WO2014006230A1

WO2014006230A1 - Il-20 epitopes and il-20 ligands

Info

Publication number: WO2014006230A1
Application number: PCT/EP2013/064414
Authority: WO
Inventors: Jesper Pass; Jes Thorn Clausen; Anders Svensson; Rune SALBO; John Rømer NIELSEN; Søren Berg PADKJÆR; Hanne Benedicte RASMUSSEN
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 2012-07-06
Filing date: 2013-07-08
Publication date: 2014-01-09
Anticipated expiration: 2015-01-06

Description

IL-20 EPITOPES AND IL-20 LIGANDS

TECHNICAL FIELD

The present invention relates to neutralizing IL-20 ligands, such as e.g. antibodies. Such ligands are suitable for treatment of inflammatory diseases such as e.g. autoimmune diseases.

BACKGROUND

Antagonizing IL-20 activity, using e.g. antagonistic IL-20 antibodies, has turned out to be a promising approach for treatment of various inflammatory diseases.

Monoclonal and polyclonal rat, murine and fully human antibodies specific for IL-20 are both commercially available from different providers such as Abgen, AbNova and AbCam and have also been previously disclosed in patent literature e.g. WO2005052000,

US20060142550, WO2010042634 and WO2007081465. WO2010000721 fully discloses the following monoclonal IL-20 antibodies: 2F6, F56, F18, C3, 5B7, 15D2, while the following antibodies are mentioned and some of their properties disclosed, without full disclosure (sequence and thereby full availability): C1 1 , 41 A6, 41 F10, 42A5, and 54F10.

There is thus a need in the art for additional potent IL-20 antibodies for treating inflammatory diseases. Such antibodies should preferably bind to IL-20 with a high affinity and inhibit/neutralize IL-20 induced effects. Preferably, such antibodies are useful in connection with various assays.

SUMMARY OF THE INVENTION

We herein define novel epitopes on IL-20, designated "BS1 " (binding site 1 or Site 1 ) and "BS2" (binding site 2 or Site2), respectively. Binding of an IL-20 ligand, e.g. an antibody, to BS1 , competes with and/or interferes with binding of IL-20R1 to IL-20 via BS1 , but does not interfere with binding of IL-20R2 to IL-20 via BS2. Likewise, binding of an IL-20 ligand, e.g. an antibody, to BS2, competes with or interferes with binding of IL-20R2 to IL-20 via BS2, but does not interfere with binding of IL-20R1 to IL-20 via BS1 . We also describe antibodies herein that inhibit formation of the IL-20:IL-20R1 :IL-20R2 complex or competes with binding of either IL-20R1 or IL-20R2 or both to IL-20 by steric hindrance. We also describe IL-20 ligands, such as antibodies, which bind specifically to the epitopes according to the invention, as well as methods for making and using such ligands.

IL-20 ligands interfering with BS1 for IL-20 biniding to IL-20R1 will interfere with a similar binding site for IL-20 biniding to IL-22R. Distinctive features of IL-20 ligands according to the invention are their ability to compete with or interfere with binding of IL-20R1 to IL-20, while IL-20 complexed with the ligand will maintain an IL-20R2 binding competent BS2 - and to compete with or interfere with binding of IL-20R2 to IL-20, while IL-20 complexed with the ligand will maintain an IL-20R1 binding competent BS1 . Accordingly, ligands of the present invention will, in the presence of IL-20, form ligand:IL-20 complexes having the ability to bind specifically, and with high affinity, to IL-20:R2, IL-20:R1 or IL-22R present on cell surfaces.

Ligands according to the invention may furthermore have the ability to block assembly of the IL-20:IL-20R1 :IL-20R2 complex or IL-20:IL-22R:IL-20R2 complex e.g. by steric hindrance. Ligands of the invention may thus have improved potency due to the combined neutralizing and receptor blocking properties.

Ligands according to the invention may furthermore have the ability to compete with binding of either IL-20R1 , IL-20R2 or IL-22F or both IL-20R1 and IL-20R2 to IL-20 or both IL- 22R and IL-20R2 by binding to a binding site different than BS1 and BS2 while mediating a steric overlap. Generally, a ligand of the invention will bind to IL-20 and form a ligand:IL-20 complex which retains a competent BS2 (and/or BS1 ) and thereby the ability to bind with high affinity to IL-20R2 and/or IL-20R1 and /or IL-22R.

SEQUENCES

SEQ ID NO 1 : mature human IL-20:

LKTLNLGSCVIATNLQEIRNGFSEIRGSVQAKDGNIDIRILRRTESLQDTKPANRCCLLRHLLRL

YLDRVFKNYQTPDHYTLRKISSLANSFLTIKKDLRLCHAHMTCHCGEEAMKKYSQILSHFEKLE

PQAAVVKALGELDILLQWMEETE SEQ ID NO 2: extracellular part of human IL-20R1 :

VPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINR TYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISWL TAPEKWKRNPEDLPVSMQQIYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCV HVESFVPGPPRRAQPSEKQCARTLKDQSSEFKAK

SEQ ID NO 3: extracellular part of human IL-20R2:

DEVAILPAPQNLSVLSTNMKHLLMWSPVIAPGETVYYSVEYQGEYESLYTSHIWIPSSWCSLT EGPECDVTDDITATVPYNLRVRATLGSQTSAWSILKHPFNRNSTILTRPGMEITKDGFHLVIEL EDLGPQFEFLVAYWRREPGAEEHVKMVRSGGIPVHLETMEPGAAYCVKAQTFVKAIGRYSAF SQTECVEVQGEAIPL

SEQ ID NO 4: Heavy chain variable part of 15D2

QVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQY SQNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGT TVTVSS SEQ ID NO 5: Light chain variable part of 15D2

AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKR

SEQ ID NO 6: Heavy chain variable part of 5B7

QVQLVQSGAEVKRPGASVKVSCKASGYTFSSHIMHWVRQAPGQRLEWMGWINAGYGNTKY SQNFQDRVTITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGELSPHDYYGMDVWGQGT TVTVSS

SEQ ID NO 7: Light chain variable part of 5B7

AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKR SEQ ID NO 8: Heavy chain variable part of 2F6

QITLKESGPTLVKPTQTLTLTCTFSGFSFSTNGVGVGWIRQPPGKALEWLALIYWNDDKRYSP SLKSRLTITKDTSKNQWLTMTNMDPLDTATYYCAHSPFIMVRGVIITFFDFWGQGTLVTVSS

SEQ ID NO 9: Light chain variable part of 2F6

VIWMTQSPSLLSASTGDRVTISCRMSQGISSYLAWYQQKPGKAPELLIYAASTLQSGVPSRFS GSGSGTDFTLTISCLQSEDFATYYCQQYYSFPLTFGGGTKVEIKR SEQ ID NO 10: Extracellular part of human IL-22R1

HAPEDPSDLLQHVKFQSSNFENILTWDSGPEGTPDTVYSIEYKTYGERDWVAKKGCQRITRK SCNLTVETGNLTELYYARVTAVSAGGRSATKMTDRFSSLQHTTLKPPDVTCISKVRSIQMIVH PTPTPIRAGDGHRLTLEDIFHDLFYHLELQVNRTYQMHLGGKQREYEFFGLTPDTEFLGTIMIC VPTWAKESAPYMCRVKTLPDRTWT

SEQ ID NO 11 : anti-IL20 7F1A1 VH (signal peptide sequence omitted)

QVQLQQSGAE LVRPGAAVTL SCKASDYTFT DFEMHWVKQT PVHGLKWIGVIDPETDGTAY NQKFKDKATL TADKSSSTAY MEIRSLTSED SAVYYCTRLLWLPFDFWGQG TTLTVSS SEQ ID NO 12: anti-IL20 7F1A1 VL (signal peptide sequence omitted)

DIVMTQSPSS LAMSVGQKVT MNCRSSQSLL NTSNQKNYLAWYQQKPGQSPKLLVHFASTR ESGVPDRFMG SGSGTDFTLT ISSVQAEDLA DYFCHQYYSTPLTFGAGTKL ELKR

SEQ ID NO 13: anti-IL20 1 1 F12 VH (signal peptide sequence omitted)

QVHLQQSGAELARPGASVKL SCKASGYTFTNYWMQWVKQRPGQGLEWIGAIYPGDGDTRY TQKFKGKATL TADKSSSTAY MQLSSLASED SAVYYCARWLLPLMDYWGQG TSVTVSS

SEQ ID NO 14: anti-IL20 1 1 F12 VL (signal peptide sequence omitted)

QIVLTQSPAI MSASPGEKVT ITCSASSSVN YIHWFQQKPG TSPKLWIYGTSKLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR SSYPYTFGGGTKLEIKR

SEQ ID NO 15: anti-IL20 1 1 F36 VH (signal peptide sequence omitted)

EVQLQESGPS LMKPSQTLSL TCSVTGDSIT SGYWYWVRKF PGNKLEYLGYISYSGSTDYN PSLKSRISIT RDTSNNQHYL QLNSVTAEDT ATYYCARPIYDGYYIPFDYW GQGTLVTVSA

SEQ ID NO 15: anti-IL20 1 1 F36 VL (signal peptide sequence omitted)

DIVMTQSQKF MSTTVGDRVS ITCKASQNVG TAVAWYQQKPGQSPKLLLYSASNRYTGVPD RFTGSGSGTD FTLTISNMQS EDLADYFCQQ YSSYPLTFGSGTKLEMKR

Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention employs unless otherwise indicated, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology and pharmacology, known to those skilled in the art. Definitions

"IL-20"/"IL20" refers, unless otherwise specifically stated, to human interleukin 20 or IL- 20 also known as Zcytol O. The amino acid sequence of mature human IL-20 is shown in SEQ ID NO 1. IL-19, IL-20, and IL-24 are members of the interleukin-10 (IL-10) cytokine family. All three interleukins bind and signal through the IL-20R1/IL-20R2 heterodimeric receptor, whereas IL-20 and IL-24 can also bind and signal through the IL-22R/IL-20R2 heterodimeric receptor. IL-22 binds and signals through IL-22RIIL:10R3 heterodimeric receptor. It has been proposed that IL-19 and IL-20 along with other IL-10 family members, form a distinct subfamily of helical cytokines where at least IL-19 and IL-20 have similar three dimensional structures. The crystal structure (see Example 1 . of present application) of human IL-20 bound to IL-20R1 and IL-20R2 has not previously been resolved (Kotenko SV. Cytokine Growth Factor Rev. 2002 Jun; 13(3):223-40.)

The term "interaction site", as used herein, is defined in the context of a molecular interaction between two molecules binding to each other. It could for example be between a receptor chain and its ligand or between two recptor chains forming a receptor complex.

Generally, "interaction site" refers to the area or region on a receptor or ligand to which a receptor specifically binds, i.e. the area or region in physical contact between the receptor and ligand, alternatively between two receptor receptor chains. Physical contact may be defined through distance criteria (e.g. a distance cut-off of 4 A) between atoms in the ligand and the receptor molecules, or between two receptor chains.

Binding Site 1 (also referred to as BS1 or Site 1 in this application) is the site of of IL-20 and IL-20R1 where the interaction between IL-20 and IL-20R1 takes place. Accordingly, Binding Site 2 (also referred to in this application as BS2 or Site 2) is the site of IL-20 and IL-20R2 where the interaction between IL-20 and IL-20R2 takes place.

And Binding Site 3 (also referred to in this application BS2 or Site 3) is the site of IL- 20R1 and IL-20R2 where interaction between IL-20R1 and IL-20R2 takes place.

Receptors as reffered to in this application are protein molecules, embedded in either the plasma membrane (cell surface receptors) or the cytoplasm or nucleus (nuclear receptors) of a cell, to which one or more specific kinds of signaling molecules may attach. A molecule which binds (attaches) to a receptor is referred to as a ligand, and may be a peptide (short protein) or other small molecule, such as a neurotransmitter, a hormone, a pharmaceutical drug, or a toxin.

Liaands interfering with IL-20R1 and/or IL-20R2 and/or IL-22R binding to IL-20:

Ligands according to the invention, that have the ability to interfere with binding of IL-20R1 and/or IL-20R2 and/or IL-22R to IL-20, does as used within this application mean ligands that bind to IL-20 and in doing so either directly competes with IL-20R1 , IL-20R2, IL-22R or both IL- 20R1 and IL-20R2 or both IL-22R and IL-20R2 for binding to IL-20, or reduces IL-20R1 , IL- 20R2, IL-22R or both IL-20R1 and IL-20R2 or both IL-22R and IL-20R2 ability to bind to or its/their affinity for IL-20. This means that ligands according to the invention may bind to an epitope that either overlaps with or is situated close enough to BS1 to provide sterical hindrance for I L-20R1 -binding or IL-22R-binding (or close enough to BS2 to provide sterical hindrance for IL-20R2) and thereby reducing its ability to bind to IL-20R1 , IL-20R2 or IL-22R by at least 25%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 90%, and most preferably at least 95%. It follows that the BS1 epitope on IL-20 is well separated from BS2. Interference with receptor binding to IL-20 can be detected by e.g. Surface Plasmon Resonance (SPR). "Interferes with/competes with" means that no significant IL-20:IL-20R1 (and/or IL-20:IL-20R2, and/or IL-20:IL-22R) binding occurs when said antibody is bound to IL-20 - preferably meaning at least a 90% reduction (preferably at least a 95% reduction) in IL-20:IL-20R1 (and/or IL-20:IL-20R2, and/or IL-20:IL-22R) binding compared to e.g. a control antibody. The same applies for the term "competes/interferes with assembly of the IL-20:IL-20R1 :IL-20R2 complex or the IL-20:IL-22R:IL-20R2 complex.

Furthermore, ligands according to the invention may have the ability to interfere with the bioactivity by binding to an epitope that either overlaps with or is situated close enough to BS1 to provide sterical hindrance for IL-20R1-binding or IL-22R-binding (or close enough to BS2 to provide sterical hindrance for IL-20R2) and thereby reducing its ability to bind to IL- 20R1 , IL-20R2 or IL-22R and hereby reduces its ability for inducing bioactivity by at least 25%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 90%, and most preferably at least 95%. Screening for binding activity (or any other desired activity) is conducted according to methods well known in the art, for instance SPR (Surface Plasmon Resonance), FACS, ELISA,Laser scanning imager etc. Screening can also be conducted by functional screenings, for instance using IL-20R transfected cells in the Baf/3 cell proliferation or STAT-3 Luciferase assays, Screening allows selection of members of a repertoire according to desired characteristics.

The term "treatment", as used herein, refers to the medical therapy of any human or other animal subject in need thereof. Said subject is expected to have undergone physical examination by a medical or veterinary medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to the health of said human or other animal subject. The timing and purpose of said treatment may vary from one individual to another, according to the status quo of the subject's health. Thus, said treatment may be prophylactic, palliative, symptomatic and/or curative. In terms of the present invention, prophylactic, palliative, symptomatic and/or curative treatments may represent separate aspects of the invention.

As used herein, an "isolated" compound is a compound that has been removed from its natural environment.

Antibodies: The term "antibody" as referred to herein refers to a poly-peptide derived from a germline immunoglobulin sequence. The term includes full-length antibodies and any antigen binding fragment as e.g. Fab fragments, and other monovalent antibodies. The term "antibody", "monoclonal antibody" and "mAb" as used herein, is intended to refer to

immunoglobulin molecules and fragments thereof that have the ability to specifically bind to an antigen. A sub-class of the immunoglobulin's of particular pharmaceutical interest are those belonging to the IgG family, which can be sub-divided into the iso-types lgG1 , lgG2, lgG3 and lgG4. IgG molecules are composed of two heavy chains interlinked by two or several disulphide bonds and two light chains, one attached to each of the heavy chains by a disulphide bond. The IgG heavy chain is composed of four Ig-domains, including the variable domain (VH) and three constant domains (CH1 , CH2, and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.

Examples of antigen-binding fragments include Fab, Fab', F (ab) 2, F (ab') 2, F (ab) S, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv; see e.g. Bird et al., Science 1988; 242:42S-426; and Huston et al. PNAS 1988; 85:5879- 5883), dsFv, Fd (typically the VH and CHI domain), and dAb (typically a VH domain) fragments; VH, VL, VhH, and V-NAR domains; monovalent molecules comprising a single VH and a single VL chain; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al. Protein Eng 1997;10:949-57); camel IgG; IgNAR; as well as one or more isolated CDRs or a functional paratope, where the isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005;2S:1 126-1 136; WO2005040219, and published U.S. Patent Applications 20050238646 and 20020161201 .

The Fc domain of an antibody according to the invention may be modified in order to modulate certain effector functions such as e.g. complement binding and/or binding to certain Fey receptors. The Fc domain may furthermore be modulated in order to increase affinity to the neonatal Fc receptor (FcRn). Mutations in positions 234, 235 and 237 (residue numbering according to the EU index) in an lgG1 Fc domain will generally result in reduced binding to the FcyRI receptor and possibly also the FcyRlla and the FcyRIII receptors. These mutations do not alter binding to the FcRn receptor, which promotes a long circulatory half life by an endocytic recycling pathway. Preferably, a modified lgG1 Fc domain of an antibody according to the invention comprises one or more of the following mutations that will result in decreased affinity to certain Fey receptors (L234A, L235E, and G237A) and in reduced C1 q-mediated complement fixation (A330S and P331 S), respectively (residue numbering according to the EU index). Alternatively, the Fc domain may be an lgG4 Fc domain optionally comprising the S241 P/S228P mutation (S241 P denotes residue numbering according to Kabat, S228P denotes residue numbering according to the EU numbering system (Edelman G.M. et AL., Proc. Natl. Acad. USA 63, 78-85 (1969).

The term "human antibody", as used herein, means antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.

However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences, e.g. the so-called "humanized antibodies" or human/mouse chimera antibodies. The term "chimeric antibody" or "chimeric antibodies" refers to antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable segments of genes from a mouse monoclonal antibody may be joined to human constant segments.

Half life extending moiety: The antibodies according to the invention may be modified in order to increase serum half-life, for example, by adding molecules - such as fatty acids or fatty acid derivates, PEG (poly ethylene glycol) or other water soluble polymers, including polysaccharide polymers to increase circulatory half-life. "Protractive groups'V'half life extending moiety" is herein understood as one or more chemical groups attached to one or more amino acid site chain functionalities such as -SH, -OH, -COOH, -CONH2, -NH2, or one or more N- and/or O-glycan structures and that can increase in vivo circulatory half life of a number of therapeutic proteins/peptides when conjugated to these proteins/peptides.

