Multispecific antibodies with binding specificity for human IL-13 and IL-17

Abstract

The present invention relates to multispecific antibodies having specificity for human IL-13, human IL-17A and / or human IL-17F. The invention further relates to methods for producing the multispecific antibodies and their therapeutic use for treating atopic dermatitis and other diseases.

Description

[Technical field]

[0001] The present invention relates to multispecific antibodies having specificity for human IL-13, human IL-17A and / or IL-17F. The present invention further relates to methods for producing the multispecific antibodies and their therapeutic use for treating atopic dermatitis and other diseases. [Background technology]

[0002] Atopic dermatitis (AD), also known as atopic eczema, is an inflammatory disease that results in intense itching, redness, swelling, weeping, cracked skin, and often thickening over time.

[0003] Since the beginning of the 20th century, many mucosal inflammatory disorders have become more common, and atopic dermatitis is a classic example of such a disease. Currently, it affects 15-30% of children and 2-10% of adults in developed countries and the United States, nearly tripling in the past 30-40 years. Over 15 million American adults and children suffer from atopic dermatitis.

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

[0005] Due to the limitations of existing pharmaceutical agents, improved treatments for atopic dermatitis are greatly needed.

[0006] WO 2013 / 102042 (Abbvie) describes dual specific binding proteins for IL-13 and IL-17 and their potential use in the treatment of a broad list of diseases. The binding proteins did not progress into clinical development.

[0007] WO 2015 / 127405 (Genentech) describes anti-IL-13 / IL-17 bispecific antibodies and methods of using them to treat moderate to severe asthma and / or eosinophilic asthma. In Phase I clinical trials, BITS7201A was associated with a high incidence of anti-drug antibodies (ADA) and was withdrawn from clinical development. Summary of the Invention [Means for solving the problem]

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

[0009] The antibodies of the present invention have improved properties, such as lower immunogenicity and / or better pharmacokinetic profile, compared to currently available antibodies. In addition, the antibodies of the present invention can be engineered so that they can be purified more efficiently using improved purification methods that include fewer steps than currently available methods, which are cost-effective and time-effective on an industrial scale. Thus, the antibodies of the present invention can have improved manufacturability.

[0010] The present invention further provides the following: ◆An isolated polynucleotide encoding a multispecific antibody. ♦ An expression vector carrying a polynucleotide. ◆ A host cell containing the vector. A method for producing a multispecific antibody, comprising culturing a host cell and recovering the antibodies produced. A pharmaceutical composition comprising the multispecific antibody described above. A multispecific antibody or pharmaceutical composition for use in a method of treatment of the human or animal body by therapy. ◆A method for treating or preventing atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergy, or eosinophilic esophagitis, comprising the step of administering a therapeutically effective amount of a multispecific antibody or pharmaceutical composition to a patient in need thereof. [Brief description of the drawings]

[0011] [Figure 1] Ab650 humanization alignment. Alignment of rat antibody (donor) V-region sequences with human germline (acceptor) V-region sequences along with the designed humanized sequence. (A) Light chain graft 650: 650=rat variable light chain sequence. 650gL8=humanized graft of 650 variable light chain using IGKV1-39 human germline as acceptor framework. CDRs are shown in bold / underlined. Donor residues are shown in bold / italic, I58 and Y71 are highlighted. (B) Heavy chain graft 650: 650=rat variable heavy chain sequence. 650gH9=humanized graft of 650 variable heavy chain using IGHV1-69 human germline as acceptor framework. CDRs are shown in bold / underlined. Donor residues are shown in bold / italic, A67, F69 and V71 are highlighted. [Diagram 2] Amino acid and DNA sequences. [Diagram 3] Purification of IL-13 / IL-17AF multispecific antibodies. (A) BEH200 SEC-UPLC analysis of purified multispecific antibodies, detected by FLR. (B) Protein samples separated by Tris-glycine SDS-PAGE under non-reducing (lane 1) or reducing (lane 2) conditions. Gels were stained with Coomassie Quick stain and destained with dH2O. Mark12 protein marker (Life Technologies) was used as standard (M). Molecular weights (MW) were measured in kilodaltons (kDa). [Figure 4] Inhibition of STAT6 signaling by IL-13 / IL-17AF multispecific antibodies. [Diagram 5](A) Inhibition of IL-6 production by IL-13 / IL-17AF multispecific antibodies in response to human or cynomolgus IL-17A in combination with TNF-α. (i) Human IL-17A. (ii) Cynomolgus IL-17A. (B) Inhibition of IL-6 production by IL-13 / IL-17AF multispecific antibodies in response to human or cynomolgus IL-17F in combination with TNF-α. (i) Human IL-17F. (ii) Cynomolgus IL-17F. [Figure 6] Simultaneous neutralization of IL-13, IL-17A and IL-17F by IL-13 / IL-17AF multispecific antibodies in NHEK CXCL1 release bioassay. Key: Circle = anti-IL-13 / IL-17AF Square = anti-IL-17A Upward triangle = anti-IL-17F Downward triangle = anti-IL-13 [Figure 7] Schematic diagram of a multispecific IL-13 / IL-17AF antibody according to the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0013] Human IL-13 is a 17 kDa glycoprotein that is produced by activated T cells of the Th2 lineage, although several non-T cell populations, such as Th0 and Th1 CD4+ T cells, CD8+ T cells, and mast cells, also produce IL-13. Functions of IL-13 include immunoglobulin isotype switching to IgE in human B cells and suppression of proinflammatory cytokine production in both humans and mice.

[0014] IL-13 binds to its cell surface receptors, IL-13R-alpha1 and IL-13R-alpha2. IL-13R-α1 binds with low affinity (K D It interacts with IL-13 with high affinity (K ) and subsequently recruits IL-4R-α D Approximately 0.4 nM) to form signaling heterodimeric receptor complexes.

[0015] The IL-4R / IL-13R-alpha1 complex is expressed in many cell types, including B cells, monocytes / macrophages, dendritic cells, eosinophils, basophils, fibroblasts, endothelial cells, airway epithelial cells, and airway smooth muscle cells. Ligation of the IL-13R-alpha / IL-4R receptor complex leads to activation of various signaling pathways, including the signal transducer and activator of transcription 6 (STAT6) and insulin receptor substrate 2 (IRS2) pathways.

[0016] Only the IL-13R-alpha2 chain has high affinity for IL-13 (K D Approximately 0.25-0.4 nM). It functions both as a decoy receptor that negatively regulates IL-13 binding and as a signaling receptor that induces TGF-β synthesis and fibrosis via the AP-1 pathway in macrophages and possibly other cell types.

[0017] IL-13 is involved in the pathogenesis of many human disorders, and therapeutic strategies are designed to inhibit or counteract IL-13 activity. In particular, there is a need for antibodies that bind to and neutralize IL-13 as a means of inhibiting IL-13 activity. However, there is a need in the art for suitable and / or improved antibodies that can bind to IL-13, particularly human IL-13, and particularly antibodies that can neutralize human IL-13.

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

[0019] Antibodies that inhibit IL-13 activity can act through several possible mechanisms of action. Bin1 represents an antibody that binds to human IL-13 and prevents the binding of IL-13Rα1, and therefore also blocks the binding of IL-4R. Bin1 antibodies can also inhibit the binding of IL-13 to IL-13Rα2. Bin2 represents an antibody that binds hIL-13 in a way that allows it to bind to IL-13Rα1 but inhibits the recruitment of IL-4R to the complex. We selected antibodies that act through Bin1.

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

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

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

[0023] In one embodiment, the multispecific antibody has a K of less than 100 pM. D It binds to human IL-13 at

[0024] IL-17 The IL-17 family of cytokines consists of six members based on structural similarity, with molecular masses of 23-36 kDa and dimeric structures. The founding member, IL-17A (still often referred to simply as IL-17 in the literature), shares 16%-50% amino acid sequence identity with the other members, IL-17B, IL-17C, IL-17D, IL-17E (also known as IL-25), and IL-17F. IL-17A and IL-17F share the greatest homology (50%) and bind to the same receptor complex, and thus shared biological activity has been observed between these two cytokines. In addition, IL-17A and IL-17F exist not only as homodimers, but also as IL-17A / F heterodimers. IL-17E (IL-25) is the least similar to IL-17A. Important and relevant to the biological activities of IL-17A and IL-17F is the finding that they share the same IL-17RA / IL-17RC receptor complex, with IL-17A having the greatest affinity for IL-17RA, whereas IL-17F binds more tightly to IL-17RC. The other family member that utilizes IL-17RA is IL-17E, which signals through the IL-17RA / IL-17RB receptor complex.

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

[0026] The cell types that respond to IL-17 cytokines are reflected by the expression of different receptors. IL-17RA is ubiquitously expressed, particularly at high levels in hematopoietic tissues, whereas IL-17RC is more highly expressed in non-immune cells of joints, liver, kidney, thyroid and prostate. This differential expression may explain the difference in the biological activity of IL-17A and IL-17F, since cells expressing high levels of IL-17RC may be more responsive to IL-17F, whereas cells with higher expression of IL-17RA than IL-17RC may respond more readily to IL-17A. Specific cell types that are responsive to IL-17A and F include fibroblasts, epithelial cells, keratinocytes, synovial cells and endothelial cells, and IL-17A has also been reported to act on T and B cells and macrophages.

[0027] The multispecific antibodies of the invention can bind to human IL-17A and / or IL-17F. Thus, the antibodies can bind to IL-17A homodimers, IL-17F homodimers and / or IL-17AF heterodimers.

[0028] 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 human IL-17A and IL-17F.

[0029] In one embodiment, the multispecific antibody has a K of less than 50 pM. D In one embodiment, the multispecific antibody binds to human IL-17A with a K of less than 25 pM. D In one embodiment, the multispecific antibody binds to human IL-17A with a K of less than 10 pM. D It binds to human IL-17A at

[0030] In one embodiment, the multispecific antibody has a K of less than 200 pM. D In one embodiment, the multispecific antibody binds to human IL-17F with a K of less than 100 pM. D It binds to human IL-17F at

[0031] albumin The high specificity and affinity of antibodies make them ideal diagnostic and therapeutic agents, especially for modulating protein:protein interactions. However, antibodies can suffer from increased clearance rates from serum, especially if they lack an Fc domain that confers long life in vivo (Medasan et al., 1997, J. Immunol. 158:2211-2217).

[0032] Means for improving the half-life of antibodies are known. One approach has been to conjugate the fragments to polymer molecules. Thus, the half-life of Fab', F(ab') in animals 2 The short circulating half-life of the fragments has been improved by conjugation to polyethylene glycol (PEG, see, e.g., WO 98 / 25791, WO 99 / 64460 and WO 98 / 37200). Another approach has been to modify antibody fragments by conjugation to agents that interact with the FcRn receptor (see, e.g., WO 97 / 34631). Yet another approach to extend half-life has been to use polypeptides that bind to serum albumin (see, e.g., Smith et al., 2001, Bioconjugate Chem. 12:750-756, EP 0486525, US Patent 6267964, WO 04 / 001064, WO 02 / 076489 and WO 01 / 45746).

[0033] Serum albumin is an abundant protein in both the vascular and extravascular compartments with a half-life in humans of approximately 19 days (Peters, 1985, Adv Protein Chem. 37:161-245), which is similar to the half-life of IgG1, which is approximately 21 days (Waldeman & Strober, 1969, Progr. Allergy, 13:1-110).

[0034] Anti-serum albumin binding single variable domains have been described along with their use as conjugates to increase the half-life of drugs, including NCE (chemical entity) drugs, proteins and peptides, see for example Holt et al., Protein Engineering, Design & Selection, vol 21, 5, pp 283-288, WO 04003019, WO 2008 / 096158, WO 05118642, WO 2006 / 0591056 and WO 2011 / 006915. Other anti-serum albumin antibodies and their use in multispecific antibody formats are described in WO 2009 / 040562, WO 2010 / 035012 and WO 2011 / 086091. In particular, the inventors have previously described anti-albumin antibodies with improved humanization in WO 2013 / 068571.

[0035] The multispecific antibodies of the invention can be engineered to bind human serum albumin in order to extend their in vivo serum half-life and provide an improved pharmacokinetic profile.

[0036] antibody Antibodies for use in the context of this disclosure include whole antibodies and functionally active fragments thereof, i.e., molecules that specifically bind to IL-13, IL-17A and / or IL-17F, also referred to as antigen-binding fragments. Features described herein with respect to antibodies also apply to antibody fragments, unless the context dictates otherwise.

[0037] Whole antibodies, also known as "immunoglobulins (Ig)", generally refer to intact or full-length antibodies, i.e., antibodies comprising two heavy and two light chain elements interconnected by disulfide bonds that assemble to define a characteristic Y-shaped three-dimensional structure. Classical natural whole antibodies are monospecific, in that they bind to one antigen type, and bivalent, in that they have two independent antigen-binding domains. The terms "intact antibody", "full-length antibody" and "whole antibody" are used interchangeably to refer to monospecific, bivalent antibodies with a structure similar to a native antibody structure, including an Fc region as defined herein.

[0038] Each light chain comprises a light chain variable region (referred to herein as V L ) and the light chain constant region (C L Each heavy chain is composed of a heavy chain variable region (herein referred to as V) depending on the Ig class. H and three constant domains, CH 1 , C.H. 2 and C.H. 3 , or four constant domains CH 1 , C.H. 2 , C.H. 3 and C.H. 4 and a heavy chain constant region (CH) composed of: IgA, IgD, IgE, IgG, and IgM. The "class" of an Ig or antibody refers to the type of constant region, and includes IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0039] V of the antibody according to the present invention H Area and V L The regions can be further subdivided into regions of hypervariability (or "hypervariable regions") that determine antigen recognition, called complementarity determining regions (CDRs), interspersed with more structurally conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: 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 an antibody or antigen-binding fragment thereof are referred to as CDR-H1, CDR-H2, and CDR-H3, and the CDRs in the light chain variable region are referred to as CDR-L1, CDR-L2, and CDR-L3. They are numbered consecutively from the N-terminus to the C-terminus of each chain.

[0040] CDRs are conventionally numbered according to the system devised by Kabat et al., which is described in Kabat et al., 1991, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al., supra"). This numbering system is used herein unless otherwise indicated.

[0041] The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to shortening or insertion of structural components of the basic variable domain structure, whether in framework or complementarity determining regions. The correct Kabat numbering of residues can be determined for a given antibody by alignment of the homologous residues in the antibody sequence with the "standard" Kabat numbering sequence.

[0042] The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia, C. and Lesk, AMJ Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus, unless otherwise indicated, "CDR-H1" as used herein is intended to refer to residues 26 to 35 as described by a combination of the Kabat numbering system and the topological loop definition of Chothia.

[0043] The CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system.

[0044] In addition to the CDR loops, a fourth loop exists between CDR-2 (CDR-L2 or CDR-H2) and CDR-3 (CDR-L3 or CDR-H3) which is formed by framework 3 (FR3). The Kabat numbering system defines framework 3 as positions 66-94 of the heavy chain and positions 57-88 of the light chain.

[0045] Numbering schemes have been proposed based on alignment of the sequences of different members of the immunoglobulin family, e.g., as described in Kabat et al., 1991, and Dondelinger et al., 2018, Frontiers in Immunology, Vol 9, article 2278.

[0046] As used herein, the terms "constant domain" and "constant region" are used interchangeably to refer to the domain of an antibody outside the variable region. The constant domain is identical in all antibodies of the same isotype, but differs from one isotype to another. Typically, the constant region of a heavy chain is a CH chain that includes three or four constant domains from the N-terminus to the C-terminus. 1 - Hinge-CH 2 -CH 3 , in some cases C.H. 4 is formed by.

[0047] The constant domains of the antibody molecules of the invention, if any, can be selected taking into account the proposed function of the antibody molecule, in particular the effector functions that may be required. For example, the constant domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, when the antibody molecule is intended for therapeutic use and antibody effector functions are required, human IgG constant domains, in particular IgG1 and IgG3 isotypes, can be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, IgG2 and IgG4 isotypes can be used. It will be understood that sequence variants of these constant domains can also be used. For example, an IgG4 molecule can be used in which the serine at position 241 (numbered according to the Kabat numbering system) has been changed to a proline as described by Angal et al. (Angal et al., 1993. A single amino acid substitution the heterogeneity of chimeric mouse / human (IgG4) antibody during SDS-PAGE analysis Mol Immunol 30, 105-108), referred to herein as IgG4P.

