Antibodies to interleukin-22

Abstract

The present invention relates to antibodies that bind to IL22 and inhibit its interaction with one or more of its natural ligands. Specific examples of such antibodies are provided. Therapeutic uses of the antibodies and methods for producing such antibodies are also provided.

Description

[Technical Field]

[0001] The present invention relates to anti-IL22 antibodies. Such antibodies provided herein are useful for treating skin inflammation, particularly atopic dermatitis. [Background technology]

[0002] Atopic dermatitis (AD), also known as atopic eczema, is an inflammatory condition that results in epidermal dysfunction and thickening, eczematous lesions, and pruritus. This condition is common among people of all ages and ethnicities and has the greatest disease burden of all skin diseases, measured by disability-adjusted life years (Laughter et al., Br. J. Dermatol. 2020; Epub ahead of print). AD is a complex condition, and its pathophysiology is influenced by multiple factors, including genetic, environmental, and immunological factors. While type 2 immune mechanisms are important in the pathogenesis of atopic dermatitis, increasing evidence supports the role of several immune pathways.

[0003] Treatments used for AD include systemic immunosuppressants such as cyclosporine, methotrexate, mycophenolate mofetil, and azathioprine. Antidepressants and naltrexone can be used to control pruritus. In 2016, crisaborole, a topical phosphodiesterase-4 inhibitor, was approved for mild to moderate eczema, and in 2017, dupilumab, a monoclonal antibody antagonist of IL-4Rα, was approved for treating moderate to severe eczema. However, current treatment options only provide temporary, incomplete, and symptomatic relief.

[0004] IL-22 is a member of the IL10 cytokine family that has multiple functions in various inflammatory and tissue responses depending on the environmental context. IL-22 is primarily produced by lymphoid cells such as T helper 1 (Th1) cells, Th17 cells, and Th22 cells, gamma delta T cells, natural killer (NK) cells, and innate lymphoid cells (ILCs), as well as non-lymphoid cells such as fibroblasts, neutrophils, macrophages, and mast cells (for a review, see Lanfranca MP, et al. J. Mol. Med. (Berl) (2016) 94(5):523-534). IL-22 signals through a heterodimeric transmembrane receptor complex composed of IL-22 receptor 1 (IL22RA1 or IL22R1, also known as interleukin-22 receptor subunit alpha-1) and IL-10 receptor 2 (IL10R2), whereas IL-10 signals through IL10R1 and IL-10R2. Like other members of the IL-10 family, IL22 mediates its effects through the IL22R1 / IL-10R2 complex, which contains Jak1, Tyk2, and STAT3, and the subsequent JAK-signal transducer and activator of transcription (STAT) signaling pathway. In contrast to IL-10, IL22 has also been reported to signal through multiple MAPK pathways, including ERK1 / 2, JNK, and p38. In the skin, IL22 acts on keratinocytes via binding to IL22R1 expressed on these cells.

[0005] Unlike other members of the IL10 cytokine family, IL22 has a soluble, secreted receptor known as IL22-binding protein (IL22BP, also known as IL22RA2 or interleukin-22 receptor subunit α2). Although IL22BP shares the highest structural homology with the IL22R1 chain, IL22BP exhibits a much higher affinity for IL22 than IL22R1, thus preventing IL22 from binding to IL22R1.

[0006] IL22BP, which is specific for IL22, has been shown to block its activity. Inhibition of total IL22 showed effective signals in patients with severe atopic dermatitis or high baseline IL22 expression (Guttman-Yassky E et al. J Am Acad Dermatol. 2018;78(5):872-881 and Brunner PM et al. J Allergy Cin Immunol. 2019;143(1):142-154). Inhibition of IL22R1 has also been proposed as a potential therapeutic option to inhibit IL22, partially blocking the effects of IL-20 and IL-24. To date, no therapeutic options exist that specifically target biologically active IL22 that is not bound to IL22BP and therefore does not affect the normal biological function of IL22BP.

[0007] Elevated expression of the Th22 cytokine IL22 is a characteristic finding in atopic dermatitis (AD). However, the specific role of IL22 in the pathogenesis of AD in vivo is not fully understood. Although the role of IL22 in the development and maintenance of AD has not been specifically investigated, it is hypothesized that IL22 plays an important role in the development of AD by impairing skin barrier function, immune dysregulation, and pruritus.

[0008] US Patent Nos. 8,906,375 and 7,901,684 disclose antibodies that bind to IL22 and the utility of such antibodies in the treatment of AD. US Patent No. 7,737,259 discloses specific anti-IL22 antibodies useful in the treatment of psoriasis. Summary of the Invention

[0009] The present invention addresses the need for new treatments for atopic dermatitis by providing anti-IL-22 antibodies that have the functional and structural properties as described herein.

[0010] The present invention provides isolated antibodies that bind to human interleukin-22 (IL22), wherein the antibodies are capable of inhibiting or attenuating IL22 binding to IL22 receptor 1 (IL22R1) and IL22 binding protein (IL22RA2).

[0011] The present invention also provides isolated antibodies that bind to human IL22 defined by a specific set of CDR sequences and / or variable region sequences.

[0012] The invention will now be described with reference to the following drawings. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows the humanization of the antibody 11041 light chain. The mutants generated for that chain are also shown. The CDR sequences are underlined.

[0014] [Figure 2] Figure 1 shows the humanization of the heavy chain of antibody 11041. The mutants generated for that chain are also shown. The CDR sequences are underlined.

[0015] [Figure 3A] Figure 1 shows the humanization of the antibody 11070 light chain. Variants of that chain are also shown. CDR sequences are underlined. [Figure 3B] Figure 1 shows the humanization of the antibody 11070 heavy chain. The mutants generated for that chain are also shown. The CDR sequences are underlined.

[0016] [Figure 4] FIG. 1 shows the IL22 peptide coverage map of the HDX-MS experiment.

[0017] [Figure 5]Figure 1 shows the results of HDX-MS analysis of 11041gL13gH14Fab. (A) Peptides showing significantly reduced deuterium uptake upon antibody binding are listed. Peptides showing similar exchange patterns in the presence and absence of antibody have insignificant deuterium uptake and are shown in light gray. (B) The determined 11041gL13gH14Fab epitope is projected onto the IL22 3D structure and highlighted in black. For reference, relative 11041gL13gH14Fab binding to IL22 from X-ray data is displayed.

[0018] [Figure 6] Figure 1 shows the results of HDX-MS analysis of 11070gL7gH16Fab. (A) Peptides that show significantly reduced deuterium uptake upon antibody binding are listed. Peptides that show similar exchange patterns in the presence and absence of antibody have insignificant deuterium uptake and are shown in light gray. (B) The 11070gL7gH16Fab epitope is determined as projected onto the IL22 3D structure and highlighted in black.

[0019] [Figure 7] Figure 1 shows the results of X-ray analysis of 11041gL13gH14 Fab binding to IL-22. (A) Illustrative representation of 11041gL13gH14 Fab binding to IL-22. (B) Detailed view of the interaction interface between IL-22 and 11041gL13gH14 Fab.

[0020] [Figure 8A] FIG. 1 shows that the 11041gL13gH14 Fab molecule interferes with the interaction of IL22 with the IL22R1 receptor (A) IL-22 (surface representation) in complex with its receptor IL22R1 (PDB:3DLQ). [Figure 8B] The light chain of 11041gL13gH14 Fab light chain blocks the interaction site between IL22 and IL22R1.

[0021] [Figure 9A]FIG. 1 shows the Cryo-EM structure of IL-22 in complex with featherkinumab and the Fab format 11070gL7gH16 Fab (VR11070). [Figure 9B] Figure 1. Model of IL-22 (VR11041) in complex with featherkinumab and 11041 Fab. The model was generated by overlaying the IL-22 / 11041gL13gH14 Fab crystal structure onto the cryo-EM structure in panel (A). This reveals that 11070gL7gH16 Fab and 11041gL13gH14 Fab have similar epitopes on IL-22.

[0022] [Figure 10A] FIG. 1 shows the superposition of the crystal structure of IL-22R1 bound to IL-22 onto the cryo-EM structures of IL-22 in complex with 11070gL7gH16 Fab and Fezakinumab Fab. [Figure 10B] The side chains of IL-22 residues known to contribute to the interaction with IL-10R2 are shown as sticks. This site is occupied by the fezakinumab Fab molecule.

[0023] [Figure 11] Figure 1 shows the activity of 11041gL13gH14 Fab (VR11041) in an in vitro human primary keratinocyte assay. (A) Example of S100A7 response in the assay. Donor lot number 438Z014, geometric mean n=3 / 6, stimuli: IL-22 at 100 ng / ml, fezakinumab Fab, and 11041gL13gH14 Fab at 50 nM; (B) Percent inhibition of S100A7 by 11041gL13gH14 Fab in the assay. Mean ± SD, n=2 donors, stimuli: IL-22 at 100 ng / ml, statistics: log(inhibitor) vs. response (three parameters). DETAILED DESCRIPTION OF THE INVENTION

[0024] [Table 1]

[0025] [Table 2]

[0026] definition The following terms are used throughout this specification.

[0027] The term "acceptor human framework" is used herein to refer to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes.

[0028] The term "affinity" refers to the strength of all non-covalent interactions between the antibody and the target protein. Unless otherwise indicated, as used herein, the term "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule for its binding partner can generally be expressed by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0029] The term "affinity maturation" in the context of antibodies refers to an antibody with one or more changes in the hypervariable regions, compared to a parent antibody that does not possess such changes, which changes result in an improvement in the affinity of the antibody for antigen.

[0030] The term "antibody" as used herein is used in the broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies, so long as they exhibit the desired antigen-binding activity. As used herein, the term antibody refers to whole (full-length) antibodies (i.e., comprising two heavy and two light chain elements) and functionally active fragments thereof (i.e., molecules comprising an antigen-binding domain that specifically binds to an antigen (also referred to as antibody fragments or antigen-binding fragments)). The characteristics described herein with respect to antibodies also apply to antibody fragments, unless the context dictates otherwise. An antibody may comprise a Fab linked to two scFvs or dsscFvs, each of which binds to the same or different targets (e.g., one scFv or dsscFv that binds to a therapeutic target and one scFv or dsscFv that extends half-life, e.g., by binding to albumin). Such antibodies are described in WO 2015 / 197772. The term "antibody" encompasses monovalent, i.e., antibodies that contain only one antigen-binding domain (e.g., one-arm antibodies comprising a full-length heavy chain and an interconnected full-length light chain, also called a "half antibody"), and multivalent, i.e., antibodies that contain two or more antigen-binding domains, e.g., bivalent antibodies.

[0031] An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay.

[0032] The term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to a mechanism of cell death that depends on the interaction of antibody-coated target cells with lytic effector cells, such as natural killer cells, monocytes, macrophages, and neutrophils, via Fc gamma receptors (FcγR) expressed on the effector cells.

[0033] The term "antigen-binding fragment" as used herein refers to a functionally active antibody-binding fragment, including, but not limited to, Fab, modified Fab, Fab', modified Fab', F(ab'), Fv, single-domain antibodies, scFv, Fv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). As used herein, a "binding fragment" refers to a fragment that can bind to a target peptide or antigen with sufficient affinity to characterize the fragment as specific for the peptide or antigen.

[0034] The term "antibody variant" refers to a polypeptide, e.g., an antibody comprising a VH and / or VL having the desired characteristics described herein and having at least about 80% amino acid sequence identity with the VH and / or VL of a reference antibody. Such antibody variants include, for example, antibodies in which one or more amino acid residues are added to or deleted from the VH and / or VL domains. Typically, the antibody variant will have at least about 80% amino acid sequence identity, or at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, with an antibody described herein. Optionally, the variant antibody will have no more than one conservative amino acid substitution compared to the antibody sequences provided herein, or alternatively, no more than about 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to the antibody sequences provided herein. In embodiments, "antibody variant" refers to an antibody or antigen-binding fragment thereof comprising a VH and / or a VL, wherein the non-CDR regions of the antibody or antigen-binding fragment thereof have at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to an antibody described herein.

[0035] The term "antigen-binding domain" as used herein refers to a portion of an antibody comprising part or all of one or more variable domains that specifically interact with a target antigen, for example, part or all of a pair of variable domains, VH and VL. In the context of the present invention, this term is used in relation to three different antigens: IL13, IL22, and albumin. Such antigen-binding domains are therefore referred to as "IL13-binding domains," "IL22-binding domains," and "albumin-binding domains." Binding domains may comprise single-domain antibodies. Each binding domain may be monovalent. Each binding domain may comprise no more than one VH and one VL.

[0036] As used herein, the term "bispecific" or "bispecific antibody" refers to an antibody with two antigen specificities.

[0037] The term "complementarity-determining region" or "CDR" generally refers to the fact that antibodies contain six CDRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia, C. and Lesk, AM J Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Therefore, unless otherwise indicated, "CDR-H1" as used herein is intended to refer to residues 26-35 as described by a combination of the Kabat numbering system and Chothia's topological loop definition. The CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) according to the Kabat numbering system. Unless otherwise indicated, CDR residues and other residues (e.g., FR residues) in the variable domain are numbered according to Kabat herein.

[0038] The term "chimeric" antibody refers to an antibody in which the variable domains (or at least a portion thereof) of the heavy and / or light chains are derived from a particular source or species, while the remainder of the heavy and / or light chains (i.e., the constant domains) are derived from a different source or species (Morrison; PNAS 81, 6851 (1984)). Chimeric antibodies can, for example, contain non-human variable domains and human constant domains. Chimeric antibodies are typically produced using recombinant DNA techniques. A subcategory of "chimeric antibodies" is "humanized antibodies."

[0039] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0040] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism of cell death in which the Fc effector domain of a target-binding antibody binds to and activates complement component C1q, which then activates the complement cascade, resulting in target cell death.

[0041] As used herein, the terms "constant domain(s)" or "constant region" are used interchangeably to refer to the domain(s) of an antibody outside the variable region. The constant domains are identical in all antibodies of the same isotype, but vary from isotype to isotype. Typically, the constant region of a heavy chain is formed from the N-terminus to the C-terminus by CH1-hinge-CH2-CH3-optionally CH4, which comprises three or four constant domains.

[0042] The term "competing antibody" or "cross-competing antibody" shall be interpreted to mean that the claimed antibody binds to either (i) the same location on the antigen as bound by the reference antibody, or (ii) a location on the antigen where the antibody sterically interferes with binding of the reference antibody to the antigen.

[0043] As used herein, the term "derivative" is intended to include reactive derivatives, e.g., thiol-selective reactive groups such as maleimides. The reactive group may be linked to the polymer directly or via a linker segment. It will be understood that the residue of such a group will, in some cases, form part of the product as the linking group between the antibody fragment and the polymer.

[0044] The term "derived" in the context of generating a variable sequence refers to the fact that the sequence used, or a sequence very similar to the sequence used, was obtained from original genetic material, such as the light or heavy chain of an antibody.

[0045] As used herein, the term "diabody" refers to two Fv pairs, a first VH / VL pair and a further VH / VL pair, with two inter-Fv linkers such that the VH of the first Fv is linked to the VL of the second Fv and the VL of the first Fv is linked to the VH of the second Fv.

[0046] As used herein, the term "DiFab" refers to two Fab molecules linked via the C-terminus of the heavy chains.

[0047] As used herein, the term "DiFab'" refers to two Fab' molecules linked via one or more disulfide bonds in their hinge regions.

[0048] As used herein, the term "dsscFv" or "disulfide-stabilized single-chain variable fragment" refers to a single-chain variable fragment stabilized by a peptide linker between the VH and VL variable domains and also containing an interdomain disulfide bond between VH and VL (see, e.g., Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012; WO 2007109254).

[0049] The term "DVD-Ig" (also known as double V-domain IgG) refers to a full-length antibody with four additional variable domains, one at the N-terminus of each heavy chain and each light chain.

[0050] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0051] As used herein, the term "effector molecule" includes, for example, anti-neoplastic agents, drugs, toxins, biologically active proteins such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides, particularly radioactive iodides, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy.

[0052] The term "epitope" or "binding site" in the context of antibodies refers to the site (or portion) on an antigen to which the antibody paratope binds or recognizes. Epitopes can be formed both from contiguous amino acids (often referred to as "linear epitopes") or from discontinuous amino acids formed by tertiary folding of a protein (often referred to as "conformational epitopes"). Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by folding are typically lost upon treatment with denaturing solvents. An epitope typically contains at least three, and more usually at least 5-10, amino acids in a unique spatial conformation. Epitopes usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific 3D structure and charge characteristics.

[0053] "EU index" or "Kabat-like EU index" or "EU numbering scheme" refers to EU antibody numbering (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85). It is generally used when referring to residues in antibody heavy chain constant regions (e.g., as reported in Kabat et al.). Unless otherwise specified, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0054] The term "Fab" as used herein refers to an antibody fragment comprising a light chain fragment containing the VL (variable light) domain and a constant domain (CL) of the light chain, and a VH (variable heavy) domain and the first constant domain (CH1) of the heavy chain. Dimerization of Fab' according to the present disclosure produces F(ab')2, where, for example, dimerization may be via a hinge.

[0055] As used herein, the term "Fab'-Fv" is analogous to FabFv, in which the Fab portion is replaced by Fab'. The format may be provided as a PEGylated version thereof.

[0056] As used herein, the term "Fab'-scFv" refers to a Fab' molecule in which an scFv is added to the C-terminus of the light or heavy chain.

[0057] As used herein, the term "Fab-dsFv" refers to a FabFv in which an intra-Fv disulfide bond stabilizes the attached C-terminal variable region. The format may be provided as a PEGylated version thereof.

[0058] As used herein, the term "Fab-Fv" refers to a Fab fragment in which the variable regions are added to the C-terminus of the heavy chain CH1 and light chain CL, respectively. The format may be provided as a PEGylated version thereof.

[0059] As used herein, the term "Fab-scFv" refers to a Fab molecule in which an scFv is added to the C-terminus of the light or heavy chain.

[0060] The terms "Fc," "Fc fragment," and "Fc region" are used interchangeably to refer to the C-terminal region of an antibody comprising the antibody's constant region, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant domains, CH2 and CH3, of IgA, IgD, and IgG, or the last three constant domains of IgE and IgM, as well as the flexible hinge N-terminal to these domains. The human IgG1 heavy chain Fc region is defined herein as comprising residue C226 through its carboxyl terminus, numbering according to the EU index. In the context of human IgG1, the lower hinge refers to positions 226-236, the CH2 domain refers to positions 237-340, and the CH3 domain refers to positions 341-447, according to the EU index. Corresponding Fc regions of other immunoglobulins can be identified by sequence alignment.

[0061] The term "framework" or "FR" refers to variable domain residues other than the hypervariable region residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0062] As used herein, the term "full-length antibody" refers to an antibody having a structure substantially similar to that of a native antibody or an antibody having a heavy chain containing an Fc region as defined herein. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (CH) composed of three constant domains, CH1, CH2, and CH3, or four constant domains, CH1, CH2, CH3, and CH4, depending on the Ig class. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0063] The term "Fv" refers to two variable domains of a full-length antibody, eg a cognate pair or affinity matured variable domains, i.e., cooperating variable domains such as a VH and VL pair.

[0064] The term "highly similar" as used in the context of amino acid sequences is intended to refer to amino acid sequences that are 95% or more similar over their entire length, such as 96, 97, 98 or 99% similar.

[0065] The term "human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0066] The term "human consensus framework" refers to a framework representing the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup such as those in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3. In some embodiments, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In some embodiments, for VH, the subgroup is subgroup III as in Kabat et al. In some embodiments, for VH, the subgroup is subgroup IV as in Kabat et al.

