Anti il22 antibody, antibody fragment and immunoconjugate of the same, and use of the same

Abstract

To provide an anti IL-22 antibody that can bind to both a human and a mouse IL-22 at high binding affinity, and can be used in order to cause the same downstream biological effect in a biological system of both a human and a mouse, and a fragment thereof.SOLUTION: Provided are an anti IL-22 antibody or an antigen-biding antibody fragment which binds to both a human IL-22 and a mammal IL-22, and a modified anti IL-22 antibody and an antibody fragment, which include a light chain variable region having an amino acid sequence having at least 90% of sequence identity with one of an amino acid sequence comprising a specific sequence, and a heavy chain variable region having an amino acid sequence having at least 90% of sequence identity with one of an amino acid sequence comprising a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to anti-IL22 antibodies, antibody fragments, variants and immunoconjugates of such antibodies and antibody fragments, and the use of the antibodies, antibody fragments, variants and immunoconjugates in diagnostic and therapeutic methods. [Background technology]

[0002] Interleukin-22 (IL-22), also known as interleukin-10-related T cell-derived inducer factor (IL-TIF), is a glycoprotein. IL-22 is expressed primarily in the thymus, brain, activated T cells and mast cells, lectin-stimulated spleen cells, interleukin-2 / interleukin-12-stimulated NK cells, and in many organs and tissues upon lipopolysaccharide (LPS) stimulation, including the intestine, liver, stomach, kidney, lung, heart, thymus, and spleen. Human IL-22 mRNA is highly expressed in peripheral T cells and T cells activated by anti-CD3 antibody or ConA, making T cells the primary source of human IL-22. Activated T cells are mostly CD4+ cells.

[0003] The IL-22 precursor contains 179 amino acid residues, and the mature IL-22 protein contains 146 amino acid residues. Dumoutier et al. cloned the mouse and human IL-22 genes (Dumoutier, et al., J. Immunol., vol. 164, pp. 1814-1819, 2000) and obtained two patents related to IL-22, U.S. Patent Nos. 6,359,117 and 6,274,710. IL-22 achieves its biological function by binding to the IL-22R1 and IL-10R2 receptors. IL-22R1 is a specific receptor for IL-22 and is expressed in the skin, kidney, digestive system (pancreas, small intestine, liver, large intestine, and colon), and respiratory system (lungs and bronchi).

[0004] IL-22 plays a key role in mucosal immunity, mediating early host defense against bacterial pathogen attack and regression (see Zheng et al., Nat. Med., vol. 14, pp. 282-289, 2008). IL-22 promotes the production of antimicrobial peptides and proinflammatory cytokines from epithelial cells. It also stimulates the proliferation and migration of colonic epithelial cells in the intestinal tract (see Kumar et al., J. Cancer, vol. 4, pp. 57-65, 2013). Upon bacterial infection, IL-22 knockout mice exhibited impaired gastrointestinal epithelial regeneration, high bacterial burden, and increased mortality (Kumar et al., supra). Similarly, influenza virus infection in IL-22 knockout mice resulted in severe weight loss and impaired regeneration of tracheal and bronchial epithelial cells. Thus, IL-22 plays a proinflammatory role in suppressing microbial infection, as well as an anti-inflammatory and protective role in epithelial regeneration during inflammatory responses.

[0005] In addition to its role in inflammatory responses, IL-22 has also been implicated in cancer (Harrison, "IL-22: linking inflammation and cancer," Nature Reviews Drug Discovery, vol. 12, pp. 504-505, 2013). Specifically, Lim and Savan ("The role of the IL-22 / IL-22R1 axis in cancer," Cytokine Growth Factor Rev., vol. 25, pp. 257-271, 2014) have shown that the IL-22 signaling pathway orchestrates mucosal immune defense and tissue regeneration through pleiotropic effects, including prosurvival signaling, cell migration, metaplasia, and angiogenesis. These functions can be hijacked by aggressive cancers to enhance tumor growth and metastasis. Thus, the role of IL-22 in cancer is complex and context-specific, as evidenced by dysregulated IL-22 expression and signaling in patients with many common cancers, including those of the intestine, skin, lung, and liver.

[0006] Lanfranca et al. ("IL-22 promotes pancreatic cancer tumorigenesis through induction of stemness and epithelial to mesenchymal transition," J Immunol., vol. 198, 1 Supplement, 66.22, 2017) disclosed that IL-22 is essential for the initiation, progression, and establishment of pancreatic cancer. Fukui et al. (British Journal of Cancer, vol. 111, pp. 763-771, 2014) found that IL-22 produced by cancer-associated fibroblasts promotes gastric cancer cell invasion via STAT3 and ERK signaling. Kobold et al. (J Thoracic Oncology, vol. 8, pp. 1032-1042, 2013) found that IL-22 is preferentially expressed in small cell and large cell lung cancer. Enhanced IL-22-R1 expression and signaling in chemotherapy-refractory lung cancer cell lines indicates a pro-tumorigenic function of IL-22 and may contribute to a more aggressive cancer phenotype.

[0007] IL-22 has been reported to treat human pancreatic diseases (see, e.g., U.S. Pat. No. 6,551,799). The use of IL-22 in reducing serum triglycerides and treating obesity has also been reported. See, e.g., WO2006 / 073508 and CN1679918. Anti-IL-22 antibodies have been developed for the treatment of inflammation-related diseases. U.S. Pat. No. 7,901,684 discloses human antibodies and antigen-binding fragments thereof that specifically bind to human IL-22. The antibodies have their V H and V L The antibodies are defined by specific amino acid sequences of the domains and are said to act as antagonists of IL-22 activity, thereby modulating the immune response to treat inflammatory disorders, autoimmune diseases, allergies, septic shock, infectious disorders, transplant rejection, cancer, and other immune system disorders.

[0008] U.S. Patent No. 7,811,567 discloses a method for treating IL-22-associated disorders by administering an antibody or antigen-binding fragment thereof that specifically binds to human IL-22. The antibody has a V domain comprising three complementarity-determining regions (CDRs) with specific amino acid sequences set forth in SEQ ID NOs: 602, 603, and 604. H V domain and three CDRs having the specific amino acid sequences of SEQ ID NOs: 605, 606, and 607. L Has a domain.

[0009] U.S. Patent No. 7,737,259 discloses a composition for the diagnosis and treatment of inflammatory and autoimmune disorders. The composition comprises an antibody that specifically binds to human IL-22. The antibody is (a) an antibody produced by a hybridoma selected from 3F11.3 (ATCC Accession No. PTA-7312), hybridoma 11H4.4 (ATCC Accession No. PTA-7315), and hybridoma 8E11.9 (ATCC Accession No. PTA-7319); (b) an affinity-matured form of the antibody of (a); (c) an antigen-binding fragment of the antibody of (a) or (b); or (d) a humanized form of the antibody of (a), (b), or (c).

[0010] WO 2005 / 000897 discloses isolated antibodies and antigen-binding fragments thereof that specifically bind to IL-22. The antibodies or fragments can reduce the binding of IL-22 to a complex containing IL-22 receptor (IL-22R) and interleukin-10 receptor 2 (IL-10R2). The antibodies or antigen-binding fragments can also reduce the direct interaction between IL-22 and the IL-22 receptor.

[0011] These antibodies were developed to bind to human IL-22 for the treatment of diseases or disorders in humans. However, their binding to non-human mammalian IL-22, such as mouse IL-22, has not been measured or studied. Antibodies intended for use in humans to bind to human IL-22 must first be tested in animals to assess their safety and efficacy. To ensure that animal testing is predictive of safety and efficacy in humans, the antibody should ideally have similar high binding affinity to both human IL-22 and at least the IL-22 of the test animal.

[0012] The present invention provides anti-IL-22 antibodies and fragments thereof that can bind to both human and mouse IL-22 with high binding affinity and can be used to elicit the same downstream biological effects in both human and mouse biological systems.

[0013] (Disclosure Summary) In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a light chain variable region comprising a light chain CDR1 having an amino acid sequence selected from SASSSVSX1MH (SEQ ID NO: 1), a light chain CDR2 having an amino acid sequence selected from X2TX3KLX4S (SEQ ID NO: 2), and a light chain CDR3 having the amino acid sequence of QQWSSNPYIT (SEQ ID NO: 3); and a heavy chain CDR1 having an amino acid sequence selected from GYIFX5SYWIH (SEQ ID NO: 4), a heavy chain CDR2 having an amino acid sequence selected from RIYPGTGX6TYYNX7KFKG (SEQ ID NO: 5), and SYX8X9SVX 10 Y (SEQ ID NO: 6), X1 is Y or K, X2 is E or K, X3 is S or R, X4 is A or L, X5 is T or R, X6 is N or R, X7 is E or R, X8 is D or M, X9 is S or Y, and X 10 is A or G, Anti-IL-22 antibodies or antibody fragments are provided.

[0014] In the above embodiment, the light chain variable region may have an amino acid sequence selected from SEQ ID NOs: 7-12, and the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs: 13-18.

[0015] In any one of the above embodiments, the antibody or antibody fragment may be selected from: an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 10 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 10 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO:8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:17; and An antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18.

[0016] In any one of the above embodiments, the antibody or antibody fragment is capable of binding to human IL-22 and to mammalian non-human IL-22.

[0017] In the above embodiments, the mammalian IL-22 may be murine IL-22.

[0018] In either one of the preceding two embodiments, the antibody or antibody fragment may bind to human IL-22 and mammalian IL-22 with an affinity within ±20%, ±15%, ±10%, or ±5% of each other.

[0019] In any one of the above embodiments, the antibody or antibody fragment is capable of inhibiting phosphorylation of Stat3.

[0020] In any one of the above embodiments, the antibody or antibody fragment is capable of inhibiting IL-22-induced cytokine production.

[0021] In any one of the above embodiments, the antibody or antibody fragment is capable of inhibiting an immune response in at least one animal or human.

[0022] In one previous embodiment, the at least one animal comprises a mammal.

[0023] In either one of the previous two embodiments, the mammal may be a mouse.

[0024] In any one of the above embodiments, the anti-IL-22 antibody or antibody fragment may be humanized.

[0025] In any one of the above embodiments, the anti-IL-22 antibody or antibody fragment may comprise an altered Fc region.

[0026] In another aspect, the invention provides an anti-IL-22 antibody or antibody fragment comprising the anti-IL-22 antibody or antibody fragment of any one of the preceding embodiments and at least one moiety selected from an oligosaccharide, a nonproteinaceous moiety, a therapeutic agent, a prophylactic agent, and a diagnostic agent.

[0027] In one previous embodiment, the at least one moiety can be selected from an oligosaccharide, at least one non-proteinaceous moiety, a therapeutic agent, a prophylactic agent, and a diagnostic agent.

[0028] In one previous embodiment, at least one non-proteinaceous moiety may be selected from a water-soluble polymer.

[0029] In either one of the previous two embodiments, the modified anti-IL-22 antibody or antibody fragment may comprise two agents selected from a therapeutic agent, a prophylactic agent, and a diagnostic agent.

[0030] In any one of the previous three embodiments, the antibody or antibody fragment and the therapeutic, prophylactic, or diagnostic agent may be covalently attached to a linker molecule.

[0031] In another aspect, the invention provides a pharmaceutical composition comprising the antibody or antibody fragment, or modified anti-IL-22 antibody or antibody fragment, of any one of the preceding embodiments and a pharmaceutically acceptable carrier.

[0032] In the previous embodiment, the pharmaceutical composition may further comprise at least one additional excipient.

[0033] In either one of the previous two embodiments, the pharmaceutical composition may further comprise at least one additional therapeutic agent, and / or a pharmaceutically acceptable preservative.

[0034] In another aspect, the invention provides a method of treating an immune-related disease or cancer, comprising administering to a subject the antibody or antibody fragment, modified anti-IL-22 antibody or antibody fragment, or pharmaceutical composition of any one of the preceding embodiments.

[0035] In another aspect, the invention provides a diagnostic or therapeutic kit comprising the antibody or antibody fragment, modified anti-IL-22 antibody or antibody fragment, or pharmaceutical composition of any one of the preceding embodiments; and instructions for using the antibody or antibody fragment, modified antibody or antibody fragment, or pharmaceutical composition for diagnosis or therapy.

[0036] In another aspect, the present invention provides an antibody or antibody fragment comprising: a light chain variable region having an amino acid sequence that has 90% or more sequence identity with one of the amino acid sequences of SEQ ID NOs: 7 to 12; and a heavy chain variable region having an amino acid sequence that has 90% or more sequence identity with one of the amino acid sequences of SEQ ID NOs: 13 to 18.

[0037] In the previous embodiment, the light chain variable region may have three complementarity determining regions identical to the complementarity determining regions of a light chain variable region having an amino acid sequence selected from SEQ ID NOs: 7-12.

[0038] In either one of the previous two embodiments, the heavy chain variable region may have three complementarity determining regions that are identical to the complementarity determining regions of a heavy chain variable region having an amino acid sequence selected from SEQ ID NOs: 13-18.