[0048] "Fc", "Fc fragment", "Fc domain" and "Fc region" are used interchangeably to refer to the C-terminal region of an antibody that comprises the constant region of the antibody excluding the first constant immunoglobulin domain. Thus, Fc refers to the C-terminal region of an antibody that comprises the last two constant domains of IgA, IgD and IgG, the CH 2 and C.H. 3 or the last three constant domains of IgE and IgM, and the flexible hinge N-terminal to these domains. The human IgG1 heavy chain Fc region is defined herein to include residues C226 to its carboxyl terminus, numbering according to the EU index as in Kabat. In the context of human IgG1, the lower hinge refers to positions 226-236, according to the EU index as in Kabat, and the CH 2 The domain refers to positions 237-340, CH 3 The domain refers to positions 341 to 447. Corresponding Fc regions of other immunoglobulins can be identified by sequence alignment.

[0049] In the context of the present disclosure, the constant region or Fc region, if present, may be native as defined above or may be modified in various ways, as long as it comprises a functional FcR binding domain, preferably a functional FcRn binding domain. Preferably, the modified constant region or Fc region improves functionality and / or pharmacokinetics. Modifications may include deletion of certain parts of the Fc fragment. Modifications may further include various amino acid substitutions that may affect the biological properties of the antibody. There may also be mutations to increase FcRn binding and thus the in vivo half-life. Modifications may further include modification of the glycosylation profile of the antibody. A native Fc fragment may comprise a CH 2 domain, with each of the two heavy chains having an N-glycan attached to the asparagine residue at position 297 (Asn297). In the context of the present disclosure, antibodies can be glycosylated, i.e. engineered, to have a particular glycosylation profile that confers, for example, improved properties, such as improved effector function, or improved serum half-life.

[0050] The antibodies described herein are isolated. An "isolated" antibody is one that has been separated (e.g., by purification means) from a component of its natural environment.

[0051] The term "antibody" encompasses monovalent, i.e., antibodies that contain only one antigen-binding domain (e.g., one-arm antibodies containing an interconnected full-length heavy chain and a full-length light chain, also called "half antibodies"), and multivalent antibodies, i.e., antibodies that contain two or more antigen-binding domains.

[0052] The term "antibody" according to the present invention also includes antigen-binding fragments of antibodies. Antigen-binding fragments of antibodies include single chain antibodies (e.g., scFv and dsscfv), Fab, Fab', F(ab') 2 , Fv, single domain antibodies or nanobodies (e.g., V H Or V L , or V HH Or V NAR Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in WO 2011 / 117648, WO 2005 / 003169, WO 2005 / 003170 and WO 2005 / 003171.

[0053] Methods for generating and producing these antibody fragments are known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0054] As used herein, the term "Fab fragment" refers to a V L (variable light chain) domain and a light chain constant domain (CL), and a light chain fragment comprising the V H (variable heavy chain) domain and the first constant domain of the heavy chain (CH 1 ) refers to an antibody fragment containing

[0055] A typical "Fab' fragment" contains a pair of heavy and light chains, the heavy chains containing the variable region VH , constant domain C-H 1 and a native or modified hinge region, and the light chain comprises a variable region V L and a constant domain CL. A Fab' dimer according to the present disclosure comprises an F(ab') 2 where, for example, dimerization can be via the hinge.

[0056] As used herein, the term "single domain antibody" refers to an antibody fragment that consists 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.

[0057] The term "Fv" refers to a peptide that combines two variable domains, e.g., a cognate pair or affinity matured variable domains, i.e., V H and V L A cooperating variable domain such as a pair of

[0058] As used herein, a "single-chain variable fragment" or "scFv" refers to a fragment of a heavy chain variable domain (V H ) and V H Variable domain and V L A light chain variable domain (V) stabilized by a peptide linker between the variable domains L V refers to a single chain variable fragment comprising or consisting of H and V L The variable domains may be in any suitable orientation, for example, V H The C-terminus of L or V L The C-terminus of H It may be linked to the N-terminus of

[0059] As used herein, a "disulfide stabilized single chain variable fragment" or "dsscFv" refers to a V H Variable domain and V L It is stabilized by a peptide linker between the variable domains, H and V L(See, e.g., Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012; WO 2007109254).

[0060] As used herein, a "disulfide stabilized variable fragment" or "dsFv" refers to a V H Variable domain and V L It does not contain a peptide linker between the variable domains, but instead contains a V H and V L It refers to a single-chain variable fragment stabilized by an interdomain disulfide bond between

[0061] In one embodiment, a multispecific antibody of the invention is an antagonist antibody. As used herein, the term "antagonist antibody" refers to an antibody that can inhibit or neutralize 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.

[0062] Antibodies for use in the present invention may be, but are not limited to, monoclonal antibodies, humanized antibodies, fully human antibodies, or chimeric antibodies.

[0063] Monoclonal antibodies can be prepared by any method known in the art, such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

[0064] Antibodies can also be produced using single lymphocyte antibody techniques, for example by cloning and expressing immunoglobulin variable region cDNA generated from a single lymphocyte selected for producing a particular antibody by the methods described in Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-7848l, WO 92 / 02551, WO 2004 / 051268 and WO 2004 / 106377.

[0065] Screening of antibodies can be performed using assays that measure binding to the antigen and / or that measure the ability to block the binding of the antigen to one or more receptors. An example of a binding assay is, for example, an ELISA using a fusion protein of IL-13 immobilized on a plate and a conjugated secondary antibody to detect anti-IL-13 antibodies bound to IL-13. An example of a blocking assay is a flow cytometry-based assay that measures blocking of IL-13 ligand protein binding to IL-13R. A fluorescently labeled secondary antibody is used to detect the amount of IL-13 ligand protein bound to IL-13R.

[0066] Humanized antibodies (including CDR-grafted antibodies) are antibody molecules having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see, e.g., U.S. Pat. No. 5,585,089, WO 91 / 09967). It will be appreciated that only the specificity determining residues of the CDRs need to be transferred, rather than the entire CDR (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). Humanized antibodies can optionally further comprise one or more framework residues from the non-human species from which the CDRs are derived.

[0067] A chimeric antibody is composed of elements derived from two different species such that the elements retain the characteristics of the species they are derived from. Generally, a chimeric antibody contains a variable region from one species, e.g., mouse, rat, rabbit, etc., and a constant region from another species, e.g., human.

[0068] Antibodies can also be generated using various phage display methods known in the art, including those described by Brinkman et al. (J. Immunol. Methods, 1995, 182:41-50), Ames et al. J. Immunol. Methods, 1995, 184:177-186), (Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 187 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280), as well as WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401, as well as U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, U.S. Pat. Including those disclosed by U.S. Patent No. 5,580,717, U.S. Patent No. 5,427,908, U.S. Patent No. 5,750,753, U.S. Patent No. 5,821,047, U.S. Patent No. 5,571,698, U.S. Patent No. 5,427,908, U.S. Patent No. 5,516,637, U.S. Patent No. 5,780,225, U.S. Patent No. 5,658,727, U.S. Patent No. 5,733,743 and U.S. Patent No. 5,969,108.

[0069] A fully human antibody is one in which the variable and constant regions (if present) of both the heavy and light chains are all of human origin or are substantially identical to sequences of human origin, but not necessarily derived from the same antibody. Examples of fully human antibodies include, for example, antibodies produced by the phage display methods mentioned above, and antibodies produced in mice in which the mouse immunoglobulin variable genes and optionally the constant region genes have been replaced by their human counterparts, as described in general terms in, for example, EP 0546073, U.S. Pat. No. 5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,770,429, EP 0438474 and EP 0463151.

[0070] multispecific antibodies The antibody of the present invention is a multispecific antibody. As used herein, "multispecific or multi-specific antibody" refers to an antibody described herein having at least two binding domains, i.e., two or more binding domains, e.g., two or three binding domains, where the at least two binding domains independently bind to two different antigens or two different epitopes on the same antigen. A multispecific antibody is generally monovalent for each specificity (antigen). The multispecific antibody described herein encompasses monovalent and multivalent, e.g., bivalent, trivalent, tetravalent, multispecific antibodies.

[0071] A paratope is a region of an antibody that recognizes and binds to an antigen. The antibody of the present invention may be a multiparatopic antibody. As used herein, "multiparatopic antibody" refers to an antibody described herein that contains two or more different paratopes that interact with different epitopes from either the same antigen or two different antigens. The multiparatopic antibodies described herein may be biparatopic, triparatopic, or tetraparatopic.

[0072] As used herein, an "antigen-binding domain" refers to a portion or all of one or more variable domains that specifically interact with a target antigen, such as a pair of variable domains V H and V L A binding domain may comprise a single domain antibody. In one embodiment, each binding domain is monovalent. Preferably, each binding domain comprises no more than one VH and one VH. L Includes.

[0073] As used herein, "specifically" refers to a binding domain that recognizes only the antigen for which it is specific, or a binding domain that has a significantly higher binding affinity for the antigen for which it is specific compared to its affinity for a non-specific antigen. Binding affinity can be measured by standard assays, for example surface plasmon resonance such as BIAcore.

[0074] A variety of multispecific antibody formats have been produced. Although various classifications have been proposed, multispecific IgG antibody formats generally include bispecific IgG, adduct 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.

[0075] Techniques for producing 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., WO 1996027011, WO 1998050431), DuoBody technology (e.g., WO 2011131746), Azymetric technology (e.g., WO 2012058768). Further techniques for producing bispecific antibodies are described, for example, in Godar et al., 2018, Therapeutic bispecific antibody formats: a patent applications review (1994-2017), Expert Opinion on Therapeutic Patents, 28: 3, 251-276. Bispecific antibodies include CrossMab antibodies, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-lgG, knobs-in-holes consensus LC, knobs-in-holes assembly, charge pair, Fab arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ-body and orthogonal Fab, among others.

[0076] Loaded IgGs classically include full-length IgGs engineered by adding additional antigen-binding domains or antigen-binding fragments to the N-terminus and / or C-terminus 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 formats include DVD-IgG, IgG(H)-scFv, scFv-(H)lgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-lgG, IgC(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-lgG, IgG-2scFv, scFv4-lg, Zybody, and DVI-IgG (four-in-one), among others, as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106.

[0077] Multispecific antibody fragments include nanobodies, nanobody-HAS, BiTEs, bispecific antibodies, DART, TandAb, sc bispecific antibodies, sc-bispecific antibodies-CH3, bispecific antibodies-CH3, triple bodies, mini antibodies, minibodies, Tri Bi minibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFV3, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, sc bispecific antibody-Fc, bispecific antibody-Fc, tandem scFv-Fc, and intrabodies, as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67 (2015): 95-106.

[0078] Multispecific fusion proteins include Dock and Lock, ImmTAC, HSabody, sc bispecific antibody-HAS, and tandem scFv-toxin.

[0079] Multispecific antibody conjugates include IgG-lgG, Cov-X-body, and scFv1-PEG-scFv2.

[0080] Further multispecific antibody formats are described, for example, in Brinkmann and Kontermann, The making of bispecific antibodies, mAbs, 9:2, 182-212 (2017), in particular in FIG. 2, such as tandem scFv, trispecific antibody, Fab-VHH, taFv-Fc, scFv 4 -Ig, scFv 2 -Fcab, scFv 4 Bispecific and trispecific antibodies and methods for their production are disclosed, for example, in WO 99 / 37791.

[0081] The present invention provides multispecific antibodies that bind to human IL-13, human IL-17A and / or human IL-17F.

[0082] In one embodiment the multispecific antibody comprises an antigen binding site that binds human IL-13, wherein the IL-13 binding site comprises 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.

[0083] In one embodiment, the multispecific antibody comprises an antigen-binding site that binds human IL-13, wherein the IL-13 binding site comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 18 for CDR-H1, the sequence set forth in SEQ ID NO: 19 for CDR-H2, and the sequence set forth in SEQ ID NO: 20 for CDR-H3.

[0084] In one embodiment, the IL-13 binding site comprises a light chain variable region comprising the sequence set forth in SEQ ID NO:27.

[0085] In one embodiment, the IL-13 binding site comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO:28.

[0086] In one embodiment, the IL-13 binding site comprises a light chain variable region comprising the sequence set forth in SEQ ID NO:31.

[0087] In one embodiment, the IL-13 binding site comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO:32.

[0088] In one aspect, the multispecific antibody comprises an antigen-binding site that binds human IL-17A and human IL-17F, the antigen-binding site comprising: It comprises a light chain variable region comprising the sequence shown in SEQ ID NO:1 for CDR-L1, the sequence shown in SEQ ID NO:2 for CDR-L2, and the sequence shown in SEQ ID NO:3 for CDR-L3.

[0089] In one aspect, the multispecific antibody comprises an antigen-binding site that binds human IL-17A and human IL-17F, the antigen-binding site comprising: It comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 4 for CDR-H1, the sequence shown in SEQ ID NO: 5 for CDR-H2, and the sequence shown in SEQ ID NO: 6 for CDR-H3.

[0090] In one aspect, the antigen binding site that binds human IL-17A and human IL-17F comprises a light chain variable region comprising the sequence shown in SEQ ID NO:7.

[0091] In one aspect, the antigen binding site that binds human IL-17A and human IL-17F comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO:9.

[0092] In one embodiment the multispecific antibody lacks an Fc domain and half-life is provided by the antigen binding site that binds serum albumin.

[0093] In one embodiment the multispecific antibody comprises the sequence shown in SEQ ID NO:57 or SEQ ID NO:59.

[0094] In one embodiment the multispecific antibody comprises the sequence shown in SEQ ID NO:61 or SEQ ID NO:63.

[0095] In one embodiment the multispecific antibody comprises the sequence set forth in SEQ ID NO:59 and the sequence set forth in SEQ ID NO:63.

[0096] In one embodiment, the multispecific antibody comprises: The polypeptide chain of formula (I): V H -CH 1 -(CH 2 ) s -(CH 3 ) t -X-(V 1 ) p , and A polypeptide chain of formula (II): V L -C L -Y.V. 2 comprising or consisting of During the ceremony, V H represents the heavy chain variable domain, CH 1 represents domain 1 of the heavy chain constant region, CH 2 represents domain 2 of the heavy chain constant region, CH 3 represents domain 3 of the heavy chain constant region, X represents a bond or a linker; V 1 represents dsscFv, dsFv, or scFv, V L represents the light chain variable domain, C L represents a domain derived from the light chain constant region, such as Ckappa, Y represents a bond or a linker; V 2 represents dsscFv, dsFv or scFv, p represents 0 or 1; s represents 0 or 1; t represents 0 or 1; wherein when p is 0, X is not present, and when q is 0, Y is not present; The polypeptide chain of formula (I) comprises a Protein A binding domain, The polypeptide chain of formula (II) does not bind to Protein A.

[0097] In one embodiment, when s is 0 and t is 0, the multispecific antibody according to the present disclosure has are provided as dimers of heavy and light chains of formula (I) and (II), respectively, V H -CH 1 The part is V L -C L Together with the portion to form a functional Fab or Fab' fragment.

[0098] In one embodiment, when s is 1 and t is 1, the multispecific antibody according to the present disclosure has The present invention is provided as a dimer of two heavy chains and two light chains of formula (I) and (II), respectively, in which the two heavy chains are linked by interchain interactions, in particular CH 2 -CH 3 The V of each heavy chain is connected at the H -CH 1 The V portion of each light chain L -C L Together with the V portion, they form a functional Fab or Fab' fragment. H -CH 1 -CH 2 -CH 3 The part has two V L -C L Together with the portion to form a functional full length antibody, in such embodiments, the full length antibody can include a functional Fc region.

[0099] V H represents a heavy chain variable domain. In one embodiment, V H In one embodiment, V is humanized. H is fully humanoid.

[0100] V L represents a light chain variable domain. In one embodiment, V L In one embodiment, V is humanized. L is fully human.

[0101] In general, V H and V L together form an antigen-binding domain. In one embodiment, H and V L form a cognate pair.

[0102] As used herein, a "cognate pair" refers to a pair of variable domains from a single antibody that has been generated in vivo, i.e., a naturally occurring pair of variable domains isolated from a host. Thus, a cognate pair is a V H and V L In one example, the cognate pair binds an antigen cooperatively.