[0067] The term "humanized" antibody refers to an antibody containing amino acid residues from non-human HVRs and human FRs. Typically, the heavy and / or light chains contain one or more CDRs (including one or more modified CDRs, if desired) from a donor antibody (e.g., a non-human antibody such as a mouse or rabbit monoclonal antibody) and are grafted onto the heavy and / or light chain variable region framework of a recipient antibody (human antibody) (see, e.g., Vaughan et al., Nature Biotechnology, 16, 535-539, 1998). The advantage of such humanized antibodies is that they retain the specificity and affinity of the parent non-human antibody while reducing immunogenicity in humans. Rather than transferring the entire CDR, only one or more of the specificity-determining residues from any one of the CDRs described hereinabove can be transferred into the human antibody framework (see, e.g., Kashmir et al., 2005, Methods, 36, 25-34). A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0068] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms structurally distinct loops ("hypervariable loops") and / or contains antigen-contacting residues ("antigen contacts").

[0069] As used herein, the term "IC50" refers to the half-maximal inhibitory concentration, which is a measure of the effectiveness of a substance, such as an antibody, in inhibiting a particular biological or biochemical function. IC50 is a quantitative measure of how much of a particular substance is required to inhibit a given biological process by 50%.

[0070] "Identity" between amino acids in sequences indicates that, at any particular position in the aligned sequences, the amino acid residue is identical between the sequences.

[0071] As used herein, the term "IgG-scFv" refers to a full-length antibody having an scFv at the C-terminus of each heavy chain or each light chain.

[0072] As used herein, the term "IgG-V" refers to a full-length antibody with a variable domain at the C-terminus of each heavy or light chain.

[0073] The term "isolated," as used throughout this specification, means that the antibody or polynucleotide, as the case may be, is present in a physical environment that is different from that in which it may occur in nature. The term "isolated" nucleic acid refers to a nucleic acid molecule that has been isolated from its natural environment or that has been produced synthetically. Isolated nucleic acid can include synthetic DNA, cDNA, genomic DNA, or any combination thereof, produced, for example, by chemical treatment.

[0074] The term "Kabat residue designation" or "Kabat" refers to the residue numbering scheme commonly used for antibodies. This does not always correspond directly to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to shortening of or insertion into structural components, whether framework or complementarity-determining regions (CDRs) of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody by aligning homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. For details, see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Unless otherwise indicated, Kabat numbering is used throughout this specification.

[0075] As used herein, the term "KD" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). Kd and Ka refer to the dissociation and association rates, respectively, of a particular antigen-antibody interaction. The KD value of an antibody can be determined using methods well established in the art.

[0076] The term "monoclonal antibody" (or "mAb") refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each individual of a monoclonal antibody preparation is identical except for possible minor variations (e.g., naturally occurring mutations). Nevertheless, certain differences in protein sequence linked to post-translational modifications (e.g., cleavage of the heavy chain C-terminal lysine, deamidation of asparagine residues, and / or isomerization of aspartate residues) may exist among the various different antibody molecules present in the composition. In contrast to polyclonal antibody preparations, each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0077] As used herein, the term "multiparatopic antibody" refers to an antibody described herein that contains two or more different paratopes that interact with different epitopes from either the same antigen or two different antigens. The multiparatopic antibodies described herein can be biparatopic, triparatopic, or tetraparatopic.

[0078] As used herein, the term "multispecific" or "multispecific antibody" refers to an antibody described herein having at least two binding domains, i.e., two or more binding domains, e.g., two or three binding domains, where the at least two binding domains independently bind to two different antigens or two different epitopes on the same antigen. Multispecific antibodies are generally monovalent for each specificity (antigen). Multispecific antibodies described herein encompass monovalent and multivalent, e.g., bivalent, trivalent, and tetravalent, multispecific antibodies.

[0079] The term "neutralizing" (or "neutralize") in the context of antibodies refers to an antibody that is able to inhibit or attenuate the biological signaling activity of its target (target protein).

[0080] The term "paratope" refers to the region of an antibody that recognizes and binds to an antigen.

[0081] The term "percent (%) sequence identity (or similarity)" with respect to polypeptide and antibody sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical (or similar) to the amino acid residues in the compared polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

[0082] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0083] The term "polyclonal antibody" refers to a mixture of different antibody molecules that bind to (or otherwise interact with) more than one epitope on an antigen.

[0084] The term "prevent" in the context of antibodies is used interchangeably herein with the term "inhibit" to refer to the effect that antibodies according to the invention have on a particular biological process or molecular interaction.

[0085] The term "sc diabody" refers to a diabody that contains intra-Fv linkers such that the molecule contains three linkers to form a normal scFv in which the VH and VL termini are each linked to one of the variable regions of an additional Fv pair.

[0086] As used herein, the term "Sc diabody-CH3" refers to two sc diabody molecules linked to each other via a CH3 domain, for example, via a hinge.

[0087] The term "Sc diabody-Fc," as used herein, refers to two sc diabodies, each attached, for example via a hinge, to the N-terminus of the CH2 domain of the constant region fragment -CH2CH3.

[0088] The term "single-chain variable fragment" or "scFv" as used herein refers to a single-chain variable fragment stabilized by a peptide linker between the VH and VL variable domains.

[0089] As used herein, the term "ScFv-Fc-scFv" refers to four scFvs, one of each attached to the N- and C-termini of both heavy chains of the CH2CH3 fragment.

[0090] As used herein, the term "scFv-IgG" refers to a full-length antibody having an scFv at the N-terminus of each heavy or light chain.

[0091] As used herein, the term "similarity" indicates that at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted for isoleucine or valine. Other amino acids that may be substituted for one another include, but are not limited to: - phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains); - lysine, arginine and histidine (amino acids with basic side chains); - aspartate and glutamate (amino acids with an acidic side chain); asparagine and glutamine (amino acids with amide side chains); and - Cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0092] The term "single domain antibody" as used herein refers to an antibody fragment consisting of a single monomeric variable domain. Examples of single domain antibodies include VH or VL or VHH or V-NAR.

[0093] The term "specific" as used herein in the context of antibodies is intended to refer to an antibody that recognizes only the antigen for which it is specific, or an antibody that has a significantly higher binding affinity, e.g., at least 5, 6, 7, 8, 9, 10 times higher, for the antigen for which it is specific compared to binding to an antigen for which it is non-specific.

[0094] As used herein, the term "steric blocking" or "sterically preventing" is intended to refer to a means of blocking an interaction between a first protein and a second protein by binding of a third protein to the first protein, which prevents the second protein from binding to the first protein due to unfavorable van der Waals or electrostatic interactions between the second and third proteins.

[0095] The term "subject" or "individual" in the context of therapy and diagnosis generally refers to mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). More specifically, the individual or subject is a human.

[0096] As used herein, the term "tandem scFv" refers to at least two scFvs linked via a single linker such that there is a single inter-Fv linker.

[0097] As used herein, the term "tandem scFv-Fc" refers to at least two tandem scFvs, each attached, for example via a hinge, to the N-terminus of the CH2 domain of the constant region fragment -CH2CH3.

[0098] As used herein, the term "target" or "antibody target" refers to the target antigen to which an antibody binds.

[0099] The term "tetrabody" as used herein refers to a format similar to a diabody, which contains four Fvs and four inter-Fv linkers.

[0100] The term "therapeutically effective amount" refers to the amount of an antibody that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The therapeutically effective amount will vary depending on the antibody, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0101] As used herein, the term "tribody" (also called Fab(scFv)2) refers to a Fab fragment with a first scFv attached to the C-terminus of the light chain and a second scFv attached to the C-terminus of the heavy chain.

[0102] As used herein, the term "trispecific or trispecific antibody" refers to an antibody with three antigen-binding specificities. For example, the antibody may have three antigen-binding domains (trivalent) that independently bind to three different antigens or three different epitopes on the same antigen, i.e., each binding domain is monovalent for each antigen. One example of a trispecific antibody format is TrYbe.

[0103] Terms such as "prevent" or "prevention" refer to achieving a prophylactic effect in that a disease or its symptoms are completely or partially prevented. Thus, preventing encompasses halting a disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease.

[0104] The terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be a partial or complete cure of a disease and / or therapeutic with respect to adverse effects resulting from a disease. Thus, treatment encompasses (a) inhibiting a disease, i.e., halting its development; and (b) relieving a disease, i.e., causing regression of a disease.

[0105] The term "TrYbe" as used herein refers to a tribody comprising two dsscFvs.dsFab, and refers to a Fab with an intra-variable region disulfide bond.

[0106] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of full-length heavy (VH) and light (VL) chains generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FRs together form the variable region. By convention, the CDRs in the heavy chain variable region of an antibody are referred to as CDR-H1, CDR-H2, and CDR-H3, and the CDRs in the light chain variable region are referred to as CDR-L1, CDR-L2, and CDR-L3. They are numbered consecutively from the N-terminus to the C-terminus of each chain. CDRs are conventionally numbered according to the system devised by Kabat.

[0107] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." The term "vector" includes "expression vectors."

[0108] The term "VH" refers to the variable domain (or sequence) of the heavy chain.

[0109] As used herein, the term "V-IgG" refers to a full-length antibody with a variable domain at the N-terminus of each heavy or light chain.

[0110] The term "VL" refers to the variable domain (or sequence) of the light chain.

[0111] Interleukin 22 (IL22) The term "interleukin-22" or "IL22" refers to a class II cytokine capable of binding to IL22R1 (also known as IL22RA1, IL22 receptor 1, or interleukin-22 receptor subunit alpha-1) and / or the receptor complex of IL22R1 and IL10RA2 (also known as IL22BP, IL22 binding protein, or interleukin-22 receptor subunit alpha 2). IL22 is also known as interleukin-10-related T-cell-derived inducer factor (IL-TIF). The term refers to naturally occurring or endogenous mammalian IL22 proteins and proteins having an amino acid sequence identical to the amino acid sequence of a naturally occurring or endogenous corresponding mammalian IL22 protein (e.g., recombinant protein, synthetic protein). Thus, as defined herein, the term includes mature IL22 protein, polymorphic or allelic variants, and other isoforms of IL22 (e.g., produced by alternative splicing or other cellular processes), as well as modified or unmodified forms of the above (e.g., lipidation, glycosylation). Naturally occurring or endogenous IL22 includes wild-type proteins, e.g., mature IL22, polymorphic or allelic variants, and other isoforms and variants that naturally occur in mammals (e.g., humans, non-human primates). These proteins, and those proteins that have the same amino acid sequence as the corresponding native or endogenous IL22, are referred to by the name of the corresponding mammal.

[0112] The amino acid sequence of mature human IL22 corresponds to amino acids 34-179 of SEQ ID NO: 1. Analysis of recombinant human IL22 reveals numerous structural domains (Nagem et al. (2002) Structure, 10:1051-62; U.S. Patent Application Publication No. 2002 / 0187512). Antibodies that bind to IL22

[0113] The present invention provides anti-IL22 antibodies that bind to IL22 (the target polypeptide) and have the functional and structural properties as further described herein.

[0114] The present disclosure provides a method for identifying an antibody that binds to IL22, the method comprising: a) immunizing an animal with human IL22; b) isolating the anti-IL22 antibodies produced from the animal; and c) i. binds to both human and cynomolgus IL22; ii. neutralize IL22-induced STAT3 phosphorylation or IL22-dependent IL-10 release; iii. binds to human IL22 and prevents the binding of IL22R1; selecting an anti-IL22 antibody; Includes:

[0115] Any suitable animal, including mice, rats, and rabbits, may be used to generate anti-IL22 antibodies. Binding and cross-linking measurements to IL22 can be performed using techniques known to those skilled in the art. Standard assays are available for measuring the neutralizing activity of identified antibodies. Examples of such assays are provided in the Examples section of this specification.

[0116] Antibodies in the context of the present invention include whole antibodies and functionally active antibody fragments (i.e., molecules that comprise an antigen-binding domain that specifically binds to an antigen (also called antigen-binding fragments)). The characteristics described herein also apply to antibody fragments, unless the context dictates otherwise. Antibodies can be (or can be derived from) polyclonal, monoclonal, multivalent, multispecific, bispecific, fully human, humanized, or chimeric.

[0117] The antibodies further described are for reference and exemplary purposes only and are not intended to limit the scope of the invention.

[0118] The antibodies used in accordance with the present invention may be monoclonal or polyclonal, preferably monoclonal. The antibodies used in accordance with the present invention may be chimeric, CDR-grafted (e.g., any suitable receptor variable region framework sequence can be used, taking into account the class / type of the donor antibody from which the CDRs are derived, including mouse, primate, and human framework regions), nanobody, human, or humanized. For the production of both monoclonal and polyclonal antibodies, the animals used to produce such antibodies are typically non-human mammals such as goats, rabbits, rats, or mice, although antibodies may also be produced in other species.

[0119] Polyclonal antibodies can be produced by routine methods such as immunizing a suitable animal with an antigen of interest, after which blood can be removed from such an animal and the antibodies produced purified.

[0120] Monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. Some exemplary methods for producing monoclonal antibodies are described herein.

[0121] For example, monoclonal antibodies can be prepared using hybridoma technology (Kohler & Milstein, 1975, Nature, 256:495-497), trioma technology, human B-cell hybridoma technology (Kozbore et al., 1983, Immunology Today, 4:72), and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

[0122] Antibodies can also be produced using single lymphocyte antibody techniques by cloning and expressing immunoglobulin variable region cDNA generated from a single lymphocyte selected for the production of specific antibodies, for example, by the methods described in WO9202551, WO2004051268, and WO2004106377.

[0123] Antibodies raised against a target polypeptide can be obtained by administering the polypeptide to an animal, preferably a non-human animal, using well-known conventional protocols, if immunization of the animal is required (see, for example, Handbook of Experimental Immunology, D.M. Weir (ed.), Vol. 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many animals can be immunized, such as rabbits, mice, rats, sheep, cattle, camels, or pigs. However, mice, rabbits, pigs, and rats are commonly used.

[0124] Monoclonal antibodies can also be generated using various phage display methods known in the art, including those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182:41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 187 9-18), and Burton et al. (Advances in Immunology, 1994, 57:191-280). In a specific phage display method, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) or randomly recombined in a phage library and screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) without immunization to provide a single source of antibodies against a wide range of non-self and self antigens, as described in Griffiths et al., EMBO J. 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol. 227:381-388 (1992).Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent No. 2005 / 0079574, U.S. Patent No. 2005 / 0119455, U.S. Patent No. 2005 / 0266000, U.S. Patent No. 2007 / 0117126, U.S. Patent No. 2007 / 0160598, U.S. Patent No. 2007 / 0237764, U.S. Patent No. 2007 / 0292936, and U.S. Patent No. 2009 / 0002360.

[0125] Antibodies can be screened using assays that measure binding to target polypeptides and / or assays that measure the ability of antibodies to block specific interactions. An example of a binding assay is, for example, an ELISA, which uses a fusion protein of the target polypeptide immobilized on a plate and a conjugated secondary antibody to detect antibodies bound to the target. An example of a blocking assay is a flow cytometry-based assay that measures the blocking of ligand proteins binding to the target polypeptide. A fluorescently labeled secondary antibody is used to detect the amount of such ligand proteins bound to the target polypeptide.

[0126] Antibodies can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics.

[0127] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments.

[0128] The antibody can be a full-length antibody. More specifically, the antibody can be of the IgG isotype. More specifically, the antibody can be an IgG1 or IgG4.

[0129] The antibody constant region domain, if present, can be selected taking into account the proposed function of the antibody molecule, particularly any effector functions that may be required. For example, the constant region domain can be a human IgA, IgD, IgE, IgG, or IgM domain. In particular, if the antibody molecule is intended for therapeutic use and antibody effector functions are required, human IgG constant region domains, particularly IgG1 and IgG3 isotypes, can be used. Alternatively, if the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, IgG2 and IgG4 isotypes may be used. It will be understood that sequence variants of these constant region domains can also be used. Those skilled in the art will also know that antibodies can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and cell culture conditions used to express the antibody. Such modifications can include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) by the action of carboxypeptidase (described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Thus, the C-terminal lysine of the antibody heavy chain may be absent.

[0130] Alternatively, the antibody is an antigen-binding fragment.

[0131] For a review of specific antigen-binding fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in *The Pharmacology of Monoclonal Antibodies*, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), and see WO 93 / 16185, U.S. Pat. No. 5,571,894, and U.S. Pat. No. 5,587,458. Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having increased in vivo half-lives are disclosed in U.S. Pat. No. 5,869,046.

[0132] Antigen-binding fragments and methods for producing them are well known in the art, see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181; Adair and Lawson, 2005. Therapeutic antibodies. Drug Design Reviews-Online 2(3):209-217. The Fab-Fv format was first disclosed in WO 2009 / 040562, its disulfide-stabilized version, Fab-dsFv, was first disclosed in WO 2010 / 035012, and the TrYbe format was disclosed in WO 2015 / 197772.

[0133] Various techniques have been developed for producing antibody fragments. Such fragments can be derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However, antibody fragments can also be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)).

[0134] F(ab')2 fragments can be directly isolated from recombinant host cell culture. The antibody can be a single-chain Fv fragment (scFv), as described in WO 93 / 16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. The antibody fragment can also be a "linear antibody," as described, for example, in U.S. Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0135] The antibody may be a Fab, Fab', F(ab')2, Fv, dsFv, scFv, or dsscFv. The antibody may be a single domain antibody or nanobody, such as a VH or VL or VHH or VNAR. The antibody may be a Fab or Fab' fragment as described in WO 2011 / 117648, WO 2005 / 003169, WO 2005 / 003170, and WO 2005 / 003171.

[0136] The antibody may be a disulfide-stabilized single-chain variable fragment (dsscFv).

[0137] The disulfide bond between the variable domains VH and VL can be between two of the residues listed below. ·V H 37+V L 95, see for example Protein Science 6,781-788 Zhu et al (1997); ·V H 44+V L 100, see, for example, Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012; ·V H 44+V L 105, see, for example, J Biochem.118,825-831 Luo et al (1995); ·V H 45+V L 87, see for example Protein Science 6,781-788 Zhu et al (1997); ·V H 55+V L 101, see e.g. FEBS Letters 377 135-139 Young et al (1995); ·V H 100+V L 50, see for example Biochemistry 29 1362-1367 Glockshuber et al (1990); ·V H 100b+V L 49, see for example Biochemistry 29 1362-1367 Glockshuber et al (1990); ·V H 98+V L For 46, see, for example, Protein Science 6, 781-788 Zhu et al (1997); ·, V H 101+V L 46, see for example Protein Science 6,781-788 Zhu et al (1997); ·V H 105+V L 43, see, for example, Proc. Natl. Acad. Sci. USA Vol. 90 pp. 7538-7542 Brinkmann et al (1993); or Proteins 19, 35-47 Jung et al (1994); ·V H 106+V L 57, see e.g. FEBS Letters 377 135-139 Young et al (1995) and the corresponding position or positions in the variable region pair located within the molecule.

[0138] A disulfide bond can be formed between positions VH44 and VL100.

[0139] The antigen-binding fragments described herein may also be characterized as monoclonal, chimeric, humanized, fully human, multispecific, bispecific, etc., and it will be understood by those skilled in the art that discussion of these terms also refers to such fragments.

[0140] multispecific antibodies The antibodies of the present invention may be multispecific antibodies.

[0141] Examples of multispecific antibodies or antigen-binding fragments thereof that are also contemplated for use in connection with the present disclosure include bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies, bibodies, and tribodies (see, e.g., Holliger and Hudson, 2005, Nature Biotech 23(9):1126-1136; Schoonjans et al. 2001, Biomolecular Engineering, 17(6), 193-202).

[0142] A variety of multispecific antibody formats have been produced. While various classifications have been proposed, multispecific IgG antibody formats generally include bispecific IgG, adducted IgG, multispecific (e.g., bispecific) antibody fragments, multispecific (e.g., bispecific) fusion proteins, and multispecific (e.g., bispecific) antibody conjugates, as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67 (2015): 95-106.

[0143] The antibody may be a bispecific antibody. In one aspect, the antibody comprises two antigen-binding domains, one binding domain binding to IL22 and the other binding domain binding to another antigen, i.e., each binding domain is monovalent for each antigen. In one embodiment, the antibody is a tetravalent bispecific antibody, i.e., the antibody comprises four antigen-binding domains, for example, two binding domains binding to IL22 and the other two binding domains binding to another antigen. In one embodiment, the antibody is a trivalent bispecific antibody.

