 of the heavy chain constant region; Y represents a bond or linker; VL represents a light chain variable domain; CL represents a domain from the light chain constant region, such as Cκ; V2 represents dsFv; wherein the polypeptide chain of formula (Ic) contains a protein A binding domain; and wherein the polypeptide chain of formula (IIc) does not bind protein A.

[0149] In this embodiment, V2, i.e., dsFv of V2, does not bind protein A. In one embodiment, the polypeptide chain of formula (Ic) contains only one protein A binding domain present in VH or CH2-CH3. In another embodiment, the polypeptide chain of formula (Ic) contains two protein A binding domains present in VH and CH2-CH3, respectively.

[0150] In one embodiment of the multispecific antibody of the present invention, VL and VH contain antigen-binding sites that bind to human IL-17A and / or human IL17F, V1 contains an antigen-binding site that binds to human serum albumin, and V2 contains an antigen-binding site that binds to human IL-13.

[0151] In one embodiment, VL includes CDR-L1 of the sequence shown in SEQ ID NO:1, CDR-L2 of the sequence shown in SEQ ID NO:2, and CDR-L3 of the sequence shown in SEQ ID NO:3; VH includes CDR-H1 of the sequence shown in SEQ ID NO:4, CDR-H2 of the sequence shown in SEQ ID NO:5, and CDR-H3 of the sequence shown in SEQ ID NO:6.

[0152] In one embodiment, V1 contains a light chain variable region comprising CDR-L1 of the sequence shown in SEQ ID NO:39, CDR-L2 of the sequence shown in SEQ ID NO:40, and CDR-L3 of the sequence shown in SEQ ID NO:41; and a heavy chain variable region comprising CDR-H1 of the sequence shown in SEQ ID NO:42, CDR-H2 of the sequence shown in SEQ ID NO:43, and CDR-H3 of the sequence shown in SEQ ID NO:44.

[0153] In one embodiment, V2 contains a light chain variable region comprising CDR-L1 of the sequence shown in SEQ ID NO:15, CDR-L2 of the sequence shown in SEQ ID NO:16, and CDR-L3 of the sequence shown in SEQ ID NO:17; and a heavy chain variable region comprising CDR-H1 of the sequence shown in SEQ ID NO:18, CDR-H2 of the sequence shown in SEQ ID NO:19, and CDR-H3 of the sequence shown in SEQ ID NO:20.

[0154] In one embodiment, VL includes the sequence shown in SEQ ID NO:7 and VH includes the sequence shown in SEQ ID NO:9.

[0155] In one embodiment, V1 contains a light chain variable region comprising the sequence shown in SEQ ID NO:45 and a heavy chain variable region comprising the sequence shown in SEQ ID NO:46.

[0156] In one embodiment, V1 contains a light chain variable region comprising the sequence shown in SEQ ID NO:49 and a heavy chain variable region comprising the sequence shown in SEQ ID NO:50.

[0157] In one embodiment, the light chain variable region and the heavy chain variable region of V1 are connected by a connector comprising the sequence shown in SEQ ID NO:68.

[0158] In one embodiment, V1 is either scFv containing the sequence shown in SEQ ID NO:53 or dsscFv containing the sequence shown in SEQ ID NO:55.

[0159] In one embodiment, V2 contains a light chain variable region comprising the sequence shown in SEQ ID NO:27 and a heavy chain variable region comprising the sequence shown in SEQ ID NO:28.

[0160] In one embodiment, V2 includes a light chain variable region comprising the sequence shown in SEQ ID NO:31 and a heavy chain variable region comprising the sequence shown in SEQ ID NO:32.

[0161] In one embodiment, the light chain variable region and the heavy chain variable region of V2 are connected by a connector comprising the sequence shown in SEQ ID NO:66.

[0162] In one embodiment, V2 is either scFv containing the sequence shown in SEQ ID NO:35 or dsscFv containing the sequence shown in SEQ ID NO:37.

[0163] In one embodiment, X is a connector containing the sequence shown in SEQ ID NO:67.

[0164] In one embodiment, Y is a connector containing the sequence shown in SEQ ID NO:65.

[0165] In one embodiment, the polypeptide chain of formula (Ia) comprises the sequence shown in SEQ ID NO:57 or SEQ ID NO:59.

[0166] In one embodiment, the polypeptide chain of formula (IIa) comprises the sequence shown in SEQ ID NO:61 or SEQ ID NO:63.

[0167] In one embodiment, the polypeptide chain of formula (Ia) comprises the sequence shown in SEQ ID NO:59 and the polypeptide chain of formula (IIa) comprises the sequence shown in SEQ ID NO:63.

[0168] It should be understood that one or more amino acid substitutions, additions, and / or deletions can be made to the sequences provided in this invention without significantly altering the antibody's ability to bind to the antigen and neutralize its biological activity. The effect of any amino acid substitution, addition, and / or deletion can be readily tested by those skilled in the art, for example by using the methods described herein, and in particular the methods illustrated in the examples, to determine the inhibition of antigen binding and biological activity.

[0169] Therefore, the present invention provides a multispecific antibody comprising a CDR defined by the sequences shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 15, 16, 17, 18, 19, 20, 39, 40, 41, 42, 43 and 44, wherein one or more of the amino acids in the CDRs have been substituted by another amino acid, such as similar amino acids defined below.

[0170] As used herein, “identity” indicates that the amino acid residues are identical at any particular position in the compared sequences. “Similarity” as used herein indicates that the amino acid residues are of similar type at any particular position in the compared sequences. For example, leucine can substitute for isoleucine or valine. Other amino acids that can often substitute for each other include (but are not limited to): - phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); - lysine, arginine, and histidine (amino acids with basic side chains); - aspartic acid and glutamic acid (amino acids with acidic side chains); - aspartic acid and glutamic acid (amino acids with amide side chains); and - cysteine ​​and methionine (amino acids with sulfur-containing side chains). The degree of consistency and similarity can be easily calculated (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., M Stockton Press, New York, 1991; BLAST™ software is available from NCBI (Altschul, SF et al., 1990, J. Mol. Biol. 215:403-410; Gish, W. & States, DJ). 1993, Nature Genet. 3:266-272. Madden, TL et al., 1996, Meth. Enzymol. 266:131-141; Altschul, SF et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, TL, 1997, Genome Res. 7:649-656).

[0171] In one embodiment, the CDR of the multispecific antibody comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequences shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 15, 16, 17, 18, 19, 20, 39, 40, 41, 42, 43 and 44.

[0172] In one embodiment, VL comprises a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:7 and VH comprises a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:9.

[0173] In one embodiment, V1 contains a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:45 and / or a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:46.

[0174] In one embodiment, V1 contains a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:49 and / or a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:50.

[0175] In one embodiment, the light chain variable region and the heavy chain variable region of V1 are connected by a connector comprising a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:68.

[0176] In one embodiment, V1 is an scFv containing a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:53, or a dsscFv containing a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:55.

[0177] In one embodiment, V2 contains a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:27 and / or a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:28.

[0178] In one embodiment, V2 contains a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:31 and / or a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:32.

[0179] In one embodiment, the light chain variable region and the heavy chain variable region of V2 are connected by a connector comprising a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:66.

[0180] In one embodiment, V2 is an scFv containing a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:35, or a dsscFv containing a sequence having at least 70%, 80%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:37.

[0181] In one embodiment, X is a connector that includes a sequence having at least 70%, 80%, 90%, 95% or 98% consistency or similarity to the sequence shown in SEQ ID NO:67.

[0182] In one embodiment, Y is a connector that includes a sequence having at least 70%, 80%, 90%, 95% or 98% consistency or similarity to the sequence shown in SEQ ID NO:65.

[0183] In one embodiment, the polypeptide chain of formula (Ia) comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence shown in SEQ ID NO:57 or SEQ ID NO:59.

[0184] In one embodiment, the polypeptide chain of formula (IIa) comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence shown in SEQ ID NO: 61 or SEQ ID NO: 63.

[0185] In one embodiment, the polypeptide chain of formula (Ia) comprises a sequence having at least 70%, 80%, 90%, 95%, or 98% similarity to the sequence shown in SEQ ID NO:59, and the polypeptide chain of formula (IIa) comprises a sequence having at least 70%, 80%, 90%, 95%, or 98% similarity to the sequence shown in SEQ ID NO:63. Antigenic determinant

[0186] An antigenic determinant is the antigenic region to which an antibody binds. An antigenic determinant can be defined as a structural or functional antigenic determinant. A functional antigenic determinant is generally a subset of a structural antigenic determinant and contains residues that directly contribute to the affinity of the interaction. An antigenic determinant can also be a configurational antigenic determinant, i.e., containing a nonlinear amino acid. In some embodiments, the antigenic determinant may include determinants of chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoyl or sulfonylurea groups, and in some embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0187] By using routine methods known in this art, it can be easily determined whether an antibody binds to the same antigenic determinant as a reference antibody or competes with a reference antibody for binding. For example, to determine whether a test antibody binds to the same antigenic determinant as the reference antibody of the present invention, the reference antibody is allowed to bind to a protein or peptide under saturation conditions. Next, the ability of the test antibody to bind to a protein or peptide is evaluated. If the test antibody can bind to a protein or peptide after saturation binding with the reference antibody, it can be concluded that the test antibody binds to an antigenic determinant different from that of the reference antibody. On the other hand, if the test antibody cannot bind to a protein or peptide after saturation binding with the reference antibody, the test antibody can bind to the same antigenic determinant as that bound by the reference antibody of the present invention.

[0188] To determine whether an antibody competes with a reference antibody for binding, the binding method described above is performed in two directions. In the first direction, the reference antibody is allowed to bind 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 direction, the test antibody is allowed to bind to the protein / peptide under saturation conditions, and then the binding of the reference antibody to the protein / peptide is evaluated. If, in both directions, only the first (saturated) antibody can bind to the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As will be apparent to those skilled in the art, an antibody competing with the reference antibody may not necessarily bind to the same antigenic determinant as the reference antibody, but the binding of the reference antibody can be spatially blocked by binding to overlapping or adjacent antigenic determinants.

[0189] Two antibodies that competitively inhibit (block) the binding of one antibody to an antigen bind to the same or overlapping antigenic determinants. That is, an excess of 1, 5, 10, 20, or 100 times that of one antibody inhibits the binding of the other antibody by at least 50%, 75%, 90%, or even 99%, as determined in a competitive binding assay (see, for example, Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, two antibodies that have the same antigenic determinant if a mutation in the antigen that reduces or eliminates substantially all amino acids binding to one antibody would reduce or eliminate the binding of the other antibody. Two antibodies that have overlapping antigenic determinants if a mutation that reduces or eliminates some amino acids binding to one antibody would reduce or eliminate the binding of the other antibody.

[0190] Further routine experiments (e.g., peptide mutation and binding assays) may then be performed to confirm whether the observed lack of binding to the test antibody is actually due to binding to the same antigenic determinant as the reference antibody or to steric hindrance (or another phenomenon) causing the observed lack of binding. Such experiments may be performed using ELISA, RIA, surface plasma resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in this technique.

[0191] The antibody may competitively bind to IL-17A or IL17F with a multispecific antibody containing the sequence combination of CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 of SEQ ID NO: 1 / 2 / 3 / 4 / 5 / 6, or bind to the same antigenic determinant as the multispecific antibody.

[0192] The antibody may competitively bind to IL-13 with a multispecific antibody containing the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequence combination of SEQ ID NO: 15 / 16 / 17 / 18 / 19 / 20, or bind to the same antigenic determinant as the multispecific antibody.

[0193] The antibody may competitively bind to serum albumin with a multispecific antibody containing the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequence combination of SEQ ID NO: 39 / 40 / 41 / 42 / 43 / 44, or bind to the same antigenic determinant as the multispecific antibody. Effector molecule

[0194] If desired, the multispecific antibody used in this invention can be bound to one or more effector molecules. It is understood that an effector molecule may comprise a single effector molecule or two or more such molecules linked in such a way that they can be linked to a single portion of the antibody of this invention. Where it is necessary to obtain an antibody fragment linked to an effector molecule, this antibody fragment can be prepared by standard chemical or recombinant DNA procedures, wherein the antibody fragment is linked directly or via a conjugating agent to the effector molecule. Techniques for binding such effector molecules to antibodies are well known in this art (see Hellstrom et al., Controlled Drug Delivery, 2nd ed., Robinson et al., 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 WO 93 / 06231, WO 92 / 22583, WO 89 / 00195, WO 89 / 01476 and WO 03031581. Alternatively, in cases where the effector molecule is a protein or polypeptide, recombinant DNA procedures can be used to achieve ligation, as described in, for example, WO 86 / 01533 and EP 0392745.