[0103] As used herein, a "variable region" or "variable domain" refers to the region of an antibody chain comprising the CDRs and frameworks, particularly appropriate frameworks.

[0104] Variable regions for use in this disclosure are generally derived from antibodies which can be produced by any method known in the art.

[0105] "Derived" as used herein refers to the fact that the sequence used, or a sequence very similar to the sequence used, is obtained from original genetic material, such as the light or heavy chain of an antibody.

[0106] As used herein, "highly similar" is intended to refer to amino acid sequences that are 95% or more similar over their entire length, such as 96, 97, 98 or 99%.

[0107] V H and V L The variable regions for use in the invention as described herein above may be from any suitable source and may for example be fully human or humanized.

[0108] In one embodiment, V H and V Lis specific for a first antigen.

[0109] In one embodiment, V 1 The binding domain of is specific for a second antigen.

[0110] In one embodiment, V 2 The binding domain of is specific for a third antigen.

[0111] In one embodiment, V H -V L , V 1 , and V 2 Each of these binds separately to its respective antigen, if present.

[0112] In one embodiment, CH 1 The domain is a naturally occurring domain 1 from an antibody heavy chain or a derivative thereof. 2 The domain is the naturally occurring domain 2 from an antibody heavy chain or a derivative thereof. 3 The domain is the naturally occurring domain 3 from an antibody heavy chain or a derivative thereof.

[0113] In one embodiment, the C in the light chain L The fragment is a constant kappa sequence or a derivative thereof. L The fragment is a constant lambda sequence or a derivative thereof.

[0114] A derivative of a naturally occurring domain, as used herein, is intended to refer to at least one amino acid in the naturally occurring sequence being substituted or deleted to optimize the properties of the domain, such as by eliminating an undesirable property, while retaining the characteristic features of the domain. In one embodiment, a derivative of a naturally occurring domain comprises the substitution or deletion of 2, 3, 4, 5, 6, 7, 8, 10, 11 or 12 amino acids compared to the naturally occurring sequence.

[0115] In one embodiment, one or more natural or engineered interchain (ie, between light and heavy chain) disulfide bonds are present in a functional Fab or Fab' fragment.

[0116] In one embodiment, the CH 1 and C. L There is a "native" disulfide bond between

[0117] C L If the domain is derived from either kappa or lambda, the natural position of the bond forming cysteine ​​is 214 in human ckappa and clamda (Kabat numbering 4th edition 1987).

[0118] CH 1 The exact location of the disulfide bond forming cysteines in the heavy chains of human IgM and IgA2B depends on the particular domain actually used. Thus, for example, in human gamma-1, the natural position of the disulfide bond is located at position 233 (Kabat numbering 4th edition 1987). The positions of the cysteine ​​forming bonds in other human isotypes such as gamma 2, 3, 4, IgM and IgD are known, e.g., position 127 for human IgM, IgE, IgG2, IgG3, IgG4, and 128 for the heavy chains of human IgD and IgA2B.

[0119] Optionally, V of the polypeptides of formula I and II H and V L There may be a disulfide bond between

[0120] In one embodiment, the multispecific antibody according to the present disclosure comprises 1 and C. L and a disulfide bond at a position equivalent or corresponding to that found in nature.

[0121] In one embodiment, CH 1 A constant region including C LThe constant region of the present invention has a disulfide bond at a non-naturally occurring position. This can be engineered into the molecule by introducing cysteines into the amino acid chain at the required position or positions. This non-naturally occurring disulfide bond is 1 and C. L In addition to, or instead of, the native disulfide bond that exists between. Cysteine ​​at the native position can be replaced with an amino acid, such as serine, that is unable to form disulfide bridges.

[0122] The introduction of engineered cysteines 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, such as QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA). Cassette mutagenesis can be performed according to Wells et al., 1985, Gene, 34:315-323.

[0123] Alternatively, variants can be generated by total gene synthesis by annealing, ligation and PCR amplification and cloning of overlapping oligonucleotides.

[0124] In one embodiment, CH 1 and C. LThe disulfide bonds between are completely absent, for example, the interchain cysteine ​​may be replaced with another amino acid such as serine. Thus, in one embodiment, there are no interchain disulfide bonds in a functional Fab fragment of the molecule. Disclosures such as WO 2005 / 003170, incorporated herein by reference, describe methods for providing Fab fragments without interchain disulfide bonds.

[0125] Examples of antibody formats for use in the present invention include adjunct IgG and adjunct Fab, where the whole IgG or Fab fragment, respectively, may contain at least one additional antigen binding domain (e.g., 1, 2, 3 or 4 additional antigen binding domains), such as single domain antibodies (e.g., V H Or V L , or VHH), scFv, dsscFv, dsFv, to the N-terminus and / or C-terminus of the light chain of the IgG or Fab, and optionally to the heavy chain of the IgG or Fab, as described, for example, in WO 2009 / 040562, WO 2010035012, WO 2011 / 030107, WO 2011 / 061492, WO 2011 / 061246 and WO 2011 / 086091, all of which are incorporated herein by reference. Added IgGs, including full length IgGs engineered by adding dsFv to the C-terminus of the IgG light chain (and optionally to the heavy chain), were first disclosed in WO 2015 / 197789, which is incorporated herein by reference.

[0126] A preferred antibody format for use in the present invention comprises a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binds a therapeutic target and one scFv or dsscFv increases half-life, e.g., by binding to albumin). Such antibody fragments are described in International Patent Application Publication No. WO 2015 / 197772, which is incorporated by reference in its entirety, particularly with respect to its discussion of antibody fragments.

[0127] V 1 represents dsscFv, dsFv or scFv.

[0128] V 2 represents dsscFv, dsFv or scFv.

[0129] In one embodiment, V 1 and / or V 2 is a dsFv or dsscFv, V 1 and / or V 2 Variable domain V H and V L The disulfide bond between is between two of the residues listed below (Kabat numbering is used in the following list unless the context indicates otherwise). When Kabat numbering is referred to, the relevant reference is Kabat et al., 1991 (5 th edition, Bethesda, Md.), in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.

[0130] In one embodiment, the disulfide bond is See, for example, Protein Science 6, 781-788 Zhu et al. (1997) H 37+V L 95C, See, for example, Weatherill et al., Protein Engineering, Design & Selection, 25 (321-329), 2012. H 44+V L 100, See, for example, J Biochem. 118, 825-831 Luo et al. (1995) H 44+V L 105, See, for example, Protein Science 6, 781-788 Zhu et al. (1997) H 45+V L 87, See, for example, FEBS Letters 377 135-139 Young et al. (1995) H 55+V L 101, See, for example, Biochemistry 29 1362-1367 Glockshuber et al. (1990) H 100+V L 50, See, for example, Biochemistry 29 1362-1367 Glockshuber et al. (1990) H 100b+V L 49, See, for example, Protein Science 6, 781-788 Zhu et al. (1997) H 98+V L 46, See, for example, Protein Science 6, 781-788 Zhu et al. (1997) H 101+V L 46, 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). H 105+V L 43, See, for example, FEBS Letters 377 135-139 Young et al. (1995) H 106+V L A position selected from the group consisting of 57 and at corresponding positions in the variable region pairs located within the molecule.

[0131] In one embodiment, the disulfide bond is at position VH 44 and position V L It is formed between 100 and 100.

[0132] The above amino acid pairs are in positions that facilitate substitution with cysteines so that disulfide bonds can be formed. Cysteines can be engineered into these desired positions by known techniques. Thus, in one embodiment, an engineered cysteine ​​according to the present disclosure refers to the case where the naturally occurring residue at a given amino acid position is replaced with a cysteine ​​residue.

[0133] The introduction of engineered cysteines 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, such as QuikChange® Site-Directed Mutagenesis Kit (Stratagen, La Jolla, CA). Cassette mutagenesis can be performed according to Wells et al., 1985, Gene, 34:315-323.

[0134] Alternatively, variants can be generated by total gene synthesis by annealing, ligation and PCR amplification and cloning of overlapping oligonucleotides.

[0135] Thus, in one embodiment, V 1 and / or V 2 is a dsFv or dsscFv, V 1Variable domain V H and V L and / or V 2 Variable domain V H and V L may be linked by a disulfide bond between two cysteine ​​residues, and the position of the pair of cysteine ​​residues is 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.

[0136] In one embodiment, V 1 and / or V 2 is a dsFv or dsscFv, V 1 Variable domain V H and V L and / or V 2 Variable domain V H and V L The two cysteine ​​residues outside the CDRs, V H One in and V L and the position of the pair of cysteine ​​residues may be 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 VL 50, V H 98 and V L 46, V H 105 and V L 43, V H 106 and V L 57.

[0137] In one embodiment, V 1 is a dsFv or dsscFv, V 1 Variable domain V H and V L One is V H 44 position and the other is V L In one embodiment, the V 2 is a dsFv or dsscFv, V 2 Variable domain V H and V L One is V H 44 position and the other is V L It is linked by a disulfide bond between two engineered cysteine ​​residues at position 100.

[0138] In one embodiment, V 1 is dsscFv, dsFv, or scFv, 1 V H The domain is attached to X.

[0139] In one embodiment, V 1 is dsscFv, dsFv, or scFv, 1 V L The domain is attached to X.

[0140] In one embodiment, V 2 is dsscFv, dsFv, or scFv, 2 V H The domain is attached to the Y.

[0141] In one embodiment, V 2is dsscFv, dsFv, or scFv, 2 V L The domain is attached to the Y.

[0142] Those skilled in the art will appreciate that 1 and / or V 2 represents a dsFv, the multispecific antibody comprises the corresponding free V that is not bound to X or Y. H Or V L It will be understood that the present invention includes a third polypeptide encoding a domain. 1 and V 2 When a Fv is a dsFv, the "free variable domains" (i.e. those linked to the remainder of the polypeptide via disulfide bonds) are common to both chains. Thus, although the actual variable domains fused or linked to the polypeptide via X or Y may be different in each polypeptide chain, the paired free variable domains are generally identical to each other.

[0143] In some embodiments, p is 1. In some embodiments, p is 0.

[0144] In some embodiments, s is 1. In some embodiments, s is 0.

[0145] In some embodiments, t is 1. In some embodiments, t is 0.

[0146] In some embodiments, s is 1 and t is 1. In some embodiments, s is 0 and t is 0.

[0147] In one embodiment, p is 1, q is 1, r is 0, s is 0, t is 0, and V 1 and V 2 and both represent dsscFv. Thus, in one embodiment, a) a polypeptide chain of formula (Ia): V H -CH 1 -XV 1 , and b) a polypeptide chain of formula (IIa): V L -C L -Y.V. 2 comprising or consisting of During the ceremony, V H represents the heavy chain variable domain, CH 1 represents domain 1 of the heavy chain constant region, X represents a bond or a linker; Y represents a bond or a linker; V 1 represents scFv, dsscFv, or dsFv, V L represents the light chain variable domain, C L represents a domain derived from the light chain constant region, such as Ckappa, V 2 represents scFv, dsscFv or dsFv, V 1 Or V 2 at least one of is a dsscFv or a dsFv, The polypeptide chain of formula (Ia) comprises a Protein A binding domain, The polypeptide chains of Formula (IIa) provide a multispecific antibody that does not bind to Protein A and that binds to human IL-13, human IL-17A and / or human IL-17F.

[0148] In such an embodiment, V 2 does not bind to Protein A, i.e., V 2 In one embodiment, the scFv, dsscFv or dsFv of V does not comprise a Protein A binding domain. 2 , i.e. V 2 In another embodiment, the scFv, dsscFv or dsFv of comprises a VH1 domain. 2 , i.e. V 2 The scFv, dsscFv or dsFv of comprises a VH3 domain that does not bind to Protein A. 2 , i.e. V 2In one embodiment, the scFv, dsscFv or dsFv of comprises a VH2 domain. 2 , i.e. V 2 In one embodiment, the scFv, dsscFv or dsFv of comprises a VH4 domain. 2 , i.e. V 2 In one embodiment, the scFv, dsscFv or dsFv of comprises a VH5 domain. 2 , i.e. V 2 In one embodiment, the scFv, dsscFv or dsFv of formula (Ia) comprises a VH6 domain. H Or V 1 In one embodiment, the polypeptide chain of formula (Ia) comprises only one Protein A binding domain present in 1 In another embodiment, the polypeptide chains of formula (Ia) each comprise a VH and a V 1 It contains two protein A binding domains present in

[0149] In another embodiment, p is 0, q is 1, r is 0, s is 1, t is 1, and V 2 is a dsscFv. Thus, in one embodiment, a) a polypeptide chain of formula (Ib): V H -CH 1 -CH 2 -CH 3 , and b) a polypeptide chain of formula (IIb): V L -C L -Y.V. 2 comprising or consisting of During the ceremony, V H represents the heavy chain variable domain, CH 1 represents domain 1 of the heavy chain constant region, CH 2 represents domain 2 of the heavy chain constant region, CH 3 represents domain 3 of the heavy chain constant region, Y represents a bond or a linker; V L represents the light chain variable domain, C L represents a domain derived from the light chain constant region, such as Ckappa, V 2 represents dsscFv, the polypeptide chain of formula (Ib) comprises a Protein A binding domain, The polypeptide chains of Formula (IIb) provide a multispecific antibody that does not bind to Protein A and that binds to human IL-13, human IL-17A and / or human IL-17F.

[0150] In such an embodiment, V 2 does not bind to Protein A, i.e., V 2 In one embodiment, the dsscFv of V does not contain a Protein A binding domain. 2 , i.e. V 2 In another embodiment, the dsscFv of comprises a VH1 domain. 2 , i.e. V 2 In one embodiment, the dsscFv of formula (Ib) comprises a VH3 domain that does not bind to Protein A. H or CH 2 -CH 3 In another embodiment, the polypeptide chains of formula (Ib) each comprise a Protein A binding domain present in VH and CH. 2 -CH 3 It contains two protein A binding domains present in

[0151] In another embodiment, p is 0, q is 1, r is 0, s is 1, t is 1, and V 2 is a dsFv. Thus, in one embodiment, a) a polypeptide chain of formula (Ic): V H -CH 1 -CH 2 -CH 3 , and b) a polypeptide chain of formula (IIc): VL -C L -Y.V. 2 comprising or consisting of During the ceremony, V H represents the heavy chain variable domain, CH 1 represents domain 1 of the heavy chain constant region, CH 2 represents domain 2 of the heavy chain constant region, CH 3 represents domain 3 of the heavy chain constant region, Y represents a bond or a linker; V L represents the light chain variable domain, C L represents a domain derived from the light chain constant region, such as Ckappa, V 2 represents dsFv, the polypeptide chain of formula (Ic) comprises a Protein A binding domain, The polypeptide chains of Formula (IIc) provide a multispecific antibody that does not bind to Protein A and binds to human IL-13, human IL-17A and / or human IL-17F.

[0152] In such an embodiment, V 2 , i.e. V 2 does not bind to Protein A. In one embodiment, the polypeptide chain of formula (Ic) is H or CH 2 -CH 3 In another embodiment, the polypeptide chains of formula (Ic) each comprise a Protein A binding domain present in H and C.H. 2 -CH 3 It contains two protein A binding domains present in

[0153] In one embodiment of the multispecific antibody of the invention: V L and V H comprises an antigen-binding site that binds human IL-17A and / or human IL-17F, V1 comprises an antigen-binding site that binds to human serum albumin, V 2 contains an antigen-binding site that binds to human IL-13.

[0154] In one embodiment, V L comprises the sequence shown in SEQ ID NO: 1 for CDR-L1, the sequence shown in SEQ ID NO: 2 for CDR-L2, and the sequence shown in SEQ ID NO: 3 for CDR-L3, H comprises the sequence shown in SEQ ID NO: 4 for CDR-H1, the sequence shown in SEQ ID NO: 5 for CDR-H2, and the sequence shown in SEQ ID NO: 6 for CDR-H3.

[0155] In one embodiment, V 1 comprises a light chain variable region comprising the sequence set forth in SEQ ID NO: 39 for CDR-L1, the sequence set forth in SEQ ID NO: 40 for CDR-L2, and the sequence set forth in SEQ ID NO: 41 for CDR-L3, and a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 42 for CDR-H1, the sequence set forth in SEQ ID NO: 43 for CDR-H2, and the sequence set forth in SEQ ID NO: 44 for CDR-H3.