[0144] Techniques for producing bispecific antibodies include, but are not limited to, CrossMab technology (Klein et al., Engineering therapeutic bispecific antibodies using CrossMab technology, Methods 154 (2019) 21-31), Knobs-in-holes engineering (e.g., WO 1996027011, WO 1998050431), DuoBody technology (e.g., WO 2011131746), and Azymetric technology (e.g., WO 2012058768). Further techniques for producing bispecific antibodies are described, for example, in Godar et al., 2018, Therapeutic bispecific antibody formats: a patent applications review (1994-2017), Expert Opinion on Therapeutic Patents, 28:3, 251-276. Bispecific antibodies include CrossMab antibodies, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-lgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body and orthogonal Fab, among others.

[0145] The antibody construct may be a trispecific antibody.

[0146] The antibody may be a multiparatopic antibody.

[0147] In one embodiment, each binding domain is monovalent. Preferably, each binding domain comprises no more than one VH and one VL.

[0148] Loaded IgGs classically comprise full-length IgGs that have been engineered by adding additional antigen-binding domains or antigen-binding fragments to the N-terminus and / or C-terminus of the IgG heavy and / or light chains. Examples of such additional antigen-binding fragments include sdAb antibodies (e.g., VH or VL), Fv, scFv, dsscFv, Fab, scFab, and additional IgG antibody formats, such as DVD-IgG, IgG(H)-scFv, scFv-(H)lgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgC(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG (four-in-one), as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106.

[0149] Multispecific antibody fragments include nanobodies, nanobody-HSA, BiTE, diabodies, DART, TandAb, scDiabody, sc-Diabody-CH3, diabody-CH3, triple body, miniantibody, minibody, tri biminibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc; and intrabodies, as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67 (2015): 95-106.

[0150] Multispecific fusion proteins include Dock and Lock, ImmTAC, HSabody, scDiabody-HSA, and Tandem scFv-Toxin.

[0151] Multispecific antibody conjugates include IgG-IgG; Cov-X-body; and scFv1-PEG-scFv2.

[0152] Further multispecific antibody formats are described, for example, in Brinkmann et al., The making of bispecific antibodies, mAbs, 9:2, 182-212 (2017), in particular Figure 2, e.g., tandem scFv, triplet, Fab-VHH, taFv-Fc, scFv4-Ig, scFv2-Fcab, scFv4-IgG. Biobodies, tribodies, and methods for producing them are disclosed, for example, in WO 99 / 37791.

[0153] An antibody for use in the invention can be a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binding to a therapeutic target and one scFv or dsscFv extending half-life, e.g., by binding to albumin). Such antibody fragments are described in WO 2015 / 197772. Another preferred antibody for use in the invention fragment comprises a Fab linked to only one scFv or dsscFv, as described, for example, in WO 2013 / 068571 and Dave et al., Mabs, 8(7) 1319-1335 (2016).

[0154] Another antibody for use in the present invention is the knobs-in-holes (KiH) antibody. This is a multispecific antibody format consisting of heavy chain homodimers for heterodimerization (e.g., for the efficient production of bispecific, multispecific, or monospecific antibodies). Generally, such techniques involve introducing a protrusion ("knob") into the interface of a first polypeptide (e.g., the first CH3 domain in the first antibody heavy chain) and a corresponding cavity ("hole") into the interface of a second polypeptide (e.g., the second CH3 domain in the second antibody heavy chain), whereby the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide (e.g., the first CH3 domain in the first antibody heavy chain) with a larger side chain (e.g., arginine, phenylalanine, tyrosine, or tryptophan). A compensatory cavity of identical or similar size to the protrusion is created at the interface of the second polypeptide (such as the second CH3 domain in a second antibody heavy chain) by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine, serine, valine, or threonine). The protrusion and cavity can be produced by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis. Further details regarding "knobs-in-holes" technology are described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; WO 2009 / 089004; U.S. Patent Application Publication No. 2009 / 0182127; Marvin md Zu, Acta Pharmacologica Sincia (2005) 26(6):649-658; Kontermann Acta Pharmacologica Sincia (2005) 26:1-9; Ridgway et al, Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001).

[0155] Humanized, human and chimeric antibodies and methods for producing such antibodies The antibodies of the present invention may be, but are not limited to, humanized antibodies, fully human antibodies, or chimeric antibodies.

[0156] In one embodiment, the antibody is humanized. More particularly, the antibody is a chimeric, human, or humanized antibody.

[0157] In certain embodiments, the antibodies provided herein are chimeric antibodies. Examples of chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0158] In one embodiment, the antibody is a humanized antibody.

[0159] Humanized antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs are derived, it being understood that only the specificity-determining residues of the CDRs need to be transferred, rather than the entire CDRs (see, e.g., Kashmir et al., 2005, Methods, 36, 25-34).

[0160] Suitably, humanized antibodies according to the invention have variable domains comprising human acceptor framework regions as well as one or more CDRs, and optionally further comprising one or more donor framework residues.

[0161] Thus, in one embodiment, a humanized antibody is provided in which the variable domains comprise human acceptor framework regions and non-human donor CDRs.

[0162] When CDRs or specificity-determining residues are grafted, any suitable receptor variable region framework sequence may be used, taking into account the class / type of the donor antibody from which the CDRs are derived, including murine, primate and human framework regions.

[0163] Examples of human frameworks that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al.). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY, and POM can be used for both the heavy and light chains. Alternatively, human germline sequences can be used, which are available at www.imgt.org. In embodiments, the recipient framework is an IGKV1D-13 human germline, an IGHV3-66 human germline, an IGKV1-12 human germline, and / or an IGHV4-31 human germline. In embodiments, the human framework comprises 1 to 5, 1 to 4, 1 to 3, or 1 to 2 donor antibody amino acid residues.

[0164] In the humanized antibodies of the present invention, the receptor heavy and light chains do not necessarily have to be derived from the same antibody, but may, if desired, comprise composite chains having framework regions derived from different chains.

[0165] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art.

[0166] A human antibody includes a heavy or light chain variable region or a full-length heavy or light chain that is the "product" of or "derived from" a particular germline sequence when the variable region or full-length chain of the antibody is obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. A human antibody or fragment thereof that is the "product" of or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with that of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is closest in sequence to the human antibody (i.e., most closely in percent identity). A human antibody that is the "product" of or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to naturally occurring somatic mutations or the deliberate introduction of site-specific mutations. However, the selected human antibody will typically be at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene when compared to the germline immunoglobulin amino acid sequence of another species (e.g., a murine germline sequence) and contain amino acid residues that identify the human antibody as human. In certain cases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, a human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0167] Structural features of antibodies The antibodies of the present invention comprise a binding domain. The binding domain generally comprises six CDRs, three from the heavy chain and three from the light chain. In one embodiment, the CDRs are within a framework and together form a variable region. Thus, an antibody has a binding domain specific for an antigen, the binding domain comprising a light chain variable region and a heavy chain variable region.

[0168] [Table 3]

[0169] In one embodiment, the present invention provides an antibody that binds to IL22, comprising: CDR-L1 comprising SEQ ID NO: 5, CDR-L2 comprising SEQ ID NO: 6, and CDR-L3 comprising SEQ ID NO:7 The present invention provides an antibody comprising a light chain variable domain comprising at least one of:

[0170] In one embodiment, the present invention provides an antibody that binds to IL22, comprising: CDR-L1 comprising SEQ ID NO: 5, CDR-L2 comprising SEQ ID NO: 6, and CDR-L3 comprising SEQ ID NO:7 The present invention provides an antibody comprising a light chain variable domain comprising:

[0171] In one embodiment, the present invention provides an antibody that binds to IL22, comprising: CDR-H1 comprising SEQ ID NO: 8, CDR-H2 comprising SEQ ID NO: 9, and CDR-H3 comprising SEQ ID NO: 10 The present invention provides an antibody comprising a heavy chain variable domain comprising at least one of:

[0172] In one embodiment, the present invention provides an antibody that binds to IL22, comprising: CDR-H1 comprising SEQ ID NO: 8, CDR-H2 comprising SEQ ID NO: 9, and CDR-H3 comprising SEQ ID NO: 10 The present invention provides an antibody comprising a heavy chain variable domain comprising:

[0173] The antibody molecules of the present invention may each comprise a complementary light chain or a complementary heavy chain.

[0174] Thus, in one embodiment, the present invention provides an antibody that binds to IL22, comprising: CDR-L1 comprising SEQ ID NO: 5, CDR-L2 comprising SEQ ID NO: 6, and CDR-L3 comprising SEQ ID NO:7 a light chain variable region comprising CDR-H1 comprising SEQ ID NO: 8, CDR-H2 comprising SEQ ID NO: 9, and CDR-H3 comprising SEQ ID NO: 10 a heavy chain variable region comprising The present invention provides an antibody comprising:

[0175] In one embodiment, an antibody of the invention comprises a light chain variable region comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:72.

[0176] In one embodiment, an antibody of the invention comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO:24 or SEQ ID NO:74.

[0177] In one embodiment, an antibody of the invention comprises a light chain variable region comprising the sequence set forth in SEQ ID NO:22 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:24.

[0178] In an alternative embodiment, an antibody of the invention comprises a light chain variable region comprising the sequence set forth in SEQ ID NO:72 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:74.

[0179] In one embodiment, the antibody of the invention comprises: CDR-L1 comprising SEQ ID NO: 5, CDR-L2 comprising SEQ ID NO: 6, and CDR-L3 comprising SEQ ID NO:7 a light chain variable region comprising CDR-H1 comprising SEQ ID NO: 8, CDR-H2 comprising SEQ ID NO: 9, and CDR-H3 comprising SEQ ID NO: 10 a heavy chain variable region comprising It is a Fab that includes.

[0180] In one embodiment, the antibody of the invention is a Fab comprising a light chain comprising the sequence set forth in SEQ ID NO:26 and a heavy chain comprising the sequence set forth in SEQ ID NO:28.

[0181] In another embodiment, the antibody of the invention is an IgG1 comprising a light chain comprising the sequence set forth in SEQ ID NO:26 and a heavy chain comprising the sequence set forth in SEQ ID NO:30.

[0182] In another embodiment, the antibody of the invention is an IgG4P comprising a light chain comprising the sequence set forth in SEQ ID NO:26 and a heavy chain comprising the sequence set forth in SEQ ID NO:32.

[0183] In an alternative embodiment, the present invention provides an antibody that binds to IL22, comprising: CDR-L1 comprising SEQ ID NO: 65, CDR-L2 comprising SEQ ID NO: 66, and CDR-L3 comprising SEQ ID NO: 67 The present invention provides an antibody comprising a light chain variable domain comprising at least one of:

[0184] In an alternative embodiment, the present invention provides an antibody that binds to IL22, comprising: CDR-L1 comprising SEQ ID NO: 65, CDR-L2 comprising SEQ ID NO: 66, and CDR-L3 comprising SEQ ID NO: 67 The present invention provides an antibody comprising a light chain variable domain comprising:

[0185] In an alternative embodiment, the present invention provides an antibody that binds to IL22, comprising: CDR-H1 comprising SEQ ID NO: 68, CDR-H2 comprising SEQ ID NO: 69, and CDR-H3 comprising SEQ ID NO: 70 The present invention provides an antibody comprising a heavy chain variable domain comprising at least one of:

[0186] In an alternative embodiment, the present invention provides an antibody that binds to IL22, comprising: CDR-H1 comprising SEQ ID NO: 68, CDR-H2 comprising SEQ ID NO: 69, and CDR-H3 comprising SEQ ID NO: 70 The present invention provides an antibody comprising a heavy chain variable domain comprising:

[0187] In an alternative embodiment, the present invention provides an antibody that binds to IL22, comprising: CDR-L1 comprising SEQ ID NO: 65, CDR-L2 comprising SEQ ID NO: 66, and CDR-L3 comprising SEQ ID NO: 67 a light chain variable region comprising CDR-H1 comprising SEQ ID NO: 68, CDR-H2 comprising SEQ ID NO: 69, and CDR-H3 comprising SEQ ID NO: 70 a heavy chain variable region comprising The present invention provides an antibody comprising:

[0188] In an alternative embodiment, the antibody of the invention CDR-L1 comprising SEQ ID NO: 65, CDR-L2 comprising SEQ ID NO: 66, and CDR-L3 comprising SEQ ID NO: 67 a light chain variable region comprising CDR-H1 comprising SEQ ID NO: 68, CDR-H2 comprising SEQ ID NO: 69, and CDR-H3 comprising SEQ ID NO: 70 a heavy chain variable region comprising It is a Fab that includes:

[0189] In yet another alternative embodiment, the antibody of the invention is a Fab comprising a light chain comprising the sequence set forth in SEQ ID NO:76 and a heavy chain comprising the sequence set forth in SEQ ID NO:78.

[0190] In another alternative embodiment, the antibody of the invention is an IgG1 comprising a light chain comprising the sequence set forth in SEQ ID NO:76 and a heavy chain comprising the sequence set forth in SEQ ID NO:80.

[0191] In another alternative embodiment, the antibody of the invention is an IgG4P comprising a light chain comprising the sequence set forth in SEQ ID NO:76 and a heavy chain comprising the sequence set forth in SEQ ID NO:82.

[0192] In embodiments, the invention provides an antibody comprising a light chain variable region comprising CDR-L1 comprising SEQ ID NO:5 or SEQ ID NO:65, CDR-L2 comprising SEQ ID NO:6 or SEQ ID NO:66, and CDR-L3 comprising SEQ ID NO:7 or SEQ ID NO:67, and a heavy chain variable region comprising CDR-H1 comprising SEQ ID NO:8 or SEQ ID NO:68, CDR-H2 comprising SEQ ID NO:9 or SEQ ID NO:69, and CDR-H3 comprising SEQ ID NO:10 or SEQ ID NO:70.

[0193] In one embodiment, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a CH1 domain and the light chain comprises a CL domain, either kappa or lambda.

[0194] Functional properties of anti-IL22 antibodies In one embodiment, the antibody of the present invention is a neutralizing antibody. Preferably, the antibody according to the present invention neutralizes IL22 activity. In particular, the antibody can neutralize IL22 binding to IL22 receptor 1 (IL22R1). The anti-IL22 antibody of the present invention also binds to IL22 and inhibits the binding of IL22 to IL22 binding protein (IL22RA2 or IL22BP). Preferably, the antibody can neutralize IL22 binding to IL22 receptor 1 (IL22R1) and IL22 binding protein (IL22RA2).

[0195] The anti-IL22 antibodies of the present invention bind to the same region on IL22 as IL22R1. In a specific embodiment, the present invention provides antibodies that bind to a region on IL22 such that the binding sterically blocks the interaction between IL22 and IL22R1. In particular, the variable region of the antibody can sterically block the interaction between IL22 and IL22R1.

[0196] In some embodiments, antibodies according to the invention bind to IL22 that is not bound to IL22BP ("free IL22"). In some embodiments, antibodies according to the invention bind to IL22 and prevent IL22 from binding to IL22BP.

[0197] The antibody according to the present invention is specific for IL22. In one embodiment, the antibody has a stronger binding affinity for IL22 compared to IL22R1, characterized by an at least 10-fold higher dissociation affinity constant (KD) for IL22 than for IL22R1 or IL22BP, as measured using BIACore technology.

[0198] In some embodiments, the antibody binds to IL22 with sufficient affinity and specificity. In certain embodiments, the antibody binds to IL22 with a specificity of about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, or 0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 In one embodiment, the antibody binds to human IL22 with a KD of less than 100 pM, including any range between these values.

[0199] In certain embodiments, the antibody is administered at a concentration of about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, or 0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10-13 M, e.g. 10 -9 M~10 -13 In one embodiment, the antibody binds to cynomolgus IL22 with a KD of less than 100 pM, including any range between these values.

[0200] Those skilled in the art will appreciate that KD values ​​may vary depending on the format and overall structure of the antibody, for example, the KD of an antibody may vary in the context of a multispecific antibody, a full-length antibody, or an antibody fragment.

[0201] The anti-IL22 antibodies of the present invention can inhibit IL22-induced IL10 release from cells.

[0202] As demonstrated by the examples, the anti-IL22 antibodies of the invention are capable of inhibiting IL22-mediated keratinocyte proliferation and differentiation.

[0203] The anti-IL22 antibodies of the present invention can also inhibit the release of IL22-induced antimicrobial peptides (eg, S100A7, etc.).

[0204] The affinity of an antibody, as well as the degree to which it inhibits binding, can be determined by one of skill in the art using conventional techniques, such as those described by Scatchard et al. (Ann. KY. Acad. Sci. 51:660-672 (1949)), or by surface plasmon resonance (SPR) using a system such as BIAcore. In surface plasmon resonance, a target molecule is immobilized on a solid phase and exposed to a ligand in a mobile phase that moves along a flow cell. Binding of the ligand to the immobilized target changes the local refractive index, resulting in a change in the SPR angle, which can be monitored in real time by detecting changes in the intensity of the reflected light. The rate of change of the SPR signal can be analyzed to obtain apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these values ​​provides the apparent equilibrium constant (affinity) (see, e.g., Wolff et al., Cancer Res. 53:2560-65 (1993)).

[0205] In one embodiment, an anti-IL22 antibody according to the invention binds to IL22 that is not bound to an IL22 binding protein ("free IL22"). More specifically, an antibody according to the invention binds to IL22 and prevents IL22 from binding to an IL22 binding protein.

[0206] Preferably, the antibody according to the invention is specific for IL22.

[0207] The disclosures herein regarding antibodies, particularly regarding binding affinity and specificity, and activity, are also applicable to antigen-binding fragments and antibody-like molecules.

[0208] Antibodies that bind to the same epitope The antibodies may compete with or bind to the same epitope as defined above with respect to the light chain, heavy chain, light chain variable region (LCVR), heavy chain variable region (HCVR) or CDR sequences for binding to IL22.

[0209] In particular, the present invention provides antibodies that compete for binding to IL22 with, or bind to the same epitope as, an antibody comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequence combination of SEQ ID NOs: 5 / 6 / 7 / 8 / 9 / 10. The antibodies may compete for binding to IL22 with, or bind to the same epitope as, an antibody comprising the LCVR and HCVR sequence pair of SEQ ID NOs: 22 / 24. The antibodies may compete for binding to IL22 with, or bind to the same epitope as, an Fab comprising the LC and HC sequence pair set forth in SEQ ID NOs: 26 / 28.

[0210] Alternatively, the present invention provides an antibody that competes for binding to IL22 with, or binds to the same epitope as, an antibody comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequence combination of SEQ ID NOs: 65 / 66 / 67 / 68 / 69 / 70. The antibody may compete for binding to IL22 with, or bind to the same epitope as, an antibody comprising the LCVR and HCVR sequence pair of SEQ ID NOs: 72 / 74. The antibody may compete for binding to IL22 with, or bind to the same epitope as, an Fab comprising the LC and HC sequence pair set forth in SEQ ID NOs: 76 / 78.

[0211] In one embodiment, the invention provides an anti-IL22 antibody that binds to an epitope on IL22, wherein the epitope comprises one or more residues VRLIGEKLFHGVSM (SEQ ID NO: 1, residues 72-85 of the amino acid sequence of IL22). In some embodiments, the invention provides an anti-IL22 antibody that binds to the polypeptide VRLIGEKLFHGVSM (SEQ ID NO: 96). In embodiments, the invention provides an antibody that binds to at least one, at least two, at least three, at least four, at least five, or all residues selected from residues 72-85 of the amino acid sequence of IL22 as defined by SEQ ID NO: 1.