Examples of protractive groups/half life extending moiety include but not limited to are:

Biocompatible fatty acids and derivatives thereof, Hydroxy Alkyl Starch (HAS) e.g. Hydroxy Ethyl Starch (HES), Poly Ethylene Glycol (PEG), Poly (Glyx-Sery)n (HAP), Hyaluronic acid (HA), Heparosan polymers (HEP), Phosphorylcholine-based polymers (PC polymer),

Fleximers, Dextran, Poly-sialic acids (PSA), an Fc domain, Transferrin, Albumin, Elastin like peptides, XTEN polymers, Albumin binding peptides, a CTP peptide, and any combination thereof.

Binning/competition binding: Antibodies binding to the same antigen can be characterized with respect to their ability to bind to their common antigen simultaneously. Antibodies may be subjected to "binning", which term in the present context refers to a method of grouping antibodies that bind to the same antigen. "Binning" of antibodies may be based on competition binding of two antibodies to their common antigen in assays based on standard techniques such as surface plasmon resonance (SPR), ELISA or flow cytometry.

A "bin" is defined by a reference antibody. If a second antibody is unable to bind to the antigen at the same time as the reference antibody, the second antibody is said to belong to the same "bin" as the reference antibody, In this case the reference and the second antibody are competing for binding to the antigen, thus the pair of antibodies is termed "competing antibodies". If a second antibody is capable of binding to the antigen at the same time as the reference antibody, the second antibody is said to belong to a separate "bin". In this case the reference and the second antibody are not competing for binding to the antigen, thus the pair of antibodies is termed "non-competing antibodies". Antibody "binning" does not provide direct information about the epitope. Competing antibodies, i.e. antibodies belonging to the same "bin" may have identical epitopes, overlapping epitopes or even separate epitopes. The latter is the case if the reference antibody bound to its epitope on the antigen takes up the space required for the second antibody to contact its epitope on the antigen ("steric hindrance"). Non-competing antibodies have separate epitopes.

Epitope, paratope and antigen: The term "epitope", as used herein, is defined in the context of a molecular interaction between an "antigen binding molecule", such as an antibody (Ab), and its corresponding "antigen" (Ag). The term antigen (Ag) may refer to the molecular entity used for immunization of an immunocompetent vertebrate to produce the antibody (Ab) that recognizes the Ag. Herein, Ag is termed more broadly and is generally intended to include target molecules that are specifically recognized by the Ab, thus including fragments or mimics of the molecule used in the immunization process for raising the Ab. Generally, "epitope" refers to the area or region on an Ag to which an Ab specifically binds, i.e. the area or region in physical contact with the Ab. Physical contact may be defined through distance criteria (e.g. a distance cut-off of 4 A) for atoms in the Ab and Ag molecules. A "discontinuous epitope" is an epitope which is formed by two or more regions of a polypeptide which are not adjacent to each other in the linear peptide sequence, but which are arranged in the three-dimensional structure of the polypeptide to form a structural epitope. Other types of epitopes include: linear peptide epitopes, conformational epitopes which consist of two or more non-contiguous amino acids located near each other in the three-dimensional structure of the antigen; and post-translational epitopes which consist, either in whole or part, of molecular structures covalently attached to the antigen, such as carbohydrate groups.

The epitope for a given antibody (Ab)/antigen (Ag) pair can be defined and

characterized at different levels of detail using a variety of experimental and computational epitope mapping methods. The experimental methods include mutagenesis, X-ray

crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy and Hydrogen deuterium exchange Mass Spectrometry (HX-MS), methods that are known in the art. As each method relies on a unique principle, the description of an epitope is intimately linked to the method by which it has been determined. Thus, depending on the epitope mapping method employed, the epitope for a given Ab/Ag pair will be described differently. At its most detailed level, the epitope for the interaction between the Ag and the Ab can be described by the spatial coordinates defining the atomic contacts present in the Ag-Ab interaction, as well as information about their relative contributions to the binding

thermodynamics. At a less detailed level, the epitope can be described by the spatial coordinates defining the atomic contacts between the Ag and Ab. At an even less detailed level the epitope can be described by the amino acid residues that it comprises as defined by a specific criteria such as the distance between atoms in the Ab and the Ag. At a further less detailed level the Ab-Ag interaction can be characterized through function, e.g. by competition binding with other Abs and "binning" although competition binding does not provide any structural information about the epitope.

In the context of an X-ray derived crystal structure defined by spatial coordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag, the term epitope is herein, unless otherwise specified or contradicted by context, specifically defined as IL20 residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of about 3.5 to about 5.0 A, such as e.g. 4 A from a heavy atom in the Ab.

From the fact that descriptions and definitions of epitopes, dependant on the epitope mapping method used, are obtained at different levels of detail, it follows that comparison of epitopes for different Abs on the same Ag can similarly be conducted at different levels of detail.

Epitopes described on the amino acid level, e.g. determined from an X-ray structure, are said to be identical if they contain the same set of amino acid residues. Epitopes are said to overlap if at least one amino acid is shared by the epitopes. Epitopes are said to be separate (unique) if no amino acid residue are shared by the epitopes.

The definition of the term "paratope" is derived from the above definition of "epitope" by reversing the perspective. Thus, the term "paratope" refers to the area or region on the Ab to which an Ag specifically binds, i.e. with which it makes physical contact to the Ag.

In the context of an X-ray derived crystal structure, defined by spatial coordinates of a complex between an Ab, such as a Fab fragment, and its Ag, the term paratope is herein, unless otherwise specified or contradicted by context, specifically defined as Ab residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of about 4 A (3.5 to 5.0 A) from a heavy atom in IL20. The term binding region of an antigen refers to a region on an antigen, defined by amino acid sequence, where to one or more antigen binding molecules, such as antibodies bind. A binding region therefore comprises at least one epitope, but can also comprise multiple epitopes of multiple antibodies, both identical epitopes, overlapping epitopes and possible separate epitopes. A binding region can be discontinious, where it is formed by two or more regions of a the antigen polypeptide which are not adjacent to each other in the linear peptide sequence, but which are arranged in the three-dimensional structure of the polypeptide to form a structural binding region. The binding region can also be linear peptide region on the antigen, or a conformational binding region which consist of two or more non-contiguous amino acids located near each other in the three-dimensional structure of the antigen; and post-translational binding region which consist, either in whole or part, of molecular structures covalently attached to the antigen, such as carbohydrate groups. Epitope, paratope and antigen: The term "epitope", as used herein, is defined in the context of a molecular interaction between an "antigen binding molecule", such as an antibody (Ab), and its corresponding "antigen" (Ag).

Binding affinity between two molecules, e.g. an antibody, or fragment thereof, and an antigen, through a monovalent interaction may be quantified by determination of the equilibrium dissociation constant (KD). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the SPR method. The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and dissociation rate constant kd (or koff), respectively. KD is related to ka and kd through the equation KD = kd / ka. Following the above definition, binding affinities associated with different molecular interactions, such as comparison of the binding affinity of different antibodies for a given antigen, may be compared by comparison of the KD values for the individual antibody/antigen complexes. Preferably, antibodies according to the present invention have a relatively high affinity, i.e. a binding affinity of 10-7 M or smaller, preferably 10-8 M or smaller, preferably 10-9 M or smaller, most preferably preferably 10-10 M or smaller.

Structure of ligands: As described above, a ligand as referred to herein may be an antibody (for example IgG, IgM, IgA, IgE) or fragment thereof (for example Fab, Fv, disulphide linked Fv, scFv, diabody) which comprises at least one heavy and a light chain variable domain which are complementary to one another and thus can associate with one another to form a VH/VL pair. It may be derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, mammalian cells, yeast or bacteria. Therapeutic Applications: The ligands/antibodies according to invention can be used in the treatment of diseases involving an inappropriate or undesired immune response (in this application reffered to as immunological disorders), such as inflammation, autoimmunity, and conditions involving such mechanisms as well as graft vs. host disease. In one embodiment, such disease or disorder is an autoimmune and/or inflammatory disease. Examples of such autoimmune and/or inflammatory diseases are Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), osteoporosis, inflammatory bowel disease (IBD) (including ulcerative colitis (UC) and Crohn's disease (CD)), multiple sclerosis (MS), scleroderma and type 1 diabetes (T1 D), and other diseases and disorders, such as PV (pemphigus vulgaris), psoriasis, psoriatic arthritis, atopic dermatitis, celiac disease, Chronic obstructive lung disease, Hashimoto's thyroiditis, Graves' disease (thyroid), Sjogren's syndrome, Guillain-Barre syndrome, Goodpasture's syndrome, Addison's disease, Wegener's granulomatosis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, polymyalgia rheumatica, Raynaud's phenomenon, temporal arteritis, giant cell arteritis, autoimmune haemolytic anaemia, pernicious anaemia, polyarteritis nodosa, behcet's disease, primary biliary cirrhosis, uveitis, myocarditis, rheumatic fever, ankylosing spondylitis, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, alopecia areata, type I diabetes, Colitis- Associated Tumorigenesis, metastasis-associated osteolysis and vitiligo.

The antibodies of the present invention may be administered in combination with other medicaments as is known in the art. The present invention further includes pharmaceutical compositions/formulations, comprising a pharmaceutically acceptable carrier and a polypeptide/ligand/antibody according to the invention as well as kits comprising such compositions. The pharmaceutical composition according to the invention may be in the form of an aqueous formulation or a dry formulation that is reconstituted in water/an aqueous buffer prior to administration. Pharmaceutical compositions comprising ligands/antibodies/polypeptides according to the invention may be supplied as a kit comprising a container that comprises the compound according to the invention. Therapeutic polypeptides can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Pharmaceutical compositions comprising compounds according to the invention are suitable for subcutaneous and/or IV administration.

Combination treatment: antibodies according to the invention may be co-administered with one or more other therapeutic agents or formulations. The other agent may be intended to treat other symptoms or conditions of the patient. For example, the other agent may be an analgesic, an immunosuppressant or an anti-inflammatory agent.

Combined administration of two or more agents may be achieved in a number of different ways. In one embodiment, the antibody and the other agent may be administered together in a single composition. In another embodiment, the antibody and the other agent may be administered in separate compositions as part of a combined therapy. For example, the modulator may be administered before, after or concurrently with the other agent.

The antibodies/proteins according to the present invention may be administered along with other drugs (e.g. methotrexate, dexamethasone, and prednisone) and/or other biological drugs. Agents already in use in autoimmunity include immune modulators such as IFNbeta, Orencia (CTLA4-lg), Humira (anti-TNF), Cimzia (anti-TNF, PEG Fab), Tysabri (a4-integrin mAb), Simponi, Rituxan/MabThera, Actemra/RoActemra, Kineret, Non-steroidal antiinflammatory drugs (NSAIDS) like Aspirin, Ibuprofen etc., Corticosteroids, disease-modifying antirheumatic drugs (DMARDS) like Plaquenil, Azulfidine, Methotrexate etc., Copaxone (glatirimer acetate), Gilneya (fingolimod), Antibiotics like Flagyl, Cipro, Topical (skin applied) medications including topical corticosteroids, vitamin D analogue creams (Daivonex), topical retinoids (Tazorac), moisturizers, topical immunomodulators (tacrolimus and pimecrolimus), coal tar, anthralin, and others, Raptiva, Ustekinumab, light therapy like PUVA, UVB, CellCept (mycophenolate mofetil).

Embodiments The following list of embodiments represents examples of embodiments of the present invention and should thus not be understood as limiting the invention.

Embodiment 1 : An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 10-50 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 130-152 of SEQ ID NO 1. Embodiment 2: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 1 1 -49 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 130-152 of SEQ ID NO 1. Embodiment 3: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 12-48 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 130-152 of SEQ ID NO 1.

Embodiment 4: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 13-47 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 130-152 of SEQ ID NO 1.

Embodiment 5: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 14-46 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 130-152 of SEQ ID NO 1.

Embodiment 6: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 15-45 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 130-152 of SEQ ID NO 1.

Embodiment 7: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 16-44 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 130-152 of SEQ ID NO 1. Embodiment 8: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 16-43 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 130-152 of SEQ ID NO 1.

Embodiment 9: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 10-50 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 131 -151 of SEQ ID NO 1.

Embodiment 10: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 10-50 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 132-150 of SEQ ID NO 1.

Embodiment 11 : An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 10-50 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 133-149 of SEQ ID NO 1.

Embodiment 12: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 10-50 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 134-148 of SEQ ID NO 1. Embodiment 13: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 10-50 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 135-147 of SEQ ID NO 1.

Embodiment 14: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 10-50 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 136-146 of SEQ ID NO 1.

Embodiment 15: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 1 1 -49 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 131 -151 of SEQ ID NO 1.

Embodiment 16: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 12-48 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 132-150 of SEQ ID NO 1. Embodiment 17: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 13-47 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 133-149 of SEQ ID NO 1. Embodiment 18: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 14-46 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 134-148 of SEQ ID NO 1.

Embodiment 19: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 15-45 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 135-147 of SEQ ID NO 1.

Embodiment 20: An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 16-44 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 136-146 of SEQ ID NO 1.

Embodiment 21 : An antibody or an antibody fragment thereof, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein the first binding part is defined by amino acids no. 16-43 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 136-146 of SEQ ID NO 1.

Embodiment 22: An antibody or an antibody fragment according to any preceding embodiment, wherein said antibody binds to both binding parts of a discontinuous binding region on IL-20, wherein first part of said discontinuous binding region comprises one or more of the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39 and Arg 43, and second part of said discontinuous binding region comprises one or more of the following amino acids of SEQ ID NO 1 : Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 23: An antibody or an antibody fragment according to any preceding embodiment, which binds to a binding region on IL-20, wherein said binding region comprises one or more (preferably two or more, preferably three or more, preferably four or more, preferably five or more, preferably six or more, preferably seven or more, preferably eight or more, preferably nine or more, preferably ten or more) of the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, and Arg 43, and one or more (preferably two or more, preferably three or more, preferably four or more, preferably five or more) of the following amino acids: Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146. Embodiment 24: An antibody or an antibody fragment according to any preceding

embodiment, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 25: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 26: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 27: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 28: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143. Embodiment 29: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 30: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 31 : An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 32: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142, and lie 143.

Embodiment 33: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146. Embodiment 34: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 35: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 36: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 37: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143. Embodiment 38: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146. Embodiment 39: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 40: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 41 : An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 42: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 43: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142, 143 and Gin 146. Embodiment 44: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 45: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 46: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly

139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 47: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 48: An antibody or an antibody fragment according to any of embodiments 1 -23, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg

19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu

140, Asp 142 and lie 143. Embodiment 49: An antibody or an antibody fragment which binds to an binding region on IL-

20, wherein said binding region comprises one or more (preferably two or more, preferably three or more, preferably four or more, preferably five or more, preferably six or more, preferably seven or more, preferably eight or more, preferably nine or more, preferably ten or more) of the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, and Arg 43.

Embodiment 50: An antibody according to embodiment 49, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, and Arg 43.

Embodiment 51 : An antibody according to embodiment 49, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, and Arg 43.

Embodiment 52: An antibody according to embodiment 49, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, and Arg 43. Embodiment 53: An antibody according to embodiment 49, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38 or Arg 39.

Embodiment 54: An antibody according to embodiment 49, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38 and Arg 39.

Embodiment 55: An antibody according to embodiment 49, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38 and Arg 39. Embodiment 56: An antibody which binds to an binding region on IL-20, wherein said binding region comprises one or more (preferably two or more, preferably three or more, preferably four or more, preferably five or more) of the following amino acids of SEQ ID NO 1 : Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 57: An antibody according to embodiment 56, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 58: An antibody according to embodiment 56, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gly 139, Glu 140, Asp 142, lie 143 and Gin 146. Embodiment 59: An antibody according to embodiment 56, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Lys 136, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 60: An antibody according to embodiment 56, wherein said binding region comprises the following amino acids of SEQ ID NO 1 : Gly 139, Glu 140, Asp 142 and lie 143. Embodiment 61 : An antibody according to any one of the preceding embodiments, wherein said antibody binds to an binding region comprising one or more or all of the the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146. Embodiment 62: An antibody according to any one of the preceding embodiments, wherein said antibody interferes with/competes with binding of IL20R1 to IL-20. Preferably, such antibodies do not interfere with/compete with IL-20R2 for binding to IL-20.

Embodiment 63: An antibody that interferes with/competes with the binding of IL-20R1 to IL- 20 or with the binding of IL-22R to IL-20. Preferably, such antibodies do not interfere with/compete with IL-20R2 for binding to IL-20.

Embodiment 64: An antibody which binds to an epitope on IL-20, wherein said epitope comprises one or more (preferably two or more, preferably three or more, preferably four or more, preferably five or more, preferably six or more, preferably seven or more, preferably eight or more, preferably nine or more, preferably ten or more) of the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, and Arg 43, and one or more (preferably two or more, preferably three or more, preferably four or more, preferably five or more) of the following amino acids: Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146. Embodiment 65: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 66: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 67: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 68: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 69: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143. Embodiment 70: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 71 : An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 72: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie

36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu 140, Asp 142 and lie 143. Embodiment 73: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp

37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142, and lie 143.

Embodiment 74: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 75: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 76: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 77: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143. Embodiment 78: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 79: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146. Embodiment 80: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 81 : An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 82: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143. Embodiment 83: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 84: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142, 143 and Gin 146.

Embodiment 85: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Gly 139, Glu 140, Asp 142 and lie 143. Embodiment 86: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 87: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 88: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146. Embodiment 89: An antibody according to embodiment 64, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 90: An antibody which binds to an epitope on IL-20, wherein said epitope comprises one or more (preferably two or more, preferably three or more, preferably four or more, preferably five or more, preferably six or more, preferably seven or more, preferably eight or more, preferably nine or more, preferably ten or more) of the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, and Arg 43. Embodiment 91 : An antibody according to embodiment 90, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, and Arg 43.

Embodiment 92: An antibody according to embodiment 90, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, and Arg 43.

Embodiment 93: An antibody according to embodiment 90, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, and Arg 43.