[0156] In one embodiment, V 2 comprises a light chain variable region comprising the sequence shown in SEQ ID NO: 15 for CDR-L1, the sequence shown in SEQ ID NO: 16 for CDR-L2, and the sequence shown in SEQ ID NO: 17 for CDR-L3, and a heavy chain variable region comprising the sequence shown in SEQ ID NO: 18 for CDR-H1, the sequence shown in SEQ ID NO: 19 for CDR-H2, and the sequence shown in SEQ ID NO: 20 for CDR-H3; In one embodiment, V L comprises the sequence shown in SEQ ID NO:7, H comprises the sequence shown in SEQ ID NO:9.

[0157] In one embodiment, V 1 comprises a light chain variable region comprising the sequence set forth in SEQ ID NO:45 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:46.

[0158] In one embodiment, V 1 comprises a light chain variable region comprising the sequence set forth in SEQ ID NO:49 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:50.

[0159] In one embodiment, V 1 the light chain variable region and the heavy chain variable region are linked by a linker, the linker comprising the sequence shown in SEQ ID NO:68.

[0160] In one embodiment, V 1 is an scFv comprising the sequence shown in SEQ ID NO:53, or a dsscFv comprising the sequence shown in SEQ ID NO:55.

[0161] In one embodiment, V 2 comprises a light chain variable region comprising the sequence set forth in SEQ ID NO:27 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:28.

[0162] In one embodiment, V 2 comprises a light chain variable region comprising the sequence set forth in SEQ ID NO:31 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:32.

[0163] In one embodiment, V 2 the light chain variable region and the heavy chain variable region are linked by a linker, the linker comprising the sequence shown in SEQ ID NO:66.

[0164] In one embodiment, V 2 is an scFv comprising the sequence shown in SEQ ID NO: 35, or a dsscFv comprising the sequence shown in SEQ ID NO: 37.

[0165] In one embodiment, X is a linker comprising the sequence set forth in SEQ ID NO:67.

[0166] In one embodiment, Y is a linker comprising the sequence set forth in SEQ ID NO:65.

[0167] In one embodiment, the polypeptide chain of formula (Ia) comprises the sequence shown in SEQ ID NO:57 or SEQ ID NO:59.

[0168] In one embodiment, the polypeptide chain of formula (IIa) comprises the sequence shown in SEQ ID NO:61 or SEQ ID NO:63.

[0169] In one embodiment, the polypeptide chain of formula (Ia) comprises the sequence set forth in SEQ ID NO:59 and the polypeptide chain of formula (IIa) comprises the sequence set forth in SEQ ID NO:63.

[0170] It will be understood that one or more amino acid substitutions, additions and / or deletions may be made to the sequences provided by the present invention without significantly altering the ability of the antibody to bind to the antigen and neutralize its biological activity. The effect of any amino acid substitution, addition and / or deletion can be readily tested by one of skill in the art by determining inhibition of antigen binding and biological activity, for example, using the methods described herein, particularly those exemplified in the examples.

[0171] The present invention therefore provides a multispecific antibody comprising CDRs defined by the sequences as set out in SEQ ID NOs: 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 of the CDRs have been replaced by another amino acid, such as a similar amino acid as defined herein below.

[0172] "Identity" as used herein indicates that at any particular position in the aligned sequences, the amino acid residue is the same between the sequences. "Similarity" as used herein indicates that at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be used in place of isoleucine or valine. Other amino acids that may be substituted for one another include: - phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains), - lysine, arginine and histidine (amino acids with basic side chains), - aspartic acid and glutamic acid (amino acids with acidic side chains), - asparagine and glutamine (amino acids with amide side chains), and - cysteine ​​and methionine (amino acids with sulfur-containing side chains). The degree of identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; BLAST™ software available from NCBI (Altschul, S. et al., Molecular Biology, vol. 1, vol. 1, pp. 1111-1115, 1997). al.,1990, J.Mol.Biol.215:403-410, Gish,W.&States,DJ1993,Nature Genet.3:266-272.Madden,TLet al.,1996,Meth.Enzymol.266:131-141, Altschul,SFet al.,1997,Nucleic Acids Res.25:3389-3402, Zhang, J. & Madden, TL1997, Genome Res.7:649-656,).

[0173] In one embodiment, the CDRs of the multispecific antibody comprise sequences having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 15, 16, 17, 18, 19, 20, 39, 40, 41, 42, 43 and 44.

[0174] In one embodiment, V L comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:7, and VH comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:9.

[0175] In one embodiment, V 1 comprises a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:45 and / or a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:46.

[0176] In one embodiment, V 1 comprises a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:49 and / or a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:50.

[0177] In one embodiment, V 1 the light chain variable region and the heavy chain variable region are linked by a linker, the linker comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:68.

[0178] In one embodiment, V 1is an scFv comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:53, or a dsscFv comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:55.

[0179] In one embodiment, V 2 comprises a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:27 and / or a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:28.

[0180] In one embodiment, V 2 comprises a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:31 and / or a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:32.

[0181] In one embodiment, V 2 the light chain variable region and the heavy chain variable region are linked by a linker, the linker comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:66.

[0182] In one embodiment, V 2 is an scFv comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO: 35, or a dsscFv comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO: 37.

[0183] In one embodiment, X is a linker comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:67.

[0184] In one embodiment Y is a linker comprising a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:65.

[0185] In one embodiment, the polypeptide chain of formula (Ia) comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence shown in SEQ ID NO:57 or SEQ ID NO:59.

[0186] In one embodiment, the polypeptide chain of formula (IIa) comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence shown in SEQ ID NO:61 or SEQ ID NO:63.

[0187] 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 set forth 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 set forth in SEQ ID NO:63.

[0188] Epitope An epitope is the region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0189] Whether an antibody binds to the same epitope as a reference antibody or competes for binding with a reference antibody can be easily determined by using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody of the present invention, the reference antibody is bound to a protein or peptide under saturating conditions. The ability of the test antibody to bind to the protein or peptide is then evaluated. If the test antibody can bind to the protein or peptide after saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind to the protein or peptide after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the present invention.

[0190] To determine whether an antibody competes for binding with a reference antibody, the above binding methodology is carried out in two directions. In the first direction, the reference antibody is bound to the protein / peptide under saturating conditions, followed by evaluating the binding of the test antibody to the protein / peptide molecule. In the second direction, the test antibody is bound to the protein / peptide under saturating conditions, followed by evaluating the binding of the reference antibody to the protein / peptide. In both directions, if only the first (saturating) antibody can bind to the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As will be understood by those skilled in the art, an antibody that competes for binding with a reference antibody does not necessarily bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0191] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, 75%, 90%, or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0192] Further routine experiments (e.g., peptide mutagenesis and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0193] The antibody may compete for binding to IL-17A or IL-17F with, or bind to the same epitope as, a multispecific antibody comprising a combination of CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences of SEQ ID NOs: 1 / 2 / 3 / 4 / 5 / 6.

[0194] The antibody may compete for binding to IL-13 with, or bind to the same epitope as, a multispecific antibody comprising a combination of CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences of SEQ ID NOs: 15 / 16 / 17 / 18 / 19 / 20.

[0195] The antibody may compete for binding to serum albumin with a multispecific antibody comprising a combination of CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences of SEQ ID NOs: 39 / 40 / 41 / 42 / 43 / 44, or may bind to the same epitope as the multispecific antibody.

[0196] Effector molecules If desired, the multispecific antibodies for use in the present invention can be conjugated to one or more effector molecules. It will be understood that the effector molecule can comprise a single effector molecule or two or more such molecules linked to form a single moiety capable of binding to the antibody of the present invention. If it is desired to obtain an antibody fragment linked to an effector molecule, it can be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment is linked to the effector molecule directly or via a coupling agent. Techniques for conjugating such effector molecules to antibodies are 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). Particular chemical procedures include, for example, those described in WO 93 / 06231, WO 92 / 22583, WO 89 / 00195, WO 89 / 01476 and WO 03031581. Alternatively, where the effector molecule is a protein or polypeptide, linkage can be achieved using recombinant DNA procedures, for example as described in WO 86 / 01533 and EP 0392745.

[0197] The term effector molecule as used herein includes, for example, anti-neoplastic agents, drugs, toxins, biologically active proteins such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides, in particular radioactive iodides, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy.

[0198] Examples of effector molecules include cytotoxins or cytotoxic agents, including any agent that is detrimental to (e.g., kills) cells, such as combrestatins, dolastatins, epothilones, staurosporines, maytansinoids, spongiostatins, rhizoxins, halichondrins, roridin, hemiasterin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.

[0199] 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., mechlorethamine, thioepaclorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicin or duocarmycin), and antimitotic agents (e.g., vincristine and vinblastine).

[0200] Other effector molecules include 111 In and 90 Y.,Lu 177 , Bismuth 213 , Californium 252 ,iridium 192 and tungsten 188 / rhenium 188 or drugs such as alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

[0201] Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteases, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin, proteins such as insulin, tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, thrombotic or antiangiogenic agents such as angiostatin or endostatin, or biological response modifiers such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factors and immunoglobulins.

[0202] Other effector molecules can include detectable substances that are useful, for example, for diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radionuclides, positron-emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents, see generally U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; suitable radionuclides include 125 I, 131 I,111 In and 99 Examples include Tc.

[0203] In another example, the effector molecule may increase the half-life of the antibody in vivo and / or reduce the immunogenicity of the antibody and / or enhance delivery of the antibody across an epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins, or albumin binding compounds, such as those described in WO 05 / 117984.

[0204] When the effector molecule is a polymer, it may generally be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer, or a branched or unbranched polysaccharide, such as a homo- or heteropolysaccharide.

[0205] Particular optional substituents which may be present on the above synthetic polymers include one or more hydroxy, methyl or methoxy groups.

[0206] Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol) poly(vinyl alcohol) or derivatives thereof, in particular optionally substituted poly(ethylene glycol), such as methoxypoly(ethylene glycol) or derivatives thereof.

[0207] Exemplary naturally occurring polymers include lactose, amylose, dextran, glycogen, or derivatives thereof.

[0208] "Derivatives" as used herein is intended to include reactive derivatives, e.g., thiol-selective reactive groups such as maleimides. The reactive groups can be linked to the polymer directly or via a linker segment. It will be understood that the residue of such a group will, in some cases, form part of the product as the linking group between the antibody fragment and the polymer.

[0209] The size of the polymer can vary as desired, but generally ranges from 500 Da to 50,000 Da, e.g., 5,000 to 40,000 Da, e.g., 20,000 to 40,000 Da, in average molecular weight. The polymer size can be selected based on, among other things, the intended use of the product, e.g., its ability to localize to a particular tissue, such as a tumor, or its ability to extend circulatory half-life (for a review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, when the product is intended to leave the circulation and penetrate into tissues, it may be advantageous to use a low molecular weight polymer, e.g., with a molecular weight of about 5,000 Da. For applications in which the product remains in the circulation, it may be advantageous to use a high molecular weight polymer, e.g., with a molecular weight in the range of 20,000 Da to 40,000 Da.

[0210] Suitable polymers include polyalkylene polymers such as poly(ethylene glycol) or especially methoxypoly(ethylene glycol) or derivatives thereof, especially those having a molecular weight in the range of about 15,000 Da to about 40,000 Da.

[0211] In one example, the antibody for use in the present invention is conjugated to a poly(ethylene glycol) (PEG) moiety. In one particular example, 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 in the antibody fragment, such as any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids may be naturally present in the antibody fragment or can be engineered into the fragment using recombinant DNA methods (see, for example, U.S. Pat. No. 5,219,996, U.S. Pat. No. 5,667,425, WO 98 / 25971). In one example, the antibody molecule of the present invention is a modified Fab fragment, and the modification is the addition of one or more amino acids to the C-terminus of its heavy chain to allow for the attachment of an effector molecule. Suitably, the additional amino acids form a modified hinge region that includes one or more cysteine ​​residues to which the effector molecule can be attached. Multiple sites can be used to attach two or more PEG molecules.

[0212] Suitably, the PEG molecule can be covalently attached via a thiol group of at least one cysteine ​​residue located in the antibody fragment. Each polymer molecule attached to the modified antibody fragment can be covalently attached to the sulfur atom of a cysteine ​​residue located in the fragment. The covalent bond is generally a disulfide bond, or in particular a sulfur-carbon bond. When a thiol group is used as the attachment point, a suitably activated effector molecule can be used, for example a thiol-selective derivative such as a maleimide or a cysteine ​​derivative. An activated polymer can be used as a starting material in the preparation of the polymer-modified antibody fragment described above. The activated polymer can be any polymer that contains a thiol-reactive group, such as an α-halo carboxylic acid or ester, for example an iodoacetamide, an imide, for example a maleimide, a vinyl sulfone or a disulfide. Such starting materials can be obtained commercially (e.g. Nektar, formerly Shearwater Polymers Inc., Huntsville, Alabama, USA) or can be prepared from commercially available starting materials using conventional chemical procedures. Particular PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly Shearwater, Rapp Polymere and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

[0213] 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, e.g. according to the methods disclosed in EP 0 948 544 or EP 1 090 037 ["Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York; "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC; and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545] In one example, PEG is attached to a cysteine ​​in the hinge region. In one example, the PEG-modified Fab fragment has a maleimide group covalently attached to a single thiol group in the modified hinge region. A lysine residue can be covalently attached to the maleimide group, and a methoxypoly(ethylene glycol) polymer having a molecular weight of about 20,000 Da can be attached to each amine group on the lysine residue. Thus, the total molecular weight of PEG attached to the Fab fragment can be about 40,000 Da.

[0214] In one embodiment, the multispecific antibody is not linked to an effector molecule.

[0215] Polynucleotides / vectors / host cells The present invention also provides isolated polynucleotides encoding the polypeptide chains of the IL-13 / IL-17AF multispecific antibody molecule.

[0216] The 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 sequences shown in the sequence listing. Typically, variants have 1-20, 1-50, 1-75 or 1-100 substitutions and / or deletions.

[0217] A suitable variant may be at least about 70% homologous, typically at least about 80 or 90%, more suitably at least about 95%, 97% or 99% homologous to any one of the polynucleotides of the nucleic acid sequences disclosed herein. A variant may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. A variant typically retains about 60% to about 99% identity, about 80% to about 99% identity, about 90% to about 99% identity or about 95% to about 99% identity. Homology and identity at these levels generally exist at least with respect to the coding region of the polynucleotide. Methods for measuring homology are known in the art, and it will be understood by the skilled artisan that in the present context, homology is calculated based on nucleic acid identity. Such homology may exist over a region of at least about 15, at least about 30, for example at least about 40, 60, 100, 200 or more contiguous nucleotides (depending on the length). Such homology may exist over the entire length of the unmodified polynucleotide sequence.

[0218] A homologue may differ from a sequence in the related polynucleotide by less than about 3, 5, 10, 15, 20 or more mutations, each of which may be a substitution, deletion or insertion. For example, a homologue may differ by between 3 and 50 mutations, often between 3 and 20 mutations. These mutations may be measured over a region of at least 30, for example at least about 40, 60 or 100 or more contiguous nucleotides of the homologue.

[0219] The DNA sequences of the present invention may include synthetic DNA, produced, for example, by chemical processes, cDNA, genomic DNA, or any combination thereof.

[0220] The general methods by which vectors can be constructed, the transfection methods and the culture methods are known to those skilled in the art. In this regard, see "Current Protocols in Molecular Biology", 1999, FMA USubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0221] Host cells are also provided that contain one or more cloning or expression vectors that contain one or more DNA sequences encoding the IL-13 / IL-17AF multispecific antibody. Any suitable host cell / vector system can be used for the expression of the DNA sequences encoding the IL-13 / IL-17AF multispecific antibody. Bacterial, such as E. coli, and other microbial systems can be used, or eukaryotic, such as mammalian, host cell expression systems can also be used. Suitable mammalian host cells include CHO cells.

[0222] The invention also provides a method for producing an IL-13 / IL-17AF multispecific antibody comprising culturing a host cell containing a vector under conditions suitable for expression of protein from DNA encoding the IL-13 / IL-17AF multispecific antibody, and isolating the IL-13 / IL-17AF multispecific antibody.