[0039] In any one of the previous three embodiments, the light and heavy chains may have at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity. [Brief explanation of the drawings]

[0040] [Figure 1]Figure 1 shows the structures of the light and heavy chains of a humanized monoclonal antibody against human IL-22 (hum10) that was used as the parent antibody to evolve the antibodies of the present invention. The CDRs are shown as thicker areas in the diagram, with the start and end positions of the light and heavy chains labeled. Some of the mutations in the CDRs and their positions that were made to the parent antibody to provide the antibodies of the present invention are indicated within the CDRs. [Figure 2] 2A-2B show sequence alignments of the light chains of hum10 and antibodies of the present invention. [Figure 3] 3A-3B show sequence alignments of the heavy chains of hum10 and antibodies of the present invention. [Figure 4] Figure 4 shows certain purified antibodies on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel as a single band under non-reducing conditions (left side) and as two bands, one heavy and one light chain, under reducing conditions (right side), demonstrating the purity of the antibody. [Figure 5] Figures 5A-5C show the single peaks obtained by size exclusion chromatography (SEC) analysis of purified antibodies (CPS02 and CPS09) compared to SEC analysis of a control human IgG antibody. [Figure 6] FIG. 6 shows the specificity of the antibodies of the invention for human and mouse IL-22 and other related antigens. [Figure 7A] FIG. 7A shows the reduced level of phosphorylation of Stat3 protein caused by the suppression of human IL-22 in HepG2 cells using an antibody of the present invention. [Figure 7B] FIG. 7B shows the reduced level of phosphorylation of Stat3 protein caused by suppression of mouse IL-22 in HepG2 cells using an antibody of the present invention. [Figure 8] FIG. 8 shows the suppression of CXCL1 protein expression in HT29 cells by antibodies of the invention. [Figure 9] 9A-9B show the plasma concentrations of antibody CPS02 in mice over time after various injection doses. [Figure 10] 10A-10B show the plasma concentrations of antibody CPS09 in mice over time after various injection doses. [Figure 11] FIG. 11 shows inhibition of IL-22-induced acute phase responses in mice by antibodies of the invention. [Figure 12] FIG. 12 shows the study design for treating the imiquimod-induced mouse psoriasis model. [Figure 13] FIG. 13 shows the change in body weight of treated mice during the treatment period according to the study design shown in FIG. [Figure 14] FIG. 14 shows the change in ear thickness of treated mice over the treatment period according to the study design shown in FIG. [Figure 15] FIG. 15 shows the change in skin erythema scores of treated mice during the treatment period according to the study design shown in FIG. [Figure 16] FIG. 16 shows the change in skin scale score of treated mice during the treatment period according to the study design shown in FIG. [Figure 17] FIG. 17 shows the change in skin thickness score of treated mice during the treatment period according to the study design shown in FIG. [Figure 18] FIG. 18 shows the change in total skin score (overall skin condition) of treated mice during the treatment period according to the study design shown in FIG.

[0041] definition To facilitate understanding of the examples provided herein, several frequently used terms are defined herein.

[0042] As used herein, the term "about" in connection with a measured quantity refers to the normal variation in that measured quantity that would be expected by one of ordinary skill in the art making the measurement and exercising a level of care commensurate with the purpose of the measurement and the precision of the measuring device being used. Unless otherwise indicated, "about" refers to a + / - 10% variation of the value provided.

[0043] As used herein, the term "affinity" or "binding affinity" refers to the strength of the total non-covalent interactions between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein 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 X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, for example, those described herein.

[0044] As used herein, the term "amino acid" refers to any organic compound containing an amino group (-NH2) and a carboxyl group (-COOH) either as a free group or, after condensation, as part of a peptide bond. The term "20 naturally occurring amino acids" is understood in the art and refers to the building blocks of natural proteins: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine ​​(cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0045] As used herein, the term "antibody" refers to intact immunoglobulin molecules and fragments of immunoglobulin molecules, such as Fab, Fab', F(ab')2, Fv, and single-chain antibody (SCAs or scFv) fragments, which are capable of binding to an epitope of an antigen. These antibody fragments, which retain some ability to selectively bind to the antigen (e.g., polypeptide antigen) of the derived antibody, can be generated using methods well known in the art and as further described below. Antibodies useful in the practice of the present invention may be IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, sIgA, IgD, or IgE. Antibodies can be used to isolate preparative quantities of antigens by immunoaffinity chromatography. Various other uses of such antibodies are for diagnosing and / or staging disease (e.g., neoplasia), as well as for therapeutic applications to treat diseases, such as neoplasia, autoimmune disease, AIDS, cardiovascular disease, infectious disease, etc. Chimeric, human-like, humanized or fully human antibodies are particularly useful for administration to human patients.

[0046] A Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule and can be produced by digestion of whole antibody molecules with the enzyme papain to yield a fragment consisting of an intact light chain and a portion of the heavy chain.

[0047] Fab' fragments of antibody molecules can be obtained by treating whole antibody molecules with pepsin, followed by reduction, to yield molecules consisting of an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained for each antibody molecule treated in this manner.

[0048] The (Fab')2 fragment of an antibody can be obtained by treating an intact antibody molecule with the enzyme pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0049] An Fv fragment is defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains.

[0050] Single-chain antibodies ("SCAs" or scFvs) are genetically engineered single-chain molecules that contain the variable region of a light chain and the variable region of a heavy chain, linked by a suitable flexible polypeptide liner, and may contain additional amino acid sequences at the amino and / or carboxyl termini. For example, single-chain antibodies may contain a tether segment for linking to an encoding polynucleotide. Functional single-chain antibodies generally contain a sufficient portion of the variable region of a light chain and sufficient region of the variable region of a heavy chain so as to retain the properties of a full-length antibody for binding to a specific target molecule or epitope.

[0051] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; monovalent antibodies; and single-chain antibody molecules (e.g., scFv). These antibody fragments, which retain some ability to selectively bind to the antigen (e.g., polypeptide antigen) of the antibody from which they are derived, can be produced using methods well known in the art.

[0052] As used herein, the terms "anti-IL-22 antibody," anti-IL-22 antibody fragment, and "antibody or antibody fragment that binds to IL-22" are used interchangeably and refer to an antibody or antibody fragment that can bind to at least one epitope of the IL-22 protein with sufficient affinity such that the antibody or antibody fragment is useful as a diagnostic, prophylactic, and / or therapeutic agent in targeting IL-22. In one embodiment, the extent of binding of an anti-IL-22 antibody or antibody fragment to an unrelated, non-IL-22 protein is less than about 5%, or less than about 10%, or less than about 20%, or less than about 50% of the binding of the antibody or antibody fragment to IL-22 as measured by ELISA. In certain embodiments, an antibody or antibody fragment that binds to IL-22 has an affinity of ≦1 μM, or ≦100 nM, or ≦10 nM, or ≦1 nM, or ≦0.1 nM, or ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, or 10 -8 M~10 -13 M, or 10 -9 M~10 -13 In certain embodiments, the anti-IL-22 antibody or antibody fragment binds to an epitope of IL-22 that is conserved among IL-22 from different species.

[0053] As used herein, the term "arthritis" means inflammation of the joints and includes, but is not limited to, osteoarthritis, gout, infection-related arthritis, Reiter's syndrome arthritis, and arthritis associated with autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis, lupus-related arthritis, spondyloarthritis, and scleroderma-related arthritis. The term "arthritic inflammation" means inflammation associated with arthritis.

[0054] As used herein, the term "autoimmune disorder" or "autoimmunity" refers to any condition in which a humoral or cell-mediated immune response is mounted against the body's own tissues. An "IL-22-mediated autoimmune disorder" is any autoimmune disorder caused, maintained, or exacerbated by IL-22 activity. The term "autoimmune inflammation" refers to inflammation associated with autoimmune disease.

[0055] As used herein, the term "binding" refers to the interaction of an antibody variable region or Fv with an antigen, where the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody variable region or Fv recognizes and binds to a specific protein structure, rather than proteins in general. As used herein, "specifically binding" or "binding specifically" means that an antibody variable region or Fv binds or associates with a specific antigen more frequently, rapidly, for a longer duration, and / or with greater affinity than with other proteins. For example, an antibody variable region or Fv specifically binds to the antigen with greater affinity, rapidly, and / or for a longer duration than it binds to other antigens. In another example, an antibody variable region or Fv binds to a cell surface protein (antigen) with significantly greater affinity than it binds to related proteins or other cell surface proteins, or to an antigen generally recognized by polyreactive natural antibodies (i.e., naturally occurring antibodies known to bind to various antigens naturally found in humans). However, "specifically binds" does not necessarily require exclusive binding or non-detectable binding of another antigen, which is what the term "selective binding" means. In one example, "specific binding" of an antibody variable region or Fv (or other binding region) to an antigen means that the antibody variable region or Fv binds to the antigen with an equilibrium constant (KD) of 100 nM or less, e.g., 50 nM or less, e.g., 20 nM or less, 15 nM or less, or 10 nM or less, or 5 nM or less, 2 nM or less, or 1 nM or less.

[0056] As used herein, the term "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia.More specific examples of such cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0057] As used herein, the terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0058] As used herein, the term "chemotherapeutic agent" refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, such as altretamine, triethylenemelamine, and triethylenephosphoramine. amide, triethylenethiophosphoramide, and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (e.g., synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcannabinol, etc.) camptothecin, scopolectin, and 9-aminocamptothecin; bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; Sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, fenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicins, e.g., dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin (carabicin), caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (e.g., ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozotocin, antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as florinic acid acid); aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidammol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinosine docetaxel ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL®), albumin-engineered nanoparticle formulations of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin;Vincas that interfere with tubulin polymerization to form microtubules, such as vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylornithine (D MF®); retinoids, such as retinoic acid, e.g., bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as the THERATOPE vaccine and gene therapy vaccines, such as the ALLOVECTIN vaccine, the LEUVECTIN vaccine, and the VAXID vaccine. trademark) vaccines; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510;Bcl-2 inhibitors, such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors, such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors, such as lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is short for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, which is short for a treatment regimen using oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™).

[0059] As used herein, the term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0060] As used herein, the term "chronic" administration refers to the administration of a drug in a continuous, as opposed to acute, mode so as to maintain the initial therapeutic effect over an extended period of time. "Intermittent" administration is treatment that is not given continuously without interruption, but rather is cyclic in nature.

[0061] As used herein, the term "chronic inflammation" refers to inflammation in which the cause of the inflammation persists and is difficult or impossible to eliminate.

[0062] As used herein, the term "constitutive," when applied to, for example, IL-22 activity, refers to continuous signaling activity that is not dependent on the presence of a ligand or other activating molecules. Some IL-22 activities may be constitutive, or the activity may be further activated by the binding of other molecules (e.g., ligands). The cellular events that lead to the activation of IL-22 activity are well known to those skilled in the art. For example, activation can include oligomerization into higher-order receptor complexes, e.g., dimerization, trimerization, etc. The complex may comprise a single type of protein, i.e., a homocomplex. Alternatively, the complex may comprise at least two different protein species, i.e., a heterocomplex. Complex formation can be induced, for example, by overexpression of a normal or mutant form of the receptor on the surface of a cell. Complex formation can also be induced by one or more specific mutations in the receptor.

[0063] As used herein, the term "detectably labeled" refers to any substance whose detection or measurement, either direct or indirect, by physical or chemical means, indicates the presence of IL-22 in a sample. Detectable labels may be detectable themselves (e.g., radioisotope labels or fluorescent labels) or, in the case of enzymatic labels, may catalyze a chemical change in a substrate compound or composition, resulting in a detectable product. Representative examples of useful detectable labels include, but are not limited to, the following: molecules or ions that are directly or indirectly detectable based on light absorption, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties; molecules or ions that are radioactively detectable; and molecules or ions that are nuclear magnetic resonance or paramagnetically detectable. The group of molecules that are indirectly detectable based on light absorption or fluorescence includes, for example, various enzymes that cause the conversion of an appropriate substrate from a non-light-absorbing to a light-absorbing molecule or from a non-fluorescent to a fluorescent molecule.

[0064] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ) in the light chain variable region (VL ) linked to a heavy chain variable region (V H (See, for example, EP 404,097; WO 1993 / 001161; Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003; and Hollinger et al., Proc. Natl. Acad. Sci. USA vol. 90, pp. 6444-6448, 1993. Triabodies and tetrabodies are also described in Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003.

[0065] The term "diagnosis" as used herein refers to determining the subject's susceptibility to disease or disorder, determining whether the subject is currently suffering from disease or disorder, prognosticating the subject suffering from disease or disorder (for example, identifying the pre-metastatic or metastatic cancerous state, the stage of cancer, or the response of cancer to treatment), and treatment strategy (for example, monitoring the subject's condition to provide information on the effectiveness or efficacy of treatment).In some embodiments, the diagnostic method of the present invention is particularly useful for detecting early cancer.

[0066] The term "diagnostic agent" as used herein refers to a molecule that can be detected directly or indirectly and used for diagnostic purposes. The diagnostic agent can be administered to a subject or sample. The diagnostic agent can be provided by itself or can be conjugated to a vehicle, such as a conditionally active antibody.

[0067] As used herein, the term "effective amount" of an anti-IL-22 antibody or antibody fragment refers to a sufficient amount of the antibody or antibody fragment to treat a disease or condition at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the antibodies or antibody fragments and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific antibody or antibody fragment used; the specific composition used, the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the specific antibody or antibody fragment used; the duration of treatment; drugs used with the specific antibody used, or drugs used in combination with or concomitantly with the specific antibody used; and factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0068] As used herein, the term "effector function" refers to the biological activity attributable to the Fc region of an antibody and varies depending on the antibody isotype. Examples of antibody effector functions include C1q 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.

