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

Abstract

The present invention relates to multispecific antibodies specific for human IL-13, human IL-17A and / or human IL-17F. The present invention also relates to methods for producing the multispecific antibodies and their therapeutic use for the treatment of atopic dermatitis and other diseases.

Description

Technical Field

[0001] This invention relates to multispecific antibodies specific to human IL-13, human IL-17A, and / or IL-17F. The invention also relates to methods for generating such multispecific antibodies and their therapeutic use in treating atopic dermatitis and other diseases. Background Technology

[0002] Atopic dermatitis (AD), also known as atopic eczema, is an inflammatory condition that causes intense itching, redness, swelling, oozing, and cracking of the skin, which often thickens over time.

[0003] Since the beginning of the 20th century, many inflammatory conditions of the mucous membranes have become more prevalent; atopic dermatitis is a prime example. Currently, in developed countries, 15-30% of children and 2-10% of adults are affected, while in the United States, this proportion has almost tripled in the past thirty to forty years. More than 15 million American adults and children suffer from atopic dermatitis.

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

[0005] Due to the limitations of existing medications, there is a great need to improve the treatment of atopic dermatitis.

[0006] WO2013 / 102042A2 (Abbvie) describes a bispecific binding protein targeting IL-13 and IL-17 and its potential use in treating a wide range of diseases. This binding protein has not entered the clinical development stage.

[0007] WO2015 / 127405A2 (Genentech) describes anti-IL-13 / IL-17 bispecific antibodies and methods for using them to treat moderate to severe asthma and / or eosinophilic asthma. BITS7201A was associated with a high incidence of anti-drug antibody (ADA) in a Phase I clinical trial and was withdrawn from clinical development. Summary of the Invention

[0008] This invention provides an improved multispecific antibody capable of binding to 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 superior pharmacokinetic profiles. Furthermore, the antibodies of the present invention can be engineered to allow for more efficient purification using improved purification methods that involve fewer steps than currently available methods, resulting in cost and time benefits at an industrial scale. Therefore, the antibodies of the present invention can have improved manufacturability.

[0010] The present invention also provides:

[0011] The isolated polynucleotide encoding this multispecific antibody.

[0012] The expression vector carrying this polynucleotide.

[0013] The host cell containing the vector.

[0014] A method for generating the multispecific antibody includes culturing host cells and recovering the resulting antibody.

[0015] A pharmaceutical composition containing this multispecific antibody.

[0016] Multispecific antibodies or pharmaceutical compositions are used in methods of treating humans or animals through therapy.

[0017] A method of treating or preventing atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergy, or eosinophilic esophagitis, comprising administering a therapeutically effective amount of the multispecific antibody or pharmaceutical composition to a patient in need of such treatment. Attached Figure Description

[0018] Figure 1. Ab650 humanization comparison.

[0019] Alignment of rat antibody (donor) V region sequence, human germline (recipient) V region sequence, and designed humanized sequence.

[0020] (A) Light chain graft 650:

[0021] 650 = Rat variable light chain sequence.

[0022] 650gL8 = Humanized grafts of 650 variable light chains using the IGKV1-39 human lineage as the recipient framework.

[0023] CDR displays text in bold / underline.

[0024] Donor residues are shown in bold / italic and highlighted: I58 and Y71.

[0025] (B) Heavy chain graft 650:

[0026] 650 = Rat variable heavy chain sequence.

[0027] 650gH9 = Humanized grafts of 650 variable heavy chains using the IGHV1-69 human lineage as the recipient framework.

[0028] CDR displays text in bold / underline.

[0029] Donor residues are shown in bold / italic and highlighted: A67, F69, and V71.

[0030] Figure 2 Amino acid and DNA sequence.

[0031] Figure 3. Purification of IL-13 / IL-17AF multispecific antibody.

[0032] (A) BEH200 SEC-UPLC analysis of purified multispecific antibodies by FLR detection.

[0033] (B) Protein samples separated by Tris-glycine SDS-PAGE under non-reducing (lane 1) or reducing (lane 2) conditions. The gels were stained with Coomassie rapid staining agent and destained in dH₂O. Mark 12 protein markers (Life Technologies) were used as standards (M). Molecular weight (MW) was measured in kilodaltons (kDa).

[0034] Figure 4. Inhibition of STAT6 signaling by IL-13 / IL-17AF multispecific antibody.

[0035] Figure 5.

[0036] (A) Response to human or cynomolgus monkey IL-17A in combination with TNF-α, with inhibition of IL-6 production via an IL-13 / IL-17AF multispecific antibody. (i) Human IL-17A. (ii) Cynomolgus monkey IL-17A.

[0037] (B) Response to human or cynomolgus monkey IL-17F in combination with TNF-α, with inhibition of IL-6 production via an IL-13 / IL-17AF multispecific antibody. (i) Human IL-17F. (ii) Cynomolgus monkey IL-17F.

[0038] Figure 6. In the NHEK CXCL1 release bioassay, IL-13, IL-17A and IL-17F were simultaneously neutralized by an IL-13 / IL-17AF multispecific antibody.

[0039] legend:

[0040] Circle = Anti-IL-13 / IL-17AF

[0041] Square = Anti-IL-17A

[0042] An upward-pointing triangle = anti-IL-17F

[0043] A downward-pointing triangle = anti-IL-13

[0044] Figure 7 . A diagrammatic illustration of the multispecific IL-13 / IL-17AF antibody according to the present invention. Invention Details

[0046] IL-13

[0047] IL-13 is a short-chain cytokine that shares 25% sequence identity with IL-4. It comprises approximately 132 amino acids, forming a secondary structure consisting of four helices spanning residues 10-21 (helix A), 43-52 (helix B), 61-69 (helix C), and 92-110 (helix D), and two β chains spanning residues 33-36 and 87-90. The solution structure of IL-13 has been resolved, revealing a predicted up-up-down-down four-helix bundle conformation, which has also been observed in IL-4 (Eisenmesser 2001).

[0048] Human IL-13 is a 17kDa 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. Functions of IL-13 include converting immunoglobulin isoforms to IgE in human B cells and inhibiting the production of inflammatory cytokines in humans and mice.

[0049] IL-13 binds to its cell surface receptors IL-13R-α1 and IL-13R-α2. IL-13R-α1 binds with low affinity (K0.05). D (Approximately 10 nM) interacts with IL-13, subsequently recruiting IL-4R-α to form a high-affinity (K) D (Approximately 0.4 nM) signal transduction heterodimer receptor complex.

[0050] 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 activates multiple signal transduction pathways, including the STAT6 signal transduction and transcription activator pathway and the insulin receptor substrate 2 (IRS2) pathway.

[0051] The standalone IL-13R-α2 chain has a high affinity for IL-13 (K D It is approximately 0.25-0.4 nM. It functions both as a trapping receptor that negatively regulates IL-13 binding and as a signal transduction receptor in macrophages and other possible cell types, inducing TGF-β synthesis and fibrosis via the AP-1 pathway.

[0052] IL-13 is involved in the pathogenesis of many human diseases, and therapeutic strategies have been devised to inhibit or counteract its activity. In particular, the search has focused on antibodies that bind to and neutralize IL-13 as a means of inhibiting its activity. However, there is a need in the art for suitable and / or improved antibodies capable of binding to IL-13, particularly human IL-13, and especially capable of neutralizing human IL-13.

[0053] This invention provides a novel family of binding proteins, CDR transplantation antibodies, humanized antibodies and their fragments, which can bind to human IL-13 with high affinity and bind to and neutralize human IL-13.

[0054] Antibodies that inhibit IL-13 activity can operate through several possible mechanisms of action. Cage 1 represents an antibody that binds to human IL-13 and prevents IL-13Rα1 binding, thus also blocking IL-4R binding. Cage 1 antibodies can also prevent IL-13 from binding to IL-13Rα2. Cage 2 represents an antibody that binds to hIL-13 in a manner that allows binding to IL-13Rα1 but prevents IL-4R recruitment into the complex. We selected the antibody that functions through Cage 1.

[0055] In one embodiment, the multispecific antibody binds to human IL-13 and prevents the binding of IL-13Rα1.

[0056] In one embodiment, the multispecific antibody binds to human IL-13 and prevents the binding of IL-13Rα2.

[0057] In one embodiment, the multispecific antibody binds to human IL-13 and prevents the binding of IL-13Rα1 and IL-13Rα2.

[0058] In one embodiment, the multispecific antibody is at <100 pM K D It binds to human IL-13.

[0059] IL-17

[0060] Based on structural similarity, the IL-17 cytokine family consists of six members with molecular weights ranging from 23 to 36 kDa and exhibiting a dimer structure. The founding member, IL-17A (often simply referred to as IL-17 in the literature), shares 16%–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 have the highest homology (50%) and bind to the same receptor complex, thus sharing common biological activities 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 lowest similarity to IL-17A. An important finding relating to the biological activities of IL-17A and IL-17F is that they share the same IL-17RA / IL-17RC receptor complex, with IL-17A exhibiting the highest affinity for IL-17RA, while IL-17F binds more strongly to IL-17RC. Another family member utilizing IL-17RA is IL-17E, which transduces signals via the IL-17RA / IL-17RB receptor complex.

[0061] IL-17A and IL-17F are produced by the Th17 subset of CD4+ T cells. In addition, other T cell subsets also produce IL-17A and IL-17F, including cytotoxic CD8+ T cells (Tc17), gdT cells, and NK T cells. Other cell populations reported to secrete IL-17A include neutrophils, monocytes, NK cells, lymphotriene-inducing factor-like (LTi-like) cells, intestinal paneth cells, and even B cells and mast cells. Furthermore, epithelial cells have also been reported to secrete IL-17F.

[0062] Cell types responding to the IL-17 cytokine are reflected by the expression of different receptors. IL-17RA is expressed ubiquitously, with particularly high levels in hematopoietic tissues, while IL-17RC is expressed more highly in non-immune cells of the joints, liver, kidneys, thyroid, and prostate. This differential expression may explain the differences in the biological activity of IL-17A and IL-17F, as cells expressing high levels of IL-17RC may be more responsive to IL-17F, while cells expressing higher levels of IL-17RA than IL-17RC may be more responsive to IL-17A. Specific cell types responding 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, as well as macrophages.

[0063] The multispecific antibody of the present invention can bind to human IL-17A and / or IL-17F. Therefore, the antibody can bind to IL-17A homodimer, IL-17F homodimer and / or IL-17AF heterodimer.

[0064] 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 IL-17F.

[0065] In one embodiment, the multispecific antibody is at <50 pM K D It binds to human IL-17A. In one embodiment, the multispecific antibody is administered at <25 pM K. D It binds to human IL-17A. In one embodiment, the multispecific antibody is administered at <10 pM K. D It binds to human IL-17A.

[0066] In one embodiment, the multispecific antibody is at <200 pM K D It binds to human IL-17F. In one embodiment, the multispecific antibody is at <100 pM K D It binds to human IL-17F.

[0067] albumin

[0068] Antibodies' high specificity and affinity make them ideal diagnostic and therapeutic agents, particularly for modulating protein-protein interactions. However, antibodies may be affected by increased serum clearance, especially when they lack the Fc domain that confers long lifespan in vivo (Medasan et al., 1997, J. Immunol. 158: 2211-2217).

[0069] Methods for improving antibody half-life are known. One approach is to conjugate the fragment with a polymer molecule. Thus, the short circulating half-life of the Fab′, F(ab′)2 fragment in animals has been improved by conjugation with polyethylene glycol (PEG; see, e.g., WO98 / 25791, WO99 / 64460, and WO98 / 37200). Another approach is to modify the antibody fragment by conjugating it with an agent that interacts with the FcRn receptor (see, e.g., WO97 / 34631). Yet another method for extending half-life is the use of peptides that bind to serum albumin (see, e.g., Smith et al., 2001, Bioconjugate Chem. 12:750-756; EP0486525; US6267964; WO04 / 001064; WO02 / 076489; and WO01 / 45746).

[0070] 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).

[0071] 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, have 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 previously described an anti-albumin antibody with improved humanization in WO2013 / 068571.

[0072] The multispecific antibody of the present invention can be engineered to bind to human serum albumin, thereby prolonging its serum half-life in vivo and improving its pharmacokinetic profile.

[0073] Antibody

[0074] Antibodies used in the context of this disclosure include whole antibodies and their functionally active fragments, i.e., molecules that specifically bind to IL-13, IL-17A, and / or IL-17F, also known as antigen-binding fragments. Unless the context otherwise requires, the characteristics of antibodies described herein also apply to antibody fragments.

[0075] Holoantibodies, also known as "immunoglobulins (Ig)," typically refer to intact or full-length antibodies, which are elements consisting of two heavy chains and two light chains linked together by disulfide bonds, assembling to form a characteristic Y-shaped three-dimensional structure. Classical natural holoantibodies are monospecific because they bind to one type of antigen and bivalent because they have two separate antigen-binding domains. The terms "intact 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.

[0076] Each light chain consists of a light chain variable region (abbreviated as V in this article). L ) and light chain constant region (C L Each heavy chain consists of a heavy chain variable region (abbreviated as V in this article). H The antibody consists of a heavy chain constant region (CH), which is composed 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 antibody constant region mediates 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 classical complement system.

[0077] V of the antibody according to the present invention H and V L The region can be further subdivided into highly variable regions (or “hypervariable regions”) that determine antigen recognition, called complementarity-determining regions (CDRs), interspersed with structurally more conserved regions called framework regions (FRs). Each V H and V LIt consists of 3 CDRs and 4 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. By 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, while 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.

[0078] CDRs are typically numbered according to the system designed by Kabat et al., described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al. (see above)"). Unless otherwise stated, this numbering system is used in this specification.

[0079] Kabat residue designations do not always directly correspond to linear amino acid residue numbers. The actual linear amino acid sequence can contain fewer or additional amino acids than a strict Kabat number, corresponding to shortening or insertion of structural components (whether framework regions or complementarity-determining regions) in a basic variable domain structure. The correct Kabat residue number for a given antibody can be determined by comparing homologous residues in the antibody sequence to a “standard” Kabat number sequence.

[0080] According to the Kabat numbering system, the CDR of the variable domain in the heavy chain is located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3). However, according to Chothia (Chothia, C. and Lesk, AM J Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Therefore, unless otherwise stated, “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 Chothia’s definition of a topological loop.

[0081] According to the Kabat numbering system, the CDRs of the variable domains in the light chain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2), and residues 89-97 (CDR-L3).

[0082] In addition to the CDR ring, a 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 66-94 in the heavy chain and positions 57-88 in the light chain.

[0083] Numbering schemes based on sequence alignment of different members of the immunoglobulin family have been proposed and described, for example, in Kabat et al., 1991 and Dondelinger et al., 2018, Frontiers in Immunology, Vol. 9, Article 2278.

[0084] As used herein, the terms "constant domain" and "constant region" are used interchangeably to refer to the domains of an antibody located outside the variable region. The constant domain is identical across all antibodies of the same isotype, but differs from isotype to isotype. Typically, the constant region of the heavy chain, from the N-terminus to the C-terminus, is formed by a CH1-hinge-CH2-CH3-optionally CH4 and contains three or four constant domains.

[0085] The constant domains of the antibody molecules of this invention, if present, can be selected based on the proposed function of the antibody molecule, particularly the effector function that may be required. For example, the constant domains can be human IgA, IgD, IgE, IgG, or IgM domains. In particular, when the antibody molecule is intended for therapeutic use and requires antibody effector function, the human IgG constant domain, especially the constant domains of the IgG1 and IgG3 isotypes, can be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and does not require antibody effector function, the IgG2 and IgG4 isotypes can be used. It should be understood that sequence variants of these constant domains can also be used. For example, an IgG4 molecule in which serine at position 241 (numbered according to the Kabat numbering system) has been changed to proline can be used, as described in Angal et al. (Angal et al., 1993. A single aminoacid 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.

[0086] The terms “Fc,” “Fc fragment,” “Fc domain,” and “Fc region” are used interchangeably to refer to the C-terminal region of an antibody, which contains the antibody’s constant regions, 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, along with the flexible hinges at the N-termini of these domains. The human IgG1 heavy chain Fc region is defined herein as containing residue C226 at its C-terminus, where the numbering is based on EU indexing as described in Kabat. In the context of human IgG1, according to EU indexing as described in Kabat, the lower hinge refers to positions 226-236, the CH2 domain to positions 237-340, and the CH3 domain to positions 341-447. The corresponding Fc regions of other immunoglobulins can be identified by sequence alignment.

[0087] In the context of this disclosure, the constant region or Fc region, when present, can be native, as defined above, or can be modified in various ways, provided that it contains a functional FcR-binding domain, and preferably a functional FcRn-binding domain. Preferably, the modified constant region or Fc region improves functionality and / or pharmacokinetics. These modifications may include the deletion of certain portions of the Fc fragment. These modifications may also include various amino acid substitutions capable of affecting the biological properties of the antibody. Mutations that enhance FcRn binding, thereby prolonging the in vivo half-life, may also be present. Modifications may further include modifications to the antibody's glycosylation characteristics. The native Fc fragment is glycosylated in the CH2 domain, and each of the two heavy chains contains an N-glycan that binds to the asparagine residue at position 297 (Asn297). In the context of this disclosure, the antibody can be glycomodified, i.e., engineered to have specific glycosylation characteristics, for example, which produces improved properties, such as improved effector function or improved serum half-life.

[0088] The antibodies described in this article are isolated. An “isolated” antibody is an antibody that has been separated from its components in its natural environment (e.g., through purification).

[0089] The term "antibody" includes 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.

[0090] The term "antibody" according to the present invention also includes an antigen-binding fragment of the 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 nanobodies (e.g., V...). H or VL Or V HH or V NAR Other antibody fragments used in this invention include the Fab and Fab' fragments described in international patent applications WO2011 / 117648, WO2005 / 003169, WO2005 / 003170 and WO2005 / 003171.

[0091] The methods used to create and prepare these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0092] As used in this article, the term "Fab fragment" refers to an antibody fragment containing a V-type light chain. L Light chain segments of (variable light chain) structural domains and constant structural domains (CL), and V of heavy chains. H (Variable heavy chain) domain and first constant domain (CH1).

[0093] A typical "Fab" fragment consists of a pair of heavy and light chains, where the heavy chain contains the variable region V. H It contains a constant structural domain CH1 and a natural or modified hinge region, and the light chain includes a variable region V. L and constant structural domain CL. The dimer of Fab' according to this disclosure produces F(ab')2, wherein dimerization can, for example, be carried out via a hinge.

[0094] As used herein, the term "single-domain antibody" refers to an antibody fragment composed of a single monomeric variable antibody domain. Examples of single-domain antibodies include V H or V L Or V H H or V-NAR.

[0095] The term "Fv" refers to two mutable domains, such as cooperating mutable domains, like cognate pairs, or mutable domains with maturing affinity, i.e., V. H and V L right.

[0096] As used in this article, "single-chain variable segment" or "scFv" refers to a single-chain variable segment that contains or constitutes a heavy-chain variable structural domain (V). H ) and light chain variable structural domain (V L ), which is composed of V H and V L Peptide linkers between variable domains provide stability. H and V LA variable domain can be in any suitable orientation, such as V. H The C-terminus can be connected to the V L The N-terminus, or V L The C-terminus can be connected to the V H The N-terminus.

[0097] As used in this article, "disulfide-stabilized single-chain variable fragment" or "dsscFv" refers to a single-chain variable fragment composed of V H and V L Peptide linkers between variable domains are used for stabilization, and also include V H and V L Interdomain disulfide bonds (see, for example, Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012 and WO2007109254).

[0098] As used in this article, "disulfide-stabilized variable fragment" or "dsFv" refers to a single-chain variable fragment that, in V H and V L The variable domains do not include peptide linkers, but are connected via V H and V L The disulfide bonds between the structural domains are used for stability.

[0099] 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, IL-17A, and / or IL-17F to their receptors.