[0195] The term effector as used herein includes, for example, antitumor agents, drugs, toxins, bioactive proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (e.g., DNA, RNA and fragments thereof), radionuclides (especially radioiodide), radioisotopes, chelated metals, nanoparticles, and reporter groups (such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy).

[0196] Examples of effector molecules may include cytotoxins or cytotoxic agents, including any agent that is harmful to cells (e.g., kills cells). Examples include combrestatins, dolastatins, epothilones, staurosporin, maytansinoids, spongistatins, rhizoxin, halichondrins, roridins, hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxyanthraquinone. dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues.

[0197] Effective molecules also include (but are not limited to) antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatin(II)(DDP)cisplatin), and anthracyclines. (e.g., daunomycin and doxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mixamycin, antramycin (AMC), calicheamicins or duocarmycins), and antimitotic agents (e.g., vincristine and vinblastine).

[0198] Other effector molecules may include chelating radionuclides such as 111In and 90Y, Lu177, bismuth-213, calix-252, iridium-192 and tungsten-188 / rhenium-188; or drugs such as (but not limited to) alkylphosphocholine, topoisomerase I inhibitors, taxoids and suramin.

[0199] Other effector molecules include proteins, peptides, and enzymes. Enzymes of interest include (but are not limited to) proteases, hydrolases, lyases, isomerases, and transferases. The proteins, polypeptides, and peptides of interest include (but are not limited to) immunoglobulins, toxins (such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin), proteins (such as insulin), tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, or plasminogen activator, thrombotic agents, or anti-angiogenic agents such as angiostatin or endostatin, or biomodifiers such as lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage community-stimulating factor (GM-CSF), granulocyte-macrophage community-stimulating factor (G-CSF), nerve growth factor (NGF), or other growth factors and immunoglobulins.

[0200] Other effector molecules may include detectable substances that can be used, for example, for diagnostic purposes. Examples of detectable substances include various enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron-emitting metals (for positron emission tomography), and non-radioactive paramagnetic metal ions. For information on metal ions that can bind to antibodies for diagnostic purposes, see U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable cofactors include streptavidin, avidin, and biotin; suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, insect luciferin, and jellyfish protein; and suitable radionuclides include 125I, 131I, 111In, and 99Tc.

[0201] In another instance, an effector molecule may increase the in vivo half-life of an antibody and / or decrease its immunogenicity and / or enhance its delivery across the epithelial barrier to the immune system. Examples of suitable effectors of this type include polymers, albumins, albumin-binding proteins, or albumin-binding compounds, such as those described in WO 05 / 117984.

[0202] When the effector molecule is a polymer, it can generally be a synthetic or naturally occurring polymer, such as linear or branched polyalkylene, polyalkylene or polyoxyalkylene polymers that are substituted as needed, or linear or unbranched polysaccharides, such as homopolysaccharides or heteropolysaccharides.

[0203] Specific optional substituents that may be present in the synthetic polymers mentioned above include one or more hydroxyl, methyl, or methoxy groups.

[0204] Specific examples of synthetic polymers include linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof that are substituted as needed, particularly poly(ethylene glycol) that are substituted as needed, such as methoxy poly(ethylene glycol) or derivatives thereof.

[0205] Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or their derivatives.

[0206] The term "derivative" as used herein is intended to include reactive derivatives, such as thiol-selective reactive groups, such as maleimine and the like. The reactive group may be attached to the polymer directly or via a linker segment. It should be understood that residues of this group may, in some cases, form part of the product as a linker group between the antibody segment and the polymer.

[0207] The polymer size can be varied as needed, but is generally within the range of 500 Da to 50,000 Da, for example 5,000 to 40,000 Da, such as 20,000 to 40,000 Da in average molecular weight. The polymer size can be selected, particularly based on the intended use of the product, such as the ability to target certain tissues (e.g., tumors) or to prolong the circulating half-life (see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545 for a review). Thus, for example, in cases where the product is intended to leave the circulation and penetrate tissues, a low molecular weight polymer, such as one with a molecular weight of about 5,000 Da, may be advantageously used. For applications where the product remains in circulation, a higher molecular weight polymer, for example, with a molecular weight in the range of 20,000 Da to 40,000 Da, may be advantageously used.

[0208] Suitable polymers include polyalkylene polymers, such as poly(ethylene glycol), or especially methoxy poly(ethylene glycol) or derivatives thereof, and especially with a molecular weight in the range of about 15,000 Da to about 40,000 Da.

[0209] In one example, the antibody system used in this invention is linked to a poly(ethylene glycol) (PEG) moiety. In a particular example, the antibody is an antibody fragment and the PEG molecule can be linked via any available amino acid side chain or terminal amino acid functional group (e.g., any free amino, imine, thiol, hydroxyl, or carboxyl group) located in the antibody fragment. Such amino acids may be naturally present in the antibody fragment or engineered into the fragment using recombinant DNA methods (see, for example, US 5,219,996; US 5,667,425; WO 98 / 25971). In one example, the antibody molecule of this invention is a modified Fab fragment, wherein the modification involves adding one or more amino acids to the C-terminus of its heavy chain to allow the linking of effector molecules. Suitably, additional amino acids form a modified hinge region containing one or more cysteine ​​residues that can link effector molecules. Multiple sites can be used to link two or more PEG molecules.

[0210] Suitablely, PEG molecules can be covalently linked via a thiol group located on at least one cysteine ​​residue in the antibody fragment. Each polymer molecule linked to the modified antibody fragment can be covalently linked to a sulfur atom on a cysteine ​​residue in the fragment. The covalent bond will generally be a disulfide bond, or more particularly a sulfur-carbon bond. Where the thiol group serves as the linker, a suitably activated effector molecule can be used, such as a thiol-selective derivative, such as maleimide and cysteine ​​derivatives. The activated polymer can be used as a starting material in the preparation of polymer-modified antibody fragments as described above. The activated polymer can be any polymer containing a thiol reactive group, such as an α-halocarboxylic acid or ester, such as iodoacetamide, amides, such as maleimide, vinyl sulfide, or disulfide. These starting materials are commercially available (e.g., from Nektar, formerly known as Shearwater Polymers Inc., Huntsville, AL, USA) or can be prepared from commercially available starting materials using known chemical procedures. Specific PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly known as Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (available from Nektar, formerly known as Shearwater).

[0211] In one embodiment, the antibody is a modified Fab fragment or diFab fragment that has been PEGylated, i.e., has PEG (poly(ethylene glycol)) covalently bound thereto, for example, according to the methods disclosed in EP 0948544 or EP 1090037. [See also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed.), Plenum Press, New York; "Poly(ethyleneglycol) Chemistry 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, PEG is linked to cysteine ​​in the hinge region. In another example, the PEG-modified Fab fragment has a maleimine group covalently linked to a single thiol group in the modified hinge region. Lysine residues may be covalently linked to the maleimine group, and each amino group on the lysine residue may be linked to a methoxylated poly(ethylene glycol) polymer with a molecular weight of about 20,000 Da. The total molecular weight of PEG linked to the Fab fragment can therefore be about 40,000 Da.

[0212] In one embodiment, the multispecific antibody is not linked to an effector molecule. The generation of multispecific antibodies...

[0213] Various methods exist for generating multispecific (especially bispecific) antibodies. Morrison et al. (Coloma and Morrison 1997, Nat Biotechnol. 15, 159-163) described the fusion of a single-chain variable fragment (scFv) to a whole antibody, such as IgG. Schoonjans et al., 2000, Journal of Immunology, 165, 7050-7057, described the fusion of scFv to an antibody Fab fragment. WO2015 / 197772 describes the fusion of a disulfide-stabilized scFv (dsscFv) to a Fab fragment.

[0214] The standard approach described in the prior art involves expressing at least two polypeptides in a host cell, each polypeptide encoding a heavy chain (HC) or a light chain (LC) of a whole antibody or an antigen-binding fragment thereof (e.g., Fab, to which another antigen-binding fragment of the antibody may be fused at the N-terminus and / or C-terminus of the heavy chain and / or light chain). When attempting to generate such a multispecific antibody by expressing two (one light chain and one heavy chain to form an additional Fab) or four polypeptides (two light chains and two heavy chains to form an additional IgG), it is generally necessary to express an excess of light chain compared to the heavy chain to ensure the correct folding of the heavy chain after its assembly with the light chain. In particular, CH1 (domain 1 of the heavy chain constant region) is prevented from folding by a BIP protein, which can be replaced by a corresponding LC; therefore, the correct folding of CH1 / HC depends on the availability of its corresponding LC (Lee et al., 1999, Molecular Biology of the Cell, Vol. 10, 2209–2219).

[0215] We have observed that methods for producing multispecific antibodies can result in the production of an excess of light chains compared to the heavy chains (which are retained in the host cell harvest), and that the excess of light chains tends to form dimer complexes (or "LC dimers"), which are present as byproducts of the production process along with the desired multispecific antibody (especially monomers), and therefore require purification to remove.

[0216] Importantly, no technical problems have yet been identified related to the formation of light chain dimers when fused to additional antigen-binding fragments at the N-terminus and / or C-terminus, and commonly used analytical methods are not yet able to detect and quantify such additional LC dimers in heterogeneous products generated during the production process. This can lead to significant bias when estimating product amounts using standard analytical methods.

[0217] Therefore, there is a need to improve the multispecific antibodies and their production methods, which allows for easy and efficient separation and removal of the additional LC dimer in the earliest step of the production process, and thus increases the yield of the protein of interest (which is a multispecific antibody, especially in its monomeric form) for use in therapy.

[0218] The multispecific antibody of the present invention has been engineered to provide an improved multispecific antibody with equivalent functionality and stability, while increasing the yield of "multispecific antibody" material (especially monomer) obtained after purification, especially after one-step purification including protein A affinity chromatography.

[0219] Advantageously, the multispecific antibody of the present invention can be purified more efficiently using a modified purification method compared to methods commonly used in the prior art. Notably, this modified method includes fewer steps, which is cost and time efficient on an industrial scale. In particular, the multispecific antibody of the present invention maximizes the amount of the protein of interest (i.e., the correct multispecific antibody form) obtained after a one-step purification method including protein A affinity chromatography, thereby simultaneously purifying the multispecific antibody of interest and removing the additional LC dimer. Advantageously, the method for generating and purifying the multispecific antibody of the present invention does not require a separate purification step to capture excess free, unbound light chains, particularly the additional LC dimer. Protein A

[0220] Protein A is a 42 kDa surface protein originally discovered in the cell wall of the bacterium Staphylococcus aureus. Protein A has been widely used for the detection, quantification, and purification of immunoglobulins. It has been reported that protein A binds to the Fab moiety of VH3 family antibodies and the Fcγ region (between the CH2 and CH3 domains) of the constant region of IgG. The crystal structure of the complex formed by protein A and Fab has been described, for example, in Graille et al., 2000, PNAS, 97(10): 5399–5404. In the context of this invention, protein A encompasses native protein A and any variants or derivatives thereof, to the extent that the variants or derivatives of protein A maintain their ability to bind the VH3 domain and / or the Fcγ domain.

[0221] The polypeptide chain of formula (I) of the present invention includes a protein A binding domain. In one embodiment, the polypeptide chain of formula (I) includes one, two or three protein A binding domains.

[0222] "Protein A binding domain" as used herein is intended to refer to a binding domain that specifically binds to protein A. A protein A binding domain may refer to the binding of protein A, that is, the VH3 domain containing the protein A binding interface or a portion thereof. This portion of the VH3 domain binding protein A does not contain the CDR of the VH3 domain, that is, the protein A binding interface of the VH3 does not involve a CDR; therefore, it should be understood that the protein A binding domain does not compete with the antigen binding domain as disclosed in this application.