Embodiment 94: An antibody according to embodiment 90, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38 or Arg 39.

Embodiment 95: An antibody according to embodiment 90, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie

36, Asp 37, lie 38 and Arg 39. Embodiment 96: An antibody according to embodiment 90, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp

37, lie 38 and Arg 39.

Embodiment 97: An antibody which binds to an epitope on IL-20, wherein said epitope comprises one or more (preferably two or more, preferably three or more, preferably four or more, preferably five or more) of the following amino acids of SEQ ID NO 1 : Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146. I -

Embodiment 98: An antibody according to embodiment 98, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 99: An antibody according to embodiment 98, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 100: An antibody according to embodiment 98, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Lys 136, Gly 139, Glu 140, Asp 142 and lie 143.

Embodiment 101 : An antibody according to embodiment 98, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Gly 139, Glu 140, Asp 142 and lie 143. Embodiment 102: An antibody according to any one of embodiments 64-101 , wherein said antibody binds to an epitope comprising one or more or all of the the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

Embodiment 103: An antibody according to any one of the preceding embodiments, wherein said antibody interferes with/competes with binding of IL20R1 to IL-20. Preferably, such antibodies do not interfere with/compete with IL-20R2 for binding to IL-20.

Embodiment 104: An antibody or an antibody fragment therof that competes with one or more of the antibodies IL20-1 F13, IL20-7F1 , IL20-1 1 F12, IL20-1 1 F25 or IL20-1 1 F36 for binding to IL-20. Embodiment 105: An antibody or an antibody fragment therof that competes with antibody IL20-1 F13 for binding to IL-20.

Embodiment 106: An antibody or an antibody fragment therof that competes with antibody IL20-7F1 for binding to IL-20.

Embodiment 107: An antibody or an antibody fragment therof that competes with antibody IL20-1 1 F12 for binding to IL-20.

Embodiment 108: An antibody or an antibody fragment therof that competes with antibody IL20-1 1 F25 for binding to IL-20.

Embodiment 109: An antibody or an antibody fragment therof that competes with antibody IL20-1 1 F36 for binding to IL-20. Embodiment 110: An antibody or an antibody fragment thereof according that competes with one or more of the antibodies produced by the hybrodoma's deposited at at Public Health England (The European Collection of Cell Cultures (ECACC)), Microbiology Services, Porton Down, Salisbury, SP4 0JG, United Kingdom under the Provisional Accession numbers 13070502, 13070501 , 13070503, 13070504 and 13070505 for binding to IL-20.

Embodiment 111 : An antibody or an antibody fragment thereof according that competes with the antibody produced by the hybrodoma deposited at at Public Health England (The

European Collection of Cell Cultures (ECACC)), Microbiology Services, Porton Down, Salisbury, SP4 0JG, United Kingdom under the Provisional Accession number 13070502 for binding to IL-20.

Embodiment 112: An antibody or an antibody fragment thereof according that competes with the antibody produced by the hybrodoma deposited at at Public Health England (The

European Collection of Cell Cultures (ECACC)), Microbiology Services, Porton Down, Salisbury, SP4 0JG, United Kingdom under the Provisional Accession number 13070501 for binding to IL-20.

Embodiment 113: An antibody or an antibody fragment thereof according that competes with the antibody produced by the hybrodoma deposited at at Public Health England (The

European Collection of Cell Cultures (ECACC)), Microbiology Services, Porton Down, Salisbury, SP4 0JG, United Kingdom under the Provisional Accession number 13070503 for binding to IL-20.

Embodiment 114: An antibody or an antibody fragment thereof according that competes with the antibody produced by the hybrodoma deposited at at Public Health England (The

European Collection of Cell Cultures (ECACC)), Microbiology Services, Porton Down, Salisbury, SP4 0JG, United Kingdom under the Provisional Accession number 13070504 for binding to IL-20.

Embodiment 115: An antibody or an antibody fragment thereof according that competes with the antibody produced by the hybrodoma deposited at at Public Health England (The

European Collection of Cell Cultures (ECACC)), Microbiology Services, Porton Down, Salisbury, SP4 0JG, United Kingdom under the Provisional Accession number 13070505 for binding to IL-20.

Antibodies according to any one of embodiments 1 -1 15 preferably have the advantage of being able to interfere with (compete with) binding of IL-20R1 to IL-20 or binding of IL-22R to IL20. Preferably, such antibodies do not interfere with IL-20R2 for binding to IL-20.

Preferably, such antibodies neutralize IL-20 induced effects or activity; have a high affinity and a high potency. The neutralizing effects of such antibodies probably arise from the inability of the IL-20: IL-20R1 :IL-20R2 complex or the IL-20: IL-22R:IL-20R2 complex to form properly. The IL-20:mAb complex formed by binding according to any one of embodiments 1-

1 15 and not interfering with binding of IL-20 to IL-20R2 function as an IL-20R2 antagonist and hereby also affect the activity of IL-19 and IL-24, which share IL-20R2 in their functional receptor complexes either Type I (IL-20R1 :IL-20R2) or Type II (IL-22R1 :IL-20R2.

The IL-20:mAb complex formed by binding according to any one of embodiments 1- 1 15 and not interfering with binding of IL-20 to IL-20R2 function as an IL-20R2 antagonist. Such antibodies might be advantageous for treatment of diseases where especially IL-22 is expressed as IL-22R will be availbale for interaction with IL-10RP which in some diseases, e.g. crohns disease leads to an anti-inflammatory signal

The IL-20:mAb complex formed by binding according to any one of embodiments 1- 1 15 and not interfering with binding of IL-20 to IL-20R2 function as an IL-20R2 antagonist. Such antibodies might be advantageous for treatment of diseases where especially IL-26 is expressed as IL-20R1 will be availbale for interaction with IL-10RP which in some diseases leads to an anti-inflammatory signal

Embodiment 116: An antibody which binds to an epitope on IL-20, wherein said epitope comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, or all of the following amino acids of SEQ ID NO 1 : Leu 1 , Asn 14, Glu 17, Leu 67, Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93, Thr 94 and Lys 97. Embodiment 1117: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Leu 1 , Asn 14, Glu 17, Leu 67, Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93, Thr 94 and Lys 97.

Embodiment 118: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 14, Glu 17, Leu 67, Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93, Thr 94 and Lys 97. Embodiment 119: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Glu 17, Leu 67, Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93, Thr 94 and Lys 97.

Embodiment 120: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Leu 67, Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93, Thr 94 and Lys 97.

Embodiment 121 : An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Leu 1 , Asn 14, Glu 17, Leu 67, Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93 and Thr 94. Embodiment 122: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asn 14, Glu 17, Leu 67, Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93 and Thr 94.

Embodiment 123: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Glu 17, Leu 67, Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93 and Thr 94.

Embodiment 124: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Leu 67, Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93 and Thr 94.

Embodiment 125: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93, Thr 94 and Lys 97.

Embodiment 126: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93 and Thr 94. Embodiment 127: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93, Thr 94 and Lys 97.

Embodiment 128: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93 and Thr 94. Embodiment 129: An antibody according to embodiment 1 16, wherein said epitope comprises the following amino acids of SEQ ID NO 1 : Leu 1 , Asn 14, Glu 17.

Embodiment 130: An antibody according to any of embodiments 1 16-129, wherein said antibody interferes with/competes with binding of IL20R2 to IL-20. Preferably, such antibodies do not interfere with/compete with IL-20R1 for binding to IL-20.

Embodiment 131 : An antibody that interferes with/competes with binding of IL-20R2 to IL-20, wherein said antibody is not antibody 15D2 and 5B7 disclosed in WO2010000721 (SEQ ID 4- 7) Preferably, such antibodies do not interfere with/compete with binding of IL-20R1 to IL-20.

Antibodies according to any one of embodiments 1 16-131 preferably have the advantage of being able to interfere with (compete with) binding of IL-20R2 to IL-20.

Preferably, such antibodies do not interfere with IL-20R1 or IL-22R for binding to IL-20.

Preferably, such antibodies neutralize IL-20 induced effects or activity; have a high affinity and a high potency. The neutralizing effects of such antibodies probably arise from the inability of both the IL-20:IL-20R1 :IL-20R2 and the IL-20:IL-22R:IL-20R2 complexes to form properly. The IL-20:mAb complex formed by binding according to any one of embodiments 1 16-131 and not interfering with binding of IL-20 to IL-20R1 or IL-20 to IL-22R might function as an IL-20R1 and IL-22R antagonist and hereby also antagonises IL-19, IL-24, IL-22 and IL-26 which share IL-20R1 or IL-22R in their functional receptor complexes as IL-22 signals via the IL-22:IL- 10Rp heterodimeric complex and IL-26 signals via the IL-20R1 :IL-10Rp heterodimeric complex.

Antibodies according to any of embodiments 1 16-131 will therefore neutralize the effect of IL-20 on cells that express both the IL-20R1 :IL-20R2 complex and the IL-22R:IL-20R2 complex or cells that express either of these. These antibodies in embodiment 1 16-131 are thus suitable for use in connection with treatment of inflammatory diseases, in particular auto- immune diseases, where the pathogenesis involves overexpression of IL-20, which acts on cells that express both the IL-20R1 :IL-20R2 complex and the IL-22R:IL-20R2 complex, either in the same cells or in different types of cells present in the inflamed tissue.

Antibodies binding to BS2 might be advantageous for treatment of diseases where proinflammatory signals derived from IL-20, IL-19, IL-24, IL-22 and IL-26 is part of the

pathogenesis whereas antibodies binding to BS1 might be advantageous for treatment pf diseases where IL-22 or IL-26 gives rise to an anti-inflamatory signal. Embodiment 132: An antibody which binds to an epitope comprising at least one (preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten, preferably at least eleven, preferably at least twelve, and preferably at least thirteen) of amino acids 1 -15 of SEQ I D NO 1 and at least one (preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten, preferably at least eleven, preferably at least twelve, preferably at least thirteen, preferably at least fifteen, preferably at least sixteen, preferably at least seventeen, preferably at least eighteen, preferably at least nineteen, and preferably at least twenty) of amino acids 83-106 of SEQ ID NO 1 , wherein said antibody is not the 2F6 (SEQ ID 8 and 9) antibody disclosed in WO2010000721.

Embodiment 133: An antibody which binds to an epitope comprising amino acids 1 -15 of SEQ ID NO 1 and amino acids 83-106 of SEQ ID NO 1 , wherein said antibody is not the 2F6 (SEQ ID 8 and 9) antibody disclosed in WO2010000721

Embodiment 134: An antibody according to embodiment 132 or 133, wherein said antibody interferes with assembly of the IL-20:IL-20R1 :IL-20R2 complex.

Embodiment 135: An antibody which binds to an epitope on IL-20, wherein binding of said antibody to IL-20 interferes with assembly of the IL-20:IL-20R1 :IL-20R2 complex, wherein the epitope of said antibody does not comprise any of the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146, and wherein said antibody does not bind any of the following amino acids of SEQ ID NO 1 : Leu 1 , Asn 14, Glu 17, Leu 67, Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93, Thr 94 and Lys 97, and wherein said antibody is not antibody "2F6" (SEQ ID 8 and 9) disclosed in

WO2010000721

Embodiment 136: An antibody which binds to an epitope on IL-20, wherein binding of said antibody to IL-20 interferes with binding of IL-20R1 or IL-20R2 to IL-20, wherein the epitope of said antibody does not comprise any of the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39, Arg 43, Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146, and wherein said antibody does not bind any of the following amino acids of SEQ ID NO 1 : Leu 1 , Asn 14, Glu 17, Leu 67, Asp 68, Phe 71 , Lys 72, His 79, Arg 83, Ser 86, Ser 87, Ala 89, Asn 90, Ser 91 , Leu 93, Thr 94 and Lys 97, and wherein said antibody is not antibody "2F6" (SEQ ID 8 and 9) disclosed in WO2010000721 .

Antibodies according to any one of embodiments 132-136 preferably have the advantage of being able to interfere with assembly of the IL-20:IL-20R1 :IL-20R2 complex or IL-20:IL-22R:IL-20R2. Preferably, such antibodies do not interfere with any of IL-20R1 or IL- 20R2 or IL-22R for binding to IL-20. Preferably, such antibodies neutralize IL-20 induced effects or activity; have a high affinity and a high potency. The neutralizing effects of such antibodies probably arise from the inability of the IL-20:IL-20R1 :IL-20R2 complex or IL-20:IL- 22R:IL-20R2 to form properly. As such antibodies do not compete with binding of the IL-20R1 and/or the IL-20R2 and/or IL-22R receptors, the neutralizing activity are thought to be explained by steric hindrance - i.e. that the "bulkiness" of said antibodies prevents IL-20R1 :IL- 20R2 or IL-22R:IL-20R2 binding via binding site 3 and thus formation of the entire IL-20:IL- 20R1 :IL-20R2 or IL-20:IL-22R:IL-20R2 complexes. Such antibodies will therefore neutralize the effect of IL-20 on cells that express both the IL-20R1 :IL-20R2 complex and the IL-22R:IL- 20R2 complex or cells that express either of these. These antibodies in embodiment 40-44 are thus suitable for use in connection with treatment of inflammatory diseases, in particular autoimmune diseases, where the pathogenesis involves overexpression of IL-20, which acts on cells that express both the IL-20R1 :IL-20R2 complex and the IL-22R:IL-20R2 complex, either in the same cells or in different types of cells present in the inflamed tissue.

Antibodies according to any one of embodiments 132-136 preferably have the advantage of being able to interfere with binding of either IL-20R1 or IL-20R2 or IL-22R or all three to IL-20 by steric hindrance. This might lead to full or partial inhibition of binding of IL- 20R1 and/or IL-20R2 and/or IL-22R, which again might lead to full or partial antagonism. Such antibodies are thus suitable for use in connection with treatment of inflammatory diseases, in particular auto-immune diseases.

Antibodies according to any one of embodiments 132-136 preferably have the advantage of being able to interfere with binding of either IL-20R1 or IL-20R2 or IL-22R or all three to IL-20 by steric hindrance will interfere with the activity of other members of IL-10 family due to sharing of receptor-chains e.g. pro-inflammatory signals derived from IL-20, IL- 19, IL-24, IL-22 and IL-26 is part of the pathogenesis.

Antibodies according to any one of embodiments 132-136 preferably have the advantage of being usable in combination with antibodies according to any one of embodiments 1-40 or antibodies according to any one of embodiments 29-39 for detection of antibodies binding to binding site 1 or binding 2 respectively. That is development of a total IL- 20 assay for detection of IL-20 and IL-20:mAb complexes in clinical samples (see example 5).

Antibodies according to the invention may be human antibodies or humanized antibodies and thus safe to use for treatment of humans. They may also have reduced effector functions.

Embodiment 137: An antibody according to any of preceding embodiments, wherein said antibody comprises a mutated Fc domain, wherein said mutations result in reduced Fey receptor binding functions.

Embodiment 138: An antibody according to embodiment 137, wherein said Fc domain is an lgG1 Fc domain, wherein said Fc domain comprises one or more of the following mutations: L234A, L235E, and G237A, A330S and P331 S (according to the Kabat numbering scheme). Embodiment 139: An antibody according to any of embodiments 1 -136, wherein said antibody comprises an lgG4 Fc domain.

Embodiment 140: An antibody according to embodiment 139, wherein said lgG4 Fc domain comprises the S241 P/S228P mutation.

Embodiment 141 : An antibody according to any one of the preceding embodiments, which is a humanized antibody.

Embodiment 142: An antibody according to any one of the preceding embodiments, which is a human antibody.

Embodiment 143: An antibody according to any one of embodiments 1 -140, which is a chimeric antibody. Embodiments 144: A fragment of the antibody of any of the preceding embodiments, which is a Fab, Fab', F(ab)2, F(ab')2, Fv, single-chain Fv, dsFv, Fd or a dAb fragment, a VH, VL, VhH, or V-NAR domains, a monovalent molecule, minibody, diabody, triabody, tetrabody or kappa body, or an IgNAR. Embodiment 145: A variant of the antibody according to any of the preceding embodiments, which is a deletion variant or an insertion variant.

Embodiment 146: A pharmaceutical composition comprising an antibody according to any one of the preceding embodiments and optionally one or more pharmaceutically acceptable excipients. Preferably, said pharmaceutical formulation is in the form of an aqueous solution - alternatively in the form of a dry powder that is reconstituted prior to administration. Administration can take place e.g. by subcutaneous or IV administration.

Embodiment 147: Use of an antibody according to any one of embodiments 1-145, or a pharmaceutical composition according to embodiment 146, for treating an immunological disorder.

Embodiment 148: Use of an antibody according to any one of embodiments 1-145, or a pharmaceutical composition according to embodiment 146, for treating an autoimmune and/or inflammatory disease.

Embodiment 149: Use of an antibody according to any one of embodiments 1-145, or a pharmaceutical composition according to embodiment 146, for treating SLE.

Embodiment 150: Use of an antibody according to any one of embodiments 1-145, or a pharmaceutical composition according to embodiment 146, for treating RA.

Embodiment 151 : Use of an antibody according to any one of embodiments 1 -145, or a pharmaceutical composition according to embodiment 146, for treating inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD).

Embodiment 152: Use of an antibody according to any one of embodiments 1-145, or a pharmaceutical composition according to embodiment 146, for treating CD.

Embodiment 153: Use of an antibody according to any one of embodiments 1-145, or a pharmaceutical composition according to embodiment 146, for treating autoimmune and/or inflammatory disease, wherein the autoimmune and/or inflammatory disease is selected from: Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), osteoporosis, multiple sclerosis (MS), scleroderma and type 1 diabetes (T1 D), and other diseases and disorders, such as PV (pemphigus vulgaris), psoriasis, psoriatic arthritis, atopic dermatitis, celiac disease, Chronic obstructive lung disease, Hashimoto's thyroiditis, Graves' disease (thyroid), Sjogren's syndrome, Guillain-Barre syndrome, Goodpasture's syndrome, Addison's disease, Wegener's granulomatosis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, polymyalgia rheumatica, Raynaud's phenomenon, temporal arteritis, giant cell arteritis, autoimmune haemolytic anaemia, pernicious anaemia, polyarteritis nodosa, behcet's disease, primary biliary cirrhosis, uveitis, myocarditis, rheumatic fever, ankylosing spondylitis, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, alopecia areata, type I diabetes, Colitis-Associated Tumorigenesis, metastasis-associated osteolysis or vitiligo.

