RU2827946C1 - Multispecific antibody with binding specificity of human il-13 and il-17 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a multispecific antibody which binds to human IL-13, human IL-17A and IL-17F, a method for production thereof, as well as to a composition containing it. Also disclosed is a recovered polynucleotide coding said antibody, as well as a vector and a cell containing it.

EFFECT: invention is effective for treating or preventing atopic dermatitis, chronic hand eczema, nasal micropolyposis or polyposis, food allergy or eosinophilic oesophagitis.

25 cl, 7 dwg, 9 tbl, 12 ex

Description

Field of technology to which the present invention relates

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

Prior art of the present invention

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

Since the early twentieth century, many inflammatory diseases of the mucous membranes have become more common; atopic dermatitis is a classic example of such a disease. It currently affects 15-30% of children and 2-10% of adults in developed countries, and in the United States it has almost tripled in the last thirty to forty years. More than 15 million adults and children in the United States suffer from atopic dermatitis.

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

Due to the limitations of existing drugs, there is a great need for improved treatment of atopic dermatitis.

WO2013/102042A2 (Abbvie) describes dual-specificity binding proteins directed against IL-13 and IL-17 and their potential use in the treatment of a wide range of diseases. The binding proteins have not been developed clinically.

WO2015/127405A2 (Genentech) describes bispecific antibodies against IL-13/IL-17 and methods for 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.

The essence of the present invention

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

The antibodies of the present invention have improved properties compared to currently available antibodies, such as lower immunogenicity and/or a better pharmacokinetic profile. In addition, the antibodies of the present invention can be designed so that they can be purified more efficiently using an improved purification method that involves fewer steps than currently available methods and is cost- and time-effective on an industrial scale. Therefore, the antibodies of the present invention can have improved manufacturability.

The invention additionally presents:

An isolated polynucleotide encoding a multispecific antibody.

An expression vector carrying a polynucleotide.

A host cell containing a vector.

A method for producing a multispecific antibody, including culturing a host cell and isolating the resulting antibody.

A pharmaceutical composition containing a multispecific antibody.

A multispecific antibody or pharmaceutical composition for use in a method of treating a human or animal by therapy.

A method for treating or preventing atopic dermatitis, chronic hand eczema, micropolyposis or nasal polyposis, food allergy or eosinophilic esophagitis, comprising administering a therapeutically effective amount of a multispecific antibody or pharmaceutical composition to a patient.

Brief description of the drawings

Fig. 1.

Alignments with Ab650 humanization.

Alignments of rat antibody V region (donor) sequences with human germline V region (acceptor) sequences along with engineered humanized sequences.

(A) 650 light chain graft:

650=rat light chain variable region sequence.

650gL8 = humanized 650 light chain variable region graft using human germline IGKV1-39 as an acceptor scaffold.

CDRs are shown in bold/underlined.

Donor residues are shown in bold/italics and highlighted: I58 and Y71.

(B) Heavy chain 650 graft:

650=rat heavy chain variable region sequence.

650gH9=humanized 650 heavy chain variable region graft using human germline IGHV1-69 as an acceptor scaffold.

CDRs are shown in bold/underlined.

Donor residues are shown in bold/italics and highlighted: A67, F69 and V71.

Fig. 2.

Amino acid sequences and DNA sequences.

Fig. 3.

Purification of the multispecific IL-13/IL-17AF antibody.

(A) BEH200 SEC-UPLC analysis of purified multispecific antibody, detected by FLR.

(B) Protein samples separated by Tris-glycine SDS-PAGE under non-reducing (lane 1) or reducing (lane 2) conditions. The gel was stained with Coomassie fast stain and destained in dH2O. Mark 12 protein markers (Life Technologies) were used as standards (M). Molecular weights (MW) were measured in kilodaltons (kDa).

Fig. 4.

Inhibition of STAT6 signaling using a multispecific IL-13/IL-17AF antibody.

Fig. 5.

(A) Inhibition of IL-6 production by IL-13/IL-17AF multispecific antibody in response to human or cynomolgus monkey IL-17A in combination with TNF-α. (i) Human IL-17A. (ii) Cynomolgus monkey IL-17A.

(B) Inhibition of IL-6 production by IL-13/IL-17AF multispecific antibody in response to human or cynomolgus monkey IL-17F in combination with TNF-α. (i) Human IL-17F. (ii) Cynomolgus monkey IL-17F.

Fig. 6.

Simultaneous neutralization of IL-13, IL-17A and IL-17F by a multispecific IL-13/IL-17AF antibody in an IL-13/IL-17AF release bioassay.

Designations:

circle=antibody against IL-13/IL-17AF

square=antibody against IL-17A

triangle with apex up=antibody against IL-17F

triangle with apex down=antibody against IL-13

Fig. 7.

Schematic representation of the IL-13/IL-17AF multispecific antibody according to the present invention.

Detailed disclosure of the present invention

IL-13

IL-13 is a short-chain cytokine sharing 25% sequence identity with IL-4. It contains approximately 132 amino acids forming a secondary structure of four helices spanning residues 10–21 (helix A), 43–52 (helix B), 61–69 (helix C), and 92–110 (helix D) along with two β strands spanning residues 33–36 and 87–90. A solution structure of IL-13 has been proposed revealing the predicted up-up-down-down four-helix bundle conformation also observed for IL-4 (Eise and Messer 2001).

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

IL-13 binds to its cell surface receptors, IL-13Ralpha1 and IL-13Ralpha2. IL-13Ralpha1 interacts with IL-13 with low affinity ( _KD ~10 nM) followed by recruitment of IL-4Ralpha to form a heterodimeric receptor signaling complex with high affinity ( _KD ~0.4 nM).

The IL-4R/IL-13Ralpha1 complex is expressed on 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-13Ralpha/IL-4R receptor complex results in activation of multiple signaling pathways, including the signal transduction and activator of transcription 6 (STAT6) and insulin receptor substrate 2 (IRS2) pathways.

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

IL-13 has been implicated in the pathogenesis of many human diseases, and therapeutic strategies have been developed to inhibit or counteract IL-13 activity. In particular, antibodies that bind and neutralize IL-13 have been sought as a means of inhibiting IL-13 activity. However, there is a need in the art for suitable and/or improved antibodies capable of binding IL-13, especially human IL-13, and in particular antibodies that are capable of neutralizing human IL-13.

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

Antibodies that inhibit IL-13 activity may act through several possible mechanisms of action. Group 1 represents an antibody that binds to human IL-13 and prevents binding of IL-13Rα1 and as a result also blocks binding of IL-4R. Group 1 antibodies can also prevent binding of IL-13 to IL-13Rα2. Group 2 represents an antibody that binds hIL-13 in a manner that allows binding to IL-13Rα1 but prevents recruitment of IL-4R to the complex. We selected antibodies that acted through Group 1.

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

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

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

In one embodiment, the multispecific antibody binds to human IL-13 with a K _D < 100 pM.

IL-17

The IL-17 cytokine family consists of 6 members based on structural similarity, with a molecular weight of 23-36 kDa and a dimeric structure. The core member IL-17A (often referred to in the literature as simply IL-17) 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 highest homology (50%) and bind to the same receptor complex, thus it has been observed that these 2 cytokines share common biological activities. Furthermore, IL-17A and IL-17F exist not only as homodimers but also as a heterodimer IL-17A/F. IL-17E (IL-25) has the least similarity to IL-17A. Significant and relevant to the biological activity of IL-17A and IL-17F is the fact that they share the same IL-17RA/IL-17RC receptor complex, where IL-17A has the highest affinity for IL-17RA, whereas IL-17F binds more strongly to IL-17RC. Another member of the IL-17RA-using family is IL-17E, which signals through the IL-17RA/IL-17RB receptor complex.

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

The cell types that respond to IL-17 cytokines are reflected by the expression of different receptors. IL-17RA is ubiquitously expressed, with particularly high levels in hematopoietic tissues, whereas IL-17RC is more highly expressed in non-immune cells of the joints, liver, kidney, thyroid, and prostate. This differential expression may explain the differences in the biological activity of IL-17A and IL-17F, as cells expressing high levels of IL-17RC may be more sensitive to IL-17F, whereas cells expressing higher levels 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, synoviocytes, and endothelial cells, where IL-17A has also been reported to act on T and B cells and macrophages.

The multispecific antibody according to the present invention is capable of binding to human IL-17A and/or IL-17F. Thus, the antibody can bind to an IL-17A homodimer, an IL-17F homodimer and/or an IL-17AF heterodimer.

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.

In one embodiment, the multispecific antibody binds to human IL-17C with a K _D < 50 pM. In one embodiment, the multispecific antibody binds to human IL-17C with a K _D < 25 pM. In one embodiment, the multispecific antibody binds to human IL-17C with a K _D < 10 pM.

In one embodiment, the multispecific antibody binds to human IL-17F with a _KD < 200 pM. In one embodiment, the multispecific antibody binds to human IL-17F with a _KD < 100 pM.

Albumen

The high specificity and affinity of antibodies make them ideal diagnostic and therapeutic tools, particularly for modulating protein–protein interactions. However, antibodies may suffer from increased clearance rates from serum, particularly when they lack the Fc domain that provides a long in vivo survival (Medasan et al . 1997, J. Immunol. 158:2211–2217).

Means for improving the half-life of antibodies are known. One approach is to conjugate the fragment to polymer molecules. Thus, the short half-life of Fab', F(ab') ₂ fragments from the bloodstream in animals was improved by conjugation with polyethyleneglycol (PEG; see, e.g., WO98/25791, WO99/64460, and WO98/37200). Another approach is to modify the antibody fragment by conjugation to an agent that interacts with the FcRn receptor (see, e.g., WO97/34631). Another approach to increasing half-life is to use polypeptides that bind serum albumin (see, e.g., Smith et al . 2001, Bioconjugate Chem. 12:750-756; EP0486525; US6267964; WO04/001064; WO02/076489; and WO01/45746).

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

Single variable domains binding serum albumin have been described along with their use as conjugates to enhance the half-life of drugs, including NCE (chemical compound) drugs, proteins and peptides, see for example Holt et al . Protein Engineering, Design & Selection, vol 21, 5, pp283-288, WO04003019, WO2008/096158, WO05118642, WO2006/0591056 and WO2011/006915. Other anti-serum albumin antibodies and their use in multispecific antibody formats have been described in WO2009/040562, WO2010/035012 and WO2011/086091. In particular, the authors previously described an anti-albumin antibody with improved humanization in WO2013/068571.

The multispecific antibodies of the present invention can be engineered to bind to human serum albumin to increase their serum half-life in vivo , resulting in an improved pharmacokinetic profile.

Antibodies

Antibodies for use in the context of the present 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. The features described herein with respect to antibodies also apply to antibody fragments, unless the context otherwise requires.

Whole antibodies, also known as "immunoglobulins (Ig)", generally refer to intact or full-length antibodies, i.e., containing elements of two heavy chains and two light chains joined together by disulfide bonds, which together form a characteristic Y-shaped three-dimensional structure. Classically, naturally occurring whole antibodies are monospecific in the sense that they bind one type of antigen and bivalent in the sense that they have two independent antigen-binding domains. The terms "intact antibody", "full-length antibody" and "whole antibody" are used interchangeably to refer to a monospecific bivalent antibody having a structure similar to the native antibody structure, including the Fc region of the present invention.

Each light chain consists of a light chain variable region (abbreviated herein as V_L) and the light chain constant region (C_L). Each heavy chain consists of a heavy chain variable region (abbreviated herein as V_H) and the heavy chain constant region (CH), consisting of three CH constant domains₁, CH₂and CH₃or four constant CH domains₁, CH_2,CH₃and CH₄depending on the Ig class. The "class" of an Ig or antibody refers to the type of constant region and includes IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4. The constant regions of antibodies can mediate the binding of immunoglobulin to tissues or host factors, including various cells of the immune system (For example,effector cells) and the first component (Clq) of the classical complement system.

The VH and VL regions of the antibody of the present invention can be further subdivided into regions of hypervariability (or "hypervariable regions") that determine antigen recognition, called complementarity determining regions (CDRs), interspersed with regions that are more structurally conserved, called framework regions (FRs). Each V _H and V _L consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FRs together form a variable region. By convention, the CDRs in the variable region of the heavy chain of an antibody or an antigen-binding fragment thereof are designated CDR-H1, CDR-H2 and CDR-H3, and in the variable regions of the light chain CDR-L1, CDR-L2 and CDR-L3. They are numbered sequentially in the direction from the N-terminus to the C-terminus of each chain.

CDRs are typically numbered according to the system developed by Kabat et al. This system 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. (See above)”). This numbering system is used in the present description unless otherwise indicated.

Kabat residue designations do not always correspond directly to the linear amino acid residue numbering. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to a truncation or insertion into a structural component, be it the framework or the complementarity determining region, of the major variable domain structure. The correct Kabat residue numbering can be determined for a given antibody by aligning the homology residues in the antibody sequence with a sequence with the "standard" Kabat numbering.

The CDRs of the variable domain of the heavy chain 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, A.M. J. 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’ within the context of the present invention refers to residues 26-35 as described by the combination of the Kabat numbering system and the Chothia definition of the topological loop.

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.

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

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

The term "constant domain(s)", "constant region" according to the present invention are used interchangeably to refer to the domain(s) of an antibody that is outside the variable regions. Constant domains are identical in all antibodies of the same isotype, but differ from one isotype to another. Typically, the constant region of a heavy chain is formed from the N to the C terminus by a CH ₁ -hinge -CH ₂ -CH ₃ -optionally CH ₄ , containing three or four constant domains.

If present, the constant domains of the antibody molecule of the present invention can be selected taking into account the intended function of the antibody molecule and in particular the effector functions that may be required. For example, the constant domains can be human IgA, IgD, IgE, IgG or IgM domains. In particular, when the antibody molecule is intended for therapeutic applications and antibody effector functions are required, human IgG constant domains, especially the IgG1 and IgG3 isotypes, can be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, the IgG2 and IgG4 isotypes can be used. It will be appreciated that sequence variants of these constant domains can also be used. For example, IgG4 molecules can be used in which the serine at position 241 (the number corresponds to the Kabat numbering system) is replaced with proline, as described in Angal et al. (Angal et al., 1993. A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody as observed during SDS-PAGE analysis Mol Immunol 30, 105-108), which are referred to herein as IgG4P.

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

In the context of the present disclosure, if present, the constant region or Fc region may be naturally occurring as defined above or may be modified in various ways, so long as it comprises a functional FcR binding domain and preferably a functional FcRn binding domain. Preferably, the modified constant region or Fc region results in improved functionality and/or pharmacokinetics. Modifications may include deletion of certain regions of the Fc fragment. Modifications may further include various amino acid substitutions capable of affecting the biological properties of the antibody. There may also be mutations to increase FcRn binding and thus the half-life in vivo. Modifications may further include modification of the glycosylation profile of the antibody. The naturally occurring Fc fragment is glycosylated in the CH ₂ domain with an N-glycan in each of the two heavy chains linked to an asparagine residue at position 297 (Asn297). In the context of the present disclosure, an antibody may be glycomodified, i.e., engineered to have a particular glycosylation profile that, for example, results in improved properties, such as improved effector function or improved serum half-life.

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.

The term "antibody" includes monovalent antibodies, i.e., antibodies containing only one antigen-binding domain (e.g., partial antibodies containing a full-length heavy chain and a full-length light chain linked together, also called a "half-antibody"), and multivalent antibodies, i.e., antibodies containing more than one antigen-binding domain.

The term "antibody" according to the invention also encompasses antigen-binding fragments of antibodies. Antigen-binding fragments of antibodies include single-chain antibodies (e.g., scFv and dsscfv), Fab, Fab', F(ab') ₂ , Fv, single-domain antibodies or nanobodies (e.g., _VH or _VL or _VHH or _VNAR ). Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in International Patent Applications WO2011/117648, WO2005/003169, WO2005/003170 and WO2005/003171.

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

The term "Fab fragment" according to the present invention refers to an antibody fragment comprising a light chain fragment comprising a VL (variable light) domain and a light chain constant domain (CL) and a V _H (variable heavy) domain and the first constant domain (CH ₁ ) of the heavy chain.

A typical "Fab'fragment" comprises a pair of heavy and light chains, in which the heavy chain comprises a V _H variable region, a CH ₁ constant domain, and a natural or modified hinge region, and the light chain comprises a V _L variable region and a CL constant domain. Fab' dimers according to the present disclosure create F(ab') ₂ , where, for example, dimerization can occur across the hinge.

The term "single domain antibody" according to the present invention refers to an antibody fragment consisting of a single monomeric variable domain of an antibody. Examples of single domain antibodies include V _H or V _L or V _H H or V-NAR.

The term "Fv" refers to two variable domains, such as combined variable domains such as a cognate pair or affinity-matured variable domains, such as a V _H and V _L pair.

"Single chain variable fragment" or "scFv" within the context of the present invention refers to a single chain variable fragment comprising or consisting of a heavy chain variable domain (V _H ) and a light chain variable domain (V _L ), which is stabilized by a peptide linker between the VH and VL variable domains. The VH and VL variable domains can be in any suitable orientation, for example the C-terminus of V _H can be linked to the N-terminus of V _L, or the C-terminus of V _L can be linked to the N-terminus of V _H .

"A disulfide-stabilized single-chain variable fragment" or "dsscFv" as used herein refers to a single-chain variable fragment that is stabilized by a peptide linker between the VH and VL variable domains and also comprises an interdomain disulfide bond between V _H and V _L. (See, e.g., Weatherill et al. Protein Engineering, Design & Selection, 25 (321-329) 2012, WO2007109254.

A "disulfide-stabilized variable fragment" or "dsFv" as used herein refers to a single-chain variable fragment that does not contain a peptide linker between the VH and VL variable domains and is instead stabilized by an interdomain disulfide bond between V _H and V _L .

In one embodiment, the multispecific antibody of the present invention is an antagonist antibody. According to the present invention, the term "antagonist antibody" describes an antibody that is capable of inhibiting or neutralizing the biological signaling activity of one or more antigens, for example by blocking the binding or reducing binding of IL-13, IL-17A and/or IL-17F to their receptors.

Antibodies for use in the present invention may be, without limitation, monoclonal, humanized, fully human, or chimeric antibodies.

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

Antibodies can also be generated using techniques for producing antibodies from individual lymphocytes by cloning and expressing immunoglobulin variable region cDNA obtained from individual lymphocytes selected for the production of specific antibodies, for example, using the methods described in Babcook, J. et al . 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-7848l; WO92/02551; WO2004/051268 and International Patent Application Number WO2004/106377.

Antibody screening can be performed using assays for measuring binding to an antigen and/or assays for measuring the ability to block the binding of an antigen to one or more of its receptors. An example of a binding assay is an ELISA, for example, using an IL-13 fusion protein that is immobilized on plates and using a conjugated secondary antibody to detect anti-IL-13 antibody bound to IL-13. An example of a blocking assay is a flow cytometric assay for measuring the blocking of IL-13 ligand protein binding to IL-13R. A fluorescently labeled secondary antibody is used to determine the amount of IL-13 ligand protein binding to IL-13R.