[0223] In one embodiment, the polypeptide chain of formula (I) includes a protein A binding domain present in VH and / or CH2-CH3 and / or V1. In one embodiment, the polypeptide chain of formula (I) includes one, two, or three protein A binding domains present in VH and / or CH2-CH3 and / or V1. In one embodiment, the polypeptide chain of formula (I) includes only one protein A binding domain present in VH or V1. In one embodiment, s is 0, t is 0, and the polypeptide chain of formula (I) includes only one protein A binding domain present in VH. In one embodiment, s is 0, t is 0, p is 0, and the polypeptide chain of formula (I) includes only one protein A binding domain present in VH. In one embodiment, the polypeptide chain of formula (I) includes only one protein A binding domain present in V1. In one embodiment, s is 0, t is 0, p is 1, and the polypeptide chain of formula (I) contains only one protein A binding domain present in V1.

[0224] In one embodiment, the polypeptide chain of formula (I) comprises two protein A binding domains. In one embodiment, the polypeptide chain of formula (I) comprises two protein A binding domains respectively located in VH and CH2-CH3. In another embodiment, the polypeptide chain of formula (I) comprises two protein A binding domains respectively located in VH and V1. In yet another embodiment, the polypeptide chain of formula (I) comprises two protein A binding domains respectively located in CH2-CH3 and V1.

[0225] In one embodiment, the polypeptide chain of formula (I) contains three protein A binding domains, each of which is present in VH, CH2-CH3 and V1.

[0226] Native protein A can specifically interact with the Fcγ region in the constant region of IgG. More specifically, protein A can interact with the binding domain between CH2 and CH3. In one embodiment, when s is 1 and t is 1, both CH2 and CH3 are naturally occurring domains of the IgG class.

[0227] In some embodiments, the protein A binding domain comprises or consists of a VH3 domain that binds protein A or a variant thereof. In some embodiments, the protein A binding domain comprises or consists of a naturally occurring VH3 domain. In some embodiments, the variant of the VH3 domain that binds protein A is a variant of a naturally occurring VH3 domain that cannot bind protein A.

[0228] The polypeptide chain of formula (II) of the present invention does not bind to protein A. In one embodiment, the binding domain of V2 does not bind to protein A.

[0229] In some embodiments, V2 comprises VH1 and / or VH2 and / or VH4 and / or VH5 and / or VH6 and does not comprise a VH3 domain. In some embodiments, V2 comprises or consists of a VH3 domain that does not bind protein A or a variant thereof. In some embodiments, V2 comprises or consists of a naturally occurring VH3 domain that does not bind protein A. In some embodiments, a variant of the VH3 domain that does not bind protein A is a variant of a naturally occurring VH3 domain that is capable of binding protein A.

[0230] The human VH3 germline gene and VH3 domain (or fragment) have been clearly characterized. Many naturally occurring VH3 domains are capable of binding protein A, but some naturally occurring VH3 domains are not capable of binding protein A (see Roben et al., 1995, J Immunol.; 154(12):6437-6445).

[0231] The VH3 domain used in this invention can be obtained by several methods. In one embodiment, the VH3 domain used in this invention is a naturally occurring VH3 domain, selected for its ability or inability to bind protein A, depending on its position in the polypeptide (I) and / or (II) of this invention. For example, an antibody set can be generated against the antigen of interest by immunizing a non-human animal, then humanized, and the humanized antibody can be screened and selected based on its ability or inability to bind protein A, for example, via a protein A affinity column. Alternatively, presentation techniques (e.g., phage presentation, yeast presentation, ribosome presentation, bacterial presentation, mammalian cell surface presentation, mRNA presentation, DNA presentation) can be used to screen antibody libraries and select antibodies containing a VH3 domain that binds (especially via a protein A binding interface not involving CDR) protein A, or does not bind protein A.

[0232] Alternatively, the VH3 domain used in this invention is a variant of naturally occurring VH3. In one embodiment, the VH3 variant comprises a sequence of naturally occurring VH3 capable of binding protein A, and further comprises at least one amino acid mutation that eliminates its ability to bind protein A. In one embodiment, the VH3 variant that binds protein A comprises a sequence of naturally occurring VH3 that cannot bind protein A, and further comprises at least one amino acid mutation. In this embodiment, the mutation is responsible for the VH3 domain gaining the ability to bind protein A, that is, the mutation helps to generate a non-naturally occurring protein A binding domain.

[0233] In one embodiment, the VH3 variant comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve amino acid mutations. In one embodiment, the VH3 variant comprises mutations at positions 15, 17, 19, 57, 59, 64, 65, 66, 68, 70, 81, or 82 on VH3, numbered according to Kabat and as defined, for example, Graille et al., 2000, PNAS, 97(10):5399–5404. Mutations may be substitutions, deletions, or insertions. In one embodiment, the VH3 variant comprises substitutions at positions 15, 17, 19, 57, 59, 64, 65, 66, 68, 70, 81, or 82 on VH3, numbered according to Kabat.

[0234] Naturally occurring VH1, VH2, VH4, VH5, and VH6 unbinding protein A. In one embodiment, the VH domain of unbinding protein A is VH1. In one embodiment, the VH domain of unbinding protein A is VH2. In one embodiment, the VH domain of unbinding protein A is VH4. In one embodiment, the VH domain of unbinding protein A is VH5. In one embodiment, the VH domain of unbinding protein A is VH6. Pharmaceutical composition, dosage, and dosing regimen.

[0235] The multispecific antibodies of the present invention can be provided as pharmaceutical compositions. Pharmaceutical compositions will generally be sterile and will generally contain pharmaceutically acceptable carriers and / or adjuvants. Pharmaceutical compositions of the present invention may additionally contain pharmaceutically acceptable adjuvants and / or carriers.

[0236] As used herein, "medically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption retardants, and the like. Carriers may be suitable for non-enteral routes of administration (e.g., intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal, or other non-enteral routes), such as by injection or infusion. Alternatively, carriers may be suitable for non-enteral administration, such as local, epidermal, or mucosal routes. Carriers may be suitable for oral administration. Depending on the route of administration, modifiers may be coated onto the material to protect the compound from acids and other natural conditions that can inactivate the compound.

[0237] The pharmaceutical compositions of the present invention may comprise one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" means a salt that retains the desired biological activity of the parent compound without conferring any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0238] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, buffered water, and saline. Other examples of carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). In many cases, it is desirable to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride.

[0239] Under the conditions of manufacture and storage, therapeutic compositions must generally be sterile and stable. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0240] The pharmaceutical composition of the present invention may contain additional active ingredients.

[0241] Kits containing the antibodies or modulators of the present invention and instructions for use are also within the scope of the present invention. Kits may further contain one or more additional reagents, such as the additional therapeutic or preventative agents discussed above.

[0242] The modulators and / or antibodies or their formulations or compositions of the present invention may be administered for preventive treatment and / or therapeutic treatment.

[0243] In therapeutic applications, a compound is administered to an individual suffering from a condition or symptom as described above in an amount sufficient to cure, alleviate, or partially prevent the condition or one or more of its symptoms. Such therapeutic treatment may result in a reduction in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this is defined as a "therapeuticly effective amount".

[0244] In preventive applications, the formulation is administered to an individual who is in or at risk of the condition as described above in an amount sufficient to prevent or mitigate the symptoms or one or more of their subsequent effects. The amount sufficient to achieve this is defined as the "preventive effective amount". The effective amount for each purpose will depend on the severity of the disease or injury and the individual's weight and general condition.

[0245] The individuals to be given can be humans or non-human animals. The term "non-human animals" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Giving to humans is typical.

[0246] The antibodies / modulators or pharmaceutical compositions of the present invention may be administered via one or more of the various methods known in the art through one or more routes of administration. As those skilled in the art will understand, the route of administration and / or mode will vary depending on the desired outcome. Examples of routes of administration for the compounds or pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal, or other non-enteric routes of administration, such as by injection or infusion. The phrase "non-enteric administration" as used herein means, other than enteral and local administration, a mode of administration typically administered by injection. Alternatively, the antibodies / modulators or pharmaceutical compositions of the present invention may be administered via non-enteric routes (such as local, epidermal, or mucosal administration). The antibodies / modulators or pharmaceutical compositions of the present invention may be administered orally.

[0247] The appropriate dosage of the antibody / modulator or pharmaceutical composition of the present invention can be determined by a skilled medical practitioner. The actual dosage of the active ingredient in the pharmaceutical composition of the present invention can be modified to achieve a non-toxic amount of the active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and administration mode. The selected dosage will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention used, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors known in medical technology.

[0248] An appropriate dose may be, for example, from about 0.01 µg / kg to about 1000 mg / kg of the patient's body weight to be treated, typically in the range of about 0.1 µg / kg to about 100 mg / kg of the patient's body weight to be treated. For example, an appropriate dose may be from about 1 µg / kg to about 10 mg / kg body weight / day or from about 10 μg / kg to about 5 mg / kg body weight / day.

[0249] The dosing regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose can be administered, several fractionated doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by an emergency situation in the treatment. As used herein, a unit dosage form refers to a physically discrete unit suitable as a unit dose for the individual to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect associated with the desired pharmaceutical delivery system.

[0250] Administration may be a single or multiple dose. Multiple doses may be administered via the same or different routes and at the same or different locations. Alternatively, the dose may be administered via a sustained-release formulation, in which case frequent administration is not required. The dose and frequency may vary depending on the half-life of the antagonist in the patient and the required duration of treatment.

[0251] As described above, the modulator / antibody or pharmaceutical composition of the present invention may be co-administered with one or more other therapeutic agents.

[0252] The combined administration of two or more agents can be achieved in a variety of different ways. Both can be administered as a single composition together, or as a single composition as part of a combination therapy. For example, one can be administered before, after, or simultaneously with the other. Therapeutic indications

[0253] The antibodies of the present invention can be used to treat, prevent or improve any condition associated with IL-13 and / or IL-17A and / or IL-17F activity; for example, any condition that is wholly or partially derived from signaling through IL-13, IL17A and / or IL17F receptors.