[0212] In one embodiment, the invention provides an anti-IL22 antibody that binds to an epitope on IL22, the epitope comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all of residues selected from the list consisting of Gln48, Glu77, Phe80, His81, Gly82, Val83, Ser84, Met85, Arg88, Leu169, Met172, Ser173, Arg175, Asn176, and Ile179 of human IL22 (SEQ ID NO: 1), as measured at a contact distance of less than 4 Å. In embodiments, the invention provides antibodies that bind to an epitope on IL22, the epitope comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all of residues selected from the list consisting of Lys44, Phe47, Gln48, Ile75, Gly76, Glu77, Phe80, His81, Gly82, Val83, Ser84, Met85, Ser86, Arg88, Leu169, Met172, Ser173, Arg175, Asn176, and Ile179 of human IL22 (SEQ ID NO: 1), as measured by a contact distance of less than 5 Å between the antibody and IL22.

[0213] In particular, the present invention provides antibodies that compete for binding to IL22 with, or can bind to the same epitope as, an antibody comprising the heavy and light chain residues listed in Tables 4 or 5 below. More specifically, the antibodies of the present invention comprise a CDR-H3 sequence comprising residues 97-104, preferably residues 99-104, of SEQ ID NO: 24, and bind to an epitope on IL22 as defined above. Even more specifically, the antibodies of the present invention comprise CDR-H1, CDR-H2, and CDR-H3 residues as defined in Table 4 or Table 5, and bind to an epitope on IL22 as defined above.

[0214] [Table 4]

[0215] [Table 5]

[0216] More specifically, the present invention provides an anti-IL22 antibody that binds to the epitope on IL22 defined above, and the antibody prevents the binding of IL22 to IL22R1 and IL22BP. More specifically, the light chain of the antibody sterically prevents the binding of IL22R1 to IL22.

[0217] Epitopes can be identified in combination with any one of the antibodies provided by the present invention using any suitable binding site mapping method known in the art. Examples of such methods include screening peptides of various lengths derived from the full-length target protein for binding to the antibody of the present invention or a fragment thereof, and identifying fragments capable of specifically binding to the antibody that contain the sequence of the epitope recognized by the antibody. The target peptide can be produced synthetically. Peptides that bind to the antibody can be identified, for example, by mass spectrometry. In another example, NMR spectroscopy or X-ray crystallography can be used to identify the epitope bound by the antibody of the present invention. Typically, when epitope determination is performed by X-ray crystallography, amino acid residues of the antigen within 4 Å of the CDR are considered to be part of the epitope. Once identified, the epitope can be useful for preparing fragments that bind to the antibody of the present invention, and, if necessary, can be used as an immunogen to obtain additional antibodies that bind to the same epitope.

[0218] In one embodiment, the epitope of the antibody is determined by X-ray crystallography.

[0219] Whether an antibody binds to the same epitope as a reference antibody or competes for binding with a reference antibody can be easily determined by using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody of the present invention, the reference antibody is bound to a protein or peptide under saturating conditions. Then, the ability of the test antibody to bind to the protein or peptide is evaluated. If the test antibody can bind to the protein or peptide after saturating binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope from the reference antibody. On the other hand, if the test antibody cannot bind to the protein or peptide after saturating binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the present invention, or the reference antibody causes a conformational change in the antigen, thus preventing the binding of the test antibody.

[0220] To determine whether an antibody competes for binding with a reference antibody, the above-described binding methodology is performed in two different experimental settings. In the first setting, the reference antibody is bound to the antigen under saturating conditions, followed by evaluation of the binding of the test antibody to the antigen. In the second setting, the test antibody is bound to the antigen under saturating conditions, followed by evaluation of the binding of the reference antibody to the protein / peptide. In both experimental settings, if only the first (saturating) antibody can bind to the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the antigen. As will be understood by those skilled in the art, an antibody that competes for binding with a reference antibody does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope, or may cause a conformational change that results in a lack of binding.

[0221] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the binding of the other to the antigen. Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0222] Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same part of the antigen as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0223] Antibody variants In certain embodiments, antibody variants are provided that have one or more amino acid substitutions, insertions, and / or deletions. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Amino acid substitutions can be introduced into an antibody of interest and screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0224] In certain embodiments, amino acid sequence variants of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of anti-IL22 antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the protein or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the anti-IL22 antibody (e.g., within one or more CDR and / or framework sequences, or within the VH and / or VL domains). Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired properties.

[0225] In certain embodiments of the variant VH and VL sequences provided herein, each HVR is unchanged or contains no more than 1, 2 or 3 amino acid substitutions.

[0226] It will be understood that one or more amino acid substitutions, additions, and / or deletions can be made to the CDRs provided by the present invention without significantly altering the ability of the antibody to bind to IL22 and neutralize IL22 activity. The effect of any amino acid substitution, addition, and / or deletion can be readily tested by one of skill in the art by demonstrating IL22 binding and inhibition of the interaction of IL22 with its receptor IL22R1 and IL22 binding proteins, for example, using the methods described herein, particularly those exemplified in the Examples.

[0227] As a result, in certain embodiments of the variant VH and VL sequences, each CDR contains no more than one, two, or three amino acid substitutions, where such amino acid substitutions are conservative and the antibody retains its binding properties to IL22 and blocks binding of IL22 to IL22R1 and IL22 binding proteins.

[0228] Thus, the present invention provides anti-IL22 antibodies comprising one or more CDRs selected from CDR-L1 (comprising SEQ ID NO:5), CDR-L2 (comprising SEQ ID NO:6), CDR-L3 (comprising SEQ ID NO:7), CDR-H1 (comprising SEQ ID NO:8), CDR-H2 (comprising SEQ ID NO:9) and CDR-H3 (comprising SEQ ID NO:10), wherein one or more amino acids in one or more CDRs are substituted with another amino acid, for example a similar amino acid as defined herein below.

[0229] In one embodiment, the present invention provides an anti-IL22 antibody comprising CDR-L1 (comprising SEQ ID NO:5), CDR-L2 (comprising SEQ ID NO:6), CDR-L3 (comprising SEQ ID NO:7), CDR-H1 (comprising SEQ ID NO:8), CDR-H2 (comprising SEQ ID NO:9), and CDR-H3 (comprising SEQ ID NO:10), wherein, for example, one or more amino acids in one or more CDRs are substituted with another amino acid, such as a similar amino acid as defined herein below. In an embodiment, the one or more amino acid substitutions in one or more CDRs replace a free cysteine ​​residue or modify a potential asparagine deamidation site. In an embodiment, the one or more amino acid substitutions in one or more CDRs modify a potential aspartate isomerization site. In an embodiment, the one or more amino acid substitutions in one or more CDRs remove a potential DP hydrolysis site. In embodiments, for CDR-L3 (SEQ ID NO: 7), the substitution is C91S or C91V; N95D; S96A; or a combination thereof, for CDR-H2 (SEQ ID NO: 9), the substitution is D54E, G55A, or a combination thereof, and for CDR-H3 (SEQ ID NO: 9), the substitution is D107E, or a combination of the listed substitutions.

[0230] In one embodiment, an anti-IL22 antibody of the invention comprises a light chain variable domain comprising three CDRs, wherein the sequence of CDR-L1 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO:5; CDR-L2 comprises a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence set forth in SEQ ID NO:6; and / or CDR-L3 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO:7.

[0231] In one embodiment, an anti-IL22 antibody of the invention comprises a heavy chain variable domain comprising three CDRs, wherein the sequence of CDR-H1 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO: 8; CDR-H2 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO: 9; and / or CDR-H3 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO: 10.

[0232] In one embodiment, an anti-IL22 antibody of the invention comprises a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO:22.

[0233] In one embodiment, an antibody of the invention comprises a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO:24.

[0234] In one embodiment, an anti-IL22 antibody of the invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to that set forth in SEQ ID NO:22, and / or the heavy chain variable region comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to that set forth in SEQ ID NO:24.

[0235] In one embodiment, an anti-IL22 antibody of the invention comprises CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences comprising SEQ ID NOs: 65 / 66 / 67 / 68 / 69 / 70, respectively, and the remainder of the light chain variable region and heavy chain variable region have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to SEQ ID NOs: 72 and 74, respectively.

[0236] In one embodiment, an anti-IL22 antibody of the invention comprises CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences comprising SEQ ID NOs: 5 / 6 / 7 / 8 / 9 / 10, respectively, and the remainder of the light chain variable region and heavy chain variable region have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to SEQ ID NOs: 22 and 24, respectively.

[0237] In one embodiment, the anti-IL22 antibody of the invention is a Fab comprising a light chain comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO:26, and a heavy chain comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO:28.

[0238] In one embodiment, the anti-IL22 antibody of the present invention is a Fab comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences shown in SEQ ID NOs: 5 / 6 / 7 / 8 / 9 / 10, respectively, and the remainder of the light and heavy chains have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to SEQ ID NOs: 26 and 28, respectively.

[0239] In one embodiment, an antibody of the invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 22, in which one or more residues at positions 91, 95 and / or 96 are substituted by another amino acid, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 24, in which one or more residues at positions 54, 55 and / or 107 are substituted by another amino acid.

[0240] The present invention provides anti-IL22 antibodies comprising one or more CDRs selected from CDR-L1 (comprising SEQ ID NO: 65), CDR-L2 (comprising SEQ ID NO: 66), CDR-L3 (comprising SEQ ID NO: 67), CDR-H1 (comprising SEQ ID NO: 68), CDR-H2 (comprising SEQ ID NO: 69) and CDR-H3 (comprising SEQ ID NO: 70), wherein one or more amino acids in one or more CDRs are substituted with another amino acid, for example a similar amino acid as defined herein below.

[0241] In one embodiment, the present invention provides an anti-IL22 antibody comprising CDR-L1 (comprising SEQ ID NO: 65), CDR-L2 (comprising SEQ ID NO: 66), CDR-L3 (comprising SEQ ID NO: 67), CDR-H1 (comprising SEQ ID NO: 68), CDR-H2 (comprising SEQ ID NO: 69), and CDR-H3 (comprising SEQ ID NO: 70), wherein, for example, one or more amino acids in one or more CDRs are substituted with another amino acid, such as a similar amino acid as defined herein below. In an embodiment, one or more amino acid substitutions in one or more CDRs replace a free cysteine ​​residue or modify a potential asparagine deamidation site. In an embodiment, one or more amino acid substitutions in one or more CDRs modify a potential aspartate isomerization site. In an embodiment, one or more amino acid substitutions in one or more CDRs remove a potential DP hydrolysis site. In an embodiment, with respect to CDR-H2 (SEQ ID NO: 69), the substitution is S61T.

[0242] In one embodiment, an anti-IL22 antibody of the invention comprises a light chain variable domain comprising three CDRs, wherein the sequence of CDR-L1 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO: 65; CDR-L2 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO: 66; and / or CDR-L3 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO: 67.

[0243] In one embodiment, an anti-IL22 antibody of the invention comprises a heavy chain variable domain comprising three CDRs, wherein the sequence of CDR-H1 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO: 68; CDR-H2 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO: 69; and / or CDR-H3 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence set forth in SEQ ID NO: 70.

[0244] In one embodiment, an anti-IL22 antibody of the disclosure comprises a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO:72.

[0245] In one embodiment, an antibody of the disclosure comprises a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO:74.

[0246] In one embodiment, an anti-IL22 antibody of the invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to that set forth in SEQ ID NO: 72, and / or the heavy chain variable region comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to that set forth in SEQ ID NO: 74.

[0247] In one embodiment, an anti-IL22 antibody of the invention comprises CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences comprising SEQ ID NOs: 65 / 66 / 67 / 68 / 69 / 70, respectively, and the remainder of the light chain variable region and heavy chain variable region have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to SEQ ID NOs: 72 and 74, respectively.

[0248] In one embodiment, an anti-IL22 antibody of the invention is a Fab comprising a light chain comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO: 76, and a heavy chain comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO: 78.

[0249] In one embodiment, the anti-IL22 antibody of the present invention is a Fab comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences set forth in SEQ ID NOs: 65 / 66 / 67 / 68 / 69 / 70, respectively, and the remainder of the light and heavy chains have at least 70%, 80%, 90%, 95%, or 98% identity or similarity to SEQ ID NOs: 76 and 78, respectively. Sequence Identity and Similarity

[0250] The degree of identity and similarity between sequences can be readily calculated. "Percent sequence identity" (or "percent sequence similarity") is calculated by: (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.); (2) determining the number of positions containing identical (or similar) amino acids (e.g., identical amino acids present in both sequences, similar amino acids present in both sequences) to obtain the number of matched positions; (3) dividing the number of matched positions by the total number of positions within the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); and (4) multiplying the result by 100 to obtain the percent sequence identity or percent sequence similarity.

[0251] Methods for alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0252] Preferred examples of algorithms suitable for determining percent sequence identity and percent sequence similarity include the BLAST and BLAST 2.0 algorithms described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990). Polypeptide sequences can also be compared using FASTA, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.

[0253] In certain embodiments, substitutions, insertions, or deletions may be made within one or more CDRs, provided that such changes do not substantially reduce the ability of the antibody to bind to its target.

[0254] For example, conservative changes can be made in the CDRs that do not substantially reduce binding affinity. Such modifications may be made outside the antigen-contacting residues in the CDRs.

[0255] Conservative substitutions are shown in Table 6, along with more substantial "exemplary substitutions."

[0256] [Table 6]

[0257] Substantial modifications in the biological properties of antibody variants can be achieved by selecting substitutions that differ significantly in their effect on the structure of the polypeptide backbone in the area of ​​the substitution, on the charge or hydrophobicity of the molecule at the target site, or on maintaining the bulk of the side chain. Amino acids can be grouped according to the similarity of their side chain properties (A.L. Lehninger, Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)).

[0258] One type of substitutional variant involves substituting one or more CDR region residues of a parent antibody (humanized or human). Generally, the resulting variant(s) selected for further study have altered specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or substantially retain specific biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using affinity maturation techniques, e.g., based on phage display. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0259] For example, alterations (e.g., substitutions) can be made to CDRs to improve antibody affinity. Such changes can be made at HVR "hotspots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or at residues that contact the antigen, and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction of a secondary library and reselection from the secondary library is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.

[0260] One method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is alanine scanning mutagenesis (Cunningham and Wells (1989) Science, 244:1081-1085). In this method, a residue or a number of target residues are identified and substituted with alanine to determine whether the interaction of the antibody with the antigen is affected. Alternatively, or in addition, an X-ray structure of an antigen-antibody complex can be used to identify contact points between the antibody and its antigen. Mutants can be screened to determine whether they contain the desired properties. Constant region mutants

[0261] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby creating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0262] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0263] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn) are described in U.S. Patent No. 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn.

[0264] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example substitutions at positions 298, 333 and / or 334 of the Fc region (EU numbering of residues).

[0265] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 234, 235, 237, 238, 265, 269, 270, 297, 327, and 329 (see, e.g., U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, where amino acid residues are numbered according to the EU numbering system.

[0266] To confirm the reduction / depletion of CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat l Acad. Sci. USA 95:652-656 (1998). Clq binding assays can also be performed to confirm that the antibody is unable to bind Clq and thus lacks CDC activity. See, e.g., the Clq and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MI Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int. Immunol. 18(12):1759-1769 (2006)).

[0267] The constant region domains of the antibody molecules of the present invention, if present, can be selected taking into account the proposed function of the antibody molecule, in particular any effector functions that may be required. For example, the constant region domains can be human IgA, IgD, IgE, IgG, or IgM domains. In particular, when the antibody molecule is intended for therapeutic use and antibody effector functions are required, human IgG constant region domains, particularly IgG1 and IgG3 isotypes, can be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, IgG2 and IgG4 isotypes may be used. It will be understood that sequence variants of these constant region domains can also be used.

[0268] Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can conveniently be accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0269] Humanized, human and chimeric antibodies The antibodies of the present invention may be, but are not limited to, humanized antibodies, fully human antibodies, or chimeric antibodies.

[0270] In one embodiment, the antibody is humanized. More specifically, the anti-IL22 antibody is a chimeric antibody, a human antibody, or a humanized antibody.

[0271] In certain embodiments, the antibodies provided herein are chimeric antibodies. Examples of chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody.

[0272] Chimeric antibodies are composed of elements derived from two different species such that the elements retain the characteristics of the species they are derived from. Generally, chimeric antibodies will contain variable regions from one species and analogous constant regions from another species, such as mouse, rat, rabbit, or human.

[0273] In certain embodiments, the chimeric antibody is a humanized antibody.

[0274] It will be appreciated that only the specificity-determining residues of the CDRs need to be transferred, rather than the entire CDR (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). Humanized antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs are derived.

[0275] Suitably, humanized antibodies according to the invention have variable domains comprising human acceptor framework regions as well as one or more CDRs, and optionally further comprising one or more donor framework residues.

[0276] In one embodiment, the antibody is a humanized antibody, wherein the variable domains comprise human acceptor framework regions and non-human donor CDRs.

[0277] When CDRs are grafted, any suitable receptor variable region framework sequence may be used, taking into account the class / type of the donor antibody from which the CDRs are derived, including murine, primate and human framework regions.

[0278] Examples of human frameworks that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al.). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY, and POM can be used for both the heavy and light chains. Alternatively, human germline sequences can be used, which are available at www.imgt.org. In embodiments, the recipient framework is an IGKV1D-13 human germline, an IGHV3-66 human germline, an IGKV1-12 human germline, and / or an IGHV4-31 human germline. In embodiments, the human framework comprises 1 to 5, 1 to 4, 1 to 3, or 1 to 2 donor antibody amino acid residues.

[0279] In the humanized antibodies of the present invention, the receptor heavy and light chains do not necessarily have to be derived from the same antibody, but may, if desired, comprise composite chains having framework regions derived from different chains.

[0280] In some embodiments, the antibody is a human antibody. Human antibodies can be produced using various techniques known in the art. More specifically, the anti-IL22 antibody comprises a human antibody heavy chain constant region and a human light chain constant region.

[0281] A human antibody includes a heavy or light chain variable region or a full-length heavy or light chain derived from a particular germline sequence when the antibody variable region or full-length chain is obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. A human antibody derived from a human germline immunoglobulin sequence can be identified by comparing the amino acid sequence of the human antibody with that of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence to the human antibody (i.e., most closely in percent identity). A human antibody derived from a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to naturally occurring somatic mutations or the intentional introduction of site-specific mutations. However, the selected human antibody will typically be at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene when compared to the germline immunoglobulin amino acid sequence of another species (e.g., a murine germline sequence) and contain amino acid residues that identify the human antibody as human. In certain cases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, a human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0282] Human antibodies can be produced by a number of methods known to those skilled in the art. Human antibodies can be produced by the hybridoma method using human myeloma or mouse-human heteromyeloma cell lines (Kozbor, J Immunol; (1984) 133:3001; Brodeur, Monoclonal Isolated Antibody Production Techniques and Applications, pp51-63, Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both of which utilize human variable region repertoires ((Winter G; (1994) Annu Rev Immunol 12:433-455, Green LL, (1999) J Immunol Methods 231:1 1-23). ​​Human antibodies can be produced, for example, in mice in which the mouse immunoglobulin variable genes, and optionally the constant region genes, have been replaced by their human counterparts, as described, for example, in U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, and 5,770,429.

[0283] Effector molecules If desired, the antibodies according to the invention may be conjugated to one or more effector molecule(s). In one embodiment, the antibody is not bound to an effector molecule.

[0284] It will be understood that an effector molecule can comprise a single effector molecule or two or more such molecules linked to form a single moiety that can be bound to an antibody of the invention. Where it is desired to obtain an antibody fragment linked to an effector molecule, this can be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment is linked to the effector molecule directly or via a coupling agent. Techniques for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Particular chemical procedures include, for example, those described in WO 93 / 06231, WO 92 / 22583, WO 89 / 00195, WO 89 / 01476 and WO 03 / 031581. Alternatively, where the effector molecule is a protein or polypeptide, linkage can be achieved using recombinant DNA procedures, for example as described in WO 86 / 01533 and EP 0 392 745.

[0285] Examples of effector molecules can include cytotoxins or cytotoxic agents, including any agent that is detrimental to (e.g., kills) cells, such as combrestatins, dolastatins, epothilones, staurosporines, maytansinoids, spongistatins, rhizoxin, halichondrin, roridin, hemiasterin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.

[0286] Effector molecules also include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepaclorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicin, or duocarmycin), and antimitotic agents (e.g., vincristine and vinblastine).