Humanized antibodies (which include 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., US 5,585,089; WO91/09967). It will be appreciated that it may be necessary to transfer only the specificity determining residues of the CDR rather than the entire CDR (see, e.g., Kashmiri et al. 2005, Methods, 36, 25-34). Humanized antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived.

Chimeric antibodies are composed of elements derived from two different species, so that the element retains the characteristics of the species from which it was derived. Typically, a chimeric antibody will contain a variable region from one species, such as mouse, rat, rabbit, etc., and a constant region from another species, such as humans.

Antibodies can also be generated using a variety of phage display techniques known in the art and include those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 187 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US 5698426; 5223409; 5403484; 5580717; 5427908; 5750753; 5821047; 5571698; 5427908; 5516637; 5780225; 5658727; 5733743 and 5969108.

Fully human antibodies are those in which the variable regions and constant regions (where 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 from the same antibody. Examples of fully human antibodies may include antibodies produced, for example, by the phage display techniques described above, and antibodies produced by mice in which the variable and optionally constant region genes of a mouse immunoglobulin have been replaced by their human counterparts, for example as described generally in EP 0 546 073, US 5 545 806, US 5 569 825, US 5 625 126, US 5 633 425, US 5 661 016, US 5 770 429, EP 0 438 474 and EP 0 463 151.

Multispecific antibodies

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

A paratope is a region of an antibody that recognizes and binds to an antigen. An antibody of the invention may be a multiparatopic antibody. A "multiparatopic antibody" as used herein 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, tetraparatopic.

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

"Specific" as used herein refers to a binding domain that recognizes only the antigen to which it is specific, or to a binding domain that has a significantly higher binding affinity for the antigen to which it is specific compared to the affinity for antigens to which it is non-specific. Binding affinity can be measured using standard assays such as surface plasmon resonance, such as BIAcore.

Many formats of multispecific antibodies have been created. Various classifications have been proposed, but multispecific IgG antibody formats generally include bispecific IgG complemented with 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.

Methods for producing bispecific antibodies include, but are not limited to, the CrossMab method (Klein et al. Engineering therapeutic bispecific antibodies using CrossMab technology, Methods 154 (2019) 21-31), the Knobs-in-holes method (e.g., WO1996027011, WO1998050431), the DuoBody method (e.g., WO2011131746), and the Azymetric method (e.g., WO2012058768). Additional methods for producing bispecific antibodies have been described, for example, in Godar et al. 2018, Therapeutic bispecific antibody formats: a patent applications review (1994-2017), Expert Opinion on Therapeutic Patents, 28:3, 251-276. Bispecific antibodies include, but are not limited to, CrossMab, DAF (two in one), DAF (four in one), DutaMab, DT-lgG, Knobs-in-holes common LC, Knobs-in-holes assembly, Charge pair, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body and orthogonal Fab antibodies.

Complemented IgG classically comprises a full-length IgG constructed by adding an additional antigen-binding domain or antigen-binding fragment to the N- and/or C-terminus of the heavy and/or light chain of IgG. Examples of such additional antigen-binding fragments include sdAb (e.g. V _H or V _L ) Fv, scFv, dsscFv, Fab, scFav antibodies. Augmented IgG antibody formats include, but are not limited to, DVD-IgG, lgG(H)-scFv, scFv-(H)lgG, lgG(L)-scFv, scFv-(L)lgG, lgG(L, H)-Fv, lgG(H)-V, V(H)-lgG, lgC(L)-V, V(L)-lgG, KIH IgG-scFab, 2scFv-lgG, lgG-2scFv, scFv4-lg, Zybody, and DVI-IgG (four in one), such as described in Spiess et al. Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106.

Multispecific antibody fragments include nanobody, nanobody-HAS, BiTEs, diabody, DART, TandAb, scDiabody, sc-Diabody-CH ₃ , Diabody-CH ₃ , Triple Body, Miniantibody; Minibody, Tri Bi minibody, scFv-CH ₃ KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFV3, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc; and intrabody, as described, for example, in Spiess et al. Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106.

Multispecific fusion proteins include Dock and Lock, ImmTAC, HSAbody, scDiabody-HAS, and Tandem scFv-Toxin.

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

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

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

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

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

In one embodiment, the IL-13 binding site comprises a light chain variable region comprising the sequence of SEQ ID NO:27.

In one embodiment, the IL-13 binding site comprises a heavy chain variable region comprising the sequence of SEQ ID NO:28.

In one embodiment, the IL-13 binding site comprises a light chain variable region comprising the sequence of SEQ ID NO:31.

In one embodiment, the IL-13 binding site comprises a heavy chain variable region comprising the sequence of SEQ ID NO:32.

In one embodiment, the multispecific antibody comprises an antigen-binding region that binds to human IL-17A and IL-17F, comprising:

a light chain variable region comprising the sequence of SEQ ID NO:1 for CDR-L1, the sequence of SEQ ID NO:2 for CDR-L2, and the sequence of SEQ ID NO:3 for CDR-L3.

In one embodiment, the multispecific antibody comprises an antigen-binding region that binds to human IL-17A and IL-17F, comprising:

a heavy chain variable region comprising the sequence of SEQ ID NO:4 for CDR-H1, the sequence of SEQ ID NO:5 for CDR-H2, and the sequence of SEQ ID NO:6 for CDR-H3.

In one embodiment, the antigen binding region that binds human IL-17A and IL-17F comprises a light chain variable region comprising the sequence of SEQ ID NO:7.

In one embodiment, the antigen binding region that binds to human IL-17A and IL-17F comprises a heavy chain variable region comprising the sequence of SEQ ID NO:9.

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

In one embodiment, the multispecific antibody comprises the sequence of SEQ ID NO:57 or SEQ ID NO:59.

In one embodiment, the multispecific antibody comprises the sequence of SEQ ID NO:61 or SEQ ID NO:63.

In one embodiment, the multispecific antibody comprises the sequence of SEQ ID NO:59 and the sequence of SEQ ID NO:63.

In one embodiment, the multispecific antibody comprises or consists of:

polypeptide chain of formula ( I ):

VH- CH ₁ -( CH ₂ )s-( CH ₃ )tX-(V1)p ; And

polypeptide chain of formula ( II ):

VL-CL-Y-V2 ;

Where:

VH represents the variable domain of the heavy chain;

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 linker;

V1 represents dsscFv, dsFv or scFv;

VL represents the variable domain of the light chain;

CL represents a domain from a light chain constant region such as Ckappa;

Y represents a bond or linker;

V2 represents dsscFv, dsFv or scFv;

p is 0 or 1;

s is 0 or 1;

t is 0 or 1;

where when p is 0, X is absent, and when q is 0, Y is absent; and

wherein the polypeptide chain of formula (I) comprises a protein A binding domain; and

wherein the polypeptide chain of formula (II) does not bind protein A.

In one embodiment, when s is 0 and t is 0, the multispecific antibody of the present disclosure is provided as a heavy and light chain dimer:

formulas (I) and (II) , respectively, where the VH-CH ₁ region together with the VL-CL region form a functional Fab or Fab' fragment.

In one embodiment, when s is 1 and t is 1, the multispecific antibody of the present disclosure is provided as a dimer of two heavy chains and two light chains:

formulas (I) and (II) , respectively, wherein the two heavy chains are connected by interchain interactions, especially at the _CH2 - _CH3 level, and wherein the VH- _CH1 region of each heavy chain, together with the VL-CL region of each light chain, form a functional Fab or Fab' fragment. In such an embodiment, the two _VH -CH1 _- CH2 _{- CH3} regions, together with the two _VL - _CL regions, form a functional full-length antibody. In such an embodiment, the full-length antibody may comprise a functional Fc region.

V _H is a variable domain of a heavy chain. In one embodiment, V _H is humanized. In one embodiment, V _H is fully human.

V _L is a light chain variable domain. In one embodiment, V _L is humanized. In one embodiment, V _L is fully human.

Typically, V _H and V _L together form an antigen-binding domain. In one embodiment, V _H and V _L form a cognate pair.

"A cognate pair" as used herein refers to a pair of variable domains from a single antibody that have been produced in vivo , i.e., a natural pair of variable domains isolated from a host. Thus, a cognate pair is a pair of V _H and V _L . In one example, a cognate pair binds an antigen together.

"Variable region" or "variable domain" as used herein refers to a region in an antibody chain comprising a CDR and a framework, particularly a suitable framework.

Variable regions for use in the present disclosure are typically obtained from an antibody, which can be produced by any method known in the art.

"Derived from" as used herein refers to the sequence used or a sequence very similar to the sequence used being derived from a starting genetic material, such as a light or heavy chain of an antibody.

“Very similar” as used herein refers to an amino acid sequence that is 95% or more similar over its entire length, such as 96%, 97%, 98% or 99% similar.

Variable regions for use in the present invention as described herein above for V_Hand V_L, may be from any suitable source and may be, for example, fully human or humanized.

In one embodiment, the binding domain formed by V_Hand V_L, is specific to the first antigen.

In one embodiment, the V ₁ binding domain is specific for a second antigen.

In one embodiment, the V ₂ binding domain is specific for a third antigen.

In one embodiment, each of the V _H -V _L , V _{1 ,} and V ₂ that is present separately binds its respective antigen.

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

In one embodiment, the CL moiety in the light chain is a kappa constant sequence or a derivative thereof. In one embodiment, the CL moiety in the light chain is a lambda constant sequence or a derivative thereof.

A derivative of a natural domain within the scope of the present invention refers to a situation where at least one amino acid in the natural sequence has been replaced or deleted, for example to optimize the properties of the domain, for example by eliminating undesirable properties, but the distinguishing feature(s) of the domain is/are retained. In one embodiment, the derivative of a natural domain comprises two, three, four, five, six, seven, eight, ten, eleven or twelve amino acid substitutions or deletions compared to the natural sequence.

In one embodiment, the functional Fab or Fab' fragment has one or more natural or engineered interchain (i.e., between the light and heavy chains) disulfide bonds.

In one embodiment, a "natural" disulfide bond is present between CH ₁ and _CL in the polypeptide chains of formula (I) and (II) .

When the C _L domain is derived from either Kappa or Lambda, the natural position for the linking cysteine is 214 in human cKappa and cLambda (numbering according to Kabat ^4th edition 1987).

The exact location of the disulfide bond-forming cysteine in CH ₁ depends on the specific domain actually used. Thus, for example, in human gamma-1, the natural location of the disulfide bond is at position 233 (numbering according to Kabat ^4th edition 1987). The position of the bond-forming cysteine is known for other human isotypes such as gamma 2, 3, 4, IgM and IgD, e.g. position 127 for human IgM, IgE, IgG2, IgG3, IgG4 and 128 of the heavy chain of human IgD and IgA2B.

Optionally, there may be a disulfide bond between V _H and V _L of the polypeptides of formula I and II.

In one embodiment, a multispecific antibody of the disclosure has a disulfide bond at a position equivalent to or corresponding to the natural position between CH ₁ and _CL .

In one embodiment, the constant region containing CH ₁ and the constant region such as _CL have a disulfide bond that is in a non-natural position. This can be engineered into the molecule by introducing cysteine(s) into the amino acid chain at the desired position or positions. This non-natural disulfide bond is in addition to or as an alternative to the natural disulfide bond present between CH ₁ and _CL . The cysteine(s) in the natural position can be replaced by an amino acid such as serine, which is incapable of forming a disulfide bridge.

The introduction of engineered cysteines can be accomplished using any method known in the art. These methods include, but are not limited to, PCR overlap extension mutagenesis, site-directed mutagenesis, or cassette mutagenesis (see, in general, Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, 1989; Ausbel et al., Current Protocols in Molecular Biology, Greene Publishing & Wiley-Interscience, NY, 1993). Site-directed mutagenesis kits are commercially available, such as the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Cassette mutagenesis can be performed based on Wells et al., 1985, Gene, 34:315-323. Alternatively, mutants can be generated by total gene synthesis by annealing, ligation and PCR amplification and cloning of overlapping oligonucleotides.

In one embodiment, the disulfide bond between CH ₁ and _CL is completely absent, for example, the interchain cysteines may be replaced by another amino acid, such as serine. Thus, in one embodiment, the functional fragment of the Fab molecule does not have interchain disulfide bonds. Disclosures such as WO2005/003170, incorporated herein by reference, describe how to provide Fab fragments without an interchain disulfide bond.

Examples of antibody formats for use in the present invention include complemented IgG and complemented Fab, wherein the entire IgG fragment or Fab, respectively, is constructed by adding at least one additional antigen-binding domain (e.g. one, two, three or four additional antigen-binding domains), e.g. a single-domain antibody (such as V _H or V _L , or VHH), scFv, dsscFv, dsFv to the N- and/or C-terminus of the light chain of said IgG or Fab and optionally to the heavy chain of said IgG or Fab, e.g. as described in WO2009/040562, WO2010035012, WO2011/030107, WO2011/061492, WO2011/061246 and WO2011/086091, all of which are incorporated herein by reference. complemented IgG, including full-length IgG constructed by adding a dsFv to the C-terminus of the light chain (and optionally to the heavy chain) of IgG, was first disclosed in WO2015/197789, incorporated herein by reference.

A preferred antibody format for use in the present invention comprises a Fab linked to two scFvs or dsscFvs, wherein each scFv or dsscFv binds the same or different targets (e.g., one scFv or dsscFv binds a therapeutic target, and one scFv or dsscFv that extends half-life by binding, for example, albumin). Such antibody fragments are described in International Patent Application Publication No. WO2015/197772, which is incorporated herein by reference in its entirety, particularly with respect to the discussion of antibody fragments.

V ₁ represents dsscFv, dsFv or scFv.

V ₂ represents dsscFv, dsFv or scFv.

In one embodiment, when V ₁ and/or V ₂ are dsFv or dsscFv, the disulfide bond between the V _H and V _L variable domains of V ₁ and/or V ₂ is between the two residues listed below (unless the context otherwise requires, the numbering in the list below is according to Kabat). Whenever reference is made to the numbering according to Kabat, the corresponding reference is Kabat et al. 1991 (5th edition, Bethesda, Md.) in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.

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

VH37+VL95C see, for example, Protein Scienc 6, 781-788 Zhu et al (1997);

VH44+VL100 see, for example; Weatherill et al. Protein Engineering, Design & Selection, 25 (321-329) 2012);

VH44+VL105 see, for example, J Biochem. 118, 825-831 Luo et al (1995);

VH45+VL87 see, for example, Protein Scienc 6, 781-788 Zhu et al (1997);

VH55+VL101 see, for example, FEBS Letters 377 135-139 Young et al (1995);

VH100+VL50 see, for example, Biochemistry 29 1362–1367 Glockshuber et al (1990);

VH100b+VL49; see, for example, Biochemistry 29 1362–1367 Glockshuber et al (1990);

VH98+VL 46; see, for example, Protein Scienc 6, 781-788 Zhu et al (1997);

VH101+VL46; see, for example, Protein Scienc 6, 781-788 Zhu et al (1997);

VH105+VL43 see, for example; Proc. Natl. Acad. Sci. USA Vol. 90 pp,7538-7542 Brinkmann et al (1993); or Proteins 19, 35-47 Jung et al (1994)

VH106+VL57 see, for example, FEBS Letters 377 135-139 Young et al (1995),

and the corresponding position in the pair of variable regions located in the molecule.

In one embodiment, a disulfide bond is formed between positions V _H 44 and V _L 100.

The pairs of amino acids listed above are in positions favorable for substitution with cysteines so that disulfide bonds can be formed. Cysteines can be engineered in these desired positions using known methods. In one embodiment, therefore, an engineered cysteine according to the present disclosure refers to a situation where a natural residue at a given amino acid position has been replaced with a cysteine residue.

The introduction of engineered cysteines can be accomplished using any method known in the art. These methods include, but are not limited to, PCR overlap extension mutagenesis, site-directed mutagenesis, or cassette mutagenesis (see, in general, Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, 1989; Ausbel et al., Current Protocols in Molecular Biology, Greene Publishing & Wiley-Interscience, NY, 1993). Site-directed mutagenesis kits are commercially available, such as the QuikChange® Site-Directed Mutagenesis Kit (Stratagen, La Jolla, CA). Cassette mutagenesis can be performed based on Wells et al., 1985, Gene, 34:315-323. Alternatively, mutants can be generated by total gene synthesis by annealing, ligation and PCR amplification and cloning of overlapping oligonucleotides.

Accordingly, in one embodiment, when _V1 and/or _V2 are a dsFv or dsscFv, the _VH and _VL variable domains of _V1 and/or the _VH and _VL variable domains of _V2 may be linked by a disulfide bond between two cysteine residues, wherein the position of the pair of cysteine residues is selected from the group consisting of: _VH37 and _VL95 , _VH44 and _VL100 , _VH44 and _VL105 , _VH45 and _VL87 , _VH100 and _VL50 , _VH100b and _VL49 , _VH98 and _VL46 , _VH101 and _VL46 , _VH105 and _VL43 , and _VH106 and _VL57 .

In one embodiment, when V₁and/or V₂are dsFv or dsscFv, variable domains V_Hand V_Lfrom V₁ and/or variable domains V_Hand V_Lfrom V₂may be are linked by a disulfide bond between two cysteine residues, one in V_Hand one in V_L, which are located outside the CDR, where the position of the pair of cysteine residues is selected from the group consisting of V_H37 and V_L95, V_H44 and V_L100, V_H44 and V_L105, V_H45 and V_L87, V_H100 and V_L50, V_H98 and V_L46, V_H105 and V_L43 and V_H106 and V_L57.

In one embodiment, when V ₁ is a dsFv or dsscFv, the V _H and V _L variable domains of V ₁ are linked by a disulfide bond between two engineered cysteine residues, one at V _H position 44 and the other at V _L 100. In one embodiment, when V ₂ is a dsFv or dsscFv, the V _H and V _L variable domains of V ₂ are linked by a disulfide bond between two engineered cysteine residues, one at V _H position 44 and the other at V _L 100.

In one embodiment, when V ₁ is a dsscFv, dsFv or scFv, the VH domain of V1 is linked to X.

In one embodiment, when V ₁ is a dsscFv, dsFv or scFv, the V _L domain of V ₁ is linked to X.

In one embodiment, when V ₂ is a dsscFv, dsFv or scFv, the VH domain of V ₂ is linked to Y.

In one embodiment, when V ₂ is a dsscFv, dsFv or scFv, the V _L domain of V ₂ is linked to Y.

It will be appreciated by those skilled in the art that when V ₁ and/or V ₂ is a dsFv, the multispecific antibody will comprise a third polypeptide encoding the corresponding free V _H or V _L domain that is not attached to an X or Y. When V ₁ and V ₂ are dsFv, the "free variable domain" (i.e., the domain linked via a disulfide bond to the rest of the polypeptide) will be common to both chains. Thus, while the actual variable domain fused or linked via an X or Y to the polypeptide may differ in each polypeptide chain, the free variable domains linked thereto will generally be identical to each other.