[0254] These diseases include primary and metastatic cancers, including breast cancer, colon cancer, rectal cancer, lung cancer, oropharyngeal cancer, laryngopharyngeal cancer, esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, gallbladder cancer and bile duct cancer, small bowel cancer, urethral cancer (including kidney cancer, bladder cancer and urothelial carcinoma), female reproductive tract cancers (including cervical cancer, uterine cancer and ovarian cancer, as well as choriocarcinoma and gestational trophoblastic disease), male reproductive tract cancers (prostate cancer, seminal vesicle cancer, testicular cancer and germ cell tumors), endocrine gland cancers (including thyroid cancer, adrenal cancer and pituitary adenocarcinoma), and skin cancer, as well as hemangioma, melanoma, sarcoma (including those arising from bone and soft tissue and Kaposi's sarcoma). Sarcoma; tumors of the brain, nerves, eyes, and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, schwannomas, and meningiomas); solid tumors originating from hematopoietic malignancies (such as leukemia), and lymphomas (Hodgkin's lymphomas and non-Hodgkin's lymphomas), rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis. Arthritis, psoriatic arthritis, reactive arthritis, spondylarthropathy, systemic lupus erythematosus, ulcerative colitis, inflammatory bowel disease, insulin-dependent diabetes mellitus, thyroiditis, allergic diseases, psoriasis, dermatitis, scleroderma, graft-versus-host disease, organ transplant rejection, acute or chronic immune diseases related to organ transplantation, sarcomatoid diseases, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the Kidneys), chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignant diseases, heart failure, Addison's disease.Diseases including: sporadic type I polygonal hypoglycemia and sporadic and type II polygonal hypoglycemia, Schmidt's syndrome, adult (acute) respiratory distress syndrome, alopecia areata, alopecia areata, joint disorders, Reiter's disease, psoriatic arthritis, ulcerative colitis arthritis, enteropathic synovitis, joint disorders associated with chlamydia, yersinia, and salmonella, atherosclerosis, atopic hypersensitivity, autoimmune bullous diseases, pemphigus vulgaris, phyllodes disease, bullous pemphigoid, linear IgA disease, autoimmune hemolytic anemia, Coombs-positive hemolytic anemia, acquired pernicious anemia, juvenile pernicious anemia, myalgic encephalitis / chronic fatigue syndrome (Royal Free) Diseases, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiency-related diseases, hepatitis B, hepatitis C, common variable immunodeficiency (common variable hypogammaglobulinemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosis alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonia, interstitial lung disease associated with connective tissue diseases, lung disease associated with mixed connective tissue diseases, interstitial lung disease associated with systemic sclerosis, interstitial lung disease associated with rheumatoid arthritis, lung disease associated with systemic lupus erythematosus, lung disease associated with dermatomyositis / polymyositis, and Sjogren's disease associated lung disease. Diseases, ankylosing spondylitis-related lung disease, vasculitis-associated lung disease, hemoglobinosis-associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation-induced fibrosis, obstructive bronchiolitis, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, post-infectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (typical autoimmune or lupus-like hepatitis), type 2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediators 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 type 1, psoriasis type 2, idiopathic leukopenia, autoimmune neutropenia, NOS nephropathy, glomerulonephritides, renal microvasculitis, Lyme disease.Diseases, discoid lupus erythematosus, idiopathic or NOS-type male infertility, sperm autoimmune disease, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestations of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren's syndrome, Takayasu's disease / arteritis, autoimmune thrombocytopenic purpura, idiopathic thrombocytopenic purpura, autoimmune thyroid disease, hyperthyroidism, goiter-related autoimmune hypothyroidism (Hashimoto's disease) Diseases including: atrophic autoimmune hypothyroidism, primary myxedema, lens uveitis, primary vasculitis, leukoplakia, acute liver disease, chronic liver disease, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis, idiopathic liver disease, drug-induced hepatitis, non-alcoholic steatohepatitis, allergies, group B streptococcal (GBS) infection, mental disorders, depression, schizophrenia, Th2 and Th1 mediated diseases, acute and chronic pain, different forms of pain, cancer, lung cancer, breast cancer, stomach cancer, bladder cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, hematopoietic malignancies, leukemia, lymphoma, abetalipoprotemia, cyanosis of the hands and feet, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (AL). L), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinoma, ectopic atrial beats, AIDS-related dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis (including seasonal allergic rhinitis), non-allergic rhinitis, allergy to transplant, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis (ALS), anemia, angina pectoris, anterior horn cell degeneration, anti-CD3 therapy, antiphospholipid syndrome, anti-receptor allergic reaction, aortic and peripheral aortic aneurysm, 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 transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma.Lymphoma, burns, arrhythmia, cardiac stunning syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation, cartilage graft rejection, cerebellar cortical degeneration, cerebellar disorders, disordered or multifocal atrial tachycardia, chemotherapy-related conditions, chronic myeloid leukemia (CML), chronic alcoholism, chronic inflammatory pathology, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylosis, colorectal cancer, congestive heart failure, conjunctivitis, contact dermatitis, pulmonary heart disease, coronary artery disease, Creutzfeldt-Jakob disease, culture-negative sepsis, cystic fibrosis, cytokine therapy-related conditions, boxer's dementia, demyelinating diseases, dengue hemorrhagic fever, dermatitis, skin disease symptoms, diabetes mellitus. Mellitus, diabetic arteriosclerosis, diffuse Lewy body disease, dilated congestive cardiomyopathy, basal ganglia disorders, Down's syndrome in middle age, drug-induced movement disorders induced by blocking CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrine disorders, epiglottitis, epstein-barr virus infection, acropause, extrapyramidal and cerebellar disorders, familial hemophagocytic lymphohistiocytosis, thymus transplant rejection, Friedreich's ataxia, functional peripheral artery disease, fungal sepsis, gas gangrene, gastric ulcer, glomerulonephritis, transplant rejection of any organ or tissue, gram-negative sepsis, gram-positive sepsis. Sepsis, granulomas caused by intracellular organisms, hairy cell leukemia, Hallervorden-Spatz disease, Hallervorden-Spatz disease, Hashimoto's thyroiditis, hay fever, heart transplant rejection, hemochromatosis, hemodialysis, hemolytic uremic syndrome / thrombolytic thrombocytopenic purpura, bleeding, hepatitis A, His bundle arrhythmias, HIV infection / HIV neuropathy, Hodgkin's disease.Diseases, hyperactivity disorder, allergic reactions, allergic pneumonia, hypertension, hypoactivity disorder, hypothalamic-pituitary-adrenal axis assessment, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, asthenia, infantile spinal muscular atrophy, aortic inflammation, influenza A, ionizing radiation exposure, iridocyclitis / uveitis / optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma. Sarcoma, kidney transplant rejection, Legionella, Leishmaniasis, Leprosy, Corticospinal disorders, lipedema, liver transplant rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcal septicemia, metabolic / idiopathic diseases, migraine, mitochondrial multisystemic disease, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple system degeneration (Mencel Dejerine, Thomas Shi-Drager, and Machado-Joseph), Mycobacterium avium intracellulare, Mycobacterium tuberculosis. Tuberculosis, myelodyplasia syndrome, myocardial infarction, myocardial ischemia, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephropathy, neurodegenerative diseases, neuromuscular atrophy, neutropenia, non-Hodgkin's lymphoma, abdominal aorta and its branches obstruction, obstructive arterial disease, OkT3 therapy, orchitis / epileptic orchitis, orchitis / vasectomy reversal, organ enlargement, osteoporosis, pancreatic transplant rejection, pancreatic cancer, paraneoplastic syndrome / malignant hypercalcemia, parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, peripheral arteriosclerosis, peripheral vascular disease, peritonitis, pernicious anemia, Pneumocystis carinii pneumonia Pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organ enlargement, endocrine disorders, monoclonal immunoglobulinemia and skin changes syndrome), post-perfusion syndrome, post-pump syndrome, post-myocardial infarction syndrome, preeclampsia, progressive supranuclear palsy, essential pulmonary hypertension, radiotherapy, Raynaud's phenomenon and related diseases, Raynaud's disease, Refsum's diseaseDiseases including: regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcoma, senile chorea, senile dementia of Lewy body type, seronegative arthropathy, shock, sickle cell anemia, skin allogeneic transplant rejection, skin change syndrome, small bowel transplant rejection, solid tumors, specific arrhythmias, spinal ataxia, spinocerebellar degeneration, streptococcal myositis, cerebellar structural lesions, subacute sclerosing panencephalitis, syncope, cardiovascular syphilis, systemic anaphylaxis, systemic inflammatory response syndrome, systemic juvenile rheumatoid arthritis, T-cell or FAB. Allergic reactions (ALL), thromboangiitis obliterans, thrombocytopenia, toxicity, transplantation, trauma / hemorrhagic reactions, type III allergic reactions, type IV allergic reactions, unstable angina, uremia, sepsis, valvular heart disease, varicose veins, vasculitis, venous disease, venous thrombosis, ventricular fibrillation, viral and fungal infections, viral encephalitis / aseptic meningitis, virus-associated hemophagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or tissue, acute coronary syndrome, acute idiopathic polyneuritis, acute inflammatory demyelinating polyradiculoneuropathy, acute ischemia, adult Still's disease. Diseases, anaphylaxis, antiphospholipid syndrome, aplastic anemia, atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmune diseases associated with streptococcal infection, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarian failure, blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular disease, catastrophic antiphospholipid syndrome, celiac disease, cervical spondylosis, chronic ischemia, cicatricial pemphigoid, clinical isolated syndrome (CIS) with risk of multiple sclerosis, childhood psychiatric disorders, dacryocystitis, dermatomyositis, diabetic retinopathy, disc herniation. Herniation, intervertebral disc prolapse, drug-induced immune hemolytic anemia, endometriosis, endophthalmitis, episcleritis, erythema multiforme, severe erythema multiforme, pemphigoid of pregnancy, Guillain-Barré syndrome (GBS), Hughes syndrome, idiopathic Parkinson's disease.Diseases including: idiopathic interstitial pneumonia, IgE-mediated allergies, immune hemolytic anemia, inclusion body myositis, infectious ocular inflammatory diseases, inflammatory demyelinating diseases, inflammatory heart diseases, inflammatory kidney diseases, IPF / UIP, iritis, keratitis, keratojuntivitis sicca, Kussmaul disease or Kussmaul-Meier disease, Landry's paralysis, Langerhans cell histiocytosis, livetioretinopathy, macular degeneration, microscopic polyangiitis, and Morbus syndrome. Bechterev, motor neuron disease, mucosal pemphigoid, multiple organ failure, myasthenia gravis, myelodylocytysis, myocarditis, radiculopathy, neuropathy, non-A, non-B hepatitis, optic neuritis, osteolysis, oligoarticular JRA, peripheral arterial occlusive disease (PAOD), peripheral vascular disease (PVD), peripheral arterial disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, canker, polyarticular JRA, polyendocrine deficiency syndrome, polymyositis, polymyalgia rheumatica (PMR), primary Parkinson's disease, prostatitis, pure red blood cell dysplasia, primary adrenal insufficiency, recurrent neuromyelitis optica, restenosis, rheumatic heart disease, sapho (synovitis, acne, impetigo, osteophyte and osteitis). Secondary amyloidosis, shock lung, scleritis, sciatica, secondary adrenal insufficiency, polysiloxane-associated connective tissue disease, Sneddon-Wilkinson dermatosis, ankylosing spondylitis, Stevens-Johnson syndrome (SJS), temporal arteritis, toxoplasmosis retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumor necrosis factor receptor), type 1 hypersensitivity reaction, type 2 diabetes mellitus, urticaria, common interstitial pneumonia (UIP), vasculitis, vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration or wound healing disorders, aspirin-sensitive asthma. Asthma, atopic asthma, chronic hand eczema, allergic bronchopulmonary aspergillosis, celiac diseaseDiseases including: Churg-Strauss syndrome (periarteritis nodosa plus atopy), eosinophilic myalgia syndrome, hypereosinophilic syndrome, edema (including episodic angioedema), helminth infections, onchocercal dermatitis, eosinophilic gastrointestinal disorders, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis, eosinophilic colitis, nasal micropolyposis and polyposis, food allergies, aspirin intolerance, and obstructive sleep apnea. apnoea), chronic asthma, Crohn's disease and endocardial myocardial fibrosis, cancer (such as glioblastoma (such as multiple glioblastoma, non-Hodgkin's lymphoma (NHL)), fibrosis, inflammatory bowel disease, pulmonary fibrosis (including idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis secondary to sclerosis), COPD and liver fibrosis.

[0255] The multispecific antibodies of the present invention are particularly useful for treating or preventing atopic dermatitis, chronic hand eczema, nasal micropolyps or polyposis, food allergies, or eosinophilic esophagitis. Therefore, in one embodiment, the multispecific antibody or pharmaceutical composition of the present invention is provided in a method of treating a human or animal body by means of a therapy. In one embodiment, the multispecific antibody or pharmaceutical composition is provided in a method of treating atopic dermatitis, chronic hand eczema, nasal micropolyps or polyposis, food allergies, or eosinophilic esophagitis. In one embodiment, the present invention provides a method of treating or preventing atopic dermatitis, chronic hand eczema, nasal micropolyps or polyposis, food allergies, or eosinophilic esophagitis, the method comprising administering a therapeutically effective amount of the multispecific antibody or pharmaceutical composition to a patient in need.

[0256] The following examples illustrate the present invention. Example 1. Production and selection of therapeutic anti-IL-13 antibody CA650

[0257] Rats were immunized with purified human IL-13 (Peprotech) or rat fibroblasts expressing human IL-13 (approximately 1 μg / ml in culture supernatant), or in some cases, a combination of both. Animals were sacrificed after 3 to 6 injections, and PBMCs, spleen, bone marrow, and lymph nodes were harvested. Serum binding to human IL-13 was monitored by ELISA, and the ability of serum to neutralize hIL-13 was also monitored by the HEK-293 IL-13R-STAT-6 reporting cell assay (HEK-Blue assay, Invivogen).