[0069] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0070] As used herein, the term "framework region" or "FR" refers to variable region residues other than those in the CDRs. The FR of a variable region generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in the following sequence in the variable region: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0071] The terms "full length antibody," "intact antibody," or "complete antibody" refer to an antibody that contains an antigen-binding variable region (V H or V L) and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Depending on the amino acid sequence of the constant domain of the heavy chain, full-length antibodies can be assigned to different "classes." There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunits and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0072] As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0073] As used herein, the term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and human framework regions. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable regions, in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. As used herein, the term "human consensus framework" refers to a chimeric antibody comprising amino acid residues from a human immunoglobulin V. Lor V H In selecting framework sequences, the framework represents the most commonly occurring amino acid residues. L or V H The selection of sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, Md. (1991), vols. 1-3. In one embodiment, V L The subgroup for V is subgroup kappa I as in Kabat et al., supra. H The subgroup for is subgroup III as in Kabat et al., supra. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and their references: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0074] As used herein, the term "IL-22," unless otherwise specified, refers to any naturally occurring IL-22 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed precursor IL-22, as well as any form of IL-22 resulting from processing in the human or animal body. The term also encompasses naturally occurring variants of IL-22, such as splice variants or allelic variants. The amino acid sequence of human IL-22 is well known in the art and is available from public databases such as GenBank.

[0075] As used herein, the term "IL-22-associated activity" refers to one or more of the biological activities of IL-22, including, but not limited to: (1) interaction, e.g., binding with an IL-22 receptor (e.g., IL-22R or IL-10R2 or complexes thereof, e.g., from a mammal, e.g., a mouse or a human); (2) binding to one or more signaling molecules; (3) stimulating phosphorylation and / or activation of protein kinases, e.g., JAK / STAT3, ERK, and MAPK; (4) modulating, e.g., stimulation, reduction, proliferation, differentiation, effector cell function, cytolytic activity, cytokine or chemokine secretion, and / or survival of IL-22-responsive cells (e.g., epithelial cells from the kidney, liver, colon, small intestine, thyroid, pancreas, skin); (5) modulating at least one parameter of the acute phase response, e.g., IL-22R or IL-10R2; For example, regulating metabolic, hepatic, hematopoietic (e.g., anemia, thrombocytosis), or neuroendocrine changes, or alterations (e.g., an increase or decrease in acute phase proteins, e.g., an increase in fibrinogen and / or serum amyloid A, or a decrease in albumin); and / or (6) regulating at least one parameter of an inflammatory state, e.g., regulating cytokine-mediated pro-inflammatory effects (e.g., fever, and / or prostaglandin synthesis, e.g., PGE2 synthesis), regulating a cellular immune response, regulating cytokine, chemokine (e.g., GRO1), or lymphokine production and / or secretion (e.g., production and / or secretion of pro-inflammatory cytokines).

[0076] As used herein, the term "immune-related disease" refers to a disease in which a component of a mammal's immune system causes, mediates, or otherwise contributes to a morbidity in the mammal. Also included are diseases in which stimulating or treating an immune response has an ameliorative effect on disease progression. Included within this term are immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, and neoplasms.

[0077] As used herein, the term "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, such as, but not limited to, a cytotoxic agent.

[0078] As used herein, the term "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, livestock 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). In some embodiments, the individual or subject is a human.

[0079] As used herein, the term "inflammation" refers to the accumulation of white blood cells and dilation of blood vessels at the site of injury or infection, typically causing pain, swelling, and redness.

[0080] As used herein, the term "inflammatory bowel disease" or "IBD" refers to a chronic disease characterized by inflammation of the digestive tract. IBD includes ulcerative colitis, which affects the large intestine and / or rectum, and Crohn's disease, which can affect the entire gastrointestinal system but more commonly affect the small intestine (ileum) and possibly the large intestine.

[0081] As used herein, the term "inhibiting cell growth or proliferation" means reducing cell growth or proliferation by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% or 100%, including inducing cell death.

[0082] As used herein, the terms "isolated" or "purified" antibody refer to an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic, prophylactic, or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody will be purified to (1) greater than 95% or greater than 99% by weight of the antibody as determined by the Lowry method, (2) sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity on an SDS-PAGE gel under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibody encompasses antibodies present in situ in recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0083] As used herein, the term "liposome" refers to a small vesicle composed of various types of lipids, phospholipids, and / or surfactants that is useful for delivery of drugs (such as nucleic acids, polypeptides, antibodies, agonists, or antagonists) to mammals, such as humans, primates, or mice. The components of the liposome are usually arranged in a two-phase formation, resembling the lipid arrangement of biological membranes.

[0084] As used herein, the term "package insert" is used to refer to instructions customarily included in the commercial packaging of a therapeutic product, which contain information about the indications, uses, dosage, administration, concomitant therapy, contraindications and / or precautions regarding the use of such therapeutic product.

[0085] As used herein, the term "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity.Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for aligning sequences, for example, any algorithm required to achieve maximum alignment across the entire length of the sequences being compared.However, for the purposes herein, the % amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is authored by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, e.g., Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0086] In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 * (X / Y) (where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B.) It is recognized that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specifically stated, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0087] As used herein, the term "pharmaceutical composition" refers to a formulation that is in a form that allows the effectiveness of the biological activity of the anti-IL-22 antibody contained therein, and that does not contain additional components that exhibit unacceptable toxicity to the subject to which the composition is administered.

[0088] As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is non-toxic to a subject exposed to the dosage and concentration used. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0089] Examples of pharmaceutically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; 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, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0090] As used herein, the term "psoriasis" refers to a condition characterized by a rash of large papules with localized, discrete and confluent, reddish, silvery scales, prominently appearing on the elbows, knees, scalp or trunk.

[0091] As used herein, the term "recombinant antibody" refers to an antibody (e.g., a chimeric, humanized, or human antibody or antigen-binding fragment thereof) expressed by a recombinant host cell containing nucleic acid encoding the antibody. Examples of "host cells" for producing recombinant antibodies include: (1) mammalian cells, such as Chinese hamster ovary (CHO) cells, COS, myeloma cells (e.g., Y0 and NS0 cells), baby hamster kidney (BHK) cells, Hela and Vero cells; (2) insect cells, such as sf9, sf21 and Tn5; (3) plant cells, such as plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, such as those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or Aspergillus (e.g., Aspergillus niger); and (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells.

[0092] As used herein, the term "single-chain Fv" ("scFv") refers to a VFv that is typically linked by a peptide-encoded linker. H and V L Covalently linked V expressed from a gene fusion containing the encoding gene H ::V L "dsFv" is a V heterodimer stabilized by disulfide bonds. H ::V L Divalent and multivalent antibody fragments can form spontaneously by association of monovalent scFvs or can be generated by coupling monovalent scFvs by peptide linkers (e.g., bivalent sc(Fv)2).

[0093] As used herein, the terms "treatment," "treat," or "treating" refer to clinical interventions that attempt to alter the natural course of the individual being treated and can be performed for prophylaxis or during the course of clinical disease. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies or antibody fragments of the invention are used to delay the onset of disease or slow the progression of disease.

[0094] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not intended to be mutually exclusive herein.

[0095] As used herein, the terms "variable region" or "variable domain" of an antibody refer interchangeably to the amino-terminal domains of the heavy or light chain of an antibody. The variable region of a heavy chain is called a "V H The variable region of the light chain can be described as "V L These variable regions are generally the most variable parts of an antibody and contain the antigen-binding site. The heavy and light chain variable regions (V and V, respectively) H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). H or V L The region may be sufficient to confer antigen-binding specificity. Furthermore, an antibody or antibody fragment that binds to a particular antigen may contain a V region from the antibody that binds to that antigen. H or V LThe complementary V L or V H Libraries of regions can be screened (see, e.g., Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993; Clarkson et al., Nature, vol. 352, pp. 624-628, 1991).

[0096] 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 and 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."

[0097] (Detailed description) For illustrative purposes, the principles of the present invention will be described with reference to various exemplary embodiments. While certain embodiments of the present invention are specifically described herein, those skilled in the art will readily recognize that the same principles are equally applicable and can be used in other systems and methods. Before describing the disclosed embodiments of the present invention in detail, it should be understood that the present invention is not limited in its application to the details of any particular embodiment shown. Furthermore, the terminology used herein is for purposes of description and not limitation. Furthermore, although certain methods are described herein with reference to steps presented in a certain order, in many cases, as one skilled in the art will recognize, these steps can be performed in any order; thus, the novel methods are not limited to the particular order of steps disclosed herein.

[0098] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The terms "comprising," "including," "having," and "consisting of" can also be used interchangeably.

[0099] Unless otherwise indicated, all numbers used in the specification and claims expressing quantities of ingredients, properties, e.g., molecular weights, percentages, ratios, reaction conditions, and the like, should be understood to be modified in all instances by the term "about," whether or not the term "about" is used. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations, which may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0100] It is to be understood that each component, compound, substituent, or parameter disclosed herein should be construed as disclosed for use alone or in combination with each and every other component, compound, substituent, or parameter disclosed herein.

[0101] It should be understood that each amount / value or amount / value range for each component, compound, substituent, or parameter disclosed herein should be construed as also being disclosed in combination with each amount / value or amount / value range disclosed for every other component, compound, substituent, or parameter disclosed herein, and thus any combination of amounts / values ​​or amount / value ranges for two or more components, compounds, substituents, or parameters disclosed herein are also disclosed in combination with each other for purposes of this detailed description.

[0102] It is further understood that each lower limit of each range disclosed herein should be interpreted as being disclosed in combination with each upper limit of each range disclosed herein for the same component, compound, substituent, or parameter. Thus, the disclosure of two ranges should be interpreted as a disclosure of four ranges derived by combining each lower limit of each range with each upper limit of each range. The disclosure of three ranges should be interpreted as a disclosure of nine ranges derived by combining each lower limit of each range with each upper limit of each range, etc. Furthermore, a specific amount / value of a component, compound, substituent, or parameter disclosed in the specification or examples should be interpreted as a disclosure of either the lower or upper limit of a range, and thus may be combined with any other lower or upper limit or specific amount / value of a range for the same component, compound, substituent, or parameter disclosed elsewhere in this application to form a range for that component, compound, substituent, or parameter.

[0103] A. Anti-IL-22 Antibody or Antibody Fragment In one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis. a light chain variable region comprising a light chain CDR1 having an amino acid sequence selected from SASSSVSX1MH (SEQ ID NO: 1), a light chain CDR2 having an amino acid sequence selected from X2TX3KLX4S (SEQ ID NO: 2), and a light chain CDR3 having the amino acid sequence of QQWSSNPYIT (SEQ ID NO: 3); and a heavy chain CDR1 having an amino acid sequence selected from GYIFX5SYWIH (SEQ ID NO: 4), a heavy chain CDR2 having an amino acid sequence selected from RIYPGTGX6TYYNX7KFKG (SEQ ID NO: 5), and SYX8X9SVX 10 Y (SEQ ID NO: 6), X1 is Y or K, X2 is E or K, X3 is S or R, X4 is A or L, X5 is T or R, X6 is N or R, X7 is E or R, X8 is D or M, X9 is S or Y, and X 10 is A or G, Anti-IL-22 antibodies or antibody fragments are provided.

[0104] In another embodiment, the anti-IL-22 antibody or antibody fragment of the invention comprises a light chain variable region having an amino acid sequence selected from SEQ ID NOs: 7-12, and a heavy chain variable region having an amino acid sequence selected from SEQ ID NOs: 13-18.

[0105] In yet another embodiment, the anti-IL-22 antibody or antibody fragment of the invention is selected from: (1) an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13; (2) an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 10 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13; (3) an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14; (4) an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15; (5) An antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15; (6) An antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 10 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15; (7) An antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16; (8) An antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17; (9) An antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17; (10) An antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18.

[0106] The anti-IL-22 antibodies or antibody fragments of the present invention are derived from a parent antibody that binds to human IL-22, designated herein as hum10, which has a light chain variable region having the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20. The parent antibody hum10 is a humanized monoclonal antibody (mAb) generated from the functional hybridoma clone 3C3 by immunizing mice with human IL-22 (hIL-22). The binding affinity of the mouse monoclonal antibody 3C3 for hIL-22 is approximately 100 pM (as measured by SPR analysis). However, the 3C3 mAb cannot bind to mouse IL-22 (mIL-22). The 3C3 mAb can block the induction of Stat3 phosphorylation by hIL2 in HepG2 cells. The 3C3 mAb was humanized to generate the humanized antibody hum10.

[0107] The parent antibody hum10 has been modified to generate mutant antibodies, including an anti-IL-22 antibody capable of binding both human and mouse IL-22 with high affinity. Human and mouse IL-22 share several completely conserved regions. The CPE™ library was constructed from the parent antibody hum10 by substituting amino acid residues at each position in all six CDRs of hum10 with at least 15 other amino acids. Some examples of substitutions are shown in Figure 1, where the CDRs are represented by bolder sections in the schematic. The CPE™ library contains 1,068 CPE antibody variants, including 472 variants with mutations in the light chain CDRs and 596 variants with mutations in the heavy chain CDRs.