In some embodiments, p is 1. In some embodiments, p is 0.

In some embodiments, s is 1. In some embodiments, s is 0.

In some embodiments, t is 1. In some embodiments, t is 0.

In some embodiments, s is 1 and t is 1. In some embodiments, s is 0 and t is 0.

In one embodiment, p is 1, q is 1, r is 0, s is 0, and t is 0, wherein both V ₁ and V ₂ are dsscFv. Thus, in one aspect, there is provided a multispecific antibody that binds human IL-13, human IL-17A and/or human IL-17F, comprising or consisting of:

a) a polypeptide chain of formula ( Ia ):

VH- CH ₁ -X-V1; and

b) polypeptide chain of formula ( IIa ):

VL-CL-Y-V2 ;

Where:

VH represents the variable domain of the heavy chain;

CH ₁ represents domain 1 of the heavy chain constant region;

X represents a bond or linker;

Y represents a bond or linker;

V1 represents scFv, dsscFv or dsFv;

VL represents the variable domain of the light chain;

CL represents a domain from a light chain constant region such as Ccappa;

V2 represents scFv, dsscFv or dsFv;

wherein at least one of V ₁ or V ₂ is dsscFv or dsFv;

wherein the polypeptide chain of formula (Ia) comprises a protein A binding domain; and

in this case, the polypeptide chain of formula (IIa) does not bind protein A.

In such an embodiment, V ₂ does not bind Protein A, i.e., the scFv, dsscFv or dsFv of V ₂ does not comprise a Protein A binding domain. In one embodiment, V ₂ , i.e., the scFv, dsscFv or dsFv of V ₂ , comprises a VH1 domain. In another embodiment, V ₂ , i.e., the scFv, dsscFv or dsFv of V ₂ , comprises a VH3 domain that does not bind Protein A. In one embodiment, V ₂ , i.e., the scFv, dsscFv or dsFv of V ₂ , comprises a VH2 domain. In one embodiment, V ₂ , i.e., the scFv, dsscFv or dsFv of V ₂ , comprises a VH4 domain. In one embodiment, V ₂ , i.e., the scFv, dsscFv, or dsFv of V ₂ , comprises a VH5 domain. In one embodiment, V ₂ , i.e., the scFv, dsscFv, or dsFv of V ₂ , comprises a VH6 domain. In one embodiment, the polypeptide chain of formula (Ia) comprises only one protein A binding domain present in V _H or V ₁ . In one embodiment, the polypeptide chain of formula (Ia) comprises only one protein A binding domain present in V ₁ . In another embodiment, the polypeptide chain of formula (Ia) comprises two protein A binding domains present in V H and V ₁ , respectively.

In another embodiment, p is 0, q is 1, r is 0, s is 1, t is 1, and V2 is dsscFv. Thus, in one aspect, there is provided a multispecific antibody that binds human IL-13, human IL-17A and/or human IL-17F and comprises or consists of:

a) polypeptide chain of formula ( Ib ):

VH- CH ₁ - CH ₂ - CH ₃ ; and

b) polypeptide chain of formula ( IIb ):

VL-CL-Y-V2 ;

Where:

VH represents the variable domain of the heavy chain;

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 linker;

VL represents the variable domain of the light chain;

CL represents a domain from a light chain constant region such as Ccappa;

V2 represents dsscFv;

wherein the polypeptide chain of formula (Ib) comprises a protein A binding domain; and

in this case, the polypeptide chain of formula (IIb) does not bind protein A.

In such an embodiment, V ₂ does not bind protein A, i.e., the dsscFv of V ₂ does not comprise a protein A binding domain. In one embodiment, V ₂ , i.e., the dsscFv of V ₂ , comprises a VH1 domain. In another embodiment, V ₂ , i.e., the dsscFv of V ₂ , comprises a VH3 domain that does not bind protein A. In one embodiment, the polypeptide chain of formula (Ib) comprises only one protein A binding domain, present in V _H or CH ₂ -CH ₃ . In another embodiment, the polypeptide chain of formula (Ib) comprises two protein A binding domains, present in VH and CH ₂ -CH ₃ , respectively.

In another embodiment, p is 0, q is 1, r is 0, s is 1, t is 1, and V2 is a dsFv. Thus, in one aspect, there is provided a multispecific antibody that binds human IL-13, human IL-17A and/or human IL-17F, comprising or consisting of:

a) polypeptide chain of formula ( Ic ):

VH- CH ₁ - CH ₂ - CH ₃ ; and

b) polypeptide chain of formula ( IIc ):

VL-CL-Y-V2 ;

Where:

VH represents the variable domain of the heavy chain;

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 linker;

VL represents the variable domain of the light chain;

CL represents a domain from a light chain constant region such as Ccappa;

V2 represents dsFv;

wherein the polypeptide chain of formula (Ic) comprises a protein A binding domain; and

in this case, the polypeptide chain of formula (IIc) does not bind protein A.

In such an embodiment, V ₂ , i.e., the dsFv of V ₂ , does not bind protein A. In one embodiment, the polypeptide chain of formula (Ic) comprises only one protein A binding domain, present in V _H or CH ₂ -CH ₃ . In another embodiment, the polypeptide chain of formula (Ic) comprises two protein A binding domains, present in V _H and CH ₂ -CH ₃ , respectively.

In one embodiment of the multispecific antibody of the invention

V _L and V _H contain an antigen-binding region that binds to human IL-17A and/or human IL-17F,

V ₁ contains an antigen-binding site that binds to human serum albumin, and

V ₂ contains an antigen-binding site that binds to human IL-13.

In one embodiment, V _L comprises the sequence of SEQ ID NO:1 for CDR-L1, the sequence of SEQ ID NO:2 for CDR-L2, and the sequence of SEQ ID NO:3 for CDR-L3; V _H comprises the sequence of SEQ ID NO:4 for CDR-H1, the sequence of SEQ ID NO:5 for CDR-H2, and the sequence of SEQ ID NO:6 for CDR-H3.

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

In one embodiment, V ₂ comprises a light chain variable region comprising the sequence of SEQ ID NO:15 for CDR-L1, the sequence of SEQ ID NO:16 for CDR-L2, and the sequence of SEQ ID NO:17 for CDR-L3; and a heavy chain variable region comprising the sequence of SEQ ID NO:18 for CDR-H1, the sequence of SEQ ID NO:19 for CDR-H2, and the sequence of SEQ ID NO:20 for CDR-H3;

In one embodiment, V _L comprises the sequence of SEQ ID NO:7 and VH comprises the sequence of SEQ ID NO:9.

In one embodiment, V ₁ comprises a light chain variable region comprising the sequence of SEQ ID NO:45 and a heavy chain variable region comprising the sequence of SEQ ID NO:46.

In one embodiment, V ₁ comprises a light chain variable region comprising the sequence of SEQ ID NO:49 and a heavy chain variable region comprising the sequence of SEQ ID NO:50.

In one embodiment, the light chain variable region and the heavy chain variable region V ₁ are connected by a linker, wherein said linker comprises the sequence of SEQ ID NO:68.

In one embodiment, V ₁ is an scFv comprising the sequence of SEQ ID NO:53 or a dsscFv comprising the sequence of SEQ ID NO:55.

In one embodiment, V ₂ comprises a light chain variable region comprising the sequence of SEQ ID NO:27 and a heavy chain variable region comprising the sequence of SEQ ID NO:28.

In one embodiment, V ₂ comprises a light chain variable region comprising the sequence of SEQ ID NO:31 and a heavy chain variable region comprising the sequence of SEQ ID NO:32.

In one embodiment, the light chain variable region and the heavy chain variable region V ₂ are connected by a linker, wherein said linker comprises the sequence of SEQ ID NO:66.

In one embodiment, V ₂ is an scFv comprising the sequence of SEQ ID NO:35 or a dsscFv comprising the sequence of SEQ ID NO:37.

In one embodiment, X is a linker comprising the sequence of SEQ ID NO:67.

In one embodiment, Y is a linker comprising the sequence SEQ ID NO:65.

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

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

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

It should be understood that one or more amino acid substitutions, additions and/or deletions can be made in the sequences provided in the present invention without significantly changing the ability of the antibody to bind to an antigen and neutralize its biological activity. The effect of any amino acid substitutions, additions and/or deletions can be readily tested by one skilled in the art, for example, using the methods described herein, in particular the methods shown in the examples, to determine antigen binding and inhibition of biological activity.

Accordingly, the present invention provides a multispecific antibody comprising CDRs with the sequences set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 15, 16, 17, 18, 19, 20, 39, 40, 41, 42, 43 and 44, in which one or more amino acids in one or more CDRs have been replaced with another amino acid, such as an analogous amino acid as defined herein below.

"Identity" according to the present invention means that at any specified position in the aligned sequences, the amino acid residue is identical in the sequences. "Similarity" according to the present invention means that at any specified position in the aligned sequences, the amino acid residue is of a similar type in the sequences. For example, leucine can be replaced by isoleucine or valine. Other amino acids that can often be replaced by each other include, but are not limited to:

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

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

- aspartate and glutamate (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, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, BLAST™ software supplied by NCBI (Altschul, S.F. et al., 1990, J. Mol. Biol. 215:403-410; Gish, W. & States, D.J. 1993, Nature Genet. 3:266-272. Madden, T.L. et al., 1996, Meth. Enzymol. 266:131-141; Altschul, S.F. et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, T.L. 1997, Genome Res. 7:649-656).

In one embodiment, the CDRs of the multispecific antibody comprise sequences that have at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequences set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 15, 16, 17, 18, 19, 20, 39, 40, 41, 42, 43, and 44.

In one embodiment, V _L comprises a sequence that has 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 that has at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence set forth in SEQ ID NO:9.

In one embodiment, V ₁ comprises a light chain variable region comprising a sequence that has 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 that has at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence set forth in SEQ ID NO:46.

In one embodiment, V ₁ comprises a light chain variable region comprising a sequence that has 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 that has at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence set forth in SEQ ID NO:50.

In one embodiment, the light chain variable region and the heavy chain variable region V ₁ are connected by a linker, wherein said linker comprises a sequence that has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:68.

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

In one embodiment, V ₂ comprises a light chain variable region comprising a sequence that has 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 that has at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence set forth in SEQ ID NO:28.

In one embodiment, V ₂ comprises a light chain variable region comprising a sequence that has 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 that has at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence set forth in SEQ ID NO:32.

In one embodiment, the light chain variable region and the heavy chain variable region V ₂ are connected by a linker, wherein said linker comprises a sequence that has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:66.

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

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

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

In one embodiment, the polypeptide chain of formula (Ia) comprises a sequence that has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:57 or SEQ ID NO:59.

In one embodiment, the polypeptide chain of formula (IIa) comprises a sequence that has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:61 or SEQ ID NO:63.

In one embodiment, the polypeptide chain of formula (Ia) comprises a sequence that has 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 that has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence set forth in SEQ ID NO:63.

Epitope

An epitope is a region of an antigen that an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and have those 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 contain determinants that are chemically active surface moieties of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments can have specific three-dimensional structural characteristics and/or specific charge characteristics.

It is easy to determine whether an antibody binds to the same epitope as a reference antibody or competes for binding with it using standard methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody of the invention, the reference antibody is allowed to bind to a protein or peptide under saturating conditions. The ability of the test antibody to bind to the protein or peptide is then assessed. If the test antibody is able to bind to the protein or peptide after binding to the reference antibody at saturation, 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 is not able to bind to the protein or peptide after binding to the reference antibody at saturation, the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the invention.

To determine whether an antibody competes for binding with a reference antibody, the above-described binding method is performed in two orientations. In the first orientation, the reference antibody is allowed to bind to the protein/peptide under saturating conditions, followed by an assessment of the binding of the test antibody to the protein/peptide molecule. In the second orientation, the test antibody is allowed to bind to the protein/peptide under saturating conditions, followed by an assessment of the binding of the reference antibody to the protein/peptide. If in both orientations only the first (saturated) antibody is able to bind to the protein/peptide, then it is concluded that the test antibody and the reference antibody compete for binding to the protein/peptide. As will be understood by one of skill in the art, an antibody that competes for binding with the reference antibody may not necessarily bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

Two antibodies bind to the same or an overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold increase in one antibody inhibits binding of the other by at least 50%, 75%, 90%, or even 99% when 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 substantially all amino acid mutations in the antigen that reduce or abolish binding of one antibody reduce or abolish binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or abolish binding of one antibody reduce or abolish binding of the other.

Further standard experiments (e.g., mutation and peptide binding assays) 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 lack of observed binding. Experiments of this type can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

The antibodies can compete for binding to IL-17A or IL-17F or bind to the same epitope as a multispecific antibody that comprises a combination of the CDR-L1/CDR-L2/CDR-L3/CDR-H1/CDR-H2/CDR-H3 sequences of SEQ ID NO: 1/2/3/4/5/6.

The antibodies can compete for binding to IL-13 or bind to the same epitope as a multispecific antibody that comprises a combination of the CDR-L1/CDR-L2/CDR-L3/CDR-H1/CDR-H2/CDR-H3 sequences of SEQ ID NO: 15/16/17/18/19/20.

The antibodies can compete for binding to serum albumin or bind to the same epitope as a multispecific antibody that comprises a combination of the CDR-L1/CDR-L2/CDR-L3/CDR-H1/CDR-H2/CDR-H3 sequences of SEQ ID NO: 39/40/41/42/43/44.

Effector molecules

If desired, a multispecific antibody for use in the present invention may be conjugated to one or more effector molecules. It will be understood that an effector molecule may comprise a single effector molecule or two or more such molecules linked to form a single fragment that can bind to the antibodies of the present invention. When it is desired to obtain an antibody fragment linked to an effector molecule, it may be obtained by standard chemical or recombinant DNA techniques in which the antibody fragment is linked either directly or via a linking agent to the effector molecule. Methods for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Particular chemical methods 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, the linkage may be provided using recombinant DNA techniques, for example, those described in WO 86/01533 and EP 0392745.

The term effector molecule according to the present invention includes, for example, antitumor agents, drugs, toxins, biologically active proteins, such as enzymes, other antibodies or antibody fragments, synthetic or natural polymers, nucleic acids and fragments thereof, such as DNA, RNA and fragments thereof, radionuclides, especially radioactive iodine, radioisotopes, chelating metals, nanoparticles and reporter groups, such as fluorescent compounds or compounds that can be detected by NMR or EPR spectroscopy.

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

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, thioepa chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines ( e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics ( e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicins or duocarmycins) and antimitotic agents ( for example, vincristine and vinblastine).

Other effector molecules may include chelated radionuclides such as ¹¹¹ In and ⁹⁰ Y, Lu ¹⁷⁷ , bismuth ²¹³ , californium ²⁵² , iridium ¹⁹² , and tungsten ¹⁸⁸ /rhenium ¹⁸⁸ ; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids, and suramin.

Other effector molecules include proteins, peptides, and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, and 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, a protein such as insulin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor or tissue plasminogen activator, a thrombotic agent or antiangiogenic agent such as angiostatin or endostatin, or a biological response modifier such as a lymphokine, 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 another growth factor and immunoglobulins.

Other effector molecules may include detectable substances useful, for example, for diagnostics. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron-emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. See generally U.S. Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-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; and Suitable radioactive nuclides include ¹²⁵ I, ¹³¹ I, ¹¹¹ In, and ⁹⁹ Tc.

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 the delivery of the antibody across the epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins or albumin binding compounds, such as those described in WO 05/117984.

When the effector molecule is a polymer, it may typically be a synthetic or natural polymer, such as an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer, or a branched or unbranched polysaccharide, such as a homo- or heteropolysaccharide.

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

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

Specific natural polymers include lactose, amylose, dextran, glycogen or their derivatives.

The term "derivatives" according to the present invention includes reactive derivatives, for example, thiol-selective reactive groups such as maleimides and the like. The reactive group can be linked directly or through a linker segment to the polymer. It is understood that the remainder of such a group in some cases forms part of the product as a linking group between the antibody fragment and the polymer.

The polymer size can be varied as desired, but is typically in the range of an average molecular weight of 500 to 50,000 Da, such as 5,000 to 40,000 Da, such as 20,000 to 40,000 Da. The polymer size can, in particular, be selected based on the intended use of the product, such as the ability to localize to specific tissues such as tumors, or to increase the half-life in the bloodstream (for a review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, when the product is intended to leave the bloodstream and penetrate into tissues, it may be preferable to use a polymer with a low molecular weight, such as one with a molecular weight of about 5,000 Da. For applications where the product remains in the bloodstream, it may be preferable to use a polymer with a higher molecular weight, such as one having a molecular weight in the range of 20,000 Da to 40,000 Da.

Suitable polymers include a polyalkylene polymer such as poly(ethylene glycol) or especially methoxypoly(ethylene glycol) or a derivative thereof and especially having a molecular weight in the range of from about 15,000 Da to about 40,000 Da.

In one example, antibodies for use in the present invention are attached to polyethylene glycol (PEG) molecules. In one specific example, the antibody is an antibody fragment, and the PEG molecules 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 can be naturally occurring in the antibody fragment or can be introduced into the fragment using recombinant DNA techniques (see, for example, US 5,219,996; US 5,667,425; WO 98/25971). In one example, an antibody molecule of the present invention is a modified Fab fragment, wherein the modification is the addition to the C-terminus of its heavy chain of one or more amino acids to allow attachment of an effector molecule. Accordingly, the additional amino acids form a modified hinge region containing 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.

Suitably, the PEG molecules can be covalently linked 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 linked to a sulfur atom of a cysteine residue located in the fragment. The covalent bond is typically a disulfide bond or, in particular, a sulfur-carbon bond. If a thiol group is used as the point of attachment, suitably activated effector molecules can be used, for example, thiol-selective derivatives such as maleimides and cysteine derivatives. The activated polymer can be used as a starting material in the preparation of polymer-modified antibody fragments as described above. The activated polymer can be any polymer containing a reactive thiol group, such as an α-halocarboxylic acid or ester, for example, iodoacetamide, an imide, for example, maleimide, vinyl sulfone or a disulfide. Such starting materials may be obtained commercially (e.g., from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or may be prepared from commercially available starting materials using conventional chemical procedures. Specific PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly Shearwater; Rapp Polymere and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

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

In one embodiment, the multispecific antibody is not attached to an effector molecule.

Polynucleotides/Vectors/Host cells

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

A variant polynucleotide 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 provided in the sequence listing. Typically, a variant has 1-20, 1-50, 1-75 or 1-100 substitutions and/or deletions.