[0100] The antibodies used in this invention may be, but are not limited to, monoclonal antibodies, humanized antibodies, fully human antibodies, or chimeric antibodies.

[0101] Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler and Milstein, 1975, Nature, 256:495-497), trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4:72) and EBV hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R Liss, Inc., 1985).

[0102] Alternatively, a monolymphocyte antibody approach can be used to generate antibodies by cloning and expressing cDNA of immunoglobulin variable regions produced from monolymphocytes selected for the production of specific antibodies, for example by the methods described below, namely 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.

[0103] Antibody screening can be performed using assays that measure binding to antigens and / or assays that measure the ability to block the binding of an antigen to one or more of its receptors. An example of a binding assay is an ELISA, for instance, using an IL-13 fusion protein immobilized on a plate and employing a conjugated secondary antibody to detect anti-IL-13 antibodies binding to IL-13. An example of a blocking assay is a flow cytometry-based assay that measures the blocking of the binding of IL-13 ligand protein to IL-13R. A fluorescently labeled secondary antibody is used to detect the amount of IL-13 ligand protein binding to IL-13R.

[0104] Humanized antibodies (including CDR-transplanted antibodies) are antibody molecules having one or more complementarity-determining regions (CDRs) derived from a 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 optionally further include one or more framework residues derived from a non-human species, the CDR of which is also derived.

[0105] Chimeric antibodies consist of elements derived from two different species, thus allowing the element to retain the characteristics of its source species. In general, a chimeric antibody will contain a variable region from one species (e.g., mouse, rat, rabbit, or similar species) and a constant region from another species (e.g., human).

[0106] Antibodies can also be generated using various phage display methods known in the art, including those disclosed by: Brinkman et al. (in 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 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.

[0107] A fully human antibody is one in which the variable and constant regions (if present) of both the heavy and light chains are of human origin, or substantially identical to human sequences, but not necessarily derived from the same antibody. Examples of fully human antibodies may include, for instance, antibodies produced by the phage display method described above, and antibodies produced by mice in which the variable and optional constant region genes of mouse immunoglobulins have been replaced with their human counterparts, such as those summarized in the following: 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.

[0108] Multispecific antibodies

[0109] The antibody of this invention is a multispecific antibody. As used herein, "multispecific or multi-specific 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 two different antigens or two different epitopes on the same antigen. For each specificity (antigen), multispecific antibodies are typically monovalent. The multispecific antibodies described herein include monovalent and multivalent, such as bivalent, trivalent, and quadrivalent multispecific antibodies.

[0110] Complementary sites are regions in an antibody that recognize and bind to antigens. The antibodies of this invention can be multi-complementary site antibodies. 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 epitopes from the same antigen or from two different antigens. Multi-complementary site antibodies described herein can be bi-complementary, tri-complementary, or tetra-complementary.

[0111] As used herein, "antigen-binding domain" refers to a portion of an antibody that contains part or all of one or more variable domains, such as a pair of variable domains V that specifically interact with the target antigen. H and V L Some or all of the binding domains may contain monovalent antibodies. In one embodiment, each binding domain is monovalent. Preferably, each binding domain contains no more than one VH and one VL.

[0112] As used herein, "specifically" refers to binding domains that recognize only their specific antigens, or binding domains that have a significantly higher binding affinity for their specific antigens compared to their affinity for non-specific antigens. Binding affinity can be measured using standard assays, such as surface plasmon resonance, like BIAcore.

[0113] A variety of multispecific antibody forms 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.

[0114] 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., WO2012058768). Other techniques for preparing bispecific antibodies have been 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-lgG, hand-in-well co-LC, hand-in-well assembly, charge pairs, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body, and orthogonal Fab.

[0115] Added IgG typically comprises full-length IgG, which is engineered by attaching an additional antigen-binding domain or antigen-binding fragment to the N- and / or C-termini of the IgG heavy and / or light chains. Examples of such additional antigen-binding fragments include sdAb antibodies (e.g., V...). H or V L), Fv, scFv, dsscFv, Fab, 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, lgC(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-lg, Zybody, 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.

[0116] Multispecific antibody fragments include nanobodies, nanobodies-HAS, BiTE, biantibodies, DART, TandAb, scDiabody, sc-biantibody-CH3, biantibody-CH3, triple bodies, microbodies; microantibodies, TriBi microantibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFV3, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, biantibody-Fc, tandem scFv-Fc; and intracellular antibodies, as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106.

[0117] Multispecific fusion proteins include Dock and Lock, ImmTAC, HSAbody, scDiabody-HAS, and tandem scFv-toxin.

[0118] Multispecific antibody conjugates include IgG-IgG; Cov-X-Body; and scFv1-PEG-scFv2.

[0119] 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, triantibodies, Fab-VHH, taFv-Fc, scFv4-Ig, scFv2-Fcab, and scFv4-IgG. For example, biantibodies, triantibodies, and methods for their preparation are disclosed in WO99 / 37791.

[0120] This invention provides a multispecific antibody that binds to human IL-13, human IL-17A and / or human IL-17F.

[0121] In one embodiment, the multispecific antibody includes an antigen-binding site that binds to human IL-13, wherein the IL-13 binding site includes a light chain variable region comprising the sequence given in SEQ ID NO:15 for CDR-L1, the sequence given in SEQ ID NO:16 for CDR-L2, and the sequence given in SEQ ID NO:17 for CDR-L3.

[0122] In one embodiment, the multispecific antibody includes an antigen-binding site that binds to human IL-13, wherein the IL-13 binding site includes a heavy chain variable region comprising the sequence given in SEQ ID NO:18 for CDR-H1, the sequence given in SEQ ID NO:19 for CDR-H2, and the sequence given in SEQ ID NO:20 for CDR-H3.

[0123] In one embodiment, the IL-13 binding site includes a light chain variable region, which is contained in the sequence given in SEQ ID NO:27.

[0124] In one embodiment, the IL-13 binding site includes a heavy chain variable region, which is contained in the sequence given in SEQ ID NO:28.

[0125] In one embodiment, the IL-13 binding site includes a light chain variable region, which is contained in the sequence given in SEQ ID NO:31.

[0126] In one embodiment, the IL-13 binding site includes a heavy chain variable region, which is contained in the sequence given in SEQ ID NO:32.

[0127] In one embodiment, the multispecific antibody includes an antigen-binding site that binds to human IL-17A and human IL-17F, the antigen-binding site comprising:

[0128] The light chain variable region includes the sequence given in SEQ ID NO:1 for CDR-L1, the sequence given in SEQ ID NO:2 for CDR-L2, and the sequence given in SEQ ID NO:3 for CDR-L3.

[0129] In one embodiment, the multispecific antibody includes an antigen-binding site that binds to human IL-17A and human IL-17F, the antigen-binding site comprising:

[0130] The heavy chain variable region includes the sequence given in SEQ ID NO:4 for CDR-H1, the sequence given in SEQ ID NO:5 for CDR-H2, and the sequence given in SEQ ID NO:6 for CDR-H3.

[0131] In one embodiment, the antigen binding site that binds to human IL-17A and human IL-17F includes a light chain variable region, which contains the sequence given in SEQ ID NO:7.

[0132] In one embodiment, the antigen-binding site that binds to human IL-17A and human IL-17F includes a heavy chain variable region, which contains the sequence given in SEQ ID NO:9.

[0133] In one implementation, the multispecific antibody lacks an Fc domain, and its half-life is provided by an antigen-binding site that binds to serum albumin.

[0134] In one embodiment, the multispecific antibody comprises the sequence given in SEQ ID NO:57 or SEQ ID NO:59.

[0135] In one embodiment, the multispecific antibody comprises the sequence given in SEQ ID NO:61 or SEQ ID NO:63.

[0136] In one embodiment, the multispecific antibody comprises the sequence given in SEQ ID NO:59 and the sequence given in SEQ ID NO:63.

[0137] In one implementation, the multispecific antibody comprises or is composed of the following:

[0138] The polypeptide chain of formula (I):

[0139] V H-CH1-(CH2)s-(CH3)tX-(V1)p; and

[0140] The polypeptide chain of formula (II):

[0141] V L -C L -Y-V2;

[0142] in:

[0143] V H Represents a heavy-chain variable structural domain;

[0144] CH1 represents domain 1 of the heavy chain constant region;

[0145] CH2 represents domain 2 of the heavy chain constant region;

[0146] CH3 represents domain 3 of the heavy chain constant region;

[0147] X represents a key or connector;

[0148] V1 represents dsscFv, dsFv, or scFv;

[0149] V L Represents a variable structural domain in a light chain;

[0150] C L Domains representing constant regions from light chains, such as Cκ;

[0151] Y represents a key or connector;

[0152] V2 represents dsscFv, dsFv, or scFv;

[0153] p represents 0 or 1;

[0154] s represents 0 or 1;

[0155] t represents 0 or 1;

[0156] Where X does not exist when p is 0, and Y does not exist when q is 0; and

[0157] The polypeptide chain of formula (I) contains a protein A binding domain; and

[0158] In formula (II), the polypeptide chain does not bind to protein A.

[0159] In one embodiment, when s is 0 and t is 0, the multispecific antibody according to this disclosure is provided as a dimer of the following heavy and light chains:

[0160] These are equations (I) and (II), respectively, where V H -CH1 part and VL -C L Some of them together form a functional Fab or Fab' fragment.

[0161] In one embodiment, when s is 1 and t is 1, the multispecific antibody according to this disclosure is provided as a dimer of the following two heavy chains and two light chains:

[0162] These are equations (I) and (II), respectively, in which two heavy chains are connected by interchain interactions, particularly at the CH2-CH3 level, and in which the V of each heavy chain... H -CH1 portion and V of each light chain L -C L Some of them together form a functional Fab or Fab' fragment. In this type of implementation, two V H The -CH1-CH2-CH3 part and two V L -C L Some of these components together form a functional full-length antibody. In this type of implementation, the full-length antibody may include a functional Fc region.

[0163] V H V represents a heavy-chain variable structural domain. In one implementation, V H It is humanized. In one implementation scheme, V H It is holistic.

[0164] V L V represents a variable structural domain of a light chain. In one implementation, V L It is humanized. In one implementation scheme, V L It is holistic.

[0165] Generally speaking, V H and V L Together they form an antigen-binding domain. In one implementation, V H and V L Form similar pairs.

[0166] As used in this article, "like-type pair" refers to a pair of variable domains derived from a single antibody, produced in vivo, that is, a naturally occurring pairing of variable domains isolated from the host. Therefore, like-type pairs are V H and V L Yes. In one example, similar pairs cooperate to bind to the antigen.

[0167] As used in this article, "variable region" or "variable structural domain" refers to the region in the antibody chain that contains CDRs and frameworks, especially suitable frameworks.

[0168] The variable regions used in this disclosure are typically derived from antibodies, which can be produced by any method known in the art.

[0169] As used in this article, “derived from / derived from” refers to the fact that the sequence used or a sequence highly similar to the sequence used was obtained from the original genetic material, such as from the light or heavy chain of the antibody.

[0170] As used in this article, “highly similar” means having 95% or higher similarity in their entire length, such as 96%, 97%, 98%, or 99% similar amino acid sequences.

[0171] As mentioned above regarding V H and V L The variable region used in this invention can come from any suitable source and can be, for example, fully human or humanized.

[0172] In one implementation scheme, by V H and V L The resulting binding domain is specific to the first antigen.

[0173] In one implementation, the binding domain of V1 is specific for the second antigen.

[0174] In one implementation, the binding domain of V2 is specific for the third antigen.

[0175] In one implementation scheme, V H -V L Each of V1 and V2, if present, binds to its respective antigen.

[0176] In one embodiment, the CH1 domain is a naturally occurring domain 1 derived from the antibody heavy chain or a derivative thereof. In one embodiment, the CH2 domain is a naturally occurring domain 2 derived from the antibody heavy chain or a derivative thereof. In one embodiment, the CH3 domain is a naturally occurring domain 3 derived from the antibody heavy chain or a derivative thereof.

[0177] In one implementation, C in the light chain L The fragment is a constant κ sequence or a derivative thereof. In one embodiment, the C in the light chain L The fragment is a constant λ sequence or its derivative.

[0178] As used herein, a derivative of a naturally occurring domain is intended to refer to a situation where at least one amino acid in the naturally occurring sequence has been substituted or deleted, for example, to optimize the properties of the domain, such as by eliminating unwanted properties, while retaining one or more of the domain's typical characteristics. In one embodiment, the derivative of the naturally occurring domain comprises 2, 3, 4, 5, 6, 7, 8, 10, 11, or 12 amino acid substitutions or deletions compared to the naturally occurring sequence.

[0179] In one implementation, one or more natural or engineered interchain (i.e., between light and heavy chains) disulfide bonds are present in the functional Fab or Fab' fragment.

[0180] In one implementation, the "natural" disulfide bonds are present at CH1 and C in the polypeptide chains of formulas (I) and (II). L between.

[0181] When C L When the domain originates from κ or λ, the native position of the cysteine ​​residue forming the bond is 214 in human cκ and cλ (Kabat No. 4, 1987).

[0182] The exact position of the cysteine ​​residue forming the disulfide bond 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 4, 1987). For other human isotypes such as γ2, 3, 4, IgM, and IgD, the positions of the cysteine ​​residues forming the bond 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.

[0183] Optionally, in the V of the polypeptides of formulas I and II H and V L Disulfide bonds can exist between them.

[0184] In one embodiment, the multispecific antibody according to this disclosure is equivalent to or corresponds to that naturally present in CH1 and C2. L There is a disulfide bond at the position between them.

[0185] In one implementation, CH1 and constant regions such as C are included. L The constant region of the molecule contains disulfide bonds located at non-naturally occurring positions. This can be engineered into the molecule by introducing one or more cysteine ​​residues into one or more desired positions in the amino acid chain. These non-natural disulfide bonds are present at CH1 and C2 positions. L The natural disulfide bond between cysteine ​​residues can be supplemented or replaced. Cysteine ​​residues in their natural position can be replaced by amino acids that cannot form disulfide bonds, such as serine.

[0186] The engineered introduction of cysteine ​​can be performed using any method known in the art. These methods include, but are not limited to, PCR extension overlap mutagenesis, site-directed mutagenesis, or cassette mutagenesis (see generally 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, for example... 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 annealing, ligation, and PCR amplification, as well as the full-gene synthesis of overlapping oligonucleotide clones.

[0187] In one implementation, CH1 and C L Inter-chain disulfide bonds are completely absent; for example, inter-chain cysteine ​​can be replaced by another amino acid such as serine. Therefore, in one embodiment, inter-chain disulfide bonds are absent in the functional Fab fragment of the molecule. How to provide Fab fragments without inter-chain disulfide bonds is described by reference to disclosures incorporated herein by reference, such as WO2005 / 003170.

[0188] Examples of antibody forms used in this invention include added IgG and added Fab, wherein at least one additional antigen-binding domain (e.g., one, two, three, or four additional antigen-binding domains), such as single-domain antibodies (e.g., V... H or V LAlternatively, VHH), scFv, dsscFv, and dsFv can be attached to the N- and / or C-terminus of the IgG or Fab light chain, and optionally to the heavy chain of the IgG or Fab, to engineer whole IgG or Fab fragments, as described, for example, in WO2009 / 040562, WO2010035012, WO2011 / 030107, WO2011 / 061492, WO2011 / 061246, and WO2011 / 086091, all of which are incorporated herein by reference. The addition of full-length IgG to an IgG fragment is first disclosed in WO2015 / 197789, which is incorporated herein by reference, wherein the full-length IgG is engineered by attaching dsFv to the C-terminus of the IgG light chain (and optionally to the heavy chain).

[0189] Preferred antibody forms used in this invention comprise Fabs 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 one scFv or dsscFv extending its half-life by binding to, for example, albumin). Such antibody fragments are described in International Patent Application Publication No. WO2015 / 197772, the entire contents of which are incorporated herein by reference, particularly concerning the discussion of antibody fragments.

[0190] V1 represents dsscFv, dsFv, or scFv.

[0191] V2 represents dsscFv, dsFv, or scFv.

[0192] In one implementation, when V1 and / or V2 are dsFv or dsscFv, the variable structural domain V of V1 and / or V2 H and V L The disulfide bond between the residues is located between the two residues listed below (unless the context otherwise requires, Kabat numbers will be used in the list below). Wherever Kabat numbers are mentioned, the relevant reference is Kabat et al., 1991 (5th edition, Bethesda, Md.), Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, NIH, USA.

[0193] In one embodiment, the disulfide bond is located at a position selected from the group consisting of:

[0194] ·V H 37+V L95C, see, for example, Protein Science 6, 781-788, Zhu et al. (1997);

[0195] ·V H 44+V L 100, see, for example, Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012;

[0196] ·V H 44+V L 105, see, for example, J Biochem. 118, 825-831, Luo et al. (1995);

[0197] ·V H 45+V L 87, see, for example, Protein Science 6, 781-788, Zhu et al. (1997);

[0198] ·V H 55+V L 101, see, for example, FEBS Letters 377 135-139 Young et al. (1995);

[0199] ·V H 100+V L 50, see, for example, Biochemistry 29 1362-1367 Glockshuber et al. (1990);

[0200] ·V H 100b+V L 49, see, for example, Biochemistry 29 1362-1367 Glockshuber et al. (1990);

[0201] ·V H 98+V L 46. ​​See, for example, Protein Science 6, 781-788, Zhu et al. (1997);

[0202] ·V H 101+V L 46. ​​See, for example, Protein Science 6, 781-788, Zhu et al. (1997);

[0203] ·V H 105+V L43. 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).

[0204] ·V H 106+V L 57, see, for example, FEBS Letters 377 135-139, Young et al. (1995),

[0205] And the corresponding positions in the variable regions located in the molecule.

[0206] In one implementation, the disulfide bond is at position V. H 44 and V L Formed between 100.

[0207] The amino acid pairs listed above are located at positions favorable for cysteine ​​substitution, thereby allowing disulfide bonds to form. Cysteines can be engineered to these desired positions using known techniques. Therefore, in one embodiment, engineered cysteine ​​according to this disclosure refers to a naturally occurring residue at a given amino acid position that has been replaced with a cysteine ​​residue.

[0208] The engineered introduction of cysteine ​​can be performed using any method known in the 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, for example... 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 annealing, ligation, and PCR amplification, as well as the full-gene synthesis of overlapping oligonucleotide clones.

[0209] Accordingly, in one implementation, when V1 and / or V2 are dsFv or dsscFv, the variable structural domain V of V1 H and V Land / or the variable structural domain V of V2 H and V L They can be linked by a disulfide bond between two cysteine ​​residues, wherein the position of the cysteine ​​residue pair is selected from the following group: V H 37 and V L 95. V H 44 and V L 100, V H 44 and V L 105, V H 45 and V L 87. V H 100 and V L 50, V H 100b and V L 49. V H 98 and V L 46. ​​V H 101 and V L 46. ​​V H 105 and V L 43 and V H 106 and V L 57.

[0210] In one implementation, when V1 and / or V2 are dsFv or dsscFv, the variable structural domain V of V1 H and V L and / or the variable structural domain V of V2 H and V L It can be passed through two cysteine ​​residues (one in V) H In the middle, one in V L The disulfide bond between the two cysteine ​​residues (CDR) is located outside the CDR, and the position of the cysteine ​​residue pair is selected from the following group: V H 37 and V L 95. V H 44 and V L 100, V H 44 and V L 105, V H 45 and V L 87. V H 100 and V L 50, V H 98 and V L 46. ​​V H 105 and V L 43 and V H 106 and V L 57.

[0211] In one implementation, when V1 is dsFv or dsscFv, the variable structural domain V of V1H and V L Linked by a disulfide bond between two engineered cysteine ​​residues, one in V H 44-bit, another in V L 100 bits. In one implementation, when V2 is dsFv or dsscFv, the variable structure domain V of V2... H and V L Linked by a disulfide bond between two engineered cysteine ​​residues, one in V H 44-bit, another in V L 100 people.