[0108] The mutant antibodies were expressed in eukaryotic hosts such as CHO cells. The sequence of each mutant antibody was verified, and the mutant antibodies were arrayed in a 96-well format.

[0109] The CPE™ library was screened for mutant antibodies that bind with high affinity to both murine and human IL-22. Ten antibodies that met the selection criteria were identified and are shown in Table 1 below. [Table 1]

[0110] Among the 10 mutant antibodies listed in Table 1, there are six different light chains. Alignment of these six different light chains with the light chain of hum10 is shown in Figures 2A-2B. Among the 10 mutant antibodies listed in Table 1, there are six different heavy chains. Alignment of these six different heavy chains with the heavy chain of hum10 is shown in Figures 3A-3B. It is observed that amino acid substitutions in the 10 mutant antibodies occur at several specific positions in the CDRs.

[0111] The ten antibodies in Table 1 were expressed in the same eukaryotic host to produce antibodies for further testing, clinical trials, and / or therapeutic, prophylactic, or diagnostic uses. The eukaryotic host can be selected from 3T3 mouse fibroblast cells; BHK21 Syrian hamster fibroblast cells; MDCK, canine epithelial cells; HeIa human epithelial cells; PtKl rat kangaroo epithelial cells; SP2 / 0 mouse plasma cells; and NSO mouse plasma cells; HEK293 human embryonic kidney cells; COS monkey kidney cells; CHO, Chinese hamster ovary cells; RI mouse embryonic cells; E14.1 mouse embryonic cells; HI human embryonic cells; H9 human embryonic cells; PER.6 human embryonic cells; S. cerevisiae yeast cells; or Pichia yeast cells. In certain embodiments, the mammalian system is CHO or HEK293.

[0112] The expressed antibody was purified from the supernatant of eukaryotic host cell culture. To verify the purity of the antibody, the purified mutant antibody was analyzed using a 10% SDS-PAGE gel. Under non-reducing conditions, the antibody showed a single band (Figure 4, left half). Under reducing conditions, the antibody separated into light and heavy chains, each appearing as two bands in its own lane (Figure 4, right half). The purified antibody was also analyzed by SEC analysis, which yielded a single peak, as shown in Figures 5A-5C, indicating that the antibody was obtained with high purity.

[0113] The specificity of the 10 antibodies in Table 1 was assayed using both human and mouse IL-22, as well as related antigens (human IL19, human IL20, human IL24, human IL26, INF alpha A, INF-gamma, INF-lambda l, INF-2, and a non-specific antigen). The 10 mutant antibodies in Table 1 were found to bind to both human and mouse IL-22 with comparable affinity, but these mutant antibodies exhibited significantly lower affinity for related antigens, as can be seen in Figure 6. This confirms that the 10 mutant antibodies in Table 1 are specific for human and mouse IL-22 due to their low affinity for other related antigens.

[0114] In one embodiment, the antibodies of the invention have similar affinities for human and mammalian IL-22 (e.g., murine IL-22), e.g., within ±20%, ±15%, ±10%, or ±5% of each other, i.e., the difference between the affinities is less than 20%, or less than 15%, or less than 10%, or less than 5% of either affinity.

[0115] The affinities of the 10 antibodies in Table 1 were measured by Surface Plasma Resonance (SPR) using a capture assay in PBS buffer, and binding curves were generated at four different concentrations of each antibody (0.05, 0.1, 0.5, and 1.0 μg / ml). The K, Kd, ​​and K of the mutant antibodies were also measured. D was calculated from the curve for each antibody.

[0116] In one embodiment, Kd is measured using a BIACORE®-2000 or BIACORE®-3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25°C with antigen immobilized on a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.). CM5 chips are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg / ml (∼0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of binding protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of antibody (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at 25°C at a flow rate of approximately 25 μl / min.

[0117] Association velocity (k on ) and dissociation rate (k offThe equilibrium dissociation constant (Kd) was calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2). off / k on For example, Chen et al., J. Mol. Biol., vol. 293, pp. 865-881, 1999. When the on-rate is 10 6 M -1 s -1 If the on-rate exceeds 100 s, the on-rate can be determined by measuring the presence of increasing antigen concentration in a spectrometer such as a spectrophotometer equipped with a stopped flow (Aviv Instruments) or an 8000 Series SLM-AMINCO® spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette, using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of a 20 nM solution of anti-antibody in PBS, pH 7.2 at 25°C.

[0118] Some representative antibodies are shown in Table 2. Two antibodies, CPS02 and CPS09, show the highest affinity to both human and mouse IL-22. LC-E049K (one of the mutant antibodies in the CPE® library) is used as a control. [Table 2]

[0119] One of the direct functions of IL-22 is to induce phosphorylation of Stat3, a protein in the IL-22 signaling pathway. The 10 antibodies listed in Table 1 were tested in a cell-based assay to block IL-22-dependent phosphorylation of Stat3 mediated by human or mouse IL-22 in HepG2 cells. The antibody hum10 and the mutant CPE-LC-E049K served as controls. Each antibody was used at four concentrations: 0.11 μg / mL, 0.33 μg / mL, 1 μg / mL, and 3 μg / mL.

[0120] Inhibition of Stat3 phosphorylation via binding to human IL-22 (hIL-22) is shown in Figure 7A, and inhibition of Stat3 phosphorylation via binding to murine IL-22 (mIL-22) is shown in Figure 7B. Inhibition of Stat3 phosphorylation was found to be dose-dependent, with increasing concentrations of the mutant antibodies resulting in greater levels of inhibition of Stat3 phosphorylation. This indicates that the mutant antibodies of the invention play a causative role in inhibiting IL-22-induced Stat3 phosphorylation in both human and murine biological systems through binding to human and murine IL-22.

[0121] HT29 cells are a human colorectal adenocarcinoma cell line with an epithelial morphology. These cells can express a wide range of cytokines, including CXCL10, CXCL11, CCL5, CXCL8, CXCL1, CCL20, and IκB. Cytokine secretion is a sign of an immune response and typically leads to inflammation in animals. The ability of anti-IL-22 antibodies to inhibit IL-22-induced CXCL1 production in HT29 cells is an indicator of the antibody's ability to suppress immune responses and inflammation. HT29 cells were treated with various concentrations of anti-IL-22 mutant antibodies, and CXCL1 production was measured (Figure 8). The suppression of CXCL1 production by the mutant antibodies was dose-dependent, indicating a causal role of the mutant antibodies in suppressing IL-22 activity and immune responses / inflammation.

[0122] The pharmacokinetics of the mutant antibodies in Table 1 were analyzed in mice. The antibodies were injected intravenously into mice at two doses, 0.3 mg / kg and 10 mg / kg. The concentration of injected antibody in plasma was measured by solid-phase ELISA. The antibodies were found to have long half-lives in mice, as shown in Figures 9A-9B and 10A-10B. The half-life of CPS02 in mice was found to be approximately 18 hours after injection at 10 mg / kg. The half-life of CPS09 was found to be approximately 16 hours after injection at 10 mg / kg.

[0123] IL-22 induces an acute phase response in animals, such as mice and humans. The mutant antibodies of the present invention can inhibit the acute phase response induced by IL-22. This acute phase response is indicated by the serum amyloid (SAA) concentration in the animal's blood, as shown in Figure 11. The SAA concentration in the mouse blood was measured 24 hours after treatment of the mice with IL-22 and / or the mutant antibody. When IL-22 alone was used, the SAA concentration in the mouse blood was significantly lowered when anti-IL-22 antibody was added, indicating that the antibodies of the present invention can effectively inhibit the acute phase response in mice.

[0124] The properties of the anti-IL-22 antibodies are exemplified by CPS02 and CPS09 and summarized in Table 3. [Table 3]

[0125] The anti-IL-22 antibodies or antibody fragments of the present invention have high binding affinity to both human and mouse IL-22. Furthermore, the anti-IL-22 antibodies or antibody fragments can induce similar biological effects in humans and mice, including inhibition of Stat3 phosphorylation and cytokine production (e.g., CXCL1 production), and inhibition of acute immune responses, and the mutant antibodies have been shown to have significantly longer half-lives in mice as well.

[0126] The present invention also extends to anti-IL-22 antibodies or antibody fragments comprising a light chain variable region and a heavy chain variable region from the ten selected antibodies of Table 1. In particular, the anti-IL-22 antibodies or antibody fragments of the present invention comprise a light chain variable region selected from light chain variable regions having the amino acid sequences of SEQ ID NOs: 7-12, and a heavy chain variable region selected from heavy chain variable regions having the amino acid sequences of SEQ ID NOs: 13-18.

[0127] The present invention also includes anti-IL-22 antibodies or antibody fragments comprising a light chain variable region having the CDRs of the light chain variable region having the amino acid sequences of SEQ ID NOs: 7-12, and a heavy chain variable region having the CDRs of the heavy chain variable region having the amino acid sequences of SEQ ID NOs: 13-18.

[0128] The antibodies of Table 1, particularly fragments of these antibodies capable of binding to both human and mouse IL-22, are also within the scope of the present invention. These antibody fragments can be produced from full-length antibodies via proteolytic digestion (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods, vol. 24, pp. 107-117, 1992; and Brennan et al., Science, vol. 229, pp. 81, 1985). These antibody fragments can also be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can be expressed in and secreted from E. coli, allowing for the facile production of large amounts of these fragments. Antibody fragments can also be isolated from the 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, vol. 10, pp. 163-167, 1992). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab'')2 fragments with increased in vivo half-lives containing salvage receptor-binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0129] In certain embodiments, the antibody is a single-chain Fv fragment (scFv). See WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. Fv and scFv are the only species with intact binding sites and lack constant regions. Therefore, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins can be constructed to allow fusion of an effector protein at either the amino or carboxy terminus of the scFv. See Antibody Engineering, ed. Borrebaeck, Oxford University Press, 1995. An antibody fragment may be a "linear antibody," e.g., as described in U.S. Patent No. 5,641,870. Such linear antibodies may be monospecific or bispecific.

[0130] In some embodiments, the antibodies of the present invention are diabodies. Diabodies can be bivalent or bispecific. For examples of diabodies, see, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. vol. 9, pp. 129-134, 2003; and Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993. Examples of triabodies and tetrabodies are also described in Hudson et al., Nat. Med., vol. 9, pp. 129-134, 2003.

[0131] In some embodiments, antibodies of the invention are single-domain antibody fragments that contain all or a portion of the heavy chain variable region or all or a portion of the light chain variable region of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see, e.g., U.S. Patent No. 6,248,516).

[0132] B. Antibody Variants In some embodiments, the present invention provides variants of the antibodies or antibody fragments described above. To derive these variants, those skilled in the art are guided by the processes described herein. Variants of these antibodies or antibody fragments can be created by introducing appropriate modifications into the nucleotide sequence encoding these antibodies or antibody fragments, or by synthesizing peptides. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody or antibody fragment. Any combination of deletions, insertions, and substitutions can be used to arrive at a variant, provided that the variant retains at least one desired property (e.g., binding to both human and mouse IL-22).

[0133] 1. Sequence variants In certain embodiments, the present invention provides antibody or antibody fragment variants having one or more amino acid substitutions, deletions, and / or insertions compared to the mutant antibodies or antibody fragments described above. Target sites for substitutional mutagenesis include the CDRs and framework regions (FRs). Conservative substitutions are shown in Table 4 under the heading "Preferred Substitutions," while more substantial changes are shown in Table 4 under the heading "Exemplary Substitutions," and further described below with reference to amino acid side chain groups determined by common side chain properties. Amino acid substitutions can be introduced into the antibody or antibody fragment of interest, and the products screened for a desired activity, e.g., binding to both human and mouse IL-22, and / or reduced immunogenicity. [Table 4]

[0134] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0135] Non-conservative substitutions involve exchanging a member of one of these classes for one from another class.

[0136] In making changes in an amino acid sequence, the hydropathic index of amino acids can be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is generally understood in the art. It is accepted that the relative hydropathic characteristics of amino acids contribute to the secondary structure of a resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0137] One type of substitutional variant involves substituting one or more CDRs of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further testing has an altered (e.g., improved) biological property relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or substantially retains a biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques, such as those described herein. For example, one or more CDR residues can be mutated, and the variant antibody can be displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0138] Sequence modifications (e.g., substitutions) can be made in the CDRs, for example, to improve antibody affinity. Such changes can be made in CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol., vol. 207, pp. 179-196, 2008), resulting in variants V H or V LThe antibodies are tested for binding affinity. Affinity maturation by library construction and selection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology, vol. 178, pp. 1-37, 2001. In some affinity maturation embodiments, 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 library containing variants is then generated. The library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scanning mutagenesis or modeling can be used to specifically identify CDR residues involved in antigen binding. LC-CDR3 and HC-CDR3 are often targeted in particular.

[0139] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody or antibody fragment to bind to IL-22. For example, conservative changes (e.g., conservative substitutions as provided herein) can be made in the CDRs that do not substantially reduce binding affinity. Such changes may be outside the "hot spots" of the CDRs. Variant V H and V L In certain embodiments of the regions, each CDR is unaltered or contains no more than one, two, or three amino acid substitutions.

[0140] In some embodiments, the modifications are introduced into non-CDR regions of the antibody. In other words, the complementarity-determining regions (CDRs) of the light chain variable region having the amino acid sequences of SEQ ID NOS: 7-12 and the heavy chain variable region having the amino acid sequences of SEQ ID NOS: 13-18 remain intact. The modifications may instead be introduced into the FR or constant region.