Suitable variants may be at least about 70% homologous to a polynucleotide of any of the nucleic acid sequences disclosed herein, typically at least about 80 or 90% homologous thereto, and more preferably at least about 95%, 97% or 99%. Variants may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Variants typically retain 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 are typically present at least with respect to the coding regions of the polynucleotides. Methods for measuring homology are well known in the art, and those skilled in the art will appreciate 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, such as at least about 40, 60, 100, 200 or more contiguous nucleotides (depending on length). Such homology may exist over the entire length of an unmodified polynucleotide sequence.

A homolog may differ from the sequence of the corresponding 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 homolog may differ by 3-50 mutations, often by 3-20 mutations. These mutations can be measured over a region of at least 30, such as at least about 40, 60 or 100 or more contiguous nucleotides of the homolog.

The DNA sequence according to the present invention may comprise synthetic DNA, such as that obtained by chemical treatment, cDNA, genomic DNA, or any combination thereof.

General methods by which vectors can be constructed, transfection methods, and culturing methods are well known to those skilled in the art. Reference is made in this regard to Current Protocols in Molecular Biology, 1999, F. M. Ausubel (ed), Wiley Interscience, New York, and the Maniatis Manual, published by Cold Spring Harbor Publishing.

Also provided is a host cell comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding the IL-13/IL-17AF multispecific antibody. Any suitable host cell/vector system can be used to express 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.

The present 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 expressing a protein from DNA encoding the IL-13/IL-17AF multispecific antibody, and isolating the IL-13/IL-17AF multispecific antibody.

Obtaining multispecific antibodies

There are a number of approaches for producing multispecific, 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, such as IgG. Schoonjans et al . 2000, JouRNAl of Immunology, 165, 7050-7057 describe the fusion of scFv to Fab fragments of an antibody. WO2015/197772 describes the fusion of disulfide-stabilized scFv (dsscFv) to Fab fragments.

Standard approaches described in the prior art involve the expression in a host cell of at least two polypeptides, each encoding a heavy chain (HC) or a light chain (LC) of a complete antibody or an antigen-binding fragment thereof, such as a Fab, to which an additional antigen-binding fragment of the antibody may be fused at the N- and/or C-terminal position of the heavy chain and/or light chain. When attempting to recombinantly produce such multispecific antibodies by expressing two (one light chain and one heavy chain to form a complemented Fab) or four polypeptides (two light chains and two heavy chains to form a complemented IgG), overexpression of the light chain is typically required compared to the heavy chain to ensure proper folding of the heavy chain when assembled with the corresponding light chain. In particular, CH ₁ (domain 1 of the heavy chain constant region) itself is prevented from folding by BIP proteins, which can be displaced by the corresponding LC; therefore, the correct folding of CH ₁ /HC depends on the accessibility of its corresponding LC (Lee et al. 1999, Molecular Biology of the Cell, Vol. 10, 2209-2219).

The authors observed that these methods of expressing multispecific antibodies can result in an excess of light chain relative to heavy chain, which persists during harvesting of host cells, and that the excess light chain tends to form dimeric complexes (or "LC dimers"), which are present as a by-product of the processing of the desired multispecific antibody, especially monomeric ones, and thus require purification.

Importantly, the technical problem associated with the formation of light chain dimers upon fusion at the N- and/or C-terminus with additional antigen-binding fragment(s) has not yet been identified, and conventional analytical methods lack the ability to detect and quantify these complemented LC dimers among heterogeneous process products. This may lead to significant bias in quantifying products using conventional analytical methods.

Thus, there is a need for improved multispecific antibodies and methods for their production that allow for easy and efficient isolation and removal of complemented LC dimers at the earliest stages of the technological process and, thus, increase the yield of the protein of interest for use in therapy, which is a multispecific antibody, in particular in its monomeric form.

The multispecific antibodies of the present invention were designed to provide improved multispecific antibodies with equivalent functionality and stability while increasing the yield of "multispecific antibody" material, especially monomeric, obtained after purification, especially after a single-step purification involving protein A affinity chromatography.

Preferably, the multispecific antibodies of the present disclosure can be purified more efficiently by a purification method that is superior to the methods generally adopted in the prior art, particularly in that the improved method involves fewer steps and is cost and time effective on an industrial scale. In particular, the multispecific antibodies of the present disclosure yield the maximum amount of proteins of interest (i.e., the correct format of the multispecific antibody) obtained after a one-step purification method comprising protein A affinity chromatography, wherein the purification of the multispecific antibodies of interest and the removal of complemented LC dimers occur simultaneously. Preferably, the methods for producing and purifying multispecific antibodies of the present disclosure do not require an additional purification step to capture excess free, unbound light chains, especially complemented LC dimers.

Protein A

Protein A is a 42 kDa surface protein originally discovered in the cell wall of Staphylococcus aureus bacteria. Protein A is widely used for the detection, quantification and purification of immunoglobulins. Protein A has been reported to bind the Fab portion of VH3 family antibodies and the Fc gamma region in a portion of the IgG constant region (between the _CH2 and _CH3 domains). The crystal structure of a 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 the present invention, protein A includes native protein A and any variant or derivative thereof, as long as the variant or derivative of protein A retains its ability to bind VH3 domains and/or Fc gamma domains.

The polypeptide chain of formula (I) according to the present invention comprises a protein A binding domain. In one embodiment, the polypeptide chain of formula (I) comprises one, two or three protein A binding domains.

"Protein A binding domain" as used herein refers to a binding domain that specifically binds to protein A. The protein A binding domain may refer to a VH3 domain or a portion of a VH3 domain that binds protein A, i.e., which comprises the protein A binding interface. The portion of a VH3 domain that binds protein A does not comprise a CDR of a VH3 domain, i.e., the VH3 protein A binding interface does not include a CDR; therefore, it will be understood that the protein A binding domain does not compete with the antigen binding domain disclosed herein.

In one embodiment, the polypeptide chain of formula (I) comprises a protein A binding domain that is present in V _H and/or CH ₂ -CH ₃ and/or V ₁ . In one embodiment, the polypeptide chain of formula (I) comprises one, two or three protein A binding domains that are/are present in V _H and/or CH ₂ -CH ₃ and/or V1. In one embodiment, the polypeptide chain of formula (I) comprises only one protein A binding domain that is present in V _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 that is present in V _H or V ₁ . In one embodiment, the polypeptide chain of formula (I) comprises only one protein A binding domain that is present in V _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 that is present in V _H . In one embodiment, the polypeptide chain of formula (I) comprises only one protein A binding domain that is present in V 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 that is present in V ₁ .

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 that are present in V _H and CH ₂ -CH ₃ , respectively. In another embodiment, the polypeptide chain of formula (I) comprises two protein A binding domains that are present in V _H and V ₁ , respectively. In another embodiment, the polypeptide chain of formula (I) comprises two protein A binding domains that are present in CH ₂ -CH ₃ and V ₁ , respectively.

In one embodiment, the polypeptide chain of formula (I) comprises three protein A binding domains, each of which is present at V _H , CH ₂ -CH ₃ and V ₁ .

Natural protein A can interact, in particular, with the Fc gamma region, in part of the constant region of IgG. More particularly, protein A can interact with the binding domain between CH ₂ and CH _3. In one embodiment, when s is 1, t is 1, and CH ₂ and CH ₃ are natural domains of the IgG class.

In some embodiments, the protein A binding domain(s) comprises or consists of a VH3 domain or variant thereof that binds protein A. In some embodiments, the protein A binding domain(s) comprises or consists of a natural VH3 domain. In some embodiments, the variant VH3 domain that binds protein A is a variant of a natural VH3 domain, wherein said natural VH3 domain is incapable of binding protein A.

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

In some embodiments, V ₂ comprises or consists of VH1 and/or VH2 and/or VH4 and/or VH5 and/or VH6 and does not comprise a VH3 domain. In some embodiments, V ₂ comprises or consists of a VH3 domain or a variant thereof that does not bind protein A. In some embodiments, V ₂ comprises or consists of a natural VH3 domain that is incapable of binding protein A. In some embodiments, a variant VH3 domain that does not bind protein is a variant of a natural VH3, wherein said natural VH3 domain is capable of binding protein A.

Human germline VH3 genes and VH3 domains (or scaffolds) are well characterized. Many natural VH3 domains have protein A binding capacity, but certain natural VH3 domains lack protein A binding capacity (see Roben et al. 1995, J Immunol.;154(12):6437-6445).

The VH3 domain for use in the present invention may be obtained in several ways. In one embodiment, the VH3 domain for use in the present invention is a naturally occurring VH3 domain selected for its ability or inability to bind Protein A, depending on its position in the polypeptide (I) and/or (II) of the present invention. For example, a panel of antibodies against an antigen of interest may be obtained by immunizing a non-human animal, then humanized, and the humanized antibodies may 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 chromatography 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 and select antibodies containing a VH3 domain that binds, in particular, via a protein A binding interface that does not contain a CDR or does not bind protein A.

Alternatively, the VH3 domain for use in the present invention is a variant of a natural VH3. In one embodiment, the VH3 variant comprises a sequence of a natural VH3 capable of binding Protein A, and further comprises at least one amino acid mutation that abolishes its ability to bind Protein A. In one embodiment, the VH3 variant that binds Protein A comprises a sequence of a natural VH3 incapable of binding Protein A, and further comprises at least one amino acid mutation. In such an embodiment, the mutation(s) is/are responsible for the acquisition of the ability of the VH3 domain to bind Protein A, i.e., the mutation(s) contribute(s) to the formation of a Protein A binding domain that does not exist in nature.

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 of VH3, numbered according to Kabat and as described in, for example, Graille et al. 2000, PNAS, 97(10): 5399-5404). The mutation may be a substitution, deletion or insertion. In one embodiment, the VH3 variant comprises a substitution at position 15, 17, 19, 57, 59, 64, 65, 66, 68, 70, 81 or 82 with VH3, numbered according to Kabat.

Natural VH1, VH2, VH4, VH5 and VH6 do not bind protein A. In one embodiment, the VH domain that does not bind protein A is VH1. In one embodiment, the VH domain that does not bind protein A is VH2. In one embodiment, the VH domain that does not bind protein A is VH4. In one embodiment, the VH domain that does not bind protein A is VH5. In one embodiment, the VH domain that does not bind protein A is VH6.

Pharmaceutical Compositions, Dosages and Treatment Regimens

The multispecific antibody according to the invention can be presented in a pharmaceutical composition. The pharmaceutical composition is usually sterile and usually contains a pharmaceutically acceptable carrier and/or an excipient. The pharmaceutical composition according to the present invention can further contain a pharmaceutically acceptable excipient and/or a carrier.

According to the present invention, a " pharmaceutically acceptable carrier " includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier may be suitable for parenteral, such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. Alternatively, the carrier may be suitable for non-parenteral administration, such as by topical, epidermal, or transmucosal 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 environmental conditions that may inactivate the compound.

The pharmaceutical compositions of the invention may contain one or more pharmaceutically acceptable salts. " Pharmaceutically acceptable salt " refers to a salt that retains the desired biological activity of the parent compound and does not impart any unnecessary toxicological effect. Examples of such salts include acid addition salts and base addition salts.

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

Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration.

The pharmaceutical composition according to the invention may contain additional active ingredients.

Also included within the scope of the present invention are kits comprising antibodies or modulating agents of the invention and instructions for use. The kit may further comprise one or more additional reagents, such as an additional therapeutic or prophylactic agent discussed above.

The modulators and/or antibodies according to the invention or dosage forms or compositions thereof can be administered for prophylactic and/or therapeutic treatment.

For therapeutic purposes, the compounds are administered to a subject already suffering from a disease or condition as described above in an amount sufficient to cure, alleviate, or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of the 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."

For prophylactic purposes, the dosage forms are administered to a subject at risk of a disease or condition as described above in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms. An amount sufficient to accomplish this is defined as a " prophylactically effective amount ." Effective amounts for each purpose will depend on the severity of the disease or condition and the weight and general condition of the subject.

The subject to be administered may be a human or a non-human animal. The term " non-human animal " includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Typically, administration to humans is considered.

The antibody/modulator or pharmaceutical composition of the invention may be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired results. 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, such as by injection or infusion. The phrase "parenteral administration" according to the present invention means routes of administration other than enteral and topical administration, typically by injection. Alternatively, the antibody/modulator or pharmaceutical composition of the invention may be administered by a non-parenteral route, such as topical, epidermal or transmucosal administration. The antibody/modulator or pharmaceutical composition of the invention may be intended for oral administration.

A suitable dosage of the antibody/modulatory agent or pharmaceutical composition of the present invention can be determined by a skilled practitioner. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present 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 route of administration, without toxicity to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular compositions of the present invention used, the route of administration, the time of administration, the rate of elimination of the particular compound used, the duration of treatment, other drugs, compounds and/or materials used in combination with the particular compositions 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 art.

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

Therapy regimens may be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose may be administered, several divided doses may be administered over time, or the dose may be proportionally decreased or increased depending on the exigencies of the therapeutic situation. The unit dosage form according to the present invention refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Administration may be single or multiple doses. Multiple doses may be administered by the same or different routes and to the same or different sites. Alternatively, doses may be administered as a sustained-release preparation, in which case less frequent administration is required. Dosage and frequency may be adjusted depending on the patient's half-life of the antagonist and the desired duration of treatment.

As mentioned above, the modulators/antibodies or pharmaceutical compositions of the invention can be administered in conjunction with one or more other therapeutic agents.

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

Therapeutic indications

The antibodies of the present invention can be used to treat, prevent or alleviate any condition associated with IL-13 and/or IL-17A and/or IL-17F activity; for example, any condition that is, in whole or in part, a result of signaling through the IL-13, IL-17A and/or IL-17F receptor.

Diseases include primary and metastatic cancers, including carcinoma of the breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder, and urothelium), female genital tract (including cervix, uterus, and ovary, and choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicle, testis, and germ cell tumors), endocrine glands (including thyroid, adrenal, and pituitary), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissue, and Kaposi's sarcoma), and tumors of the brain, nerves, eyes, and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, schwannomas, and meningiomas), solid tumors arising from hematopoietic malignancies such as leukemias and lymphomas (both Hodgkin's and non-Hodgkin's lymphomas), 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, scleroderma dermatitis, graft-versus-host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schönlein purpura, microscopic renal vasculitis, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, septic syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignant neoplasms, heart failure, Addison's disease, sporadic, polyglandular deficiency type I and polyglandular deficiency type II, Schmidt syndrome, acute respiratory distress syndrome in adulthood, alopecia, alopecia areata, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitis arthropathy, enteropathic synovitis, chlamydia, Yersinia and Salmonella arthropathy, atheromatous disease/atherosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune hemolytic anemia, Coombs test-positive hemolytic anemia, acquired pernicious anemia, juvenile pernicious anemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiency diseases, hepatitis B, hepatitis C, unclassifiable variable immunodeficiency (unclassifiable variable hypogammaglobulinemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic pulmonary disease, cryptogenic fibrosing alveolitis, postinflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated with interstitial lung disease, mixed connective tissue disease associated with lung disease, interstitial lung disease associated with systemic sclerosis, interstitial lung disease associated with rheumatoid arthritis, lung disease associated with systemic lupus erythematosus, lung disease associated with dermatomyositis/polymyositis, lung disease associated with Sjogren's disease, lung disease associated with ankylosing spondylitis, diffuse vasculitic pulmonary disease, lung disease associated with hemosiderosis, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, autoimmune hepatitis type 1 (classical autoimmune or lupus hepatitis), autoimmune hepatitis type 2 (hepatitis with LKM antibodies), autoimmune-mediated hypoglycemia, insulin resistance type B with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthritis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leukopenia, autoimmune neutropenia, renal disease not otherwise specified (NOS), glomerulonephritis, microscopic renal vasulitis, Lyme disease, discoid lupus erythematosus, idiopathic male infertility NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestations of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren's 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, Alcoholic liver disease, Cholestasis, Idiosyncratic liver disease, Drug-induced hepatitis, Nonalcoholic steatohepatitis, Allergy, Group B Streptococcus (GBS) infection, Mental disorders, Depression, Schizophrenia, Th2 and Th1-mediated diseases, Acute and chronic pain, Various forms of pain, Cancer, Lung cancer, Breast cancer, Gastric cancer, Bladder cancer, Colon cancer, Pancreatic cancer, Ovarian cancer, Prostate cancer, Rectal cancer, Hematopoietic malignancies, Leukemia, Lymphoma, Abetalipoproteemia, Acrocyanosis, Acute and chronic parasitic or infectious processes, Acute leukemia, Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Acute or chronic bacterial infection, Acute pancreatitis, Acute renal failure, adenocarcinoma, atrial extrasystoles, AIDS-dementia complex, alcoholic hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis (including seasonal allergic rhinitis), nonallergic 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, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B-cell lymphoma, bone graft rejection, graft rejection bone marrow failure (BMT), bundle branch block, Burkitt's lymphoma, burns, cardiac arrhythmias, stunned myocardial syndrome, cardiac tumors, cardiomyopathy, inflammatory reaction to cardiopulmonary bypass, cartilage graft rejection, cerebellar cortical degeneration, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy-related disorders, chronic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory disorders, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, ischemic heart disease, Creutzfeldt-Jakob disease, culture-negative sepsis, cystic fibrosis, cytokine therapy-related disorders, dementia boxer, demyelinating diseases, dengue hemorrhagic fever, dermatitis, dermatologic disorders, diabetes, diabetes mellitus, diabetic atherosclerotic disease, diffuse Lewy body disease, dilated congestive cardiomyopathy, basal ganglia disorder, middle-aged Down syndrome, drug-induced movement disorders due to drugs that block central dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, Epstein-Barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hematophagocytic lymphohistiocytosis, rejection embryonic thymus implant, Friedreich's ataxia, peripheral arterial dysfunction, fungal sepsis, gas gangrene, gastric ulcer, glomerular nephritis, any organ or tissue transplant rejection, gram-negative sepsis, gram-positive sepsis, granulomas due to intracellular microorganisms, hairy cell leukemia, Hallervorden-Spatz disease, Hashimoto's thyroiditis, hay fever, cardiac transplant rejection, hemochromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis A, His bundle branch arrhythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitivity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, assessment hypothalamic-pituitary-adrenal axis, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody-mediated cytotoxicity, asthenia, childhood spinal muscular atrophy, otitis media, influenza A, radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, renal allograft rejection, legionellosis, leishmaniasis, leprosy, corticospinal system disorder, fatty edema, liver allograft rejection, lymphedema, malaria, malignant lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic migraine syndrome, idiopathic migraine, mitochondrial multisystem disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple system degenerations (Menzel, Dejerine-Thomas, Shy-Drager, and Machado-Joseph), Mycobacterium avium intracellulare infection, Mycobacterium tuberculosis infection, myelodysplastic syndrome, myocardial infarction, myocardial ischemic disorders, nasopharyngeal carcinoma, chronic lung disease of newborns, nephritis, nephrosis, neurodegenerative diseases, neurogenic muscular atrophy, neutropenic fever, non-Hodgkin's lymphoma, abdominal aortic occlusion, arterial occlusive lesions, OKT3 therapy, orchitis/epididymitis, orchitis/procedures Vasectomy reversal, organomegaly, osteoporosis, pancreatic allograft rejection, pancreatic carcinoma, paraneoplastic syndrome/malignant hypercalcemia, parathyroid allograft rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, peripheral atherosclerotic disease, peripheral vascular disease, peritonitis, pernicious anemia, pneumocystis pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), postperfusion syndrome, posthemodialysis syndrome, postcardiotomy syndrome after myocardial infarction, preeclampsia, progressive supranuclear palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynaud's disease, Refsum disease, tachycardia with regular narrow QRS complexes, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, senile chorea, senile dementia with Lewy bodies, seronegative arthropathies, shock, sickle cell anemia, cutaneous allograft rejection, skin change syndrome, small bowel graft rejection, solid tumors, specific arrhythmias, spinal ataxia, spinocerebellar degenerations, streptococcal myositis, structural cerebellar lesions, subacute sclerosing panencephalitis, syncope, cardiovascular syphilis, systemic anaphalaxis, systemic inflammatory response syndrome, juvenile idiopathic arthritis of systemic onset, T-cell or FAB ALL telangiectasia, thromboangiitis obliterans, thrombocytopenia, intoxication, transplants, trauma/hemorrhage, 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, viral encephalitis/aseptic meningitis, virus-associated hemophagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, organ or tissue xenograft rejection, acute coronary syndromes, acute idiopathic polyneuritis, acute inflammatory demyelinating polyradiculoneuropathy, acute ischemia, adult-onset Still's disease, anaphylaxis, antiphospholipid antibody syndrome, aplastic anemia, atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmune disease associated with streptococcal infection, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarian failure, blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular disease, catastrophic antiphospholipid syndrome, celiac disease, cervical spondylosis, chronic ischemia, cicatricial pemphigoid, clinically isolated syndrome (cis) with risk of multiple sclerosis, childhood-onset mental disorder, dacryocystitis, dermatomyositis, diabetic retinopathy, intervertebral disc herniation, disc prolapse, drug-induced immune hemolytic anemia, endometriosis, endophthalmitis, episcleritis, erythema multiforme, large erythema multiforme, 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 inflammatory eye disease, inflammatory demyelinating disease, inflammatory heart disease, inflammatory kidney disease, IPF/UIP, iritis, keratitis, keratojunctivitis sicca, Kussmaul disease or Kussmaul-Meyer disease, Landry's palsy, Langerhans cell histiocytosis, livedo reticularis, macular degeneration, microscopic polyangiitis, ankylosing spondylitis, motor neuron disease, mucous membrane pemphigoid, multiple organ failure, myasthenia gravis, myelodysplastic syndrome, myocarditis, nerve root disease, neuropathy, non-A-non-B hepatitis, optic neuritis, osteolysis, polyarticular juvenile rheumatoid arthritis JRA, peripheral arterial occlusive disease (PAOD), peripheral vascular disease (PVD), peripheral arterial disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, poliosis, polyarticular JRA, polyendocrine deficiency syndrome, polymyositis, polymyalgia rheumatica (PMR), primary parkinsonism, prostatitis, pure red cell aplasia, primary adrenal insufficiency, relapsing neuromyelitis optica spectrum disorder, restenosis, rheumatic carditis, sapho (synovitis, acne, pustulosis, hyperostosis, and osteitis), secondary amyloidosis, shock lung, scleritis, radiculitis, secondary adrenal insufficiency, silicone-related connective tissue disease, Sneddon-Wilkinson dermatosis, ankylosing spondylitis, Stevens-Johnson syndrome (SJS), temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumor necrosis factor receptor), allergic reaction type 1, diabetes type 2, urticaria, usual interstitial pneumonia (UIP), vasculitis, vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration, wound healing, aspirin-sensitive asthma, atopic asthma, chronic hand eczema, allergic bronchopulmonary aspergillosis, celiac disease, Churg-Strauss syndrome (periarteritis nodosa plus atopy), eosinophilic myalgia syndrome, hypereosinophilic syndrome, edema reactions including episodic angioedema, helminthiasis, onchocercal dermatitis, eosinophil-associated gastrointestinal diseases, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis, eosinophilic colitis, micropolyposis and nasal polyposis, food allergy, aspirin intolerance and obstructive sleep apnea, chronic asthma, Crohn's disease and endomyocardial fibrosis, cancer (eg, glioblastoma (eg, 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 liver fibrosis.