[0212] In one implementation, when V1 is dsscFv, dsFv, or scFv, V1's V H The structural domain is attached to X.

[0213] In one implementation, when V1 is dsscFv, dsFv, or scFv, V1's V L The structural domain is attached to X.

[0214] In one implementation, when V2 is dsscFv, dsFv, or scFv, V2's V H The structural domain is attached to Y.

[0215] In one implementation, when V2 is dsscFv, dsFv, or scFv, V2's V L The structural domain is attached to Y.

[0216] Technicians will understand that when V1 and / or V2 represent dsFv, the multispecific antibody will contain the corresponding free V. H or V L The third polypeptide of the V domain H or V L The domain is not attached to X or Y. When V1 and V2 are dsFv, then the "free variable domain" (i.e., the domain that is linked to the rest of the polypeptide via a disulfide bond) will be common to both chains. Therefore, although the actual variable domain fused or linked to the polypeptide via X or Y may differ in each polypeptide chain, the free variable domain that it is paired with is usually the same to each other.

[0217] In some implementations, p is 1. In some implementations, p is 0.

[0218] In some implementations, s is 1. In some implementations, s is 0.

[0219] In some implementations, t is 1. In some implementations, t is 0.

[0220] In some implementations, s is 1 and t is 1. In some implementations, s is 0 and t is 0.

[0221] 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 aspect, a multispecific antibody binding human IL-13, human IL-17A and / or human IL-17F is provided, comprising or composed of the following:

[0222] a) Polypeptide chain of formula (Ia):

[0223] V H -CH1-X-V1; and

[0224] b) Polypeptide chain of formula (IIa):

[0225] V L -C L -Y-V2;

[0226] in:

[0227] V H Represents a heavy-chain variable structural domain;

[0228] CH1 represents domain 1 of the heavy chain constant region;

[0229] X represents a key or connector;

[0230] Y represents a key or connector;

[0231] V1 represents scFv, dsscFv, or dsFv;

[0232] V L Represents a variable structural domain in a light chain;

[0233] C L Domains representing the constant regions of light chains, such as Cκ;

[0234] V2 represents scFv, dsscFv, or dsFv;

[0235] At least one of V1 or V2 is dsscFv or dsFv;

[0236] The polypeptide chain of formula (Ia) contains a protein A binding domain; and

[0237] In formula (IIa), the polypeptide chain does not bind to protein A.

[0238] In such embodiments, V2 does not bind protein A, meaning that the scFv, dsscFv, or dsFv of V2 does not contain a protein A-binding domain. In one embodiment, V2, i.e., the scFv, dsscFv, or dsFv of V2, contains a VH1 domain. In another embodiment, V2, i.e., the scFv, dsscFv, or dsFv of V2, contains a VH3 domain that does not bind protein A. In one embodiment, V2, i.e., the scFv, dsscFv, or dsFv of V2, contains a VH2 domain. In one embodiment, V2, i.e., the scFv, dsscFv, or dsFv of V2, contains a VH4 domain. In one embodiment, V2, i.e., the scFv, dsscFv, or dsFv of V2, contains a VH5 domain. In one embodiment, V2, i.e., the scFv, dsscFv, or dsFv of V2, contains a VH6 domain. In one implementation, the polypeptide chain of formula (Ia) contains only one present in V. H Or a protein A binding domain in V1. In one embodiment, the polypeptide chain of formula (Ia) contains only one protein A binding domain present in V1. In another embodiment, the polypeptide chain of formula (Ia) contains a protein A binding domain present in V1, respectively. H It binds to the two protein A domains in V1.

[0239] In another embodiment, p is 0, q is 1, r is 0, s is 1, t is 1, and V2 is dsscFv. Therefore, in one aspect, a multispecific antibody binding human IL-13, human IL-17A, and / or human IL-17F is provided, and it comprises or is composed of the following:

[0240] a) Polypeptide chain of formula (Ib):

[0241] V H -CH1-CH2-CH3; and

[0242] b) Polypeptide chain of formula (IIb):

[0243] V L -C L -Y-V2;

[0244] in:

[0245] V H Represents a heavy-chain variable structural domain;

[0246] CH1 represents domain 1 of the heavy chain constant region;

[0247] CH2 represents domain 2 of the heavy chain constant region;

[0248] CH3 represents domain 3 of the heavy chain constant region;

[0249] Y represents a key or connector;

[0250] V L Represents a variable structural domain in a light chain;

[0251] C L Domains representing the constant regions of light chains, such as Cκ;

[0252] V2 stands for dsscFv;

[0253] The polypeptide chain of formula (Ib) contains a protein A binding domain; and

[0254] The polypeptide chain of formula (IIb) does not bind to protein A.

[0255] In such embodiments, V2 does not bind protein A, meaning the dsscFv of V2 does not contain a protein A-binding domain. In one embodiment, V2, i.e., the dsscFv of V2, contains a VH1 domain. In another embodiment, V2, i.e., 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 domain present in V... H Or a protein A binding domain in CH2-CH3. In another embodiment, the polypeptide chain of formula (Ib) comprises a protein A binding domain present in V. H It binds to two protein A domains in CH2-CH3.

[0256] In another embodiment, p is 0, q is 1, r is 0, s is 1, t is 1, and V2 is dsFv. Therefore, in one aspect, a multispecific antibody binding human IL-13, human IL-17A, and / or human IL-17F is provided, comprising or composed of the following:

[0257] a) Polypeptide chain of formula (Ic):

[0258] V H -CH1-CH2-CH3; and

[0259] b) Polypeptide chain of formula (IIc):

[0260] V L -C L -Y-V2;

[0261] in:

[0262] V H Represents a heavy-chain variable structural domain;

[0263] CH1 represents domain 1 of the heavy chain constant region;

[0264] CH2 represents domain 2 of the heavy chain constant region;

[0265] CH3 represents domain 3 of the heavy chain constant region;

[0266] Y represents a key or connector;

[0267] V L Represents a variable structural domain in a light chain;

[0268] C L Domains representing the constant regions of light chains, such as Cκ;

[0269] V2 represents dsFv;

[0270] The polypeptide chain of formula (Ic) contains a protein A binding domain; and

[0271] In formula (IIc), the polypeptide chain does not bind to protein A.

[0272] In this type of implementation, V2, i.e., dsFv of V2, does not bind to protein A. In one implementation, the polypeptide chain of formula (Ic) contains only one protein present in V. H Or a protein A binding domain in CH2-CH3. In another embodiment, the polypeptide chain of formula (Ic) comprises a protein A binding domain present in V. H It binds to two protein A domains in CH2-CH3.

[0273] In one embodiment of the multispecific antibody of the present invention,

[0274] V L and V H It contains antigen-binding sites that bind to human IL-17A and / or human IL-17F.

[0275] V1 contains an antigen-binding site that binds to human serum albumin, and

[0276] V2 contains an antigen-binding site that binds to human IL-13.

[0277] In one implementation scheme, V L Includes the sequence given in SEQ ID NO:1 for CDR-L1, the sequence given in SEQ ID NO:2 for CDR-L2, and the sequence given in SEQ ID NO:3 for CDR-L3; V H It includes the sequence given in SEQ ID NO:4 for CDR-H1, the sequence given in SEQ ID NO:5 for CDR-H2, and the sequence given in SEQ ID NO:6 for CDR-H3.

[0278] In one embodiment, V1 includes a light chain variable region and a heavy chain variable region, the light chain variable region including the sequence given in SEQ ID NO:39 for CDR-L1, the sequence given in SEQ ID NO:40 for CDR-L2, and the sequence given in SEQ ID NO:41 for CDR-L3; the heavy chain variable region including the sequence given in SEQ ID NO:42 for CDR-H1, the sequence given in SEQ ID NO:43 for CDR-H2, and the sequence given in SEQ ID NO:44 for CDR-H3.

[0279] In one embodiment, V2 includes a light chain variable region and a heavy chain variable region, the light chain variable region including the sequence given in SEQ ID NO:15 for CDR-L1, the sequence given in SEQ ID NO:16 for CDR-L2, and the sequence given in SEQ ID NO:17 for CDR-L3; the heavy chain variable region including the sequence given in SEQ ID NO:18 for CDR-H1, the sequence given in SEQ ID NO:19 for CDR-H2, and the sequence given in SEQ ID NO:20 for CDR-H3.

[0280] In one implementation scheme, V L Contains the sequence given in SEQ ID NO:7, and V H The sequence is included in SEQ ID NO:9.

[0281] In one embodiment, V1 includes a light chain variable region and a heavy chain variable region, the light chain variable region comprising the sequence given in SEQ ID NO:45 and the heavy chain variable region comprising the sequence given in SEQ ID NO:46.

[0282] In one embodiment, V1 includes a light chain variable region and a heavy chain variable region, the light chain variable region comprising the sequence given in SEQ ID NO:49 and the heavy chain variable region comprising the sequence given in SEQ ID NO:50.

[0283] In one embodiment, the light chain variable region and the heavy chain variable region of V1 are connected by a connector, the connector being contained in the sequence given in SEQ ID NO:68.

[0284] In one embodiment, V1 is either the scFv containing the sequence given in SEQ ID NO:53 or the dsscFv containing the sequence given in SEQ ID NO:55.

[0285] In one embodiment, V2 includes a light chain variable region and a heavy chain variable region, the light chain variable region comprising the sequence given in SEQ ID NO:27 and the heavy chain variable region comprising the sequence given in SEQ ID NO:28.

[0286] In one embodiment, V2 includes a light chain variable region and a heavy chain variable region, the light chain variable region comprising the sequence given in SEQ ID NO:31 and the heavy chain variable region comprising the sequence given in SEQ ID NO:32.

[0287] In one embodiment, the light chain variable region and the heavy chain variable region of V2 are connected by a connector, the connector being contained in the sequence given in SEQ ID NO:66.

[0288] In one embodiment, V2 is either the scFv containing the sequence given in SEQ ID NO:35 or the dsscFv containing the sequence given in SEQ ID NO:37.

[0289] In one embodiment, X is a connector contained in the sequence given in SEQ ID NO:67.

[0290] In one embodiment, Y is a connector contained in the sequence given in SEQ ID NO:65.

[0291] In one embodiment, the polypeptide chain of formula (Ia) comprises the sequence given in SEQ ID NO:57 or SEQ ID NO:59.

[0292] In one embodiment, the polypeptide chain of formula (IIa) comprises the sequence given in SEQ ID NO:61 or SEQ ID NO:63.

[0293] In one embodiment, the polypeptide chain of formula (Ia) comprises the sequence given in SEQ ID NO:59, and the polypeptide chain of formula (IIa) comprises the sequence given in SEQ ID NO:63.

[0294] 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 antigens and neutralize their biological activity. Those skilled in the art can readily test the effects of any amino acid substitution, addition, and / or deletion, for example, by using the methods described herein, particularly those illustrated in the examples, to determine antigen binding and inhibition of biological activity.

[0295] Accordingly, the present invention provides a multispecific antibody comprising a CDR defined by the sequences given 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 amino acids in one or more CDRs have been substituted for another amino acid, such as similar amino acids as defined below.

[0296] As used herein, “identity” means that the amino acid residues at any particular position in the aligned sequences are identical between sequences. As used herein, “similarity” means that the amino acid residues at any particular position in the aligned sequences are of a similar type between sequences. For example, leucine can substitute for isoleucine or valine. Other amino acids that can frequently substitute for each other include, but are not limited to:

[0297] - Phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains);

[0298] - Lysine, arginine, and histidine (amino acids with basic side chains);

[0299] - Aspartic acid and glutamic acid (amino acids with acidic side chains);

[0300] - Asparagine and glutamine (amino acids with amide side chains); and

[0301] - Cysteine ​​and methionine (amino acids with sulfur-containing side chains). The degree of identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, AM, eds., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, eds., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991, NCBI BLAST).TM Software (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)).

[0302] 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 given in SEQ ID NO:1, 2, 3, 4, 5, 6, 15, 16, 17, 18, 19, 20, 39, 40, 41, 42, 43, and 44.

[0303] In one implementation scheme, V L Contains sequences having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:7, and V H It includes sequences that have at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:9.

[0304] In one embodiment, V1 includes a light chain variable region and / or a heavy chain variable region, the light chain variable region containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:45, and the heavy chain variable region containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:46.

[0305] In one embodiment, V1 includes a light chain variable region and / or a heavy chain variable region, the light chain variable region containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:49, and the heavy chain variable region containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:50.

[0306] 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% identity or similarity to the sequence given in SEQ ID NO:68.

[0307] In one embodiment, V1 is scFv or dsscFv, the scFv containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:53, and the dsscFv containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:55.

[0308] In one embodiment, V2 includes a light chain variable region and / or a heavy chain variable region, the light chain variable region containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:27, and the heavy chain variable region containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:28.

[0309] In one embodiment, V2 includes a light chain variable region and / or a heavy chain variable region, the light chain variable region containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:31, and the heavy chain variable region containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:32.

[0310] 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% identity or similarity to the sequence given in SEQ ID NO:66.

[0311] In one embodiment, V2 is an scFv or dsscFv, the scFv containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:35, and the dsscFv containing a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:37.

[0312] In one embodiment, X is a connector that contains a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:67.

[0313] In one embodiment, Y is a connector that comprises a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:65.

[0314] 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 given in SEQ ID NO:57 or SEQ ID NO:59.

[0315] 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 given in SEQ ID NO:61 or SEQ ID NO:63.

[0316] 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 given in SEQ ID NO:59, and the polypeptide chain of formula (IIa) comprises a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence given in SEQ ID NO:63.

[0317] Epitope

[0318] An epitope is an antigenic region that an antibody binds to. An epitope can be defined as a structural or functional epitope. Functional epitopes are typically a subset of structural epitopes and contain residues that directly contribute to the interaction affinity. Epitopes can also be conformational epitopes, i.e., composed of nonlinear amino acids. In some embodiments, an epitope may comprise a determinant cluster as a chemically active surface cluster of a molecule (such as an amino acid, sugar side chain, phosphoryl group, or sulfonyl group), and in some embodiments, may have specific three-dimensional structural features and / or specific charge features.

[0319] Using conventional methods known in the art, it is readily possible to determine whether an antibody binds to the same epitope as a reference antibody, or whether it competes with the reference antibody for epitope binding. For example, to determine whether a test antibody binds to the same epitope as the reference antibody of the present invention, the reference antibody is allowed to bind to a protein or peptide under saturation conditions. The ability of the test antibody to bind to the protein or peptide is then evaluated. If, after saturation binding with the reference antibody, the test antibody is still able to bind to the protein or peptide, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if, after saturation binding with the reference antibody, the test antibody cannot bind to the protein or peptide, then the test antibody may bind to the same epitope as the reference antibody of the present invention.

[0320] To determine whether an antibody competitively binds to a reference antibody, 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 only the first (saturated) antibody can bind to the protein / peptide in both directions, it can be concluded that the test antibody competitively binds to the protein / peptide with the reference antibody. As understood by those skilled in the art, an antibody competitively binding to the reference antibody does not necessarily bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0321] If two antibodies competitively inhibit (block) the binding of another antibody to an antigen, then the two antibodies bind to the same or overlapping epitopes. 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 measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, if reducing or eliminating substantially all amino acid mutations in the antigen bound by one antibody reduces or eliminates the binding of the other antibody, then the two antibodies have the same epitope. If reducing or eliminating some amino acid mutations in the binding of one antibody reduces or eliminates the binding of the other antibody, then the two antibodies have overlapping epitopes.

[0322] Further routine experiments (e.g., peptide mutation and binding assays) can then be performed to confirm whether the observed lack of binding to the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric hindrance (or other phenomena) is the cause of the observed lack of binding. Such experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0323] The antibody can competitively bind to IL-17A or IL-17F, or bind to the same epitope, of the following multispecific antibodies comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequence combination of SEQ ID NO:1 / 2 / 3 / 4 / 5 / 6.

[0324] The antibody can competitively bind to IL-13, or bind to the same epitope as the following multispecific antibodies, which comprise 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.

[0325] The antibody can competitively bind to serum albumin, or bind to the same epitope as the following multispecific antibodies, which comprise 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.

[0326] Effector molecules

[0327] If desired, the multispecific antibodies used in this invention can be conjugated to one or more effector molecules. It should be understood that the effector molecule may comprise a single effector molecule or two or more such molecules linked together such that a single portion can be attached to the antibody of this invention. In cases where it is desired to obtain an antibody fragment linked to an effector molecule, it can be prepared by standard chemical or recombinant DNA procedures, wherein the antibody fragment is linked to the effector molecule directly or via a conjugating agent. Techniques for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58; and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemical procedures include, for example, those described in WO 93 / 06231, WO 92 / 22583, WO 89 / 00195, WO 89 / 01476, and WO 03031581. Alternatively, in the case where the effector molecule is a protein or polypeptide, ligation can be achieved using recombinant DNA procedures, as described, for example, in WO 86 / 01533 and EP 0392745.

[0328] As used herein, the term "effector molecule" 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 radioiodides), radioisotopes, chelated metals, nanoparticles, and reporter groups (such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy).

[0329] Examples of effector molecules can include cytotoxins or cytotoxic agents, including any agent that is harmful to cells (e.g., kills cells). Examples include comprestatins, slug toxin, epothilone, asteroidin, maytansine, spongistatin, rhizoxin, halichondrin, roridins, hemiasterlins, paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, ipecacine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, photomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues and homologues.

[0330] Effector molecules also include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., dichloromethyldiethylamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatin(II)(DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., dermatomycin (formerly known as actinomycin), bleomycin, photomycin, amiodarone (AMC), calicheamicin or pyromycin), and antimitotic agents (e.g., vincristine and vinblastine).

[0331] Other effector molecules may include chelating radionuclides, such as 111 In and 90 Y, Lu 177 ,bismuth 213 ,californium 252 ,iridium 192 and tungsten 188 / rhenium 188 Or some drugs, such as, but not limited to, alkylphosphocholine, topoisomerase I inhibitors, taxanes and suramin.

[0332] Other effector molecules include proteins, peptides, and enzymes. Enzymes of interest include, but are not limited to, proteases, hydrolases, lyases, isomerases, and transferases. Proteins, polypeptides, and peptides of interest include, but are not limited to, immunoglobulins, toxins (such as abrinogen, 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 tissue plasminogen activator, thrombolytic agents or anti-angiogenic agents (e.g., angiostatin or endostatin), or biological response modulators such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), nerve growth factor (NGF), or other growth factors, and immunoglobulins.

[0333] Other effector molecules may include, for example, detectable substances useful in diagnostics. 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. Generally, for metal ions that can be conjugated with antibodies as diagnostic agents, 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, luciferin, and jellyfish luminescent protein; and suitable radionuclides include 125 I, 131 I, 111 In and 99 Tc.

[0334] In another example, effector molecules can prolong the half-life of antibodies in vivo and / or reduce the immunogenicity of antibodies and / or enhance antibody delivery across the epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumins, albumin-binding proteins, or albumin-binding compounds, such as those described in WO 05 / 117984.

[0335] When the effector molecule is a polymer, it can typically be a synthetic or naturally occurring polymer, such as optionally substituted linear or branched polyalkylene, polyolefin or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homopolysaccharides or heteropolysaccharides.

[0336] Specific optional substituents that may be present on the above-mentioned synthetic polymers include one or more hydroxyl, methyl, or methoxy groups.

[0337] Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), or derivatives thereof, especially optionally substituted poly(ethylene glycol) such as methoxylated poly(ethylene glycol) or derivatives thereof.

[0338] Specific naturally occurring polymers include lactose, amylose, dextran, glycogen, or their derivatives.