[0287] Other effector molecules may include chelated radionuclides such as In and Y, Lu, bismuth, californium, iridium, and tungsten / rhenium; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids, and suramin.

[0288] Other effector molecules include proteins, peptides, and enzymes. Enzymes of interest include, but are not limited to, proteases, hydrolases, lyases, isomerases, and transferases. Proteins, polypeptides, and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin, proteins such as insulin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, or tissue plasminogen activator, thrombotic or anti-angiogenic agents such as angiostatin or endostatin, or biological response modifiers such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), nerve growth factor (NGF), or other growth factors and immunoglobulins.

[0289] Other effector molecules may include, for example, detectable substances useful in diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron-emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. See generally U.S. Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radionuclides include 125I, 131I, 111In, and 99Tc.

[0290] In another example, the effector molecule may increase the half-life of the antibody in vivo and / or reduce the immunogenicity of the antibody and / or enhance delivery of the antibody across an epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin-binding proteins, or albumin-binding compounds such as those described in WO 2005 / 117984.

[0291] When the effector molecule is a polymer, it may generally be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer, or a branched or unbranched polysaccharide, such as a homo- or heteropolysaccharide.

[0292] Particular optional substituents which may be present on the above synthetic polymers include one or more hydroxy, methyl or methoxy groups.

[0293] Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, particularly optionally substituted poly(ethylene glycol), such as methoxypoly(ethylene glycol) or derivatives thereof.

[0294] Specific naturally occurring polymers include lactose, amylose, dextran, glycogen, or derivatives thereof.

[0295] In one embodiment, the polymer is albumin or a fragment thereof, for example, human serum albumin or a fragment thereof.

[0296] The size of the polymer can be varied as desired, but generally ranges from 500 Da to 50,000 Da, e.g., 5,000 to 40,000 Da, e.g., 20,000 to 40,000 Da, in average molecular weight. The polymer size can be selected based, inter alia, on the intended use of the product, e.g., its ability to localize to a specific tissue, such as a tumor, or its ability to extend circulatory half-life (for a review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, if the product is intended to leave the circulation and penetrate tissue, e.g., for use in treating tumors, it may be advantageous to use a low molecular weight polymer, e.g., having a molecular weight of about 5,000 Da. For applications in which the product remains in the circulation, it may be advantageous to use a high molecular weight polymer, e.g., having a molecular weight in the range of 20,000 Da to 40,000 Da.

[0297] Suitable polymers include polyalkylene polymers such as poly(ethylene glycol) or especially methoxypoly(ethylene glycol) or derivatives thereof, especially those having a molecular weight in the range of about 15,000 Da to about 40,000 Da.

[0298] In one example, an antibody according to the present invention is conjugated to a poly(ethylene glycol) (PEG) moiety. In a specific embodiment, an antigen-binding fragment according to the present invention and a PEG molecule can be attached via any available amino acid side chain or terminal amino acid functional group located in the antibody fragment, such as any free amino, imino, thiol, hydroxyl, or carboxyl group. Such amino acids can occur naturally in the antibody fragment or can be engineered into the fragment using recombinant DNA methods (see, e.g., U.S. Pat. No. 5,219,996; U.S. Pat. No. 5,667,425; WO 98 / 25971; WO 2008 / 038024). In one example, an antibody molecule of the present invention is a modified Fab fragment, in which the modification is the addition of one or more amino acids to the C-terminus of its heavy chain to allow attachment of an effector molecule. Suitably, the additional amino acids form a modified hinge region containing one or more cysteine ​​residues to which an effector molecule can be attached. Multiple sites can be used to attach two or more PEG molecules.

[0299] Suitably, the PEG molecule is covalently attached via the thiol group of at least one cysteine ​​residue located in the antibody fragment. Each polymer molecule attached to the modified antibody fragment may be covalently attached to the sulfur atom of a cysteine ​​residue located in the fragment. The covalent bond is generally a disulfide bond, or more particularly, a sulfur-carbon bond. When a thiol group is used as the attachment point, an appropriately activated effector molecule, such as a thiol-selective derivative such as maleimide or cysteine ​​derivative, can be used. Activated polymers can be used as starting materials in the preparation of the above-described polymer-modified antibody fragments. Activated polymers can be any polymer containing a thiol-reactive group, such as an α-halocarboxylic acid or ester, e.g., iodoacetamide, imide, e.g., maleimide, vinyl sulfone, or disulfide. Such starting materials can be commercially obtained (e.g., from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or prepared from commercially available starting materials using conventional chemical procedures. Particular PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

[0300] In one embodiment, the antibody is PEGylated, i.e., a modified Fab fragment, Fab' fragment, or diFab, to which PEG (poly(ethylene glycol)) has been covalently attached, e.g., according to the methods disclosed in EP 0948544 or EP 1090037 (see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York; "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC; and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545). In one example, PEG is bound to a cysteine ​​in the hinge region. In one example, the PEG-modified Fab fragment has a maleimide group covalently bound to a single thiol group in the modified hinge region. A lysine residue may be covalently bound to the maleimide group, and a methoxypoly(ethylene glycol) polymer having a molecular weight of about 20,000 Da may be bound to each amine group on the lysine residue. Thus, the total molecular weight of PEG bound to the Fab fragment may be about 40,000 Da.

[0301] In one embodiment, the antibody is a modified Fab' fragment having at the C-terminus of its heavy chain a modified hinge region containing at least one cysteine ​​residue to which an effector molecule is attached. Suitably, the effector molecule is PEG (attached using the methods described in WO 98 / 25971 and WO 2004072116 or WO 2007 / 003898). Effector molecules can be attached to the antibody fragment using the methods described in International Patent Applications WO 2005 / 003169, WO 2005 / 003170 and WO 2005 / 003171.

[0302] In one embodiment, the antibody is not conjugated to an effector molecule.

[0303] Polynucleotides and Vectors The present invention also provides an isolated polynucleotide encoding an antibody or portion thereof according to the present invention. Isolated polynucleotides according to the present invention may comprise synthetic DNA, produced for example by chemical processing, cDNA, genomic DNA, or any combination thereof.

[0304] [Table 7]

[0305] Examples of suitable sequences are provided herein. Thus, in one embodiment, the invention provides an isolated polynucleotide encoding an antibody comprising the sequence set forth in SEQ ID NOs: 23, 25, 27, 29, 31, 33, 73, 75, 77, 79, 81 and 83.

[0306] In one embodiment, the invention provides an isolated polynucleotide encoding an antibody Fab fragment or a heavy chain of an IgG1 or IgG4P antibody of the invention comprising the sequence shown in SEQ ID NOs: 29, 31, 33, respectively.

[0307] Also provided is an isolated polynucleotide encoding the light chain of an antibody Fab fragment or an IgG1 or IgG4 antibody of the invention comprising the sequence set forth in SEQ ID NOs: 79, 81, 83, respectively.

[0308] In another embodiment, the invention provides isolated polynucleotides encoding the heavy and light chains of a Fab antibody of the invention, wherein the polynucleotide encoding the heavy chain comprises the sequence set forth in SEQ ID NO: 29 or 79, and the polynucleotide encoding the light chain comprises the sequence set forth in SEQ ID NO: 27 or 77.

[0309] The present invention also provides cloning or expression vectors comprising one or more polynucleotides described herein. In one example, a cloning or expression vector according to the present invention comprises one or more isolated polynucleotides comprising a sequence selected from SEQ ID NOs: 23, 25, 27, 29, 31, 33, 73, 75, 77, 79, 81, and 83.

[0310] Standard techniques of molecular biology can be used to prepare DNA sequences encoding the antibodies of the present invention or antigen-binding fragments thereof. The desired DNA sequence can be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques can be used as needed.

[0311] General methods by which vectors can be constructed, transfection methods, and culture methods are well known to those skilled in the art, see, in this regard, "Current Protocols in Molecular Biology", 1999, FMA Usubel (ed), Wiley Interscience, New York, and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0312] Host cells for producing antibodies and antigen-binding fragments thereof Host cells comprising one or more cloning or expression vectors comprising one or more isolated polynucleotide sequences according to the invention, or one or more isolated polynucleotide sequences encoding an antibody of the invention, are also provided. Any suitable host cell / vector system can be used for expression of polynucleotide sequences encoding the antibodies or antigen-binding fragments thereof of the invention. Bacterial, e.g., E. coli, and other microbial systems may be used, or eukaryotic, e.g., mammalian, host cell expression systems may be used. Suitable mammalian host cells include CHO, myeloma, or hybridoma cells.

[0313] In a further embodiment, a host cell comprising such a nucleic acid(s) or vector(s) is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an anti-IL22 antibody and an amino acid sequence comprising the VH of an anti-IL22 antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an anti-IL22 antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an anti-IL22 antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, or Sp20 cell). In one embodiment, the host cell is a prokaryotic cell, such as an Escherichia coli (E. coli) cell. In one embodiment, a method of producing an anti-IL22 antibody is provided, comprising culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0314] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0315] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including "humanized" fungal and yeast strains in which the glycosylation pathway results in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0316] Suitable types of Chinese hamster ovary (CHO cells) for use in the present invention can include CHO and CHO-K1 cells, including dhfr-CHO cells such as CHO-DG44 cells and CHO-DXB11 cells, which can be used with a DHFR selectable marker, or CHOK1-SV cells, which can be used with a glutamine synthetase selectable marker. Other cell types used to express antibodies include lymphoid cell lines, e.g., NS0 myeloma cells and SP2 cells, COS cells. Host cells can be stably transformed or transfected with the isolated polynucleotide sequences or expression vectors of the present invention.

[0317] Antibody manufacturing process The present invention also provides a method for producing an antibody according to the invention, which comprises culturing a host cell according to the invention under conditions suitable for producing an antibody according to the invention, and isolating the antibody.

[0318] An antibody may contain only heavy or light chain polypeptides, in which case only the heavy or light chain polypeptide coding sequence needs to be used to transfect the host cell. To produce an antibody or antigen-binding fragment thereof containing both heavy and light chains, a cell line can be transfected with two vectors: a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector containing sequences encoding both light and heavy chain polypeptides can be used.

[0319] Thus, a process is provided for culturing a host cell, expressing an antibody, isolating the antibody, and optionally purifying the antibody to provide an isolated antibody. In one embodiment, the process further comprises conjugating an effector molecule to the isolated antibody.

[0320] The present invention also provides a method for producing an antibody according to the invention, which comprises culturing a host cell containing a vector of the invention under conditions suitable for resulting in expression of a protein from DNA encoding an antibody molecule of the invention, and isolating the antibody molecule.

[0321] An antibody molecule may contain only heavy or light chain polypeptides, in which case only the heavy or light chain polypeptide coding sequence needs to be used to transfect the host cell. For the production of a product containing both heavy and light chains, a cell line can be transfected with two vectors: a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector containing sequences encoding both light and heavy chain polypeptides can be used.

[0322] The antibodies according to the invention are expressed at good levels from host cells and therefore the properties of the antibodies are likely to be optimized for commercial processing.

[0323] purified antibody In one embodiment, purified antibodies, such as humanized antibodies, particularly antibodies according to the invention, are provided which are substantially purified from endotoxins and / or host cell proteins or DNA, in particular free or substantially free from endotoxins and / or host cell proteins or DNA.

[0324] Substantially free of endotoxin is generally intended to refer to an endotoxin content of 1 EU / mg of antibody product or less, for example 0.5 or 0.1 EU / mg of product.

[0325] Substantially free of host cell proteins or DNA is generally intended to refer to a host cell protein and / or DNA content of 400 μg or less, for example 100 μg / mg or less, in particular 20 μg / mg or less, as appropriate, of host cell protein and / or DNA per mg of antibody product.

[0326] In vitro and ex vivo uses of antibodies The present invention also provides an in vitro or ex vivo method for inhibiting IL-22-induced STAT3 phosphorylation, comprising contacting and incubating keratinocyte cells with an antibody of the present invention. Any keratinocyte cells and their derivatives can be used, including, for example, HaCaT cells.

[0327] The present invention further provides an in vitro or ex vivo method for inhibiting IL-22-induced IL-10 release, the method comprising contacting and incubating epithelial cells with an antibody according to the present invention. More specifically, COLO205 cells can be used.

[0328] An in vitro or ex vivo method of inhibiting IL22-induced s100A7 release is provided, the method comprising contacting and incubating keratinocytes with an antibody according to the invention.

[0329] Also provided is an in vitro or ex vivo method for inhibiting IL22-induced epidermal or corneal thickening associated with abnormal keratinocyte differentiation and parakeratosis, comprising contacting and incubating a reconstituted epithelium consisting of keratinocytes and dermal fibroblasts with an antibody according to the invention, wherein, in particular, abnormal keratinocyte proliferation and differentiation, as evidenced by IL22-induced epidermal / corneal thickening and parakeratosis, are inhibited.

[0330] The cells are generally incubated for a period of time sufficient for the antibody to bind to the IL22 and elicit a biological effect.

[0331] The examples provide a description of methods involving anti-IL22 antibodies that can be used to achieve specific biological effects.

[0332] Therapeutic Uses of Antibodies The antibodies, formulations or pharmaceutical compositions thereof of the invention can be administered for prophylactic and / or therapeutic treatments.

[0333] The present invention provides the anti-IL22 antibody of the present invention or a pharmaceutical composition thereof for use as a pharmaceutical.

[0334] In prophylactic applications, antibodies, formulations or compositions are administered to a subject at risk of a disorder or condition as described herein in an amount sufficient to prevent or lessen the subsequent effects of the condition or one or more of its symptoms.

[0335] In therapeutic applications, antibodies are administered to a subject already suffering from a disorder or condition described herein in an amount sufficient to cure, alleviate, or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of disease symptoms, or an increase in the frequency or duration of symptom-free periods.

[0336] The subjects to be treated may be animals. Preferably, the pharmaceutical compositions according to the invention are adapted for administration to human subjects.

[0337] The invention provides a method of treating a disorder or condition described herein in a subject in need thereof, the method comprising administering to the subject an antibody according to the invention, wherein such antibody is administered in a therapeutically effective amount.

[0338] The present invention also provides an antibody of the present invention for use in the treatment of a disorder or condition as described herein.

[0339] Treatment indications The antibodies of the invention may be used in the treatment, prevention, or amelioration of any condition associated with IL22 or IL22R1 activity, for example, any condition that results in whole or in part from signaling through the IL22R1 receptor.

[0340] High levels of IL22 have been found in human psoriatic plaques (Boniface et al., Clin Exp Immunol. 150:407-415 (2007)), and the involvement of this cytokine in the pathogenesis of psoriasis has been demonstrated in a mouse model of skin inflammation (Van Belle et al. J Immunol. January 1;188(1):462-9 (2012)). Ligands such as IL22 that signal through IL22R1 are involved in many diseases, and because IL22R1 is primarily expressed in epithelial and stromal cells, important diseases are, for example, those affecting the stroma and epithelium of the skin.

[0341] The antibodies and compositions of the invention can be used to treat inflammatory skin conditions. In certain embodiments, the inflammatory skin condition is selected from psoriasis, psoriatic arthritis, contact dermatitis, chronic hand eczema, or atopic dermatitis. Specifically, the inflammatory skin condition is atopic dermatitis.

[0342] In particular, the antibodies and compositions of the invention can be used to inhibit IL22-mediated epidermal thickening associated with abnormal keratinocyte differentiation and parakeratosis in subjects diagnosed with inflammatory skin conditions by reducing abnormal IL22-mediated keratinocyte proliferation and differentiation.

[0343] Accordingly, the present invention provides a method for attenuating skin barrier dysfunction and / or parakeratosis and / or release of cytokines and / or antimicrobial peptides, such as S100A7, in a subject diagnosed with an inflammatory skin condition, the method comprising administering to the subject an antibody provided by the present invention.

[0344] In yet another embodiment, the invention provides an antibody of the invention for use in attenuating skin barrier dysfunction and / or parakeratosis, and / or release of cytokines and / or antimicrobial peptides, such as S100A7, in a subject diagnosed with an inflammatory skin condition.

[0345] In yet another embodiment, the invention provides the use of an antibody of the invention for the manufacture of a medicament for attenuating skin barrier dysfunction and / or parakeratosis and / or release of cytokines and / or antimicrobial peptides, such as S100A7, in a subject diagnosed with a skin inflammatory disease.

[0346] In particular, such attenuation of the impaired barrier function of the skin is achieved by reducing aberrant IL-22-mediated keratinocyte proliferation and differentiation.

[0347] The antibodies and compositions of the invention can be used to directly or indirectly inhibit the proliferation, differentiation, and / or survival of immune cells or hematopoietic cells (e.g., myeloid, lymphoid, or erythroid cells, or their precursor cells, etc.).

[0348] The antibodies and compositions of the invention can be used to treat a variety of immune and hyperproliferative disorders.

[0349] Examples of immune disorders that can be treated include autoimmune disorders such as arthritis (including rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus-related arthritis, or ankylosing spondylitis), scleroderma, systemic lupus erythematosus, HIV, Sjogren's syndrome, vasculitis, multiple sclerosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, colitis, diabetes mellitus (Type 1); disorders of the skin (e.g., psoriasis), the cardiovascular system (e.g., atherosclerosis), and the immune system (e.g., vasculitis). These conditions include, but are not limited to, inflammatory conditions of the nervous system (e.g., Alzheimer's disease), liver (e.g., hepatitis), kidney (e.g., nephritis), and pancreas (e.g., pancreatitis); cardiovascular disorders, e.g., cholesterol metabolism disorders, oxygen free radical damage, ischemia; disorders associated with wound healing; respiratory disorders, e.g., asthma and COPD (e.g., cystic fibrosis); acute inflammatory conditions (e.g., endotoxemia, sepsis and septicaemia, toxic shock syndrome, and infectious diseases); transplant rejection, and allergies.

[0350] The antibodies and compositions of the invention can be used to treat IL-22-associated disorders, such as arthritic disorders, e.g., rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or ankylosing spondylitis; respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD)); cardiovascular system (e.g., atherosclerosis), nervous system (e.g., Alzheimer's disease), liver (e.g., hepatitis), kidney (e.g., nephritis), pancreas (e.g., pancreatitis), and gastrointestinal tract, e.g., colitis, Crohn's disease, and IBD; acute inflammatory conditions, e.g., endotoxemia, sepsis, toxic shock syndrome, and infections; multiple organ failure; respiratory diseases (ARDs); amyloidosis; nephropathies, e.g., glomerulosclerosis, membranous neuropathy, renal arteriosclerosis, glomerulonephritis, fibroproliferative diseases of the kidney, and other kidney failures and kidney tumors.

[0351] The anti-IL-22 antibodies and compositions of the invention can be used to treat epithelial cancers, such as carcinomas, melanomas, etc. IL-22 inhibition in these and other disease states is disclosed in WO 03 / 083062.

[0352] The antibodies and compositions of the invention can similarly be used to treat multiple sclerosis in humans. In mouse models of multiple sclerosis (Tuohy et al. (J. Immunol. (1988) 141:1126-1130), Sobel et al. (J. Immunol. (1984) 132:2393-2401), and Traugott (Cell Immunol. (1989) 119:114-129), treatment of mice with anti-IL22 antibodies can greatly delay the onset of disease.

[0353] The antibodies and compositions of the present invention can be used to treat rheumatoid arthritis (RA) or other arthritic diseases. In RA synovial biopsies, IL22 protein is detected in vimentin+ synovial fibroblasts and some CD68+ macrophages, and IL22R1 is detected in synovial fibroblasts. Inhibitors of IL22 ameliorate the symptoms of rheumatoid arthritis (WO 2005 / 000897; U.S. Pat. No. 6,939,545).

[0354] The antibodies and compositions of the invention can be used to reduce the mixed lymphocyte reaction (MLR) and treat IL-22-dependent transplant rejection and related disorders (e.g., graft-versus-host disease). MLR and transplantation models are described in Current Protocols in Immunology, Second Edition, Coligan et al. eds., John Wiley & Sons, 1994; Kasaian et al. (Immunity (2002) 16:559-569).