[0258] In the Applied Biosystems FMAT assay, B cell cultures were set up and the ability of the supernatant to bind hIL-13 was first screened in a bead-based assay. This was a homogenization assay using biotinylated human IL-13 and a goat anti-rat Fc-Cy5 conjugate coated on streptavidin beads as indicators. Positive results from this assay were then subjected to the HEK-293 IL-13R-STAT-6 reporter cell assay (HEK-Blue assay, Invivogen) to identify the neutralizing agent. The neutralized supernatant was then profiled in Biacore to estimate the off-rate and characterize the neutralization mechanism. Neutralization was classified into bin 1 or bin 2. Bin 1 represents an antibody that binds to human IL-13 and prevents the binding of IL-13Rα1, and thus also blocks the binding of IL-4R. Bin 1 antibodies also prevent IL-13 from binding to IL-13Rα2. Cell 2 represents an antibody that binds to hIL-13, thereby allowing binding to IL-13Rα1 but preventing IL-4R recruitment into the complex. We selected an antibody that acts via Cell 1.

[0259] Approximately 7500 IL-13-specific positives were identified in the primary FMAT screening of a total of 27 x 100-plate SLAM assays. Neutralization was confirmed in 800 wells by the HEK-blue assay. 170 wells exhibited the desired Biacore profile, indicating a dissociation rate of < 5 x 10⁻⁴ s⁻¹ for the V region antibody. Variable region selection was attempted from these 170 wells, and 160 wells successfully generated fluorescent lesions. Following reverse transcription (RT)-PCR, 100 wells yielded heavy and light chain variable region gene pairs. These V region genes were selected as full-length mouse IgG1 antibodies and re-expressed in the HEK-293 transient expression system. Sequence analysis revealed 27 unique anti-human IL-13 antibody families. Then, in cell-based assays, the ability of these recombinant antibodies to block recombinant hIL-13 (derived from E. coli and mammals), recombinant variant hIL-13 (R130Q) (derived from E. coli), natural wild-type and variant hIL-13 (derived from human donors), and cynomolgus monkey IL-13 (derived from mammals) was retested. The ability of the recombinant antibodies to bind variant human IL-13 (R130Q) and cynomolgus monkey IL-13 was also tested in Biacore. Following this characterization analysis, antibody families meeting our criteria, i.e., sub-100 pM antibodies, were selected, exhibiting the least decrease in titer and affinity against all human and cynomolgus monkey IL-13 formulations.

[0260] Based on the neutralizing titer, affinity, and donor content in the humanized graft (see below), humanized CA650 was selected for further progress. Example 2. Humanization of antibody CA650

[0261] Antibody 650 was humanized by grafting a CDR from the rat V region onto the framework of the human germline antibody V region. To restore antibody activity, many framework residues from the rat V region were also retained in the humanized sequence. These residues were selected using a protocol outlined by Adair et al. (1991) (Humanised antibodies. WO91 / 09967). Figure 1 shows the alignment of the rat antibody (donor) V region sequence with the human germline (recipient) V region sequence, and the designed humanized sequence. (Figure 1(A) Light graft 650 and Figure 1(B) Heavy graft 650). The CDR grafted from the donor to the recipient sequence was defined by Kabat (Kabat et al., 1987), except for CDR-H1, which was defined using the combination Chothia / Kabat (see Adair et al., 1991 Humanised antibodies. WO 91 / 09967).

[0262] Genes encoding the initial V region sequence were designed and constructed by Entelechon GmbH using an automated synthesis method, and grafted forms gL8 and gH9 were generated by site-directed oligonucleotide mutagenesis. The gL8 sequence was then selected and colonized into the UCB Celltech human light chain expression vector pVhCK, which contains DNA encoding the human C-κ constant region (Km3 allotype). The gH9 sequence was then selected and colonized into pVhg1Fab, which contains DNA encoding the human heavy chain γ-1 CH1 constant region.

[0263] The human V region IGKV1-39 plus the JK2 J region (International Immunogenetics Information System® IMGT, http: / / www.imgt.org) was selected as the receptor for the CDR of the antibody 650 light chain. All light chain framework residues in the grafted gL8 were derived from human germline genes, except for residues 58 and 71 (encoded according to Kabat), of which donor residues isoleucine (I58) and tyrosine (Y71) were retained, respectively. The retention of residues I58 and Y71 is crucial for the intact titer of the humanized antibody.

[0264] The human V region IGHV1-69 plus the JH4 J region (IMGT, http: / / www.imgt.org) was selected as the receptor for the heavy chain CDR of antibody 650. All heavy chain framework residues in the grafted gH9 were derived from human germline genes, except for residues 67, 69, and 71 (encoded according to Kabat), which retained donor residues alanine (A67), phenylalanine (F69), and valine (V71), respectively. The retention of residues A67, F69, and V71 was crucial for the intact titer of the humanized antibody. Glutamic acid residues at position 1 of the human framework were replaced with glutamic acid (E1) to represent and purify the homogenate: the conversion of N-terminal glutamic acid to pyroglutamic acid in antibodies and antibody fragments has been widely reported. The finally selected variable grafting sequences gL8 and gH9 are shown in Figure 1(A) and Figure 1(B), respectively.

[0265] The CDR, heavy and light chain variable regions, scFv and dsscFV amino acid and DNA sequences encoding antibody 650 are shown in Figure 2. Example 3. Production of anti-IL-17AF antibody 496.g3

[0266] The production of antibody CA028_00496.g3 (also referred to herein as antibody 496.g3), which is anti-human IL-17A and human IL-17F, has been previously described in WO2012 / 095662. This antibody binds to human IL-17A, IL-17F, and IL-17A / F heterodimers with pM affinity. The CDR, heavy and light chain variable regions, and the amino acid and DNA sequences of the heavy and light chains in Fab form of antibody 496.g3 are shown in Figure 2. The Fab constant region of 496.g3 (IL-17A / F binding) contains the human C-κ constant region (K1m3 allotype) and the human γ-1 CH1 constant region and hinge (G1m17 allotype). Example 4. Production of anti-human albumin antibody 645

[0267] The production of anti-human albumin antibody 645 has been previously described in WO2013 / 068571. The CDR, heavy and light chain variable regions, amino acids in scFv and dsscFV forms, and DNA sequence encoding antibody 645 are shown in Figure 2. Example 5. Multispecific antibody IL13 / IL17AF—Construction of transient plasmids and its expression in cells.

[0268] The multispecific antibody was designed to fix the anti-IL17AF V region (496.g3) at the Fab site; and to recombine the anti-albumin V region (645gL4gH5) and IL13 (1539gL8gH9) into disulfide-linked scFvs in HL orientation (dsHL) and link them to the C-terminus of each light chain and heavy chain constant region via 11 amino acid-rich glycine-serine linkers.

[0269] Light and heavy chain genes were independently selected and colonized into mammalian expression vectors for transient expression under the control of the hCMV promoter. Using the commercial ExpiCHO expifectamine transient expression kit (Thermo Scientific), equal ratios of the two plastids were transfected into the CHO-S XE cell line (UCB). Cultures were incubated in Corning roller flasks with vented lids at 37°C, 8.0% CO2, and 190 rpm. After 18 to 22 hours, an appropriate volume of CHO enhancer was added to the culture, following the HiTiter method provided by the manufacturer. Cultures were then incubated for another 10 to 12 days at 32°C, 8.0% CO2, and 190 rpm. The supernatant was harvested by centrifugation at 4000 rpm for 1 hour at 4°C and then filtered through a 0.45 µm followed by a 0.2 µm filter for sterilization. Performance titers were quantified using protein G HPLC with a 1 ml GE HiTrap protein G column (GE Healthcare) and internally generated Fab standards. Performance titers are summarized in Table 1. Table 1: Performance Titrations of Transient Performance in CHO S-XE Cell Lines Antibody Concentration (mg / L) IL17AF-IL13 multispecific antibody 160 Example 6. IL13 / IL17AF multispecific antibody – mammalian cell line development.

[0270] To confirm the stable expression of the IL13 / IL17AF multispecific antibody, a stable mammalian cell line was established. CHO cell lines were transfected with a vector containing 496.g 3 Fab, 1539g H9gL8 dsscFv HL (LC, INS0025609), 645g H5gL4 dsscFv HL (HC, INS0025306), and an optional label. Cell lines were selected and evaluated for suitability for a suitable manufacturing process. To assess protein quality and quantity and ensure optimal cell line selection, the cell line was evaluated in a small-scale batch bioreactor model. CHO cell lines expressing the IL13 / IL17AF multispecific antibody at concentrations greater than 1.8 g / L and monomer concentrations greater than 75% were selected. Example 7. Purification process of the IL13 / IL17AF multispecific antibody.

[0271] The multispecific antibody protein was purified by a native protein A capture step followed by a preparative size exclusion stripping step. The clear supernatant from the standard transient CHO expression was loaded into a MabSelect (GE Healthcare) column, given a 5-minute contact time, and washed with binding buffer (20 mM Hepes pH 7.4 + 150 mM NaCl). The binding material was eluted with 0.1 M sodium citrate pH 3.1 and neutralized with 2 M Tris / HCl pH 8.5, and quantified by absorbance at 280 nm.

[0272] Size exclusion chromatography (SE-UPLC) was used to determine the purity of the eluted product. The antibody (approximately 2 µg) was loaded onto a BEH200, 200 Å, 1.7 µm, 4.6 mm ID x 300 mm column (Waters ACQUITY) and developed with an isocratic gradient of 0.2 M phosphate at pH 7 at 0.35 mL / min. Serial detection was performed using absorbance at 280 nm and a multichannel fluorescence (FLR) detector (Waters). The eluted multispecific antibody was found to be 72% monomeric.

[0273] The neutralized sample was concentrated using an Amicon Ultra-15 concentrator (10 kDa molecular weight cutoff membrane) and centrifuged at 4000 xg with a spin rotor. The concentrated sample was applied to an XK16 / 60 Superdex200 column (GE Healthcare) equilibrated in PBS (pH 7.4) and developed with isocratic PBS (pH 7.4) at 1 ml / min. The dissolved fraction was collected and analyzed by size exclusion chromatography on a BEH200, 200 Å, 1.7 µm, 4.6 mm ID x 300 mm column (Aquity) and developed with isocratic gradient 0.2 M phosphate at pH 7 at 0.35 mL / min. Detection was performed by absorbance at 280 nm and a multichannel fluorescence (FLR) detector (Waters). The selected monomer fragments were combined, sterile filtered through 0.22 µm, and the final sample concentration was analyzed by A280 scanning on a DropSense96 (Trinean). Endotoxin concentrations were less than 1.0 EU / mg, as assessed using Charles River's EndoSafe® portable testing system with a Limulus Amebocyte Lysate (LAL) test cartridge.

[0274] The monomeric state of the final multispecific antibody was determined by size exclusion chromatography on a BEH200, 200 Å, 1.7 µm, 4.6 mm ID x 300 mm column (Aquity) with isocratic gradient 0.2 M phosphate at pH 7 at 0.35 mL / min, and detected by absorbance at 280 nm and a multichannel fluorescence (FLR) detector (Waters). As shown in Figure 3(A), the final multispecific antibody was found to be >99% monomeric.

[0275] For analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), samples were prepared by adding approximately 5 µg of purified protein to 4 x Novex NuPAGE LDS sample buffer (Life Technologies) and 10X NuPAGE sample reducing agent (Life Technologies) or 100 mM N-ethylmaleimide (Sigma-Aldrich), and heating to 100°C for 3 minutes. Samples were loaded onto 10-well Novex 4-20% Tris-glycine 1.0 mm SDS-polyacrylamide gels (Life Technologies) and separated at a constant voltage of 225 V for 40 minutes in Tris-glycine SDS running buffer (Life Technologies). Novex Mark 12 broad-range protein standards (Life Technologies) were used as standards. The gels were stained with Coomassie rapid staining agent (Generon) and destained in distilled water.