[0339] As used herein, “derivative” is intended to include reactive derivatives, such as thiol-selective reactive groups, like maleimide. Reactive groups can be linked to the polymer directly or via linker segments. It should be understood that residues of such groups may, in certain cases, form part of the product as a linking group between the antibody fragment and the polymer.

[0340] The size of polymers can vary as needed, but typically the average molecular weight ranges from 500 Da to 50,000 Da, for example, 5,000 to 40,000 Da, or 20,000 to 40,000 Da. The size of the polymer can be selected in particular based on the intended use of the product, such as the ability to target specific tissues like tumors or to prolong the circulating half-life (for a review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Therefore, for example, in cases where the product is intended to leave the circulation and penetrate tissues, using a low molecular weight polymer, such as one with a molecular weight of approximately 5,000 Da, may be advantageous. For applications where the product remains in the circulation, using a higher molecular weight polymer, such as one with a molecular weight range of 20,000 Da to 40,000 Da, may be advantageous.

[0341] Suitable polymers include polyalkylene polymers, such as poly(ethylene glycol) or, in particular, methoxy poly(ethylene glycol) or derivatives thereof, and especially polymers with a molecular weight range of about 15,000 Da to about 40,000 Da.

[0342] In one example, the antibody used in this invention is attached to a poly(ethylene glycol) (PEG) portion. In a particular instance, the antibody is an antibody fragment, and the PEG molecule can be attached via any available amino acid side chain or terminal amino acid functional group located on the antibody fragment, such as any free amino, imine, thiol, hydroxyl, or carboxyl group. 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 is the addition of one or more amino acids to the C-terminus of its heavy chain to allow attachment of an effector molecule. Suitablely, the additional amino acid forms a modified hinge region containing one or more cysteine ​​residues to which the effector molecule can attach. Multiple sites may be used to attach two or more PEG molecules.

[0343] Suitable PEG molecules can be covalently linked via a thiol group located on at least one cysteine ​​residue in the antibody fragment. Each polymer molecule attached to the modified antibody fragment can be covalently linked to a sulfur atom on a cysteine ​​residue in the fragment. The covalent link is typically a disulfide bond, or more particularly a sulfur-carbon bond. When using a thiol group as an attachment point, appropriately activated effector molecules can be used, such as thiol-selective derivatives like maleimide and cysteine ​​derivatives. The activated polymer can be used as a starting material for preparing polymer-modified antibody fragments as described above. The activated polymer can be any polymer containing a thiol reactive group, such as α-halocarboxylic acids or esters (e.g., iodoacetamide), imides (e.g., maleimide), vinyl sulfones, or disulfides. Such starting materials are commercially available (e.g., from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA), or can be prepared from commercially available starting materials using conventional chemical procedures. Specific PEG molecules include 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).

[0344] In one embodiment, the antibody is a modified Fab fragment or diFab that is PEGylated, i.e., has PEG (poly(ethylene glycol)) covalently attached thereto, for example according to the methods disclosed in EP 0948544 or EP 1090037 [see also, “Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications”, 1992, J. Milton Harris (ed.), Plenum Press, New York; “Poly(ethyleneglycol) Chemistry and Biological Applications”, 1997, J. Milton Harris and S. Zalipsky (ed.), American Chemical Society, Washington DC; and “Bioconjugation Protein Coupling Techniques for the Biomedical Sciences”, 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545]. In one example, PEG is attached to a cysteine ​​residue in the hinge region. In another example, the PEG-modified Fab fragment has a maleimide group covalently linked to a single thiol group in the modified hinge region. Lysine residues can be covalently linked to the maleimide group, and each amino group on the lysine residue can be attached to a methoxylated poly(ethylene glycol) polymer with a molecular weight of approximately 20,000 Da. Therefore, the total molecular weight of the PEG attached to the Fab fragment may be approximately 40,000 Da.

[0345] In one implementation, the multispecific antibody does not attach to the effector molecule.

[0346] Polynucleotide / carrier / host cell

[0347] The present invention also provides an isolated polynucleotide encoding a polypeptide chain of an IL-13 / IL-17AF multispecific antibody molecule.

[0348] Variant polynucleotides may contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleic acid substitutions and / or deletions from the sequence given in the sequence listing. In general, variants have 1-20, 1-50, 1-75 or 1-100 substitutions and / or deletions.

[0349] Suitable variants may share at least about 70% homology with any of the nucleic acid sequences disclosed herein, typically at least about 80% or 90% homology, and more preferably at least about 95%, 97%, or 99% homology. Variants may maintain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Variants typically maintain about 60%–about 99% identity, about 80%–about 99% identity, about 90%–about 99% identity, or about 95%–about 99% identity. These levels of homology and identity are typically present, at least for the coding regions of the polynucleotides. Methods for measuring homology are well known in the art, and those skilled in the art will understand that, in this context, homology is calculated based on nucleic acid identity. This homology can exist in regions of at least about 15, at least about 30, such as at least about 40, 60, 100, 200 or more consecutive nucleotides (depending on length). This homology can also exist along the entire length of an unmodified polynucleotide sequence.

[0350] The sequence differences between a homolog and the associated polynucleotide may be less than about 3, 5, 10, 15, 20 or more mutations (each of which can be a substitution, deletion or insertion). For example, homologs may have 3-50 mutation differences, typically 3-20 mutation differences. These mutations can be measured in regions of at least 30, such as at least about 40, 60 or 100 or more consecutive nucleotides in the homolog.

[0351] The DNA sequence of the present invention may include synthetic DNA (e.g., produced by chemical processing), cDNA, genomic DNA, or any combination thereof.

[0352] Conventional methods for constructing vectors, transfection methods, and culture methods are well known to those skilled in the art. For further information, please refer to "Current Protocols in Molecular Biology," 1999, F.M. Usubel (ed.), Wiley Interscience, New York, and Maniatis Manual produced by Cold Spring Harbor Publishing.

[0353] Host cells containing one or more clones or expression vectors are also provided, said vectors containing one or more DNA sequences encoding IL-13 / IL-17AF multispecific antibodies. Any suitable host cell / vector system can be used to express DNA sequences encoding IL-13 / IL-17AF multispecific antibodies. Bacterial (e.g., *E. coli*) and other microbial systems can be used, or eukaryotic (e.g., mammalian) host cell expression systems can be used. Suitable mammalian host cells include CHO cells.

[0354] The present invention also provides a method for generating IL-13 / IL-17AF multispecific antibodies, comprising culturing a vector-containing host cell under conditions suitable for expressing a protein from DNA encoding the IL-13 / IL-17AF multispecific antibody, and isolating the IL-13 / IL-17AF multispecific antibody.

[0355] Production of multispecific antibodies

[0356] Numerous methods exist for generating multispecific antibodies, particularly bispecific antibodies. Morrison et al. (Coloma and Morrison 1997, Nat Biotechnol. 15, 159-163) described the fusion of a single-chain variable fragment (scFv) with a complete antibody (such as IgG). Schoonjans et al., 2000, Journal of Immunology, 165, 7050-7057, described the fusion of scFv with an antibody Fab fragment. WO2015 / 197772 describes the fusion of a disulfide-stabilized scFv (dsscFv) with a Fab fragment.

[0357] Standard methods described in the prior art involve expressing at least two polypeptides in a host cell, each polypeptide encoding a heavy chain (HC) or light chain (LC) of a whole antibody or its antigen-binding fragment (e.g., Fab), at the N- and / or C-terminal positions of the heavy and / or light chains, whereby an additional antigen-binding fragment of the antibody may be fused to the whole antibody or its antigen-binding fragment. When attempting to recombinantly generate such multispecific antibodies 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 overexpress the light chain relative to the heavy chain to ensure proper folding during assembly of the heavy chain with its corresponding light chain. In particular, the BIP protein prevents the self-folding of CH1 (domain 1 of the heavy chain constant region), which can be replaced by the corresponding LC; therefore, the proper 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).

[0358] We have observed that methods for expressing multispecific antibodies may result in the production of light chains exceeding that of heavy chains, which are retained in the host cell harvest. Excess light chains tend to form dimer complexes (or “LC dimers”), which are present as byproducts of the production process along with the desired multispecific antibody (particularly the monomer) and therefore need to be purified.

[0359] Importantly, the technical issues associated with light chain dimer formation when fused to additional antigen-binding fragments at the N- and / or C-termini remain unresolved, and commonly used analytical methods do not allow for the detection and quantification of those additional LC dimers in the heterogeneous products of the generation process. This can lead to significant bias when estimating product quantities using standard analytical methods.

[0360] Therefore, there is a need to improve multispecific antibodies and their production methods so as to easily and efficiently separate and remove additional LC dimers in the earliest steps of the production process, thereby increasing the yield of proteins of interest for therapeutic purposes, said proteins being multispecific antibodies, particularly in their monomeric form.

[0361] The multispecific antibodies of the present invention have been engineered to provide improved multispecific antibodies with equivalent functionality and stability, while increasing the yield of "multispecific antibody" material (especially monomers) obtained after purification (especially after one-step purification including protein A affinity chromatography).

[0362] Advantageously, the multispecific antibodies of this disclosure can be purified more efficiently using a purification method that improves upon methods commonly used in the prior art, particularly because the improved method involves fewer steps, resulting in cost and time benefits on an industrial scale. In particular, the multispecific antibodies of this disclosure maximize 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 additional LC dimers. Advantageously, the method for generating and purifying the multispecific antibodies of this disclosure does not require additional purification steps to capture excess free, unbound light chains, particularly additional LC dimers.

[0363] Protein A

[0364] Protein A is a 42 kDa surface protein originally discovered in the cell wall of *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 derived from VH3 family antibodies and the Fcγ region (located between the CH2 and CH3 domains) in 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 disclosure, protein A includes native protein A and any variants or derivatives thereof, provided that the protein A variant or derivative retains its ability to bind the VH3 domain and / or the Fcγ domain.

[0365] 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.

[0366] As used herein, "protein A-binding domain" refers to a binding domain that specifically binds to protein A. A protein A-binding domain can refer to the VH3 domain of protein A or a portion thereof, i.e., it includes the protein A-binding interface. The portion of the VH3 domain that binds protein A does not include the CDR of the VH3 domain; that is, the protein A-binding interface of VH3 does not involve the CDR. Therefore, it should be understood that the protein A-binding domain does not compete with the antigen-binding domains disclosed in this application.

[0367] In one embodiment, the polypeptide chain of formula (I) contains substances present in V H And / or the protein A binding domain in CH2-CH3 and / or V1. In one embodiment, the polypeptide chain of formula (I) contains one, two, or three protein A binding domains, which are present in V H and / or CH2-CH3 and / or V1. In one embodiment, the polypeptide chain of formula (I) contains only one present in V. H Or the protein A binding domain in V1. In one embodiment, s is 0, t is 0, and the polypeptide chain of formula (I) contains only one domain present in V. H Or the protein A binding domain in V1. In one embodiment, the polypeptide chain of formula (I) contains only one domain present in V. H The protein A binding domain in [the protein]. In one embodiment, s is 0, t is 0, p is 0, and the polypeptide chain of formula (I) contains only one domain present in V. HThe protein A binding domain is present in V1. In one embodiment, the polypeptide chain of formula (I) contains 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.

[0368] 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 domains respectively present in V H And two protein A binding domains in CH2-CH3. In another embodiment, the polypeptide chain of formula (I) contains respectively present in V H The polypeptide chain of formula (I) contains two protein A binding domains located in CH2-CH3 and V1, respectively. In another embodiment, the polypeptide chain of formula (I) comprises two protein A binding domains located in CH2-CH3 and V1, respectively.

[0369] In one embodiment, the polypeptide chain of formula (I) comprises three protein A-binding domains, each domain being present in V. H In CH2-CH3 and V1.

[0370] Native protein A can interact in particular 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 IgG.

[0371] In some embodiments, the protein A binding domain comprises or is configured as a VH3 domain or a variant thereof that binds protein A. In some embodiments, the protein A binding domain comprises or is configured as 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.

[0372] The polypeptide chain of this disclosure (II) does not bind to protein A. In one embodiment, the binding domain of V2 does not bind to protein A.

[0373] In some embodiments, V2 comprises or is composed of VH1 and / or VH2 and / or VH4 and / or VH5 and / or VH6, and does not contain a VH3 domain. In some embodiments, V2 comprises or is composed of a VH3 domain or a variant thereof that does not bind protein A. In some embodiments, V2 comprises or is composed of a naturally occurring VH3 domain that cannot bind protein A. In some embodiments, the variant of the VH3 domain that does not bind protein A is a variant of a naturally occurring VH3 domain that can bind protein A.

[0374] Human VH3 germline genes and VH3 domains (or frameworks) have been clearly characterized. Many naturally occurring VH3 domains have the ability to bind protein A, but some naturally occurring VH3 domains do not (see Roben et al., 1995, J Immunol.; 154(12):6437-6445).

[0375] The VH3 domain used in this disclosure can be obtained by several methods. In one embodiment, the VH3 domain used in this disclosure is a naturally occurring VH3 domain, selected based on its ability to bind or not bind protein A, depending on its position in the polypeptide (I) and / or (II) of this disclosure. For example, a panel of antibodies against an antigen of interest can be generated by immunizing a non-human animal, which is then humanized, and the humanized antibodies can be screened and selected based on their ability to bind or not bind protein A through the humanized VH3 domain (e.g., against a protein A affinity column). Alternatively, display techniques (e.g., phage display, yeast display, ribosome display, bacterial display, mammalian cell surface display, mRNA display, DNA display) can be used to screen antibody libraries and select antibodies containing a VH3 domain that binds (particularly through a protein A binding interface not involving a CDR) or does not bind protein A.

[0376] Alternatively, the VH3 domain used in this disclosure 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 protein A-binding VH3 variant comprises a naturally occurring VH3 that cannot bind protein A, and further comprises at least one amino acid mutation. In such embodiments, the mutation is responsible for enabling the VH3 domain to bind protein A; that is, the mutation contributes to the creation of a protein A-binding domain that is not naturally occurring.

[0377] In one embodiment, the VH3 variant contains mutations at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids. In one embodiment, the VH3 variant contains mutations at positions 15, 17, 19, 57, 59, 64, 65, 66, 68, 70, 81, or 82 on VH3, numbered according to Kabat and described, for example, in Graille et al., 2000, PNAS, 97(10): 5399–5404. Mutations can be substitutions, deletions, or insertions. In one embodiment, the VH3 variant contains substitutions at positions 15, 17, 19, 57, 59, 64, 65, 66, 68, 70, 81, or 82 on VH3, numbered according to Kabat.

[0378] Naturally occurring VH1, VH2, VH4, VH5, and VH6 do not bind to protein A. In one embodiment, VH1 does not bind to protein A. H The domain is VH1. In one implementation, V does not bind to protein A. H The domain is VH2. In one implementation, V does not bind to protein A. H The domain is VH4. In one implementation, V does not bind to protein A. H The domain is VH5. In one implementation, V does not bind to protein A. H The structural domain is VH6.

[0379] Drug composition, dosage and dosage regimen

[0380] The multispecific antibodies of the present invention can be provided in pharmaceutical compositions. The pharmaceutical compositions are generally sterile and typically include pharmaceutically acceptable loads and / or adjuvants. The pharmaceutical compositions of the present invention may additionally contain pharmaceutically acceptable adjuvants and / or loads.

[0381] As used herein, a “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. The carrier may be suitable for parenteral administration routes, such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, intraspinal, or other parenteral routes, such as by injection or infusion. Alternatively, the carrier may be suitable for non-parenteral administration routes, such as local, epidermal, or mucosal administration routes. The carrier may be suitable for oral administration. Depending on the route of administration, the modifier may be coated with a material to protect the compound from acids and other natural conditions that may inactivate the compound.

[0382] The pharmaceutical compositions of the present invention may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound without conferring any unwanted toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0383] 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, etc.) 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.

[0384] Under production and storage conditions, therapeutic compositions must generally be sterile and stable. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations.

[0385] The pharmaceutical compositions of the present invention may contain additional active ingredients.

[0386] Kits containing the antibodies or modulators of the present invention, along with instructions for use, are also within the scope of this invention. The kit may further contain one or more additional reagents, such as the additional therapeutic or prophylactic agents discussed above.

[0387] The modulators and / or antibodies or their formulations or compositions of the present invention may be applied for preventive and / or therapeutic treatment.

[0388] In therapeutic applications, a compound is administered to a subject already suffering from the condition or illness described above in an amount sufficient to cure, alleviate, or partially suppress the condition or one or more of its symptoms. This 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 purpose is defined as a “therapeuticly effective amount.”

[0389] In prophylactic applications, the preparation is administered to subjects at risk of the condition or illness as described above in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms. The amount sufficient to achieve this purpose is defined as the "preventive effective amount". The effective amount for each purpose will depend on the severity of the disease or injury, as well as the subject's weight and general condition.

[0390] The subject of administration can be a human or a non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Administration to humans is typical.

[0391] The antibody / modulator or pharmaceutical composition of the present invention can be administered using one or more of a variety of methods known in the art, via one or more routes of administration. As those skilled in the art will understand, the route and / or mode of administration 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 parenteral administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and local administration, typically by injection. Alternatively, the antibody / modulator or pharmaceutical composition of the present invention can be administered via non-parenteral routes, such as local, epidermal, or mucosal administration routes. The antibody / modulator or pharmaceutical composition of the present invention can be used for oral administration.

[0392] The appropriate dosage of the antibody / modulator or pharmaceutical composition of the present invention can be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may vary to obtain a specific amount of active ingredient that effectively achieves the desired therapeutic response without toxicity to the patient for a particular patient, composition, and administration method. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the specific composition of the present invention used, route of administration, time of administration, excretion rate of the specific compound used, duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health status, and medical history of the patient receiving treatment, and similar factors well known in the medical field.

[0393] For example, for patients awaiting treatment, an appropriate dose range could be from about 0.01 μg / kg to about 1000 mg / kg body weight, typically from about 0.1 μg / kg to about 100 mg / kg body weight. For example, an appropriate dose could be from about 1 μg / kg to about 10 mg / kg body weight daily, or from about 10 μg / kg to about 5 mg / kg body weight daily.

[0394] Dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose can be administered, or several separate doses can be administered over a period of time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. The dosage unit form used herein refers to a physically discrete unit suitable as a unit dose for use on a subject to be treated; each unit contains a predetermined amount of the active compound, which is calculated to correlate with the desired drug delivery system to produce the desired therapeutic effect.

[0395] Administration can be a single dose or multiple doses. Multiple doses can be administered via the same or different routes and at the same or different sites. Alternatively, administration can be via a sustained-release formulation, in which case fewer doses are required. Dosage and frequency can vary depending on the antagonist's half-life in the patient and the desired duration of treatment.

[0396] As described above, the modulator / antibody or pharmaceutical composition of the present invention can be co-administered with one or more other therapeutic agents.

[0397] The combined administration of two or more drugs can be achieved in a variety of different ways. They can be administered together in a single composition, or they can be administered in separate compositions as part of a combination therapy. For example, one can be administered before, after, or simultaneously with the other.

[0398] Treatment indications

[0399] 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 wholly or partially caused by signaling through IL-13, IL-17A and / or IL-17F receptors.