Embodiment 154: A DNA molecule encoding an antibody according to any one of embodiments 1-145. Embodiment 155: An expression vector comprising a DNA molecule according to embodiment 154.

Embodiment 156: A host cell comprising an expression vector according to embodiment 155.

Embodiment 157: A method of making an antibody according to any one of embodiments 1 - 145, wherein said method comprises the step of culturing a host cell according to embodiment 156 under conditions suitable for expressing said antibody.

Embodiment 158: A kit comprising an antibody according to any one of embodiments 1-145 or a pharmaceutical formulation according to embodiment 146.

Embodiment 159: A kit according to embodiment 158, wherein said kit furthermore comprises a device for administrating said antibody or said pharmaceutical formulation. Embodiment 160: A method of treating an immunological disorder, wherein said method comprises administering to a person in need thereof, a therapeutically effective amount of an antibody according to any one of embodiments 1 -145, or a pharmaceutical composition according to embodiment 146.

Embodiment 161 : A method of treating an autoimmune and/or inflammatory disease, wherein said method comprises administering to a person in need thereof, a therapeutically effective amount of an antibody according to any one of embodiments 1-145, or a pharmaceutical composition according to embodiment 146.

Embodiment 162: A method according to embodiment 161 , wherein said autoimmune and/or inflammatory disease is selected from SLE, CD, and RA. Embodiment 163: A method according to embodiment 161 , wherein said autoimmune and/or inflammatory disease is selected from: Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), osteoporosis, multiple sclerosis (MS), scleroderma and type 1 diabetes (T1 D), and other diseases and disorders, such as PV (pemphigus vulgaris), psoriasis, psoriatic arthritis, atopic dermatitis, celiac disease, Chronic obstructive lung disease, Hashimoto's thyroiditis, Graves' disease (thyroid), Sjogren's syndrome, Guillain-Barre syndrome,

Goodpasture's syndrome, Addison's disease, Wegener's granulomatosis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, polymyalgia rheumatica, Raynaud's phenomenon, temporal arteritis, giant cell arteritis, autoimmune haemolytic anaemia, pernicious anaemia, polyarteritis nodosa, behcet's disease, primary biliary cirrhosis, uveitis, myocarditis, rheumatic fever, ankylosing spondylitis, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, alopecia areata, type I diabetes, Colitis- Associated Tumorigenesis, metastasis-associated osteolysis or vitiligo.

EXAMPLES

Example 1 : Crystal structure of the ternary IL-20/IL-20R1/IL-20R2 complex

In this Example the sequence numbering of IL-20 is following the numbering of the crystallographically determined IL-20/IL-20R1/IL-20R2 complex X-ray structure. In the X-ray structure IL-20, IL-20R1 and IL-20R2 have has been numbered as if including a signal peptide but also shows a Gly residue from a tag preceding the mature IL-20 protein chain. To obtain the same sequence numbering as of SEQ ID NO: 1 all IL-20 sequence numbering in this Example needs to have its sequence number subtracted by 24. To obtain the same sequence numbering as of SEQ ID NO: 2 all IL-20R1 sequence numbering in this Example needs to have its sequence number subtracted by 29. To obtain the same sequence numbering as of SEQ ID NO: 3 all IL-20R2 sequence numbering in this Example needs to have its sequence number subtracted by 29. For example: Gin 40 of IL-20 of this example is equivalent to Gin 16 of SEQ ID NO: 1 . Phe 74 of IL-20R1 of this example is equivalent of Phe 45 of SEQ ID NO:2. Tyr 70 of IL-20R2 of this example is equivalent of Tyr 41 of SEQ ID NO:3.

IL-20/IL-20R1/IL-20R2 ternary complex formation is mediated by 3 protein interfaces, IL-20/IL-20R1 (Site 1 ), IL-20/IL-20R2 (Site 2), and IL-20R1/IL-20R2 (Site 3). The crystals used for the structure determination contained two independent ternary complexes per

crystallographic asymmetric unit. The direct contacts between the IL-20, IL-20R1 , and IL-20R2 were identified by running the CONTACT software of the CCP4 program suite (Bailey, 1994) using a cut-off distance of 4.0 A between the two molecules in each run, respectively.

Site 1 interaction:

The results from the IL-20/IL-20R1 interaction site of the crystal structure using the CONTACT software are shown in Table 1 and 2. The resulting IL-20 interaction residues for IL-20R1 was found to comprise the following residues of IL-20, when combining the two crystallographically independent sites as found in the IL-20/IL-20R1/IL-20R2 crystal: Gin 40, Arg 43, Asn 44, Ser 47, Arg 50, Gin 54, Asp 57, lie 60, Asp 61 , lie 62, Arg 63, Arg 67, Lys 160, Gly 163, Glu 164, Asp 166, lie 167 and Gin 170.

The IL-20R1 residues in interaction with IL-20 comprise residues Phe 74, Tyr 76, Gly 77, Gin 78, Trp 81 , Asp 103, Glu 105, His 106, Gin 107, Tyr 109, Arg 128, Tyr 130, Phe 132, Leu 133, Glu 134, Gly 224, Pro 225 and Pro 226 of IL-20R1. The IL-20/IL-20R1 interactions and the residues involved in hydrogen-binding can also be extracted from Table 1 and 2.

Thus, the IL-20 interaction with IL-20R1 consists of two contact surfaces, were the first is formed by IL-20R1 L2-L4 loops that contact a small cavity on IL-20 located at the

intersection of helix F and AB loop. In the second part of the contact surface the contacts occur between IL-20R1 L6 and the N-terminus of helix A.

Table 1 shows IL-20 interactions with IL-20R1 , first crystallographically independent ternary complex given by chains A and R respectively. A distance cut-off of 4.0 A was used. The contacts were identified by the CONTACT computer software program of the CCP4 suite (Bailey, Acta Crystallogr. Sect. D-Biol. Crystallogr, 50:760-763 (1994)).

Table 1 . IL-20 interaction with IL-20R1

IL-20 IL-20R1

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Gin 40A CD Gly 224 R O 3.52

Gin 40A OE1 Gly 224R N 3.92 *

Gly 224 R CA 3.76 IL-20 IL-20R1

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Gly 224R C 3.69

Gly 224R 0 3.31 _*

Gin 40A NE2 Gly 224R C 3.90

Gly 224 R 0 3.04 _***

Arg 43A CD Pro 225R N 3.86

Arg 43A NE Pro 225R N 3.88 _*

Pro 225R CD 3.63

Arg 43A CZ Pro 225R CD 3.53

Arg 43A NH1 Pro 225R CD 3.87

Arg 43A NH2 Pro 225R CD 3.81

Asn 44A OD1 Pro 226R CD 3.60

Pro 226R CG 3.81

Ser 47A OG Pro 226R CG 3.78

Arg 50A NH1 Glu 134R OE2 4.00 _*

Gin 54A CG Arg 128R NH1 3.86

Gin 54A CD Arg 128R NH1 3.73

Gin 54A OE1 Arg 128R NH1 3.50 _*

Gin 54A NE2 Arg 128R 0 3.73 _*

Arg 128R CB 3.45

Asp 57A CG Arg 128R NH2 3.75

Asp 57A OD2 Tyr 76R OH 3.79 _*

Arg 128R CZ 3.96

Arg 128R NH2 2.65 _*** lie 60A CA Tyr 76R CD1 3.99

lie 60A CB Tyr 109R CE1 3.86

lie 60A CG1 Phe 74 R CE1 3.94

Phe 74 R CD2 3.97

Phe 74 R CE2 3.59

Phe 74 R CZ 3.57

lie 60A CD1 Phe 74 R CG 3.52

Phe 74 R CD1 3.50 IL-20 IL-20R1

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Phe 74 R CE1 3.61

Trp 81 R CZ2 3.91

Phe 74 R CD2 3.63

Phe 74 R CE2 3.73

Phe 74 R CZ 3.71

lie 60A C Tyr 76R CD1 3.93

lie 60A 0 Tyr 76R CA 3.61

Tyr 76R C 3.78

Gly 77R N 2.98 _***

Gly 77R CA 3.95

Phe 74 R CE2 3.31

Phe 74 R CZ 3.87

Tyr 76R CD1 3.37

Tyr 76R CE1 3.98

Asp 61A CA Gin 78R 0 3.37

Asp 61A CB Gin 78R 0 3.39

Asp 61A C Gly 77R 0 3.94

Gin 78R 0 3.97

Gly 77R N 3.99

Gly 77R CA 3.59

Gly 77R C 3.81

Asp 61A 0 Gly 77R 0 2.96 _***

Gin 78R 0 3.66 _*

Gly 77R CA 3.31

Gly 77R C 3.14

Gin 78R N 3.95 _* lie 62A 0 Tyr 76R CZ 3.69

Tyr 76R OH 3.33 _*

Tyr 76R CE1 3.81

Arg 63A N Gly 77R CA 3.73

Arg 63A CB Gly 77R CA 3.77 IL-20 IL-20R1

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Arg 63A CG Tyr 76R CE2 3.71

Arg 63A CD Tyr 76R CD2 3.91

Tyr 76R CE2 3.97

Arg 63A CZ Glu 105R 0 3.84

Arg 63A NH1 Glu 105R 0 3.09 _***

Gin 107R NE2 3.54 _*

Arg 63A NH2 Glu 105R 0 3.69 _*

Arg 67A CD Glu 105R CB 3.92

Arg 67A NE Glu 105R CD 3.98

Glu 105R OE1 3.47 _*

Glu 105R CB 3.97

Arg 67A CZ His 106R CE1 3.60

His 106R NE2 3.56

Arg 67A NH1 His 106R CE1 3.60

His 106R NE2 3.18 _***

Arg 67A NH2 Asp 103R OD2 3.95 _*

His 106R CE1 3.59

His 106R NE2 3.98 _*

Lys 160A CG Tyr 76R OH 3.78

Lys 160A CD Tyr 76R OH 3.90

Lys 160A CE Tyr 76R OH 3.96

Lys 160A NZ Tyr 76R CZ 3.71

Tyr 76R OH 2.89 _***

Tyr 76R CE1 3.64

Gly 163A C Arg 128R NH1 3.93

Gly 163A 0 Arg 128R NH1 3.30 _*

Glu 164A CG Tyr 76R OH 3.93

Glu 164A CD Tyr 76R OH 3.63

Glu 164A 0E2 Tyr 76R CE2 3.59

Tyr 76R CZ 3.50

Tyr 76R OH 2.60 _*** IL-20 IL-20R1

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Asp 166A CB Leu 133R CD1 3.96

Asp 166A OD2 Tyr 130R CD2 3.67

Asp 166A 0 Leu 133R CD1 3.83

lie 167A CG1 Gin 107R NE2 3.70

Gin 107R OE1 3.80

lie 167A CD1 Gin 107R NE2 3.56

Gin 107R CD 4.00

Gin 107R OE1 3.58

Gin 170A CG Tyr 130R OH 3.99

Gin 170A CD Phe 132R CE2 3.77

Gin 170A OE1 Phe 132R CE2 3.93

Leu 133R CD2 3.17

Gin 170A NE2 Phe 132R CE2 3.55

^*** indicates a strong possibility for a hydrogen bond at this contact (distance < 3.3 A) as calculated by CONTACT

^* indicates a weak possibility (distance > 3.3 A).

Blank indicates that the program considered there to be no possibility of a hydrogen bond. Hydrogen-bonds are specific between a donor and an acceptor, are typically strong, and are easily identifiable.

Table 2. demonstres IL-20 interactions with IL-20R1 , second crystallographically independent ternary complex given by chain C and E respectively. A distance cut-off of 4.0 A was used. The contacts were identified by the CONTACT computer software program of the CCP4 suite suite (Bailey, Acta Crystallogr. Sect. D-Biol. Crystallogr, 50:760-763 (1994)).

Table 2. IL-20 interaction with IL-20R1

IL-20 IL-20R1

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Gin 40C CD Gly 224E C 3.92 IL-20 IL-20R1

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Gly 224E 0 3.59

Gin 40C OE1 Gly 224E CA 3.49

Gly 224E C 3.36

Pro 225E N 3.97 _*

Gly 224E 0 3.44 _*

Gin 40C NE2 Gly 224E 0 3.48 _*

Pro 226E CD 3.16

Gin 54C OE1 Arg 128E CD 3.88

Arg 128E NH1 3.76 _*

Gin 54C NE2 Arg 128E CB 3.67

Arg 128E 0 3.90 _*

Asp 57C OD2 Tyr 76E OH 3.81 _*

Arg 128E NH2 3.22 _*** lie 60C CB Tyr 109E CE1 3.87

lie 60C CG2 Tyr 109E CE1 3.91

Tyr 109E CZ 3.75

Tyr 109E OH 3.95

lie 60C CG1 Phe 74 E CE2 3.65

Phe 74 E CZ 3.74

lie 60C CD1 Phe 74 E CG 3.77

Phe 74 E CD1 3.82

Phe 74 E CD2 3.79

Phe 74 E CE1 3.91

Phe 74 E CE2 3.87

Phe 74 E CZ 3.93

lie 60C C Phe 74 E CE2 3.99

lie 60C 0 Tyr 76E CA 3.73

Phe 74 E CE2 3.26

Phe 74 E CZ 3.90

Tyr 76E CD1 3.61

Tyr 76E C 3.95 IL-20 IL-20R1

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Gly 77E N 3.15 _***

Asp 61 C CA Gin 78E 0 3.21

Asp 61 C CB Gin 78E 0 3.28

Asp 61 C C Gin 78E 0 3.77

Asp 61 C 0 Gly 77E 0 3.08 _***

Gin 78E 0 3.44 _*

Gly 77E CA 3.73

Gly 77E C 3.38

lie 62C 0 Tyr 76E CE1 3.93

Tyr 76E CZ 3.80

Tyr 76E OH 3.59 _*

Arg 63C N Gly 77E CA 3.94

Arg 63C CB Tyr 76E CE2 3.95

Gly 77E CA 3.69

Arg 63C CG Tyr 76E CD2 3.71

Tyr 76E CE2 3.38

Arg 63C CD Tyr 76E CD2 3.60

Tyr 76E CE2 3.78

Arg 63C NH1 His 106E CD2 3.99

Glu 105E 0 3.57 _*

Gin 107E NE2 3.84 _*

Arg 67C NE Glu 105E OE1 2.91 _***

Glu 105E CB 3.47

Glu 105E CD 3.52

Arg 67C CZ Glu 105E OE1 3.19

Asp 103E OD2 3.83

Glu 105E CB 3.58

Arg 67C NH 1 Asp 103E OD2 3.72 _*

His 106E ND1 3.34 _*

His 106E CE1 2.81

His 106E NE2 2.97 _*** IL-20 IL-20R1

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

His 106E CG 3.79

His 106E CD2 3.59

Arg 67C NH2 Glu 105E OE1 2.64 _***

Asp 103E CG 3.59

Asp 103E OD1 3.44 _*

Asp 103E OD2 3.09 _***

Glu 105E CB 3.44

Glu 105E CD 3.70

Lys 160C CG Tyr 76E OH 3.72

Lys 160C CD Tyr 76E OH 3.84

Lys 160C CE Tyr 76E OH 3.90

Lys 160C NZ Tyr 76E CE1 3.61

Tyr 76E CZ 3.63

Tyr 76E OH 2.83 _***

Gly 163C 0 Arg 128E NH1 3.89 _*

Glu 164C CG Tyr 76E OH 3.44

Glu 164C CD Tyr 76E OH 3.29

Glu 164C OE2 Tyr 76E CE2 3.48

Tyr 76E CZ 3.34

Tyr 76E OH 2.44 _***

Asp 166C OD2 Tyr 130E CD2 3.91

lie 167C CG1 Gin 107E NE2 3.82

lie 167C CD1 Gin 107E OE1 3.84

Gin 107E NE2 3.96

Gin 170C CG Tyr 130E OH 3.75

Gin 170C CD Phe 132E CE2 3.93

Gin 170C OE1 Phe 132E CE2 3.98

indicates a strong possibility for a hydrogen bond at this contact (distance < 3.3 A) as calculated by CONTACT

* indicates a weak possibility (distance > 3.3 A). Blank indicates that the program considered there to be no possibility of a hydrogen bond. Hydrogen-bonds are specific between a donor and an acceptor, are typically strong, and are easily identifiable. Site 2 interaction:

The results from the IL-20/IL-20R2 interaction site of the crystal structure using the CONTACT software are shown in Table 3 and 4. The resulting IL-20 interaction residues for IL-20R2 was found to comprise the following residues of IL-20, when combining the two crystallographically independent sites as found in the IL-20/IL-20R1/IL-20R2 crystal: Gly 24, Leu 25, Asn 38, Glu 41 , Leu 91 , Asp 92, Phe 95, Lys 96, His 103, Arg 107, Ser 1 10, Ser 1 1 1 , Ala 1 13, Asn 1 14, Ser 1 15, Leu 1 17, Thr 1 18 and Lys 121 .

The IL-20R2 residues in interaction with IL-20 comprise residues Tyr 70, Gly 72, Glu 73, Tyr 74, Glu 75, Tyr 78, Thr 79, lie 84, Pro 85, Ser 87, Asp 102, Thr 104, Ala 105, Thr 106, Val 107, Arg 133, Gin 162, Glu 164, Lys 210 and Ala 21 1 of IL-20R2. The IL-20/IL-20R2 interactions and the residues involved in hydrogen-binding can also be extracted from Table 3 and 4.

The site 2 IL-20/IL-20R2 interface is centred on IL-20 helix D, which is surrounded by IL-20R2 L2 and L3. The IL-20R2 L2 loop also makes significant contacts with helix C, via Tyr-74 of the IL-20R2 chain, while residues on L4, and L5 loops form hydrogen bonds with helix A and the IL-20 N-terminus.

Table 3 demonstrates the IL-20 interactions with IL-20R2, first crystallographically independent ternary complex given by chain A and B respectively. A distance cut-off of 4.0 A was used. The contacts were identified by the CONTACT computer software program of the CCP4 suite suite (Bailey, Acta Crystallogr. Sect. D-Biol. Crystallogr., 50:760-763 (1994)).