[0141] A useful method for identifying amino acid residues or regions of an antibody that can be targeted for mutagenesis is "alanine scanning mutagenesis," as described by Cunningham and Wells, Science, vol. 244, pp. 1081-1085, 1989. In this method, a residue or group of target residues (e.g., charged residues, such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody or antibody fragment and the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody or antibody fragment and the antigen. Such contact residues and adjacent residues can be targeted or excluded as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

[0142] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0143] Amino acid sequence modification(s) of the antibodies described herein may improve the binding affinity and / or other biological properties of the antibody.

[0144] Further aspects of the present invention include function-conservative variants of antibodies or antibody fragments. Function-conservative variants are those in which a given amino acid residue in an antibody is altered without altering the overall conformation and function of the antibody, including, but not limited to, substitution with an amino acid having similar properties, such as polarity, hydrogen bonding potential, acidicity, basicity, hydrophobicity, aromaticity, etc. Amino acids other than those shown as conserved may differ between proteins such that the percent protein or amino acid sequence similarity between any two proteins with similar functions may vary, for example, from 70% to 99% as determined according to an alignment scheme such as the cluster method, where similarity is based on the MEGALIGN algorithm. "Function-conservative variants" also include antibodies that have at least 60% amino acid identity as determined by BLAST or FASTA algorithms, or at least 75%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity with a parent antibody or antibody fragment, and have the same or substantially similar properties or functions as the antibodies or antibody fragments identified according to the present invention, in particular good binding affinity for both human and mouse IL-22.

[0145] 2. Humanized antibodies or antibody fragments In some embodiments, the present invention provides humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable regions in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody. A humanized antibody may also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to, for example, restore or improve the specificity or affinity of the antibody.

[0146] Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically taken from an "import" variable domain. Humanization can essentially be performed according to the method of Winter et al. (Jones et al. (1986) Nature, vol. 321, pp. 522-525; Riechmann et al. (1988) Nature, vol. 332, pp. 323-327; Verhoeyen et al. (1988) Science, vol. 239, pp. 1534-1536) by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Such "humanized" antibodies are therefore chimeric antibodies (U.S. Pat. No. 4,816,567), in which substantially less than intact human variable region sequences have been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0147] The choice of human variable region, both heavy and light, to use in making a humanized antibody can be important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable region of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework of the humanized antibody (Sims et al. (1993) J. Immunol., vol. 151, pp. 2296; Chothia et al. (1987) J. Mol. Biol., vol. 196, p. 901). Another method is to use a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, vol. 89, p. 4285; Presta et al. (1993) J. Immunol., vol. 151, p. 2623).

[0148] More generally, it is desirable for antibodies to be humanized with high affinity for the antigen and retention of other favorable biological properties. To this end, one method involves preparing humanized antibodies by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display the probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient to import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen. In general, CDR residues are directly and most substantially involved in influencing antigen binding.

[0149] Humanized antibodies and methods for their production are reviewed, for example, by Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008, and further described, for example, by Riechmann et al., Nature, vol. 332, pp. 323-329, 1988; Queen et al., Proc. Nat'l Acad. Sci. USA, vol. 86, pp. 10029-10033, 1989; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. al., Methods, vol. 36, pp. 25-34, 2005 (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol., vol. 28, pp. 489-498, 1991 (describing "resurfacing"); Dall'Acqua et al., Methods, vol. 36, pp. 43-60, 2005 (describing "FR shuffling"); and Osbourn et al., Methods, vol. 36, pp. 61-68, 2005 and Klimka et al., Br. J. Cancer, vol. 83, pp. 252-260, 2000 (describing a "guided selection" approach to FR shuffling).

[0150] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol., vol. 151, p. 2296, 1993); framework regions derived from consensus sequences of human antibodies in specific subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, vol. 89, p. 4285, 1992; and Presta et al. J. Immunol., vol. 151, p. 2623, 1993), human mature (somatically mutated) framework regions, or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem., vol. 272, pp. 10678-10684, 1997 and Rosok et al., J. Biol. Chem., vol. 271, pp. 22611-22618, 1996).

[0151] V of antibodies derived from non-human animals H and V L Only the CDRs in the V H and V L It is known that when a humanized antibody is produced by simply grafting a V FR of a non-human antibody, the antibody binding affinity is reduced compared to that of the original antibody derived from a non-human animal. H and V L It is considered that some amino acid residues in the V of a human antibody, not only in the CDR but also in the FR, are directly or indirectly related to the antibody binding affinity. H and V L Substitution of these amino acid residues with different amino acid residues from the FR of the human antibody may reduce binding affinity. To overcome this problem, in an antibody onto which human FRs are grafted, the V H and VL Among the amino acid sequences of the FRs of the antibody, one must attempt to identify amino acid residues that are directly involved in binding to the antibody, that interact with amino acid residues of the CDRs, or that maintain the three-dimensional structure of the antibody and are directly involved in binding to the antigen. The reduced antigen-binding affinity can be improved by replacing the identified amino acids with amino acid residues of the original antibody derived from a non-human animal.

[0152] 3. Glycosylation variants In certain embodiments, the antibodies or fragments thereof provided herein are modified by adding one or more oligosaccharides to the antibody, thereby increasing or decreasing the degree to which the antibody is glycosylated. The modification is performed by adding oligosaccharides to one or more glycosylation sites. Adding or deleting glycosylation sites from an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0153] If the antibody contains an Fc region, the carbohydrate attached thereto can be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, generally attached via an N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH, vol. 15, pp. 26-32, 1997. Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention can be made to generate antibody variants with improved properties.

[0154] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region; however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations within antibodies. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Application Publication No. 2003 / 0157108 (Presta, L.); US Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent Application Publication No. 2002 / 0164328; U.S. Patent Application Publication No. 2004 / 0093621; U.S. Patent Application Publication No. 2004 / 0132140; U.S. Patent Application No. Publication No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 0110282; U.S. Patent Application Publication No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol., vol. 336, pp. 1239-1249, 2004; Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004. Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys., vol. 249, pp. 533-545, 1986; U.S. Patent Application Publication No. 2003 / 0157108A; and WO 2004 / 056312A1, particularly Example 11), as well as knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8 knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004; Kanda, Y. et al. al., Biotechnol. Bioeng., vol. 94, pp. 680-688, 2006; and WO 2003 / 085107).

[0155] Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody, bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; U.S. Pat. No. 6,602,684; and U.S. Patent Application Publication No. 2005 / 0123546. Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0156] 4. Fc region variants In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can 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.

[0157] In certain embodiments, the present invention provides antibody variants with Fc regions that retain some, but not all, effector functions, making them desirable candidates for applications where in vivo antibody half-life is important and certain effector functions (e.g., ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. 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. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 5 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., vol. 9, pp. 457-492, 1991. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see also, e.g., Hellstrom et al. Proc. Nat'l Acad. Sci. USA, vol. 83, pp. 7059-7063, 1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, vol. 82, pp. 1499-1502, 1985; U.S. Pat. No. 5,821,337 (see also, Bruggemann et al., J. Exp. Med., vol. 166, pp. 1351-1361, 1987). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model, such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA, vol. 95, pp. 652-656, 1998. A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods, vol. 202, pp. 163-171, 1996; Cragg, MS et al., Blood, vol. 101, pp. 1045-1052, 2003; and Cragg, MS, and MJ Glennie, Blood, vol. 103, pp. 2738-2743, 2004). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol., vol. 18, pp. 1759-1769, 2006).

[0158] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include the so-called "DANA" Fc mutant, which contains substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, with alanine substitutions of residues 265 and 297 (U.S. Pat. No. 7,332,581).

[0159] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem., vol. 9, pp. 6591-6604, 2001).

[0160] In one embodiment, 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 (EU numbering of residues) of the Fc region.

[0161] In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or diminished) C1q binding and / or complement-dependent cytotoxicity (CDC), such as those described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol., vol. 164, pp. 4178-4184, 2000.

[0162] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., vol. 117, pp. 587-593, 1976 and Kim et al., J. Immunol., vol. 24, p. 249, 1994), are described in U.S. Patent Application Publication No. 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Pat. No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351.

[0163] 5. Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine-engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are thereby located at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates as further described herein. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and 5400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0164] 6. Antibody derivatives In certain embodiments, the antibodies or antibody fragments provided herein may be modified antibodies or antibody fragments containing additional non-proteinaceous moieties readily available in the art. Suitable moieties for derivatizing antibodies or antibody fragments include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water.

[0165] The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody or antibody fragment can vary, and when two or more polymers are attached, they can be the same or different molecules. In general, the number and / or type of polymers used in antibody derivatization can be determined based on considerations, such as, but not limited to, the particular properties or functions of the antibody or antibody fragment to be improved, whether the derivative will be used in therapy under specified conditions, etc.

[0166] In certain embodiments, the antibody or antibody fragment and the non-protein moiety can be selectively bonded using heating followed by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA, vol. 102, pp. 11600-11605, 2005). The radiation can be of any wavelength, including but not limited to, a wavelength that does not harm normal cells but heats the non-protein moiety to a temperature that kills cells in proximity to the antibody-non-protein moiety.

[0167] The anti-IL-22 antibody of the present invention can be a chimeric antibody. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 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 in which the class or subclass of the antibody has been changed relative to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0168] In some embodiments, the anti-IL-22 antibodies of the present invention are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one binding specificity is for IL-22 and the other is for another antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of IL-22. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing IL-22. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0169] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, vol. 305, pp. 537-540, 1983; WO 93 / 08829; and Traunecker et al., EMBO J., vol. 10, pp. 3655-3659, 1991), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be produced by engineering electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, vol. 229, pp. 81-83, 1985); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp. 1547-1553, 1992); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., vol. 90, pp. 6444-6448, 1993). al., J. Immunol., vol. 152, pp. 5368-5374, 1994); and, for example, by preparing trispecific antibodies as described in Tutt et al. J. Immunol., vol. 147, pp. 60-69, 1991.

[0170] Engineered antibodies having three or more functional antigen binding sites, such as "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576A1).

[0171] Antibodies or antibody fragments also include "dual acting Fabs" or "DAFs" that contain antigen binding sites that bind to IL-22 and another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0172] C. Immunoconjugates In another aspect, the present invention also provides immunoconjugates comprising the anti-IL-22 antibodies or antibody fragments described herein modified by conjugation to one or more therapeutic agents, prophylactic agents, diagnostic agents, detectable labels, chelators, and imaging agents. In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including, but not limited to, maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and EP 0425235 B1); auristatins, e.g., monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,645, ... 483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res., vol. 53, pp. 3336-3342, 1993; and Lode et al., Cancer Res., vol. 58, pp. 2925-2928, 1998); anthracyclines, such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem., vol. 13, pp. 477-523, 2006; Jeffrey et al., Bioorganic & Med. Chem. Letters, vol. 16, pp. 358-362, 2006; Torgov et al., Bioconj. Chem., vol. 16, pp. 717-721, 2005; Nagy et al., Proc. Natl. Acad. Sci. USA, vol. 97, pp. 829-834, 2000; Dubowchik et al. al.,Bioorg.&Med.Chem.Letters,vol.12,vol.1529-1532,2002;King et al.,J.Med.Chem.,vol.45,pp.4336-4343, 2002; and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as decetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0173] In one embodiment, the binding agent is a cytotoxic agent selected from a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope.

[0174] In another embodiment, the binding agent is an enzymatically active toxin or fragment thereof, including, by way of example and not limitation, diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, or curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0175] In another embodiment, the conjugate is At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive atoms including radioactive isotopes of Lu. When a radioactive conjugate is used for detection, it can be used with a radioactive atom for scintigraphy studies, such as tc99m or I123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, as well as iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese.

[0176] Immunoconjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science, vol. 238, p. 1098, 1987. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent or chelator for conjugating radionucleotides to antibodies. See International Publication No. WO 1994 / 11026. The linker can be a "cleavable linker" that facilitates the release of cytotoxic drugs in cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992; U.S. Patent No. 5,208,020) can be used.

[0177] Immunoconjugates may be prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0178] Exemplary embodiments of ADCs include an antibody (Ab) that targets tumor cells, a drug moiety (D), and a linker moiety (L) that attaches the Ab to D. In some embodiments, the antibody is attached to the linker moiety (L) via one or more amino acid residues, e.g., lysine and / or cysteine. ADCs have the formula Ab-(LD) p where p is from 1 to about 20.

[0179] In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of free cysteine ​​residues. In some embodiments, free cysteine ​​residues are introduced into the antibody amino acid sequence by the methods described herein. Exemplary ADCs include, but are not limited to, antibodies with one, two, three, or four engineered cysteine ​​amino acids (Lyon et al., Methods in Enzym., vol. 502, pp. 123-138, 2012). In some embodiments, one or more free cysteine ​​residues already exist in the antibody without engineering, and in this case, the existing free cysteine ​​residues can be used to conjugate the antibody to a drug. In some embodiments, the antibody is exposed to reducing conditions to generate one or more free cysteine ​​residues before conjugation of the antibody.

[0180] Linkers are used to attach moieties to antibodies to form immunoconjugates such as ADCs. Suitable linkers are described in WO2017 / 180842.