[0276] On non-reducing SDS-PAGE, a multispecific antibody with a theoretical molecular weight (MW) of approximately 100 kDa migrated to approximately 120 kDa. When the multispecific antibody protein was reduced, both chains migrated at rates close to their respective theoretical MWs: approximately 52 kDa for the heavy chain (HC) and approximately 51 kDa for the light chain (LC). An additional band at approximately 45 to 50 kDa in the unreduced gel represented "free" LC and HC with disulfide bonds missing from the Fab moiety of the molecule. These did not migrate to the same positions as LC and HC in lane 2 because they were incompletely reduced (Fig. 3(B)). Example 8. Antigen binding of IL-13 / IL-17AF / albumin multispecific antibody molecules. (i) Antigen binding affinity

[0277] The binding kinetics of IL-13, IL-17AA, AF, FF and albumin in humans and cynomolgus monkeys were evaluated by surface plasma resonance (Biacore T200).

[0278] Goat anti-human IgG, F(ab')2 fragment-specific antibodies (Jackson ImmunoResearch) were immobilized on a CM5 sensor chip at a concentration of approximately 5000 RU using amine conjugation chemicals. Each analysis cycle consisted of the following: capturing IL-13 / IL-17AF / albumin multispecific antibody molecules onto the anti-F(ab')2 surface, injecting the analyte (at 25°C, at a flow rate of 30 µl / min), followed by surface regeneration. Human and cynomolgus monkey analytes were injected using serially diluted 2-fold buffers in HBS-EP+ (GE Healthcare) at concentrations of 10 nM to 0.3125 nM for IL-13, 5 nM to 0.156 nM for IL-17-AA, AF, FF, and 100 nM to 3.125 nM for albumin. Antigens are prepared internally, except for human IL-13 (R&D Systems), cynomolgus monkey IL-13 (Sinobiologicals), and human serum albumin (Jackson ImmunoResearch). Blank buffer injection is included to reduce instrument noise and drift.

[0279] The kinetic parameters were determined using the Biacore T200 evaluation software (version 3.0) with a 1:1 combined model and are summarized in Tables 2(1) and 2(2). When the measured dissociation rate (kd) was less than 1.0 x 10⁻⁵, it was fixed to 1.0 x 10⁻⁵ (the detection limit of the Biacore T200 instrument, defined by the manufacturer GE Healthcare) to calculate the affinity (KD).

[0280] The results confirmed that the multispecific antibodies effectively bound to human and cynomolgus monkey IL-17AA, IL-17AF, IL-17FF, IL-13 and albumin. Table 2(1): Kinetic constants of IL-13 / IL-17AF / albumin multispecific antibodies binding to human antigens. Analytes k a (1 / Ms) k d (1 / s) K D (pM) n= Human IL-17AA 2.26E+06 1.57E-05 9 7 Human IL-17AF 3.70E+06 4.54E-05 15 4 Human IL-17FF 1.67E+06 1.98E-04 128 7 Human IL-13 1.46E+06 3.84E-05 26 3 Human albumin 6.65E+04 1.54E-04 2508 3

[0281] Results represent the average of a specified number of measurements. Table 2(2): Kinetic constants of IL-13 / IL-17AF / albumin multispecific antibody binding to cynomolgus monkey antigen. Analytes k a (1 / Ms) k d (1 / s) K D (pM) n= Crab-eating macaque IL-17AA 1.65E+06 1.24E-05 7 4 Crab-eating macaque IL-17AF 2.25E+06 2.87E-05 14 4 Crab-eating macaque IL-17FF 1.14E+06 2.82E-04 283 4 Crab-eating macaque IL-13 9.48E+05 1.48E-04 156 3 Crab-eating macaque albumin 8.18E+04 2.04E-04 2473 3

[0282] The result represents the average of the specified number of measurements. (ii) Simultaneous antigen binding

[0283] Surface plasma resonance was used to confirm that IL-17AA, IL-13 and albumin can bind to UCBXXXX simultaneously in human or cynomolgus monkey forms of the protein.

[0284] The method for evaluating the simultaneous binding of analytes to UCBXXXX involves capturing the antibody sample onto a fixed anti-human IgG F(ab')2 fragment-specific antibody. Human or cynomolgus monkey IL-13, IL-17A, and albumin are then injected individually or as a mixture of the three analytes (at a rate of 30 μl / min for 300 seconds) onto the captured UCBXXXX (final concentrations of 30 nM IL-13, 15 nM IL-17AA, and 150 nM albumin).

[0285] At the end of each cycle, the surface was regenerated by injecting 50 mM HCl for 60 seconds at a flow rate of 10 μl / min, followed by injecting 5 mM NaOH for 30 seconds and finally injecting 50 mM HCl for 60 seconds.

[0286] Determine the binding reaction of each antigen when injected alone, and compare the sum of the individual reactions with the binding reaction when a mixture of all three antigens is injected.

[0287] The average binding response of a mixture of human IL-17AA, IL-13, and albumin to UCBXXXX was 100% of the sum of individual binding responses (summarized in Table 3), indicating that UCBXXXX can bind to each antigen simultaneously and independently. Table 3: Simultaneous binding of UCBXXXX to human IL-17AA, IL-13, and albumin Analytes Combined, n=1 (RU) Combined, n=2 (RU) Combined, n=3 (RU) average value IL-13 12 19 19 IL-17AA 33 53 51 albumin 36 60 59 IL-13 + IL-17AA + Albumin 81 131 129 The sum of individual binding reactions 81 132 129 The percentage (%) of the mixture as a single binding reaction. 100 99 100 100

[0288] The average binding response of a mixture of cynomolgus macaque IL-17AA, IL-13, and albumin to UCBXXXX was 97% of the sum of individual binding responses (summarized in Table 4), indicating that UCBXXXX can bind to each antigen simultaneously and independently. Table 4: Simultaneous binding of UCBXXXX to cynomolgus macaque IL-17AA, IL-13, and albumin Analytes Combined, n=1 (RU) Combined, n=2 (RU) Combined, n=3 (RU) average value IL-13 31 30 30 IL-17AA 57 56 60 albumin 63 61 60 IL-13 + IL-17AA + Albumin 146 141 145 The sum of individual binding reactions 151 146 150 The percentage (%) of the mixture as a single binding reaction. 97 96 97 97 Example 9. Neutralizing IL13 using anti-IL13 / IL17AF multispecific antibodies.

[0289] The activity of anti-IL13 / IL17AF multispecific antibodies in neutralizing IL13 was evaluated using the HEK 293 human IL4 / IL13 SEAP reporting cell line assay. SEAP secretion was measured after activation of the STAT6 pathway to assess IL13 response.

[0290] HEK 293 human IL4 / IL13 SEAP reporter cells (#hkb-il413) were obtained from Invivogen, San Diego. The cell line was cultured, frozen, and maintained in accordance with the manufacturer’s agreement.

[0291] Recombinant human IL13 lineage obtained from R&D Systems, Minneapolis, MN. (#213-ILB)

[0292] Cells were seeded on plates so that about 80% of the cells merged after stimulation in 96-well flat plates.

[0293] The cells were treated with the antibody in duplicate. The antibody was pre-cultured with 250 pg / mL IL-13 for 30 minutes at 37°C, 5% CO2, and 100% humidity 24 hours before the cells were added.

[0294] 24 hours later, according to the manufacturer’s instructions, 20 µL of supernatant was aspirated from the cell stimulation and 180 µL of Quanti-Blue #rep-qbs (Invivogen, San Diego) was added.

[0295] The assay was developed until a visible color gradient was observed, and the absorbance was read using a spectrophotometer at 620 nm. The IC50 was calculated using a Graphpad Prism (San Diego, CA). Figure 4 shows a representative graph of the % inhibition of STAT6 signaling by the anti-IL13 / IL-17AF multispecific antibody. Table 5: IC50 values ​​of STAT6 signaling inhibition by the anti-IL13 / IL17AF multispecific antibody. pM + / - SEM ng / ml + / - SEM n= 4.118 1.319 0.4118 0.1319 3 Example 10. Neutralization of IL-6 responses to IL-17A and IL-17F in humans and cynomolgus monkeys via human skin fibroblasts by anti-IL13 / IL17AF multispecific antibodies.

[0296] The aim of this study was to determine the neutralizing capacity of a multispecific anti-IL13 / IL17AF antibody against human and cynomolgus monkey IL-17A and IL-17F in the human primary cell system. IL-17, when present in combination with other cytokines such as TNF-α, induces a pro-inflammatory response. Therefore, this synergistic effect was utilized to assess the release of IL-6 from primary normal neonatal human dermal fibroblasts (nHDF) stimulated by IL-17 and TNF-α.

[0297] This assay measures the ability of anti-IL13 / IL17AF multispecific antibodies to inhibit IL-17-induced release of IL-6 from nHDF. Specifically, nHDF was stimulated with a combination of human or cynomolgus monkey IL-17A (50 pM) or IL-17F (25,000 pM) and TNF-α (25 pM) in the presence of anti-IL13 / IL17AF multispecific antibodies (concentration range of 5000 pM to 0.25 pM for IL-17A studies; 500,000 pM to 25 pM for IL-17F studies). The resulting IL-6 response was then measured using homogeneous time-resolved FRET (HTRF).

[0298] nHDF cells (Sigma #106-05n) were cultured in complete medium (DMEM + 10% FCS + 2 mM L-glutamic acid) and maintained in tissue culture flasks using standard techniques. Cells were harvested from the tissue culture flasks using a TrypLE (Invitrogen #12605036). The TrypLE was neutralized with complete medium (45 ml) and the cells were centrifuged at 300 xg for 3 minutes. The cells were resuspended in complete medium (3 to 5 ml), counted, and adjusted to a concentration of 3.125 x 10⁴ cells / mL. The cells were then added at 40 µl / well to a 384-well assay plate (Corning #3701). The cells were cultured at 37°C / 5% CO₂ for 3 hours to allow adhesion to the plate. The anti-IL13 / IL17AF multispecific antibody was serially diluted in complete medium in a 384-well dilution plate (Greiner #781281) to a final concentration range of 5000 pM to 0.25 pM for IL-17A assessment and 500,000 pM to 25 pM for IL-17F assessment. A mixture of TNF-α and IL-17 cytokines was prepared in complete medium to a final concentration of TNF-α of 25 pM, human or cynomolgus monkey IL-17A of 50 pM, or IL-17-F of 25,000 pM. 30 µl / well of these solutions were then added to a 384-well reagent plate (Greiner #781281). 10 µl from the serially diluted anti-IL13 / IL17AF multispecific antibody plate was then transferred to a reagent plate containing 30 µl of diluted cytokines. Then, the anti-IL13 / IL17AF multispecific antibody was incubated with the cytokine mixture at 37°C / 5% CO2 for one hour. After incubation, 10 μl was transferred from the reagent plate to the test plate containing cells. The test plate was then incubated at 37°C / 5% CO2 for 18 hours ± 2 hours. After incubation, europium cryptate and Alexa 665 antibody (Cisbio #62IL6PEB) from the Cisbio IL-6 HTRF kit were diluted in rehydration buffer and mixed 1:1 according to the kit insert. Subsequently, 10 µl / well of this antibody mixture was added to a white low-volume 384-well HTRF plate (Greiner #784075). Then, the supernatant from the test plate was transferred to the HTRF plate at 10 µl / well. The HTRF plate was then incubated at room temperature with gentle shaking for 2 hours. Then, the HTRF board was read on the Synergy Neo 2 board reader according to the manufacturer's instructions, and the fluorescence was measured at readings of 330 / 620 nm and 330 / 665 nm.The ratio value was then calculated using the formula (330 / 665 nm divided by 330 / 620 nm) x 10,000 and used to determine the relative inhibition percentage compared to the control well using Microsoft Excel. GraphPad Prism 7.0 was used for 4PL curve fitting and IC50 value calculation.

[0299] The results shown are the average of three independent experiments (+ / - SEM). The IC50 values ​​of the anti-IL13 / IL17AF multispecific antibody were calculated to be 42 pM for human IL-17A and 49 pM for cynomolgus monkey IL-17A. The IC50 values ​​of the anti-IL13 / IL17AF multispecific antibody were calculated to be 28,030 pM for human IL-17F and 34,320 pM for cynomolgus monkey IL-17F (Figure 5). Table 6: Overview of the titer, efficacy, and hill slope values ​​of the anti-IL13 / IL17AF multispecific antibody.