[0355] The antibodies and compositions of the invention can also be used to treat hyperproliferative disorders associated with abnormal activity of IL22-responsive cells and IL22R1 / IL10R2-responsive cells by administering the antibodies in an amount sufficient to inhibit or reduce hyperproliferation of IL22- and / or IL22R1- and / or IL10R2-responsive cells in a subject.

[0356] Thus, the antibodies of the present invention may be used to treat neoplasms such as squamous cell carcinoma, basal cell carcinoma, transitional cell papilloma and carcinoma, adenoma, adenocarcinoma, plasma membrane tumor, insulinoma, glucagonoma, gastrinoma, VIPoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, carcinoid tumor of the appendix, prolactinoma, oncocytoma, Hurthle cell adenoma, renal cell carcinoma, Grawitz tumor, multiple endocrine adenoma, endometrioid adenoma, appendicular and cutaneous appendicular neoplasms, mucoepidermoid neoplasm, cystic, mucocutaneous neoplasm, mucocutaneous neoplasm, epidermoid neoplasm, cystic, mucocutaneous ... It may be used to inhibit the progression of liquid and serous neoplasms, cystadenoma, pseudomyxoma, peritoneal, ductal, lobular and medullary neoplasms, glandular cell neoplasms, composite epithelial neoplasms, Warthin's tumor, thymoma, specialized stromal neoplasms, gonadal cord tumor, meningioma, granulosa cell tumor, virilizing tumor, Sertoli-Leydig cell tumor, paraganglioma, pheochromocytoma, glomus tumor, pigmented nevus, malignant melanoma, melanoma, nodular melanoma, dysplastic nevus, lentigo maligna, superficial spreading melanoma, or acral lentiginous melanoma.

[0357] The antibodies and compositions of the invention can also be used to reduce the acute phase response in a subject.

[0358] The antibodies and compositions of the invention may also be used to treat Sjogren's syndrome, or a cancer selected from liver cancer, liposarcoma, oral squamous cell carcinoma, colon and colorectal cancer, pancreatic cancer, small cell and large cell lung cancer, breast cancer, glioblastoma, cutaneous T-cell lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma.

[0359] Diagnostic uses of antibodies and antigen-binding fragments thereof The present invention also provides the use of the antibodies of the present invention as diagnostic active agents or in diagnostic assays, for example, for diagnosing inflammatory skin diseases or their severity.

[0360] Diagnosis may preferably be performed on a biological sample. A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in diagnostic or monitoring assays. The definition includes cerebrospinal fluid, blood, such as plasma and serum, and other liquid samples of biological origin, such as urine and saliva, solid tissue samples, such as biopsy specimens or tissue cultures, or cells derived therefrom and their progeny. The definition also includes samples that have been manipulated in some way after their procurement, for example, by treatment with reagents, solubilization, or enrichment for particular components, such as polynucleotides.

[0361] Diagnostic tests may preferably be performed on biological samples that have not come into contact with the human or animal body. Such diagnostic tests are also called in vitro tests. In vitro diagnostic tests may rely on in vitro methods to detect free IL22 (e.g., not bound to IL22BP) in a biological sample obtained from a subject.

[0362] Pharmaceutical and diagnostic compositions The antibodies of the present invention may be provided in pharmaceutical compositions, which are usually sterile and may further comprise pharmaceutically acceptable adjuvants and / or carriers.

[0363] Since the antibodies of the invention are useful in the treatment, diagnosis and / or prevention of disorders or conditions such as those described herein, the present invention also provides pharmaceutical or diagnostic compositions comprising an antibody or antigen-binding fragment thereof according to the invention in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0364] In particular, the antibody or antigen-binding fragment thereof is provided as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, diluents or carriers.

[0365] These compositions may contain, in addition to the therapeutically active ingredient(s), pharmaceutically acceptable excipients, carriers, diluents, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredients.

[0366] Also provided are compositions (including pharmaceutical formulations) comprising anti-IL22 antibodies of the invention or polynucleotides comprising sequences encoding the antibodies of the invention. In certain embodiments, the compositions comprise one or more antibodies of the invention or one or more polynucleotides comprising sequences encoding one or more antibodies of the invention. These compositions may further comprise suitable carriers known in the art, such as adjuvants, including pharmaceutically acceptable excipients and / or buffers.

[0367] Pharmaceutical compositions of the antibodies of the present invention are prepared by mixing such antibodies having the desired purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution.

[0368] Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0369] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0370] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations containing a histidine-acetate buffer.

[0371] The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0372] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0373] Formulations to be used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0374] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations containing a histidine-acetate buffer.

[0375] The pharmaceutical compositions of the invention may include one or more pharmaceutically acceptable salts.

[0376] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, buffered water, and physiological saline. Other examples of carriers include ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and their suitable mixtures, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. In many cases, it is desirable to include isotonic agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0377] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0378] In one embodiment, an antibody of the invention is the sole active ingredient. In another embodiment, an antibody of the invention is combined with one or more additional active ingredients. Alternatively, a pharmaceutical composition comprising an antibody of the invention as the sole active ingredient may be administered separately to a patient in combination (e.g., simultaneously, sequentially, or separately) with other agents, drugs, or hormones.

[0379] The exact nature of the carrier or other material may depend on the route of administration, for example, oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, and intraperitoneal. For example, solid oral forms may contain, together with the active substance, diluents such as lactose, dextrose, saccharose, cellulose, corn starch, or potato starch; lubricants such as silica, talc, stearic acid, magnesium stearate, or calcium stearate, and / or polyethylene glycol; binders such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, or polyvinylpyrrolidone; disaggregating agents such as starch, alginic acid, alginates, or sodium starch glycolate; effervescent mixtures; dyes; sweeteners; wetting agents such as lecithin, polysorbates, lauryl sulfate, and non-toxic and pharmacologically inert substances generally used in pharmaceutical formulations. Such pharmaceutical preparations can be prepared in known manner, for example, by mixing, granulating, tableting, sugar-coating, or film-coating processes.

[0380] Oral formulations contain commonly employed excipients such as, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders and contain 10% to 95%, preferably 25% to 70%, of the active ingredient. If the pharmaceutical composition is lyophilized, the lyophilized material can be reconstituted (e.g., as a suspension) prior to administration. Reconstitution is preferably carried out in a buffer solution.

[0381] The solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile aqueous isotonic saline solutions.

[0382] Preferably, the pharmaceutical or diagnostic composition comprises a humanized antibody according to the invention.

[0383] Determining Therapeutically Effective Amounts and Dosages The antibodies and pharmaceutical compositions of the present invention can be appropriately administered to patients to determine the therapeutically effective amount required. For any antibody, the therapeutically effective amount can be initially estimated either in cell culture assays or animal models, usually rodents, rabbits, dogs, pigs, or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful dosages and routes of administration in humans.

[0384] The precise therapeutically effective amount for a human subject will depend on the severity of the disease state, the subject's general health, the subject's age, weight, and sex, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance / response to treatment. Compositions can be conveniently provided in unit dose forms containing a predetermined amount of an active agent of the present disclosure per dose. Dose ranges and regimens for any of the embodiments described herein include, but are not limited to, doses ranging from 1 mg to 1000 mg unit doses.

[0385] Appropriate dosages of antibodies or pharmaceutical compositions of the present invention can be determined by those skilled in the art. The actual dosage level of the active ingredient in a pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is not toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0386] Suitable dosages may range, for example, from about 0.01 μg / kg to about 1000 mg / kg body weight of the patient being treated, typically from about 0.1 μg / kg to about 100 mg / kg body weight.

[0387] Dosage regimens can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0388] Administration of pharmaceutical compositions or formulations The antibodies described herein or formulations or compositions thereof can be administered for prophylactic and / or therapeutic treatments.

[0389] An antibody or pharmaceutical composition of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Examples of routes of administration for an antibody or pharmaceutical composition of the present invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, e.g., by injection or infusion. Alternatively, an antibody or pharmaceutical composition of the present invention can be administered via a parenteral route, such as a topical, epidermal, or mucosal route of administration. An antibody or pharmaceutical composition of the present invention can be for oral administration.

[0390] Forms suitable for administration include those suitable for parenteral administration, for example, by injection or infusion, e.g., by bolus injection or continuous infusion, in intravenous, inhalable, or subcutaneous form. When the product is for injection or infusion, it can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and can contain additional agents such as suspending agents, preservatives, stabilizers, and / or dispersing agents. Alternatively, the antibody or antigen-binding fragment thereof according to the invention can be in a dry form for reconstitution with an appropriate sterile liquid before use. Solid forms suitable for dissolution or suspension in a liquid vehicle before injection can also be prepared.

[0391] Once formulated, the pharmaceutical compositions of the invention can be administered directly to a subject. Accordingly, there is provided herein the use of an antibody or antigen-binding fragment thereof according to the invention for the manufacture of a medicament.

[0392] Products and Kits The present disclosure also provides kits comprising an anti-IL22 antibody of the invention and instructions for use. The kits may further comprise one or more additional reagents, such as additional therapeutic or prophylactic agents discussed above.

[0393] The present invention provides use of an antibody according to the present invention or a pharmaceutical composition thereof for producing a medicament.

[0394] The invention also provides the use of an antibody of the invention for the manufacture of a medicament for the treatment of a disorder or condition as described herein.

[0395] In certain embodiments, the article of manufacture or kit comprises a container containing one or more antibodies of the invention or compositions described herein. In certain embodiments, the article of manufacture or kit comprises a container containing nucleic acid(s) encoding one or more antibodies or compositions described herein. In some embodiments, the kit comprises cells or cell lines that produce the antibodies described herein.

[0396] In certain embodiments, the article of manufacture or kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds the composition, either by itself or in combination with another therapeutically, prophylactically, and / or diagnostically effective composition, and may have a sterile access port. At least one agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat inflammatory skin conditions, more specifically, atopic dermatitis.

[0397] It should be noted that the above embodiments are illustrative rather than limiting of the present invention, and that those skilled in the art can design many alternative embodiments without departing from the scope of the claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the scope of the claims.

[0398] The sequences encompassed by the present invention are shown in Tables 8-10:

[0399] [Table 8]

[0400] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6]

[0401] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5]

[0402] example Example 1. Generation and selection of therapeutic anti-IL22 antibodies 11041 and 11070 Numerous animals across different species (including mice, rats, and rabbits) were immunized with either purified, homemade human IL-22 or commercially available human IL-22 (R&D systems). After 3-5 injections, animals were sacrificed and PBMCs, spleens, bone marrow, and lymph nodes were harvested. Serum was monitored for binding to human and cynomolgus IL-22 by ELISA.

[0403] For 11041, memory B cell cultures were prepared and supernatants were first screened for their ability to bind human and cynomolgus IL22 in a bead-based assay on the TTP Labtech Mirrorball system. This was a homogeneous multiplex assay using biotinylated human IL22 and biotinylated cynomolgus IL22 coated on Sol-R streptavidin beads (TTP Labtech) and goat anti-rabbit Fc-FITC conjugate as revealing agent.

[0404] Approximately 4500 IL22-specific positive hits were identified in the primary mirror ball screen from a total of 12 x (164-400) Plate B culture experiments. Positive supernatants from this assay were then progressed for further characterization by: ELISA to confirm binding to human and cynomolgus monkey IL-22 Progression to an IL22-dependent HACAT phospho-STAT3 HTRF cell assay (CisBio) to identify neutralizing agents, and Profiling in BIAcore to estimate off-rates and characterize the mode of action of neutralization.

[0405] Neutralization was classified into either bin 1 or bin 2. Bin 1 represents antibodies that bind to human IL22 and prevent IL22R1 binding. Bin 2 represents antibodies that bind to human IL22 but allow IL22R1 binding. We selected antibodies that acted through bin 1. Wells demonstrating neutralization in the phospho-STAT3 HTRF assay and / or with desirable BIAcore profiles were advanced for V-region recovery using fluorescence focus.

[0406] Bone marrow-derived plasma cells were also directly screened for their ability to bind human IL-22 using a fluorescent focusing method (related to 11070). Here, B cells secreting IL-22-specific antibodies were collected on biotinylated human IL-22 immobilized on streptavidin beads using a goat anti-rat Fc-FITC conjugate revealing reagent. Approximately 300 direct foci were selected.

[0407] Following reverse transcription (RT) and PCR of sorted cells, "transcriptionally activated PCR" (TAP) products encoding the antibody V regions were generated and used to transiently transfect HEK-293 cells. The resulting TAP supernatants containing recombinant antibodies bind to human IL-22 and cynomolgus IL-22, block IL-22R1 binding in BIAcore, and neutralize IL-22 in a HACAT phospho-STAT3 HTRF cell assay.

[0408] Heavy and light chain variable region gene pairs from interesting TAP products were then cloned as rabbit or mouse Fab antibodies and re-expressed in a HEK-293 transient expression system. A total of 131 V regions were cloned and sequenced. The recombinant cloned antibodies were then retested for their ability to bind human and cynomolgus monkey IL22, block IL22R1 binding in BIAcore, and neutralize IL22-dependent IL-10 release in the COLO205 IL-10 HTRF cell-based assay (CisBio). After this characterization, two antibodies met the criteria: rabbit-derived 11041 and rat-derived 11070.

[0409] Based on neutralization potency, affinity for both human and cynomolgus IL22, donor content in the humanized graft (see below) and expression data, rabbit-derived 11041 was selected for further progression.

[0410] Example 2. Binding of rabbit 11041 to human, cynomolgus monkey, and mouse IL22 The affinity of purified 11041 rabbit Fab for human, cynomolgus monkey, and mouse IL22 was assessed using a Biacore T200 instrument (GE Healthcare) by capturing rabbit 11041 Fab on immobilized anti-rabbit IgG F(ab')2 followed by titration of IL22 from each species. Affinipure goat anti-rabbit IgG-F(ab')2 fragment-specific (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip via amine coupling chemistry to a capture level of approximately 5000 response units (RU). HBS-EP+ buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, GE Healthcare) was used as the running buffer at a flow rate of 10 μL / min. A 10 μL injection of 0.5 μg / mL 11041 Fab was used for capture by the immobilized goat anti-rabbit Fab. Human IL22, CynoIL22, and murine IL22 were titrated against captured 11041Fab (at 0, 0.6, 1.8, 5.5, 16.6, and 50 nM) at a flow rate of 30 μL / min to assess affinity. Blockade of human IL22R1 was assessed by injecting 100 nM IL-22 (30 μL / min for 180 seconds) followed by human IL22R1 (50 nM for 180 seconds).

[0411] Surfaces were generated with 2 x 10 μL injections of 50 mM HCl and interspersed with 10 μL injections of 5 mM NaOH at a flow rate of 10 μL / min. Background-subtracted binding curves were analyzed using Biacore T200 evaluation software according to standard procedures. Kinetic parameters were determined from a fitting algorithm. Kinetic parameters for the binding of purified 11041 to human, cynomolgus monkey, and mouse IL22 are shown in Table 11.

[0412] Table 11. Kinetic parameters of rabbit 11041 binding to human, cynomolgus monkey, and mouse IL22 [Table 11]

[0413] Example 3. Humanization of 11041 Antibody 11041 was humanized by grafting CDRs from rabbit V regions onto a human germline antibody V region framework. To restore antibody activity, many framework residues from the rabbit V region were also retained in the humanized sequence. These residues were selected using the protocol outlined by Adair et al. (1991) (WO 91 / 09967). Alignments of the rabbit antibody (donor) V region sequence with the human germline (receptor) V region sequence, along with the designed humanized sequence, are shown in Figures 1 and 2. The CDRs grafted from the donor to the recipient sequence are as defined by Kabat (Kabat et al., 1987), except for CDR-H1, for which the combined Chothia / Kabat definition is used (see Adair et al., WO 91 / 09967).

[0414] The human V region IGKV1D-13+IGKJ4J region (IMGT, www.imgt.org / ) was selected as the recipient for the antibody 11041 light chain CDRs. All light chain framework residues in the humanized graft variants are derived from human germline genes, except for the absence, one, or two residues from the group containing residues 2 and 3 (see SEQ ID NO: 38 gL1), where donor residues valine (V2) and valine (V3), respectively, are retained. In some humanized graft variants, the unpaired / free cysteine ​​residue at position 91 of CDRL3 was removed by mutation to either valine (C91V) or serine (C91S); free cysteine ​​residues can undergo post-translational modifications, such as cysteinylation, which can contribute to covalent aggregation and reduced stability. Mutation of this residue resulted in an unexpected 15- to 50-fold increase in binding affinity, as measured by surface plasmon resonance (Table 12, gL1gH1 (642 pM) compared to gL1(C91V)gH1 (41.9 pM) or gL1(C91S)gH1 (12.4 pM), respectively). In several humanized graft variants, potential asparagine deamidation sites in CDRL3 were modified by substituting the asparagine residue at position 95 with aspartic acid (N95D) or the serine residue at position 96 with alanine (S96A). Modification of the deamidation site with the S96A mutation significantly reduced the basal level of deamidation.

[0415] The human V region IGHV3-66+IGHJ4J region (IMGT, www.imgt.org / ) was selected as the receptor for the heavy chain CDR of antibody 11041. Like many rabbit antibodies, the VH gene of antibody 11041 is shorter than the selected human receptor. When aligned with the human receptor sequence, framework 1 of the VH region of antibody 11041 lacks the N-terminal residues that are retained in the humanized antibody (Figure 2). Framework 3 of the 11041 rabbit VH region also lacks two residues (75 and 76, with reference to SEQ ID NO: 49 gH1) in the loop between beta-sheet strands D and E: in the humanized graft variant, the gap is filled with the corresponding residues (lysine 75, K75; asparagine 76, N76) from the selected human receptor sequence (Figure 2). All heavy chain framework residues in the humanized graft variants are derived from human germline genes, except for residues 24, 48, 49, 73, and 78 (see SEQ ID NO: 49 gH1), where donor residues valine (V24), isoleucine (I48), glycine (G49), serine (S73), and valine (V78), respectively, were retained. Retention of donor residues V24, I48, G49, and V78 was essential for maximal affinity binding to human IL-22, as measured by surface plasmon resonance. In some humanized graft variants, potential aspartic acid isomerization sites in CDRH2 were altered by substituting the aspartic acid residue at position 54 with glutamic acid (D54E) or the glycine residue at position 55 with alanine (G55A). In some humanized grafted variants, a potential hydrolysis site in CDRH3 was modified by substituting the aspartic acid residue at position 107 with glutamic acid (D107E).

[0416] [Table 12]

[0417] Example 4. Humanization of 11070 Antibody 11070 was humanized by grafting CDRs from rat V regions onto a human germline antibody V region framework. To restore antibody activity, many framework residues from the rat V region were also retained in the humanized sequence. These residues were selected using the protocol outlined by Adair et al. (1991) (WO 91 / 09967). An alignment of the rat antibody (donor) V region sequence with the human germline (receptor) V region sequence, along with the designed humanized sequence, is shown in Figures 3A and 3B. The CDRs grafted from the donor to the recipient sequence are as defined by Kabat (Kabat et al., 1987), except for CDR-H1, for which the combined Chothia / Kabat definition is used (see Adair et al., WO 91 / 09967).

[0418] The human V region IGKV1-12 + IGKJ2 J region (IMGT, www.imgt.org / ) was selected as the recipient for the antibody 11070 light chain CDR. All light chain framework residues in the humanized grafted variants are derived from human germline genes, except for one or more residues from the group comprising residues 3, 44, 58, and 68 (see SEQ ID NO: 88 gL1), where donor residues valine (V3), asparagine (N44), threonine (T58), and serine (S68), respectively, are retained. Retention of donor residue N44 was essential for maximal affinity binding to human IL-22, as measured by surface plasmon resonance (SPR) (Table 13).