Table 3. IL-20 interaction with IL-20R2

IL-20 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Gly 24A C Lys 210B CD 3.74

Lys 210B CE 3.84

Gly 24A O Lys 210B CD 3.98

Lys 210B CE 3.79 IL-20 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Gly 24A N Glu 164B CD 3.87

Glu 164B 0E1 2.90 _***

Lys 210B CE 3.42

Leu 25A N Lys 210B CD 3.76

Leu 25A CD2 Lys 210B CD 3.86

Asn 38A ND2 Ala 21 1 B CA 3.96

Ala 21 1 B CB 3.73

Glu 41A CD Arg 133B NH2 3.54

Thr 104B 0 3.18

Glu 41A OE1 Arg 133B NH2 3.32 _*

Thr 104B 0 3.30 _***

Glu 41A OE2 Gin 162B NE2 3.70 _*

Arg 133B NH2 2.96 _***

Thr 104B C 3.91

Thr 104B 0 3.12 _***

Arg 133B CZ 3.52

Arg 133B NH1 3.23 _***

Leu 91A CB Tyr 78B OH 3.90

Leu 91A CD2 Tyr 74 B CD1 3.88

Tyr 74 B CE1 3.74

Leu 91A C Tyr 78B CE1 3.85

Tyr 74 B OH 3.79

Leu 91A 0 Tyr 78B CD1 3.38

Tyr 78B CE1 3.48

Tyr 74 B CE1 3.52

Tyr 74 B CZ 3.63

Tyr 74 B OH 2.82 _***

Asp 92A OD1 Tyr 78B CE1 3.24

Phe 95A CB Tyr 74 B OH 3.70

Lys 96A N Tyr 74 B OH 3.18 _***

Lys 96A CA Tyr 74 B OH 3.76 IL-20 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Lys 96A CB Tyr 74 B OH 3.28

Thr 79B CG2 3.69

Lys 96A CG Tyr 74 B OH 3.81

Lys 96A CD Tyr 74 B OH 3.82

Thr 79B CG2 4.00

Arg 107A CA Tyr 70B OH 3.94

Arg 107A CB Tyr 70B OH 3.71

Arg 107A CG lie 84 B CG2 3.47

Tyr 70B OH 3.32

Arg 107A CD Tyr 70B OH 3.90

Pro 85B 0 3.77

Asp 102B OD1 3.78

Ser 87B CB 3.79

Arg 107A NE Pro 85B 0 3.61 _*

Ser 87B CA 3.81

Ser 87B CB 3.78

Arg 107A CZ Ser 87B CA 3.99

Ser 87B CB 3.76

Arg 107A NH1 Ser 87B CB 3.68

Ser 1 10A CA Glu 75B OE2 3.64

Ser 1 10A CB Glu 75B OE2 3.21

Ser 1 10A OG Glu 75B OE2 2.46 _***

Glu 75B CD 3.15

Glu 75B OE1 3.33 _*

Ser 1 1 1A CA Thr 104B OG1 3.55

Ser 1 1 1A CB Thr 104B OG1 3.19

Ser 1 1 1A OG Thr 104B CB 3.64

Thr 104B OG1 2.64 _***

Thr 104B CG2 3.86

Ala 1 13A CB Tyr 74 B CD2 3.52

Tyr 74 B CE2 3.71 IL-20 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Tyr 74 B CG 3.77

Asn 1 14A CB Ala 105B CB 3.46

Asn 1 14A CG Tyr 74 B CB 3.66

Tyr 74 B N 3.93

Glu 75B OE1 3.92

Asn 1 14A OD1 Tyr 74 B CB 3.33

Gly 72 B C 3.42

Glu 73B N 3.30 _***

Glu 73B CA 3.97

Glu 73B C 3.88

Tyr 74 B N 2.90 _***

Tyr 74 B CA 3.68

Val 107B CG2 3.61

Gly 72 B CA 3.51

Glu 75B OE1 3.85 _*

Asn 1 14A ND2 Tyr 74 B CB 3.77

Glu 75B OE2 3.64 _*

Glu 75B CD 3.79

Glu 75B OE1 3.12 _***

Asn 1 14A C Ala 105B CB 3.74

Ser 1 15A N Ala 105B CB 3.67

Ser 1 15A OG Ala 105B CB 3.95

Thr 104B 0 3.83 _*

Leu 1 17A CD2 Glu 73B OE1 3.69

Glu 73B CB 3.88

Glu 73B CG 3.47

Glu 73B C 3.99

Tyr 74 B N 3.65

Tyr 74 B CA 3.95

Thr 1 18A CG2 Thr 106B 0 3.72

Val 107B CG1 3.50 IL-20 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Lys 121A CD Glu 73B OE1 3.79

Lys 121A CE Glu 73B OE1 3.61

Lys 121A NZ Glu 73B CD 3.82

Glu 73B OE1 2.69 _***

^* indicates a weak possibility (distance > 3.3 A).

Table 4 shows IL-20 interactions with IL-20R2, second crystallographically independent ternary complex given by chain C and D respectively. A distance cut-off of 4.0 A was used. The contacts were identified by the CONTACT computer software program of the CCP4 suite suite (Bailey, Acta Crystallogr. Sect. D-Biol. Crystallogr., 50:760-763 (1994)).

Table 4. IL-20 interaction with IL-20R2

IL-20 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Gly 24C C Lys 210D CD 3.78

Lys 210D CE 3.88

Gly 24C N Glu 164D OE1 3.24 _***

Lys 210D CE 3.59

Leu 25C N Lys 210D CD 3.35

Lys 210D CE 3.94

Leu 25C CA Lys 210D CD 3.90

Leu 25C CG Lys 210D CG 3.98

Lys 210D CD 3.42

Lys 210D CB 3.73 IL-20 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Leu 25C CD1 Lys 210D CB 3.97

Lys 210D 0 3.54

Leu 25C CD2 Lys 210D CD 3.56

Asn 38C OD1 Lys 210D NZ 3.49 _*

Asn 38C ND2 Ala 21 1 D CB 3.99

Glu 41 C CG Thr 104D 0 3.85

Glu 41 C CD Arg 133D NH2 3.25

Thr 104D 0 3.1 1

Glu 41 C OE1 Arg 133D NH2 2.96 _***

Thr 104D 0 3.14 _***

Ala 105D CA 3.92

Glu 41 C OE2 Arg 133D CZ 3.45

Arg 133D NH1 3.26 _***

Arg 133D NH2 2.79 _***

Gin 162D NE2 3.90 _*

Thr 104D 0 3.23 _***

Leu 91 C CB Tyr 78D OH 3.99

Tyr 78D CE1 3.97

Leu 91 C CD2 Tyr 74 D CD1 3.59

Tyr 74 D CE1 3.49

Leu 91 C C Tyr 78D CE1 3.96

Leu 91 C 0 Tyr 78D CD1 3.55

Tyr 78D CE1 3.58

Tyr 74 D CE1 3.58

Tyr 74 D CZ 3.74

Tyr 74 D OH 3.00 _***

Asp 92C OD1 Tyr 78D CE1 3.49

Phe 95C CB Tyr 74 D CZ 3.97

Tyr 74 D OH 3.50

Phe 95C C Tyr 74 D OH 3.95

Lys 96C N Tyr 74 D OH 3.1 1 _*** _«9_

IL-20 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Lys 96C CA Tyr 74 D OH 3.75

Lys 96C CB Tyr 74 D OH 3.51

Thr 79D CG2 3.71

Lys 96C CG Tyr 74 D OH 3.65

Lys 96C CD Tyr 74 D OH 3.93

His 103C CD2 Pro 85D CG 3.77

Arg 107C CA Tyr 70D OH 3.80

Arg 107C CB Tyr 70D OH 3.72

Arg 107C CG Tyr 70D OH 3.19

lie 84 D CG2 3.29

Pro 85D 0 3.88

Arg 107C CD Asp 102D OD1 3.49

Ser 87D N 3.89

Ser 87D CA 3.99

Ser 87D CB 3.73

Pro 85D 0 3.52

Arg 107C NE Ser 87D N 3.91 _*

Ser 87D CA 3.71

Ser 87D CB 3.77

Pro 85D 0 3.25 _***

Arg 107C CZ Ser 87D CA 3.83

Ser 87D CB 3.70

Arg 107C NH1 Ser 87D CB 3.50

Arg 107C 0 Tyr 70D OH 3.94 _*

Ser 1 10C CA Glu 75D OE2 3.50

Ser 1 10C CB Glu 75D CD 4.00

Glu 75D OE2 3.12

lie 84 D CD1 3.98

Ser 1 10C OG Glu 75D CD 3.44

Glu 75D OE1 3.45 _*

Glu 75D OE2 2.95 _*** IL-20 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Tyr 70D CE1 3.82

lie 84 D CD1 3.88

Ser 1 1 1 c CA Thr 104D OG1 3.58

Ser 1 1 1 c CB Thr 104D OG1 3.24

Ser 1 1 1 c OG Thr 104D CB 3.67

Thr 104D OG1 2.77 _***

Thr 104D CG2 3.83

Ala 1 13C CB Tyr 74 D CG 3.81

Tyr 74 D CD2 3.49

Tyr 74 D CE2 3.67

Asn 1 14C CB Ala 105D CB 3.82

Asn 1 14C CG Tyr 74 D N 3.75

Tyr 74 D CB 3.61

Gly 72 D CA 3.90

Glu 73D N 3.98

Glu 75D OE1 3.78

Asn 1 14C OD1 Tyr 74 D N 2.77 _***

Tyr 74 D CA 3.60

Tyr 74 D CB 3.36

Glu 73D CA 3.70

Glu 73D C 3.69

Val 107D CG2 3.63

Gly 72 D CA 3.46

Gly 72 D C 3.27

Glu 73D N 2.91 _***

Glu 75D OE1 3.94 _*

Asn 1 14C ND2 Tyr 74 D CB 3.76

Gly 72 D CA 3.76

Glu 75D CD 3.62

Glu 75D OE1 2.78 _***

Glu 75D OE2 3.70 _* IL-20 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Asn 1 14C C Ala 105D CB 3.77

Asn 1 14C 0 Ala 105D CB 3.91

Ser 1 15C N Ala 105D CB 3.77

Ser 1 15C OG Thr 104D 0 3.99 _*

Ala 105D CB 3.99

Leu 1 17C CD2 Tyr 74 D N 3.19

Tyr 74 D CA 3.41

Tyr 74 D CB 3.76

Glu 73D CB 3.90

Glu 73D CG 3.98

Glu 73D C 3.73

Thr 1 18C CG2 Thr 106D 0 3.63

Val 107D CG1 3.61

Lys 121 C CD Glu 73D OE1 3.97

Lys 121 C CE Glu 73D OE1 3.81

Lys 121 C NZ Glu 73D CD 3.76

Glu 73D OE1 2.82 _***

Glu 73D OE2 3.94 _*

* indicates a weak possibility (distance > 3.3 A).

Site 3 interaction:

The results from the IL-20R1/IL-20R2 interaction site of the crystal structure using the

CONTACT software are shown in Table 5 and 6. The resulting IL-20R1 interaction residues for IL-20R2 was found to comprise the following residues of IL-20R1 , when combining the two crystallographically independent sites as found in the IL-20/IL-20R1/IL-20R2 crystal: Asn 187, Ser 190, Arg 192, Thr 193, Trp 194, Ser 195, Gin 196, Thr 206, Trp 207, Leu 208 and Glu 209. 5-

The IL-20R2 residues in interaction with IL-20R1 comprise residues Asp 148, Gly 149, Phe 150, His 151 , Val 153, lie 188, Pro 189, Val 190, His 191 , Thr 194, Glu 196 and Pro 197 of IL-20R2. The IL-20R1/IL-20R2 interactions and the residues involved in hydrogen-binding can also be extracted from Table 5 and 6.

Thus, the site 3 interface is formed from IL-20R1 D2 residues on β-strand C, the CC loop, and the EF loop, which contact IL-20R2 residues on the AB loop, β-strand E, and the EF loop.

Table 5 demonstrates IL-20R1 interactions with IL-20R2, first crystallographically independent ternary complex given by chain R and B respectively. A distance cut-off of 4.0 A was used. The contacts were identified by the CONTACT computer software program of the CCP4 suite suite (Bailey, Acta Crystallogr. Sect. D-Biol. Crystallogr, 50:760-763 (1994)).

Table 5. IL-20R1 interaction with IL-20R2

IL-20R1 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Asn 187R ND2 Asp 148B CG 3.99

Asp 148B OD1 3.45 _*

Asp 148B OD2 3.64 _*

Ser 190R OG Asp 148B OD2 3.1 1 _***

Arg 192R CB Asp 148B OD2 3.46

Arg 192R CG Asp 148B OD2 3.67

Arg 192R CD Asp 148B OD2 3.83

Thr 193R O Pro 189B CB 3.54

Pro 189B CG 3.93

Trp 194R CA Pro 189B O 3.15

Trp 194R CB Pro 189B O 3.48

Val 153B CG2 3.97

Trp 194R CG Val 153B CG2 3.92

Trp 194R CE3 His 191 B CB 3.47

His 191 B CG 3.85

His 191 B CD2 3.95

Trp 194R CD1 Val 153B CG2 3.86 D-

IL-20R1 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Trp 194R CZ3 His 191 B CB 3.98

His 191 B CG 3.91

His 191 B CD2 3.89

Trp 194R C Pro 189B 0 3.45

Ser 195R N Pro 189B 0 2.82 _***

Ser 195R CA Pro 189B 0 3.92

Ser 195R CB lie 188B CG2 3.92

Ser 195R OG lie 188B CG2 3.75

Ser 195R 0 His 191 B CB 3.84

Pro 189B 0 3.99 _*

Val 190B CA 3.47

Val 190B CG1 3.84

Val 190B C 3.76

His 191 B N 3.07 _***

Gin 196R CD His 191 B ND1 3.96

Gin 196R OE1 His 191 B ND1 3.71 _*

Gin 196R NE2 His 191 B ND1 3.80 _*

Thr 206R OG1 Thr 194B OG1 3.96 _*

Thr 194B CG2 3.55

Thr 206R C His 151 B NE2 3.96

Thr 206R 0 His 151 B CD2 3.99

His 151 B CE1 3.30

His 151 B NE2 2.77 _***

Thr 194B CG2 3.93

Trp 207R CB Thr 194B CG2 3.73

Phe 150B CB 3.86

Phe 150B CD1 3.63

Trp 207R CE2 Glu 196B OE1 3.82

Trp 207R CE3 Phe 150B CG 3.79

Phe 150B CD1 3.46

Phe 150B CD2 3.99 IL-20R1 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Phe 150B CE1 3.37

Phe 150B CE2 3.92

Phe 150B CZ 3.61

Trp 207R NE1 Glu 196B OE2 3.94 *

Trp 207R CZ2 Glu 196B CD 3.94

Glu 196B OE1 3.12

Trp 207R CZ3 Phe 150B CZ 3.81

Pro 197B CD 3.64

Pro 197B CG 3.72

Trp 207R CH2 Glu 196B OE1 3.70

Pro 197B CD 3.60

Pro 197B CG 3.98

Trp 207R 0 His 151 B CD2 3.78

His 151 B NE2 3.62 *

Phe 150B CD1 3.77

Leu 208R CD2 His 151 B NE2 3.83

Glu 209R OE2 Gly 149B CA 3.87

* indicates a weak possibility (distance > 3.3 A).

Table 6 demonstrates IL-20R1 interactions with IL-20R2, second crystallographically independent ternary complex given by chain E and D respectively. A distance cut-off of 4.0 A was used. The contacts were identified by the CONTACT computer software program of the CCP4 suite suite (Bailey, Acta Crystallogr. Sect. D-Biol. Crystallogr., 50:760-763 (1994)). Table 6. IL-20R1 interaction with IL-20R2

IL-20R1 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Asn 187E CB Asp 148D OD1 3.82

Asn 187E ND2 Asp 148D OD1 3.37 _*

Asp 148D CG 3.89

Asp 148D OD2 3.58 _*

Ser 190E CB Asp 148D OD2 3.84

Ser 190E OG Asp 148D CG 3.97

Asp 148D OD2 2.78 _***

Arg 192E CB Asp 148D OD2 3.36

Arg 192E CG Asp 148D OD2 3.54

Arg 192E CD Asp 148D OD2 3.77

Thr 193E 0 Pro 189D CB 3.44

Trp 194E CA Pro 189D 0 3.47

Trp 194E CB Val 153D CG2 3.62

Pro 189D 0 3.83

Trp 194E CG Val 153D CG2 3.65

Trp 194E CD2 His 191 D CD2 3.92

Trp 194E CE3 His 191 D CB 3.66

His 191 D CG 3.87

His 191 D CD2 3.59

Trp 194E CD1 Val 153D CG2 3.70

Trp 194E CZ2 His 151 D CG 3.99

Trp 194E CZ3 His 191 D CD2 3.60

Trp 194E CH2 His 191 D CD2 3.94

Trp 194E C Pro 189D 0 3.79

Ser 195E N Pro 189D 0 3.13 _***

Ser 195E OG Pro 189D 0 3.65 _*

lie 188D CG2 3.62

Ser 195E 0 His 191 D CA 3.90

His 191 D CB 3.63

His 191 D N 3.00 _*** IL-20R1 IL-20R2

Res. Res. # Atom Res. Res. # Atom Distance Possibly

Type and name Type and name [A] H-bond

Chain Chain

Val 190D CA 3.70

Val 190D C 3.81

Gin 196E 0E1 His 191 D CG 3.87

Thr 206E 0G1 Thr 194D CG2 3.84

Thr 206E C His 151 D NE2 3.87

Thr 206E 0 His 151 D CE1 2.87

Thr 194D CG2 3.59

His 151 D NE2 2.68 _***

His 151 D CD2 3.98

Trp 207E CB Thr 194D CG2 3.67

Phe 150D CB 3.90

Trp 207E CE2 Glu 196D OE1 3.40

Trp 207E CE3 Phe 150D CG 3.96

Phe 150D CD2 3.87

Phe 150D CE2 3.90

Trp 207E CD1 Thr 194D CG2 3.94

Trp 207E NE1 Glu 196D OE1 3.63 _*

Trp 207E CZ2 Glu 196D OE1 3.08

Trp 207E CZ3 Pro 197D CG 3.94

Pro 197D CD 3.99

Trp 207E CH2 Glu 196D OE1 3.81

Pro 197D CD 3.90

Trp 207E C His 151 D NE2 3.74

Trp 207E 0 His 151 D NE2 3.25 _***

Phe 150D CD1 3.70

Phe 150D CB 3.85

His 151 D CD2 3.56

Leu 208E CD2 His 151 D NE2 3.87

Glu 209E 0E2 Phe 150D CD1 3.92

Phe 150D CE1 3.98

Gly 149D CA 3.54 ^*** indicates a strong possibility for a hydrogen bond at this contact (distance < 3.3 A) as calculated by CONTACT

^* indicates a weak possibility (distance > 3.3 A).