[0181] Some drug moieties that can be attached to antibodies are described in WO2017 / 180842.

[0182] Drug moieties also include compounds with nucleolytic activity (eg, ribonucleases or DNA endonucleases).

[0183] In certain embodiments, the immunoconjugate may comprise a spin label for nuclear magnetic resonance (NMR) imaging, such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. For example, zirconium-89 can be complexed to various metal chelators and conjugated to antibodies for PET imaging (WO 2011 / 056983).

[0184] Radiolabels or other labels can be incorporated into the immunoconjugate using known techniques. For example, the antibody or fragment can be biosynthesized or chemically synthesized using suitable amino acid precursors, e.g., containing one or more fluorine-19 atoms in place of one or more hydrogen atoms. In some embodiments, the label, e.g., Tc 99 , I 123 ,Re 186 ,Re 188 , and In 111can be attached via a cysteine ​​residue in the antibody. In some embodiments, yttrium-90 can be attached via a lysine residue in the antibody. In some embodiments, iodine-123 can be incorporated using the IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun., vol. 80, pp. 49-57, 1978). "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes certain other methods.

[0185] In certain embodiments, the immunoconjugate may comprise an antibody conjugated to a prodrug-activating enzyme. The prodrug-activating enzyme can convert a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81 / 01145) into an active drug, e.g., an anticancer drug. Such immunoconjugates are useful in antibody-dependent enzyme-mediated prodrug therapy ("ADEPT"). Enzymes that can be conjugated to the antibody include, but are not limited to, alkaline phosphatase, which is useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase, which is useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase, which is useful for converting non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; and proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidase, and cathepsins (e.g., cathepsins B and L), which are useful for converting peptide-containing prodrugs into free drugs. D-alanylcarboxypeptidase, which is useful for converting prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes, such as β-galactosidase and neuraminidase, which are useful for converting glycosylated prodrugs into free drugs; β-lactamase, which is useful for converting drugs that are derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase and penicillin G amidase, which are useful for converting drugs that are derivatized with phenoxyacetyl or phenylacetyl groups at the amine nitrogen into free drugs. In some embodiments, enzymes can be covalently bound to antibodies by recombinant DNA techniques well known in the art. For example, see Neuberger et al., Nature, vol. 312, pp. 604-608, 1984.

[0186] D. Pharmaceutical Compositions In some embodiments, the present invention provides pharmaceutical compositions comprising anti-IL-22 antibodies, antibody fragments, variants, derivatives, or immunoconjugates thereof. The anti-IL-22 antibodies or antibody fragments have anti-inflammatory activity. Furthermore, IL-22 is known to be involved in the initiation or progression of cancer. Therefore, anti-IL-22 antibodies, antibody fragments, variants, derivatives, or immunoconjugates thereof can be used in pharmaceutical compositions for treating immune-related or proliferative diseases associated with IL-22 expression. The antibodies, fragments, variants, derivatives, or immunoconjugates thereof can be used as the sole active agent in the pharmaceutical composition or in combination with any suitable agent or other conventional treatment.

[0187] The antibody or antibody fragment may be formulated into a pharmaceutical composition to deliver about 0.0001 to 10.0 milligrams, or about 0.001 to 5 milligrams, or about 0.001 to 1 milligram, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or about 10 milligrams of antibody or antibody fragment. Multiple administrations at selected time intervals are possible.

[0188] For treating or reducing the severity of immune disorders, the appropriate dose of the compositions of the present invention will depend on the type of disorder being treated, the severity and course of the disorder, whether the composition is being administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the compounds, and the discretion of the attending physician.

[0189] For example, depending on the type and severity of the disease, about 1 μg / kg to about 15 mg / kg (e.g., 0.1 to 20 mg / kg) of antibody or antibody fragment is an initial candidate dose for administration to a patient, whether by one or more separate administrations or by continuous infusion. A typical daily dose can be about 1 μg / kg to about 100 mg / kg or more of antibody or antibody fragment, depending on the factors described above. For repeated administrations over several days or longer, depending on the condition, treatment can be sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be monitored by conventional techniques and assays.

[0190] Pharmaceutical compositions containing anti-IL-22 antibodies, antibody fragments, or immunoconjugates can be formulated according to known methods for preparing pharmaceutical compositions by mixing the antibody or antibody fragment having the desired purity, in the form of a lyophilized formulation or an aqueous solution, with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)).

[0191] Pharmaceutically acceptable carriers are employed in dosages and concentrations that are generally non-toxic to recipients and include, but are not limited to, the following: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as 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 (fewer than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; a sugar such as sucrose, mannitol, trehalose, or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., Zn-protein complex); and / or a non-ionic surfactant such as polyethylene glycol (PEG).

[0192] Pharmaceutically acceptable carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption, for example, aluminum monostearate and gelatin, in the composition.

[0193] In some embodiments, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for the formulation to be injected. These can be present in a particular isotonic sterile saline solution (monosodium or disodium phosphate, sodium, potassium, calcium, or magnesium chloride, etc., or a mixture of such salts), or in a dried, particularly lyophilized, composition that allows the constitution of an injectable solution upon addition of, for example, sterile water or saline.

[0194] In some embodiments, pharmaceutically acceptable tonicity agents, sometimes known as "stabilizers," are present to adjust or maintain the isotonicity of the liquid in the composition. When used with large charged biomolecules, such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with the charged groups on amino acid side chains, thereby reducing the potential for inter- and intramolecular interactions. Tonicity agents can be present in any amount from 0.1% to 25% by weight of the pharmaceutical composition, or from 1 to 5%. Exemplary tonicity agents include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0195] Additional pharmaceutically acceptable excipients that can be included in pharmaceutical compositions include agents that can function as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Excipients include polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polysaccharides, and the like. Examples of suitable reducing agents include polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides (e.g., raffinose); and polysaccharides (e.g., dextrin or dextran).

[0196] The pharmaceutical composition can include a pharmaceutically acceptable non-ionic surfactant or detergent (also known as a "wetting agent") to help dissolve the antibody or antibody fragment and to protect the antibody or antibody fragment from agitation-induced aggregation, thereby allowing the composition to be exposed to shear surface stresses without causing denaturation of the antibody or antibody fragment. The non-ionic surfactant can be present in a concentration range of about 0.05 mg / ml to about 1.0 mg / ml, or about 0.07 mg / ml to about 0.2 mg / ml.

[0197] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyol, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0198] Antibodies or antibody fragments can be formulated into pharmaceutical compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as potassium, ammonium, calcium, or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0199] In some embodiments, pharmaceutical compositions comprise a combination of at least one anti-IL-22 antibody or antibody fragment with at least one other therapeutic or prophylactic agent administered in combination therapy, which may be a cytokine inhibitor, growth factor inhibitor, immunosuppressant, anti-inflammatory agent, metabolic inhibitor, enzyme inhibitor, cytotoxic agent, and cytostatic agent, as described in more detail below. In one embodiment, the other therapeutic agent is a standard of care for arthritis, including, but not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs); corticosteroids, including prednisolone, prednisone, cortisone, and triamcinolone; and disease-modifying antirheumatic drugs (DMARDs), such as methotrexate, hydroxychloroquine (Plaquenil), and sulfasalazine, leflunomide (Arava); tumor necrosis factor inhibitors, including etanercept (Enbrel), infliximab (Remicade) (with or without methotrexate), and adalimumab (Humira), anti-CD20 antibodies (such as Rituxan), soluble interleukin-1 receptors, such as anakinra (Kineret), gold, minocycline (Minocin), penicillamine, and cytotoxic agents, including azathioprine, cyclophosphamide, and cyclosporine. Such combination therapies may advantageously utilize lower doses of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with various monotherapies. Additionally, other therapeutic agents disclosed herein may act on pathways in addition to or different from the IL-22 / IL-22R / IL-10R2 pathway and are therefore expected to enhance and / or synergize the effects of anti-IL-22 antibodies.

[0200] In some embodiments, other therapeutic or prophylactic agents used in combination with an anti-IL-22 antibody or fragment may be therapeutic agents that interfere with various stages of the autoimmune response and subsequent inflammatory response. In one embodiment, at least one anti-IL-22 antibody described herein may be formulated and / or co-administered with at least one cytokine and / or growth factor antagonist. Antagonists may include soluble receptors, peptide inhibitors, small molecules, ligand fusions, antibodies and binding fragments thereof (which bind to cytokines or growth factors or their receptors or other cell surface molecules), and "anti-inflammatory cytokines" and agonists thereof.

[0201] Non-limiting examples of these therapeutic or prophylactic agents include antagonists of at least one interleukin (e.g., IL-1, IL-2, IL-6, IL-7, IL-8, IL-12 (or one of its subunits p35 or p40), IL-13, IL-15, IL-16, IL-17A-F (including, for example, the IL-17A / IL-17F heterodimer), IL-18, IL-19, IL-20, IL-21, and IL-23 (or one of its subunits p19 or p40)); cytokines (e.g., TNFα, LT, EMAP-II, and GM-CSF); and growth factors (e.g., FGF and PDGF). Therapeutic agents also include, but are not limited to, antagonists of at least one receptor for interleukins, cytokines, and growth factors. Anti-IL-22 antibodies can further be used in combination with inhibitors (e.g., antibodies or binding fragments thereof) against cell surface molecules such as CD2, CD3, CD4, CD8, CD20 (e.g., Rituxan), CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, or their ligands (e.g., CD154 (gp39, CD40L)), or LFA-1 / ICAM-1 and VLA-4 / VCAM-1 (Yusuf-Makagiansar et al. (2002) Med Res Rev 22(2):146-67)).

[0202] In certain embodiments, antagonists that can be used in combination with the anti-IL-22 antibodies described herein can include antagonists of IL-1, IL-12 (or one of its subunits p35 or p40), TNFα, IL-15, IL-17A-F (including heterodimers thereof, e.g., the IL-17A / IL-17F heterodimer), IL-18, IL-19, IL-20, IL-21, and IL-23 (or one of its subunits p19 or p40), and their receptors.

[0203] In one embodiment, the other therapeutic or prophylactic agent is selected from an IL-12 antagonist (such as an antibody that binds to IL-12 (see, e.g., WO 00 / 56772) or one of its subunits p35 or p40); an IL-12 receptor inhibitor (such as an antibody against the IL-12 receptor); and a soluble IL-12 receptor and fragments thereof. In one embodiment, the other therapeutic agent is selected from an IL-15 antagonist, e.g., IL-15 or its receptor, a soluble fragment of the IL-15 receptor, and an IL-15 binding protein. In one embodiment, the other therapeutic agent is selected from an IL-12 antagonist, such as an antibody against IL-18, a soluble fragment of the IL-18 receptor, and an IL-18 binding protein (IL-18BP, Mallet et al. (2001) Circ. Res. 28). In one embodiment, the other therapeutic agent is selected from an IL-1 antagonist, such as an interleukin-1 converting enzyme (ICE) inhibitor (e.g., Vx740), an IL-1 antagonist (e.g., IL-1RA (ANIKINRA, AMGEN)), sIL-1RII (Immunex), and an anti-IL-1 receptor antibody.

[0204] In one embodiment, the other therapeutic or prophylactic agent is an antibody against TNF (e.g., human TNFα), such as D2E7 (human anti-TNFα antibody, U.S. Pat. No. 6,258,562, Humira®); CDP-571 / CDP-870 / BAY-10-3356 (humanized anti-TNFα antibody, Celltech / Pharmacia); cA2 (chimeric anti-TNFα antibody, Remicade®, Centocor); and anti-TNF antibody fragments (e.g., CPD870). Other examples are soluble TNF receptor (e.g., human p55 or p75) fragments and derivatives, such as p55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein, Lenercept®) and 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, Enbrel®, Immunex, see, e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest.Med.(1996) Vol.44, 235A). Further examples include enzyme antagonists (e.g., TNFα-converting enzyme inhibitors, such as α-sulfonylhydroxamic acid derivatives (WO 01 / 55112) or N-hydroxyformamide inhibitors (GW 3333, -005, or -022) and TNF-bp / s-TNFR (soluble TNF-binding proteins, see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S284; and Am.J. Physiol.Heart Circ.Physiol.(1995) Vol.268, pp.37-42). TNF antagonists include soluble TNF receptor (e.g., human p55 or p75) fragments and 75 derivatives such as kdTNFR-IgG; and TNFα-converting enzyme (TACE) inhibitors.

[0205] In one embodiment, the other therapeutic or prophylactic agent is selected from an IL-13 antagonist, such as a soluble IL-13 receptor and / or an anti-IL-13 antibody; and an IL-2 antagonist, such as an IL-2 fusion protein (e.g., DAB 486-IL-2 and / or DAB 389-IL-2, Theragen (see, Arthritis & Rheumatism (1993) Vol. 36, 1223) and an anti-IL-2R antibody (e.g., anti-Tac (humanized antibody, Protein Design Labs, see Cancer Res. 1990 Mar. 1; 50(5):1495-502)).