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

The following examples illustrate the invention.

EXAMPLES

EXAMPLE 1. Production and selection of the therapeutic antibody CA650 against IL-13

Rats were immunized with either purified human IL-13 (Peprotech) or rat fibroblasts expressing human IL-13 (expressing approximately 1 μg/ml in cell culture supernatant), or in some cases a combination of the two. After 3-6 injections, animals were sacrificed and PBMCs, spleen, bone marrow, and lymph nodes were collected. Serum binding to human IL-13 in an ELISA and the ability to neutralize hIL-13 in a HEK-293 IL-13R-STAT-6 reporter cell assay (HEK-Blue assay, Invivogen) were monitored.

B cell cultures were prepared 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 the detection agent. Positive results from this assay were then carried over to a HEK-293 IL-13R-STAT-6 reporter cell assay (HEK-Blue assay, Invivogen) to identify neutralizers. Neutralizing supernatants were then analyzed in Biacore to assess the extent of dissociation as well as to characterize the mechanism of action of neutralization. Neutralization was classified as either Group 1 or Group 2. Group 1 is an antibody that binds to human IL-13 and prevents IL-13Rα1 binding, and as a result also blocks IL-4R binding. Group 1 antibodies can also prevent IL-13 binding to IL-13Rα2. Group 2 is an antibody that binds hIL-13 in a way that allows binding to IL-13Rα1 but prevents IL-4R recruitment to the complex. The authors selected antibodies that were active in Group 1.

Approximately 7500 IL-13-specific positives were identified in the primary FMAT screen from 27 SLAM experiments on 100 plates. 800 wells demonstrated neutralization in the HEK-blue assay. 170 wells had the desired Biacore profiles, i.e., group 1 antibodies with an off rate of < 5x 10 ^-4 s ^-1 . An attempt was made to clone the variable region from these 170 wells and fluorescent foci were successfully obtained in 160 wells. In 100 wells, pairs of heavy and light chain variable region genes were obtained after reverse transcription (RT)-PCR. These V region genes were cloned as full-length mouse IgG1 antibodies and re-expressed in the HEK-293 transient expression system. Sequence analysis revealed that there are 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 hIL-13 variant (R130Q) (E. coli-derived), native wild-type and variant hIL-13 (derived from a human donor), and cynomolgus macaque IL-13 (mammalian-derived) in a cell-based assay. The recombinant antibodies were also tested for their ability to bind human variant IL-13 (R130Q) and cynomolgus macaque IL-13 in Biacore. Following this characterization, antibody families meeting the authors' criteria were selected, i.e., antibodies <100 pM with minimal loss of potency and affinity for all human and cynomolgus macaque IL-13 preparations.

Based on the neutralizing activity, affinity, and donor content of humanized grafts (see below), humanized CA650 was selected for further development.

EXAMPLE 2. Humanization of CA650 Antibody

The 650 antibody was humanized by grafting the CDRs from the rat V region onto the human germline antibody V region frameworks. To restore antibody activity, a number of framework residues from the rat V region were also retained in the humanized sequence. These residues were selected using the protocol described by Adair et al. (1991) (Humanised antibodies. WO91/09967). The alignments of the rat antibody (donor) V region sequences with the human germline (acceptor) V region sequences are shown in Fig. 1 along with the engineered humanized sequences. (Fig. 1(A) 650 light chain graft and Fig. 1(B) 650 heavy chain graft). The CDRs grafted from the donor to the acceptor sequence correspond to the Kabat definitions (Kabat et al., 1987), with the exception of CDR-H1, where the combined Chothia/Kabat definition is used (see Adair et al., 1991 Humanised antibodies. WO91/0996).

The genes encoding the original V region sequences were designed and constructed using the Entelechon GmbH automated synthesis approach and modified to produce grafted versions of gL8 and gH9 using 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 subcloned into pVhg1Fab, which contains DNA encoding the human _CH1 gamma-1 heavy chain constant region.

The V region of IGKV1-39 plus the J region of human JK2 (International Immunogenetics Information System® IMGT, http://www.imgt.org) were chosen as the acceptor for the light chain CDR of antibody 650. The framework residues of the light chain in the gL8 graft are all from the human germline gene except residues 58 and 71 (Kabat numbering), where the donor residues of isoleucine (I58) and tyrosine (Y71), respectively, were retained. Conservation of residues I58 and Y71 was necessary for the full potency of the humanized antibody.

The V region of IGHV1-69 plus the J region of human JH4 (IMGT, http://www.imgt.org) were chosen as the acceptor for the heavy chain CDRs of antibody 650. All heavy chain framework residues in the gH9 grafts are from the human germline except for residues 67, 69, and 71 (Kabat numbering), which retain the donor residues alanine (A67), phenylalanine (F69), and valine (V71), respectively. Retention of residues A67, F69, and V71 are required for full activity of the humanized antibody. The glutamine residue at position 1 of the human framework was replaced by glutamic acid (E1) to allow expression and purification of a homogeneous product: the conversion of glutamine to pyroglutamate at the N terminus of antibodies and antibody fragments has been widely reported. The final selected variable sequences of the gL8 and gH9 graft are shown in Fig. 1(A) and Fig. 1(B), respectively.

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. 2.

EXAMPLE 3. Production of antibody 496.g3 against IL-17AF

The production of antibody CA028_00496.g3 (also referred to herein as antibody 496.g3) against human IL-17A and human IL-17F was previously described in WO 2012/095662. The antibody binds human IL-17A, IL-17F and the IL-17A/F heterodimer with pM affinity. The amino acid and DNA sequences 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 constant regions of Fab 496.g3 (binding IL-17A/F) contained the human C-kappa constant region (K1m3 allotype) and the human gamma-1 CH ₁ constant region and hinge (G1m17 allotype).

EXAMPLE 4. Obtaining of anti-human albumin antibody 645

The production of the anti-human albumin antibody 645 was previously described in WO 2013/068571. The amino acid and DNA sequences encoding the CDRs, heavy and light chain variable regions, scFv and dsscFV formats of the antibody 645 are shown in Fig. 2.

EXAMPLE 5. Multispecific antibody IL-13/IL-17AF - construction of transient plasmids and expression in cells

Multispecific antibodies were constructed with the anti-IL-17AF (496.g3) V region fixed in the Fab position; the antialbumin (645gL4gH5) and IL-13 (1539gL8gH9) V region were reformatted into disulfide-linked scFv in the HL orientation (dsHL) and attached to the C-termini of the corresponding Fab heavy and light chain constant regions via 11-amino acid glycine- and serine-rich linkers (Fig. 7). The amino acid and DNA sequences encoding the full-length heavy chain and light chain of the multispecific antibody are shown in Fig. 2.

The light and heavy chain genes were independently cloned into mammalian expression vectors for transient expression under the control of the hCMV promoter. Both plasmids were transfected in equal ratios into the CHO-S XE cell line (UCB) using the commercial ExpiFectamine ExpiCHO Transient Expression Kit (Thermo Scientific). Cultures were incubated in Corning roller bottles with vented caps at 37°C, 8.0% _CO2 , 190 rpm. After 18-22 h, the cultures were fed appropriate volumes of CHO enhancer and HiTiter nutrients as specified by the manufacturer. Cultures were reincubated at 32°C, 8.0% _CO2 , 190 rpm for an additional 10-12 days. The supernatant was collected by centrifugation at 4000 rpm for 1 h at 4°C before filter sterilization through a 0.45 μm filter and then a 0.2 μm filter. Expression titers were quantified by Protein G HPLC using a 1 ml GE HiTrap Protein G column (GE Healthcare) and in-house Fab standards. Expression titers are shown in Table 1.

Table 1: Expression titers from transient expression in the S-XE CHO cell line

Antibody Concentration (mg/l) Multispecific antibody IL-17AF-IL-13 160

EXAMPLE 6. Multispecific antibody IL-13/IL-17AF - development of a mammalian cell line.

To demonstrate stable expression of the IL-13/IL-17AF multispecific antibody, a stably expressing mammalian cell line was generated. The CHO cell line was transfected with a vector containing 496.g3 Fab, 1539gH9gL8 dsscFv HL (LC, INS0025609), 645gH5gL4 dsscFv HL (HC, INS0025306) and a selectable marker. The cell lines were cloned and evaluated for appropriate manufacturing process. To assess protein quality and quantity and to ensure selection of the optimal cell line, the cell line was evaluated in a small-scale batch bioreactor model of production. A CHO cell line expressing IL-13/IL-17AF multispecific antibodies at levels greater than 1.8 g/L and greater than 75% monomer was selected.

EXAMPLE 7. Method for purification of IL-13/IL-17AF multispecific antibody

The multispecific antibody protein was purified by a native protein A capture step followed by a final preparative size exclusion chromatography step. Purified supernatants from standard CHO transient expression were loaded onto a MabSelect column (GE Healthcare) with a contact time of 5 min and washed with binding buffer (20 mM Hepes pH 7.4 + 150 mM NaCl). Bound material was eluted by step elution with 0.1 M sodium citrate, pH 3.1, and neutralized with 2 M Tris/HCl, pH 8.5, and quantified by absorbance at 280 nm.

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

Neutralized samples were concentrated using an Amicon Ultra-15 concentrator (10 kDa molecular weight cutoff membrane) and centrifuged at 4000×g in a swinging swing rotor. Concentrated samples were loaded onto an XK16/60 Superdex200 column (GE Healthcare) equilibrated in PBS, pH 7.4, and chromatographed with an isocratic gradient of PBS, pH 7.4, at a flow rate of 1 ml/min. Fractions were pooled and analyzed by size exclusion chromatography on a BEH200, 200Å, 1.7 μm, 4.6 mm ID x 300 mm column (Aquity) and chromatographed over an isocratic gradient of 0.2 M phosphate, pH 7, at 0.35 mL/min with absorbance detection at 280 nm and a multichannel fluorescence (FLR) detector (Waters). Selected monomeric fractions were pooled, sterile filtered with 0.22 μm pore size, and final samples analyzed for concentration by A280 scanning on a DropSense96 (Trinean). Endotoxin levels were less than 1.0 EU/mg as assessed by the Charles River EndoSafe® Portable Test System with Limulus Amebocyte Lysate (LAL) test cartridges.

The monomer state of the final multispecific antibody was determined by size exclusion chromatography on a BEH200, 200 Å, 1.7 μm, 4.6 mm ID x 300 mm column (Aquity) and chromatographed with an isocratic gradient of 0.2 M phosphate, pH 7 at 0.35 ml/min with detection 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).

For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, samples were prepared by adding 4x Novex NuPAGE LDS sample buffer (Life Technologies) and either 10x NuPAGE sample reduction solution (Life Technologies) or 100 mM N-ethylmaleimide (Sigma-Aldrich) to ~5 μg purified protein and heating to 100°C for 3 min. Samples were loaded onto a 10-well Novex 4-20% Tris-glycine 1.0 mM SDS-polyacrylamide gel plate (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. The gel was stained with Coomassie Quick Stain (Generon) and decolorized in distilled water.

On non-reducing SDS-PAGE, the multispecific antibody with a theoretical molecular weight (MW) of ~100 kDa migrated to ~120 kDa. When the multispecific antibody protein was reduced, both chains migrated at mobility velocities approaching their respective theoretical MWs, heavy chain (HC) ~52 kDa and light chain (LC) ~51 kDa. The additional bands on the non-reducing gel at ~45-50 kDa represent “free” LC and HC without the disulfide bond in the Fab portion of the molecule; they do not migrate to the same position as LC and HC in lane 2 because they are not fully reduced (Fig. 3(B)).

EXAMPLE 8. Antigen binding by the IL-13/IL-17AF multispecific antibody molecule.

(i) Antigen Binding Affinity

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

Goat anti-human IgG antibody specific for the F(ab') ₂ fragment (Jackson ImmunoResearch) was immobilized on the CM5 sensor chip using amine coupling chemistry to a level of approximately 5000 RU. Each assay cycle consisted of capture of the IL-13/IL-17AF/albumin multispecific antibody molecule onto the anti-F(ab') ₂ surface, analyte injection (at 25°C with a flow rate of 30 μl/min), followed by surface regeneration. Human and cynomolgus monkey 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, 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 monkey IL-13 (Sinobiologicals), and human serum albumin (Jackson ImmunoResearch). A buffer blank was injected to subtract instrument noise and drift.

The 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). If the dissociation rate (K _D ) was measured to be less than 1.0×10 ^-5 , it was fixed at 1.0×10 ^-5 (the detection limit of the Biacore T200 instrument determined by the manufacturer GE Healthcare) for the calculation of the affinity (K _D ).

The results showed that the multispecific antibody efficiently bound to IL-17A, IL-17AF, IL-17F, IL-13 and albumin from humans and cynomolgus monkeys.

Table 2(1): Kinetic Binding Constants of the Multispecific Antibody IL-13/IL-17AF to Human Antigens

Analyte k _a (1/ms) K _D (1/s) K _D (pM) n= Human IL-17A 2.26E+06 1,57E-05 9 7 Human IL-17AF 3.70E+06 4,54E-05 15 4 Human IL-17F 1.67E+06 1,98E-04 128 7 Human IL-13 1.46E+06 3.84E-05 26 3 Human albumin 6.65E+04 1,54E-04 2508 3

Results show the average value from the specified number of determinations.

Table 2(2): Kinetic constants of binding of the multispecific antibody IL-13/IL-17AF to cynomolgus monkey antigens.

Analyte k _a (1/ms) K _D (1/s) K _D (pM) n= Cyno IL-17A 1.65E+06 1,24E-05 7 4 Cyno IL-17AF 2.25E+06 2,87E-05 14 4 Cyno IL-17F 1.14E+06 2,82E-04 283 4 Cyno IL-13 9.48E+05 1,48E-04 156 3 Cyno albumin 8.18E+04 2.04E-04 2473 3

Results show the average value from the specified number of determinations.

(ii) Simultaneous binding of antigens

Surface plasmon resonance was used to demonstrate that IL-17A, IL-13, and albumin can simultaneously bind to the IL-13/IL-17AF multispecific antibody using human or cynomolgus monkey forms of the proteins.

The assay format for assessing simultaneous binding of analytes to the IL-13/IL-17AF multispecific antibody consisted of capturing the antibody sample with an immobilized antibody specific for the F(ab') ₂ fragment of human IgG. IL-13, IL-17A, and human or cynomolgus monkey albumin were then injected individually or in a mixed solution of all three analytes (30 μl/min for 300 s) over the captured IL-13/IL-17AF multispecific antibody (final concentrations 30 nM IL-13, 15 nM IL-17A, 150 nM albumin).