[0223] Production of multispecific antibodies There are several approaches to produce multispecific antibodies, especially bispecific antibodies. Morrison et al. (Coloma and Morrison 1997, Nat Biotechnol. 15, 159-163) describe the fusion of single chain variable fragments (scFv) to whole antibodies, e.g. IgG. Schoonjans et al., 2000, Journal of Immunology, 165, 7050-7057 describe the fusion of scFv to antibody Fab fragments. WO 2015 / 197772 describes the fusion of disulfide stabilized scFv (dsscFv) to Fab fragments.

[0224] The standard approach described in the prior art involves the expression in a host cell of at least two polypeptides, each encoding the whole antibody or an antigen-binding fragment thereof, e.g. a heavy chain (HC) or a light chain (LC) of a Fab, and additional antigen-binding fragments of the antibody can be fused to the N- and / or C-terminal positions of the heavy and / or light chains. If one wishes to recombinantly produce such multispecific antibodies by expressing two (one light and one heavy chain to form an additional Fab) or four polypeptides (two light and two heavy chains to form an additional IgG), it is usually necessary to express the light chains in excess of the heavy chains to ensure proper folding of the heavy chains upon assembly with their corresponding light chains. In particular, the CH 1 (domain 1 of the heavy chain constant region) is prevented from folding on itself by the BIP protein, which can be replaced by the corresponding LC, thus forming a CH 1 Correct folding of the / HC depends on the availability of its corresponding LC (Lee et al., 1999, Molecular Biology of the Cell, Vol. 10, 2209-2219).

[0225] The inventors have observed that those methods of expressing multispecific antibodies can result in the production of an excess of light chains over heavy chains that remain in the host cell harvest, and that the excess light chains tend to form dimeric complexes (or "LC dimers") that are present as a by-product of the production process with the desired multispecific antibodies, especially the monomers, and therefore need to be purified away.

[0226] Importantly, technical problems related to the formation of dimers of light chains when fused at the N- and / or C-terminus to additional antigen-binding fragments have not been identified so far, and commonly used analytical methods do not allow the detection and quantification of additional LC dimers in the heterogeneous product of the production process, which may result in a large bias when estimating the amount of product using standard analytical methods.

[0227] There is therefore a need for improved multispecific antibodies and methods for their production, which allow easy and efficient isolation and removal of the adjunct LC dimer at the earliest stage of the production process, thus improving the yield of the protein of interest, i.e. the multispecific antibody, for therapeutic use, particularly the protein in its monomeric form.

[0228] The multispecific antibodies of the present invention have been engineered to provide improved multispecific antibodies with comparable functionality and stability while increasing the yield of "multispecific antibody" material, particularly monomeric, obtained after purification, particularly after a single purification step involving Protein A affinity chromatography.

[0229] Advantageously, the multispecific antibodies of the present disclosure can be purified more efficiently with improved purification methods than those commonly used in the prior art, in particular in that the improved methods involve fewer steps, which is cost- and time-efficient on an industrial scale. In particular, the multispecific antibodies of the present disclosure maximize the amount of protein of interest (i.e., the correct multispecific antibody format) obtained after a one-step purification method involving Protein A affinity chromatography, thereby simultaneously purifying the multispecific antibody of interest and removing the attached LC dimer. Advantageously, the multispecific antibody production and purification method of the present disclosure does not require an additional purification step to capture free unbound light chains in excess, in particular the attached LC dimer.

[0230] Protein A Protein A is a 42 kDa surface protein first found in the cell wall of the bacterium Staphylococcus aureus. Protein A is widely used to detect, quantify and purify immunoglobulins. Protein A is a 42 kDa surface protein that is derived from the Fab portion of the VH3 family of antibodies and the constant region portion of IgG (CH 2 Domain and CH 3 It has been reported that Protein A binds to the Fc gamma region of the VH3 domain (between the VH3 domain and the Fc gamma domain). The crystal structure of the complex formed by Protein A and Fab is described, for example, in Graille et al., 2000, PNAS, 97(10):5399-5404. In the context of this disclosure, Protein A encompasses native Protein A and any variant or derivative thereof, so long as the Protein A variant or derivative retains the ability to bind to the VH3 domain and / or the Fc gamma domain.

[0231] The polypeptide chains of formula (I) of the invention comprise a Protein A binding domain. In one embodiment, the polypeptide chains of formula (I) comprise one, two or three Protein A binding domains.

[0232] A "protein A binding domain" as used herein is intended to refer to a binding domain that specifically binds to protein A. A protein A binding domain can refer to a VH3 domain or a portion of a VH3 domain that binds protein A, i.e. comprises the protein A binding interface. It will be understood that the portion of a VH3 domain that binds protein A does not include the CDRs of the VH3 domain, i.e. the protein A binding interface of VH3 does not include the CDRs, and therefore the protein A binding domain does not compete with the antigen binding domains disclosed in the present application.

[0233] In one embodiment, the polypeptide chain of formula (I) is H and / or CH 2 -CH 3 and / or V 1 In one embodiment, the polypeptide chain of formula (I) comprises a Protein A binding domain present in H and / or CH 2 -CH 3 and / or one, two or three Protein A binding domains present in V1. In one embodiment, the polypeptide chain of formula (I) comprises one, two or three Protein A binding domains present in V2, V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, V13, V14, V15, V16, V17, V18, V19, V20, V21, V22, V23, V24, H Or V 1 In one embodiment, s is 0, t is 0, and the polypeptide chain of formula (I) comprises only one Protein A binding domain present in H Or V 1 In one embodiment, the polypeptide chain of formula (I) comprises only one Protein A binding domain present in H In one embodiment, s is 0, t is 0, p is 0 and the polypeptide chain of formula (I) comprises only one Protein A binding domain present in H In one embodiment, the polypeptide chain of formula (I) comprises only one Protein A binding domain present in 1 In one embodiment, s is 0, t is 0, p is 1 and the polypeptide chain of formula (I) comprises only one Protein A binding domain present in 1 It contains only one Protein A binding domain present in

[0234] In one embodiment, the polypeptide chain of formula (I) comprises two Protein A binding domains. In one embodiment, the polypeptide chain of formula (I) comprises two Protein A binding domains, each of which is V H and C.H. 2 -CH 3 In another embodiment, the polypeptide chains of formula (I) each comprise two Protein A binding domains present in H and V 1 In another embodiment, the polypeptide chains of formula (I) each comprise two Protein A binding domains present in 2 -CH 3 and V 1 It contains two protein A binding domains present in

[0235] In one embodiment, the polypeptide chains of formula (I) each have the structure V H , C.H. 2 -CH 3 and V 1 It contains three protein A binding domains present in

[0236] Native protein A can interact with the constant region of IgG, particularly with the Fc gamma region. More specifically, protein A binds to the CH 2 and C.H. 3 In one embodiment, when s is 1 and t is 1, the CH 2 and C.H. 3 Both are naturally occurring domains of the IgG class.

[0237] In some embodiments, the Protein A binding domain comprises or consists of a VH3 domain or a variant thereof that binds Protein A. In some embodiments, the Protein A binding domain comprises or consists of a naturally occurring VH3 domain. In some embodiments, the variant of the VH3 domain that binds Protein A is a variant of a naturally occurring VH3 domain, which is incapable of binding Protein A.

[0238] The polypeptide chain of formula (II) of the present disclosure does not bind to Protein A. In one embodiment, V 2 The binding domain of does not bind to Protein A.

[0239] In some embodiments, V 2 In some embodiments, VH1 and / or VH2 and / or VH4 and / or VH5 and / or VH6 are included or consist of VH1 and / or VH2 and / or VH4 and / or VH5 and / or VH6, and do not include a VH3 domain. 2 comprises or consists of a VH3 domain or a variant thereof that does not bind Protein A. In some embodiments, the V 2 comprises or consists of a naturally occurring VH3 domain that is unable to 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 is able to bind Protein A.

[0240] Human VH3 germline genes and VH3 domains (or frameworks) have been well characterized. Many naturally occurring VH3 domains have the ability to bind Protein A, but certain naturally occurring VH3 domains do not have the ability to bind Protein A (see Roben et al., 1995, J Immunol.; 154(12):6437-6445).

[0241] VH3 domains for use in the present disclosure can be obtained by several methods. In one embodiment, the VH3 domains for use in the present disclosure are naturally occurring VH3 domains that are selected for their ability or inability to bind Protein A depending on their location in the polypeptides (I) and / or (II) of the present disclosure. For example, a panel of antibodies can be generated against an antigen of interest by immunization of a non-human animal, then humanized, and the humanized antibodies can be screened and selected based on their ability or inability to bind Protein A via the humanized VH3 domain, for example, against a Protein A affinity column. Alternatively, display technologies (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 to select antibodies that contain VH3 domains that do or do not bind Protein A, particularly via a Protein A binding interface that does not contain the CDRs.

[0242] Alternatively, a VH3 domain for use in the present disclosure is a variant of a naturally occurring VH3. In one embodiment, a VH3 variant comprises the sequence of a naturally occurring VH3 capable of binding to protein A and further comprises at least one amino acid mutation that abolishes its ability to bind protein A. In one embodiment, a VH3 variant that binds protein A comprises the sequence of a naturally occurring VH3 that is unable to bind protein A and further comprises at least one amino acid mutation. In such an embodiment, the mutation is responsible for conferring the VH3 domain the ability to bind protein A, i.e., the mutation contributes to the generation of a protein A binding domain that did not exist in nature.

[0243] In one embodiment, the VH3 variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid mutations. In one embodiment, the VH3 variant comprises a mutation at position 15, 17, 19, 57, 59, 64, 65, 66, 68, 70, 81, or 82 on VH3, numbering according to Kabat, e.g., Graille et al., 2000, PNAS, 97(10):5399-5404. The mutations may be substitutions, deletions, or insertions. In one embodiment, the VH3 variant comprises a substitution at position 15, 17, 19, 57, 59, 64, 65, 66, 68, 70, 81, or 82 on VH3, numbering according to Kabat.

[0244] Naturally occurring VH1, VH2, VH4, VH5 and VH6 do not bind Protein A. In one embodiment, VH1, VH2, VH4, VH5 and VH6 do not bind Protein A. H In one embodiment, the VH domain is a VH1 domain that does not bind Protein A. H In one embodiment, the V domain is VH2. H In one embodiment, the V domain is VH4. H In one embodiment, the V domain is VH5. H The domain is VH6.

[0245] Pharmaceutical Compositions, Dosages and Administration Regimens The multispecific antibodies of the present invention can be provided in a pharmaceutical composition. The pharmaceutical composition is usually sterile and typically comprises a pharma- ceutically acceptable carrier and / or adjuvant. The pharmaceutical composition of the present invention can further comprise a pharma- ceutically acceptable adjuvant and / or carrier.

[0246] As used herein, a "pharmaceutical acceptable carrier" includes any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. The carrier may be suitable for parenteral administration, for example, intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example, by injection or infusion. Alternatively, the carrier may be suitable for parenteral administration, such as topical, epidermal or mucosal routes of administration. The carrier may be suitable for oral administration. Depending on the route of administration, the modulator may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0247] The pharmaceutical composition of the present invention can contain one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0248] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Suitable examples of 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, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and their appropriate mixtures, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. In many cases, it is desirable to include isotonic agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0249] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0250] The pharmaceutical compositions of the invention may contain additional active ingredients.

[0251] Kits comprising the antibodies or modulators of the invention and instructions for use are also within the scope of the invention. The kits can further comprise one or more additional reagents, such as additional therapeutic or prophylactic agents as described above.

[0252] The modulators and / or antibodies of the invention or formulations or compositions thereof can be administered for prophylactic and / or therapeutic treatments.

[0253] In therapeutic applications, compounds are administered to subjects already suffering from the above disorders or conditions in an amount sufficient to cure, alleviate or partially halt the condition or one or more of its symptoms. Such therapeutic treatment can result in a decrease in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this is defined as a "therapeutically effective amount."

[0254] In prophylactic applications, the formulations are administered to subjects at risk of the above disorders or conditions in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms. An amount sufficient to accomplish this is defined as a "prophylactically effective amount." Effective amounts for each purpose depend on the severity of the disease or injury, as well as the weight and general state of the subject.

[0255] The subject of administration may be a human or a non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Administration to humans is typical.

[0256] The antibody / modulator or pharmaceutical composition of the invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those of skill in the art, the route and / or mode of administration will vary depending on the desired outcome. Examples of routes of administration of the compounds or pharmaceutical compositions of the invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically by injection. Alternatively, the antibody / modulator or pharmaceutical composition of the invention can be administered via a parenteral route, such as a topical, epidermal or mucosal route of administration. The antibody / modulator or pharmaceutical composition of the invention may be for oral administration.

[0257] The appropriate dosage of the antibody / modulator or pharmaceutical composition of the invention can be determined by one of skill in the art. The actual dosage level of the active ingredient in the pharmaceutical composition of the invention can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0258] A suitable dose may be, for example, in the range of about 0.01 μg / kg to about 1000 mg / kg body weight of the patient to be treated, typically about 0.1 μg / kg to about 100 mg / kg body weight. For example, a suitable dosage may be about 1 μg / kg to about 10 mg / kg body weight / day or about 10 μg / kg to about 5 mg / kg body weight / day.

[0259] The dosage regimen can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0260] Administration may be a single dose or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different locations. Alternatively, the dose may be via sustained release formulation, in which case less frequent administration is required. Dosage and frequency may vary depending on the half-life of the antagonist in the patient and the desired duration of treatment.

[0261] As noted above, the modulators / antibodies or pharmaceutical compositions of the invention may be co-administered with one or more other therapeutic agents.

[0262] The co-administration of two or more agents can be accomplished in a number of different ways. Both can be administered together in a single composition, or they can be administered in separate compositions as part of a combination therapy. For example, one can be administered before, after, or simultaneously with the other.

[0263] Treatment indications The antibodies of the invention can be used in the treatment, prevention or amelioration of any condition associated with IL-13 and / or IL-17A and / or IL-17F activity, for example, any condition that results in whole or in part from signaling via the IL-13, IL-17A and / or IL-17F receptors.