[0206] In one embodiment, the other therapeutic or prophylactic agent is selected from a non-depleting anti-CD4 inhibitor, such as IDEC-CE9.1 / SB 210396 (anti-CD4 antibody, IDEC / SmithKline). In one embodiment, the other therapeutic agent is selected from antagonists (such as antibodies, soluble receptors, or antagonists) of costimulatory molecules, such as CD80 (B7.1) and CD86 (B7.2); ICOSL, ICOS, CD28, and CTLA4 (e.g., CTLA4-1g); P-selectin glycoprotein ligand (PSGL); and anti-inflammatory cytokines and agonists (e.g., antibodies) thereof. Anti-inflammatory cytokines may include IL-4 (DNAX / Schering); IL-10 (SCH 52000, recombinant IL-10, DNAX / Schering); IL-13; and TGF.

[0207] In one embodiment, the other therapeutic or prophylactic agent is selected from anti-inflammatory agents, immunosuppressants, metabolic inhibitors, and enzyme inhibitors. Non-limiting examples of agents or inhibitors that can be used in combination with the IL-22 antagonists described herein include, but are not limited to, at least one of the following: nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, Tenidap (see, e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S280)), naproxen (e.g., Neuro Report (1996) Vol. 7, pp. 1209-1213), meloxicam, piroxicam, diclofenac, and indomethacin); sulfasalazine (e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S281), corticosteroids (such as prednisolone), cytokine suppressive anti-inflammatory drugs (CSAIDs), inhibitors of nucleotide biosynthesis (such as inhibitors of purine biosynthesis (e.g., folate antagonists such as methotrexate)), and inhibitors of pyrimidine biosynthesis (such as dihydroorotate dehydrogenase (DHODH) inhibitors such as leflunomide (see, e.g., Arthritis & Rheumatism, vol. 39, No. 9 (supplement), S131, 1996; Inflammation Research, vol. 45, pp. 103-107, 1996)).

[0208] The other therapeutic or prophylactic agent conjugated to the antibody is at least one of the following: corticosteroids (oral, inhaled, and local injection); immunosuppressants (such as cyclosporine and tacrolimus (FK-506)); mTOR inhibitors (sirolimus (rapamycin) or rapamycin derivatives (ester rapamycin derivatives such as CCI-779 (Elit. L. (2002) Current Opinion Investig. Drugs 3(8):1249-53; Huang, S. et al. (2002) Current Opinion Investig. Drugs 3(2):295-304)); TNFα and IL-1 (e.g., IRAK, IKK, p38, or MAP kinase inhibitors); COX2 inhibitors (e.g., celecoxib and its variants (MK-966) Arthritis & Rheumatism, vol. 39, No. 9 (supplement), S81 1996); phosphodiesterase inhibitors (see, e.g., R973401, Arthritis & Rheumatism, vol. 39, No. 9 (supplement), S282 1996); phospholipase inhibitors such as trifluoromethyl ketone analogs (e.g., cPLA2) Patent No. 6,350,892); inhibitors of vascular endothelial growth factor (VEGF); and inhibitors of angiogenesis.

[0209] The pharmaceutical composition may be suitable for injection into a subject. Such compositions may be sterile aqueous solutions or dispersions, including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The compositions must be sterile and fluid enough to allow easy syringeability. They must be stable under the conditions of manufacture and storage and must be free from the contamination of microorganisms such as bacteria and fungi.

[0210] Sterile injectable solutions are prepared by incorporating the required amount of antibody in a suitable solvent, if necessary, with one or more of the other ingredients discussed above, and then sterile filtered.Generally, dispersions are prepared by incorporating various sterilized antibody solutions into a sterile vehicle containing a basic dispersion medium and the other ingredients required from those listed above.In the case of sterile powders for preparing sterile injectable solutions, exemplary preparation methods are vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient and any additional desired ingredients from its solution that has previously been sterile filtered.

[0211] The pharmaceutical composition may be in the form of liposomes and / or nanoparticles for the introduction of antibodies or antibody fragments into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art. Liposomes are formed from phospholipids dispersed in an aqueous medium, spontaneously forming multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 Å, containing aqueous solution in their cores. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0212] Anti-IL-22 antibodies, antibody fragments, or immunoconjugates can be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization. For example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules can be used in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Nanocapsules can generally entrap compounds in a stable and reproducible manner. To avoid side effects caused by intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) are generally designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be easily produced. Sustained-release formulations can also be prepared.

[0213] Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antibody fragment, which matrices may be in the form of shaped articles, eg, films, or microcapsules.

[0214] Upon formulation, pharmaceutical compositions will be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The compositions are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although drug release capsules and the like can also be used.

[0215] E. Treatment and Prevention In one aspect, the present invention provides therapeutic and prophylactic methods using anti-IL-22 antibodies, antibody fragments, variants, derivatives, immunoconjugates, or any of the above pharmaceutical compositions. Such methods include in vitro, ex vivo, and in vivo therapeutic and prophylactic methods. In one embodiment, a method for inhibiting an IL-22-mediated signaling pathway is provided. A method for stimulating or inhibiting ThIL-17 cell function is provided. A method for treating inflammatory and / or autoimmune disorders is also provided. A method for treating disorders associated with IL-22 signaling is further provided. A method for treating ThIL-17-mediated disorders is also provided.

[0216] In one embodiment, a method of inhibiting an IL-22-mediated signaling pathway in a biological system is provided, comprising providing an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions to the biological system.

[0217] In another embodiment, a method for inhibiting ThIL-17 cell function is provided. The method includes exposing ThIL-17 cells to an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions. Exemplary ThIL-17 cell functions include, but are not limited to, stimulating cell-mediated immunity (delayed-type hypersensitivity); recruiting innate immune cells, such as myeloid cells (e.g., monocytes and neutrophils), to sites of inflammation; and stimulating inflammatory cell infiltration into tissues.

[0218] Anti-IL-22 antibodies, antibody fragments, variants, derivatives, immunoconjugates, or any of the above pharmaceutical compositions can be used to treat immune-related diseases, thrombotic diseases (thrombosis and atherothrombosis), and cardiovascular diseases. Immune diseases include arthritis, autoimmune diseases, chronic inflammation, inflammatory bowel disease, psoriasis, and T cell-mediated diseases. Anti-IL-22 antibodies or antibody fragments act as antagonists to IL-22, indirectly modulating downstream immune responses by acting on epithelial cells in solid tissues, e.g., T HThe antibody may modulate at least one IL-22-mediated immune response, such as blocking the expansion of T cell subsets, including IL-17 T cells. In one embodiment, the antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above pharmaceutical compositions is used in a method for modulating an immune response. The method comprises contacting IL-22 with an antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions of the invention, thereby modulating an immune response. In one embodiment, the immune response comprises cell proliferation, cytolytic activity, cytokine secretion, or chemokine secretion.

[0219] Thus, the antibodies, antibody fragments, variants, derivatives, immunoconjugates, or any of the above-described pharmaceutical compositions of the invention can be used to directly or indirectly inhibit the activity (e.g., proliferation, differentiation, and / or survival) of immune cells or hematopoietic cells (e.g., myeloid, lymphoid, or erythroid cells, or their precursor cells), and thus can be used in approaches to treat a variety of immune and hyperproliferative disorders. Non-limiting examples of treatable immune disorders include, for example, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, 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 (Type 1); skin (e.g., psoriasis), cardiovascular system (e.g., atherosclerosis), 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 related to 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 infections); transplant rejection and allergies.In one embodiment, the IL-22-associated disorder is an arthritic disorder, e.g., a disorder selected from one or more of rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or ankylosing spondylitis; a respiratory disorder, e.g., asthma, chronic obstructive pulmonary disease (COPD); or an IL-22-associated disorder, e.g., a disorder of the skin (e.g., psoriasis), the cardiovascular system (e.g., atherosclerosis), the nervous system (e.g., Alzheimer's disease), the liver (e.g., hepatitis), the kidney (e.g., nephritis), the pancreas (e.g., urinary tract infection), or the like. (e.g., pancreatitis), and gastrointestinal tract, e.g., colitis, Crohn's disease, and IBD; acute inflammatory conditions, e.g., endotoxemia, sepsis, and septicaemia, toxic shock syndrome and infections, multiple organ failure, respiratory disease (ARD), renal disorders such as amyloidosis, glomerulosclerosis, membranous nephropathy, renal arteriosclerosis, glomerulonephritis, fibroproliferative diseases of the kidney, and other renal dysfunction and tumors. Because IL-22 acts on epithelia, anti-IL-22 antibodies can be used to treat epithelial cancers, e.g., carcinoma, melanoma, and others. For a discussion of the rationale for IL-22 inhibition in these and other diseases, see WO 2003 / 083062 (pp. 58-75).

[0220] In yet another embodiment, a method of treating an autoimmune disorder is provided. The method comprises administering to a mammal in need of such treatment an effective amount of an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions. Autoimmune diseases include, but are not limited to, connective tissue diseases, multiple sclerosis, systemic lupus erythematosus, inflammatory arthritis (e.g., rheumatoid arthritis), autoimmune pulmonary inflammation, Guillain-Barré syndrome, autoimmune thyroiditis, insulin-dependent diabetes mellitus, uveitis, myasthenia gravis, graft-versus-host disease, autoimmune inflammatory eye disease, psoriasis, autoimmune-associated arthritis (e.g., rheumatoid arthritis), autoimmune inflammation of the brain, and inflammatory bowel disease. In one embodiment, the autoimmune disorder is an IL-23-mediated autoimmune disorder.

[0221] In one embodiment, a method for treating psoriasis and / or disorders characterized by psoriatic symptoms is provided. Psoriasis is considered an autoimmune disease in which T cells of the immune system recognize proteins in the skin and attack the areas where the proteins are found, causing the rapid proliferation of new skin cells and painful, raised, scaly lesions. These lesions are characterized by the hyperproliferation of keratinocytes and the accumulation of activated T cells in the epidermis of psoriatic lesions. While the initial molecular cause is unknown, genetic linkages have been mapped to at least seven psoriasis susceptibility loci (Psor1:6p21.3, Psor2:17q, Psor3:4q, Psor4:1cent-q21, Psor5:3q21, Psor6:19p13, and Psor7:1p). Some of these loci are associated with other autoimmune / inflammatory diseases, including rheumatoid arthritis, atopic dermatitis, and inflammatory bowel disease (IBD). Current approaches to the treatment of psoriasis include the administration of IL-12 or TNF-α antagonists. See, for example, Nickoloff et al. (2004) J. Clin. Invest., vol. 113, pp. 1664-1675; Bowcock et al. (2005) Nat. Rev. Immunol., vol. 5, pp. 699-711; Kauffman et al. (2004) J. Invest. Dermatol., vol. 123, pp. 1037-1044. It is known that a different IL-23 / IL-22 signaling pathway is involved in the pathogenesis of psoriasis. Therefore, therapeutic agents that modulate this signaling pathway may provide an alternative or complement other approaches to the treatment of psoriasis.

[0222] In one embodiment, a method for treating psoriasis comprises administering to a patient an effective amount of an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions. In various embodiments, the method further comprises administering (either in the same pharmaceutical composition or a separate pharmaceutical composition) at least one additional therapeutic agent. In one such embodiment, the additional therapeutic agent is an antagonist of at least one cytokine selected from IL-19, IL-20, and IL-24. Such antagonists include, but are not limited to, antibodies that bind to IL-19, IL-20, IL-24, IL-20Ra, IL-20Rb, or IL-10R2. Any number of such antibodies may be selected in any combination. In another embodiment, the additional therapeutic agent is an agent known to be effective in treating psoriasis. Identification of such therapeutic agents is described, for example, in Nickoloff et al. (2004) J. Clin. Invest. 113:1664-1675; Bowcock et al. (2005) Nat. Rev. Immunol. 5:699-711; and Kauffman et al. (2004) J. Invest. Dermatol. 123:1037-1044. Such therapeutic agents include, but are not limited to, therapeutic agents that target T cells, such as efalizumab and / or alefacept; IL-12 antagonists, such as blocking antibodies that bind to IL-12 or its receptor; and TNF-α antagonists, such as blocking antibodies that bind to TNF-α or its receptor.

[0223] In one embodiment, a method for treating multiple sclerosis is provided. The method comprises administering to a patient an effective amount of an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions. Multiple sclerosis is a central nervous system disease characterized by inflammation and loss of myelin (the fatty substance that insulates nerves and is required for proper nerve function).

[0224] In one embodiment, a method for treating arthritis comprises administering to a patient an effective amount of an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above pharmaceutical compositions. Arthritis is a disease characterized by inflammation of the joints. Rheumatoid arthritis is the most common form of arthritis and involves inflammation of the connective tissue and synovium (the membrane that covers the joints). The inflamed synovium often infiltrates the joint, damaging articular cartilage and bone. IL-22 and IL-22R protein and / or transcripts are associated with both human diseases. In RA synovial biopsies, IL-22 protein is expressed in the vimentin + Synovial fibroblasts and some CD68 + IL-22R is detected in macrophages, while IL-22R is detected in synovial fibroblasts. Treatment of synovial fibroblasts with IL-22 induces the production of monocyte chemoattractant protein-1 (MCP-1) and systemic metabolic activity (Ikeuchi, H., et al., (2005) Arthritis Rheum., vol. 52, pp. 1037-46). IL-22 inhibitors improve the symptoms of rheumatoid arthritis (WO 2005 / 000897 A2; U.S. Patent No. 6,939,545). Increased secretion of inflammatory cytokines and chemokines, and more importantly, increased disease caused by IL-22-dependent immune responses, can be treated by the methods of the present invention. Similarly, the methods of the present invention can be used to treat RA or other arthritic diseases in humans.