At the end of each cycle, the surface was regenerated at a flow rate of 10 μl/min using a 60 s infusion of 50 mM HCl, followed by a 30 s infusion of 5 mM NaOH and a final 60 s infusion of 50 mM HCl.

The binding response of each antigen upon injection was determined separately, and the sum of the individual responses was compared with the binding response upon injection of a mixture of all three antigens.

The mean binding response of the mixture of IL-17A, IL-13, and human 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 simultaneously and independently bind each antigen.

Table 3: Simultaneous Binding of IL-13/IL-17AF Multispecific Antibody to IL-17A, IL-13 and Human Albumin

Analyte Linking, n=1 (RU) Linking, n=2 (RU) Linking, n=3 (RU) Average value IL-13 12 19 19 IL-17A 33 53 51 Albumen 36 60 59 IL-13+IL-17A+Albumin 81 131 129 Sum of individual binding responses 81 131 129 Mixture binding as a percentage of individual binding responses (%) 100 100 100 100

The mean binding response of the mixture of cynomolgus macaque IL-17A, IL-13, and albumin to the IL-13/IL-17AF multispecific antibody was 97% of the sum of the individual binding responses (shown in Table 4), indicating that the IL-13/IL-17AF multispecific antibody is capable of binding each antigen simultaneously and independently.

Table 4: Simultaneous Binding of IL-13/IL-17AF Multispecific Antibody to IL-17A, IL-13, and Cynomolgus Monkey Albumin

Analyte Linking, n=1 (RU) Linking, n=2 (RU) Linking, n=3 (RU) Average value IL-13 31 30 30 IL-17A 57 56 60 Albumen 63 61 60 IL-13+IL-17A+Albumin 146 141 145 Sum of individual binding responses 151 146 150 Mixture binding as a percentage of individual binding responses (%) 97 96 97 97

EXAMPLE 9. Neutralization of IL-13 using the multispecific antibody IL-13/IL-17AF.

The IL-13 neutralizing activity of the IL-13/IL-17AF multispecific antibody was assessed using the HEK 293 Human IL4/IL-13 SEAP reporter cell line assay. SEAP secretion was measured upon activation of the STAT6 pathway to assess IL-13 responses.

Human IL4/IL-13 SEAP HEK 293 reporter cells (#hkb-il413) were obtained from Invivogen, San Diego. Cell line culture, freezing, and maintenance were performed according to the manufacturer's protocol.

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

Cells were seeded such that when stimulated in 96-well flat-bottomed plates, the cells were approximately 80% confluent.

Cells were treated in duplicate with antibodies pre-incubated with 250 pg/ml IL-13 for 30 min at 37°C, 5% _CO2 , 100% humidity before addition to cells for 24 h.

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

The assay was continued until a visible gradient of color change was observed and the absorbance was read using a spectrophotometer at 620 nm. IC ₅₀ was calculated by nonlinear regression using a Graphpad prism (San Diego, CA). Figure 4 shows a representative plot of % inhibition of STAT6 signaling by the IL-13/IL-17AF multispecific antibody.

Table 5: IC ₅₀ value for inhibition of STAT6 signaling by IL-13/IL-17AF multispecific antibody.

p.m. +/- SEM ng/ml +/- SEM n= 4,118 1,319 0.4118 0.1319 3

EXAMPLE 10. Neutralizing effect of the multispecific antibody IL-13/IL-17AF on the IL-6 responses of human dermal fibroblasts to human and cynomolgus macaque IL-17A and IL-17F.

The aim of this study was to determine the neutralizing capacity of the multispecific IL-13/IL-17AF antibody against human and cynomolgus monkey IL-17A and IL-17F in a human primary cell system. A proinflammatory response is induced when IL-17 is present in combination with other cytokines such as TNF-α. Therefore, this synergistic effect was used to perform an IL-6 release assay from primary normal neonatal skin fibroblasts (nHDF) stimulated with IL-17 and TNF-α.

In this assay, the ability of the IL-13/IL-17AF multispecific antibody to inhibit IL-17-induced IL-6 release from nHDF was measured. Specifically, nHDF were stimulated with human or cynomolgus monkey IL-17A (50 pM) or IL-17F (25,000 pM) in combination with TNF-α (25 pM) in the presence of titrating IL-13/IL-17AF multispecific antibody (concentration range, 5,000 pM to 0.25 pM for IL-17A; 500,000 to 25 pM for IL-17F). The resulting IL-6 response was then measured using homogeneous time-resolved FRET (HTRF).

nHDF cells (Sigma #106-05n) were cultured in complete media (DMEM+10% FCS+2 mM L-glutamine) and maintained in tissue culture flasks using standard techniques. Cells were harvested from the tissue culture flask using TrypLE (Invitrogen #12605036). TrypLE was neutralized using complete media (45 ml) and cells were centrifuged at 300×g for 3 min. Cells were resuspended in complete media (3-5 ml), counted and adjusted to a concentration of 3.125× ¹⁰⁴ cells/ml before addition to a 384-well assay plate (Corning #3701) at 40 μl/well. Cells were incubated for three hours at 37°C/5% _CO2 to allow adherence to the plate. The IL-13/IL-17AF multispecific antibody was serially diluted in complete media in a 384-well dilution plate (Greiner #781281) to a final concentration range of 5,000 pM to 0.25 pM for assays against IL-17A and 500,000 pM to 25 pM for assays against IL-17F. Mixtures of TNF-α and IL-17 cytokine were prepared in complete media to final concentrations of 25 μM TNF-α with 50 pM human or cynomolgus monkey IL-17A or 25,000 pM IL-17F. 30 μL/well of these solutions were then added to a 384-well reagent plate (Greiner #781281). 10 µl from the IL-13/IL-17AF multispecific antibody serial dilution plate was then transferred to a reagent plate containing 30 µl of the diluted cytokines. The IL-13/IL-17AF multispecific antibody was then incubated with the cytokine mixture for one hour at 37°C/5% CO ₂ . Following incubation, 10 µl was transferred from the reagent plate to the assay plate containing the cells. The assay plate was then incubated for 18 hours ± 2 hours at 37°C/5% CO ₂ . After completion of the incubation, europium cryptate and Alexa 665 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 mixture was then added to a white low volume 384-well HTRF plate (Greiner #784075). The supernatant from the assay plate was then transferred to the HTRF plate at 10 µl/well. The HTRF plate was then incubated at room temperature for 2 hours with gentle shaking. The HTRF plates were then read on a Synergy Neo 2 plate reader according to the manufacturer's instructions, measuring fluorescence at the 330/620 nm and 330/665 nm reads. Ratio values were then calculated using the following equation (330/665 nm divided by 330/620 nm) × 10,000 and used to determine the relative percent inhibition compared to the control wells using Microsoft Excel. 4PL curve fitting and IC ₅₀ value calculation were performed using GraphPad Prism 7.0.

The results shown represent the mean (+/- SEM) of three independent experiments. The _IC50 value of the IL-13/IL-17AF multispecific antibody was calculated as 42 pM for human IL-17A and 49 pM for cynomolgus monkey IL-17A. The _IC50 value of the IL-13/IL-17AF multispecific antibody was calculated as 28,030 pM for human IL-17F and 34,320 pM for cynomolgus monkey IL-17F. (Fig. 5).

Table 6: Summary of potency, efficacy and Hill coefficient values for the IL-13/IL-17AF multispecific antibody.

Values were calculated from three independent experiments.

Analysis IC ₅₀ (pM) Emax (%) Coefficient Human IL-17A 42 100 2 IL-17A cynomolgus macaque 49 100 2.4 Human IL-17F 28030 100 3.2 IL-17F cynomolgus macaque 34320 100 2.6

EXAMPLE 11. Simultaneous neutralization of IL-13, IL-17A and IL-17F by a multispecific IL-13/IL-17AF antibody in an IL-13/IL-17AF release bioassay

The aim of this assay was to evaluate the ability of the IL-13/IL-17AF multispecific antibody to simultaneously neutralize IL-13, IL-17A, and IL-17F in a primary cell system. When NHEK are treated with IL-13 or IL-17A or IL-17F alone, they induce the secretion of CXCL1, a chemokine responsible for recruiting cells to inflammatory sites. In the context of atopic dermatitis, there is evidence that CXCL1 plays a role in sensitizing neurons so that they have a lower threshold for excitation. This observation may be relevant to the itch experienced by patients (Yang TB and Kim BS 2019; “Pruritus in allergy and immunology” J Allergy Clin Immunol 144(2): 353–360).

NHEK (PromoCell, Heidelberg) were stored, cultured and used according to the manufacturer's protocol. Cells were seeded to be 100% confluent upon stimulation in 48-well flat-bottomed plates. Cells were pre-incubated with increasing concentrations of anti-IL-13, anti-IL-17A, anti-IL-17F or IL-13/IL-17AF multispecific antibody, respectively, for 30 min before treatment with 100 ng/ml IL-13 and 100 ng/ml IL-17A, 1 μg/ml IL-17F for 72 h. After the specified time period, 50 μl of cell-free supernatant was collected for quantification of CXCL1 concentration by ELISA according to the manufacturer's protocol (R&D Systems). The IC ₅₀ of each experimental group was calculated by nonlinear regression using a Graphpad prism (San Diego, CA).

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

EXAMPLE 12. Comparison of IL-13/IL-17 Multispecific Antibody with Prior Art IL-13/IL-17 Antibodies

Introduction

An example of a multispecific IL-13/IL-17AF antibody according to the present invention is shown in Fig. 7. It comprises a Fab domain with dual specificity for IL-17A and IL-17F linked to 2 scFv domains, one specific for IL-13 and the other for albumin. The anti-albumin domain provides the multispecific antibody with an increased half-life.

Bispecific antibodies that bind to IL-13 and IL-17 have been previously described by Abbvie (WO2013/102042A2) and Genentech (WO2015/127405A2). However, little is known about how they bind and to what extent they bind to IL-13, IL-17A, and IL-17F. To compare these properties, we prepared antibodies described in the prior art and examined their binding behavior for the following characteristics:

Affinity to IL-13

Interaction of the molecule with IL-13 and IL-13Rα1

Affinity to IL-17A

Affinity to IL-17F

Obtaining comparison antibodies

BITS7201A (Genentech) was constructed using the sequences described in Example 6 of WO2015/127405A2.

DVD2166 and DVD2174 (Abbvie) were constructed using the sequences described in WO2013/102042A2, Table 6 and Table 7. These molecules were selected based on reports of activity of individual arms of bispecific antibodies against IL-13 and IL-17 that are nearly equivalent or equivalent to their respective parent antibody (WO2013/102042A2, Example 4, page 83, paragraph 0195).

DNA constructs were transfected into CHO-SXE cells using the high titer protocol of the ExpiCHO transfection system (ThermoFisher Scientific). After harvest, cell cultures were centrifuged for at least 1 h at 4000 rpm and supernatants were clarified by filtration using 0.22 μM Stericup filters.

Cleaning DVD2166+DVD2174

DVD-IgG proteins were purified by loading the purified supernatants onto a 10 ml MabSelect Sure column and washing with PBS, pH 7.4, for 3 column volumes (CV). Proteins were eluted from the column using a step elution of 0.1 M sodium citrate, pH 3.6, and neutralized with 2 M Tris-HCl, pH 8.5. Monomeric proteins were isolated by loading onto a HiLoad 16×60 Superdex 200 pg column (Sigma) equilibrated with PBS, pH 7.4. Fractions containing monomeric proteins were pooled, sterile filtered, and stored at 4°C.

Cleaning BITS7201A

The knob and hole stock proteins were purified by loading the clarified supernatants onto a 10 ml MabSelect Sure column and washing with 3 CVs PBS, pH 7.4. The proteins were eluted from the column with 0.1 M sodium citrate, pH 3.6. To neutralize and stabilize the protein, samples were diluted 1:1 in 1 M arginine/succinate buffer, pH 8.7. The stock antibodies were then loaded onto a HiLoad 26×60 Superdex 200 pg column (Sigma) equilibrated with 0.15 M sodium acetate, 0.5 M arginine buffer, pH 8.5. The bispecific material was then prepared by mixing the stock antibodies 1:1 in the presence of 5 mM cysteamine and incubating overnight at room temperature. A second step of preparative gel filtration was performed to remove any high molecular weight species from the exchangeable bispecific material by loading onto a HiLoad 26×60 Superdex 200 pg column equilibrated with PBS, pH 7.4. Fractions containing monomeric bispecific proteins were pooled, sterile filtered and stored at 4°C.

Comparison of binding characteristics

The binding kinetics of human IL-13, IL-17A, and IL-17F binding to the IL-13/IL-17AF multispecific antibody, designated "UCBXXXX", and prior art antibodies were assessed using 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 UCCBXXXX or reference molecules to IL-13 resulted in blocking the interaction of IL-13 with IL-13 receptors.

Goat anti-human IgG antibody specific for the F(ab') ₂ fragment (Jackson ImmunoResearch) was immobilized on the CM5 sensor chip using amine coupling chemistry to a level of approximately 5000 RU. For affinity assessment, each assay run consisted of capturing the IL-13/IL-17AF multispecific antibody or bispecific reference molecule onto the anti-F(ab') ₂ surface (50 to 100 RU), introducing the analyte (at 25°C at a flow rate of 30 μl/min for 180 s), and then monitoring the dissociation 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 at a flow rate of 10 μl/min using 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. 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 from 5 nM to 0.156 nM for IL-17A and IL-17F. IL-17A and IL-17F were obtained in-house, whereas human IL-13 was obtained from R&D Systems. A buffer blank injection was provided to subtract instrument noise and drift.

Kinetic parameters were determined using a 1:1 binding model using Biacore T200 Evaluation software (version 3.0). The results are summarized in Table 7.

Table 7: Comparison of kinetic constants for binding to IL-13, IL-17A and IL-17F.

Molecule Analyte k _a (1/ms) K _D (1/s) K _D (pM) UCBXXXX* IL-13 3.61E+06 1,11E-04 25.8 BITS7210A* 2.05E+06 5,69E-05 27.5 DVD2166 8.16E+05 1,50E-05 18.35 DVD2174 6.50E+05 3,66E-05 56.34 UCBXXXX* IL-17A 3.20E+06 3.45E-05 8.2 BITS7210A* 1.27E+07 1,06E-03 84.4 DVD2166 5.59E+05 7,68E-05 137.4 DVD2174 4.76E+05 1,28E-04 268.4 UCBXXXX* IL-17F 2.66E+06 2,19E-04 82.1 BITS7210A* 7.71E+06 5.74E-04 74.4 DVD2166 ←←←←←←←No Binding→→→→→→→→ DVD2174 ←←←←←←←No Binding→→→→→→→→ *Results represent the average of two technical replicates in the same experiment.

To assess IL-13Rα1 receptor blockade, each antibody molecule was captured on goat anti-human F(ab') ₂ IgG (approximately 50-100 RU) followed by IL-13 (25 nM for 180 sec at 10 μl/min) and IL-13Rα1 (R&D Systems, 100 nM for 300 sec at 10 μl/min). Control injections of both IL-13 and IL-13Rα1 were provided to subtract any drift or background responses. As shown in Table 8, UCBXXXX was able to block the interaction of IL-13 with IL-13Rα1, whereas BITS7210A, DVD2166, and DVD 2174 did not.

Table 8: Blockade of IL-13Rα1 receptor by IL-13/IL-17AF multispecific antibody and comparator molecules.

Molecule Capture (RU) IL-13 binding (RU) IL-13Rα1 binding (RU) IL-13Rα1 inhibitor UCBXXXX 65.4 7.3 0.2 Yes BITS7210A 86 8.6 15.7 No DVD2166 115.2 11.7 13.8 No DVD2174 115.3 11.3 16.2 No

An additional experiment was performed to assess the ability of the IL-13/IL-17AF multispecific antibody to block binding to IL-13Rα1 and IL-13Rα2. In this experiment, approximately 260 RU UCBXXXX were captured with immobilized mouse anti-human _CH1 antibody (own UCB), followed by IL-13 (25 nM for 180 sec at 10 μl/min), followed by either IL-13Rα1 or IL-13Rα2 (R&D Systems, 100 nM for 300 sec at 10 μl/min). Control injections of IL-13, IL-13Rα1, and IL-13Rα2 were provided to subtract any drift or background responses. The results showed that UCXXXX was able to block the interaction of IL-13 with both IL-13Ra1 and IL-13Ra2 (Table 9).

Table 9: Blockade of IL-13Rα1 and IL-13Rα2 receptor by IL-13/IL-17AF multispecific antibody.

Capture (RU) IL-13 binding (RU) IL-13Rα1 binding (RU) IL-13Rα2 binding (RU) 264.4 27.0 0.8 0.3

Discussion

A comparative study showed that the IL-13/IL-17AF multispecific antibody UCBXXXX, the bispecific antibody BITS7210A, and the dual variable domain antibodies DVD2166 and DVD2174 were able to bind to IL-13 with high affinity. However, the characteristics of the binding interactions were very different. UCXXXX was able to block the interaction of IL-13 with IL-13-Rα1, whereas BITS7210A, DVD2166, and DVD2174 did not.

The comparative study also demonstrated that the multispecific antibody UCXXXX, the bispecific antibody BITS7210A, and the dual variable domain antibodies DVD2166 and DVD2174 were able to bind to 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 anti-DVD antibodies. UCXXXX and BITS7210A bound IL-17F with similar affinity, while the anti-DVD antibodies were unable to bind IL-17F at all.

The interaction between IL-13 and IL-13Rα1 in the presence of these antibodies is important in the context of reducing potential immunogenicity. Current evidence suggests that immunogenicity, particularly anti-drug antibody (ADA) formation, should be carefully considered when exploring potential new bispecific antibody therapeutics. The main driving force for ADA formation is the interaction and subsequent internalization of the therapeutic antibody with target antigens expressed on the cell surface (Schellekens, H., 2002; Clin Ther. 24(11):1720-40). Internalized therapeutic antibody/target antigen complexes transit through various intracellular compartments and can either recycle back to the cell surface or undergo degradation (St Pierre et al, 2011); which may lead to peptide presentation by antigen-presenting molecules and ADA formation.

In the bispecific antibody BITS7201A, the IL-13 F(ab) portion of the molecule is the same as that of the anti-IL-13 antibody lebrikizumab (see Example 6 of WO2015/127405A2). Lebrikizumab is thought to bind to IL-13 at a site that allows IL-13 to bind to its receptors IL-13Rα1 and IL-13Rα2, but blocks interaction with the IL-4Rα receptor (Popovic et al., 2017; J Mol. Biol., 429(2):208-19). This particular type of interaction may promote internalization of the antibody/target antigen/receptor complex via IL-13Rα2 and thus increase 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.

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

To reduce the potential risk of immunogenicity, UCBXXXX has been specifically designed to interact with the corresponding target antigens IL-13, IL-17A and IL-17F to prevent their interaction with receptors on cells, thereby reducing the likelihood of internalization, degradation and the likelihood of ADA formation. The incidence of ADA with UCBXXXX in humans will only be known once clinical data are available.