[0264] Such diseases include primary and metastatic cancers, including cancer of the breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile duct, small intestine, urinary tract (including kidney, bladder and urothelium), female reproductive organs (including cervix, uterus and ovaries and choriocarcinoma and gestational trophoblastic disease), male reproductive organs (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including thyroid, adrenal and pituitary glands) and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissue and Kaposi's sarcoma), tumors of the brain, nerves, eyes and meninges. (including astrocytoma, glioma, glioblastoma, retinoblastoma, neuroma, neuroblastoma, Schwannoma and meningioma), solid tumors arising from hematopoietic malignancies such as leukemia and lymphoma (both Hodgkin's and non-Hodgkin's lymphoma), rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, ulcerative colitis, inflammatory bowel disease, insulin-dependent diabetes mellitus, thyroiditis, allergic diseases, psoriasis, dermatitis, scleroderma, graft-versus-host disease, organ transplant rejection, acute myelopathy associated with organ transplantation or chronic immune disorders, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Graves' disease, renal syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schönlein's disease, renal microvasculitis, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, septic syndrome, cachexia, infectious diseases, parasitic diseases, acquired immune deficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignant tumors, heart failure, Addison's disease, sporadic polyglandular deficiency type I and polyglandular deficiency type I Type I, Schmidt syndrome, Adult (acute) respiratory distress syndrome, Alopecia, Alopecia areata, Arthropathy, Reiter's disease, Psoriatic arthropathy, Ulcerative colitis arthropathy, Enteropathic synovitis, Chlamydia, Yersinia and Salmonella-associated arthropathy, Atherosclerosis / arteriosclerosis, Atopic allergy, Autoimmune bullous disease, Pemphigus vulgaris, Pemphigus foliaceus, Pemphigoid, Linear IgA disease, Autoimmune hemolytic anemia, Coombs positive hemolytic anemia, Acquired pernicious anemia, Juvenile pernicious anemia, Myalgic encephalitis / Chronic fatigue syndrome, Chronic mucocutaneous candidiasis, Giant cell arteritis, Primary sclerosing hepatitis, Idiopathic autoimmune hepatitis,Acquired immunodeficiency-related diseases, Hepatitis B, Hepatitis C, Common variable immunodeficiency (Common variable hypogammaglobulinemia), Dilated cardiomyopathy, Female infertility, Ovarian failure, Premature ovarian failure, Fibrotic lung disease, Idiopathic fibrosing alveolitis, Postinflammatory interstitial lung disease, Interstitial pneumonia, Connective tissue disease-associated interstitial lung disease, Mixed connective tissue disease-associated lung disease, Systemic sclerosis-associated interstitial lung disease, Rheumatoid arthritis-associated interstitial lung disease, Systemic lupus erythematosus-associated lung disease, Dermatomyositis / Polymyositis-associated lung disease, Sjogren's disease-associated lung Disease, ankylosing spondylitis-associated lung disease, vasculitic diffuse lung disease, hemosiderosity-associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, post-infectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type 2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disorders associated with organ transplantation, organ Chronic immune diseases associated with organ transplantation, osteoarthritis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leukopenia, autoimmune neutropenia, renal disease NOS, glomerulonephritis, microscopic vasculitis of the kidney, Lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestations of polyarteritis nodosa, acute rheumatic fever, rheumatic spondylitis, Still's disease, systemic sclerosis, rheumatoid arteritis ... Egren's syndrome, Takayasu's disease / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxedema, phacogenic uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver disease, alcoholic cirrhosis, alcohol-induced liver injury, cholestasis, idiopathic liver disease, drug-induced hepatitis, nonalcoholic steatohepatitis, allergies, group B streptococcus (GBS) Infectious diseases, psychiatric disorders, depression, schizophrenia, Th2 and Th1 mediated diseases, acute and chronic pain, different forms of pain, cancer, lung cancer, breast cancer, stomach cancer, bladder cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, hematopoietic malignancies, leukemia, lymphoma, abetalipoproteinemia, acrocyanosis, acute and chronic parasitic or infectious processes,Acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infections, acute pancreatitis, acute renal failure, adenocarcinoma, air ectopic pulses, AIDS dementia complications, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis (including seasonal allergic rhinitis), non-allergic rhinitis, allograft rejection, alpha-I-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti-CD3 therapy, antiphospholipid syndrome, antireceptor hypersensitivity reactions, aortic and peripheral aneurysms, aortic dissection, arterial hypertension, arterial hypertension, atherosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (persistent or paroxysmal), atrial flutter, atrioventricular block, B-cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, burns, arrhythmias, cardiac dysfunction syndromes, cardiac tumors, cardiomyopathy, inflammatory response to cardiopulmonary bypass, cartilage graft rejection, cerebellar cortical degeneration, cerebellar disease, chaotic or multifocal atrial tachycardia, chemotherapy-related disorders, chronic myeloid leukemia (CML), chronic alcoholism, chronic inflammatory conditions, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate poisoning, colorectal cancer, congestive heart failure, Conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture-negative sepsis, cystic fibrosis, cytokine therapy-associated disorders, dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, skin diseases, diabetes mellitus, diabetic atherosclerotic disease, diffuse Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's syndrome in middle age, drug-induced movement disorders induced by drugs that block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrine disorders, epiglottitis, Epstein-Barr virus infection, erythromelalgia, extrapyramidal disorders and Cerebellar disorders, familial hemophagocytic lymphohistiocytosis, fetal thymus graft rejection, Friedreich's ataxia, functional peripheral arterial disease, fungal sepsis, gas gangrene, gastric ulcer, glomerulonephritis, any organ or tissue graft rejection, gram-negative sepsis, gram-positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallervorden-Spatz disease, Hashimoto's disease, hay fever, cardiac transplant rejection, hemochromatosis, hemodialysis, hemolytic uremic syndrome / thrombolytic thrombocytopenic purpura, bleeding, hepatitis A, His bundle arrhythmia, HIV infection / HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorder,Hypersensitivity reactions, hypersensitivity pneumonitis, hypertension, hypokinesia, hypothalamic-pituitary-adrenal axis, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, asthenia, infantile spinal muscular atrophy, aortic inflammation, influenza, ionizing radiation exposure, iridocyclitis / uveitis / optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, Legionella, leishmaniasis, leprosy, corticospinal system lesions, liver transplant rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic / idiopathic, migraine, mitochondrial multisystem disease, mixed connective tissue disease, monoclonal hypergammaglobulinemia, multiple myeloma, multiple system degeneration (Mencel Dejerine-Thomas Shi-Drager and Machado-Joseph), Mycobacterium avium intracellulare, Mycobacterium tuberculosis, Myelodysplastic syndrome, Myocardial infarction, Myocardial ischemic disorder, Nasopharyngeal carcinoma, Chronic lung disease of the newborn, Nephritis, Nephrosis, Neurodegenerative disease, Neurogenic muscular atrophy, Neutropenic fever, Non-Hodgkin's lymphoma, Obstruction of the abdominal aorta and its branches, Obstructive arterial disease, OKT3 treatment, Orchitis / epididymitis, Orchitis / vasectomy reversal procedure, Organ enlargement, Osteoporosis, Pancreatic transplant rejection, Pancreatic cancer, Paraneoplastic syndrome / hypercalcemia associated with malignant tumor, Parathyroid transplant rejection, Pelvic inflammatory disease, Perennial rhinitis, Pericardial disease, Peripheral arteriosclerosis, Peripheral vascular disease, Peritonitis, Pernicious anemia, Pneumocystis carinii pneumonia, Pneumonia, POEMS syndrome (multiple myeloma) vascular disorders, organomegaly, endocrine disorders, monoclonal hypergammaglobulinemia and skin change syndrome), post-perfusion syndrome, post-pump syndrome, post-cardiotomy syndrome, pre-eclampsia, progressive supranuclear palsy, primary pulmonary hypertension, radiation therapy, Raynaud phenomenon and disease, Raynaud's disease, Refsum's disease, regular tachycardia with normal QRS complexes, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcoma, senile chorea, senile dementia with Lewy bodies, seronegative arthropathy, shock, sickle cell anemia, skin allograft rejection, skin manifestation syndrome, small bowel transplant rejection, solid tumors, specific arrhythmias, spinal ataxia, spinocerebellar degeneration, streptococcal myositis, structural lesions of the cerebellum, subacute sclerosing panencephalitis, syncope, cardiovascular syphilis, systemic unmyelinated axis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis,T cell or FAB ALL telangiectasia, thromboangiitis obliterans, thrombocytopenia, toxicity, grafts, trauma / bleeding, type III hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, valvular heart disease, varicose veins, vasculitis, venous disease, venous thrombosis, ventricular fibrillation, viral and fungal infections, vital encephalitis / aseptic meningitis, vitalassociated hemaphagocytic syndrome syndrome), Wernicke-Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or tissue, acute coronary syndrome, acute idiopathic polyneuropathy, acute inflammatory demyelinating polyneuropathy, acute ischemia, adult Still's disease, anaphylaxis, antiphospholipid syndrome, aplastic anemia, atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmune disorders associated with streptococcal infections, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative disorders Syndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarian failure, blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular disease, idiopathic antiphospholipid syndrome, celiac disease, cervical spondylosis, chronic ischemia, cicatricial pemphigoid, clinically isolated syndrome with risk of multiple sclerosis (CIS), childhood-onset psychiatric disorders, dacryocystitis, dermatomyositis, diabetic retinopathy, herniated disc, disc bulge, drug-induced immune hemolytic anemia, endometriosis, endophthalmitis, episcleritis, erythema multiforme, multiforme severe erythema pemphigoid, gestational pemphigoid, Guillain-Barré syndrome (GBS), Hughes syndrome, idiopathic Parkinson's disease, idiopathic interstitial pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion body myositis, infectious ocular inflammatory disease, inflammatory demyelinating disease, inflammatory heart disease, inflammatory kidney disease, IPF / UIP, iritis, keratitis, keratoconjunctivitis sicca, Kussmaul or Kussmaul-Meyer disease, Landry palsy, Langerhans cell histiocytosis, livedo reticularis, macular degeneration, microscopic polyangiitis, ankylosing spondylitis, motor neuron disorder, mucous membrane pemphigoid, multiple organ failure, myasthenia gravis, myelodysplastic syndrome, myocarditis, radiculopathy, neuropathy, non-A non-B hepatitis, optic neuritis, osteolysis, oligoarticular JRA, peripheral arterial occlusive disease (PAOD), peripheral vascular disease (PVD), peripheral arterial disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, polio, polyarticular JRA, multiple endocrine deficiency syndrome, polymyositis,Polymyalgia rheumatica (PMR), primary parkinsonism, prostatitis, pure red cell hypoplasia, primary adrenal insufficiency, recurrent neuromyelitis optica, restenosis, rheumatic heart disease, SAPHO (synovitis, acne, pustulosis, osteophytosis and osteitis), secondary amyloidosis, shock lung, scleritis, sciatica, Secondary adrenal insufficiency, silicone-associated connective tissue disease, Snidden-Wilkinson dermatosis, ankylosing spondylitis, Stevens-Johnson syndrome (SJS), temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumor necrosis factor receptor 1, type 1 allergic reactions, type II diabetes mellitus, urticaria, usual interstitial pneumonia (UIP), vasculitis, vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration or wound healing, aspirin hypersensitivity asthma, atopic asthma, chronic hand eczema, allergic bronchopulmonary aspergillosis, celiac disease, Churg-Strauss syndrome (nodular musculoskeletal syndrome), perivascular inflammation + atopy), eosinophilia myalgia syndrome, hypereosinophilic syndrome, edematous reactions including episodic angioedema, helminth infections, hair follicle dermatitis, eosinophil-associated gastrointestinal disorders, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis, eosinophilic colitis, nasal micropolyposis and polyposis, food allergies, aspirin intolerance, and obstructive sleep apnea, chronic asthma, Crohn's disease and endomyocardial fibrosis, cancer (e.g., glioblastoma (such as glioblastoma multiforme), non-Hodgkin's lymphoma (NHL)), fibrosis, inflammatory bowel disease, pulmonary fibrosis (including idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis secondary to sclerosis), COPD, and hepatic fibrosis.

[0265] The multispecific antibodies of the invention may be particularly useful for the treatment or prevention of atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergies, or eosinophilic esophagitis. Thus, in one embodiment, a multispecific antibody or pharmaceutical composition of the invention is provided for use in a method of treatment of the human or animal body by therapy. In one embodiment, a multispecific antibody or pharmaceutical composition is provided for use in a method of treating atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergies, or eosinophilic esophagitis. In one embodiment, the invention provides a method of treating or preventing atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergies, or eosinophilic esophagitis, comprising administering a therapeutically effective amount of the multispecific antibody or pharmaceutical composition to a patient in need thereof.

[0266] The following examples illustrate the invention.

[0267] example Example 1. Generation and selection of therapeutic anti-IL-13 antibody CA650 Rats were immunized with either purified human IL-13 (Peprotech) or rat fibroblasts expressing human IL-13 (expressing approximately 1ug / ml in culture supernatant), or in some cases a combination of the two. After 3-6 injections, animals were sacrificed and PBMCs, spleens, bone marrow and lymph nodes were harvested. Serum was monitored for binding to human IL-13 in an ELISA and for the ability to neutralize hIL-13 in a HEK-293 IL-13R-STAT-6 reporter cell assay (HEK-Blue assay, Invivogen).

[0268] B cell cultures were set up and supernatants were first screened for their ability to bind hIL-13 in a bead-based assay in the Applied Biosystems FMAT assay. This was a homogeneous assay using biotinylated human IL-13 coated on streptavidin beads and goat anti-rat Fc-Cy5 conjugate as a revealing agent. Positives from this assay were then carried forward to a HEK-293 IL-13R-STAT-6 reporter cell assay (HEK-Blue assay, Invivogen) to identify neutralizing agents. Neutralized supernatants were then profiled on Biacore to estimate off-rates and to characterize the mode of action of neutralization. Neutralization was classified as either Bin 1 or Bin 2. Bin1 represents antibodies that bind human IL-13 and prevent binding of IL-13Rα1, which in turn also blocks binding of IL-4R. Bin1 antibodies can also inhibit binding of IL-13 to IL-13Rα2. Bin2 represents an antibody that binds to hIL-13 in a manner that allows binding to IL-13Rα1 but inhibits recruitment of IL-4R to the complex. We selected antibodies that act through Bin1.

[0269] Approximately 7500 IL-13 specific positives were identified in the primary FMAT screen from a total of 27 x 100 plate SLAM experiments. 800 wells demonstrated neutralization in the HEK-blue assay. 170 wells showed the desired Biacore profile, i.e., 5 x 10 -4 s -1All wells had bin 1 antibodies with off-rates of less than 1000. Variable region cloning from these 170 wells was attempted, and 160 were successful in obtaining fluorescent focus. 100 wells generated heavy and light chain variable region gene pairs after reverse transcription (RT)-PCR. These V region genes were cloned as mouse IgG1 full-length antibodies and re-expressed in HEK-293 transient expression system. Sequence analysis revealed the presence of 27 unique families of anti-human IL-13 antibodies. These recombinant antibodies were then retested for their ability to block recombinant hIL-13 (E. coli-derived and mammalian-derived), recombinant mutant hIL-13 (R130 Q) (E. coli-derived), native wild-type and mutant hIL-13 (human donor-derived) and cynomolgus IL-13 (mammalian-derived) in cell-based assays. The recombinant antibodies were also tested for their ability to bind mutant human IL-13 (R130Q) and cynomolgus IL-13 in Biacore. After this characterization, a family of antibodies was selected to meet our criteria, namely antibodies with minimal loss of potency and affinity to all human and cynomolgus IL-13 preparations, less than 100 pM.

[0270] Based on neutralization potency, affinity and donor content in humanized grafts (see below), humanized CA650 was selected for further development.

[0271] Example 2 Humanization of antibody CA650 Antibody 650 was humanized by grafting the CDRs from the rat V-region into a human germline antibody V-region framework. To restore the activity of the antibody, 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. WO 91 / 09967). An alignment of the rat antibody (donor) V-region sequence with the human germline (acceptor) V-region sequence, along with the designed humanized sequence, is shown in Figure 1. (Figure 1(A) Light chain graft 650 and Figure 1(B) Heavy chain graft 650). The CDRs grafted from the donor to the acceptor sequence are as defined by Kabat (Kabat et al., 1987), except for CDR-H1, where the combined Chothia / Kabat definition is used (see Adair et al., 1991 Humanised antibodies. WO 91 / 09967).

[0272] Genes encoding the initial V-region sequences were designed and constructed by an automated synthesis approach by Entelechon GmbH and modified to generate the grafted versions gL8 and gH9 by oligonucleotide-directed mutagenesis. The gL8 sequence was subcloned into the UCB Celltech human light chain expression vector pVhCK, which contains DNA encoding the human C-kappa constant region (Km3 allotype). The gH9 sequence was cloned into the human heavy chain gamma-1 CH 1 It was subcloned into pVhg1Fab, which contains DNA encoding the constant region.

[0273] The human V region IGKV1-39+JK2 J region (International Immunogenetics Information System® IMGT, http: / / www.imgt.org) was selected as the acceptor for the antibody 650 light chain CDRs. All light chain framework residues in graft gL8 are derived from human germline genes, except for residues 58 and 71 (numbering according to Kabat), where donor residues isoleucine (I58) and tyrosine (Y71), respectively, were retained. Retention of residues I58 and Y71 was essential for full potency of the humanized antibody.

[0274] The human V region IGHV1-69+JH4 J region (IMGT, http: / / www.imgt.org) was selected as the acceptor for the heavy chain CDRs of antibody 650. All heavy chain framework residues in graft gH9 are derived from human germline genes, except for residues 67, 69 and 71 (numbering according to Kabat), where donor residues alanine (A67), phenylalanine (F69) and valine (V71) were retained, respectively. Retention of residues A67, F69 and V71 was essential for full potency of the humanized antibody. The glutamine residue at position 1 of the human framework was replaced with glutamic acid (E1) to result in expression and purification of homogeneous products, and conversion of glutamine to pyroglutamic acid at the N-terminus of antibodies and antibody fragments has been widely reported. The final selected variable graft sequences gL8 and gH9 are shown in Figure 1(A) and Figure 1(B), respectively.

[0275] The amino acid and DNA sequences encoding the CDRs, heavy and light variable regions, scFv and dsscFV formats of antibody 650 are shown in FIG.