Example 2: Determination of interaction interface between IL-20 and the antibodies 2F6, 5B7, 7F1 , 9F1 , 11 F12, 11 F25, and 11 F36

This example identifies the interaction surface on IL-20 when binding the antibodies 2F6, 5B7/15D2, 7F1 , 9F1 , 1 1 F12, 1 1 F25, 1 1 F36, respectively.

Hydrogen exchange mass spectrometry (HX-MS) exploits that the change in mass when hydrogen is exchanged with deuterium can be followed over time using mass

spectrometry. When an antibody binds to IL-20, amino acid residues located in the binding interface are protected from hydrogen exchange with the solvent. Pepsin digestion allows identification of peptides that are part of the epitope and show slower rates of hydrogen exchange when the antibody is bound. HX-MS can also reveal other features such as structural flexibility/stabilization and changes in these features upon binding of an antibody. These experiments were performed using IL-20 with an added N-terminal methionine residue (position -1 ).

Solutions of IL-20 alone or in the presence of one of the antibodies 2F6, 5B7, or 9F1 were diluted 13-fold in 95% deuterated PBS buffer (10 mM sodium phosphate, 2.68 mM potassium chloride, 140 mM sodium chloride, pH 7.4). A 25-fold dilution was used in the experiments with the antibodies 7F1 , 1 1 F12, 1 1 F25, and 1 1 F36. Non-deuterated controls were prepared by diluting into protiated PBS buffer. The hydrogen exchange experiments were performed on a nanoAcquity UPLC system with HDX technology (Waters Corporation, Milford, MA, USA) which includes the HD-x PAL auto sampler (LEAP Technologies Inc., Carrboro, NC, USA) for automated sample preparation and an ultra-high performance liquid chromatography (UPLC) system. The UPLC tubing, pre- and analytical columns and switching valves were located in a chamber cooled to 1 °C. The trypsin digestion column was stored at 25°C.

Hydrogen exchange reactions were performed at 20°C. Mass analysis was performed online using a Waters SYNAPT G2 HDMS mass spectrometer. A volume containing 100 pmol of I L-20 with or without 120 pmol of one of the antibodies was diluted into deuterated PBS buffer. At the time intervals from 15 seconds to 8 hours 49 μΙ of the sample was quenched in 50 μΙ 1 .35 mM Tris (2-carboxyethyl) phosphine adjusted to pH 2.7 and held at 3°C. 99 μΙ of the quenched solution was immediately injected and passed over a Porozyme immobilized pepsin column (2.1 mm x 30 mm) (Applied

Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) and trapped on a Waters VanGuard BEH C18 1 .7 μηη (2.1 mm 5 mm) column using a 5% methanol, 0.1 % formic acid mobile phase and a 100 μΙ/min flow rate. The peptides were separated on a Waters UPLC BEH C18 1 .7 μηη (1 .0 mm x 100 mm) column using a 10 - 40 % acetonitrile gradient containing 0.1 % formic acid at a 40 μΙ/min flow-rate.

The mass spectrometer was run in positive ion mode with ion mobility separation enabled. The instrument parameters used were 3.2 kV capillary, 25 V sample cone, and 4 V extraction cone offsets, 850 ml/min flow of desolvation gas and 50 ml/min cone gas flow. The source block was heated to 120°C and the desolvation gas to 350°C. Lock-mass correction data using acquired using the 1 + ion of Leucine-enkephalin (m/z 556.2771 ) as reference compound and applied during data analysis. For peptide identification MS^E-type experiments using trap collision offsets of 6 V (low-energy) and 15-45 V ramp (elevated energy) were performed. Deuterated samples were analysed using the 6 V low energy trap collision offset only. For further details see Andersen et al, Int. J. Mass Spectrom. 302:139-148 (201 1 ). The MS^E-data was analysed using Waters ProteinLynx Global Server 2.5 and peptides of I L-20 were identified that covered 99 % of the protein sequence (see table 7). The HX-MS data files were analysed using Waters DynamX 1 .0 or Waters DynamX 2.0 that automatically applies lock-mass correction and determines the degree of deuterium incorporation in each peptide. In addition, all data was manually inspected to ensure correct peak assignment and calculation of deuterium incorporation.

Results

Epitope mapping of the antibodies 2F5, 5B7/15D2, and 9F1

Amide hydrogen exchange of 44 peptides covering 99 % of the I L-20 protein sequence were followed in the presence or absence of one of the antibodies, 2F6, 5B7, or 9F1 . The peptides could be divided into groups of 1 ) peptides that displayed an exchange pattern that was largely unaffected by binding of the antibody, 2) Peptides whose exchange pattern was markedly affected by antibody binding indicating that most of the peptide is part of the epitope or that the structure in this region is stabilized by antibody binding, 3) peptides whose exchange pattern was affected in a small, but significant, degree by antibody binding indicating that only a few residues in the peptides are part of the epitope, and 4) Peptides where the changes in exchange pattern upon antibody binding could not be determined due to noise in the data.

Some peptides that are not part of the epitope showed protection from amide hydrogen exchange upon binding of the antibodies due to structural stabilization. The peptides lie 38 - Leu 58, Gin 48 - Leu 58, Thr 107 - Phe 125, His 109 - Phe 125, and Val 134 - Leu 141 , and Val 134 - Asp 142 were significantly protected against hydrogen exchange due to structural stabilization upon binding of either the 2F6, 5B7, or 9F1 antibody.

The data for the peptides Gin 16 - Phe 22 and Gin 16 - Phe 23 contained substantial noise and it was not possible de confirm or exclude that antibody binding protected them from hydrogen exchange. However, as the peptides, Gin 16 - Gly 27, Gin 16 - Gin 30, and Gin 16 - Asp 37 also cover this part of the IL-20 sequence and show no protection from hydrogen exchange upon antibody binding, protection in this region can be excluded.

5B7/15D2 epitope on IL-20

There is a strong protection from amide hydrogen exchange in the peptides that cover the region from Tyr 66 - Leu 93 and these peptides form the binding epitope. This epitope is located in the interface between IL-20 and IL-20R2 (see example 1 ).

2F6 epitope on IL-20

There is a strong protection from amide hydrogen exchange in the peptides from residue Met -1 - Leu 15 and from residue Arg 83 - Met 106. This epitope is not located in the any of the interfaces between IL-20 and IL-20R1 or IL-20R2 (see example 1 ). However, binding of the antibody may inhibit receptor binding sterically. Weak protection was observed in peptides covering the sequence from Tyr 66 - Lys 73.

9F1 epitope on IL-20

There is a strong protection from amide hydrogen exchange in the peptides from residue Met -1 - Leu 15 and from residue Leu 93 - Met 106. This epitope is not located in the any of the interfaces between IL-20 and IL-20R1 or IL-20R2 (see example 1 ). However, binding of the antibody may inhibit receptor binding sterically. Weak protection was observed in peptides covering the sequence from Tyr 66 - Lys 73. Table 7. Classification of HXMS experiments on 2F6, 5B7 and 9F1

Peptide 2F6 5B7 9F1

M-1-L6 EX N EX

M-1 -S8 EX N EX

M-1-C9 EX N EX

M-1 -V10 EX N EX

M-1-L15 EX N EX

Q16-F22 ND ND ND

Q16-F23 ND ND ND

Q16-G27 N N N

Q16-Q30 N N N

Q16-D37 N N N

S23-D37 N N N

I38-L47 N N N

I38-L58 W W W

Q48-L58 W W W

L59-L65 N N N

Y66-L82 W EX W

Y66-S87 W EX W

Y66-L88 W EX W

Y66-F92 W EX W

D68-L82 N EX N

D68-L88 W EX W

D68-F92 W EX W

N73-L93 N EX N

R83-F92 EX EX N

L93-L101 EX N EX

L93-A104 EX N EX

L93-M106 EX N EX

L93-E1 12 EX N EX

L93-E1 13 EX N EX

L93-F125 EX N EX

T94-M106 EX N EX

T94-E126 W N W

I95-M106 EX N EX Peptide 2F6 5B7 9F1

T107-F125 W W W

H109-F125 W W W

E1 13-F125 N N N

A1 14-L122 N N N

A1 14-F125 N N N

M1 15-F125 N N N

E126-A133 N N N

E126-L141 N N N

V134-L141 W W EX

V134-D142 W W EX

L145-K152 N N N

EX: Epitope region indicated by hydrogen exchange protection upon antibody binding. (EX>0.6)

W: Weak protection from exchange upon antibody binding. (0.3<EX<0.6)

N: No protection from exchange upon antibody binding. (EX<0.3)

NA: Not analysable.

Epitope mapping of antibodies 7F1 , 1 1 F12, 1 1 F25, and 1 1 F36

49 peptides covering 100 % of the sequence were analysed and the amount of deuterium incorporation was determined in the presence or absence of one of the antibodies, 7F1 , 1 1 F12, 1 1 F25, and 1 1 F36. The peptides could be divided into groups of 1 ) peptides that displayed an exchange pattern that was largely unaffected by binding of the antibody, 2) Peptides whose exchange pattern was markedly affected by antibody binding indicating that most of the peptide is part of the epitope or that the structure in this region is stabilized by antibody binding, 3) Peptides that showed increased uptake of deuterium upon antibody binding, thus indicating conformational changes, increased dynamics, or less hydrogen bonding. 4) Peptides where the changes in exchange pattern upon antibody binding could not be determined due to noise in the data.

Binding epitope mapping of mAb 1 1 F12 on human IL-20

When binding mAb 1 1 F12, IL-20 either showed no significant protection of hydrogen exchange or increased exchange as shown in table 8. Increased hydrogen exchange indicates that the protein has undergone a conformational change where parts of the protein becomes 5- more exposed to solvent or that back-bone amide hydrogens participate less in hydrogen bonds. Increased hydrogen exchange can also be a result of increased dynamics in that part of the protein.

The binding epitope is most likely found in the region where there is no observed protection. These regions are from residue M-1 -11 1 , S23-L65, R83-E1 13, L145-E152. The region from V134-L144 only showed low intrinsic exchange. However, this low exchange was markedly reduced upon binding of mAb 1 1 F12, and this region is therefor also likely to be part of the binding epitope. These regions include the IL-20 receptor-1 binding-site, and the hydrogen exchange results are in agreement with 1 1 F12 having its binding epitope in the IL-20 receptor-1 binding-site. However, the high degree of conformational changes observed may also in itself cause IL-20 to not be able to bind to IL-20 receptor-1 .

Table 8. Hydrogen exchange protection upon binding of mAbs 1 1 F12, 1 1 F25, and

1 1 F36

First End

11 F residue residue Sequence 11 F12 11 F25

36 no. no.

-1 6 MLKTLNL NP^* p_** P

-1 8 MLKTLNLGS NP P P

-1 9 MLKTLNLGSC NP P P

-1 10 MLKTLNLGSCV NP P P

-1 11 MLKTLNLGSCVI NP P P

-1 15 MLKTLNLGSCVIATNL P P

9 15 CVIATNL N P P

16 22 QEIRNGF N P P

16 30 QEIRNGFSEIRGSVQ NP P P

16 37 QEIRNGFSEIRGSVQAKDGNID NP P P

38 47 IRILRRTESL NP P P

38 57 IRILRRTESLQDTKPANRCC NP P P

38 58 IRILRRTESLQDTKPANRCCL NP P P

38 65 IRILRRTESLQDTKPANRCCLLRHLL

N P P RL

42 58 RRTESLQDTKPANRCCL NP NP NP

43 58 RTESLQDTKPANRCCL NP NP NP

59 65 LRHLLRL NP LE/P

66 82 YLDRVFKNYQTPDHYTL N P P

66 83 YLDRVFKNYQTPDHYTLR N P P

66 87 YLDRVFKNYQTPDHYTLRKISS N P P

66 88 YLDRVFKNYQTPDHYTLRKISSL N P P First End

11 F residue residue Sequence 11 F12 11 F25

36 no. no.

66 92 YLDRVFKNYQTPDHYTLRKISSLANS

N P P

F

66 93 YLDRVFKNYQTPDHYTLRKISSLANS

N P P FL

67 82 LDRVFKNYQTPDHYTL N P P

67 83 LDRVFKNYQTPDHYTLR N P P

67 88 LDRVFKNYQTPDHYTLRKISSL N P P

67 92 LDRVFKNYQTPDHYTLRKISSLANSF N P P

68 82 DRVFKNYQTPDHYTL N P P

68 83 DRVFKNYQTPDHYTLR N P P

68 88 DRVFKNYQTPDHYTLRKISSL N P P

68 92 DRVFKNYQTPDHYTLRKISSLANSF N P P

83 92 RKISSLANSF NP P P

83 93 RKISSLANSFL NP P P

93 101 LTIKKDLRL NP P P

93 106 LTIKKDLRLCHAHM NP P P

93 113 LTIKKDLRLCHAHMTCHCGEE NP P P

93 125 LTIKKDLRLCHAHMTCHCGEEAMKK

N P P YSQILSHF

94 101 TIKKDLRL NP P P

94 106 TIKKDLRLCHAHM NP P P

107 125 TCHCGEEAMKKYSQILSHF N P P

109 125 HCGEEAMKKYSQILSHF N P P

113 125 EAMKKYSQILSHF N P P

114 125 AMKKYSQILSHF N P P

115 125 MKKYSQILSHF N P P

126 141 EKLEPQAAVVKALGEL N P P

134 141 VVKALGEL LE/P LE/P LE/P

134 144 VVKALGELDIL LE/P LE/P LE/P

135 141 VKALGEL LE/P LE/P LE/P

145 152 LQWMEETE NP P NP

^*NP: No Protection: Insignificant or no protection against hydrogen exchange of less than 0.5 deuterons in the peptide.

^**P: Protection: Protection against hydrogen exchange by more than 0.5 deuteron in the peptide.

^***N: Negative: Increased rate of hydrogen exchange when mAb is bound

^****Low exchange/protection: IL-20 absent of mAb exchanges less than 1 hydrogen for a peptide, but binding of the mAb decreases hydrogen exchange to less than 0.5 hydrogens.

Binding epitope mapping of mAb 1 1 F25 on human IL-20

Upon binding mAb 1 1 F25, protection from hydrogen exchange is observed throughout the sequence of IL-20 (Table 8). This is an indication that 1 1 F25 binding stabilizes the dynamics of IL-20 throughout the majority of the structure. Only in the regions R42-L65 and L145-E152 there is no observed protection from hydrogen exchange upon binding of mAb 1 1 F25. These results are in agreement with 1 1 F25 having its binding epitope in the IL-20 receptor-1 binding-site. Binding epitope mapping of mAb 1 1 F36 on human IL-20

Upon binding mAb 1 1 F36, protection from hydrogen exchange is observed throughout the sequence of IL-20 (Table 8). This is an indication that 1 1 F36 binding stabilizes the dynamics of IL-20 throughout the majority of the structure. Only in the region R42-L65, there is no observed protection from hydrogen exchange upon binding of mAb 1 1 F36. These results are in agreement with 1 1 F36 having its binding epitope in the IL-20 receptor-1 binding-site.

Binding epitope mapping of mAb 7F1 on human IL-20

When an antibody binds it is expected that protection against hydrogen exchange is observed on peptides that cover the epitope. For 7F1 , no significant protection against hydrogen exchange was observed on any peptide upon binding on human IL-20. It was therefore not possible to determine the epitope using HXMS with the conditions used. This could be caused by a low binding affinity of mAb 7F1 . Stabilizing IL-20 by binding multiple mAbs

Binding of one or more other antibodies might mitigate the structural stabilization of IL- 20 when mAb 1 1 F25 and 1 1 F36 bind. Thus, mAb 1 1 F25 1 1 F36 each were bound to IL-20 in the presence of both mAb 2F6 and mAb 5B7 and the hydrogen exchange pattern was compared to both IL-20 absent of any mAbs and IL-20 bound to mAb 2F6 and mAb 5B7 simultaneously as shown in Table 13. In the regions M-1-L15 and L93-M1 15 where mAb 2F6 has part of its binding epitope and the region Y90-F1 16 where 5B7 has its epitope, no further protection was observed upon binding of 1 1 F25 or 1 1 F36. This shows that mAbs 2F6, 5B7, and either 1 1 F25 or 1 1 F36 all are bound to IL-20 simultaneously and that the structural stabilization of IL-20 upon binding of 1 1 F25 or 1 1 F36 is mitigated in these regions when mAbs 2F6 and 5B7 are bound. The structural stabilization in the region I38-L58 observed when 1 1 F36 was bound alone was not observed when mAbs 2F6 and 5B7 also were bound. Thus, this segment could be excluded from the binding 1 1 F36 epitope. These results are in agreement with 1 1 F25 and 1 1 F36 binding to the IL-20 receptor-1 binding site. Table 9. Binding of mAbs 1 1 F25 and 1 1 F36 while both 2F6 and 1 1 F25 are bound.

^*P: Protection: Protection against hydrogen exchange by more than 0.5 deuteron in the peptide.

^**NP: No Protection: Insignificant or no protection against hydrogen exchange of less than 0.5 deuterons in the peptide. Example 3: Immunization strategy for Site 1 specific antibodies

Making monoclonal antibodies is a standard technique and fairly easy to perform by a person skilled in the art.

Making monoclonal antibodies employing the hybridoma technology requires immunization of mice or other rodents. The essence of this technology is to immortalize a B- lymphocyte, which is producing an antigen-specific antibody. This is done by fusing the B- lymphocyte with a myeloma cell. The myeloma cell has the ability to replicate in vitro and is giving this feature on to the fusion-product, the hybridoma. The hybridoma will produce the antibody originally coded for by the B-lymphocyte. To be able to make good high-affinity antigen-specific antibodies we therefore need to immunize/vaccinate mice to induce a strong immune response towards the antigen in question in the mice. When immunizing mice the antigen have to be administered in adjuvant for slow sustained release of the antigen over several days. The adjuvant normally also have components which stimulates the immune system. These components can be of bacterial origin.