[0419] The human V region IGHV4-31+IGHJ6 J region (IMGT, www.imgt.org / ) was selected as the recipient for the heavy chain CDRs of antibody 11070. All heavy chain framework residues in the humanized graft variants were derived from human germline genes, except for one or more residues from the group including residues 37, 41, 48, 67, 71, 76, and 78 (see SEQ ID NO: 89 gH1), where the donor residues valine (V37), serine (S41), methionine (M48), leucine (L67), arginine (R71), serine (S76), and valine (V78), respectively, were retained. The glutamine residue at position 1 of the human framework was replaced with glutamic acid (E1) to obtain expression and purification of homogeneous products; the conversion of glutamine to pyroglutamate at the N-terminus of antibodies and antibody fragments has been widely reported. Retention of donor residues V37, L67, R71, and V78 was essential for maximal affinity binding to human IL-22 as measured by surface plasmon resonance (Table 13). In some humanized graft variants, a potential asparagine deamidation site in CDRH2 was engineered by substituting the serine residue at position 61 with threonine (S61T).

[0420] [Table 13]

[0421] Example 5. Cloning and generation of mutants Genes encoding different variants of the heavy and light chain V region sequences were designed and constructed using an automated synthesis approach by ATUM (Newark, CA). Additional variants of the heavy and light chain V regions were generated by modifying the VH and VK genes by oligonucleotide-directed mutagenesis, in some cases including mutations within the CDRs. For transient expression in mammalian cells, the humanized light chain V region genes were cloned into the UCB light chain expression vector pMhCK, which contains DNA encoding the human kappa chain constant region (Km3 allotype). The humanized heavy chain V region genes were cloned into the UCB human gamma-1 Fab heavy chain expression vector pMhFabnh, which contains DNA encoding the human gamma-1 CH1 hinge domain. The resulting heavy and light chain vectors were cotransfected into Expi293™ suspension cells, resulting in the expression of humanized recombinant antibodies in the human Fab format. The variant humanized Fab antibodies were evaluated for their binding affinity to human IL-22 compared to the parent antibody, their potency in in vitro assays, their biophysical properties and suitability for downstream processing.

[0422] Example 6. Binding characteristics of humanized 11041 antibody Humanized samples for 11,041 antibodies were tested by capturing samples on immobilized anti-human IgG-F(ab')2 and then titrating human IL-22 on the captured surface. Assays were performed on a Biacore 8K instrument (GE Healthcare), and BIA (Biomolecular Interaction Analysis) was performed using Biacore 8000 evaluation software. Affinpure goat anti-human IgG-F(ab')2 fragment-specific (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip via amine coupling chemistry to a capture level of approximately 5,000 response units (RU). HBS-EP+ buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, GE Healthcare) was used as the running buffer at a flow rate of 10 μL / min. For capture with immobilized goat anti-human Fab IgG, a 10 μL injection of a 0.5 μg / mL humanized sample of 11041 antibody was used. Human IL22 (50 nM, 16.7 nM, 5.6 nM, 1.9 nM, and 617 pM) was titrated across the captured 11041 antibody at a flow rate of 30 μL / min.

[0423] Surfaces were generated by two 10 μL injections of 50 mM HCl and interspersed with 5 μL injections of 5 mM NaOH at a flow rate of 10 μL / min. Background-subtracted binding curves were analyzed using Insight evaluation software according to standard procedures. Kinetic parameters were determined from a fitting algorithm. IL22 affinity determined from a single experiment is shown in Table 14 and was shown to be less than 100 pM.

[0424] [Table 14]

[0425] Example 7.11041 Blockade of IL22 / IL22R1 interaction by Fab Cross-blocking assays were performed on a Biacore4000 (GE Healthcare) to determine whether the 11041 rabbit antibody binds to the same binding site as IL22R1.

[0426] A CM5 sensor chip was prepared by activation with a 7-minute injection (10 μL min-1) of a mixture of EDC / NHS (GE Healthcare), followed by a 7-minute injection of rabbit Fab-specific goat Fab'2 (Jackson Immuno Research) at 50 μg / ml in acetate buffer pH 5.0 (GE Healthcare) to achieve an immobilization level of approximately 5500 RU. Finally, a 7-minute injection (10 μL min-1) of 1 M ethanolamine hydrochloride-NaOH (pH 8.5) was performed to inactivate the surface. A reference surface was prepared as above, omitting the rabbit Fc-specific capture antibody.

[0427] Cross-competition was performed in HBS-EP+ buffer (GE Healthcare) at 25°C. Each analytical cycle involved the injection of the variable region formatted as rabbit Fab, followed by the injection of human IL-22 at 100 nM, and finally the injection of IL-22R1 at 50 nM. Binding responses were calculated after subtracting buffer blanks and no capture control samples. A positive response to IL-22R1 would indicate that the 11041 antibody binds to a different epitope than IL-22R1. A lack of response to IL-22R1 could indicate that the binding site of the 11041 antibody on IL-22 overlaps with the IL-22R1 binding site. After each cycle, the surface was regenerated by injecting the following: 50 mM HCl for 60 seconds, 5 mM NaOH for 60 seconds, and HCl for 60 seconds, all at 10 μL / min.

[0428] As shown in Table 15, a clear binding response was observed when human IL-22 was injected into 11041, but no response was seen with IL22R1 injection, demonstrating that the 11041 antibody and IL-22R1 have overlapping binding sites.

[0429] [Table 15]

[0430] Example 8. Evaluation of blockade of the IL22BP binding site on IL22 Surface plasmon resonance (Biacore T200) was used to assess whether 11041gL13gH14Fab (as part of a bispecific antibody) or fezakinumab could block the IL22BP binding site on IL22.

[0431] A goat anti-human IgG Fab specific antibody (Jackson ImmunoResearch) was immobilized to a level of approximately 6000 RU on a CM5 Sensorchip via amine coupling chemistry.

[0432] Each analytical cycle consisted of capturing 11041gL13gH14Fab or Fezakinumab molecules on the anti-Fab surface, followed by an injection of IL22 (prepared in-house) at 20 nM, followed by an injection of IL22BP at 100 nM, each injected at 30 μL / min for 180 s. At the end of each cycle, the surface was regenerated using a 60 s injection of 50 mM HCl, followed by a 30 s injection of 5 mM NaOH, and a final 60 s injection of 50 mM HCl at a flow rate of 10 μL / min. Background binding and drift were subtracted using a control cycle consisting of buffer capture or buffer analyte injection.

[0433] [Table 16]

[0434] When IL22 bound to surface-captured 11041gL13gH14 Fab, IL22BP was unable to bind to IL22. When IL22 bound to surface-captured fezakinumab, IL22BP was still able to bind to IL22. In conclusion, 11041gL13gH14 Fab (as part of a bispecific antibody) blocks the IL22BP binding site on IL22, but fezakinumab does not.

[0435] Example 9. Purification of IL22. A His-tagged version of IL-22 was purified largely as described by Nagem et al. [Nagem et al. Structure. 2002 Aug;10(8):1051-62.] The BL21(DE3) Escherichia coli (E. coli) strain was transformed by heat shock with an expression construct encoding His-tagged IL-22.

[0436] The encoded protein sequence is: MGSSHHHHHHSSGENLYFQGSQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI (SEQ ID NO: 3)

[0437] IL-22 protein sequence after TEV cleavage (see below): GSQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI (SEQ ID NO: 4)

[0438] Cells were grown in the presence of 100 μg / ml ampicillin, and protein expression was induced by adding IPTG to a concentration of 1 mM when the cells reached an optical density of 1 (measured at 600 nM). After 4 hours, cells were harvested by centrifugation. After cell lysis using a high-pressure cell homogenizer, inclusion bodies containing IL-22 were collected by high-speed centrifugation. The inclusion bodies were washed with 50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.5% (w / v) DOC (pH 8), and then washed again with the same buffer without detergent. The washed inclusion bodies were solubilized overnight at 4°C in a buffer containing 50 mM MES, 10 mM EDTA, 1 mM DTT, and 8 M urea. Insoluble material was separated by centrifugation, and IL-22 in the soluble fraction was refolded by dilution to 0.1 mg / ml in 100 mM Tris-HCl, 2 mM EDTA, 0.5 M arginine, 1 mM reduced glutathione, and 0.1 mM oxidized glutathione, with a final pH of 8.0. After incubation at 4°C for 72 hours, the protein was concentrated and purified by size-exclusion chromatography on a HiLoad26 / 600 Superdex 75 pg column equilibrated with 25 mM MES pH 5.4 and 150 mM NaCl. The protein was then frozen at -80°C until further use.

[0439] The His tag was removed by incubating IL-22 protein with TEV protease overnight at 4° C. After diluting the protein in PBS containing 25 mM imidazole, the cleaved protein was passed through a 5 ml HisTrap™ High Performance column (GE Healthcare) and collected in the flow-through.

[0440] Example 10. HDX-MS of IL22 in the presence of 11041gL13gH14 Fab and 11070gL7gH16 Fab Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used for epitope mapping of IL22 to 11041gL13gH14 Fab and 11070gL7gH16 Fab.

[0441] Sample preparation and data acquisition For HDX-MS analysis, 30 μM IL22 (prepared as described in Example 9) complexed with 90 μM of either 11041gL13gH14 Fab or 11070gL7gH16 Fab was prepared and incubated for 1 h at 4°C. 4 μl of IL22, IL22 / 11041gL13gH14 Fab or IL22 / 11070gL7gH16 The Fab complex was diluted to 57 μL of 10 mM phosphate in H2O (pH 7.0) or 10 mM phosphate in D2O (pH 7.0) at 25 °C. The deuterated samples were then incubated at 25 °C for 0.5, 2, 15, and 60 minutes. After the reaction, all samples were quenched by mixing 1:1 with quench buffer (4 M guanidine hydrochloride, 250 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 100 mM phosphate) at 1 °C. The mixed solution had a final pH of 2.5. The mixture was then loaded onto a nanoAcquity HDX module (Waters) for digestion. The peptides were immediately injected into a column (Waters, Inc.). Peptide digestion was then performed online using an Enzymatic online digestion column (Waters) in 0.2% formic acid in water at 20°C and a flow rate of 100 μL / min. All deuterated time points and undeuterated controls were performed in triplicate, with blank runs between each data point.

[0442] Peptide fragments were then captured using an Acquity BEH C18 1.7 μM VANGUARD chilled precolumn for 3 min. Peptides were then eluted onto a chilled Acquity UPLC BEH C18 1.7 µM 1.0 × 100 column using the following gradient: 0 min, 5% B; 6 min, 35% B; 7 min, 40% B; 8 min, 95% B; 11 min, 5% B; 12 min, 95% B; 13 min, 5% B; 14 min, 95% B; 15 min, 5% B (A: 0.2% HCOOH in HO; B: 0.2% HCOOH in acetonitrile). Peptide fragments were ionized by positive electrospray into a Synapt G2-Si mass spectrometer (Waters). Data acquisition was performed in ToF-only mode over an m / z range of 50–2000 Th using MSe (low collision energy, 4 V; high collision energy: ramp from 18 V to 40 V). The Glu-1-fibrinopeptide B peptide was used for internal lock mass correction.

[0443] HDX-MS data processing MSE data from undeuterated control samples of IL22, IL22 / 11041gL13gH14 Fab, or IL22 / 11070gL7gH16 Fab complexes were used for sequence identification using Waters Protein Lynx Global Server 2.5.1 (PLGS). Peptide searches were performed against the IL22 sequence-only database, using a precursor intensity threshold of 500 counts and three matched product ions required for assignment. Ion accounting files from the three control samples were combined into a peptide list imported into Dynamx v3.0 software.

[0444] Peptides were further filtered using DynamX. The filtering parameters used were a minimum and maximum peptide sequence length of 4 and 25, respectively, a minimum intensity of 1000, a minimum MS / MS product of 2, a minimum product per amino acid of 0.2, and a maximum MH+ error threshold of 10 ppm. DynamX v3.0 was used to quantify the isotopic envelope resulting from deuterium incorporation for each peptide at each time point. Furthermore, all spectra were visually inspected to ensure accurate assignment of m / z peaks, and only peptides with high signal-to-noise ratios were used for HDX-MS analysis.

[0445] After manual filtering in Dynamx, statistical analysis and filtering were performed using Deuteros (www.academic.oup.com / bioinformatics / article / 35 / 17 / 3171 / 5288775), which uses the statistical analysis published by Houde et al., 2011 (www.ncbi.nlm.nih.gov / pubmed / 21491437). Deuteration generates a Wood plot displaying peptide length, start and end residues, global coverage, and the y-axis metric, which is absolute uptake (in Daltons). This is the difference in uptake in the presence of the ligand (bound) and the apo form. The Wood plot first applies confidence filtering to all peptides at each time point. Peptides with deuteration differences outside the selected confidence limits are not significant and are shown in light gray. Significant peptides are shown in dark gray and black. An in-house algorithm was used to filter the results and identify epitopes. Data presented are after 0.5 min of deuteration incubation.

[0446] IL22 coverage map HDX analysis of IL22, containing 11041gL13gH14Fab and 11070gL7gH16Fab, was performed in a single experiment. 47 peptides yielded a total coverage of 91.3% for the HDX-MS experiment. After filtering and analysis, the peptide redundancy was 3.48 (Figure 4).

[0447] HDX-MS of IL22 in the presence of 11041gL13gH14 Fab Seven peptides (i.e., potential epitopes) were observed to exhibit a statistically significant decrease in deuterium uptake upon antibody binding, six of which were consistent with the SPEED analysis (Figure 5A, highlighted in black on the Wood plot): 72VRLIGEKLFHGVS84, 72VRLIGEKLFHGVSM85, 75IGEKLFHGVS84, 75IGEKLFHGVSM85, 76GEKLFHGVS84, and 80FHGVSM85. Increased deuterium uptake (i.e., potential conformational changes) was observed in three peptides: 101EEVLFPQSDRF111, 103VLFPQSDRFQPYM115, and 103VLFPQSDRFQPYMQE117. The 11041gL13gH14 Fab epitope is projected onto the structure of IL22 (Figure 5B). Other regions that are protected or deprotected upon antibody binding due to conformational changes are shown in Figure 5B and highlighted in dark grey.

[0448] In conclusion, the conserved region representing the epitope region of 11041gL13gH14 Fab is residues 72, -85 (VRLIGEKLFHGVSM).

[0449] HDX-MS of IL22 in the presence of 11070gL7gH16 Fab Four peptides (i.e., potential epitopes) were observed to show a statistically significant decrease in deuterium uptake upon antibody binding, three of which were consistent with the SPEED analysis (Figure 6A, highlighted in black on the Wood plot): 72VRLIGEKLFHGVSM85, 75IGEKLFHGVSM85, and 80FHGVSM85. Increased deuterium uptake (i.e., potential conformational changes) was observed in two peptides: 43DKSNFQQPYITNRTFM58 and 105FPQSDRFQPYMQE117. The 11070gL7gH16 Fab epitope is projected onto the structure of IL22 (Figure 6B). Other regions protected or deprotected upon antibody binding due to conformational changes are shown in Figure 6B and highlighted in dark gray.

[0450] In conclusion, the conserved region representing the epitope region of 11070gL7gH16Fab is residues 72, -85 (VRLIGEKLFHGVSM).

[0451] [Table 17]

[0452] Example 11. Purification and structural analysis of the IL-22 / 11041gL13gH14 complex IL-22 was purified as described in Example 9.

[0453] Cleaved IL-22 was mixed with 11041gL13gH14 Fab and purified by size exclusion chromatography on a HiLoad® 26 / 600 Superdex® 75pg column (GE Healthcare) equilibrated with 10 mM Tris pH 7.4 and 150 mM NaCl.

[0454] The IL-22 / 11041gL13gH14 Fab complex was concentrated to 10.1 mg / ml. Crystallization conditions for the complex were identified using several commercially available crystallization screens. These were performed in a sitting-drop format using Swissci 96-well, two-droplet MRC crystallization plates (supplied by Molecular Dimensions, catalog number MD11-00-100). First, reservoirs were filled with 75 μL of each crystallization condition in the screen using a Microlab STAR liquid handling system (Hamilton). Then, 300 nL of IL-22 / Fab complex and 300 nL of reservoir solution were dispensed into the wells of the crystallization plate using a Mosquito liquid handler (TTP LabTech). Initial crystallization conditions were identified as condition 59 of the Nextal Tubes JCSG+ screen (Qiagen catalog number: 130720), containing 0.16 M calcium acetate hexahydrate, 0.08 M sodium cacodylate pH 6.5, 14.4% PEG 8000, and 20% glycerol. This condition is further referred to as JCSG+59. Optimized crystals were obtained by adding 0.01 M yttrium(III) chloride hexahydrate, included in the supplementation screen (Hampton Research catalog number HR2-138), to JCSG+59, supplied by Molecular Dimensions (catalog number MDSR-37-E11). Optimized crystals were grown in MRC Maxi 48-well Crystallization Plates (Swissci) using a reservoir volume of 250 μL and droplets consisting of 2 μL of reservoir solution mixed with 2 μL of IL-22 / Fab complex. Prior to flash freezing in liquid nitrogen, the crystals were transferred to a 4 μL drop of cryoprotectant solution, prepared by mixing 40 μL of the optimized reservoir solution with 10 μL of CryoMixes™ 7 solution included in the CryoProtX™ kit (Molecular Dimensions MD1-61).CryoMixes™ 7 contains 12.5% ​​v / v diethylene glycol, 12.5% ​​v / v ethylene glycol, 25% v / v 1,2-propanediol, 12.5% ​​v / v dimethyl sulfoxide and 12.5% ​​v / v glycerol.

[0455] Diffraction data were collected on beamline I04 (Diamond Light Source, UK). Data were indexed and integrated using XDS [Kabsch, W. Acta Cryst. D 66, 125-132 (2010)] and subsequently scaled using [Evans et al. Acta Crystallogr D Biol Crystallogr. 2013; 69 (Pt 7): 1204-1214]. The IL-22 / Fab structure was solved by molecular replacement using Phaser [McCoy et al. J. Appl. Cryst. (2007). 40, 658-674] in the Phenix software suite [Adams et al. Methods. 2011; 55 (1): 94-106]. In this procedure, the IL-22 structure 1YKB [Xu et al. Acta Crystallogr D Biol Crystallogr. 2005 Jul;61(Pt 7):942-50] and Fab structure 5BVJ [Rondeau et al. MAbs. 2015;7(6):1151-60] were used as molecular replacement templates. Coot [P. Emsley et al. (2010). Acta Crystallographica. D 66:486-501] and phenix.refine [PVAfonine et al. Acta Crystallogr D Biol Crystallogr 68,352-67(2012)] were used in the following cycles of manual model completion and refinement. MolProbity [Williams et al. (2018) Protein Science 27:293-315] was used to analyze the quality of the final model.

[0456] The 3IL-22 / 11041gL13gH14 Fab complex is observed in the crystal asymmetric unit.

[0457] Figure 7A shows the interaction of 11041gL13gH14 Fab with IL-22, with a detailed view of the interaction interface (Figure 7B). The epitope on IL-22 recognized by the Fab molecule was determined using NCONT in the CCP4 software suite [Winn MD et al. Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42]. IL-22 amino acid numbering is based on UnitProtKB entry Q9GZX6.

[0458] Within contact distances of less than 4 Å with the Fab molecule, the IL-22 epitope is composed of residues Gln48, Glu77, Phe80, His81, Gly82, Val83, Ser84, Met85, Arg88, Leu169, Met172, Ser173, Arg175, Asn176, and Ile179.

[0459] Within contact distances of less than 5 Å with the Fab molecule, the IL-22 epitope is composed of residues Lys44, Phe47, Gln48, Ile75, Gly76, Glu77, Phe80, His81, Gly82, Val83, Ser84, Met85, Ser86, Arg88, Leu169, Met172, Ser173, Arg175, Asn176, and Ile179.

[0460] [Table 18]

[0461] [Table 19]

[0462] The 11041gL13gH14 Fab molecule prevents the interaction of IL-22 with the IL22R1 receptor because the Fab light chain binds to the same region on IL-22 (Figure 8).

[0463] Example 11. Structural determination of IL-22 in complex with fezakinumab and VR11070 by cryo-EM After structural analysis of the IL22 / 11041gL13gH14 complex, we used cryo-EM techniques to determine the structure of IL22 in complex with fezakinumab and 11070gL7gH16 Fab (VR11070).