[0276] Example 3: Generation of anti-IL-17 AF antibody 496.g3 The production of antibody CA028_00496.g3 (also referred to herein as antibody 496.g3) against human IL-17A and human IL-17F has been previously described in WO 2012 / 095662. The antibody binds to human IL-17A, IL-17F and the IL-17A / F heterodimer with pM affinity. The amino acid sequence and DNA sequence encoding the CDRs, heavy and light variable regions and light and heavy chains of the Fab format of antibody 496.g3 are shown in FIG. 2. The 496.g3 (IL-17A / F binding) Fab constant region contains a human C-kappa constant region (K1m3 allotype) and a human gamma-1 CH constant region. 1 It contained the constant region and hinge (G1m17 allotype).

[0277] Example 4: Preparation of anti-human albumin antibody 645 The production of anti-human albumin antibody 645 has been previously described in WO 2013 / 068571. The amino acid and DNA sequences encoding the CDRs, heavy and light variable regions, scFv and dsscFV formats of antibody 645 are shown in FIG.

[0278] Example 5 Multispecific antibody IL-13 / IL-17AF-transient plasmid construction and expression in cells. A multispecific antibody was designed with the anti-IL-17 A FV region (496.g3) fixed in the Fab position, and the anti-Albumin V region (645gL4gH5) and IL-13 (1539gL8gH9) reformatted into disulfide-linked scFvs in the HL orientation (dsHL) and linked to the C-terminus of the respective heavy and light chain constant regions of the Fab via 11 amino acid glycine-serine rich linkers (Figure 7). The amino acid and DNA sequences encoding the full-length heavy and light chains of the multispecific antibody are shown in Figure 2.

[0279] The light and heavy chain genes were independently cloned into mammalian expression vectors for transient expression under the control of the hCMV promoter. Equal ratios of both plasmids were transfected into the CHO-S XE cell line (UCB) using the commercially available ExpiCHO Expifectamine transient expression kit (Thermo Scientific). Cultures were incubated at 37°C, 8.0% CO in Corning roller bottles with vented caps. 2 The cultures were incubated at 32°C, 8.0% CO, and 190 rpm. After 18-22 hours, the cultures were fed with the appropriate volumes of CHO enhancer and HiTiter feed provided by the manufacturer. The cultures were incubated at 32°C, 8.0% CO 2 The cells were incubated at 100° C. for 1 h at 4000 rpm and then re-incubated at 190 rpm for an additional 10-12 days. The supernatant was collected by centrifugation at 4000 rpm for 1 h at 4° C. and then filter sterilized through a 0.45 μm 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 Fab standards generated by the inventors. Expression titers are shown in Table 1. [Table 1]

[0280] Example 6. IL-13 / IL-17AF Multispecific Antibody-Mammalian Cell Line Development. To demonstrate stable expression of IL-13 / IL-17AF multispecific antibodies, stable expressing mammalian cell lines were generated. CHO cell lines were transfected with vectors containing 496.g3 Fab, 1539gH9gL8 dsscFv HL (LC, INS0025609), 645gH5gL4 dsscFv HL (HC, INS0025306) and a selection marker. Cell lines were cloned and evaluated for suitability for the appropriate manufacturing process. Cell lines were evaluated in a small-scale model of a production fed-batch bioreactor to assess protein quality and quantity and ensure that the optimal cell line was selected. A CHO cell line was selected that expressed IL-13 / IL-17AF multispecific antibodies at >1.8g / L and >75% monomer.

[0281] Example 7. Methods for purifying IL-13 / IL-17AF multispecific antibodies. The multispecific antibody proteins were purified by a native Protein A capture step followed by a preparative size-exclusion polishing step. Clarified supernatant from standard transient CHO expression was loaded onto a MabSelect (GE Healthcare) column for 5 minutes and washed with binding buffer (20 mM Hepes pH 7.4 + 150 mM NaCl). Bound material was eluted with a 0.1 M sodium citrate pH 3.1 step elution, neutralized with 2 M Tris / HCl pH 8.5 and quantified by absorbance at 280 nm.

[0282] Size exclusion chromatography (SE-UPLC) was used to determine the purity status of the eluted product. Antibody (approximately 2 μg) was loaded onto a BEH200, 200 Å, 1.7 μm, 4.6 mm ID x 300 mm column (Waters ACQUITY) and developed with an isocratic gradient of 0.2 M phosphate pH 7 at 0.35 mL / min. Sequential detection was by absorbance at 280 nm and a multichannel fluorescence (FLR) detector (Waters). The eluted multispecific antibody was found to be 72% monomeric.

[0283] Neutralized samples were concentrated using Amicon Ultra-15 concentrators (10 kDa molecular weight cut-off membrane) and centrifuged at 4000xg in a swing-out rotor. The concentrated samples were applied to an XK16 / 60 Superdex200 column (GE Healthcare) equilibrated with PBS, pH 7.4 and developed with an isocratic gradient of PBS, pH 7.4 at 1 ml / min. Fractions were collected and analyzed by size exclusion chromatography on a BEH200, 200 Å, 1.7 μm, 4.6 mm ID x 300 mm column (Aquity), developed with an isocratic gradient of 0.2 M phosphate pH 7 at 0.35 mL / min, and detected by absorbance at 280 nm and a multichannel fluorescence (FLR) detector (Waters). Selected monomeric fractions were pooled, 0.22 μm sterile filtered, and the final sample was assayed for concentration by A280 scanning on a DropSense96 (Trinean). Endotoxin levels were <1.0 EU / mg as assessed by Charles River's EndoSafe® portable testing system equipped with a Limulus Amebocyte Lysate (LAL) test cartridge.

[0284] The monomeric state of the final multispecific antibody was determined by size-exclusion chromatography on a BEH200, 200 Å, 1.7 μm, 4.6 mm ID×300 mm column (Aquity) developed with an isocratic gradient of 0.2 M phosphate pH 7 at 0.35 mL / min and detected by absorbance at 280 nm and a multichannel fluorescence (FLR) detector (Waters). The final multispecific antibody was found to be >99% monomeric as shown in FIG. 3(A).

[0285] For analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), samples were prepared by adding 4× Novex NuPAGE LDS sample buffer (Life Technologies) and either 10× NuPAGE sample reducing agent (Life Technologies) or 100 mM N-ethylmaleimide (Sigma-Aldrich) to approximately 5 μg of purified protein and heated to 100 °C for 3 min. 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 min in Tris-glycine SDS running buffer (Life Technologies). Novex Mark12 broad range protein standards (Life Technologies) were used as standards. Gels were stained with Coomassie Quick stain (Generon) and destained in distilled water.

[0286] In non-reducing SDS-PAGE, the theoretical molecular weight (MW) of the multispecific antibody is approximately 100 kDa and migrated at approximately 120 kDa. When the multispecific antibody protein was reduced, both chains migrated with mobilities approaching their respective theoretical molecular weights, heavy chain (HC) approximately 52 kDa and light chain (LC) approximately 51 kDa. Further bands on the non-reducing gel at approximately 45-50 kDa are "free" LC and HC that lack the disulfide bonds in the Fab portion of the molecule and do not migrate at the same position as the LC and HC in lane 2, as they are not fully reduced. (Figure 3(B))

[0287] Example 8. Antigen binding of IL-13 / IL-17AF multispecific antibody molecules. (i) Antigen binding affinity The binding kinetics of human and cynomolgus IL-13, IL-17A, AF, F and albumin were assessed by surface plasmon resonance (Biacore T200).

[0288] Goat anti-human IgG, F(ab') 2Fragment-specific antibodies (Jackson ImmunoResearch) were immobilized on a CM5 sensor chip via amine coupling chemistry at a level of approximately 5000 RU. Each analytical cycle consisted of an anti-F(ab') 2 It consisted of capture of IL-13 / IL-17AF / albumin multispecific antibody molecules onto the surface, injection of analytes (25° C., flow rate 30 μl / min), followed by surface regeneration. Human and cynomolgus analytes were injected in two-fold serial dilutions in HBS-EP+ running buffer (GE Healthcare) at concentrations of 10 nM to 0.3125 nM for IL-13, 5 nM to 0.156 nM for IL-17A, AF, F, and 100 nM to 3.125 nM for albumin. Antigens were prepared in-house, except for human IL-13 (R&D Systems), cynomolgus IL-13 (Sinobiologicals), and human serum albumin (Jackson ImmunoResearch). Buffer blank injections were included to subtract instrument noise and drift.

[0289] Kinetic parameters were determined using a 1:1 binding model using Biacore T200 evaluation software (version 3.0) and are summarized in Tables 2(1) and 2(2). Dissociation rates (k d ) is 1.0 × 10 -5 If they are measured as less than 1.0 x 10 -5 (detection limit of the Biacore T200 instrument as defined by the manufacturer GE Healthcare) and affinity (K D ) was calculated.

[0290] The results demonstrated that the multispecific antibodies effectively bound to human and cynomolgus IL-17A, IL-17AF, IL-17F, IL-13 and albumin. [Table 2] [Table 3]

[0291] (ii) simultaneous antigen binding; Using surface plasmon resonance, we demonstrated that IL-17A, IL-13 and albumin can be bound simultaneously to an IL-13 / IL-17AF multispecific antibody using either the human or cynomolgus forms of the proteins.

[0292] The method format for evaluating simultaneous binding of analytes to IL-13 / IL-17AF multispecific antibodies was to capture antibody samples on immobilized anti-human IgG F(ab')2 fragment specific antibodies. Human or cynomolgus IL-13, IL-17A and albumin were then injected (300 s at 30 μl / min) either alone or in a mixed solution of all three analytes over the captured IL-13 / IL-17AF multispecific antibodies (final concentrations 30 nM IL-13, 15 nM IL-17A, 150 nM albumin).

[0293] At the end of each cycle, the surface was regenerated with a 60 second injection of 50 mM HCl, followed by a 30 second injection of 5 mM NaOH and a final 60 second injection of 50 mM HCl at a flow rate of 10 μl / min.

[0294] The binding response of each antigen when injected alone was determined and the sum of the individual responses was compared to the binding response when a mixture of all three antigens was injected.

[0295] 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 was able to bind each antigen simultaneously and independently. [Table 4]

[0296] The average binding response of the mixture of cynomolgus 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 was able to bind each antigen simultaneously and independently. [Table 5]

[0297] Example 9 Neutralization of IL-13 by IL-13 / IL-17AF multispecific antibodies. The activity of IL-13 / IL-17AF multispecific antibodies to neutralize IL-13 was assessed using a HEK293 human IL4 / IL-13SEAP reporter cell line assay. IL-13 responses were assessed by measuring SEAP secretion upon activation of the STAT6 pathway.

[0298] HEK293 human IL4 / IL-13 SEAP reporter cells (#hkb-il413) were obtained from Invivogen, San Diego. The manufacturer's protocol was followed for culturing, freezing and maintenance of the cell line.

[0299] Recombinant human IL-13 was obtained from R&D Systems, Minneapolis, Minnesota (#213-ILB).

[0300] Cells were seeded in 96 flat-bottom wells so that they were approximately 80% confluent at the time of stimulation.

[0301] Cells were treated in duplicate with antibodies preincubated with 250 pg / mL IL-13 for 30 min at 37°C, 5% CO2, 100% humidity, and then added to the cells for 24 h.

[0302] After 24 h, 20 μL of supernatant was pipetted from the cell stimulation and added to 180 μL of Quanti-blue#rep-qbs (Invivogen, San Diego) according to the manufacturer's instructions.

[0303] The assay was developed until there was a visible color change gradient and the absorbance was read at 620 nm using a spectrophotometer. IC50 was calculated by non-linear regression using Graphpad Prism (San Diego, CA). Figure 4 shows a representative graph of % inhibition of STAT6 signaling by IL-13 / IL-17AF multispecific antibodies. [Table 6]

[0304] Example 10 Neutralizing effect of IL-13 / IL-17AF multispecific antibodies on IL-6 responses by human skin fibroblasts to human and cynomolgus IL-17A and IL-17F The aim of this study was to determine the neutralizing capacity of IL-13 / IL-17AF multispecific antibodies against human and cynomolgus IL-17A and IL-17F in human primary cell lines. When IL-17 is present in combination with other cytokines such as TNF-α, a proinflammatory response is induced. Therefore, we exploited this synergistic effect to create an IL-6 release assay from primary normal neonatal human dermal fibroblasts (nHDFs) stimulated with IL-17 and TNF-α.

[0305] The ability of IL-13 / IL-17AF multispecific antibodies to inhibit IL-17-induced IL-6 release from nHDFs was measured in this assay. Specifically, nHDFs were stimulated with human or cynomolgus IL-17A (50 pM) or IL-17F (25,000 pM) in combination with TNF-α (25 pM) in the presence of titers of IL-13 / IL-17AF multispecific antibodies (concentration range of 5000 pM to 0.25 pM for IL-17A studies, and 500,000 pM to 25 pM for IL-17F studies). The resulting IL-6 response was then measured using homogeneous time-resolved FRET (HTRF).

[0306] nHDF cells (Sigma #106-05n) were cultured in complete medium (DMEM + 10% FCS + 2 mM L-glutamine) and maintained in tissue culture flasks using standard techniques. Cells were harvested from tissue culture flasks using TrypLE (Invitrogen #12605036). Complete medium (45 ml) was used to neutralize the TrypLE and cells were centrifuged at 300 x g for 3 min. Cells were resuspended in complete medium (3-5 ml), counted and yielded 3.125 x 10 4 After adjusting the concentration to cells / mL, 40 μl / well was added to a 384-well assay plate (Corning #3701). Cells were incubated at 37°C / 5% CO 2 The plates were incubated for 3 hours at 4°C to allow adhesion to the plates. IL-13 / IL-17AF multispecific antibodies were serially diluted in complete medium in a 384-well dilution plate (Greiner#781281) to a final concentration range of 5000pM to 0.25pM for evaluation against IL-17A, and 500,000pM to 25pM for evaluation against IL-17F. Mixtures of TNF-α and IL-17 cytokines were prepared in complete medium to a final concentration of TNF-α 25pM, along with either human or cynomolgus IL-17A 50pM or IL-17F 25,000pM. 30μl / well of these solutions were then added to a 384-well reagent plate (Greiner#781281). 10μl from the IL-13 / IL-17AF multispecific antibody serial dilution plate was then transferred to the reagent plate containing 30μl of diluted cytokines. The IL-13 / IL-17AF multispecific antibody was then incubated with the cytokine mixture at 37°C / 5% CO 2 The cells were incubated at 4°C for 1 hour. After incubation, 10 μl was transferred from the reagent plate to the assay plate containing the cells. The assay plate was then incubated at 37°C / 5% CO 2The plates were incubated at RT for 18 hours ± 2 hours. After incubation was complete, europium cryptate and Alexa665 antibodies from the Cisbio IL-6 HTRF kit (Cisbio#62IL6PEB) were diluted in reconstitution buffer and mixed 1:1 according to the kit insert. 10 μl / well of this antibody mix was then added to a white low volume 384-well HTRF plate (Greiner#784075). Supernatants from the assay plate were then transferred to the HTRF plate at 10 μl / well. The HTRF plate was then incubated at room temperature for 2 hours with gentle shaking. The HTRF plate was then read on a Synergy Neo2 plate reader according to the manufacturer's instructions, measuring fluorescence at 330 / 620nm and 330 / 665nm readings. Ratio values ​​were then calculated using the following formula (330 / 665 nm ÷ 330 / 620 nm) × 10,000, which was used to determine the relative inhibition compared to control wells using Microsoft Excel. 4PL Curve Fitting and IC 50 Values ​​were calculated using GraphPad Prism 7.0.

[0307] Results shown are the mean (+ / - SEM) of three independent experiments. The IC50 values ​​of the IL-13 / IL-17AF multispecific antibody were calculated as 42 pM for human IL-17A and 49 pM for cynomolgus IL-17A. The IC50 values ​​of the IL-13 / IL-17AF multispecific antibody were calculated as 28,030 pM for human IL-17F and 34,320 pM for cynomolgus IL-17F (Figure 5). [Table 7]

[0308] Example 11 Simultaneous Neutralization of IL-13, IL-17A, and IL-17F by IL-13 / IL-17AF Multispecific Antibodies in the NHEK CXCL1 Release Bioassay The purpose of this assay was to evaluate the ability of IL-13 / IL-17AF multispecific antibodies to simultaneously neutralize IL-13, IL-17A and IL-17F in primary cell lines. When NHEKs are treated individually with IL-13 or IL-17A or IL-17F, they induce the secretion of CXCL1, a chemokine involved in cell recruitment to sites of inflammation. In the context of atopic dermatitis, there is evidence that CXCL1 has a role in sensitizing neurons to have a lower excitation threshold. This observation may be related to the itch experienced by patients (Yang TB and Kim BS2019;''Pruritus in allergy and immunology''J Allergy Clin Immunol 144(2):353-360).