[0300] Values ​​calculated from three independent experiments. Inspection IC50 (pM) Emax (%) slope Human IL-17A 42 100 2 Crab-eating macaque IL-17A 49 100 2.4 Human IL-17F 28030 100 3.2 Crab-eating macaque IL-17F 34320 100 2.6 Example 11. In the NHEK CXCL1 release bioassay, IL-13, IL-17AA and IL-17FF were simultaneously neutralized by a multispecific antibody against IL13 / IL17AF.

[0301] The purpose of this test is to assess the ability of anti-IL13 / IL17AF multispecific antibodies in the primary cellular system to simultaneously neutralize IL-13, IL-17AA, and IL-17FF. When NHEK is treated with IL-13, IL-17AA, or IL-17FF, it induces the secretion of CXCL1 (a cytokine responsible for recruiting cells to the site of inflammation). In the case of atopic dermatitis, evidence suggests that CXCL1 plays a role in sensitized neurons and therefore has a lower excitation threshold. This observation may be related to the itch experienced by the patient (Yang TB and Kim BS 2019; "Pruritus in allergy and immunology" J Allergy Clin Immunol 144(2): 353-360).

[0302] NHEK (PromoCell, Heidelberg) cells were stored, cultured, and used according to the manufacturer's protocol. Cell plates were seeded to ensure 100% confluence upon stimulation in 48-well plates. Cells were pre-cultured for 30 minutes with increased concentrations of anti-IL-13, anti-IL-17AA, anti-IL-17FF, or anti-IL13 / IL17AF multispecific antibodies, respectively, and then treated with 100 ng / mL IL-13, 100 ng / mL IL-17AA, and 1 µg / mL IL-17FF for 72 hours. After this period, 50 µl of cell-free supernatant was collected by ELISA according to the manufacturer's protocol (R&D Systems) for quantification of CXCL1 concentration. The IC50 values ​​for each experimental group were calculated using nonlinear regression with Graphpad Prism (San Diego, CA).

[0303] Results confirmed that the anti-IL13 / IL17AF multispecific antibody simultaneously neutralized the activities of IL-13, IL-17AA, and IL-17FF (Figure 6). When normalized to the number of binding sites available for assay, the anti-IL13 / IL17AF multispecific antibody (81.3%) inhibited CXCL1 release more effectively than anti-IL17A (52.7%), anti-IL-17F (0.7%), or anti-IL-13 (48.8%). Increasing the concentration of anti-IL-17AA, anti-IL-17FF, or anti-IL-13 alone did not improve the maximum inhibition achieved. The results highlight the benefit of simultaneous inhibition of IL-13, IL-17AA, and IL-17FF compared to single-cell cytokine neutralization. Example 12. Comparison of multispecific IL13 / IL17 antibodies with prior art IL13 / IL17 antibodies. Introduction

[0304] UCBXXXX is an example of a multispecific IL13 / IL17 antibody according to the present invention. It comprises a Fab domain linked to a 2 scFv domain, which has dual specificity for IL-17A and IL-17F, one specific for IL-13 and the other specific for albumin. The anti-albumin domain gives UCBXXXX an extended half-life (Figure 7).

[0305] Previously, Abbvie (WO2013 / 102042A2) and Genentech (WO2015 / 127405A2) described bispecific antibodies binding to IL-13 and IL-17. However, little has been revealed about how these antibodies bind to IL-13, IL-17A, and IL-17F, and the extent to which they bind to IL-13, IL-17A, and IL-17F. To compare these properties, we prepared antibodies described in the prior art and investigated their binding behavior for the following characteristics: ● Affinity to IL-13 ● Interaction of the molecule with IL-13 and IL-13Rα1 ● Affinity to IL-17A ● Affinity to IL-17F Comparison of antibody production

[0306] Use the sequence construction BITS7201A (Genentech) described in Example 6 of WO2015 / 127405A2.

[0307] DVD2166 and DVD2174 (abbreviations) were constructed using the sequences described in Tables 6 and 7 of WO2013 / 102042A2. These molecules were selected based on the nearly equivalent or equivalent activity reported by their respective parent antibodies to each group having dual specificity against IL-13 and IL-17 (WO2013 / 102042A2 Example 4, page 83, paragraph 0195).

[0308] DNA constructs were transfected into CHO-SXE cells using the high-titer protocol of the ExpiCHO transfection system (ThermoFisher Scientific). After harvesting, the cell culture was centrifuged at 4000 RPM for at least one hour, and the supernatant was clarified by filtration using a 0.22 µM Stericup filter unit. Purification was performed using DVD2166 + DVD2174.

[0309] DVD-IgG protein was purified by applying clarified supernatant to a 10 ml MabSelect Sure column and washing three times with PBS at pH 7.4 (column volume CV). The protein was eluted from the column with 0.1 M sodium citrate at pH 3.6 and neutralized with 2 M Tris-HCl at pH 8.5. Monomeric proteins were then separated by applying the solution to a HiLoad 16 x 60 Superdex 200 pg column (Sigma) equilibrated with PBS at pH 7.4. The fractions containing the monomeric proteins were pooled, aseptically filtered, and stored at 4°C. (BITS7201A purification)

[0310] Parental bulge and cave proteins were purified by applying clarified supernatant to a 10 mL MabSelect Sure column and washing with 3 CV PBS, pH 7.4. Proteins were removed by elution from the column with 0.1 M sodium citrate, pH 3.6. To neutralize and stabilize the proteins, the sample was diluted 1:1 with 1 M arginine / succinate buffer, pH 8.7. The parental antibody was then applied to a HiLoad 26 x 60 Superdex 200 pg column (Sigma) equilibrated with 0.15 M sodium acetate, 0.5 M arginine buffer, pH 8.5. Bispecificity was subsequently generated by mixing the parental antibody at a 1:1 ratio in the presence of 5 mM cysteine ​​and incubating overnight at room temperature. A second preparative gel filtration step was performed to remove any high molecular weight substances by applying the gel to a HiLoad 26 x 60 Superdex 200 pg column equilibrated with PBS at pH 7.4, after the exchange of dual-specificity substances. The dissolved fractions containing monomeric dual-specific proteins were combined, aseptically filtered, and stored at 4°C. Comparison of binding properties was then conducted.

[0311] The binding kinetics of human IL-13, IL-17A, and IL-17F to UCBXXXX and prior art antibodies were evaluated and compared directly in a single experiment using surface plasma resonance (Biacore T200). Furthermore, surface plasma resonance was used to assess whether the binding of UCBXXXX or comparative molecules to IL-13 resulted in the blocking of the interaction between IL-13 and the IL-13 receptor.

[0312] Goat anti-human IgG, F(ab')2 fragment-specific antibody (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip at a concentration of approximately 5000 RU using amine conjugation chemicals. For affinity assessment, each analytical cycle consisted of the following: capturing UCBXXXX or comparative bispecific molecules onto the anti-F(ab')2 surface (50 to 100 RU), injecting the analyte (at 25°C, at a flow rate of 30 µl / min for 180 seconds), followed by monitoring dissociation: 1200 seconds for IL-13 and IL-17A, and 600 seconds for IL-17F. At the end of each cycle, the surface was regenerated by injecting 50 mM HCl at a flow rate of 10 μl / min for 60 seconds, followed by 5 mM NaOH for 30 seconds, and finally 50 mM HCl for 60 seconds. The analytes were injected in HBS-EP+ run buffer (GE Healthcare) with serial dilutions of 2-fold, at concentrations ranging from 10 nM to 0.3125 nM for IL-13 and from 5 nM to 0.156 nM for IL-17A and IL-17F. IL-17A and IL-17F were prepared in-house, while human IL-13 was derived from R&D Systems. Blank buffer was included to reduce instrument noise and drift.

[0313] The kinetic parameters were determined using the Biacore T200 evaluation software (version 3.0) with a 1:1 bound model. The results are summarized in Table 7. Table 7: Comparison of kinetic constants bound to IL-13, IL-17A and IL-17F. molecular Analytes k a (1 / Ms) k d (1 / s) K D (pM) UCBXXXX* IL13 3.61E+06 1.11E-04 25.8 BITS7210A* 2.05E+06 5.69E-05 27.5 DVD2166 8.16E+05 1.50E-05 18.35 DVD2174 6.50E+05 3.66E-05 56.34 UCBXXXX* IL17A 3.20E+06 3.45E-05 8.2 BITS7210A* 1.27E+07 1.06E-03 84.4 DVD2166 5.59E+05 7.68E-05 137.4 DVD2174 4.76E+05 1.28E-04 268.4 UCBXXXX* IL17F 2.66E+06 2.19E-04 82.1 BITS7210A* 7.71E+06 5.74E-04 74.4 DVD2166 ←←←←←←←←←No combination→→→→→→→→→ DVD2174 ←←←←←←←←←No combination→→→→→→→→→ *Results represent the average of two technical repetitions in the same experiment.

[0314] To evaluate IL13Rα1 receptor blockade, each antibody molecule was captured onto the surface of goat anti-human IgG and F(ab')2 (to approximately 50 to 100 RU), followed by injection of IL-13 (25 nM for 180 seconds, 10 µl / min) and IL13Rα1 (R&D Systems, 100 nM for 300 seconds, 10 µl / min). Blank injections including both IL13 and IL13Rα1 were performed to subtract any drift or background reaction. As summarized in Table 8, UCBXXXX blocked the interaction between IL13 and IL13Rα1, while BITS7210A, DVD2166, and DVD 2174 molecules did not. Table 8: IL13Rα1 receptor blockade by UCBXXXX and comparison molecules. molecular Capture (RU) IL13 binding (RU) IL13Rα1 binding (RU) IL13Rα1 blockers UCBXXXX 65.4 7.3 0.2 yes BITS7210A 86 8.6 15.7 no DVD2166 115.2 11.7 13.8 no DVD2174 115.3 11.3 16.2 no

[0315] Another experiment was conducted to evaluate the ability of UCBXXXX to block binding to IL13Rα1 and IL13Rα2. In this experiment, approximately 260 RU of UCBXXXX was captured onto a fixed mouse anti-human CH1 antibody (internal UCB), followed by injection of IL13 (25 nM for 180 seconds, 10 µl / min), and then injection of either IL13Rα1 or IL13Rα2 (R&D Systems, 100 nM for 300 seconds, 10 µl / min). Blank injections including IL13, IL13Rα1, and IL13Rα2 were included to subtract any drift or background response. The results confirmed that UCBXXXX could block the interaction between IL13 and both IL13Ra1 and IL13Ra2 (Table 9). Table 9: Blockade of IL13Rα1 and IL13Rα2 receptors by UCBXXXX Capture (RU) IL-13 binding (RU) IL13Rα1 binding (RU) IL13Rα2 binding (RU) 264.4 27.0 0.8 0.3 discuss

[0316] Comparative studies have confirmed that the multispecific antibody UCBXXXX, the bispecific antibody BITS7210A, and the dual variable domain antibodies DVD2166 and DVD2174 can bind to IL13 with high affinity. However, the characteristics of their binding interactions are very different. UCBXXXX can block the interaction between IL13 and IL13-Rα1, while BITS7210A, DVD2166, and DVD2174 cannot.

[0317] Comparative studies further confirmed that the multispecific antibody UCBXXXX, the bispecific antibody BITS7210A, and the dual variable-domain antibodies DVD2166 and DVD2174 can bind to IL17. However, the binding interaction characteristics are very different. UCBXXXX has a significantly higher affinity for IL17A than BITS7210A and the DVD antibody. UCBXXXX and BITS7210A bind to IL17F with similar affinities, while the DVD antibody cannot bind to IL17F at all.

[0318] In the presence of such antibodies, the interaction between IL-13 and IL-13Rα1 is important in reducing potential immunogenicity. Evidence suggests that immunogenicity, particularly the generation of anti-drug antibodies (ADAs), should be closely considered when investigating potential novel bispecific antibody therapeutics. The main driver of ADA generation is the binding of therapeutic antibodies to target antigens expressed on the cell surface and their subsequent internalization (Schellekens, H., 2002; Clin Ther. 24(11):1720-40). Internalized therapeutic antibody / target antigen complexes are transported through various intracellular compartments and can be recycled back to the cell surface or degraded (St Pierre et al., 2011); this can lead to the presentation of antigen-presenting molecules and peptides generated by ADAs.