The data obtained above demonstrate that the IL-13/IL-17AF multispecific antibody UCCBXXXX has the right properties to become an effective therapeutic IL-13/IL-17 antibody, with increased efficacy and reduced risk of immunogenicity.

--->

SEQUENCE LIST

<110> UCB Biopharma SRL

<120> Multispecific antibody with specificity

binding of human IL-13 and IL-17

<130> PF0208-WO-PCT

<150>GB 1919061.0

<151> 2019-12-20

<160> 68

<170> PatentIn version 3.5

<210> 1

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 1

Arg Ala Asp Glu Ser Val Arg Thr Leu Met His

1 5 10

<210> 2

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 2

Leu Val Ser Asn Ser Glu Ile

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 3

Gln Gln Thr Trp Ser Asp Pro Trp Thr

1 5

<210> 4

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 4

Gly Phe Thr Phe Ser Asp Tyr Asn Met Ala

1 5 10

<210> 5

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 5

Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp Ser Val Lys

1 5 10 15

Gly

<210> 6

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 6

Pro Pro Gln Tyr Tyr Glu Gly Ser Ile Tyr Arg Leu Trp Phe Ala His

1 5 10 15

<210> 7

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 7

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Asp Glu Ser Val Arg Thr Leu

20 25 30

Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Val Ser Asn Ser Glu Ile Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Arg Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Trp Ser Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 8

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 8

gcaatccagc tcacccagag tccaagcagt ctctccgcca gcgtaggcga ccgtgtgact 60

attacctgta gagcggacga gtcggtcagg actctcatgc actggtatca acagaagcct 120

ggtaaagctc ctaaactgct catctatctg gtgtccaact cggagatagg tgtgccagat 180

cggtttagtg ggtctggttc aggcactgat ttcagactga ccatatcatc tctacagcca 240

gaggacttcg ccacatatta ctgtcagcaa acctggagtg acccgtggac tttcggccag 300

ggcactaaag tagaaattaa a 321

<210> 9

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Pro Gln Tyr Tyr Glu Gly Ser Ile Tyr Arg Leu Trp Phe

100 105 110

Ala His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 10

<211> 375

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 10

gaagttcagc tggtcgagtc tggaggtggc cttgtccaac ctggagggag cctgcgtctc 60

tcttgtgcag caagcggatt cacgttttct gattacaata tggcttgggt tagacaggca 120

ccgggtaagg gccttgaatg ggttgcgacg attacatacg aaggcagaaa tacctattac 180

agggactcag taaaagggcg gtttaccata agccgagata atgctaaaaa cagtctgtat 240

ttgcaaatga acagcctacg agctgaagac actgccgtgt attactgcgc gagtccacct 300

cagtattatg aaggatcaat ctatcgcctc tggttcgcac attggggaca ggggaccctt 360

gtgacagtct cgagt 375

<210> 11

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 11

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Asp Glu Ser Val Arg Thr Leu

20 25 30

Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Val Ser Asn Ser Glu Ile Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Arg Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Trp Ser Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 12

<211> 642

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 12

gcaatccagc tcacccagag tccaagcagt ctctccgcca gcgtaggcga ccgtgtgact 60

attacctgta gagcggacga gtcggtcagg actctcatgc actggtatca acagaagcct 120

ggtaaagctc ctaaactgct catctatctg gtgtccaact cggagatagg tgtgccagat 180

cggtttagtg ggtctggttc aggcactgat ttcagactga ccatatcatc tctacagcca 240

gaggacttcg ccacatatta ctgtcagcaa acctggagtg acccgtggac tttcggccag 300

ggcactaaag tagaaattaa acgtacggtg gccgctccct ccgtgttcat cttcccaccc 360

tccgacgagc agctgaagtc cggcaccgcc tccgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480

gaatccgtca ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccaggc 600

ctgtccagcc ccgtgaccaa gtccttcaac cggggcgagt gc 642

<210> 13

<211> 228

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Pro Gln Tyr Tyr Glu Gly Ser Ile Tyr Arg Leu Trp Phe

100 105 110

Ala His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys

225

<210> 14

<211> 684

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 14

gaagttcagc tggtcgagtc tggaggtggc cttgtccaac ctggagggag cctgcgtctc 60

tcttgtgcag caagcggatt cacgttttct gattacaata tggcttgggt tagacaggca 120

ccgggtaagg gccttgaatg ggttgcgacg attacatacg aaggcagaaa tacctattac 180

agggactcag taaaagggcg gtttaccata agccgagata atgctaaaaa cagtctgtat 240

ttgcaaatga acagcctacg agctgaagac actgccgtgt attactgcgc gagtccacct 300

cagtattatg aaggatcaat ctatcgcctc tggttcgcac attggggaca ggggaccctt 360

gtgacagtct cgagtgcgtc cacaaagggc ccatcggtct tccccctggc accctcctcc 420

aagagcacct ctggggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480

ccagtgacgg tgtcgtggaa ctcaggtgcc ctgaccagcg gcgttcacac cttcccggct 540

gtcctacagt cttcaggact ctactccctg agcagcgtgg tgaccgtgcc ctccagcagc 600

ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtcgat 660

aagaaagttg agcccaaatc ttgt 684

<210> 15

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 15

Lys Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp

1 5 10

<210> 16

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 16

Tyr Thr Asp Ile Leu Gln Thr

1 5

<210> 17

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 17

Tyr Gln Tyr Tyr Ser Gly Tyr Thr

1 5

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 18

Gly Tyr Ser Phe Thr Ser Tyr Tyr Ile His

1 5 10

<210> 19

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 19

Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 20

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 20

Phe His Tyr Asp Gly Ala Asp

1 5

<210> 21

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Pro Val Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Leu Ser Cys Lys Ala Ser Gln Asn Ile Asn Glu Asn

20 25 30

Leu Asp Trp Tyr His Gln Lys His Gly Glu Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Asp Ile Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr

85 90 95

Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 22

<211> 318

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 22

gacatccaga tgacccagtc tcctccagtc ctgtctgcat ctgtgggaga cagagtcact 60

ctcagttgca aagcaagtca gaatattaat gagaacttag actggtatca tcaaaagcat 120

ggcgaagctc caaaactcct gatatattat acagacattt tgcaaacggg catcccatca 180

aggttcagtg gcagtggatc tggtacagat tacacactca ccatcagcag cctgcagcct 240

gaagatgttg ccacatatta ctgctatcag tattacagtg ggtacacgtt tggacctggg 300

accaagctgg aaataaaa 318

<210> 23

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 23

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Val Asp Lys Tyr Phe Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Ser Pro Glu Asp Thr Ala Val Phe Tyr Cys

85 90 95

Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 24

<211> 348

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 24

caggtacaac tgcagcagtc tggagctgag ttggtgaagc ctgggtcttc agtgaagatg 60

tcctgcaagg cttctggcta cagtttcacc agctactaca tacactggat aaagcagagg 120

cctggacagg gccttgagtg gattgggcgt attggtcctg gaagtggaga tattaattac 180

aatgagaagt tcaagggcaa ggccacattt actgtggaca aatatttcag cacagcctac 240

atgcaactca gcagcctgtc acctgaggac actgcggtct tttactgtgc aagatttcac 300

tatgatgggg ctgactgggg ccaaggcact ctggtcacag tctcgagc 348

<210> 25

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 26

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210> 27

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Glu Asn

20 25 30

Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Asp Ile Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 28

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 28

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 29

<211> 318

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 29

gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga cagggtgacc 60

atcacctgca aggcctccca gaacatcaac gagaacctgg actggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctactac accgacatcc tgcagaccgg catcccctcc 180

aggttctccg gctccggctc cggcaccgac tacaccctga ccatctcctc cctgcagccc 240

gaggacttcg ccacctacta ctgctaccag tactactccg gctacacctt cggccagggc 300

accaagctgg agatcaag 318

<210> 30

<211> 348

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 30

gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60

tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180

aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240

atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300

tacgacggcg ccgactgggg ccagggcacc ctggtgaccg tctcgagc 348

<210> 31

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 31

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Glu Asn

20 25 30

Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Asp Ile Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr

85 90 95

Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 32

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 32

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Met

35 40 45

Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 33

<211> 318

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 33

gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga cagggtgacc 60

atcacctgca aggcctccca gaacatcaac gagaacctgg actggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctactac accgacatcc tgcagaccgg catcccctcc 180

aggttctccg gctccggctc cggcaccgac tacaccctga ccatctcctc cctgcagccc 240

gaggacttcg ccacctacta ctgctaccag tactactccg gctacacctt cggctgcggc 300

accaagctgg agatcaag 318

<210> 34

<211> 348

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 34

gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60

tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120

cccggccagt gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180

aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240

atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300

tacgacggcg ccgactgggg ccagggcacc ctggtgaccg tgtcctcc 348

<210> 35

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 35

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro

130 135 140

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys

145 150 155 160

Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp Trp Tyr Gln Gln Lys Pro

165 170 175

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asp Ile Leu Gln Thr

180 185 190

Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr

195 200 205

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

210 215 220

Tyr Gln Tyr Tyr Ser Gly Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 36

<211> 726

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 36

gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60

tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180

aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240

atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300

tacgacggcg ccgactgggg cccaggcacc ctggtgaccg tgtcctccgg aggtggcggt 360

tctggcggtg gcggttccgg tggcggtgga tcgggaggtg gcggttctga catccagatg 420

acccagtccc cctcctccct gtccgcctcc gtgggcgaca gggtgaccat cacctgcaag 480

gcctcccaga acatcaacga gaacctggac tggtaccagc agaagcccgg caaggccccc 540

aagctgctga tctactacac cgacatcctg cagaccggca tcccctccag gttctccggc 600

tccggctccg gcaccgacta caccctgacc atctcctccc tgcagcccga ggacttcgcc 660

acctactact gctaccagta ctactccggc tacaccttcg gccagggcac caagctggag 720

atcaag 726

<210> 37

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 37

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Met

35 40 45

Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro

130 135 140

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys

145 150 155 160

Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp Trp Tyr Gln Gln Lys Pro

165 170 175

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asp Ile Leu Gln Thr

180 185 190

Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr

195 200 205

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

210 215 220

Tyr Gln Tyr Tyr Ser Gly Tyr Thr Phe Gly Cys Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 38

<211> 726

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 38

gaggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60

tcctgcaagg cctccggcta ctccttcacc tcctactaca tccactgggt gaggcaggcc 120

cccggccagt gcctggagtg gatgggcagg atcggccccg gctccggcga catcaactac 180

aacgagaagt tcaagggcag ggccaccttc accgtggaca agtccacctc caccgcctac 240

atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggttccac 300

tacgacggcg ccgactgggg cccaggcacc ctggtgaccg tgtcctccgg aggtggcggt 360

tctggcggtg gcggttccgg tggcggtgga tcgggaggtg gcggttctga catccagatg 420

acccagtccc cctcctccct gtccgcctcc gtgggcgaca gggtgaccat cacctgcaag 480

gcctcccaga acatcaacga gaacctggac tggtaccagc agaagcccgg caaggccccc 540

aagctgctga tctactacac cgacatcctg cagaccggca tcccctccag gttctccggc 600

tccggctccg gcaccgacta caccctgacc atctcctccc tgcagcccga ggacttcgcc 660

acctactact gctaccagta ctactccggc tacaccttcg gctgcggcac caagctggag 720

atcaag 726

<210> 39

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 39

Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser

1 5 10

<210> 40

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 40

Glu Ala Ser Lys Leu Thr Ser

1 5

<210> 41

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 41

Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr Thr

1 5 10

<210> 42

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 42

Gly Ile Asp Leu Ser Asn Tyr Ala Ile Asn

1 5 10

<210> 43

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 43

Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly

1 5 10 15

<210> 44

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 44

Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu

1 5 10

<210> 45

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 45

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn

20 25 30

Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile

85 90 95

Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 46

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 46

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 47

<211> 330

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 47

gatattcaga tgacgcaatc accttcgagc gtatccgcct cggtgggaga cagggtgaca 60

atcacttgtc agtcatcccc ctcagtctgg agcaactttt tgtcatggta tcagcagaag 120

cccggaaagg ctccgaaatt gctgatctac gaggcatcga agttgacgag cggtgtacca 180

agcagattct ccggttcggg gtcgggaact gacttcaccc ttacgatctc atcgctgcag 240

ccggaggatt ttgcgaccta ctactgtggg ggtgggtatt cgtcgatttc cgacacaaca 300

ttcggggggcg gcacgaaagt ggaaatcaag 330

<210> 48

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 48

gaagtgcagt tgctggagtc aggtggaggg ctggtgcagc ccggaggatc gctgcggttg 60

tcatgcgcgg tgtccggtat tgatttgtcc aattacgcca tcaattgggt acgccaagcg 120

ccagggaagg gccttgagtg gattggcatc atctgggcgt cggggacgac cttttatgct 180

acttgggcca aaggaagatt cacaatctcc cgagacaact cgaagaacac cgtgtatctt 240

caaatgaact cgctcagggc cgaggacacg gcggtctact actgtgcacg gacagtgccg 300

ggttattcaa cggcacctta ctttgatctt tggggccagg ggaccctcgt gactgtctca 360

agt 363

<210> 49

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 49

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn

20 25 30

Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile

85 90 95

Ser Asp Thr Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 50

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 50

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 51

<211> 330

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 51

gatattcaga tgacgcaatc accttcgagc gtatccgcct cggtgggaga cagggtgaca 60

atcacttgtc agtcatcccc ctcagtctgg agcaactttt tgtcatggta tcagcagaag 120

cccggaaagg ctccgaaatt gctgatctac gaggcatcga agttgacgag cggtgtacca 180

agcagattct ccggttcggg gtcgggaact gacttcaccc ttacgatctc atcgctgcag 240

ccggaggatt ttgcgaccta ctactgtggg ggtgggtatt cgtcgatttc cgacacaaca 300

ttcgggtgcg gcacgaaagt ggaaatcaag 330

<210> 52

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 52

gaagtgcagt tgctggagtc aggtggaggg ctggtgcagc ccggaggatc gctgcggttg 60

tcatgcgcgg tgtccggtat tgatttgtcc aattacgcca tcaattgggt acgccaagcg 120

ccagggaagt gccttgagtg gattggcatc atctgggcgt cggggacgac cttttatgct 180

acttgggcca aaggaagatt cacaatctcc cgagacaact cgaagaacac cgtgtatctt 240

caaatgaact cgctcagggc cgaggacacg gcggtctact actgtgcacg gacagtgccg 300

ggttattcaa cggcacctta ctttgatctt tggggccagg ggaccctcgt gactgtctca 360

agt 363

<210> 53

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 53

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser

165 170 175

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu

180 185 190

Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

195 200 205

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

210 215 220

Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 54

<211> 753

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 54

gaagtgcagt tgctggagtc aggtggaggg ctggtgcagc ccggaggatc gctgcggttg 60

tcatgcgcgg tgtccggtat tgatttgtcc aattacgcca tcaattgggt acgccaagcg 120

ccagggaagg gccttgagtg gattggcatc atctgggcgt cggggacgac cttttatgct 180

acttgggcca aaggaagatt cacaatctcc cgagacaact cgaagaacac cgtgtatctt 240

caaatgaact cgctcagggc cgaggacacg gcggtctact actgtgcacg gacagtgccg 300

ggttattcaa cggcacctta ctttgatctt tggggccagg ggaccctcgt gactgtctca 360

agtggaggtg gcggttctgg cggtggcggt tccggtggcg gtggatcggg aggtggcggt 420

tctgatattc agatgacgca atcaccttcg agcgtatccg cctcggtggg agacaggtg 480

acaatcactt gtcagtcatc cccctcagtc tggagcaact ttttgtcatg gtatcagcag 540

aagcccggaa aggctccgaa attgctgatc tacgaggcat cgaagttgac gagcggtgta 600

ccaagcagat tctccggttc ggggtcggga actgacttca cccttacgat ctcatcgctg 660

cagccggagg attttgcgac ctactactgt gggggtgggt attcgtcgat ttccgacaca 720

acattcgggg gcggcacgaa agtggaaatc aag 753

<210> 55

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 55

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser

165 170 175

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu

180 185 190

Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

195 200 205

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

210 215 220

Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr

225 230 235 240

Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys

245 250

<210> 56

<211> 753

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 56

gaagtgcagt tgctggagtc aggtggaggg ctggtgcagc ccggaggatc gctgcggttg 60

tcatgcgcgg tgtccggtat tgatttgtcc aattacgcca tcaattgggt acgccaagcg 120

ccagggaagt gccttgagtg gattggcatc atctgggcgt cggggacgac cttttatgct 180

acttgggcca aaggaagatt cacaatctcc cgagacaact cgaagaacac cgtgtatctt 240

caaatgaact cgctcagggc cgaggacacg gcggtctact actgtgcacg gacagtgccg 300

ggttattcaa cggcacctta ctttgatctt tggggccagg ggaccctcgt gactgtctca 360

agtggaggtg gcggttctgg cggtggcggt tccggtggcg gtggatcggg aggtggcggt 420

tctgatattc agatgacgca atcaccttcg agcgtatccg cctcggtggg agacaggtg 480

acaatcactt gtcagtcatc cccctcagtc tggagcaact ttttgtcatg gtatcagcag 540

aagcccggaa aggctccgaa attgctgatc tacgaggcat cgaagttgac gagcggtgta 600

ccaagcagat tctccggttc ggggtcggga actgacttca cccttacgat ctcatcgctg 660

cagccggagg attttgcgac ctactactgt gggggtgggt attcgtcgat ttccgacaca 720

acattcgggt gcggcacgaa agtggaaatc aag 753

<210> 57

<211> 492

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 57

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Pro Gln Tyr Tyr Glu Gly Ser Ile Tyr Arg Leu Trp Phe