Proteins from other species than mouse will normally be seen as 'foreign' and the mouse will mount an immune response resulting in specific antibodies against these proteins.

IL-20 binds to two different receptor complexes IL-20R1/IL-20R2 and IL22R1/IL-20R2. Binding of IL-20 to the IL20R1/IL-20R2 complex has been described in example 1 ). Two non- overlapping binding sites are defined, one to the IL-20R1 (Site 1 ) and one to IL-20R2 (Site 2). The binding epitope of antibody 15D215D2 disclosed in WO2010000721 are overlapping with the IL-20 interface to IL-20R2 i.e. BS22.

Using the modelling of the IL20/IL-20R1/IL-20R2 complex we have designed a strategy to make monoclonal antibodies specific to the surface area of IL-20 binding to the IL-20R1 receptor chain i.e. BS1 .1 .

Studying the model of the receptor complex we discovered that the interface of IL-20 tolL-20R1 receptor is mainly restricted to a highly flexible peptide not engaged in coiled coil nor engaged in a beta sheet. This peptide is spanning from nr. Gin 30 - Asn 54 (SEQ ID NO

1 -) The remaining of the interface of IL-20 to IL-20R1 is done by a more rigid peptide (Val

134 - Glu 152, SEQ ID NO 1 ). Taking advantage of these two observations we have synthesised the following peptides for immunization covering the first Gin 30 - Asn 54 (SEQ ID NO 1 ) area, listed in table 10.

Table 10. Peptides for immunization of mice

The peptides for the following for the second Val 134 - Glu 152 area of mature human IL-20 (SEQ ID NO 1 ) are shown in table 1 1.

Table 1 1. Peptides for immunization of mice

All peptides are coupled to the carrier molecules before immunization of mice.

The site 2 epitope defined by the interface of IL-20 to IL-20R2 seems to be an immune- dominant epitope. To be able to 'blind' the immune system to this epitope we scrambled the epitope by exchanging several amino acids (hydrophilic to hydrophobic) in the Site 2 area. Other analogues are where the Site 2 is deleted from the molecule by deleting domains if possible other IL-20 analogues we exchanged amino acids in the Site 1 to create an analogue suitable to be used in a subtraction immunization regiment (see later). In order to present the Site 1 epitope in its proper three dimensional structure to the immune system we have made hybrid molecules taking advantage of the large similarity between IL-20 and its family member IL-19.

The hybrid molecule is molecule made in such a way that the binding sites 1 and 2 consist of the IL-IL-20 binding site 1 and of the 11-19 binding site 2 amino acids respectively.

When protein molecules used for immunization are too small (peptides or fragments), unstable, homologous to the mouse variant of the molecule or simply weak immunogens, immunologist often use larger protein as carrier molecules. These molecules are chemically coupled to the antigen either randomly to primary amines, specifically via cysteine bridges or randomly via glutaraldehyde coupling. In this project we have used cBSA (negatively charged Bovine Serum Albumin), mcKLH (Keyhole Limpet Hemocyanin) and PPD ( Derivative of B.turberculosis vaccine).

The mostly used adjuvant for immunization is Freunds Adjuvant. Freund's adjuvant is a solution of antigen emulsified in mineral oil and used as an immune potentiator (booster). The complete form, Freund's Complete Adjuvant, (CFA or FCA) is composed of inactivated and dried mycobacteria (usually M. tuberculosis), whereas the incomplete form (I FA or FIA) lacks the mycobacterium components (hence just the water in oil emulsion).

We also use Ribi adjuvant. Ribi adjuvant is oil-in-water emulsions where antigens are mixed with small volumes of a metabolizable oil (squalene) which are then emulsified with saline containing the surfactant Tween 80. This system also contains refined mycobacterium products (cord factor, cell wall skeleton) as immune stimulants and bacterial monophosphoryl lipid A.

Other adjuvant systems/paradigms such as inorganic adjuvants, organic adjuvants, oil- based adjuvants, virosomes or antigen presenting cell (APC) targeting modules can be used. Inorganic salts as Aluminium hydroxide or phosphate can be used as adjuvants. In organic adjuvants squalene is often used as an immune-potentiator. Virosomes contain a membrane- bound hemagglutinin and neuraminidase derived from the influenza virus. Antigens can be targeted to APC by coupling the antigen to monoclonal antibodies specific for various surface proteins of the APC.

Immunization Protocols

In our regular immunization protocol we inject 20 ug of antigen in CFA subcutaneously followed by two injections of the same amount in I FA every two weeks. This protocol always gives high titers of antibody. If titers are low a further two injection in IFA is sufficient to bring up the titers.

In order to overcome and/or exclude an immune reaction towards IL-20 Site 2 we performed subtraction immunization. This protocol tolerates the mice to the Site 2 epitope by stimulating the immune system by immunizing with an IL-20 analogue with deleted or scrambled Site 1 but fully intact Site 2 epitope. The mice are immunized three times every two weeks but 3 days after each injection the mice are given cyclophosphamide, which eradicated the stimulated and proliferating immune cells. After a further 2 weeks the mice are immunized with a Site 2 scrambled or deleted IL-20 analogue with an intact Site 1 analogue.

This protocol can also be done by employing Site 2 specific peptides as the tolerizing object and then followed by regular immunization with the IL-20 molecule or analogues thereof.

Example 4: Competition and screening for site specific anti-IL20 mAbs

Monoclonal antibodies targeting a desired region on the antigen can be selected by careful screening using various assays. Such methods include, but are not limited to, ELISA and Surface Plasmon Resonance (SPR) analysis. These screening assays are in general established as competition assays, measuring simultaneous binding of e.g. two mAbs or mAb/ligand to an antigen. The data obtained from such assays does not provide the exact epitope for the test compound (mAb or ligand), but serves to describe the ability of this compound to bind the antigen simultaneously with a second compound. Therefore, narrowing down the region to which e.g. a specific mAb binds, often require screening of this against multiple compounds with known binding region on the antigen. Use of competition ELISA is often challenging due to the requirement for labelled detection reagents, whereas SPR analysis can be performed on unlabelled reagents. In SPR analysis the protein interactions are monitored in real time, thus providing additional information on the binding profiles for the individual analytes.

A competition ELISA can be established by coating immunosorbent plates with a mAb with known binding site. After incubation with the antigen, the mAb of interest is added.

Detection of binding of the mAb of interest can be performed by a species specific labelled secondary polyclonal Ab, if the two mAb originates from different species. However, as this is rarely the case, labelling of the mAb of interest is required. This label could be biotin or horse radish peroxidase (HRP). If a colour reaction is detected, the two mAbs are binding simultaneously to the antigen.

As an alternative approach, a ligand for the antigen can be coated. Thereby, mAbs binding to the same region as the ligand will not be able to bind the ligand/antigen complex. As an example of using the ELISA methods described for screening for mAbs potentially binding to site 1 on IL-20, an ELISA could be designed using as coating an antibody binding to site 2. Mabs positive for binding in this assay could be tested further in an ELISA using a mAb binding to an epitope outside site 1 and 2, e.g. mAb anti-IL20-9F1. Positive binders should be further characterized e.g. by HX-MS, in order to identify their epitope. In another example, an ELISA could be designed using recombinant IL-20R1 or free

IL-20 as coating, and detect for mAbs not binding the IL20/IL20R1 complex but to free IL-20.

An SPR analysis can be performed e.g. on a Biacore instrument. A competition, or "binning", experiment can be performed by immobilizing specific mAbs or ligands to the individual flow cells on a sensor chip. Subsequently the antigen is injected across all flow cells, followed by injection of the mAb of interest. This approach does not require a detection step, as the binding profile is monitored in real time.

As an example of using SPR analysis to identify mAbs binding to site 1 on IL-20, mAbs binding to regions outside site 1 (e.g. Site 2 mAbs and mAb anti-IL20-9F1 ) are immobilized in individual flow cells. IL-20 is injected across all flow cells resulting in the formation of a mAb/IL- 20 complex. Subsequently the mAb of interest is injected across all flow cells. Binding of the mAb of interest indicates that it binds to a region outside those of the immobilized mAbs.

In another example, recombinant IL-20R1 is immobilized and IL-20 followed by the mAb's of interest are subsequently injected. MAbs not binding to the IL-20/IL-20R1 complex potentially binds to site 1 . In another example, the mAb of interest is captured by an immobilized anti-Fc pAb.

Subsequently IL20 is injected, followed by IL20R1 .

As an alternative approach, the mAbs of interest can be screened by SPR analysis for the binding to intact antigen, as well as peptide fragments covering the region of interest. As an example, mAbs binding to site 1 in IL-20 can be identified by immobilizing or capturing the mAb of interest, followed by injection of either intact IL-20 or peptides covering the region of site 1 . Example 5: Total IL-20 Assay

IL-20 can be measured in samples from clinical trials by the use of either a Free IL-20 assay or a Total IL-20 assay.

It is commonly known that when the mAb binds to free ligand the clearance of the complex from circulation is usually slower than the clearance of the free ligand, leading to an accumulation of the complex and the total ligand levels.

Therefore the accumulation of either to drug-ligand complex or the total ligand concentration can be incorporated into PK-PD modelling for prediction of the free ligand concentration. The measurement of Free IL-20 is an assay technical challenge due to low baseline concentrations of IL-20 in blood circulation which drops further when 15D2 is administrated. The Free IL-20 assay has been set-up using a non-competing epitope from 15D2 as capture antibody (9F1 ) and the 15D2 as detection antibody.

The Total IL-20 assay measures all forms of IL-20 including free and complexed IL-20. The assay requires two anti-IL-20 antibodies that bind to non-competing epitopes from 15D2, and from each other. The challenge is to find these antibodies. An assay build up with a site 1 as coating antibody and an antibody with a binding site different from site 1 and site 2 as detection, can be feasible for detection of e.g. antibodies binding to site 2.

Example 6: Epitope diversity assessed by BLI analysis

Antibody binding competition assays were performed by Bio-Layer Interferometry (BLI) analysis in order to investigate whether anti-IL-20 mAbs bound simultaneously to IL-20. An inability of mAbs to bind simultaneously indicates common or overlapping epitopes, though factors such as steric hindrance and conformational changes may contribute. A panel of 96 monoclonal antibodies generated against recombinant human IL-20, were tested for binding to a region of IL-20 not covered by mAbs 9F1 and 5B7 (see example 2).

The BLI studies were performed using a ForteBio OctetRed384 instrument (Pall). The purified anti-IL-20 mAbs 9F1 and 5B7 were immobilized on AR2G sensors using standard amine coupling. In the first step, the sensors were incubated in a fixed concentration of IL-20 in HBS-EP buffer (10mM HEPES, 150mM NaCI, 3mM EDTA and 0.005% Polysorbat P20, pH7.4). Following a short dissociation period in HBS-EP, the sensors were subsequently incubated with the specific second mAb. Out of the 96 mAbs tested, 15 mAbs were identified as binders to a region on IL-20 not influenced by binding of mAbs 9F1 or 5B7, and serves therefor as potential site-1 binders.

Competition binding experiments for compunds competing with the binding of identified potential site-1 binders could be performed by eg. ELISA or SPR analysis as described in example 4.

Example 7: Selection of mAbs binding to site-1 by peptide ELISA

In order to screen for mAbs binding to site-1 on IL20, an ELISA was established based on the site-1 peptides described in example 3.

The peptides covering site-1 were coated in individual wells on immunoplates

(Maxisorb, Nunc), and incubated for 1 hr. with the primary mAb. Following a washing step,detection was carried out with a HRP-conjugated anti-mouse polyclonal antibody.

The mAbs identified to bind to a region on IL-20 different from mAbs 9F1 and 5B7 ( see example 6) were tested in the site-1 peptide ELISA.

Of the mAbs tested, the mAbs: IL20-7F1 , IL20-1 1 F12, IL20-1 1 F25 and IL20-1 1 F36 demonstrated binding to the binding site 1 peptides of table 12. Table 12. Binding site 1 peptides used for ELISA

The above mentioned four antibodies: IL20-7F1 , IL20-1 1 F12, IL20-1 1 F25 and the antibody IL20-1 F13, were deposited at Public Health England (previously known as The European Collection of Cell Cultures (ECACC)), Microbiology Services, Porton Down, Salisbury, SP4 OJG, United Kingdom under the following accession numbers as listed in table 13.

Table 13. Accession Numbers for deposited antibody hybridoma, deposited at Public Health England (previously The European Collection of Cell Cultures (ECACC)), Microbiology Services, Porton Down, Salisbury, SP4 OJG, United Kingdom.

Example 8: Anti-IL20 site-1 mAbs interfering with the interaction between IL20 and IL20Ra.

SPR analysis was performed on a BiacoreT200 instrument (GE Healthcare) in order to investigate the effect of anti-IL20 site-1 mAbs on the interaction between IL20 and a recombinant IL20Ra-Fc construct.

In this experiment the effect of site-1 mAbs IL20-7F1 , IL20-1 1 F12 and IL20-1 1 F36 was measured. Anti-IL20 mAb 5B7 binding to an epitope outside site-1 was included as control. The recombinant IL20Ra-Fc was covalently coupled via free amine groups to the carboxy methylated dextrane membrane on the sensor chip (CM5) surface, to a level of 2000 Resonance Units (RU).

IL20 (588 nM) in HBS-EB (100 mM Hepes, 100 mM NaCI, 3 mM EDTA and 0,02% tween20, pH 7,4) was pre-incubated with the individual mAbs (667nM) for 15 min. IL20 +/- mAb was injected across the immobilized IL20Ra-Fc, followed by a dissociation period with constant buffer flow over the sensor chip surface. Injection time was 60 sec, followed by 60 sec. dissociation. Flowrate was 30 ul/min, and the binding level in RU was measured 5 sec. prior to injection stop. Binding to IL20Ra-Fc was observed for IL20 alone (See Table 14). Pre-incubation of

IL20 with mAbs IL20-7F1 , IL20-1 1 F12 or IL20-1 1 F36 resulted in a reduction in the binding signal to IL20Ra-Fc, indicating that these mAbs interfere with the interaction between IL20 and IL20Ra-Fc.

In contrast, an increase in binding signal was observed when injecting IL20 pre- incubated with mAb 5B7. This indicates binding of an IL20/5B7 complex to the IL20Ra-Fc.

Table 14 shows binding response to IL20Ra-Fc by either IL20 alone, or IL20 pre- incubated with specific anti-IL20 site-1 mAbs. The relative response in RU is measured 5 sec. prior to injection stop.

Table 14: SPR analysis of effect of IL20 site-1 mAbs on the IL20/IL20Ra-Fc interaction.

Example 9: Sitel binding Antibodies in IL-20 reporter gene assay

In order to generate two IL-20 responsive reporter cell lines the BHK-21 cell line (ATCC #CCL-10) was either transfected with IL-22R and IL-20R-beta expression vectors and a STAT3-luciferase reporter plasmid or IL-20R-alpha and IL-20R-beta expression vectors and a STAT3-luciferase reporter . During selection with 600 migrogram/ml G418, 200 microgram/ml Hygromycin B and 5 microgram/ml Puromycin stable transfectants were established. Single clones were isolated by limited dilution. The reporter cells were seeded out in a black viewplate (Nunc), 20,000 cells/well. The antibodies in serial dilutions were incubated with 20 nanogram/ml (final concentration) IL-20 for fifteen minutes and then added to the reporter cell. After four hour stimulation SteadyGLO (Promega) Luciferase substrate was added and after 10 minutes incubation the plate was read on the TopCount NXT (Perkin Elmer) Instrument. Of the five sitel binding antibodies tested in IL-22R/IL-20R-beta (Man42) and IL-20R- alpha/IL-20R-beta reporter gene assay, one is not inhibiting IL-20 signalling (1 1 F12) and two are very poor (1 F13 and 7F1 ), one has an IC50 of 6 nM (1 1 F25) and one has an IC50 of 3 nM (1 1 F36).

The results are shown in Table 15. Numbers from the TopCount read were processed and IC50 were determined or predicted by GraphPad prism. The IL-20 concentration was 20 nanogram/ml.

Table 15. IC50 of sitel antibodies inhibiting the activity of the IL-22R/IL-20R-beta reporter cell line (Man42) and the IL-20R-alpha/IL-20R-beta reporter cell line (Man40).

IC50 nM

IL-22R/IL-20R- IL-20R-alpha/IL- beta (Man42) 20R-beta (Man40)

11 F12 NA NA

11 F25 6,0 6,6

7F1 83, 1 95,9

11 F36 3,2 3,2

1 F13 37,7 69,0

1 F139F1 6,3 7,4

5B7 1,4 1,0 Example 10: Selection strategy for Site 1 specific binders using antibody phage display

Analogous to example 3, using modelling of the IL20/IL-20R1/IL-20R2 complex we have designed a strategy using antibody phage display to generate specific binders to the surface area of I L-20 binding to the I L-20R1 receptor chain i.e. BS1 .1.

We used peptide 2, 3, 4 and 5, listed in Table 8, covering the Gln30-Asn54 (SEQ ID N01 ) area of I L-20, and peptide 6 and 7, both listed in Table 9, covering the Val134-Glu152 (SEQ ID N01 ) area of I L-20, for selection purposes. All peptides were C-terminally coupled to biotin to allow for capturing of the peptides using streptavidin-coated magnetic beads during the selection process.

Phage selection protocol

For each of the 6 peptides, streptavidin-coated M280 magnetic beads (Invitrogen) were washed three times using PBS, and subsequently used to capture 2 g of the biotinylated peptides. After peptide capture the beads were washed 6 times using PBS/0.1 % Tween-20 and blocked at room temperature in PBS containing 3% BSA and 0.1 % Tween-20 for 30 minutes.