[0400] These diseases include primary and metastatic cancers, including breast cancer, colon cancer, rectal cancer, lung cancer, oropharyngeal cancer, hypopharyngeal cancer, esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, gallbladder 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 (including prostate cancer, seminal vesicle cancer, testicular cancer, and germ cell tumors), endocrine gland cancers (including thyroid cancer, adrenal cancer, and pituitary cancer), and skin cancer, as well as hemangiomas, melanomas, sarcomas (including sarcomas originating 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 caused by hematopoietic malignancies such as leukemia and lymphomas (Hodgkin's lymphoma and non-Hodgkin's lymphoma), rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, suppurative arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthritis, systemic lupus erythematosus, ulcerative colitis, inflammatory bowel disease, insulin-dependent diabetes mellitus, thyroiditis, allergic diseases, psoriasis, scleroderma, graft-versus-host disease, organ transplant rejection, acute or chronic immune diseases associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis. Granulomatosis, Henoch-Schoenlein purpurea, microscopic renal vasculitis, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's disease, Parkinson's disease, Alzheimer's disease, stroke, primary cholecystitis, hemolytic anemia, malignancy, heart failure, Addison's disease, sporadic polyglandular dysplasia type I and II, Schmidt's syndrome, adult-onset (acute) respiratory distress syndrome, alopecia areata, alopecia areata, arthropathy, Reiter's diseaseDiseases, psoriatic arthritis, ulcerative colitis arthritis, enteropathic synovitis, chlamydia, Yersinia and Salmonella-associated arthritis, atherosclerosis / arteriosclerosis, atopic dermatitis, autoimmune bullous diseases, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, linear IgA disease, autoimmune hemolytic anemia, Coombs test 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 variant immunodeficiency (common variant hypogammaglobulinemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonia, interstitial lung disease associated with connective tissue diseases, interstitial lung disease associated with mixed connective tissue diseases, interstitial lung disease associated with systemic sclerosis, interstitial lung disease associated with rheumatoid arthritis, interstitial lung disease associated with systemic lupus erythematosus, dermatomyositis / polymyositis associated with dermatomyositis, Sjogren's disease. Lung diseases associated with ankylosing spondylitis, diffuse vasculitis, hemosiderin deposition-related lung diseases, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, obliterative bronchiolitis, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, post-infectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (classical autoimmune or lupus-like hepatitis), type 2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune-mediated hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune diseases associated with organ transplantation, chronic immune diseases associated with organ transplantation, osteoarthritis, primary sclerosing cholangitis, type 1 psoriasis, type 2 psoriasis, idiopathic leukopenia, autoimmune neutropenia, NOS type nephropathy, glomerulonephritis, renal microvasculitis, Lyme disease. Diseases including discoid lupus erythematosus, idiopathic or NOS-type male infertility, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue diseases, Goodpasture's syndrome, pulmonary manifestations of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren's syndrome, and Takayasu's disease.Diseases including arteritis, autoimmune thrombocytopenic purpura, idiopathic thrombocytopenic purpura, autoimmune thyroid disease, hyperthyroidism, goiter-related autoimmune hypothyroidism (Hashimoto's disease). Diseases, atrophic autoimmune hypothyroidism, primary myxedema, lens-derived uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver disease, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis, idiosyncratic liver disease, drug-induced hepatitis, non-alcoholic steatohepatitis, allergy, Group B streptococcal (GBS) infection, mental illness, depression, schizophrenia, Th2 and Th1 mediated diseases, acute and chronic pain, different forms of pain, cancer, lung cancer, breast cancer, gastric cancer, bladder cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, hematopoietic malignancies, leukemia, lymphoma, abeta-lipoproteinemia, acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (ALL). AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinoma, atrial ectopic 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 rejection, α-I-antitrypsin deficiency, amyotrophic lateral sclerosis (ALS), anemia, angina pectoris, anterior horn cell degeneration, anti-CD3 therapy, antiphospholipid syndrome, receptor hypersensitivity, aortic and peripheral artery aneurysm, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (persistent or paroxysmal), atrial fibrillation, atrioventricular block, B-cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma. Lymphoma, burns, arrhythmias, cardiac stunning syndrome, cardiac tumors, cardiomyopathy, inflammatory response to cardiopulmonary bypass, cartilage graft rejection, cerebellar cortical degeneration, cerebellar disorders, disordered or multifocal atrial tachycardia, chemotherapy-related conditions, chronic myeloid leukemia (CML), chronic alcoholism, chronic inflammatory diseases, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylates poisoning, 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 conditions, polyuria, diabetes, diabetic arteriosclerosis, diffuse Lewy body disease, dilated congestive cardiomyopathy, basal ganglia disorders, Down syndrome in middle age.Syndrome, drug-induced motor disorders caused by drugs that block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrine disorders, epiglottitis, Epstein-Barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hemophagocytic lymphohistiocytosis, fetal 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, granulomas caused by intracellular organisms, hairy cell leukemia, Hallervorden-Spatz disease. Diseases, Hashimoto's thyroiditis, hay fever, heart transplant rejection, hemoglobinemia, hemodialysis, hemolytic uremic syndrome / thrombolytic thrombocytopenic purpura, bleeding, hepatitis A, His bundle arrhythmia, HIV infection / HIV neuropathy, Hodgkin's disease, hyperactivity disorder, hypersensitivity reaction, allergic pneumonia, hypertension, hypokinetic dyskinesia, 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, Legionnaires' disease, leishmaniasis, leprosy, corticospinal disorders, lipedema, liver transplant rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcal blood disease, metabolic / idiopathic diseases, migraine, mitochondrial multisystem disorders, mixed connective tissue diseases, monoclonal gammopathy, multiple myeloma, Mencel Dejerine-Thomas disease.Shi-Drager and Machado-Joseph), avian intracellular mycobacteria, Mycobacterium tuberculosis, myelodysplastic syndrome, myocardial infarction, ischemic myocardial disease, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephropathy, neurodegenerative diseases, neurogenic muscular atrophy, neutropenic fever, non-Hodgkin's lymphoma, abdominal aorta and its branches obstruction, obstructive arterial disease, OkT3 therapy, orchitis / epididymitis, orchitis / vasectomy recanalization, 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, POEMS syndrome (polyneuropathy, organomegaly, endocrine disorders, monoclonal gammopathy and skin changes syndrome), post-perfusion syndrome, post-pump syndrome, MI cardiotomy syndrome, preeclampsia, progressive supranuclear palsy, essential pulmonary hypertension, radiotherapy, Raynaud's phenomenon and diseases, Raynaud's disease, Refsum's disease Diseases, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcoma, senile chorea, Lewy body type Alzheimer's disease, 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. ALL telangiectasia, thromboangiitis obliterans, thrombocytopenia, toxicity, transplantation, trauma / hemorrhagic reaction, type III hypersensitivity, type IV hypersensitivity, unstable pharyngitis, uremia, urosepsis, valvular heart disease, varicose veins, vasculitis, venous disease, venous thrombosis, ventricular fibrillation, viral and fungal infections, fatal encephalitis / aseptic meningitis, vital signs-related hemophagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's diseaseDisease, xenograft rejection of any organ or tissue, acute coronary syndrome, acute idiopathic polyneuritis, acute inflammatory demyelinating polyradiculoneuropathy, acute ischemia, adult-onset Stiebel syndrome, allergy, antiphospholipid antibody 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, clinically isolated syndrome (CIS) with risk of multiple sclerosis, childhood-onset mental disorders, dacryocystitis, dermatomyositis, diabetic retinopathy, herniated disc, disc prolapse, drug-induced immune hemolytic anemia, endometriosis, endophthalmitis, episcleritis, erythema multiforme, severe erythema multiforme, pemphigoid of pregnancy, Guillain-Barré syndrome (GBS), Hughes syndrome. Syndrome), idiopathic Parkinson's disease, idiopathic interstitial pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion body myositis, infectious ocular inflammatory diseases, inflammatory demyelinating diseases, inflammatory heart disease, inflammatory nephropathy, IPF / UIP, iritis, keratitis, keratoconjunctivitis sicca, Kussmauldisease or Kussmaul-Meier disease, Landry's paralysis, Langerhans cell histiocytosis, livetioretinopathy, macular degeneration, microscopic polyangiitis, Morbus syndrome.bechterev), motor neuron disorder, mucosal pemphigoid, multiple organ failure, myasthenia gravis, myelodysplastic syndrome, myocarditis, radiculopathy, neuropathy, non-A, non-B hepatitis, optic neuritis, osteolysis, oligoarticular JRA, peripheral artery occlusive disease (PAOD), peripheral vascular disease (PVD), peripheral artery disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, canities, polyarticular JRA, polyendocrine disorders, polymyositis, polymyalgia rheumatica (PMR), primary Parkinson's disease, prostatitis, pure red blood cells Aplastic disorders, 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, silicone-related connective tissue disorders, Sneddon-Wilkinson dermatosis, ankylosing spondylitis, Stephens-Johnson syndrome (SJS), temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumor necrosis factor). Receptors, type 1 allergic reactions, type 2 diabetes, urticaria, common interstitial pneumonia (UIP), vasculitis, vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration or wound healing, aspirin-sensitive asthma, atopic asthma, chronic hand eczema, allergic bronchopulmonary aspergillosis, celiac disease, Churg-Strauss syndrome (nodular and atopic periarteritis), eosinophilic myalgia syndrome, hypereosinophilic syndrome, edema (including paroxysmal angioedema), helminth infection, onchocerciasis. Dermatitis, eosinophil-related gastrointestinal disorders, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis, eosinophilic colitis, nasal micropolyps and polyposis, food allergies, aspirin intolerance and obstructive sleep apnea, chronic asthma, Crohn's disease and endocardial myocardial fibrosis, cancers (e.g., glioblastoma (such as glioblastoma multiforme), non-Hodgkin lymphoma (NHL)), fibrosis, inflammatory bowel disease, pulmonary fibrosis (including idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis secondary to sclerosis), COPD, and liver fibrosis.

[0401] The multispecific antibodies of the present invention are particularly useful for the treatment or prevention of atopic dermatitis, chronic hand eczema, nasal micropolyps or polyps, food allergies, or eosinophilic esophagitis. Therefore, in one embodiment, the multispecific antibody or pharmaceutical composition of the present invention is provided for use in a method of therapeutically treating a human or animal body. In one embodiment, a multispecific antibody or pharmaceutical composition is provided for use in a method of treating atopic dermatitis, chronic hand eczema, nasal micropolyps or polyps, 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 polyps, food allergies, or eosinophilic esophagitis, comprising administering a therapeutically effective amount of the multispecific antibody or pharmaceutical composition to a patient in need.

[0402] The following examples illustrate the present invention. Example

[0403] Example 1. Generation and selection of therapeutic anti-IL-13 antibody CA650

[0404] 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. After 3 to 6 injections, animals were sacrificed 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 monitored by the HEK-293IL-13R-STAT-6 reporter cell assay (HEK-Blue assay, Invivogen).

[0405] B cell cultures were established, and the ability of the supernatant to bind hIL-13 was screened first in a bead-based assay using the Applied Biosystems FMAT assay. This is a homogeneous assay that uses biotinylated human IL-13 coated on streptavidin beads and a goat anti-rat Fc-Cy5 conjugate as a display agent. Positive cells from this assay were then subjected to the HEK-293IL-13R-STAT-6 reporter cell assay (HEK-Blue assay, Invivogen) to identify neutralizers. The neutralization supernatant was then analyzed in Biacore to estimate the dissociation rate and characterize the mode of neutralization. Neutralizers were classified as either Cylinder 1 or Cylinder 2. Cylinder 1 represents antibodies that bind human IL-13 and prevent the binding of IL-13Rα1 (and thus also block the binding of IL-4R). Cylinder 1 antibodies also prevent the binding of IL-13 to IL-13Rα2. Cavity 2 represents an antibody that binds to hIL-13 in a manner that allows binding to IL-13Rα1 but prevents IL-4R from being recruited into the complex. We selected an antibody that functions through Cavity 1.

[0406] In the primary FMAT screening, approximately 7500 IL-13-specific positives were identified from a total of 27 × 100 SLAM assays. 800 wells showed neutralizing activity in the HEK-blue assay. 170 wells exhibited the desired Biacore characteristic, namely a dissociation rate of <5 x 10⁻⁶ for the IL-1 antibody. -4 s -1 An attempt was made to clone the variable regions from these 170 wells, of which 160 successfully produced fluorescent foci. Heavy and light chain variable region gene pairs were generated from 100 wells following reverse transcription (RT)-PCR. These V region genes were cloned into full-length mouse IgG1 antibodies and re-expressed in the HEK-293 transient expression system. Sequence analysis revealed the presence of a unique family of 27 anti-human IL-13 antibodies. The ability of these recombinant antibodies to block recombinant hIL-13 (from E. coli and mammals), recombinant variant hIL-13 (R130Q) (from E. coli), natural wild-type and variant hIL-13 (from human donors), and cynomolgus monkey IL-13 (from mammals) was then retested in cell-based assays. The ability of the recombinant antibodies to bind variant human IL-13 (R130Q) and cynomolgus monkey IL-13 was also tested in Biacore. Based on this characterization, antibody families were selected to meet our criteria: antibodies below 100 pM and with minimal drop-offs in potency and affinity for all human and cynomolgus monkey IL-13 preparations.

[0407] Based on neutralizing efficacy, affinity, and donor content in humanized grafts (see below), humanized CA650 was selected for further study.

[0408] Example 2. Humanization of antibody CA650

[0409] Antibody 650 was humanized by transplanting a CDR from the rat V region onto the human antibody V region framework. To restore antibody activity, some framework residues from the rat V region were also retained in the humanized sequence. These residues were selected using the protocol outlined by Adair et al. (1991) (Humanised antibodies.WO91 / 09967). Figure 1 shows the alignment of the rat antibody (donor) V region sequence with the human (recipient) V region sequence and the designed humanized sequence. (Figure 1(A) Light chain graft 650 and Figure 1(B) Heavy chain graft 650). The CDRs transplanted from the donor to the recipient sequence were as defined by Kabat (Kabat et al., 1987), except for CDR-H1, where the Chothia / Kabat combination definition was used (see, Adair et al., 1991 Humanised antibodies.WO91 / 09967).

[0410] The gene encoding the initial V region sequence was designed by Entrechon GmbH and constructed using automated synthesis methods, and modified by oligonucleotide directed mutagenesis to produce transplantable forms gL8 and gH9. The gL8 sequence was subcloned into the UCBelltech human light chain expression vector pVhCK, which contains DNA encoding the human C-κ constant region (Km3 allotype). The gH9 sequence was subcloned into pVhg1Fab, which contains DNA encoding the human heavy chain γ-1 CH1 constant region.

[0411] Select V zone IGKV1-39 and JK2 J zone (International Immunogenetics Information) IMGT (http: / / www.imgt.org) serves as the acceptor for the light chain CDR of antibody 650. The light chain framework residues in the graft gL8 are all derived from human germline genes, except for residues 58 and 71 (numbered according to Kabat), which retain the donor residues isoleucine (I58) and tyrosine (Y71), respectively. The retention of residues I58 and Y71 is essential for the full potency of the humanized antibody.

[0412] Human V region IGHV1-69 and JH4 J region (IMGT, http: / / www.imgt.org) were selected as acceptors for the antibody 650 heavy chain CDR. All heavy chain framework residues in the graft gH9 were derived from human germline genes, except for residues 67, 69, and 71 (numbered according to Kabat), which retained the donor residues alanine (A67), phenylalanine (F69), and valine (V71), respectively. The retention of residues A67, F69, and V71 is essential for the complete potency of the humanized antibody. The glutamine residue at position 1 of the human framework was replaced with glutamate (E1) to provide homogenized product expression and purification: the conversion of glutamine at the N-terminus of antibodies and antibody fragments to pyroglutamate is widely reported. The finally selected alternative graft sequences gL8 and gH9 are shown in Figure 1(A) and Figure 1(B), respectively.

[0413] Figure 2 shows the amino acid and DNA sequences that encode the CDR, heavy and light chain variable regions, scFv, and dsscFV forms of antibody 650.

[0414] Example 3. Generation of anti-IL-17AF antibody 496.g3

[0415] The production of antibody CA028_00496.g3 (also referred to herein as antibody 496.g3) against human IL-17A and human IL-17F was previously described in WO2012 / 095662. This antibody binds to human IL-17A, IL-17F, and the IL-17A / F heterodimer with a pM affinity. The amino acid and DNA sequences, encoding the CDR, heavy and light chain variable regions, and 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-1CH1 constant region, as well as the hinge (G1m17 allotype).

[0416] Example 4. Generation of anti-human albumin antibody 645

[0417] The production of anti-human albumin antibody 645 has been previously described in WO2013 / 068571. The amino acid and DNA sequences encoding the CDR, heavy and light chain variable regions, scFv, and dsscFV forms of antibody 645 are shown in Figure 2.

[0418] Example 5. Construction and expression in cells of the multispecific antibody IL-13 / IL-17AF transient plasmid.

[0419] Multispecific antibodies were designed in which the anti-IL-17AF V region (496.g3) was immobilized at the Fab site; the anti-albumin V region (645gL4gH5) and anti-IL-13 (1539gL8gH9) were reconstructed into disulfide-linked scFvs in the HL direction (dsHL), and then linked to the C-terminus of the heavy and light chain constant regions of the Fab via 11 amino acid-rich glycine-serine linkers, respectively. Figure 7 The amino acid and DNA sequences of the full-length heavy and light chains encoding multispecific antibodies are shown in Figure 2.

[0420] Light and heavy chain genes were independently cloned into mammalian expression vectors for transient expression under the control of the hCMV promoter. Using the commercially available ExpiCHO expifectamine transient expression kit (Thermo Scientific), equal proportions of the two plasmids were transfected into the CHO-S XE cell line (UCB). Cultures were incubated in Corning roller flasks with vented caps at 37°C, 8.0% CO2, and 190 rpm for 18–22 h. After 18–22 h, an appropriate volume of CHO enhancer and feed (such as the manufacturer's suggested feed for the HiTiter method) was added to the culture. The culture was then incubated for another 10–12 days at 32°C, 8.0% CO2, and 190 rpm. The supernatant was harvested by centrifugation at 4000 rpm for 1 h at 4°C, and then sterilized by filtration through a 0.45 μm filter followed by a 0.2 μm filter. Expression titers were quantified by protein G HPLC using a 1 ml GE HiTrap protein G column (GE Healthcare) and in-house manufactured Fab standards. The expression titers are shown in Table 1.

[0421] Table 1: Transient expression titers in CHO S–XE cell lines

[0422]

[0423]

[0424] Example 6. Development of IL-13 / IL-17AF multispecific antibody in mammalian cell lines

[0425] To demonstrate stable expression of the IL-13 / IL-17AF multispecific antibody, a stable mammalian cell line was established. CHO cell lines were transfected with a vector containing 496.g 3 Fab, 1539g H9g L8 dsscFv HL (LC, INS0025609), 645g H5g L4 dsscFv HL (HC, INS0025306), and selectable markers. Cell lines were cloned, and their suitability for a suitable production process was assessed. To evaluate protein quality and quantity and ensure the selection of the optimal cell line, the cell line was evaluated in a small-scale model of a fed-batch bioreactor. CHO cell lines expressing greater than 1.8 g / L of the IL-13 / IL-17AF multispecific antibody and greater than 75% monomer were selected.

[0426] Example 7. Purification process of IL-13 / IL-17AF multispecific antibody

[0427] Multispecific antibody proteins were purified using a natural protein A capture step followed by preparative size exclusion purification. The clarified supernatant from standard transient CHO expression was loaded onto a MabSelect (GE Healthcare) column, allowed a 5-minute contact time, and washed with binding buffer (20 mM Hepes pH 7.4 + 150 mM NaCl). The bound material was eluted with 0.1 M sodium citrate (pH 3.1) elution buffer, neutralized with 2 M Tris / HCl (pH 8.5), and quantified by absorbance at 280 nm.

[0428] Size exclusion chromatography (SE-UPLC) was used to determine the purity of the eluted products. The antibody (approximately 2 μg) was loaded onto a BEH200 plate. The antibody was developed on a 1.7 μm, 4.6 mm ID x 300 mm column (Waters ACQUITY) using an isocratic gradient of 0.2 M phosphate (pH 7) at a rate of 0.35 mL / min. Serial detection was performed using absorbance at 280 nm and a multichannel fluorescence (FLR) detector (Waters). It was found that 72% of the eluted multispecific antibody was monomeric.