100 105 110

Ala His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser Glu

225 230 235 240

Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

245 250 255

Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr Ala

260 265 270

Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly

275 280 285

Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly

290 295 300

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln

305 310 315 320

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

325 330 335

Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln

340 345 350

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

355 360 365

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

370 375 380

Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr

385 390 395 400

Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser Trp

405 410 415

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu Ala

420 425 430

Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

435 440 445

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

450 455 460

Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr Thr

465 470 475 480

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

485 490

<210> 58

<211> 1476

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 58

gaagttcagc tggtcgagtc tggaggtggc cttgtccaac ctggagggag cctgcgtctc 60

tcttgtgcag caagcggatt cacgttttct gattacaata tggcttgggt tagacaggca 120

ccgggtaagg gccttgaatg ggttgcgacg attacatacg aaggcagaaa tacctattac 180

agggactcag taaaagggcg gtttaccata agccgagata atgctaaaaa cagtctgtat 240

ttgcaaatga acagcctacg agctgaagac actgccgtgt attactgcgc gagtccacct 300

cagtattatg aaggatcaat ctatcgcctc tggttcgcac attggggaca ggggaccctt 360

gtgacagtct cgagtgcgtc cacaaagggc ccatcggtct tccccctggc accctcctcc 420

aagagcacct ctggggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480

ccagtgacgg tgtcgtggaa ctcaggtgcc ctgaccagcg gcgttcacac cttcccggct 540

gtcctacagt cttcaggact ctactccctg agcagcgtgg tgaccgtgcc ctccagcagc 600

ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtcgat 660

aagaaagttg agcccaaatc ttgtagcggt ggcggtggca ccggaggtgg cggttcagaa 720

gtgcagttgc tggagtcagg tggagggctg gtgcagcccg gaggatcgct gcggttgtca 780

tgcgcggtgt ccggtattga tttgtccaat tacgccatca attgggtacg ccaagcgcca 840

gggaagggcc ttgagtggat tggcatcatc tgggcgtcgg ggacgacctt ttatgctact 900

tgggccaaag gaagattcac aatctcccga gacaactcga agaacaccgt gtatcttcaa 960

atgaactcgc tcagggccga ggacacggcg gtctactact gtgcacggac agtgccgggt 1020

tattcaacgg caccttactt tgatctttgg ggccagggga ccctcgtgac tgtctcaagt 1080

ggaggtggcg gttctggcgg tggcggttcc ggtggcggtg gatcgggagg tggcggttct 1140

gatattcaga tgacgcaatc accttcgagc gtatccgcct cggtgggaga cagggtgaca 1200

atcacttgtc agtcatcccc ctcagtctgg agcaactttt tgtcatggta tcagcagaag 1260

cccggaaagg ctccgaaatt gctgatctac gaggcatcga agttgacgag cggtgtacca 1320

agcagattct ccggttcggg gtcgggaact gacttcaccc ttacgatctc atcgctgcag 1380

ccggaggatt ttgcgaccta ctactgtggg ggtgggtatt cgtcgatttc cgacacaaca 1440

ttcggggggcg gcacgaaagt ggaaatcaag cgtacc 1476

<210> 59

<211> 492

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 59

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Pro Gln Tyr Tyr Glu Gly Ser Ile Tyr Arg Leu Trp Phe

100 105 110

Ala His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser Glu

225 230 235 240

Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

245 250 255

Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr Ala

260 265 270

Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile Gly

275 280 285

Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly

290 295 300

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln

305 310 315 320

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

325 330 335

Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln

340 345 350

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

355 360 365

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

370 375 380

Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr

385 390 395 400

Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser Trp

405 410 415

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu Ala

420 425 430

Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

435 440 445

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

450 455 460

Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr Thr

465 470 475 480

Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Arg Thr

485 490

<210> 60

<211> 1476

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 60

gaagttcagc tggtcgagtc tggaggtggc cttgtccaac ctggagggag cctgcgtctc 60

tcttgtgcag caagcggatt cacgttttct gattacaata tggcttgggt tagacaggca 120

ccgggtaagg gccttgaatg ggttgcgacg attacatacg aaggcagaaa tacctattac 180

agggactcag taaaagggcg gtttaccata agccgagata atgctaaaaa cagtctgtat 240

ttgcaaatga acagcctacg agctgaagac actgccgtgt attactgcgc gagtccacct 300

cagtattatg aaggatcaat ctatcgcctc tggttcgcac attggggaca ggggaccctt 360

gtgacagtct cgagtgcgtc cacaaagggc ccatcggtct tccccctggc accctcctcc 420

aagagcacct ctggggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480

ccagtgacgg tgtcgtggaa ctcaggtgcc ctgaccagcg gcgttcacac cttcccggct 540

gtcctacagt cttcaggact ctactccctg agcagcgtgg tgaccgtgcc ctccagcagc 600

ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtcgat 660

aagaaagttg agcccaaatc ttgtagcggt ggcggtggca ccggaggtgg cggttcagaa 720

gtgcagttgc tggagtcagg tggagggctg gtgcagcccg gaggatcgct gcggttgtca 780

tgcgcggtgt ccggtattga tttgtccaat tacgccatca attgggtacg ccaagcgcca 840

gggaagtgcc ttgagtggat tggcatcatc tgggcgtcgg ggacgacctt ttatgctact 900

tgggccaaag gaagattcac aatctcccga gacaactcga agaacaccgt gtatcttcaa 960

atgaactcgc tcagggccga ggacacggcg gtctactact gtgcacggac agtgccgggt 1020

tattcaacgg caccttactt tgatctttgg ggccagggga ccctcgtgac tgtctcaagt 1080

ggaggtggcg gttctggcgg tggcggttcc ggtggcggtg gatcgggagg tggcggttct 1140

gatattcaga tgacgcaatc accttcgagc gtatccgcct cggtgggaga cagggtgaca 1200

atcacttgtc agtcatcccc ctcagtctgg agcaactttt tgtcatggta tcagcagaag 1260

cccggaaagg ctccgaaatt gctgatctac gaggcatcga agttgacgag cggtgtacca 1320

agcagattct ccggttcggg gtcgggaact gacttcaccc ttacgatctc atcgctgcag 1380

ccggaggatt ttgcgaccta ctactgtggg ggtgggtatt cgtcgatttc cgacacaaca 1440

ttcgggtgcg gcacgaaagt ggaaatcaag cgtacc 1476

<210> 61

<211> 469

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 61

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Asp Glu Ser Val Arg Thr Leu

20 25 30

Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Val Ser Asn Ser Glu Ile Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Arg Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Trp Ser Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

225 230 235 240

Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser

245 250 255

Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

260 265 270

Met Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys

275 280 285

Phe Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala

290 295 300

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

305 310 315 320

Cys Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu

325 330 335

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

340 345 350

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

355 360 365

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

370 375 380

Lys Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp Trp Tyr Gln Gln Lys

385 390 395 400

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asp Ile Leu Gln

405 410 415

Thr Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr

420 425 430

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

435 440 445

Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr Phe Gly Gln Gly Thr Lys Leu

450 455 460

Glu Ile Lys Arg Thr

465

<210> 62

<211> 1407

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 62

gcaatccagc tcacccagag tccaagcagt ctctccgcca gcgtaggcga ccgtgtgact 60

attacctgta gagcggacga gtcggtcagg actctcatgc actggtatca acagaagcct 120

ggtaaagctc ctaaactgct catctatctg gtgtccaact cggagatagg tgtgccagat 180

cggtttagtg ggtctggttc aggcactgat ttcagactga ccatatcatc tctacagcca 240

gaggacttcg ccacatatta ctgtcagcaa acctggagtg acccgtggac tttcggccag 300

ggcactaaag tagaaattaa acgtacggtg gccgctccct ccgtgttcat cttcccaccc 360

tccgacgagc agctgaagtc cggcaccgcc tccgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480

gaatccgtca ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccaggc 600

ctgtccagcc ccgtgaccaa gtccttcaac cggggcgagt gcagcggtgg cggtggctcc 660

ggaggtggcg gttcagaggt gcagctggtg cagtccggcg ccgaggtgaa gaagcccggc 720

tcctccgtga aggtgtcctg caaggcctcc ggctactcct tcacctccta ctacatccac 780

tgggtgaggc aggcccccgg ccagggcctg gagtggatgg gcaggatcgg ccccggctcc 840

ggcgacatca actacaacga gaagttcaag ggcagggcca ccttcaccgt ggacaagtcc 900

acctccaccg cctacatgga gctgtcctcc ctgaggtccg aggacaccgc cgtgtactac 960

tgcgccaggt tccactacga cggcgccgac tggggccagg gcaccctggt gaccgtgtcc 1020

tccggaggtg gcggttctgg cggtggcggt tccggtggcg gtggatcggg aggtggcggt 1080

tctgacatcc agatgaccca gtccccctcc tccctgtccg cctccgtggg cgacagggtg 1140

accatcacct gcaaggcctc ccagaacatc aacgagaacc tggactggta ccagcagaag 1200

cccggcaagg cccccaagct gctgatctac tacaccgaca tcctgcagac cggcatcccc 1260

tccaggttct ccggctccgg ctccggcacc gactacaccc tgaccatctc ctccctgcag 1320

cccgaggact tcgccaccta ctactgctac cagtactact ccggctacac cttcggccag 1380

ggcaccaagc tggagatcaa gcgtacc 1407

<210> 63

<211> 469

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 63

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Asp Glu Ser Val Arg Thr Leu

20 25 30

Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Val Ser Asn Ser Glu Ile Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Arg Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Trp Ser Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

225 230 235 240

Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser

245 250 255

Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp

260 265 270

Met Gly Arg Ile Gly Pro Gly Ser Gly Asp Ile Asn Tyr Asn Glu Lys

275 280 285

Phe Lys Gly Arg Ala Thr Phe Thr Val Asp Lys Ser Thr Ser Thr Ala

290 295 300

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

305 310 315 320

Cys Ala Arg Phe His Tyr Asp Gly Ala Asp Trp Gly Gln Gly Thr Leu

325 330 335

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

340 345 350

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

355 360 365

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

370 375 380

Lys Ala Ser Gln Asn Ile Asn Glu Asn Leu Asp Trp Tyr Gln Gln Lys

385 390 395 400

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asp Ile Leu Gln

405 410 415

Thr Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr

420 425 430

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

435 440 445

Cys Tyr Gln Tyr Tyr Ser Gly Tyr Thr Phe Gly Cys Gly Thr Lys Leu

450 455 460

Glu Ile Lys Arg Thr

465

<210> 64

<211> 1407

<212> DNA

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 64

gcaatccagc tcacccagag tccaagcagt ctctccgcca gcgtaggcga ccgtgtgact 60

attacctgta gagcggacga gtcggtcagg actctcatgc actggtatca acagaagcct 120

ggtaaagctc ctaaactgct catctatctg gtgtccaact cggagatagg tgtgccagat 180

cggtttagtg ggtctggttc aggcactgat ttcagactga ccatatcatc tctacagcca 240

gaggacttcg ccacatatta ctgtcagcaa acctggagtg acccgtggac tttcggccag 300

ggcactaaag tagaaattaa acgtacggtg gccgctccct ccgtgttcat cttcccaccc 360

tccgacgagc agctgaagtc cggcaccgcc tccgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480

gaatccgtca ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccaggc 600

ctgtccagcc ccgtgaccaa gtccttcaac cggggcgagt gcagcggtgg cggtggctcc 660

ggaggtggcg gttcagaggt gcagctggtg cagtccggcg ccgaggtgaa gaagcccggc 720

tcctccgtga aggtgtcctg caaggcctcc ggctactcct tcacctccta ctacatccac 780

tgggtgaggc aggcccccgg ccagtgcctg gagtggatgg gcaggatcgg ccccggctcc 840

ggcgacatca actacaacga gaagttcaag ggcagggcca ccttcaccgt ggacaagtcc 900

acctccaccg cctacatgga gctgtcctcc ctgaggtccg aggacaccgc cgtgtactac 960

tgcgccaggt tccactacga cggcgccgac tggggccagg gcaccctggt gaccgtgtcc 1020

tccggaggtg gcggttctgg cggtggcggt tccggtggcg gtggatcggg aggtggcggt 1080

tctgacatcc agatgaccca gtccccctcc tccctgtccg cctccgtggg cgacagggtg 1140

accatcacct gcaaggcctc ccagaacatc aacgagaacc tggactggta ccagcagaag 1200

cccggcaagg cccccaagct gctgatctac tacaccgaca tcctgcagac cggcatcccc 1260

tccaggttct ccggctccgg ctccggcacc gactacaccc tgaccatctc ctccctgcag 1320

cccgaggact tcgccaccta ctactgctac cagtactact ccggctacac cttcggctgc 1380

ggcaccaagc tggagatcaa gcgtacc 1407

<210> 65

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 65

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 66

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 66

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 67

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 67

Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser

1 5 10

<210> 68

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant sequence

<400> 68

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<---

Claims (119)

1. A multispecific antibody that binds to human IL-13, human IL-17A and human IL-17F, comprising an IL-13-binding region that comprises: variable region of the light chain containing CDR-L1 with the sequence SEQ ID NO:15, CDR-L2 with the sequence SEQ ID NO:16 and CDR-L3 with the sequence SEQ ID NO:17, and variable region of the heavy chain containing CDR-H1 with the sequence SEQ ID NO:18, CDR-H2 with the sequence SEQ ID NO:19, and CDR-H3 with the sequence SEQ ID NO:20, wherein said antibody comprises an antigen-binding region that binds to human IL-17A and human IL-17F, which comprises: variable region of the light chain containing CDR-L1 with the sequence SEQ ID NO:1, CDR-L2 with the sequence SEQ ID NO:2 and CDR-L3 with the sequence SEQ ID NO:3, and variable region of the heavy chain containing CDR-H1 with the sequence SEQ ID NO:4, CDR-H2 with the sequence SEQ ID NO:5, and CDR-H3 with the sequence SEQ ID NO:6, wherein said antibody comprises: 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 ; Where: V _H represents the variable domain of the heavy chain; CH ₁ represents domain 1 of the heavy chain constant region; X represents a bond or linker; Y represents a bond or linker; V ₁ is scFv, dsscFv or dsFv; V _L represents the variable domain of the light chain; C _L represents a domain from a light chain constant region such as Ccappa; V ₂ is scFv, dsscFv or dsFv; wherein the polypeptide chain of formula (Ia) comprises a protein A binding domain; and wherein the polypeptide chain of formula (IIa) does not bind to protein A; Where V _L and V _H contain an antigen-binding region that binds to human IL-17A and human IL17F, V ₂ contains an antigen-binding site that binds to human IL-13, and V ₁ contains an antigen-binding site that binds to human serum albumin; Where V _L contains CDR-L1 with the sequence SEQ ID NO:1, CDR-L2 with the sequence SEQ ID NO:2 and CDR-L3 with the sequence SEQ ID NO:3, and V _H contains CDR-H1 with the sequence SEQ ID NO:4, CDR-H2 with the sequence SEQ ID NO:5 and CDR-H3 with the sequence SEQ ID NO:6; Where V ₁ contains variable region of the light chain containing CDR-L1 with the sequence SEQ ID NO:39, CDR-L2 with the sequence SEQ ID NO:40 and CDR-L3 with the sequence SEQ ID NO:41, and variable region of the heavy chain containing CDR-H1 with the sequence SEQ ID NO:42, CDR-H2 with the sequence SEQ ID NO:43 and CDR-H3 with the sequence SEQ ID NO:44; and Where V ₂ contains variable region of the light chain containing CDR-L1 with the sequence SEQ ID NO:15, CDR-L2 with the sequence SEQ ID NO:16 and CDR-L3 with the sequence SEQ ID NO:17, and variable region of the heavy chain containing CDR-H1 with the sequence SEQ ID NO:18, CDR-H2 with the sequence SEQ ID NO:19 and CDR-H3 with the sequence SEQ ID NO:20. 2. The multispecific antibody of claim 1, wherein the IL-13-binding region comprises a light chain variable region comprising the sequence SEQ ID NO:27, and a heavy chain variable region comprising the sequence SEQ ID NO:28. 3. The multispecific antibody of claim 1, wherein the IL-13-binding region comprises a light chain variable region comprising the sequence SEQ ID NO:31, and a heavy chain variable region comprising the sequence SEQ ID NO:32. 4. The multispecific antibody of any one of claims 1 to 3, wherein the antigen-binding region that binds to human IL-17A and human IL-17F comprises a light chain variable region comprising the sequence SEQ ID NO:7, and a heavy chain variable region comprising the sequence SEQ ID NO:9. 5. A multispecific antibody according to any one of claims 1-4, wherein V _L contains the sequence SEQ ID NO:7, and V _H contains the sequence SEQ ID NO:9. 6. A multispecific antibody according to any one of claims 1 to 5, wherein V ₂ comprises a light chain variable region comprising the sequence SEQ ID NO:27, and a heavy chain variable region comprising the sequence SEQ ID NO:28. 7. A multispecific antibody according to any one of claims 1 to 5, wherein V ₂ comprises a light chain variable region comprising the sequence SEQ ID NO:31, and a heavy chain variable region comprising the sequence SEQ ID NO:32. 8. A multispecific antibody according to any one of claims 1 to 7, wherein V ₁ comprises a light chain variable region comprising the sequence of SEQ ID NO:45, and a heavy chain variable region comprising the sequence SEQ ID NO:46. 9. A multispecific antibody according to any one of claims 1 to 8, wherein V ₁ comprises a light chain variable region comprising the sequence SEQ ID NO:49, and a heavy chain variable region comprising the sequence SEQ ID NO:50. 10. A multispecific antibody according to any one of claims 1-8, where the variable region of the light chain and the variable region of the heavy chain V ₂ are connected by a linker, wherein said linker comprises the sequence SEQ ID NO:66. 11. The multispecific antibody of claim 10, wherein V ₂ is scFv comprising the sequence SEQ ID NO:35, or dsscFv containing the sequence SEQ ID NO:37. 12. A multispecific antibody according to any one of claims 1-11, where the variable region of the light chain and the variable region of the heavy chain V ₁ are connected by a linker, wherein said linker comprises the sequence SEQ ID NO:68. 13. The multispecific antibody according to claim 12, where V ₁ represents scFv comprising the sequence SEQ ID NO:53, or dsscFv containing the sequence SEQ ID NO:55. 14. A multispecific antibody according to any one of claims 1-13, wherein Y is a linker comprising the sequence SEQ ID NO:65. 15. A multispecific antibody according to any one of claims 1-14, wherein X is a linker comprising the sequence SEQ ID NO:67. 16. A multispecific antibody according to any preceding paragraph, comprising the sequence of SEQ ID NO:57 or SEQ ID NO:59. 17. A multispecific antibody according to any preceding paragraph, comprising the sequence of SEQ ID NO:61 or SEQ ID NO:63. 18. A multispecific antibody according to any preceding paragraph, comprising the sequence of SEQ ID NO:59 and the sequence of SEQ ID NO:63. 19. An isolated polynucleotide encoding a multispecific antibody according to any one of claims 1-18, wherein said polynucleotide is a DNA sequence for expressing said multispecific antibody. 20. An expression vector carrying the polynucleotide according to claim 19 for expressing the said multispecific antibody. 21. A host cell for expressing said multispecific antibody, wherein the cell contains the vector according to claim 20. 22. A method for producing a multispecific antibody according to any one of claims 1-15, comprising culturing the host cell according to claim 21 under conditions that ensure the production of the antibody, and isolation of the obtained antibody. 23. The method according to claim 22, including the step of purifying protein A. 24. A pharmaceutical composition for the treatment or prevention of atopic dermatitis, chronic hand eczema, micropolyposis or nasal polyposis, food allergy or eosinophilic esophagitis, wherein the composition comprises an antibody according to any one of claims 1-18 and a pharmaceutically acceptable auxiliary agent and/or carrier. 25. A method for treating or preventing atopic dermatitis, chronic hand eczema, micropolyposis or nasal polyposis, food allergy or eosinophilic esophagitis, comprising administering a therapeutically effective amount of a multispecific antibody according to claims 1-21 or a pharmaceutical composition according to claim 24 to a patient.