For each selection FAB310 phage library aliquots (approximately 2x10¹² cfu), obtained from Dyax Corporation, were blocked for 1 hour at room temperature in PBS containing 3% BSA and 0.1 % Tween-20. After 5 consecutive rounds of deselection on streptavidin-coated M280 magnetic beads, deselected phage were incubated for 1 hour at room temperature with the peptides captured on streptavidin beads under constant rotation. Non-specific phage were eliminated by 12 washes using PBS/0.1 % Tween-20 and 2 washes with PBS, each wash step was incubated for 10 minutes at room temperature under constant rotation. To recover the remaining peptide bound phage and generate an amplified phage library for subsequent rounds of selections, the washed bead mixture was used to infect 5ml of exponentially growing E. Coli TG1 cells. After 30 minutes of incubation at 37^°C, 1.5x10¹⁰ pfu of M13K07 helper phage (Invitrogen) was added and incubated for an additional 30 minutes at 37^°C, after which 25ml fresh 2xTY media containing 100ug/ml Carbenicillin and 50ug/ml Kanamycin was added and cells were grown overnight at 30^°C. The next day phage were purified and concentrated from the culture supernatant by precipitation with one-fifth v/v of 20%PEG8000/2.5M NaCI

(Teknova), centrifugation and resuspension in PBS. For each peptide three rounds of selections were performed as described above, after which individual clones were obtained and subjected to phage ELISAs.

Phage ELISA

To examine individual clones after three rounds of selection, peptide bound phage were recovered by infecting E.coli TG1 cells, as described above, with the exception that before the addition of helper phage an 180ul aliquot of infected TG1 cells was taken aside. This aliquot of infected TG1 cells was titrated and spread on 2xTY agar plates containing 2% glucose and 100ug/ml Carbenicillin and incubated overnight at 37°C. For each peptide selection two 96-well plates of individual colonies were picked and grown at 37^°C for 5hrs, M13K07 helper phage was added and after 30 minutes plates were transferred to 30^°C. The next day the culture supernatants, containing the phage particles, were separated from the cells by centrifugation. The phage culture supernatants were diluted 1 :10 in PBS containing 3%BSA and 0.1 %Tween-20.

To examine the phage culture supernatants for specific binding to site 1 of hulL-20, they were tested for binding to four different targets, i.e. their respective target peptide, captured via streptavidin, streptavidin (Pierce) alone, hulL20 coated directly, and an irrelevant protein KLH (Sigma) coated directly to Maxisorb 96-well plates (Nunc). All proteins were coated at 1 g/ml overnight at 4^°C. For the direct ELISAs, the next day plates were blocked with PBS containing 3% BSA and 0.1 % Tween-20. For the sandwich ELISAs biotin-conjugated peptides were captured before the plates were blocked. Diluted phage culture supernatants were added and phage binding was determined by using anti-M13-HRP (GE Healthcare). All ELISAs were carried out in a volume of 100 I and four washes with PBS containing 0.1 % Tween-20 were carried out in between each step. Incubations were all carried out for 1 hour at room temperature unless specified. ELISAs were visualized with the addition of TMB substrate (Surmodics) and after the addition of 2N H₂S0₄ stop solution (Surmodics) absorbance at 450nm was detected.

Selections using peptide 2, 3 and 4 did not generate any specific binders that recognized both the target peptide and IL-20. As summarized in Table 15, selections using target peptide 5 generated 1 specific binder, clone M2.407.G04, that bound both peptide 5 and hulL-20. Selections performed using target peptide 6 generated 7 unique and specific binders, clones M2.405.D08, M2.405.G05, M2.405.G10, M2.405.H08, M2.405.H10, M2.406.D02, and M2.406.F06, which bound both peptide 6 and hulL-20. Additionally selections using target peptide 7 identified 7 unique and specific binders, M2.401.A05, M2.401 .C01 , M2.401.C06, M2.401 .H08, M2.402.D09, M2.402.F12, and M2.402.H02, which bound both peptide 7 and hulL-20. None of these clones bound control proteins KLH or streptavidin (see Table 15).

Table 15. Summary of binding specificity of sitel binders generated via antibody phage display.

Sequence analysis

The VH and VL sequences of the 15 clones summarized in Table 14 were analysed by classical Sanger sequencing. First, PCR was performed on individual phage cultures using the vector primer NN0370 (5 '-AGC GG ATAAC AATTTC ACACAG G A) and CH 1 primer NN0367 (5 - GTCCTTGACCAGGCAGCCCAGGGC), subsequently the clones were sequenced using the same primers. Sequences were analysed using the Geneious assemble software (Biomatters Ltd). Example 11 : Cloning and sequencing of mouse anti-IL20 mAbs

This example describes cloning and sequencing of the murine heavy chain and light chain sequences of anti-IL20 antibodies: anti-IL20 7F1A1 , anti-IL20 1 F13, anti-IL20 1 1 F12, anti-IL20 1 1 F25 and anti-IL20 1 1 F36.

Total RNA was extracted from hybridoma cells using the RNeasy-Mini Kit from Qiagen and used as template for cDNA synthesis. cDNA was synthesized in a 5'-RACE reaction using the SMARTer™ RACE cDNA amplification kit from Clontech. Subsequent target amplification of HC and LC sequences was performed by PCR using Phusion Hot Start polymerase

(Finnzymes) and the universal primer mix (UPM) included in the SMARTer™ RACE kit as forward primer. A reverse primer with the following sequence was used for HC (VH domain) amplification:

5'-CCCTTGACCAGGCATCCCAG-3' A reverse primer with the following sequence was used for LC amplification:

5'-GCTCTAGACTAACACTCATTCCTGTTGAAGCTCTTG-3'

PCR products were separated by gel electrophoresis, extracted using the GFX PCR DNA & Gel Band Purification Kit from GE Healthcare Bio-Sciences and cloned for sequencing using a Zero Blunt TOPO PCR Cloning Kit and chemically competent TOP10 E.coli

(Invitrogen). Colony PCR was performed on selected colonies using an AmpliTaq Gold Master Mix from Applied Biosystems and M13uni/M13rev primers. Colony PCR clean-up was performed using the ExoSAP-IT enzyme mix (USB). Sequencing was performed at Eurofins MWG Operon, Germany using M13uni(-21 )/M13rev(-29) sequencing primers. Sequences were analyzed and annotated using the VectorNTI program. All kits and reagents were used according to the manufacturer's instructions.

anti-IL20 7F1A1

A single unique murine kappa type LC was identified. A single unique murine HC, subclass mlgG1 was identified. Amino acid sequences are listed below, the leader peptide sequences are not included. The CDR's of the antibody are indicated bold and underlined. anti-IL20 7F1A1 VH amino acid sequences (signal peptide sequence omitted): 1 QVQLQQSGAE LVRPGAAVTL SCKASDYTFT DFEMHWVKQT PVHGLKWIGV 51 IDPETDGTAY NQKFKDKATL TADKSSSTAY MEIRSLTSED SAVYYCTRLL

101 WLPFDFWGQG TTLTVSS

anti-IL20 7F1A1 VL amino acid sequence (signal peptide sequence omitted):

1 DIVMTQSPSS LAMSVGQKVT MNCRSSQSLL NTSNQKNYLA WYQQKPGQSP 51 KLLVHFASTR ESGVPDRFMG SGSGTDFTLT ISSVQAEDLA DYFCHQYYST

101 PLTFGAGTKL ELKR

anti-IL20 1 F13

A single unique murine kappa type LC was identified. Three murine HCs, subclass mlgG1 (A & C) or mlgG2b (B) were identified. Amino acid sequences are listed below, the leader peptide sequences are not included. The CDR's of the antibody are indicated bold and underlined. anti-IL20 1 F13 VH (A) amino acid sequences (signal peptide sequence omitted, mlgG1 ): 1 EVQLVESGGG LVKPGGSLKL SCAASGFTFS DYYMYWVRQT PEKRLEWVAT 51 ISDGGSYTYY PDSVKGRFTI SRDNAKNNLY LQMSSLKSED TAMYYCARGF 101 ITTVRGFAYW GQGTLVTVSA

anti-IL20 1 F13 VH (B) amino acid sequences (signal peptide sequence omitted, mlgG2b): 1 EVQLVESGGG LVRPGGSLKL SCAASGFTFS DYYMYWVRQT PEKRLEWVAT 51 ISDGGTYTYY PDSVKGRFTI SRDNAKNNLY LQMSSLKSED TAMYYCARGG 101 SSTMIRGFAY WGQGTLVTVS A anti-IL20 1 F13 VH (C) amino acid sequences (signal peptide sequence omitted, mlgG1 ): 1 QVQLLQSGAE LVRPGASVKL SCTPSGNTST NYWISWVKQR PGQGLEWIGN 51 IHPSDSYSNY NQKFKDKATL TVDKSSSTVY MQLSGPSSED SAVYYCTSGI 101 YYDYDRVDVW GAGTTVTVSS

anti-IL20 1 F13 VL amino acid sequence (signal peptide sequence omitted):

1 DIVMTQSPSS LTVTAGEKVT MSCKSSQSLL SSGNQKNYLT WYQQKPGQPP 51 KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCQNDYSY

101 PLTFGAGTKL ELKR

anti-IL20 1 1 F12

A single unique murine kappa type LC was identified. A single unique murine HC, subclass mlgG1 was identified. Amino acid sequences are listed below, the leader peptide sequences are not included. The CDR's of the antibody are indicated bold and underlined. anti-IL20 1 1 F12 VH amino acid sequences (signal peptide sequence omitted):

1 QVHLQQSGAE LARPGASVKL SCKASGYTFT NYWMQWVKQR PGQGLEWIGA 51 IYPGDGDTRY TQKFKGKATL TADKSSSTAY MQLSSLASED SAVYYCARWL 101 LPLMDYWGQG TSVTVSS

anti-IL20 1 1 F12 VL amino acid sequence (signal peptide sequence omitted):

1 QIVLTQSPAI MSASPGEKVT ITCSASSSVN YIHWFQQKPG TSPKLWIYGT 51 SKLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR SSYPYTFGGG 101 TKLEIKR anti-IL20 1 1 F25

Five murine kappa type LC were identified. Three murine HCs, subclass mlgG1 (A & C) or mlgG2b (B) were identified. Amino acid sequences are listed below, the leader peptide sequences are not included. The CDR's of the antibody are indicated bold and underlined. anti-IL20 1 1 F25 VH (A) amino acid sequences (signal peptide sequence omitted, mlgG1 ): 1 EVQLQQSGTE LVKPGASVKL SCTASGFNIE DSYIHWVNQR PEQGLEWIGR 51 IDPASGRANY DPKFQGKATI TADTSSNTAY LQLSSLTSED TAVYYCARPY 101 YYGTSRNYFD FWGQGTTLTV SS

anti-IL20 1 1 F25 VH (B) amino acid sequences (signal peptide sequence omitted, mlgG2b) 1 EVQLQQSGAE LVKPGASVKL SCTASGFNIR DTFMYWVKQR PEQGLEWIGR 51 IDPGNGNTKY DSKFQGKATI TADTSSNTAY LQLSSLTSED TAVYYCTKVR 101 DWGRGFFYWG QGTLVTVSA

anti-IL20 1 1 F25 VH (C) amino acid sequences (signal peptide sequence omitted, mlgG1 ) 1 QIQLVQSGPE LKKPGETVKI SCKASGYTFT NYGMNWVKQA PGKGLKWMGW 51 INTNTGEPTY AEEFKGRFAF SLETSASTAY LQINNLKNED TATYFCARGD

101 YGYDYYVMDY WGQGTSVTVS S

anti-IL20 1 1 F25 VL (A) amino acid sequence (signal peptide sequence omitted)

1 SIVMTQTPKF LLISPGDRVT ITCKASQRVT NDVAWYQQKP GQSPKLLIYY 51 ASNRYTGVPD RFTGSGYGTD FTFTINTVQT EDLAVYFCQQ DYSSLTFGAG

101 TKLELKR anti-IL20 1 1 F25 VL (B) amino acid sequence (signal peptide sequence omitted) 1 DIVLTQSPTS LAVSLGQRAT ISCRASESVD NYGITFMHWY QQKPGQPPRL 51 LIYLVSNLES GVPARFSGSG SGTDFTLTIN PVETDDVATY YCQQSNIYPW

101 TFGGGTKLEI KR

anti-IL20 1 1 F25 VL (C) amino acid sequence (signal peptide sequence omitted)

1 DIVMTQSPAT LSVTPGDRVS LSCRASQSIS DYLHWYQQKS HESPRLLIRY 51 ASQSISGIPS RFSGSGSGSD FTLSINSVEP EDVGVYYCQN GHSFPLTFGA

101 GTKLEIKR

anti-IL20 1 1 F25 VL (D) amino acid sequence (signal peptide sequence omitted)

1 DIVMTQSQKF MSTTVGDRVS ITCKASQNVG AAVAWYQQKP GQSPKLLIHS 51 PSTRYSGVPD RFTGSGSGTD FTLTISNMQS EDLAAYFCHQ YSSYPFTFGS

101 GTKLEIKR

anti-IL20 1 1 F25 VL (E) amino acid sequence (signal peptide sequence omitted)

1 DIQMTQSPSS LSASLGERVS LTCRASQELS GYLSWLQQKP DGTIKRLIYA 51 ASTLDSGVPN RFSGSRSGSD FSLTISSLES EDFADYYCLQ YANYPYTFGG

101 GTKLEIKR

anti-IL20 1 1 F36

A single unique murine kappa type LC was identified. A single unique murine HC, subclass mlgG2b was identified. Amino acid sequences are listed below, the leader peptide sequences are not included. anti-IL20 1 1 F36 VH amino acid sequences (signal peptide sequence omitted) 1 EVQLQESGPS LMKPSQTLSL TCSVTGDSIT SGYWYWVRKF PGNKLEYLGY 51 ISYSGSTDYN PSLKSRISIT RDTSNNQHYL QLNSVTAEDT ATYYCARPIY 101 DGYYIPFDYW GQGTLVTVSA

anti-IL20 1 1 F36 VL amino acid sequence (signal peptide sequence omitted)

1 DIVMTQSQKF MSTTVGDRVS ITCKASQNVG TAVAWYQQKP GQSPKLLLYS 51 ASNRYTGVPD RFTGSGSGTD FTLTISNMQS EDLADYFCQQ YSSYPLTFGS 101 GTKLEMKR

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Claims

1. An antibody or an antibody fragment thereof, wherein said antibody binds to both

binding parts of a discontineous binding region on IL-20, wherein the first binding part is defined by amino acids no. 10-50 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 130-152 of SEQ ID NO 1.

2. An antibody or an antibody fragment thereof according to claim 1 , wherein said

antibody binds to both binding parts of a discontineous binding region on IL-20, wherein the first binding part is defined by amino acids no. 15-45 of SEQ ID NO 1 and the second binding part is defined by amino acids no. 135-147 of SEQ ID NO 1.

3. An antibody or an antibody fragment thereof according to claim 2, wherein said

antibody binds to both binding parts of a discontineous binding region on IL-20, wherein the first binding part is defined by amino acids no. 16-43 of SEQ ID NO 1 , and the second binding part is defined by amino acids no. 136-146 of SEQ ID NO 1.

4. An antibody or an antibody fragment according to claim 1 or 2, wherein said antibody binds to both binding parts of a discontineous binding region on IL-20, wherein first part of said discontineous binding region comprises one or more of the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39 and Arg 43, and second part of said discontineous binding region comprises one or more of the following amion acids of SEQ ID NO 1 : Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

5. An antibody or an antibody fragment according to claim 1 or 2, wherein said antibody binds to a discontinious epitope situated on the binding region of any of claims 1-4, wherein said epitope comprises one or more of the following amino acids of SEQ ID NO 1 : Gin 16, Arg 19, Asn 20, Ser 23, Arg 26, Gin 30, Asp 33, lie 36, Asp 37, lie 38, Arg 39 and Arg 43, and one or more of the following amion acids of SEQ ID NO 1 : Lys 136, Gly 139, Glu 140, Asp 142, lie 143 and Gin 146.

6. An antibody or an antibody fragment therof according to any of claims 1-5 that

competes with one of more of antibodies I L20-1 F13, IL20-7F1 , IL20-1 1 F12, IL20-

1 1 F25 or IL20-1 1 F36 for binding to IL-20.

7. An antibody or an antibody fragment therof according to any of claims 1-6, that binds to a epitope/binding region not influenced by binding of antibodies 9F1 or 5B7

8. An antibody or an antibody fragment thereof according to any of claim 1-6, wherein said antibody interferes with binding of IL20R1 to IL-20.

9. An antibody or an antibody fragment thereof according to any preceeding claim that competes with one or more of the antibodies produced by the hybrodoma's deposited at at Public Health England (previously The European Collection of Cell Cultures (ECACC)), Microbiology Services, Porton Down, Salisbury, SP4 OJG, United Kingdom under the Provisional Accession numbers 13070502, 13070501 , 13070503, 13070504 and 13070505 for binding to IL-20.

10. A pharmaceutical composition comprising an antibody or an antibody fragment thereof, according to any one of the preceding claims, and optionally one or more pharmaceutically acceptable excipients.

1 1 . Use of an antibody or an antibody fragment therof, according to any one of claims 1-7, or a pharmaceutical composition according to claim 8, for treating an immunological disorder.

12. Use of an antibody or an antibody fragment therof, according to any one of claims 1-7, or a pharmaceutical composition according to claim 8, for threating autoimmune and/or inflammatory disease.

13. Use of an antibody or an antibody fragment thereof, according to to claim 10, wherein the autoimmune and/or inflammatory disease is selected from: Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), osteoporosis, multiple sclerosis (MS), scleroderma and type 1 diabetes (T1 D), and other diseases and disorders, such as PV (pemphigus vulgaris), psoriasis, psoriatic arthritis, atopic dermatitis, celiac disease, Chronic obstructive lung disease, Hashimoto's thyroiditis, Graves' disease (thyroid), Sjogren's syndrome, Guillain-Barre syndrome, Goodpasture's syndrome, Addison's disease, Wegener's granulomatosis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, polymyalgia rheumatica, Raynaud's phenomenon, temporal arteritis, giant cell arteritis, autoimmune haemolytic anaemia, pernicious anaemia, polyarteritis nodosa, behcet's disease, primary biliary cirrhosis, uveitis, myocarditis, rheumatic fever, ankylosing spondylitis, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, alopecia areata, type I diabetes, Colitis-Associated Tumorigenesis, metastasis- associated osteolysis or vitiligo.