[0464] IL-22 was expressed using Expi293 cells fused to an N-terminal human Fc tag. After centrifugation to remove cells, the supernatant was loaded onto a 5 ml HiTrap Protein A column (Cytiva). The protein was eluted with a buffer gradient from PBS to 0.1 M sodium citrate at pH 2.0. The hFc tag was cleaved using TEV protease, and IL-22 was separated from the cleaved tag by a separate passage by gravity flow over 4 ml of packed Protein A resin. After elution from the resin, IL-22 was further purified on a HiLoad 26 / 600 Superdex 75 pg column (Cytiva) equilibrated in PBS.

[0465] Seventy microliters of VR11070 Fab at 12.1 mg / ml, 153 microliters of fezakinumab Fab at 11.5 mg / ml, and 153 microliters of IL-22 at 1.36 mg / ml were mixed. 55 microliters was injected onto a Superdex 200 5 / 150 column equilibrated in 10 mM Hepes pH 7.4 and 150 mM NaCl. Fractions containing 1.7 mg / ml of the IL-22 + VR11070 + fezakinumab complex were collected and used to prepare cryo-EM grids.

[0466] Quantifoil® R1.2 / 1.3 holey carbon grids (SPT Labtech) were glow-discharged for 45 seconds at 22 mA in a Pelco easyGlow™ filter immediately before use. The gel-filtered IL22 with 11070gL7gH16 Fab and fezakinumab Fab was applied to a fresh glow-discharged grid in a Vitrobot Mark IV (Thermo Fisher Scientific) for 2 seconds in a 100% humidity and 4°C chamber. The grid was then blotted onto fresh filter paper at power 7 for 4 seconds and placed in liquid ethane. The grid was initially screened for ice thickness and particle distribution on an in-house Glacios system operated at 200 keV and equipped with a Falcon 3 camera. Data were then collected on a Cambridge Consortium Krios 2 system equipped with a Falcon 4 and operated at an accelerating voltage of 300 keV. The 5700 videos were recorded using EPU software with a defocus range of -1 to -2.5 μm, a pixel size of 0.67 Å, ​​and a resolution of 49.36 e - / Å 2 The final electron flux was automatically collected in counting mode with a 12.2-second exposure, distributed across 42 fractions. All subsequent data analysis was performed with Cryosparc, version 2.15 (Structura Biotechnology Inc.). Videos were aligned using patch motion, contrast transfer function parameters (CTFs) were estimated using the patch CTFs, and particles were initially picked with a blob picker, yielding a total of 5.5 million particles. The selected particles were binned twice into 300-pixel box sizes and subjected to an initial two-dimensional classification, which resulted in the selection of 488,000 particles with distinct features. Five initial models were generated, two of which differed from each other in the glycosylation site of IL22. These two classes were pooled together, for a total of 240,000 particles, and heterogeneous refinement yielded a resolution estimate of 3.4 Å using the gold standard FSC 0.143 criterion.

[0467] The two Fab molecules and the IL22 structure were fitted to cryo-EM densities using UCSF Chimera [Pettersen, et al. J. Comput. Chem. 25(13):1605-1612(2004)]. Further manual model building was performed using Coot [Emsley et al. (2010) Acta Crystallographica. D66:486-501]. Maps were then sharpened using the Autosharpen tool [Terwilliger, (2018) Acta Cryst. D74,545-559] in Phenix [Liebschner et al. Acta Cryst. D75,861-877(2019)], and the model was further refined using the Real-space refinement tool in Phenix [Afonine et al. Acta Cryst. D74,531-544(2018)].

[0468] The epitopes on IL22 recognized by the 11070gL7gH16 Fab and fezakinumab Fab molecules were determined using NCONT in the CCP4 software suite [Winn et al. Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt4):235-42]. The IL22 amino acid numbering below is based on UnitProtKB entry Q9GZX6.

[0469] Within contact distances of less than 4 Å with the 11070gL7gH16 Fab molecule, the IL22 epitope is composed of residues: Glu77, Lys78, His81, Ser84, Met85, Ser86, Arg88, Asn176, Ala177.

[0470] Within a contact distance of less than 5 Å with the 11070gL7gH16 Fab molecule, the IL22 epitope is composed of residues: Ile75, Gly76, Glu77, Lys78, Phe80, His81, Ser84, Met85, Ser86, Arg88, Leu169, Met172, Ser173, Asn176, and Ala177.

[0471] Within contact distances of less than 4 Å with the fezakinumab Fab molecule, the IL22 epitope is composed of residues Gln49, Tyr51, Phe105, Ser108, ​​Asp109, Gln112, Pro113, Tyr114, Gln116, Glu117, Pro120, Ala123, and Arg124.

[0472] At a contact distance of <5 Å with the fezakinumab Fab molecule, the IL22 epitope is composed of residues: Gln49, Pro50, Tyr51, Ile52, Arg55, Phe105, Pro106, Ser108, ​​Asp109, Gln112, Pro113, Tyr114, Gln116, Glu117, Val119, Pro120, Phe121, Ala123, and Arg124.

[0473] Structural analysis reveals that 11070 Fab has a distinct epitope on IL-22 from that of fezakinumab. Furthermore, 11070gL7gH16 Fab (VR11070) has an epitope similar to that of 11041gL13gH14 Fab (VR11041) on IL-22 (Figures 9A and 9B). Similar to 11041gL13gH14 Fab, 11070gL7gH16 Fab blocks IL-22 signaling by preventing its interaction with the IL-22R1 receptor (Figure 10A). In contrast, fezakinumab blocks IL-22 signaling by preventing its interaction with IL-22R2 (Figure 10B).

[0474] Example 12. COLO205 IL-10 release assay The antibodies were tested for activity against human IL-22 in an in vitro cell assay. The COLO205 cell line is a human colorectal cancer epithelial cell line. IL-22 binds to IL-22R1 and IL-10R2 on the cell surface, inducing STAT3 phosphorylation and downstream cytokine release (e.g., IL-10). In this assay, COLO205 cells are stimulated with IL-22 with or without anti-IL-22 antibodies. The resulting IL-10 response is then measured in the cell culture supernatant using a homogeneous time-resolved FRET (HTRF) kit (Cisbio).

[0475] COLO205 cells were seeded at 25,000 cells / well in tissue-culture-treated, flat-bottom 96-well plates. Human IL-22 (30 pM final assay concentration) was preincubated with antibody (3 nM–1.4 pM final assay concentration) for 1 hour at 37°C. The antibody / cytokine complex was then transferred to COLO205 cells and incubated at 37°C, 5% CO2 for 48 hours. Cell-free cell culture supernatants were then collected and stored at -80°C. The cell culture supernatants were thawed on ice, and IL-10 levels were determined by HTRF.

[0476] Samples were run either singly or in duplicate.

[0477] 11041Fab Results The results confirm that 11041 Fab inhibits the IL-22-induced IL-10 response of COLO205 cells in the COLO205 IL-10 release assay. 11041 Fab had an IC50 of 56.78 pM, as determined by the geometric mean of duplicate assays (Table 20). These measurements are considered reliable, as the range of IC50s measured in each replicate of the assay was found to vary by less than three-fold in each case.

[0478] [Table 20]

[0479] 11041 gL13gH14 Fab Results The results confirm that 11041 gL13gH14 Fab inhibits the IL-22-induced IL-10 response of COLO205 cells in the COLO205 IL-10 release assay. 11041 gL13gH14 Fab had an IC50 of 42.7 pM, as determined by the geometric mean of duplicate assays (Table 21). These measurements are considered reliable, as the range of IC50s measured in each replicate of the assay was found to vary by less than three-fold in each case.

[0480] [Table 21]

[0481] Example 13. In vitro human primary keratinocyte cell-based activity of 11041gL13gH14 Fab against human IL22 The antibody molecule 11041gL13gH14 Fab was tested in an in vitro cell assay for the activity of human IL-22 (manufactured in-house). Primary human neonatal epidermal keratinocytes (NHEK) derived from foreskin were ethically sourced from a donor (Promocell, catalog number C-12001), expanded in culture, and used in the assay. NHEK cells respond to IL-22 stimulation by secreting soluble molecules, including S100A7 (psoriasin), which can be detected in the cell supernatant (Figure 11A). S100A7 was used in the assay as a biomarker to assess the activity of 11041gL13gH14 Fab.

[0482] NHEK cells from two donors at passages 2 and 3 were cultured at 10 in Dermal Basal Medium (LGC, cat. no. #ATCC-PCS-200-030) containing Keratinocyte Growth Kit (LGC, cat. no. #ATCC-PCS-200-040) in 48-well plates (Corning, Costar® Clear TC-Treated Plates, cat. no. 3548) pre-coated with extracellular matrix (TheromoFisher, cat. no. R011K). 4Keratinocytes were cultured under standard conditions (37°C, 5% CO2, 100% humidity) until confluence was reached. On day 3, growth medium was aspirated from all wells, and cells were washed with 200 μl of basal dermal medium to remove any dead cells and growth factors. Anti-IL-22 antibody 11041gL13gH14 Fab at concentrations ranging from 50 nM to 0.01 nM (2500-1 ng / ml) was pre-incubated with 100 ng / ml IL-22 in basal dermal medium for 30 minutes at 37°C. Additionally, 50 nM (2500 ng / ml) of fezakinumab (anti-IL-22 antibody, in-house stock, batch number #PB7490) in Fab format was pre-incubated with 100 ng / ml IL-22 in basal dermal medium for 30 minutes at 37°C. 400 μl / well of antibody / cytokine solution was then transferred to the cells. After 48 hours of stimulation, supernatants were collected and S100A7 levels were measured using ELISA (LSBio, Cat. No. LS-F50031).

[0483] The increase in S100A7 was measured after IL22 stimulation (Figure 11A). 50 nM Fezakinumab Fab completely inhibited IL22-induced S100A7 signaling. 50 nM 11041gL13gH14 Fab also showed complete inhibition of S100A7 (Figure 11A). 11041gL13gH14 Fab showed concentration-dependent inhibition of S100A7 (Figure 11B).

[0484] In conclusion, 11041gL13gH14 Fab tested in a human primary keratinocyte assay showed dose-dependent inhibition of an IL-22-dependent biomarker (S100A7).

[0485] Example 14. IL22 phospho-STAT3 method Hacat cells were added to 96-well flat-bottom tissue culture plates at 150,000 cells / well in 100 μl of DMEM + 10% FBS + 2 mM L-glutamine / well and incubated overnight at 37°C and 5% CO2. Anti-IL22 antibody was diluted in mock supernatant medium to a final assay concentration of 18.75 nM, and 60 μl was added to columns 1 and 12 of a 96-well polypropylene V-bottom plate as a minimum signal control. 60 μl of mock supernatant medium was added to columns 2 and 11 of a 96-well polypropylene V-bottom plate as a maximum signal control. Samples were titrated 1:3 into mock supernatant medium, leaving a final volume of 60 μl in columns 3–10 of the 96-well polypropylene V-bottom plate. 30 μl of IL22 solution was added to all wells to obtain a final assay concentration of 30 ng / ml FAC. The plate was preincubated at 37°C for 1 hour. 75 μl of culture medium from the cell culture plate was transferred to the cell plate, leaving 25 μl in the plate. 75 μl of sample titration / control + il22 was transferred to the cell plate. These plates were incubated at 37°C for 30 minutes. The supernatant was removed. The remaining cells were lysed using the Cisbio STAT3 PhosphoY705 kit, and HTRF signals were generated from the lysates in each well. The plates were sealed and incubated overnight at room temperature on a shaker for 18 hours. Well signals were measured using the HTRF protocol on a Synergy Neo2 plate reader. Antibodies 11041 and 11070 demonstrated clear inhibition of IL22-induced STAT3 phosphorylation.

[0486] All references cited herein, including patents, patent applications, articles, textbooks, etc., and the references cited therein, are incorporated herein by reference in their entirety, unless already present. [Sequence List Free Text]

[0487] Sequence Listing 3 <223> His-tagged IL22 Sequence Listings 5-21 <223> CDR sequences Sequence Table 22-23 <223> 11041 gL6 C91S S96A(gL13)V area Sequence Listings 24-25 <223> 11041 gH5 D54E(gH14)V area Sequence Table 26-27 <223> 11041gL13 light chain Sequence Listings 28-29 <223> 11041gH14 Fab heavy chain Sequence Table 30-31 <223> 11041gH14 heavy chain (IgG1) Sequence Table 32-33 <223> 11041 gH5 D54E (gH14) heavy chain (IgG4P) Sequence Table 34-35 <223> Rabbit 11041 VL region Sequence Table 36-37 <223> Rabbit 11041 VH region Sequence Listing 38 <223> 11041 gL1 V area Sequence Listing 39 <223> 11041 gL1 C91S V area Sequence Listing 40 <223> 11041 gL1 C91V V area (gL3) Sequence Listing 41 <223> 11041gL6 V area Sequence Listing 42 <223> 11041gL7 V area Sequence Listing 43 <223> 11041 gL1 N95D V area(gL8) Sequence Listing 44 <223> 11041 gL1 S96A V area (gL9) Sequence Listing 45 <223> 11041 gL1 C91S S96A V area (gL10) Sequence Listing 46 <223> 11041 gL6 C91S V area (gL11) Sequence Listing 47 <223> 11041 gL7 C91S V area (gL12) Sequence Listing 48 <223> 11041 gL7 C91S S96A V area (gL14) Sequence Listing 49 <223> 11041gH1 V area Sequence Listing 50 <223> 11041 gH1 G55A V area(gH2) Sequence Listing 51 <223> 11041 gH1 D54E V area(gH3) Sequence Listing 52 <223> 11041 gH1 D107E V area(gH4) Sequence Listing 53 <223> 11041gH5 V area Sequence Listing 54 <223> 11041gH8 V area Sequence Listing 55 <223> 11041gH9 V area Sequence Listing 56 <223> 11041gH11 V area Sequence Listing 57 <223> 11041gH12 V area Sequence Listing 58 <223> 11041 gH8 D54E V area (gH15) Sequence Listing 59 <223> 11041 gH11 D54E V area (gH17) Sequence Listing 60 <223> 11041 gH12 D54E V area (gH18) Sequence Table 61-62 <223> Human IGKV1D-13 IGKJ4 receptor framework Sequence Table 63-64 <223> Human IGHV3-66 IGHJ4 receptor framework Sequence Listing 71 <223> 11070 CDRH2 (no mutation) Sequence Table 72-73 <223> 11070gL7 V area Sequence List 74-75 <223> 11070gH16 ​​V area Sequence Table 76-77 <223> 11070gL7 light chain Sequence Listing 78-79 <223> 11070gH16 ​​Fab heavy chain Sequence Table 80-81 <223> 11070gH16 ​​IgG1 heavy chain Sequence Table 82-83 <223> 11070gH16 ​​IgG4P heavy chain Sequence Table 84-85 <223> Rat Ab 11070 VL region Sequence Table 86-87 <223> Rat Ab 11070 VH region Sequence Listing 88 <223> 11070gL1 V area Sequence Listing 89 <223> 11070gH1 V area Sequence Listing 90 <223> 11070gH13 V area (gH1 S61T) Sequence Table 91-92 <223> Human IGKV1-12 IGKJ2 receptor framework Sequence List 93-94 <223> Human IGHV4-31 IGHJ6 receptor framework Sequence Listing 95 <223> Peptides 72-84 Sequence Listing 96 <223> IL22 peptides 72-85 Sequence Listing 97 <223> IL22 peptides 75-84 Sequence Listing 98 <223> IL22 peptide 75-85 Sequence Listing 99 <223> IL22 peptides 76-84 Sequence Listing 100 <223> IL22 peptide 80-85 Sequence Listing 101 <223> IL22 peptides 126-139 Sequence Listing 102 <223> IL22 peptides 101-111 Sequence Listing 103 <223> IL22 peptides 103-115 Sequence Listing 104 <223> IL22 peptides 103-117 Sequence Listing 105 <223> IL22 peptides 43-58 Sequence Listing 106 <223> IL22 peptides 105-117

Claims

1. 1. An isolated antibody that binds to interleukin 22 (IL22) and inhibits or attenuates binding of IL22 to IL22 receptor 1 (IL22R1), CDR-L1 comprising SEQ ID NO: 5, CDR-L2 comprising SEQ ID NO: 6, and CDR-L3 comprising SEQ ID NO:7 a light chain variable region comprising CDR-H1 comprising SEQ ID NO: 8, CDR-H2 comprising SEQ ID NO: 9, and CDR-H3 comprising SEQ ID NO: 10 a heavy chain variable region comprising The antibody described above.

2. The antibody of claim 1, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:

22.

3. The antibody of claim 1, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO:

24.

4. 2. The antibody of claim 1, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 22, or a sequence at least 90% identical thereto, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 24, or a sequence at least 90% identical thereto.

5. the antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region comprises the sequence set forth in SEQ ID NO:22, wherein one or more residues at positions 91, 95, and / or 96 are substituted with another amino acid, and the amino acid substitutions are C91S, C91V, N95D, and / or S96A; the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 24, wherein the residue at position 54 is replaced by glutamic acid (E); The antibody described in claim 1.

6. 2. The antibody of claim 1, wherein the framework regions of the light chain variable region and the heavy chain variable region as defined by Kabat have at least 90% identity to SEQ ID NOs: 22 and 24, respectively.

7. The antibody of any one of claims 1 to 6, which is a full-length antibody.

8. The antibody of any one of claims 1 to 6, which is an antibody fragment.

9. The antibody fragment may be Fab, Fab', F(ab') ２ 9. The antibody of claim 8, which is an Fv, dsFv, scFv, or dsscFv.

10. the antibody is a Fab comprising a light chain comprising the sequence set forth in SEQ ID NO: 26 and a heavy chain comprising the sequence set forth in SEQ ID NO: 28; or The antibody is a Fab comprising CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 having the sequences shown in SEQ ID NOs: 5 / 6 / 7 / 8 / 9 / 10, respectively; 10. The antibody of claim 9, wherein the Kabat-defined framework regions and constant domains of the light and heavy chains have at least 90% identity to SEQ ID NOs: 26 and 28, respectively.

11. The antibody is an IgG1 comprising a light chain comprising the sequence set forth in SEQ ID NO: 26 and a heavy chain comprising the sequence set forth in SEQ ID NO: 30, or The antibody is an IgG1 comprising CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3, each of which comprises the sequences shown in SEQ ID NOs: 5 / 6 / 7 / 8 / 9 / 10, 8. The antibody of claim 7, wherein the Kabat-defined framework regions and constant domains of the light and heavy chains have at least 90% identity to SEQ ID NOs: 26 and 30, respectively.

12. an IgG4P comprising a light chain comprising the sequence set forth in SEQ ID NO: 26 and a heavy chain comprising the sequence set forth in SEQ ID NO: 32; or The antibody is an IgG4P comprising CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 having the sequences shown in SEQ ID NOs: 5 / 6 / 7 / 8 / 9 / 10, respectively; 8. The antibody of claim 7, wherein the Kabat-defined framework regions and constant domains of the light and heavy chains have at least 90% identity to SEQ ID NOs: 26 and 32, respectively.

13. An isolated polynucleotide encoding the antibody of any one of claims 1 to 12.

14. An expression vector carrying the polynucleotide of claim 13.

15. A host cell comprising the vector of claim 14.

16. 15. A method for producing an antibody according to any one of claims 1 to 12, comprising culturing a host cell according to claim 14 under conditions that allow the production of said antibody, and recovering the produced antibody.

17. A pharmaceutical composition comprising the antibody of any one of claims 1 to 12 and a pharmaceutically acceptable adjuvant or carrier.

18. 18. A pharmaceutical composition according to claim 17 for use in the treatment of an inflammatory skin condition.

19. 19. The pharmaceutical composition of claim 18, wherein the inflammatory skin condition is psoriasis, psoriatic arthritis, contact dermatitis, chronic hand eczema, or atopic dermatitis.

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