[0309] NHEKs (PromoCell, Heidelberg) were stored, cultured and used according to the manufacturer's protocol. Cells were seeded in 48 flat-bottom well plates to 100% confluence at the time of stimulation. Cells were preincubated with increasing concentrations of anti-IL-13, anti-IL-17A, anti-IL-17F or IL-13 / IL-17AF multispecific antibodies for 30 min each, and then treated with 100 ng / mL IL-13, 100 ng / mL IL-17A, 1 μg / mL IL-17F for 72 h. After the elapsed time, 50 μl of cell-free supernatant was collected for quantification of CXCL1 concentration by ELISA according to the manufacturer's protocol (R&D Systems). IC of each experimental group was 0.01 mg / mL. 50 was calculated by nonlinear regression using Graphpad Prism (San Diego, CA).

[0310] The results demonstrated that the IL-13 / IL-17AF multispecific antibody simultaneously neutralized IL-13, IL-17A and IL-17F activity (Figure 6). When normalized for the number of binding sites available in the assay, the IL-13 / IL-17AF multispecific antibody (81.3%) was more effective at inhibiting CXCL1 release than anti-IL-17A (52.7%), anti-IL-17F (0.7%) or anti-IL-13 (48.8%). 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 advantage of simultaneously inhibiting IL-13, IL-17A and IL-17F compared to single cytokine neutralization.

[0311] Example 12. Comparison of multispecific IL-13 / IL-17 antibodies with prior art IL-13 / IL-17 antibodies. Introduction An example of an IL-13 / IL-17AF multispecific antibody according to the invention is shown in Figure 7. It comprises a Fab domain with dual specificity for IL-17A and IL-17F linked to two scFv domains, one specific for IL-13 and the other for albumin. The anti-albumin domain confers the multispecific antibody with extended half-life.

[0312] Bispecific antibodies binding to IL-13 and IL-17 have been previously described by Abbvie (WO 2013 / 102042) and Genentech (WO 2015 / 127405). However, little has been disclosed about how and to what extent they bind to IL-13, IL-17A and IL-17F. To compare their properties, the inventors generated antibodies described in the prior art and examined their binding behavior for the following characteristics: Affinity for IL-13 ·Molecule interaction with IL-13 and IL-13Rα1 Affinity for IL-17A Affinity for IL-17F

[0313] Preparation of comparative antibodies BITS7201A (Genentech) was constructed using the sequence described in Example 6 of WO 2015 / 127405.

[0314] DVD2166 and DVD2174 (Abbvie) were constructed using sequences set out in WO 2013 / 102042, Tables 6 and 7. These molecules were selected on the basis that the reported activities of the individual arms of the bispecific against IL-13 and IL-17 were similar or comparable to the respective parent antibodies (WO 2013 / 102042, Example 4, p. 83, 0195).

[0315] DNA constructs were transfected into CHO-SXE cells using the high titer protocol of the ExpiCHO transfection system (ThermoFisher Scientific). Upon harvesting, cell cultures were centrifuged at 4000 RPM for at least 1 hour and the supernatants were clarified by filtration using a 0.22 μM Stericup filter unit.

[0316] Purification of DVD2166+DVD2174 DVD-IgG protein was purified by applying the clarified supernatant to a 10 ml MabSelect Sure column and washed with 3 column volumes (CV) of PBS, pH 7.4. The protein was eluted from the column with a 0.1 M sodium citrate pH 3.6 step elution and neutralized with 2 M Tris-HCl, pH 8.5. Monomeric protein was isolated by application to a HiLoad 16x60 Superdex200pg column (Sigma) equilibrated with PBS, pH 7.4. Fractions containing monomeric protein were pooled, sterile filtered and stored at 4°C.

[0317] Purification of BITS7201A The parent knob and hole proteins were purified by applying the clarified supernatant to a 10 mL MabSelect Sure column and washed with 3 CV of PBS, pH 7.4. The protein was eluted from the column with 0.1 M sodium citrate pH 3.6. To neutralize and stabilize the protein, the sample was diluted 1:1 with 1 M arginine / succinate buffer, pH 8.7. The parent antibody was then applied to a HiLoad 26x60 Superdex200pg column (Sigma) equilibrated with 0.15 M sodium acetate, 0.5 M arginine buffer, pH 8.5. The bispecific material was subsequently generated by mixing the parent antibody in a 1:1 ratio in the presence of 5 mM cysteamine and incubating overnight at room temperature. A second preparative gel filtration step was performed to remove any high molecular weight species from the exchanged bispecific material by applying to a HiLoad 26x60 Superdex200pg column equilibrated with PBS, pH 7.4. Fractions containing the monomeric bispecific protein were pooled, sterile filtered and stored at 4°C.

[0318] Comparison of binding properties The binding kinetics of human IL-13, IL-17A and IL-17F to the IL-13 / IL-17AF multispecific antibody designated "UCBXXXX" and to prior art antibodies were assessed by surface plasmon resonance (Biacore T200) and directly compared within a single experiment. In addition, surface plasmon resonance was used to assess whether binding of the IL-13 / IL-17AF multispecific antibody UCBXXXX or a comparison molecule to IL-13 resulted in blocking of IL-13 interaction with the IL-13 receptor.

[0319] Goat anti-human IgG, F(ab') 2 Fragment-specific antibodies (Jackson ImmunoResearch) were immobilized on a CM5 sensor chip via amine coupling chemistry at a level of approximately 5000 RU. For affinity evaluation, each analytical cycle consisted of a single antibody, anti-F(ab') 2It consisted of capture of IL-13 / IL-17AF polyspecific antibodies or comparative bispecific molecules onto the surface (50-100 RU), injection of analyte (25° C., flow rate 30 μl / min for 180 s), after which dissociation was monitored for 1200 s for IL-13 and IL-17A and 600 s for IL-17F. At the end of each cycle, the surface was regenerated with a 60 s injection of 50 mM HCl, followed by a 30 s injection of 5 mM NaOH and a final 60 s injection of 50 mM HCl at a flow rate of 10 μl / min. Analytes were injected in 2-fold serial dilutions in HBS-EP+ running buffer (GE Healthcare) at concentrations ranging from 10 nM to 0.3125 nM for IL-13 and 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 supplied by R&D Systems. A buffer blank injection was included to subtract instrument noise and drift.

[0320] Kinetic parameters were determined using a 1:1 binding model using Biacore T200 evaluation software (version 3.0) and the results are summarized in Table 7. [Table 8]

[0321] To assess IL-13Rα1 receptor blocking, each antibody molecule was incubated with goat anti-human IgG, F(ab') 2 IL-13 was captured on the surface (approximately 50-100 RU) followed by injection of IL-13 (25 nM, 10 μl / min for 180 sec) and IL-13Rα1 (R&D Systems, 100 nM, 10 μl / min for 300 sec). Blank injections of both IL-13 and IL-13Rα1 were included to subtract any drift or background responses. As summarized in Table 8, UCBXXXX was able to block the interaction of IL-13 with IL-13Rα1, whereas BITS7210A, DVD2166 and DVD2174 molecules did not. [Table 9]

[0322] Further experiments were performed to evaluate the ability of IL-13 / IL-17AF multispecific antibodies to block binding to IL-13Rα1 and IL-13Rα2. In this experiment, approximately 260 RU of UCBXXXX was captured onto immobilized mouse anti-human CH1 antibody (UCB inventors), followed by injection of IL-13 (25 nM, 10 μl / min for 180 sec) and then injection of either IL-13Rα1 or IL-13Rα2 (R&D Systems, 100 nM, 10 μl / min for 300 sec). Blank injections of IL-13, IL-13Rα1 and IL-13Rα2 were included to subtract any drift or background responses. The results demonstrated that UCBXXXX can block the interaction of IL-13 with both IL-13Ra1 and IL-13Ra2. (Table 9) [Table 10]

[0323] Consideration Comparative studies demonstrated 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 the binding interaction were very different. UCBXXXX could block the interaction of IL-13 with IL-13-Rα1, whereas BITS7210A, DVD2166, and DVD2174 could not.

[0324] Comparative studies further demonstrated that the multispecific antibody UCBXXXX, the bispecific antibody BITS7210A, and the dual variable domain antibodies DVD2166 and DVD2174 were able to bind IL-17. However, again, the characteristics of the binding interactions were very different. The binding affinity of UCBXXXX to IL-17A was significantly higher than that of BITS7210A and the DVD antibodies. UCBXXXX and BITS7210A bound with similar affinity to IL-17F, whereas the DVD antibodies were unable to bind to IL-17F at all.

[0325] The interaction between IL-13 and IL-13Rα1 in the presence of these antibodies is important in terms of reducing potential immunogenicity. Evidence suggests that immunogenicity, especially the generation of anti-drug antibodies (ADA), should be closely considered when investigating potential novel bispecific antibody therapeutics. The main driver of ADA generation is the association of therapeutic antibodies with target antigens expressed on the cell surface and subsequent internalization (Schellekens, H., 2002; Clin Ther. 24(11):1720-40). The internalized therapeutic antibody / target antigen complex can undergo trafficking through various intracellular compartments and recycle to the cell surface or undergo degradation (St Pierre et al., 2011), which can lead to peptide presentation by antigen-presenting molecules and ADA generation.

[0326] In the bispecific antibody BITS7201A, the IL-13 F(ab) portion of the molecule is the same as the anti-IL-13 antibody lebrikizumab (see Example 6 of WO 2015 / 127405). Lebrikizumab is believed to bind IL-13 at a site that allows IL-13 to bind to its receptors IL-13Rα1 and IL-13Rα2, but blocks interaction with the IL-4Rα receptor (Popovic et al., 2017; J Mol Biol. 429(2):208-19). This particular type of interaction may allow internalization of the antibody / target antigen / receptor complex via IL-13Rα2, thus increasing the likelihood of an immunogenic response. In Phase I clinical trials, BITS7201A was associated with a high incidence of anti-drug antibodies (ADA) and was withdrawn from clinical development.

[0327] Similarly, the dual variable domain antibodies DVD2166 and DVD2174 were unable to block the interaction of IL-13 with IL-13-Rα1.

[0328] To mitigate potential immunogenicity risks, UCBXXXX has been specifically designed to prevent its respective target antigens IL-13, IL-17A and IL-17F from interacting with receptors on cells upon association, thus reducing the chance of internalization, degradation and the likelihood of ADA generation. The incidence of ADA in humans due to UCBXXXX can only be determined with certainty once clinical data are available.

[0329] The data generated above indicates that the IL-13 / IL-17AF multispecific antibody UCBXXXX has the right properties to be an effective IL-13 / IL-17 antibody therapeutic with improved efficacy and low risk of immunogenicity. [Sequence List Free Text]

[0330] Sequence Listing 1-68 <223> Recombination Sequence

Claims

1. A multispecific antibody that binds to human IL-13, human IL-17A and / or human IL-17F, a) a polypeptide chain of formula (Ia): V Ｈ -CH １ -X-V １ と、 b) a polypeptide chain of formula (IIa): V Ｌ -C Ｌ -Y-V ２ と Including, During the ceremony, V Ｈ represents the heavy chain variable domain, CH １ represents domain 1 of the heavy chain constant region, X represents a bond or a linker; Y represents a bond or a linker; V １ represents a scFv, a dsscFv, or a dsFv; V Ｌ represents the light chain variable domain, C Ｌ represents a domain derived from the light chain constant region, V ２ represents scFv, dsscFv or dsFv, The polypeptide chain of formula (Ia) comprises a Protein A binding domain, and the polypeptide chain of formula (IIa) does not bind to Protein A, V Ｌ and V Ｈ comprises an antigen-binding site that binds human IL-17A and human IL-17F, V ２ comprises an antigen-binding site that binds to human IL-13, V １ comprises an antigen-binding site that binds to human serum albumin, V Ｌ comprises the sequence shown in SEQ ID NO:1 for CDR-L1, the sequence shown in SEQ ID NO:2 for CDR-L2, and the sequence shown in SEQ ID NO:3 for CDR-L3, V Ｈ comprises the sequence shown in SEQ ID NO: 4 for CDR-H1, the sequence shown in SEQ ID NO: 5 for CDR-H2, and the sequence shown in SEQ ID NO: 6 for CDR-H3; V １ comprises a light chain variable region comprising the sequence shown in SEQ ID NO: 39 for CDR-L1, the sequence shown in SEQ ID NO: 40 for CDR-L2, and the sequence shown in SEQ ID NO: 41 for CDR-L3, and a heavy chain variable region comprising the sequence shown in SEQ ID NO: 42 for CDR-H1, the sequence shown in SEQ ID NO: 43 for CDR-H2, and the sequence shown in SEQ ID NO: 44 for CDR-H3; V ２ comprises a light chain variable region comprising the sequence shown in SEQ ID NO: 15 for CDR-L1, the sequence shown in SEQ ID NO: 16 for CDR-L2, and the sequence shown in SEQ ID NO: 17 for CDR-L3, and a heavy chain variable region comprising the sequence shown in SEQ ID NO: 18 for CDR-H1, the sequence shown in SEQ ID NO: 19 for CDR-H2, and the sequence shown in SEQ ID NO: 20 for CDR-H3, A multispecific antibody that binds to human IL-13, human IL-17A and / or human IL-17F.

2. V Ｌ comprises the sequence shown in SEQ ID NO:7, and V Ｈ The multispecific antibody of claim 1, wherein said multispecific antibody comprises the sequence shown in SEQ ID NO:

9.

3. V ２ 3. The multispecific antibody according to claim 1 or 2, comprising a light chain variable region comprising the sequence as set forth in SEQ ID NO: 27 and a heavy chain variable region comprising the sequence as set forth in SEQ ID NO:

28.

4. V ２ 3. The multispecific antibody of claim 1 , wherein the multispecific antibody comprises a light chain variable region comprising the sequence set forth in SEQ ID NO: 31 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:

32.

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

46.

6. V １ 5. The multispecific antibody according to claim 1 , wherein the multispecific antibody comprises a light chain variable region comprising the sequence as set forth in SEQ ID NO: 49 and a heavy chain variable region comprising the sequence as set forth in SEQ ID NO:

50.

7. V ２ 7. The multispecific antibody of claim 1 , wherein the light chain variable region and the heavy chain variable region are linked by a linker, and the linker comprises the sequence shown in SEQ ID NO:

66.

8. V ２ The multispecific antibody of claim 7, wherein said scFv comprises the sequence shown in SEQ ID NO: 35, or said dsscFv comprises the sequence shown in SEQ ID NO:

37.

9. V １ 9. The multispecific antibody according to claim 1 , wherein the light chain variable region and the heavy chain variable region are linked by a linker, and the linker comprises the sequence shown in SEQ ID NO:

68.

10. V １ is an scFv comprising the sequence shown in SEQ ID NO:53 or a dsscFv comprising the sequence shown in SEQ ID NO:

55.

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

65.

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

67.

13. 13. A multispecific antibody according to any one of claims 1 to 12, comprising the sequence shown in SEQ ID NO: 57 or SEQ ID NO:

59.

14. 14. A multispecific antibody according to any one of claims 1 to 13, comprising the sequence shown in SEQ ID NO: 61 or SEQ ID NO:

63.

15. 15. A multispecific antibody according to any one of claims 1 to 14, comprising the sequence shown in SEQ ID NO: 59 and the sequence shown in SEQ ID NO:

63.

16. 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 a vector as defined in claim 17.

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

20. 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 pharma- ceutically acceptable adjuvant and / or carrier.

22. 22. A pharmaceutical composition according to claim 21 for use in a method of treatment of the human or animal body by therapy.

23. 23. A pharmaceutical composition according to claim 22 for use in the treatment or prevention of atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergies, or eosinophilic esophagitis.

24. 20. Use of a multispecific antibody according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment or prevention of atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergy, or eosinophilic esophagitis.

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