[0429] The neutralized sample was concentrated using an Amicon Ultra-15 concentrator (10 kDa molecular weight cutoff membrane) and centrifuged at 4000 x g in a spin rotor. The concentrated sample was loaded onto an XK16 / 60 Superdex 200 column (GE Healthcare), which was equilibrated in PBS at pH 7.4 and developed with an isocratic gradient of PBS at pH 7.4 at a rate of 1 ml / min. Fractions were collected and analyzed using a BEH200... Analysis was performed by size exclusion chromatography on a 1.7 μm, 4.6 mm ID x 300 mm column (Aquity), with development using an isocratic gradient of 0.2 M phosphate (pH 7) at 0.35 mL / min. Detection was performed by absorbance at 280 nm and a multichannel fluorescence (FLR) detector (Waters). Selected monomer fractions were pooled, sterilized by 0.22 μm filtration, and the final sample concentration was determined by A280 scanning on a DropSense96 (Trinean). Limulus amoeba-like cell lysate (LAL) assay kits were used via Charles River. The endotoxin level was assessed using a portable testing system and was found to be below 1.0 EU / mg.

[0430] In BEH200, The monomeric state of the final multispecific antibody was determined by size exclusion chromatography on a 1.7 μm, 4.6 mm ID x 300 mm column (Aquity), and the development was performed at 0.35 mL / min with an isocratic gradient of 0.2 M phosphate (pH 7). Detection was performed using 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.

[0431] To perform analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), samples were prepared as follows: 4 x Novex NuPAGE LDS sample buffer (Life Technologies) and 10X NuPAGE sample reducing agent (Life Technologies) or 100 mM N-ethylmaleimide (Sigma-Aldrich) were added to approximately 5 μg of purified protein, and the mixture was heated 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.

[0432] On non-reducing SDS-PAGE, multispecific antibodies with a theoretical molecular weight (MW) of approximately 100 kDa migrate to approximately 120 kDa. When the multispecific antibody protein is reduced, both chains migrate at rates close to their respective theoretical MWs (heavy chain (HC) approximately 52 kDa, light chain (LC) approximately 51 kDa). The additional bands at approximately 45–50 kDa on the non-reducing gel are “free” LC and HC, lacking the disulfide bonds in the Fab moiety of the molecule; they do not migrate to the same positions as LC and HC in lane 2 because they are not completely reduced (Figure 3(B)).

[0433] Example 8. Antigen binding of IL-13 / IL-17AF multispecific antibody molecules

[0434] (i) Antigen binding affinity

[0435] The binding kinetics of human and cynomolgus monkey IL-13, IL-17A, AF, F and albumin were evaluated using surface plasmon resonance (Biacore T200).

[0436] Goat anti-human IgG, F(ab')2 fragment-specific antibody (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip using amine coupling chemistry, achieving a level of approximately 5000 RU. Each analysis cycle consisted of the following: IL-13 / IL-17AF / albumin multispecific antibody molecules were captured onto the anti-F(ab')2 surface, analytes were injected (at 25°C and a flow rate of 30 μl / min), followed by surface regeneration. Human and cynomolgus monkey analytes were injected in serially diluted 2-fold HBS-EP+ running buffer (GE Healthcare), with IL-13 concentrations ranging from 10 nM to 0.3125 nM, IL-17A, AF, and F concentrations ranging from 5 nM to 0.156 nM, and albumin concentrations ranging from 100 nM to 3.125 nM. Apart from human IL-13 (R&D Systems), cynomolgus monkey IL-13 (Sinobiologicals), and human serum albumin (Jackson ImmunoResearch), all other antigens were prepared internally. This included buffer blank injection to reduce instrument noise and drift.

[0437] 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 (k... d Less than 1.0 × 10 -5 At that time, it was fixed at 1.0 × 10 -5 (The detection limit of the Biacore T200 instrument as specified by the manufacturer GE Healthcare) to calculate affinity (KD ).

[0438] The results showed that the multispecific antibody effectively bound to human and cynomolgus monkey IL-17A, IL-17AF, IL-17F, IL-13 and albumin.

[0439] Table 2(1): Kinetic constants of binding between IL-13 / IL-17AF multispecific antibodies and human antigens

[0440]

[0441] The result represents the mean from a specified number of measurements.

[0442] Table 2(2): Kinetic constants of binding between IL-13 / IL-17AF multispecific antibody and cynomolgus monkey antigen

[0443]

[0444] The result represents the mean from a specified number of measurements.

[0445] (ii) Simultaneous antigen binding

[0446] Surface plasmon resonance was used to demonstrate that IL-17A, IL-13, and albumin can simultaneously bind to IL-13 / IL-17AF multispecific antibodies using proteins in human or cynomolgus monkey forms.

[0447] The method for evaluating the simultaneous binding of analytes to IL-13 / IL-17AF multispecific antibodies involves capturing the antibody sample onto an immobilized anti-human IgG F(ab')2 fragment-specific antibody. Human or cynomolgus monkey IL-13, IL-17A, and albumin, either individually or in a mixture of all three analytes, are then injected (at 30 μl / min for 300 s) through the captured IL-13 / IL-17AF multispecific antibody (final concentrations of 30 nM IL-13, 15 nM IL-17A, and 150 nM albumin).

[0448] 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 injecting 5 mM NaOH for 30 seconds, and finally injecting 50 mM HCl for 60 seconds.

[0449] The binding response of each antigen when injected alone was determined, and the sum of the individual responses was compared with the binding response when a mixture of all three antigens was injected.

[0450] The average binding response of the mixture of human IL-17A, IL-13 and albumin to the IL-13 / IL-17AF multispecific antibody was 100% of the sum of the individual binding responses (summarized in Table 3), indicating that the IL-13 / IL-17AF multispecific antibody can bind to each antigen simultaneously and independently.

[0451] Table 3: Simultaneous binding of IL-13 / IL-17AF multispecific antibodies to human IL-17A, IL-13, and albumin

[0452]

[0453] The average binding response of the mixture of cynomolgus monkey IL-17A, IL-13 and albumin to the IL-13 / IL-17AF multispecific antibody was 97% of the sum of the individual binding responses (summarized in Table 4), indicating that the IL-13 / IL-17AF multispecific antibody can bind to each antigen simultaneously and independently.

[0454] Table 4: Simultaneous binding of IL-13 / IL-17AF multispecific antibody to cynomolgus monkey IL-17A, IL-13, and albumin

[0455]

[0456] Example 9. Neutralization of IL-13 by an IL-13 / IL-17AF multispecific antibody

[0457] The activity of the IL-13 / IL-17AF multispecific antibody in neutralizing IL-13 was assessed using the HEK 293 human IL4 / IL-13 SEAP reporter cell line assay. SEAP secretion was measured after activation of the STAT6 pathway to evaluate the IL-13 response.

[0458] HEK 293 human IL4 / IL-13 SEAP reporter cells (#hkb-il413) were obtained from Invivogen, San Diego. The cell line was cultured, frozen, and maintained according to the manufacturer's protocol.

[0459] Recombinant human IL-13 was obtained from R&D Systems, Minneapolis, MN (#213-ILB).

[0460] Cells were plated so that approximately 80% of the cells congregated in a 96-well flat-bottom plate upon stimulation.

[0461] The cells were treated with two copies of the antibody. Before adding the antibody to the cells for 24 hours of treatment, the cells were pre-incubated with 250 pg / mL IL-13 for 30 minutes at 37°C, 5% CO2, and 100% humidity.

[0462] 24 hours later, following the manufacturer's instructions, 20 μL of the supernatant was aspirated from the cell stimulation and added to 180 μL of Quanti-blue#rep-qbs (Invivogen, San Diego).

[0463] Measurements were performed until a visible color change gradient was observed, and absorbance was read at 620 nm using a spectrophotometer. The IC50 was calculated using nonlinear regression with Graphpad Prism (San Diego, CA). Figure 4 shows a representative graph of the percentage inhibition of STAT6 signaling by the IL-13 / IL-17AF multispecific antibody.

[0464] Table 5: IC50 values ​​of IL-13 / IL-17AF multispecific antibodies inhibiting STAT6 signaling.

[0465] pM + / -SEM ng / ml + / -SEM n＝ 4.118 1.319 0.4118 0.1319 3

[0466] Example 10. IL-13 / IL-17AF multispecific antibody against human and cynomolgus monkey IL-17A and IL-17F via human true... Neutralizing effect of IL-6 response produced by epidermal fibroblasts

[0467] The aim of this study was to determine the neutralizing capacity of the IL-13 / IL-17AF multispecific antibody against IL-17A and IL-17F in human and cynomolgus monkeys in a primary human cell system. IL-17 induces a pro-inflammatory response when combined with other cytokines such as TNF-α. Therefore, this synergistic effect was utilized to measure IL-6 release from primary normal neonatal human dermal fibroblasts (nHDF) stimulated with IL-17 and TNF-α.

[0468] In this assay, the ability of the IL-13 / IL-17AF multispecific antibody to inhibit IL-17-induced IL-6 release from nHDF was measured. Specifically, in the presence of IL-13 / IL-17AF multispecific antibody titration (concentration range of 5000 pM to 0.25 pM for IL-17A studies; concentration range of 500,000 pM to 25 pM for IL-17F studies), nHDF was stimulated with human or cynomolgus monkey IL-17A (50 pM) or IL-17F (25,000 pM) in combination with TNF-α (25 pM). The resulting IL-6 response was then measured using homogeneous time-resolved FRET (HTRF).

[0469] nHDF cells (Sigma#106-05n) were cultured in complete medium (DMEM + 10% FCS + 2mM L-glutamine) using standard techniques and maintained in tissue culture flasks. Cells were harvested from the tissue culture flasks using TrypLE (Invitrogen#12605036). The TrypLE was neutralized with complete medium (45 ml), and the cells were centrifuged at 300 x g for 3 minutes. The cells were resuspended in complete medium (3-5 ml), counted, and adjusted to a concentration of 3.125 × 10⁻⁶ cells / mL. 4Cells / mL were collected and then added at 40 μl / well to a 384-well assay plate (Corning #3701). Cells were incubated at 37°C / 5% CO2 for 3 hours to allow adhesion to the plate. In a 384-well dilution plate (Greiner #781281), the IL-13 / IL-17AF multispecific antibody was serially diluted with complete medium to a final concentration range of 5000 pM to 0.25 pM for anti-IL-17A assessment and 500,000 pM to 25 pM for anti-IL-17F assessment. A mixture of TNF-α and IL-17 cytokines was prepared in complete medium to a final concentration of 25 pM for TNF-α, 50 pM for human or cynomolgus monkey IL-17A, or 25,000 pM for IL-17F. Then, 30 μl / well of these solutions were added to a 384-well reagent plate (Greiner #781281). Next, 10 μl from the serially diluted IL-13 / IL-17AF multispecific antibody plate was transferred to a reagent plate containing 30 μl of the diluted cytokine. The IL-13 / IL-17AF multispecific antibody and cytokine mixture were then incubated at 37°C / 5% CO2 for one hour. After incubation, 10 μl was transferred from the reagent plate to an assay plate containing cells. The assay plate was then incubated at 37°C / 5% CO2 for 18 hours ± 2 hours. After incubation, the europium cryptate and Alexa 665 antibody from the Cisbio IL-6 HTRF kit (Cisbio #62IL6PEB) were diluted 1:1 in reconstitution buffer and mixed according to the instructions on the kit insert. Subsequently, 10 μl / well of this antibody mixture was added to a white low-capacity 384-well HTRF plate (Greiner #784075). The supernatant from the assay plate was then transferred to the HTRF plate at 10 μl / well. The HTRF plate was then incubated at room temperature for 2 hours with gentle agitation. The HTRF plate was then read on a Synergy Neo 2 plate reader according to the manufacturer's instructions, and fluorescence was measured at 330 / 620 nm and 330 / 665 nm readings. The ratio was then calculated using the formula (330 / 665 nm divided by 330 / 620 nm) × 10,000 and used to determine the relative percentage of inhibition compared to the control wells using Microsoft Excel. 4PL curve fitting and IC50 values ​​were calculated using GraphPad Prism 7.0.

[0470] The results shown are the average of three independent experiments (+ / - SEM). The calculated IC50 values ​​for the IL-13 / IL-17AF multispecific antibody were 42 pM for human IL-17A and 49 pM for cynomolgus monkey IL-17A. The calculated IC50 values ​​for the IL-13 / IL-17AF multispecific antibody were 28,030 pM for human IL-17F and 34,320 pM for cynomolgus monkey IL-17F (Figure 5).

[0471] Table 6: Summary of potency, efficacy, and curve slope values ​​of IL-13 / IL-17AF multispecific antibodies.

[0472] The values ​​were calculated from three independent experiments.

[0473]

[0474] Example 11. In the NHEK CXCL1 release bioassay, simultaneous administration of IL-13 / IL-17AF multispecific antibodies Neutralizes IL-13, IL-17A and IL-17F

[0475] The purpose of this assay was to evaluate the ability of the IL-13 / IL-17AF multispecific antibody to simultaneously neutralize IL-13, IL-17A, and IL-17F in primary cell systems. When NHEK was treated with IL-13, IL-17A, or IL-17F, respectively, they induced the secretion of CXCL1, a chemokine responsible for recruiting cells to sites of inflammation. In the case of atopic dermatitis, there is evidence that CXCL1 plays a role in sensitizing neurons, thereby giving neurons a lower excitation threshold. This observation may be related to the pruritus experienced by patients (Yang TB and Kim BS 2019; "Pruritus in allergy and immunology" J Allergy Clin Immunol 144(2):353-360).

[0476] NHEK (PromoCell, Heidelberg) cells were stored, cultured, and used according to the manufacturer's instructions. Cells were plated to achieve 100% confluence in 48-well flat-bottomed plates upon stimulation. Cells were pre-incubated for 30 minutes with increasing concentrations of anti-IL-13, anti-IL-17A, anti-IL-17F, or IL-13 / IL-17AF multispecific antibodies, followed by treatment with 100 ng / mL IL-13, 100 ng / mL IL-17A, and 1 μg / mL IL-17F for 72 hours. After this period, 50 μl of cell-free supernatant was collected according to the manufacturer's instructions (R&D Systems) for ELISA quantification of CXCL1 concentration. The IC50 for each experimental group was calculated using nonlinear regression with Graphpadprism (San Diego, CA).50 .

[0477] The results showed that the IL-13 / IL-17AF multispecific antibody simultaneously neutralized the activities of IL-13, IL-17A, and IL-17F (Figure 6). When normalized to the number of binding sites available in the assay, the IL-13 / IL-17AF multispecific antibody (81.3%) was more effective than anti-IL-17A (52.7%), anti-IL-17F (0.7%), or anti-IL-13 (48.8%) in inhibiting CXCL1 release. Increasing the concentration of anti-IL-17A, anti-IL-17F, or anti-IL-13 alone did not improve the maximum inhibition achieved. The results highlight the benefit of simultaneous inhibition of IL-13, IL-17A, and IL-17F compared to neutralization by a single cytokine.

[0478] Example 12. Comparison of Multispecific IL-13 / IL-17 Antibody with Prior Art IL-13 / IL-17 Antibodies (Introduction)

[0479] Figure 7 An example of an IL-13 / IL-17AF multispecific antibody according to the present invention is shown. It comprises a Fab domain with bispecificity for IL-17A and IL-17F, which is linked to two scFv domains, one specific for IL-13 and the other for albumin. The anti-albumin domain gives this multispecific antibody a longer half-life.

[0480] Abbvie (WO2013 / 102042A2) and Genentech (WO2015 / 127405A2) have previously described bispecific antibodies that bind to IL-13 and IL-17. However, little has been disclosed about how they bind 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:

[0481] • Affinity to IL-13

[0482] • Interactions between molecules and IL-13 and IL-13Rα1

[0483] • Affinity to IL-17A

[0484] • Affinity for IL-17F

[0485] Production of reference antibody

[0486] BITS7201A (Genentech) was constructed using the sequence described in Example 6 of WO2015 / 127405A2.

[0487] DVD2166 and DVD2174 (Abbvie) were constructed using the sequences described in Tables 6 and 7 of WO2013 / 102042A2. These molecules were selected based on the reported bispecific activity of each arm against IL-13 and IL-17, which is substantially equivalent to or equivalent to their respective parent antibodies (WO2013 / 102042A2, Example 4, page 83, paragraph 0195).

[0488] The DNA construct was transfected into CHO-SXE cells using a high-titer protocol with the ExpiCHO transfection system (ThermoFisher Scientific). After harvesting, the cell culture was centrifuged at 4000 RPM for at least 1 hour, and the supernatant was clarified by filtration using a 0.22 μM Stericup filter unit.

[0489] DVD2166+DVD2174 Purification

[0490] DVD-IgG protein was purified by loading the clarified supernatant onto a 10 ml MabSelect Sure column and washing with 3 column volumes (CV) of PBS (pH 7.4). The protein was eluted from the column with 0.1 M sodium citrate (pH 3.6) and neutralized with 2 M Tris-HCl (pH 8.5). Monomeric proteins were separated by loading them onto a HiLoad 16x60 Superdex 200 pg column (Sigma) equilibrated with PBS (pH 7.4). Fractions containing monomeric proteins were combined, filtered sterilely, and stored at 4°C.

[0491] BITS7201A Purification

[0492] The parental handle and pore proteins were purified by loading the clarified supernatant onto a 10 mL MabSelect Sure column and washing with 3x CV PBS (pH 7.4). The proteins were eluted from the column with 0.1 M sodium citrate (pH 3.6). To neutralize and stabilize the proteins, the samples were diluted 1:1 with 1 M arginine / succinate buffer at pH 8.7. The parental antibody was then loaded onto a HiLoad 26x60 Superdex 200 pg column (Sigma) equilibrated with 0.15 M sodium acetate and 0.5 M arginine buffer at pH 8.5. Bispecific material was then 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 by loading the material onto a HiLoad 26x60 Superdex 200pg column equilibrated with PBS (pH 7.4) to remove any high molecular weight species from the exchanged bispecific material. Fractions containing monomeric bispecific proteins were combined, filtered sterilely, and stored at 4°C.

[0493] Comparison of combined features

[0494] The binding kinetics of human IL-13, IL-17A, and IL-17F to the IL-13 / IL-17AF multispecific antibody (referred to as "UCBXXXX") and prior art antibodies were evaluated using surface plasmon resonance (Biacore T200) and direct comparisons in a single experiment. Furthermore, surface plasmon resonance was used to assess whether binding of the IL-13 / IL-17AF multispecific antibody UCBXXXX or a reference molecule to IL-13 would lead to the blockade of IL-13-IL-13 receptor interaction.

[0495] Goat anti-human IgG and F(ab')2 fragment-specific antibodies (Jackson ImmunoResearch) were immobilized on a CM5 sensor chip using amine coupling chemistry, achieving a level of approximately 5000 RU. For affinity assessment, each analysis cycle consisted of the following: 50 to 100 RU of IL-13 / IL-17AF multispecific antibody or reference bispecific molecule was captured onto the anti-F(ab')2 surface; analyte was injected (at 25°C, at a flow rate of 30 μl / min for 180 s); then, the dissociation of IL-13 and IL-17A was monitored for 1200 s, and the dissociation of IL-17F was monitored for 600 s. 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 s, followed by injecting 5 mM NaOH for 30 s, and finally injecting 50 mM HCl for 60 s. Analytes were injected in HBS-EP+ run buffer (GE Healthcare) at 2-fold serial dilutions, 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 sourced from R&D Systems. Buffer blank injections were included to mitigate instrument noise and drift.

[0496] The dynamic parameters were determined using Biacore T200 evaluation software (version 3.0) and a 1:1 combined model. The results are summarized in Table 7.

[0497] Table 7: Comparison of kinetic constants for binding with IL-13, IL-17A and IL-17F.