[0319] In the bispecific antibody BITS7201A, the IL-13 F(ab) portion of the molecule is identical to that of the anti-IL-13 antibody lebrikizumab (see Example 6 of WO2015 / 127405A2). It is believed that lebrikizumab binds to IL-13 at a site that allows IL-13 to bind to its receptors IL-13Rα1 and IL-13Rα2 but blocks interaction with the IL-4Rα receptor (Popovic et al. 2017; J Mol Biol. 429(2):208-19). This particular type of interaction can allow the antibody / target antigen / receptor complex to be internalized through IL-13Rα2 and thus increase the likelihood of an immunogenic response. In a phase I clinical trial, BITS7201A was associated with a high incidence of anti-drug antibody (ADA) and clinical development was withdrawn.

[0320] Similarly, the dual variable domain antibodies DVD2166 and DVD2174 were unable to block the interaction between IL13 and IL13-Rα1.

[0321] To mitigate potential immunogenic risks, UCBXXXX has been specifically designed to prevent its interaction with receptors on cells after binding to its respective target antigens IL-13, IL-17A, and IL-17F, thereby reducing the chance of internalization, degradation, and the likelihood of ADA production. Once clinical data are available, the incidence of ADA in humans using UCBXXXX will only be known in certain cases.

[0322] The data generated above indicate that UCBXXXX has the appropriate properties to be an effective IL-13 / IL-17 antibody therapeutic agent with improved efficacy and reduced immunogenicity risk. [Simplified Explanation of the Diagram]

[0323] Figure 1. Humanization alignment of Ab650. Alignment of rat antibody (donor) V region sequence with human germline (receptor) V region sequence, and the designed humanized sequence. (A) Light linker 650: 650 = rat variable light chain sequence. 650gL8 = humanized grafting of the 650 variable light chain using the IGKV1-39 human germline as the receptor framework. CDRs are shown in bold / underlined. Donor residues are shown in bold / italic and highlighted: I58 and Y71. (B) Heavy linker 650: 650 = rat variable heavy chain sequence. 650gH9 = humanized grafting of the 650 variable heavy chain using the IGHV1-69 human germline as the receptor framework. CDRs are shown in bold / underlined. Donor residues are shown in bold / italic and highlighted: A67, F69, and V71. Figure 2. Amino acid and DNA sequences. Figure 3. Purification of the IL13 / IL17AF multispecific antibody. (A) BEH200 SEC-UPLC analysis of the purified multispecific antibody by FLR detection. (B) Protein samples separated by Tris-glycine SDS-PAGE under non-reducing (lane 1) or reducing (lane 2) conditions. Gels were stained with Coomassie quick stain and destained in dH2O. Mark 12 protein markers (Life Technologies) were used as standards (M). Molecular weight (MW) was measured in kilodaltons (kDa). Figure 4. Inhibition of STAT6 signaling by the IL-13 / IL-17AF multispecific antibody. Figure 5. (A) Inhibition of IL-6 production by the IL-13 / IL-17AF multispecific antibody in response to the combination of human or cynomolgus monkey IL-17A and TNF-α. (i) Human IL17-A. (ii) Cynomolgus monkey IL-17A. (B) In response to a combination of human or cynomolgus monkey IL-17F and TNF-α, inhibition of IL-6 production by an IL-13 / IL-17AF multispecific antibody. (i) Human IL17-F. (ii) Cynomolgus monkey IL-17F. Figure 6. In the NHEK CXCL1 release bioassay, simultaneous neutralization of IL-13, IL-17AA, and IL-17FF by an IL13 / IL17AF multispecific antibody. Legend: Circle = anti-IL13 / IL17AF Square = anti-IL-17A Upward-pointing triangle = anti-IL-17F Downward-pointing triangle = anti-IL-13 Figure 7. Schematic illustration of the multispecific IL13 / IL17 antibody according to the present invention. [Sequence List]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

Claims

1. A multispecific antibody that binds to human IL-13, human IL-17A, and / or human IL-17F, comprising an IL-13 binding site, the binding site comprising: a light chain variable region comprising CDR-L1 of the sequence shown in SEQ ID NO:15, CDR-L2 of the sequence shown in SEQ ID NO:16, and CDR-L3 of the sequence shown in SEQ ID NO:17; and a heavy chain variable region comprising CDR-H1 of the sequence shown in SEQ ID NO:18, CDR-H2 of the sequence shown in SEQ ID NO:19, and CDR-H3 of the sequence shown in SEQ ID NO:

20.

2. The multispecific antibody of claim 1, wherein the IL-13 binding site contains a light chain variable region comprising the sequence shown in SEQ ID NO:27 and a heavy chain variable region comprising the sequence shown in SEQ ID NO:

28.

3. The multispecific antibody of claim 1, wherein the IL-13 binding site contains a light chain variable region comprising the sequence shown in SEQ ID NO:31 and a heavy chain variable region comprising the sequence shown in SEQ ID NO:

32.

4. A multispecific antibody as claimed in any of claims 1 to 3, comprising an antigen-binding site for binding to human IL-17A and human IL-17F, the antigen-binding site comprising: a light chain variable region comprising CDR-L1 of the sequence shown in SEQ ID NO:1, CDR-L2 of the sequence shown in SEQ ID NO:2, and CDR-L3 of the sequence shown in SEQ ID NO:3; and a heavy chain variable region comprising CDR-H1 of the sequence shown in SEQ ID NO:4, CDR-H2 of the sequence shown in SEQ ID NO:5, and CDR-H3 of the sequence shown in SEQ ID NO:

6.

5. The multispecific antibody of claim 4, wherein the antigen binding site binding to human IL-17A and human IL-17F contains a light chain variable region comprising the sequence shown in SEQ ID NO:7 and a heavy chain variable region comprising the sequence shown in SEQ ID NO:

9.

6. A multispecific antibody as claimed in any of the preceding claims, which binds to human IL-13, human IL-17A, and / or human IL-17F, wherein the multispecific antibody comprises: a) a polypeptide chain of formula (Ia): VH-CH1-X-V1; and b) a polypeptide chain of formula (IIa): VL-CL-Y-V2; wherein: VH represents the variable domain of the heavy chain; CH1 represents domain 1 of the constant region of the heavy chain; X represents a bond or linker; Y represents a bond or linker; V1 represents scFv, dsscFv, or dsFv; VL represents the variable domain of the light chain; CL represents a domain of the constant region of the light chain, such as Cκ; V2 represents scFv, dsscFv, or dsFv; wherein the polypeptide chain of formula (Ia) contains a protein A binding domain; and wherein the polypeptide chain of formula (IIa) does not bind protein A.

7. As in request item 6, a multispecific antibody, wherein: VL and VH contain antigen-binding sites binding to human IL-17A and human IL-17F, V2 contains an antigen-binding site binding to human IL-13, and V1 contains an antigen-binding site binding to human serum albumin; wherein VL contains CDR-L1 of the sequence shown in SEQ ID NO:1, CDR-L2 of the sequence shown in SEQ ID NO:2, and CDR-L3 of the sequence shown in SEQ ID NO:3, and VH contains CDR-H1 of the sequence shown in SEQ ID NO:4, CDR-H2 of the sequence shown in SEQ ID NO:5, and CDR-H3 of the sequence shown in SEQ ID NO:6; wherein V1 contains a light chain variable region containing CDR-L1 of the sequence shown in SEQ ID NO:39, CDR-L2 of the sequence shown in SEQ ID NO:40, and CDR-L3 of the sequence shown in SEQ ID NO:41; and a heavy chain variable region containing CDR-H1 of the sequence shown in SEQ ID NO:42, CDR-H2 of the sequence shown in SEQ ID NO:43, and SEQ ID NO:6; The CDR-H3 of the sequence shown in SEQ ID NO:44; and V2 therein comprising a light chain variable region comprising CDR-L1 of the sequence shown in SEQ ID NO:15, CDR-L2 of the sequence shown in SEQ ID NO:16, and CDR-L3 of the sequence shown in SEQ ID NO:17; and a heavy chain variable region comprising CDR-H1 of the sequence shown in SEQ ID NO:18, CDR-H2 of the sequence shown in SEQ ID NO:19, and CDR-H3 of the sequence shown in SEQ ID NO:

20.

8. A multispecific antibody as claimed in claim 6 or claim 7, wherein VL contains the sequence shown in SEQ ID NO:7 and VH contains the sequence shown in SEQ ID NO:

9.

9. A multispecific antibody as claimed in any of claims 6 to 8, wherein V2 contains a light chain variable region comprising the sequence shown in SEQ ID NO:27 and a heavy chain variable region comprising the sequence shown in SEQ ID NO:

28.

10. A multispecific antibody as claimed in any of claims 6 to 8, wherein V2 contains a light chain variable region comprising the sequence shown in SEQ ID NO:31 and a heavy chain variable region comprising the sequence shown in SEQ ID NO:

32.

11. A multispecific antibody as claimed in any one of claims 6 to 10, wherein V1 contains a light chain variable region comprising the sequence shown in SEQ ID NO:45 and a heavy chain variable region comprising the sequence shown in SEQ ID NO:

46.

12. A multispecific antibody as claimed in any one of claims 6 to 10, wherein V1 contains a light chain variable region comprising the sequence shown in SEQ ID NO:49 and a heavy chain variable region comprising the sequence shown in SEQ ID NO:

50.

13. A multispecific antibody as claimed in any of claims 6 to 12, wherein the light chain variable region and the heavy chain variable region of V2 are linked by a linker comprising the sequence shown in SEQ ID NO:

66.

14. The multispecific antibody of claim 13, wherein V2 is an scFv containing the sequence shown in SEQ ID NO:35 or a dsscFv containing the sequence shown in SEQ ID NO:

37.

15. A multispecific antibody as claimed in any of claims 6 to 14, wherein the light chain variable region and the heavy chain variable region of V1 are linked by a linker comprising the sequence shown in SEQ ID NO:

68.

16. The multispecific antibody of claim 15, wherein V1 is an scFv containing the sequence shown in SEQ ID NO:53 or a dsscFv containing the sequence shown in SEQ ID NO:

55.

17. A multispecific antibody as claimed in any of claims 6 to 16, wherein Y is a linker comprising the sequence shown in SEQ ID NO:

65.

18. A multispecific antibody as claimed in any of claims 6 to 17, wherein X is a linker comprising the sequence shown in SEQ ID NO:

67.

19. A multispecific antibody as claimed in any of the preceding claims, comprising the sequence shown in SEQ ID NO:57 or SEQ ID NO:

59.

20. A multispecific antibody as claimed in any of the preceding claims, comprising the sequence shown in SEQ ID NO:61 or SEQ ID NO:

63.

21. A multispecific antibody as claimed in any of the preceding claims, comprising the sequences shown in SEQ ID NO:59 and SEQ ID NO:

63.

22. An isolated polynucleotide encoding a multispecific antibody as defined in any one of claims 1 to 21.

23. An expression vector carrying a polynucleotide as claimed in claim 22.

24. A host cell comprising a vector as defined in claim 23.

25. A method for preparing a multispecific antibody as defined in any one of claims 1 to 21, the method comprising culturing a host cell as described in claim 24 under conditions that allow the antibody to be produced, and recovering the produced antibody.

26. The method of claim 25, wherein the method includes a protein A purification step.

27. A pharmaceutical composition comprising an antibody as defined in any one of claims 1 to 21 and a pharmaceutically acceptable adjuvant and / or carrier.

28. An antibody as defined in any one of claims 1 to 21 or a pharmaceutical composition as defined in claim 27, and a method of using it to treat a human or animal body by means of a therapy.

29. The antibody or pharmaceutical composition of claim 28, used to treat or prevent atopic dermatitis, chronic hand eczema, nasal micropolyps or nasal polyps, food allergies, or eosinophilic esophagitis.

30. A method for treating or preventing atopic dermatitis, chronic hand eczema, nasal micropolyps or nasal polyps, food allergies or eosinophilic esophagitis, the method comprising administering to a patient in need a therapeutically effective amount of a multispecific antibody as claimed in claims 1 to 21 or a pharmaceutical composition as claimed in claim 27.