[0498]

[0499] To evaluate IL-13Rα1 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 at 10 μl / min for 180 s) and IL-13Rα1 (R&D Systems, 100 nM at 10 μl / min for 300 s). Blank injections including both IL-13 and IL-13Rα1 were performed to subtract any drift or background response. As summarized in Table 8, UCBXXXX blocked the interaction between IL-13 and IL-13Rα1, while BITS7210A, DVD2166, and DVD 2174 molecules did not.

[0500] Table 8: Blocking of IL-13Rα1 receptor by IL-13 / IL-17AF multispecific antibody and reference molecule.

[0501]

[0502] Further experiments were conducted to evaluate the ability of the IL-13 / IL-17AF multispecific antibody to block the binding of IL-13Rα1 and IL-13Rα2. In this experiment, approximately 260 RU of UCBXXXX was captured by immobilized mouse anti-human CH1 antibody (inside UCB), followed by injection of IL-13 (25 nM at 10 μl / min for 180 s), then injection of either IL-13Rα1 or IL-13Rα2 (R&D Systems, 100 nM at 10 μl / min for 300 s). Blank injections including IL-13, IL-13Rα1, and IL-13Rα2 were used to subtract any drift or background response. The results showed that UCBXXXX effectively blocked the interaction of IL-13 with both IL-13Rα1 and IL-13Rα2 (Table 9).

[0503] Table 9: Blocking IL-13Rα1 and IL-13Rα2 receptors with IL-13 / IL-17AF multispecific antibodies.

[0504]

[0505] discuss

[0506] Comparative studies showed that the IL-13 / IL-17AF multispecific antibody UCBXXXX, the bispecific antibody BITS7210A, and the dual variable domain antibodies DVD2166 and DVD2174 could bind to IL-13 with high affinity. However, the characteristics of their binding interactions differed significantly. UCBXXXX blocked the interaction between IL-13 and IL-13-Rα1, while BITS7210A, DVD2166, and DVD2174 did not.

[0507] Comparative studies further demonstrated that the multispecific antibody UCBXXXX, the bispecific antibody BITS7210A, and the dual variable domain antibodies DVD2166 and DVD2174 can bind to IL-17. However, the binding interactions exhibited vastly different characteristics. UCBXXXX showed a significantly higher affinity for IL-17A than BITS7210A and the DVD antibody. UCBXXXX and BITS7210A bound to IL-17F with similar affinities, while the DVD antibody failed to bind to IL-17F at all.

[0508] In the presence of these 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 primary driver of ADA generation is the binding of the therapeutic antibody to the target antigen expressed on the cell surface and subsequent internalization (Schellekens, H., 2002; Clin Ther. 24(11):1720-40). Internalized therapeutic antibody / target antigen complexes are transported through different intracellular compartments, either recycled back to the cell surface or degraded (St Pierre et al., 2011); this can lead to peptide presentation via antigen-presenting molecules and the generation of ADAs.

[0509] In the bispecific antibody BITS7201A, the IL-13F(ab) moiety is identical to that of the anti-IL-13 antibody lebrikizumab (see Example 6 of WO2015 / 127405A2). It is believed that the lebrikizumab binding site allows IL-13 to bind to its receptors IL-13Rα1 and IL-13Rα2, but blocks its interaction with the IL-4Rα receptor (Popovic et al., 2017; J Mol Biol. 429(2):208-19). This particular type of interaction may allow the antibody / target antigen / receptor complex to be internalized via IL-13Rα2, thereby increasing the likelihood of an immunogenic response. BITS7201A was associated with a high incidence of anti-drug antibody (ADA) in a Phase I clinical trial and was withdrawn from clinical development.

[0510] Similarly, the dual variable domain antibodies DVD2166 and DVD2174 could not block the interaction between IL-13 and IL-13-Rα1.

[0511] To reduce potential immunogenic risks, UCBXXXX has been specially designed to prevent its binding to its respective target antigens IL-13, IL-17A, and IL-17F from interacting with receptors on cells, thereby reducing the chance of internalization, degradation, and the likelihood of ADA formation. The incidence of ADA in humans using UCBXXXX can only be determined after clinical data are obtained.

[0512] The data presented above indicate that the IL-13 / IL-17AF multispecific antibody UCBXXXX possesses the appropriate characteristics to be an effective IL-13 / IL-17 antibody therapeutic agent, with improved efficacy and a lower risk of immunogenicity. sequence list <110> UCB Biopharma SRL <120> Multispecific antibodies with binding specificity to human IL-13 and IL-17 <130> PF0208-WO-PCT <150> GB 1919061.0 <151> 2019-12-20 <160> 68 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 1 Arg Ala Asp Glu Ser Val Arg Thr Leu Met His 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 2 Leu Val Ser Asn Ser Glu Ile 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 3 Gln Gln Thr Trp Ser Asp Pro Trp Thr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 4 Gly Phe Thr Phe Ser Asp Tyr Asn Met Ala 1 5 10 <210> 5 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 5 Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp Ser Val Lys 1 5 10 15 Gly <210> 6 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 6 Pro Pro Gln Tyr Tyr Glu Gly Ser Ile Tyr Arg Leu Trp Phe Ala His 1 5 10 15 <210> 7 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 7 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Asp Glu Ser Val Arg Thr Leu 20 25 30 Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Val Ser Asn Ser Glu Ile Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Arg Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Trp Ser Asp Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 8 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 8 gcaatccagc tcacccagag tccaagcagt ctctccgcca gcgtaggcga ccgtgtgact 60 attacctgta gagcggacga gtcggtcagg actctcatgc actggtatca acagaagcct 120 ggtaaagctc ctaaactgct catctatctg gtgtccaact cggagatagg tgtgccagat 180 cggtttagtg ggtctggttc aggcactgat ttcagactga ccatatcatc tctacagcca 240 gaggacttcg ccacatatta ctgtcagcaa acctggagtg acccgtggac tttcggccag 300 ggcactaaag tagaaattaa a 321 <210> 9 <211> 125 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Pro Gln Tyr Tyr Glu Gly Ser Ile Tyr Arg Leu Trp Phe 100 105 110 Ala His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 10 <211> 375 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 10 gaagttcagc tggtcgagtc tggaggtggc cttgtccaac ctggagggag cctgcgtctc 60 tcttgtgcag caagcggatt cacgttttct gattacaata tggcttgggt tagacaggca 120 ccgggtaagg gccttgaatg ggttgcgacg attacatacg aaggcagaaa tacctattac 180 agggactcag taaaagggcg gtttaccata agccgagata atgctaaaaa cagtctgtat 240 ttgcaaatga acagcctacg agctgaagac actgccgtgt attactgcgc gagtccacct 300 cagtattatg aaggatcaat ctatcgcctc tggttcgcac attggggaca ggggaccctt 360 gtgacagtct cgagt 375 <210> 11 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 11 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Asp Glu Ser Val Arg Thr Leu 20 25 30 Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Val Ser Asn Ser Glu Ile Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Arg Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Trp Ser Asp Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 12 <211> 642 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 12 gcaatccagc tcacccagag tccaagcagt ctctccgcca gcgtaggcga ccgtgtgact 60 attacctgta gagcggacga gtcggtcagg actctcatgc actggtatca acagaagcct 120 ggtaaagctc ctaaactgct catctatctg gtgtccaact cggagatagg tgtgccagat 180 cggtttagtg ggtctggttc aggcactgat ttcagactga ccatatcatc tctacagcca 240 gaggacttcg ccacatatta ctgtcagcaa acctggagtg acccgtggac tttcggccag 300 ggcactaaag tagaaattaa acgtacggtg gccgctccct ccgtgttcat cttcccaccc 360 tccgacgagc agctgaagtc cggcaccgcc tccgtcgtgt gcctgctgaa caacttctac 420 ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480 gaatccgtca ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540 ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600 ctgtccagcc ccgtgaccaa gtccttcaac cggggcgagt gc 642 <210> 13 <211> 228 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Pro Gln Tyr Tyr Glu Gly Ser Ile Tyr Arg Leu Trp Phe 100 105 110 Ala His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys 225 <210> 14 <211> 684 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 14 gaagttcagc tggtcgagtc tggaggtggc cttgtccaac ctggagggag cctgcgtctc 60 tcttgtgcag caagcggatt cacgttttct gattacaata tggcttgggt tagacaggca 120 ccgggtaagg gccttgaatg ggttgcgacg attacatacg aaggcagaaa tacctattac 180 agggactcag taaaagggcg gtttaccata agccgagata atgctaaaaa cagtctgtat 240 ttgcaaatga acagcctacg agctgaagac actgccgtgt attactgcgc gagtccacct 300 cagtattatg aaggatcaat ctatcgcctc tggttcgcac attggggaca ggggaccctt 360 gtgacagtct cgagtgcgtc cacaaagggc ccatcggtct tccccctggc accctcctcc 420 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480 ccagtgacgg tgtcgtggaa ctcaggtgcc ctgaccagcg gcgttcacac cttcccggct 540 gtcctacagt cttcaggact ctactccctg agcagcgtgg tgaccgtgcc ctccagcagc 600 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtcgat 660 aagaaagttg agcccaaatc ttgt 684 <210> 15 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 15 Lys Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp 1 5 10 <210> 16 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 16 Tyr Thr Asp Ile Leu Gln Thr 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 17 Tyr Gln Tyr Tyr Ser Gly Tyr Thr 1 5 <210> 18 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 18 Gly Tyr Ser Phe Thr Ser Tyr Tyr Ile His 1 5 10 <210> 19 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 19 Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 20 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 20 Phe His Tyr Asp Gly Ala Asp 1 5 <210> twenty one <211> 106 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> twenty one Asp Ile Gln Met Thr Gln Ser Pro Pro Val Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Leu Ser Cys Lys Ala Ser Gln Asn Ile Asn Glu Asn 20 25 30 Leu Asp Trp Tyr His Gln Lys His Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Asp Ile Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr 85 90 95 Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 22 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 22 gacatccaga tgacccagtc tcctccagtc ctgtctgcat ctgtgggaga cagagtcact 60 ctcagttgca aagcaagtca gaatattaat gagaacttag actggtatca tcaaaagcat 120 ggcgaagctc caaaactcct gatatattat acagacattt tgcaaacggg catcccatca 180 aggttcagtg gcagtggatc tggtacagat tacacactca ccatcagcag cctgcagcct 240 gaagatgttg ccacatatta ctgctatcag tattacagtg ggtacacgtt tggacctggg 300 accaagctgg aaataaaa 318 <210> 23 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 23 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Val Asp Lys Tyr Phe Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ser Pro Glu Asp Thr Ala Val Phe Tyr Cys 85 90 95 Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 24 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 24 caggtacaac tgcagcagtc tggagctgag ttggtgaagc ctgggtcttc agtgaagatg 60 tcctgcaagg cttctggcta cagtttcacc agctactaca tacactggat aaagcagagg 120 cctggacagg gccttgagtg gattgggcgt attggtcctg gaagtggaga tattaattac 180 aatgagaagt tcaagggcaa ggccacattt actgtggaca aatatttcag cacagcctac 240 atgcaactca gcagcctgtc acctgaggac actgcggtct tttactgtgc aagatttcac 300 tatgatgggg ctgactgggg ccaaggcact ctggtcacag tctcgagc 348 <210> 25 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 26 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 26 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 27 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Glu Asn 20 25 30 Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Asp Ile Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 28 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 28 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 29 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 29 gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga cagggtgacc 60 atcacctgca aggcctccca gaacatcaac gagaacctgg actggtacca gcagaagccc 120 ggcaaggccc ccaagctgct gatctactac accgacatcc tgcagaccgg catcccctcc 180 aggttctccg gctccggctc cggcaccgac tacaccctga ccatctcctc cctgcagccc 240 gaggacttcg ccacctacta ctgctaccag tactactccg gctacacctt cggccagggc 300 accaagctgg agatcaag 318 <210> 30 <211> 348 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 30 gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60 tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120 cccggccagg gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180 aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240 atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300 tacgacggcg ccgactgggg ccagggcacc ctggtgaccg tctcgagc 348 <210> 31 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 31 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Glu Asn 20 25 30 Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Asp Ile Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr 85 90 95 Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 32 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Met 35 40 45 Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 33 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 33 gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga cagggtgacc 60 atcacctgca aggcctccca gaacatcaac gagaacctgg actggtacca gcagaagccc 120 ggcaaggccc ccaagctgct gatctactac accgacatcc tgcagaccgg catcccctcc 180 aggttctccg gctccggctc cggcaccgac tacaccctga ccatctcctc cctgcagccc 240 gaggacttcg ccacctacta ctgctaccag tactactccg gctacacctt cggctgcggc 300 accaagctgg agatcaag 318 <210> 34 <211> 348 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 34 gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60 tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120 cccggccagt gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180 aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240 atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300 tacgacggcg ccgactgggg ccagggcacc ctggtgaccg tgtcctcc 348 <210> 35 <211> 242 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 35 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 130 135 140 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys 145 150 155 160 Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asp Ile Leu Gln Thr 180 185 190 Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 210 215 220 Tyr Gln Tyr Tyr Ser Gly Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys <210> 36 <211> 726 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 36 gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60 tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120 cccggccagg gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180 aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240 atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300 tacgacggcg ccgactgggg ccagggcacc ctggtgaccg tgtcctccgg aggtggcggt 360 tctggcggtg gcggttccgg tggcggtgga tcgggaggtg gcggttctga catccagatg 420 acccagtccc cctcctccct gtccgcctcc gtgggcgaca gggtgaccat cacctgcaag 480 gcctcccaga acatcaacga gaacctggac tggtaccagc agaagcccgg caaggccccc 540 aagctgctga tctactacac cgacatcctg cagaccggca tcccctccag gttctccggc 600 tccggctccg gcaccgacta caccctgacc atctcctccc tgcagcccga ggacttcgcc 660 acctactact gctaccagta ctactccggc tacaccttcg gccagggcac caagctggag 720 atcaag 726 <210> 37 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 37 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Met 35 40 45 Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 130 135 140 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys 145 150 155 160 Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asp Ile Leu Gln Thr 180 185 190 Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 210 215 220 Tyr Gln Tyr Tyr Ser Gly Tyr Thr Phe Gly Cys Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys <210> 38 <211> 726 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 38 gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60 tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120 cccggccagt gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180 aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240 atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300 tacgacggcg ccgactgggg ccagggcacc ctggtgaccg tgtcctccgg aggtggcggt 360 tctggcggtg gcggttccgg tggcggtgga tcgggaggtg gcggttctga catccagatg 420 acccagtccc cctcctccct gtccgcctcc gtgggcgaca gggtgaccat cacctgcaag 480 gcctcccaga acatcaacga gaacctggac tggtaccagc agaagcccgg caaggccccc 540 aagctgctga tctactacac cgacatcctg cagaccggca tcccctccag gttctccggc 600 tccggctccg gcaccgacta caccctgacc atctcctccc tgcagcccga ggacttcgcc 660 acctactact gctaccagta ctactccggc tacaccttcg gctgcggcac caagctggag 720 atcaag 726 <210> 39 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 39 Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser 1 5 10 <210> 40 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Recombinant sequences <400> 40 Glu Ala Ser Lys Leu Thr Ser 1 5 <210> 41 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 41 Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr Thr 1 5 10 <210> 42 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 42 Gly Ile Asp Leu Ser Asn Tyr Ala Ile Asn 1 5 10 <210> 43 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 43 Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15 <210> 44 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 44 Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu 1 5 10 <210> 45 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 45 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn 20 25 30 Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile 85 90 95 Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 46 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 46 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 47 <211> 330 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 47 gatattcaga tgacgcaatc accttcgagc gtatccgcct cggtgggaga cagggtgaca 60 atcacttgtc agtcatcccc ctcagtctgg agcaactttt tgtcatggta tcagcagaag 120 cccggaaagg ctccgaaatt gctgatctac gaggcatcga agttgacgag cggtgtacca 180 agcagattct ccggttcggg gtcgggaact gacttcaccc ttacgatctc atcgctgcag 240 ccggaggatt ttgcgaccta ctactgtggg ggtgggtatt cgtcgatttc cgacacaaca 300 ttcgggggcg gcacgaaagt ggaaatcaag 330 <210> 48 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 48 gaagtgcagt tgctggagtc aggtggaggg ctggtgcagc ccggaggatc gctgcggttg 60 tcatgcgcgg tgtccggtat tgatttgtcc aattacgcca tcaattgggt acgccaagcg 120 ccagggaagg gccttgagtg gattggcatc atctgggcgt cggggacgac cttttatgct 180 acttgggcca aaggaagatt cacaatctcc cgagacaact cgaagaacac cgtgtatctt 240 caaatgaact cgctcagggc cgaggacacg gcggtctact actgtgcacg gacagtgccg 300 ggttattcaa cggcacctta ctttgatctt tggggccagg ggaccctcgt gactgtctca 360 agt 363 <210> 49 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 49 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn 20 25 30 Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile 85 90 95 Ser Asp Thr Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 50 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequences <400> 50 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile 35 40 45 Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 51 <211> 330 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 51 gatattcaga tgacgcaatc accttcgagc gtatccgcct cggtgggaga cagggtgaca 60 atcacttgtc agtcatcccc ctcagtctgg agcaactttt tgtcatggta tcagcagaag 120 cccggaaagg ctccgaaatt gctgatctac gaggcatcga agttgacgag cggtgtacca 180 agcagattct ccggttcggg gtcgggaact gacttcaccc ttacgatctc atcgctgcag 240 ccggaggatt ttgcgaccta ctactgtggg ggtgggtatt cgtcgatttc cgacacaaca 300 ttcgggtgcg gcacgaaagt ggaaatcaag 330 <210> 52 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 52 gaagtgcagt tgctggagtc aggtggaggg ctggtgcagc ccggaggatc gctgcggttg 60 tcatgcgcgg tgtccggtat tgatttgtcc aattacgcca tcaattgggt acgccaagcg 120 ccagggaagt gccttgagtg gattggcatc atctgggcgt cggggacgac cttttatgct 180 acttgggcca aaggaagatt cacaatctcc cgagacaact cgaagaacac cgtgtatctt 240 caaatgaact cgctcagggc cgaggacacg gcggtctact actgtgcacg gacagtgccg 300 ggttattcaa cggcacctta ctttgatctt tggggccagg ggaccctcgt gactgtctca 360 agt 363 <210> 53 <211> 251 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 53 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln 130 135 140 Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val 145 150 155 160 Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser 165 170 175 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu 180 185 190 Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 210 215 220 Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr 225 230 235 240 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 245 250 <210> 54 <211> 753 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 54 gaagtgcagt tgctggagtc aggtggaggg ctggtgcagc ccggaggatc gctgcggttg 60 tcatgcgcgg tgtccggtat tgatttgtcc aattacgcca tcaattgggt acgccaagcg 120 ccagggaagg gccttgagtg gattggcatc atctgggcgt cggggacgac cttttatgct 180 acttgggcca aaggaagatt cacaatctcc cgagacaact cgaagaacac cgtgtatctt 240 caaatgaact cgctcagggc cgaggacacg gcggtctact actgtgcacg gacagtgccg 300 ggttattcaa cggcacctta ctttgatctt tggggccagg ggaccctcgt gactgtctca 360 agtggaggtg gcggttctgg cggtggcggt tccggtggcg gtggatcggg aggtggcggt 420 tctgatattc agatgacgca atcaccttcg agcgtatccg cctcggtggg agacagggtg 480 acaatcactt gtcagtcatc cccctcagtc tggagcaact ttttgtcatg gtatcagcag 540 aagcccggaa aggctccgaa attgctgatc tacgaggcat cgaagttgac gagcggtgta 600 ccaagcagat tctccggttc ggggtcggga actgacttca cccttacgat ctcatcgctg 660 cagccggagg attttgcgac ctactactgt gggggtgggt attcgtcgat ttccgacaca 720 acattcgggg gcggcacgaa agtggaaatc aag 753 <210> 55 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 55 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile 35 40 45 Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln 130 135 140 Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val 145 150 155 160 Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser 165 170 175 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu 180 185 190 Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 210 215 220 Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr 225 230 235 240 Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys 245 250 <210> 56 <211> 753 <212> DNA <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 56 gaagtgcagt tgctggagtc aggtggaggg ctggtgcagc ccggaggatc gctgcggttg 60 tcatgcgcgg tgtccggtat tgatttgtcc aattacgcca tcaattgggt acgccaagcg 120 ccagggaagt gccttgagtg gattggcatc atctgggcgt cggggacgac cttttatgct 180 acttgggcca aaagagatt cacaatctcc cgagacaact cgaagaacac cgtgtatctt 240 aaatgaact cgctcagggc cgaggacacg gcggtctact actgtgcacg gacagtgccg 300 ggttattcaa cggcacctta ctttgatctt tggggccagg ggaccctcgt gactgtctca 360 agtggaggtg gcggttctgg cggtggcggt tccggtggcg gtggatcggg aggtggcggt 420 tctgatattc agatgacgca atcaccttcg agcgtatccg cctcggtggg agacagggtg 480 acaatcactt gtcagtcatc cccctcagtc tggagcaact ttttgtcatg gtatcagcag 540 aagccccgaa aggctccgaa attgctgatc tacgaggcat cgaagttgac gagcggtgta 600 ccaagcagat tctccggttc ggggtcggga actgacttca cccttacgat ctcatcgctg 660 cagccggagg attttgcgac ctactactgt gggggtgggt attcgtcgat ttccgacaca 720 acattcgggt gcggcacgaa agtggaaatc aag 753 <210> 57 <211> 492 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Pro Gln Tyr Tyr Glu Gly Ser Ile Tyr Arg Leu Trp Phe 100 105 110 Ala His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser Glu 225 230 235 240 Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 245 250 255 Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr Ala 260 265 270 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 275 280 285 Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly 290 295 300 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln 305 310 315 320 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 325 330 335 Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln 340 345 350 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 355 360 365 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met 370 375 380 Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr 385 390 395 400 Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser Trp 405 410 415 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu Ala 420 425 430 Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 435 440 445 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 450 455 460 Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr Thr 465 470 475 480 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 485 490 <210> 58 <211> 1476 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 58 gaagttcagc tggtcgagtc tggaggtggc cttgtccaac ctggagggag cctgcgtctc 60 tcttgtgcag caagcggatt cacgttttct gattacaata tggcttgggt tagacaggca 120 ccgggtaagg gccttgaatg ggttgcgacg attacatacg aaggcagaaa tacctattac 180 agggactcag taaaagggcg gtttaccata agccgagata atgctaaaaa cagtctgtat 240 ttgcaaatga acagcctacg agctgaagac actgccgtgt attactgcgc gagtccacct 300 cagtattatg aaggatcaat ctatcgcctc tggttcgcac attggggaca ggggaccctt 360 gtgacagtct cgagtgcgtc cacaaagggc ccatcggtct tccccctggc accctcctcc 420 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480 ccagtgacgg tgtcgtggaa ctcaggtgcc ctgaccagcg gcgttcacac cttcccggct 540 gtcctacagt cttcaggact ctactccctg agcagcgtgg tgaccgtgcc ctccagcagc 600 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtcgat 660 aagaaagttg agcccaaatc ttgtagcggt ggcggtggca ccggaggtgg cggttcagaa 720 gtgcagttgc tggagtcagg tggagggctg gtgcagcccg gaggatcgct gcggttgtca 780 tgcgcggtgt ccggtattga tttgtccaat tacgccatca attgggtacg ccaagcgcca 840 gggaagggcc ttgagtggat tggcatcatc tgggcgtcgg ggacgacctt ttatgctact 900 tgggccaaag gaagattcac aatctcccga gacaactcga agaacaccgt gtatcttcaa 960 atgaactcgc tcagggccga ggacacggcg gtctactact gtgcacggac agtgccgggt 1020 tattcaacgg caccttactt tgatctttgg ggccagggga ccctcgtgac tgtctcaagt 1080 ggaggtggcg gttctggcgg tggcggttcc ggtggcggtg gatcgggagg tggcggttct 1140 gatattcaga tgacgcaatc accttcgagc gtatccgcct cggtgggaga cagggtgaca 1200 atcacttgtc agtcatcccc ctcagtctgg agcaactttt tgtcatggta tcagcagaag 1260 cccggaaagg ctccgaaatt gctgatctac gaggcatcga agttgacgag cggtgtacca 1320 agcagattct ccggttcggg gtcgggaact gacttcaccc ttacgatctc atcgctgcag 1380 ccggaggatt ttgcgaccta ctactgtggg ggtgggtatt cgtcgatttc cgacacaaca 1440 ttcgggggcg gcacgaaagt ggaaatcaag cgtacc 1476 <210> 59 <211> 492 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 59 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Pro Gln Tyr Tyr Glu Gly Ser Ile Tyr Arg Leu Trp Phe 100 105 110 Ala His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser Glu 225 230 235 240 Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 245 250 255 Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr Ala 260 265 270 Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile Gly 275 280 285 Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly 290 295 300 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln 305 310 315 320 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 325 330 335 Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln 340 345 350 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 355 360 365 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met 370 375 380 Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr 385 390 395 400 Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser Trp 405 410 415 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu Ala 420 425 430 Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 435 440 445 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 450 455 460 Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr Thr 465 470 475 480 Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Arg Thr 485 490 <210> 60 <211> 1476 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 60 gaagttcagc tggtcgagtc tggaggtggc cttgtccaac ctggagggag cctgcgtctc 60 tcttgtgcag caagcggatt cacgttttct gattacaata tggcttgggt tagacaggca 120 ccgggtaagg gccttgaatg ggttgcgacg attacatacg aaggcagaaa tacctattac 180 agggactcag taaaagggcg gtttaccata agccgagata atgctaaaaa cagtctgtat 240 ttgcaaatga acagcctacg agctgaagac actgccgtgt attactgcgc gagtccacct 300 cagtattatg aaggatcaat ctatcgcctc tggttcgcac attggggaca ggggaccctt 360 gtgacagtct cgagtgcgtc cacaaagggc ccatcggtct tccccctggc accctcctcc 420 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480 ccagtgacgg tgtcgtggaa ctcaggtgcc ctgaccagcg gcgttcacac cttcccggct 540 gtcctacagt cttcaggact ctactccctg agcagcgtgg tgaccgtgcc ctccagcagc 600 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtcgat 660 aagaaagttg agcccaaatc ttgtagcggt ggcggtggca ccggaggtgg cggttcagaa 720 gtgcagttgc tggagtcagg tggagggctg gtgcagcccg gaggatcgct gcggttgtca 780 tgcgcggtgt ccggtattga tttgtccaat tacgccatca attgggtacg ccaagcgcca 840 gggaagtgcc ttgagtggat tggcatcatc tgggcgtcgg ggacgacctt ttatgctact 900 tgggccaaag gaagattcac aatctcccga gacaactcga agaacaccgt gtatcttcaa 960 atgaactcgc tcagggccga ggacacggcg gtctactact gtgcacggac agtgccgggt 1020 tattcaacgg caccttactt tgatctttgg ggccagggga ccctcgtgac tgtctcaagt 1080 ggaggtggcg gttctggcgg tggcggttcc ggtggcggtg gatcgggagg tggcggttct 1140 gatattcaga tgacgcaatc accttcgagc gtatccgcct cggtgggaga cagggtgaca 1200 atcacttgtc agtcatcccc ctcagtctgg agcaactttt tgtcatggta tcagcagaag 1260 cccggaaagg ctccgaaatt gctgatctac gaggcatcga agttgacgag cggtgtacca 1320 agcagattct ccggttcggg gtcgggaact gacttcaccc ttacgatctc atcgctgcag 1380 ccggaggatt ttgcgaccta ctactgtggg ggtgggtatt cgtcgatttc cgacacaaca 1440 ttcgggtgcg gcacgaaagt ggaaatcaag cgtacc 1476 <210> 61 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 61 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Asp Glu Ser Val Arg Thr Leu 20 25 30 Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Val Ser Asn Ser Glu Ile Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Arg Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Trp Ser Asp Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 225 230 235 240 Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser 245 250 255 Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 260 265 270 Met Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys 275 280 285 Phe Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala 290 295 300 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 305 310 315 320 Cys Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu 325 330 335 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 340 345 350 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 355 360 365 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 370 375 380 Lys Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp Trp Tyr Gln Gln Lys 385 390 395 400 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asp Ile Leu Gln 405 410 415 Thr Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr 420 425 430 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 435 440 445 Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr Phe Gly Gln Gly Thr Lys Leu 450 455 460 Glu Ile Lys Arg Thr 465 <210> 62 <211> 1407 <212> DNA <213> Artificial sequence <220> <223> Recombinant sequence <400> 62 gcaatccagc tcacccagag tccaagcagt ctctccgcca gcgtaggcga ccgtgtgact 60 attacctgta gagcggacga gtcggtcagg actctcatgc actggtatca acagaagcct 120 ggtaaagctc ctaaactgct catctatctg gtgtccaact cggagatagg tgtgccagat 180 cggtttagtg ggtctggttc aggcactgat ttcagactga ccatatcatc tctacagcca 240 gaggacttcg ccacatatta ctgtcagcaa acctggagtg acccgtggac tttcggccag 300 ggcactaaag tagaaattaa acgtacggtg gccgctccct ccgtgttcat cttcccaccc 360 tccgacgagc agctgaagtc cggcaccgcc tccgtcgtgt gcctgctgaa caacttctac 420 ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480 gaatccgtca ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540 ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600 ctgtccagcc ccgtgaccaa gtccttcaac cggggcgagt gcagcggtgg cggtggctcc 660 ggaggtggcg gttcagaggt gcagctggtg cagtccggcg ccgaggtgaa gaagcccggc 720 tcctccgtga aggtgtcctg caaggcctcc ggctactcct tcacctccta ctacatccac 780 tgggtgaggc aggcccccgg ccagggcctg gagtggatgg gcaggatcgg ccccggctcc 840 ggcgacatca actacaacga gaagttcaag ggcagggcca ccttcaccgt ggacaagtcc 900 acctccaccg cctacatgga gctgtcctcc ctgaggtccg aggacaccgc cgtgtactac 960 tgcgccaggt tccactacga cggcgccgac tggggccagg gcaccctggt gaccgtgtcc 1020 tccggaggtg gcggttctgg cggtggcggt tccggtggcg gtggatcggg aggtggcggt 1080 tctgacatcc agatgaccca gtccccctcc tccctgtccg cctccgtggg cgacagggtg 1140 accatcacct gcaaggcctc ccagaacatc aacgagaacc tggactggta ccagcagaag 1200 cccggcaagg cccccaagct gctgatctac tacaccgaca tcctgcagac cggcatcccc 1260 tccaggttct ccggctccgg ctccggcacc gactacaccc tgaccatctc ctccctgcag 1320 cccgaggact tcgccaccta ctactgctac cagtactact ccggctacac cttcggccag 1380 ggcaccaagc tggagatcaa gcgtacc 1407 <210> 63 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 63 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Asp Glu Ser Val Arg Thr Leu 20 25 30 Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Val Ser Asn Ser Glu Ile Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Arg Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Trp Ser Asp Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 225 230 235 240 Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser 245 250 255 Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp 260 265 270 Met Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys 275 280 285 Phe Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala 290 295 300 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 305 310 315 320 Cys Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu 325 330 335 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 340 345 350 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 355 360 365 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 370 375 380 Lys Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp Trp Tyr Gln Gln Lys 385 390 395 400 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asp Ile Leu Gln 405 410 415 Thr Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr 420 425 430 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 435 440 445 Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr Phe Gly Cys Gly Thr Lys Leu 450 455 460 Glu Ile Lys Arg Thr 465 <210> 64 <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 64 gcaatccagc tcacccagag tccaagcagt ctctccgcca gcgtaggcga ccgtgtgact 60 attacctgta gagcggacga gtcggtcagg actctcatgc actggtatca acagaagcct 120 ggtaaagctc ctaaactgct catctatctg gtgtccaact cggagatagg tgtgccagat 180 cggtttagtg ggtctggttc aggcactgat ttcagactga ccatatcatc tctacagcca 240 gaggacttcg ccacatatta ctgtcagcaa acctggagtg acccgtggac tttcggccag 300 ggcactaaag tagaaattaa acgtacggtg gccgctccct ccgtgttcat cttcccaccc 360 tccgacgagc agctgaagtc cggcaccgcc tccgtcgtgt gcctgctgaa caacttctac 420 ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480 gaatccgtca ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540 ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600 ctgtccagcc ccgtgaccaa gtccttcaac cggggcgagt gcagcggtgg cggtggctcc 660 ggaggtggcg gttcagaggt gcagctggtg cagtccggcg ccgaggtgaa gaagcccggc 720 tcctccgtga aggtgtcctg caaggcctcc ggctactcct tcacctccta ctacatccac 780 tgggtgaggc aggcccccgg ccagtgcctg gagtggatgg gcaggatcgg ccccggctcc 840 ggcgacatca actacaacga gaagttcaag ggcagggcca ccttcaccgt ggacaagtcc 900 acctccaccg cctacatgga gctgtcctcc ctgaggtccg aggacaccgc cgtgtactac 960 tgcgccaggt tccactacga cggcgccgac tggggccagg gcaccctggt gaccgtgtcc 1020 tccggaggtg gcggttctgg cggtggcggt tccggtggcg gtggatcggg aggtggcggt 1080 tctgacatcc agatgaccca gtccccctcc tccctgtccg cctccgtggg cgacagggtg 1140 accatcacct gcaaggcctc ccagaacatc aacgagaacc tggactggta ccagcagaag 1200 cccggcaagg cccccaagct gctgatctac tacaccgaca tcctgcagac cggcatcccc 1260 tccaggttct ccggctccgg ctccggcacc gactacaccc tgaccatctc ctccctgcag 1320 cccgaggact tcgccaccta ctactgctac cagtactact ccggctacac cttcggctgc 1380 ggcaccaagc tggagatcaa gcgtacc 1407 <210> 65 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 65 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser 1 5 10 <210> 66 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Sequence <400> 66 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 67 <211> 11 <212> PRT <213> Synthetic sequence <220> <223> Recombinant sequence <400> 67 Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser 1 5 10 <210> 68 <211> 20 <212> PRT <213> Synthetic sequence <220> <223> Recombinant sequence <400> 68 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20

Claims

1. A multispecific antibody that binds human IL-13, human IL-17A, and / or human IL-17F, comprising: a) Polypeptide chain of formula (Ia): V H -CH1-X-V1; and b) Polypeptide chain of formula (IIa): V L -C L -Y-V2; in: V H Represents a heavy-chain variable structural domain; CH1 represents domain 1 of the heavy chain constant region; X represents a key or connector; Y represents a key or connector; V1 represents scFv, dsscFv, or dsFv; V L Represents a variable structural domain in a light chain; C L Domains representing the structural regions derived from the constant region of the light chain; V2 represents scFv, dsscFv, or dsFv; The polypeptide chain of formula (Ia) contains a protein A binding domain; and In formula (IIa), the polypeptide chain does not bind to protein A; in: V L and V H It contains antigen-binding sites that bind to human IL-17A and human IL-17F. V2 contains an antigen-binding site that binds to human IL-13, and V1 contains an antigen-binding site that binds to human serum albumin; in V L The sequence containing CDR-L1 (SEQ ID NO: 1), CDR-L2 (SEQ ID NO: 2), and CDR-L3 (SEQ ID NO: 3) is... V H The sequence containing CDR-H1 (SEQ ID NO: 4), CDR-H2 (SEQ ID NO: 5), and CDR-H3 (SEQ ID NO: 6); in V1 includes a light chain variable region and a heavy chain variable region. The light chain variable region includes the sequence SEQ ID NO: 39 of CDR-L1, the sequence SEQ ID NO: 40 of CDR-L2, and the sequence SEQ ID NO: 41 of CDR-L3. The heavy chain variable region includes the sequence SEQ ID NO: 42 of CDR-H1, the sequence SEQ ID NO: 43 of CDR-H2, and the sequence SEQ ID NO: 44 of CDR-H3. in V2 includes a light chain variable region and a heavy chain variable region. The light chain variable region includes the sequence SEQ ID NO: 15 of CDR-L1, the sequence SEQ ID NO: 16 of CDR-L2, and the sequence SEQ ID NO: 17 of CDR-L3. The heavy chain variable region includes the sequence SEQ ID NO: 18 of CDR-H1, the sequence SEQ ID NO: 19 of CDR-H2, and the sequence SEQ ID NO: 20 of CDR-H3.

2. The multispecific antibody according to claim 1, wherein V L It contains the sequence SEQ ID NO: 7, and V H Contains sequence SEQ ID NO:

9.

3. The multispecific antibody according to claim 1, wherein V2 comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising sequence SEQ ID NO: 27, and the heavy chain variable region comprising sequence SEQ ID NO:

28.

4. The multispecific antibody according to claim 1, wherein V2 comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising sequence SEQ ID NO: 31, and the heavy chain variable region comprising sequence SEQ ID NO:

32.

5. The multispecific antibody according to claim 1, wherein V1 comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising sequence SEQ ID NO: 45, and the heavy chain variable region comprising sequence SEQ ID NO:

46.

6. The multispecific antibody according to claim 1, wherein V1 comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising sequence SEQ ID NO: 49, and the heavy chain variable region comprising sequence SEQ ID NO:

50.

7. The multispecific antibody according to claim 1, wherein the light chain variable region and the heavy chain variable region of V2 are connected by a adapter, the adapter comprising the sequence SEQ ID NO:

66.

8. The multispecific antibody according to claim 7, wherein V2 is an scFv containing the sequence SEQ ID NO: 35 or a dsscFv containing the sequence SEQ ID NO:

37.

9. The multispecific antibody according to claim 1, wherein the light chain variable region and the heavy chain variable region of V1 are connected by a adapter, the adapter comprising the sequence SEQ ID NO:

68.

10. The multispecific antibody according to claim 9, wherein V1 is an scFv containing the sequence SEQ ID NO: 53 or a dsscFv containing the sequence SEQ ID NO:

55.

11. The multispecific antibody according to claim 1, wherein Y is a linker comprising the sequence SEQ ID NO:

65.

12. The multispecific antibody according to claim 1, wherein X is a linker comprising the sequence SEQ ID NO:

67.

13. The multispecific antibody according to claim 1, wherein the polypeptide chain of formula (Ia) comprises the sequence SEQ ID NO:57 or SEQ ID NO:

59.

14. The multispecific antibody according to claim 1, wherein the polypeptide chain of formula (IIa) comprises the sequence SEQ ID NO:61 or SEQ ID NO:

63.

15. The multispecific antibody according to claim 1, wherein the polypeptide chain of formula (Ia) comprises the sequence SEQ ID NO:59 and the polypeptide chain of formula (IIa) comprises the sequence SEQ ID NO:

63.

16. An isolated polynucleotide encoding a multispecific antibody as defined in any one of claims 1 to 15.

17. An expression vector carrying the polynucleotide of claim 16.

18. A host cell comprising the vector as defined in claim 17.

19. A method for producing a multispecific antibody as defined in any one of claims 1-15, comprising culturing the host cell of claim 18 under conditions that allow antibody production, and recovering the produced antibody.

20. The method of claim 19, comprising a protein A purification step.

21. A pharmaceutical composition comprising an antibody as defined in any one of claims 1 to 15 and a pharmaceutically acceptable adjuvant.

22. The pharmaceutical composition of claim 21, wherein it comprises a pharmaceutically acceptable carrier.

23. An antibody as defined in any one of claims 1-15 or a pharmaceutical composition as defined in claim 21, used in a method of treating a human or animal body by means of a therapy.

24. The antibody or pharmaceutical composition according to claim 23, for the treatment or prevention of atopic dermatitis.

25. Use of the multispecific antibody according to claims 1-15 in the preparation of a medicament for the treatment or prevention of atopic dermatitis.

Images