[0225] In one embodiment, a method for treating transplant rejection comprises administering to a patient an effective amount of an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the pharmaceutical compositions described above. Transplant rejection is an immunological phenomenon in which donor-derived tissue is specifically "attacked" by host immune cells, primarily T cells, whose T cell receptors recognize donor MHC molecules as "foreign." This recognition activates the T cells, which proliferate and secrete various cytokines and cytolytic proteins, ultimately destroying the graft. Mixed lymphocyte reaction (MLR) and transplantation models have been described by Current Protocols in Immunology, Second Edition, Coligan et al., eds., John Wiley & Sons, 1994; Kasaian et al. (Immunity (2002) 16: 559-569); Fulmer et al. (Am. J. Anat. (1963) 113: 273-285); and Lenschow et al. (Science (1992) 257: 789-792). The methods of the invention can be used to reduce MLR and treat transplant rejection and related diseases (e.g., graft-versus-host disease) in humans that are dependent on IL-22.

[0226] In one embodiment, the invention provides a method of treating a hyperproliferative disorder associated with aberrant activity of IL-22- and IL-22R / IL-10R2-responsive cells, comprising administering an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions to inhibit or reduce hyperproliferation of IL-22- and / or IL-22R- and / or IL-10R2-responsive cells in a subject. IL-22 and IL-22R expression is constitutive in epithelial cells of many tissues, including, but not limited to, the pancreas, lung, skin, intestine, liver, and kidney (Kotenko, SV et al. (2001) J. Biol. Chem., vol. 276, pp. 2725-32; Xie, MH et al. (2000) J. Biol. Chem., vol. 275, pp. 31335-9; Wolk, K. et al. (2004) Immunity, vol. 21, pp. 241-54). Furthermore, the IL-22 receptor complex is also expressed on the surface of fibroblasts derived from diseased joints and normal intestine (Ikeuchi, H. et al. (2005) Arthritis Rheum., vol. 52, pp. 1037-46; Andoh, A. et al. (2005) Gastroenterology, vol. 129, pp. 969-84). Neoplastic derivatives of these cell types are hyperresponsive to IL-22, which modulates their in vivo survival capacity.Thus, the methods of the present invention can be used to inhibit the progression of neoplasms such as squamous cell carcinoma, basal cell carcinoma, transitional cell papilloma and transitional cell carcinoma, adenoma, adenocarcinoma, linitis plastica, insulinoma, glucagonoma, gastrinoma, vipoma, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, adenoid, Carcinoid tumor of the appendix, prolactinoma, oncocytoma, Hürthle cell adenoma, renal cell carcinoma, Grawitz tumor, multiple endocrine adenoma, endometrioid tumor, adnexal and cutaneous adnexal neoplasms, mucoepidermoid neoplasms, cystic, mucinous, and serous neoplasms, cyst adenoma, pseudomyxoma peritonei, ductal, lobular, and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, Warthin tumor, thymoma, specialized gonadal tumors, sex cord-stromal tumors, thecoma, granulosioma, ovarian male germinoma, Sertoli-Leydig cell tumor, paraganglioma, pheochromocytoma, glomus tumor, melanocytic nevus, malignant melanoma, melanoma, nodular melanoma, dysplastic nevus, malignant melanoma, superficial spreading melanoma, or acral lentiginous melanoma.

[0227] In yet another embodiment, a method of inhibiting tumor progression is provided, comprising administering to a mammal an effective amount of an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above pharmaceutical compositions.

[0228] In another embodiment, the invention provides a method for reducing, inhibiting, or decreasing an acute phase response in a subject. The method comprises administering to the subject an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions in an amount sufficient to reduce, inhibit, or decrease the acute phase response in the subject. In one embodiment, the subject is a mammal, e.g., a human, suffering from an IL-22-associated disorder described herein, including, e.g., a respiratory disorder, an inflammatory disorder, and an autoimmune disorder. In one embodiment, the IL-22-binding agent is administered locally, e.g., topically, subcutaneously, or otherwise not into the systemic circulation.

[0229] In another embodiment, the present invention provides a method for administering an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions simultaneously or sequentially with an antibody against another immune disease-related or tumor-associated antigen, such as an antibody that binds CD20, CD11a, CD18, ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein may be co-administered to a patient. In certain embodiments, it may also be beneficial to administer one or more cytokines to a patient. In certain embodiments, an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions of the present invention is co-administered with a growth inhibitory agent. For example, the growth inhibitory agent can be administered before, after, or simultaneously with the administration of the composition. Suitable doses of the growth inhibitory agent are those currently used and may be reduced due to the combined action (synergism) of the growth inhibitory agent and the composition.

[0230] In each and all of the above methods, the antibodies, antibody fragments, variants, derivatives, or any of the above pharmaceutical compositions of the present invention can be used in therapy alone, as immunoconjugates, or in combination with other agents. For example, the antibodies, antibody fragments, variants, derivatives, or any of the above pharmaceutical compositions of the present invention can be used in therapy alone, as immunoconjugates, or in combination with other agents. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is an anti-angiogenic agent. In some embodiments, the additional therapeutic agent is a VEGF antagonist (in some embodiments, an anti-VEGF antibody, e.g., bevacizumab). In some embodiments, the additional therapeutic agent is an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, the additional therapeutic agent is a chemotherapeutic agent and / or a cytostatic agent. In some embodiments, the additional therapeutic agent is a taxoid (e.g., paclitaxel) and / or a platinum agent (e.g., carboplatinum). In some embodiments, the additional therapeutic agent is an agent that enhances the patient's immunity or immune system.

[0231] Such combination therapy as described above encompasses combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, where administration of the antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above pharmaceutical compositions can occur before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. The antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above pharmaceutical compositions can also be used in combination with radiation therapy.

[0232] F. Diagnostic Methods In some embodiments, the anti-IL-22 antibodies, antibody fragments, variants, derivatives, immunoconjugates, or any of the above pharmaceutical compositions provided herein can be used to quantitatively or qualitatively detect the presence of IL-22 in a biological sample. In certain embodiments, the biological sample comprises cells or tissues, such as breast, pancreatic, esophageal, lung, and / or brain cells or tissues.

[0233] In exemplary embodiments, the antibody, antibody fragment, variant, derivative, immunoconjugate or any of the above pharmaceutical compositions of the present invention can be labeled with a detectable molecule or substance, such as the above-mentioned fluorescent molecules, radioactive molecules or any other label known in the art. For example, the antibody of the present invention can be labeled with a radioactive molecule. Suitable radioactive molecules include, but are not limited to, radioactive atoms used in scintigraphy tests, e.g., 123 I, 124 I, 111 In, 186 Re, and 188 Alternatively, the antibody or antibody fragment of the present invention can be labeled with a spin label for nuclear magnetic resonance (NMR) imaging, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. After administration of the antibody, the distribution of the radiolabeled antibody within the patient is detected. Any suitable known method can be used. Some non-limiting examples include computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence, and ultrasound diagnosis.

[0234] In certain embodiments, labels include labels or moieties that are directly detected (e.g., fluorescent labels, chromophores, electron-dense, chemiluminescent, and radioactive labels), and moieties such as enzymes or ligands that are indirectly detected, e.g., via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C.125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, enzymes that oxidize dye precursors using hydrogen peroxide, such as heterocyclic oxidases coupled with HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0235] In one embodiment, a method for diagnosing psoriasis in a mammal is provided, comprising detecting the expression level of a gene encoding an IL-22 or IL-22R polypeptide in a test sample of tissue cells obtained from the mammal, wherein a higher expression level in the test sample compared to a control sample (e.g., a sample of known normal tissue cells of the same cell type) indicates the presence of psoriasis in the mammal from which the test sample was obtained. Detection may be qualitative or quantitative. In one embodiment, the test sample comprises blood or serum. In one embodiment, detecting the expression level of a gene encoding an IL-22 or IL-22R polypeptide comprises (a) contacting an anti-IL-22 antibody or anti-IL-22R antibody or antibody fragment with the test sample obtained from the mammal, and (b) detecting the formation of a complex between the antibody and the IL-22 or IL-22R polypeptide in the test sample. The antibody may be linked to a detectable label. Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluorometry, or other techniques known in the art. A test sample can be obtained from an individual suspected of having psoriasis.

[0236] The antibodies, antibody fragments, variants, derivatives, immunoconjugates, or any of the above pharmaceutical compositions of the present invention may be useful for the diagnosis and staging of cancers and diseases associated with IL-22 overexpression, including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, sarcoma, blood cancer (leukemia), astrocytoma, various types of head and neck cancer, and other hyperproliferative diseases of IL-22 expression or overexpression.

[0237] The antibodies, antibody fragments, variants, derivatives, immunoconjugates, or any of the above-mentioned pharmaceutical compositions of the present invention can be used in methods for diagnosing and staging cancers and diseases associated with IL-22 overexpression. The antibodies, antibody fragments, variants, derivatives, immunoconjugates, or any of the above-mentioned pharmaceutical compositions of the present invention can be used in methods for diagnosing immune system diseases in which IL-22 expression is increased or decreased. In certain embodiments, the present invention provides methods for diagnosing diseases associated with IL-22 expression or overexpression. Examples of such diseases include immune-related diseases, thrombotic diseases (thrombosis and atherothrombosis), and cardiovascular diseases. Immune-related diseases include arthritis, autoimmune diseases, chronic inflammation, inflammation, inflammatory bowel disease, psoriasis, and T-cell-mediated diseases.

[0238] In one embodiment, an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above pharmaceutical compositions is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of IL-22 in a biological sample is provided. In a further aspect, a method for quantifying the amount of IL-22 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above pharmaceutical compositions described herein under conditions that allow binding to IL-22, and detecting whether a complex forms. Such a method can be performed in vitro or in vivo. In one embodiment, an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above pharmaceutical compositions is used to select subjects eligible for treatment. In some embodiments, the treatment will involve administering to the subject an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above pharmaceutical compositions.

[0239] In another embodiment, the present invention relates to a diagnostic kit comprising an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above pharmaceutical compositions in suitable packaging. The kit may also include instructions for using the composition to detect IL-22 or IL-22R polypeptide. In one aspect, the diagnostic kit is for diagnosing psoriasis.

[0240] G. Products and Kits In another aspect of the present invention, an article of manufacture containing an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-mentioned pharmaceutical compositions useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders is provided. The article of manufacture comprises 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 by itself or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-mentioned pharmaceutical compositions. The label or package insert indicates that the composition is used for treating the selected condition. Additionally, the article of manufacture may include (a) a first container containing a composition comprising an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions; and (b) a second container containing a composition comprising an additional cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0241] It is understood that any of the above articles of manufacture may include one or more anti-IL-22 antibodies, antibody fragments, variants, derivatives, immunoconjugates, or any of the above pharmaceutical compositions.

[0242] Finally, the present invention also provides kits comprising at least one anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions of the present invention. The kits can be used in detecting IL-22 expression (increase or decrease) or in therapeutic or diagnostic assays. The kits of the present invention can contain an anti-IL-22 antibody, antibody fragment, variant, derivative, immunoconjugate, or any of the above-described pharmaceutical compositions coupled to a solid support, such as a tissue culture plate or beads (e.g., sepharose beads). Kits containing antibodies for in vitro detection and quantification of IL-22, for example, in ELISA or Western blot, can be provided. Such antibodies useful for detection can be provided with a label, such as a fluorescent or radiolabel.

[0243] The kit further contains instructions for its use. In some embodiments, the instructions include those required by the US Food and Drug Administration for in vitro diagnostic kits. In some embodiments, the kit further includes instructions for diagnosing the presence or absence of cerebrospinal fluid in a sample based on the presence or absence of IL-22 in the sample. In other embodiments, the kit further includes one or more enzymes, enzyme inhibitors, or enzyme activators. In yet other embodiments, the kit further includes one or more chromatography compounds. In yet other embodiments, the kit further includes one or more compounds used to prepare a sample for a spectrophotometric assay. In further embodiments, the kit further includes a reference material for interpreting the presence or absence of IL-22 according to the intensity, color spectrum, or other physical attribute of the indicator.

[0244] The following examples are illustrative, but not limiting, of the IL-22 antibodies of the present disclosure. Other suitable modifications and adaptations of the variety of conditions and parameters normally found in the art and obvious to those skilled in the art are within the scope of the present disclosure. [Example]

[0245] Example 1: Anti-IL-22 antibodies Mice were immunized with human IL-22 to generate a functional hybridoma clone, 3C3, expressing a mouse monoclonal antibody (3C3 mAb) that binds to human IL-22 (hIL-22). The binding affinity of 3C3 mAb to hIL-22 is approximately 100 pM (as measured by SPR analysis). However, 3C3 mAb was unable to bind to mouse IL-22 (mIL-22). 3C3 mAb can block the induction of Stat3 phosphorylation by hIL2 in HepG2 cells. The 3C3 mAb was humanized to generate the humanized antibody hum10.

[0246] Hum10 was selected for comprehensive positional evolution (CPE®) to generate a CPE® library containing Hum10 variants. Each position in the six CDRs of hum10 was substituted with at least 15 amino acids to generate antibody variants. The CDR positions in hum10 are shown in Figure 1, with some exemplary substitutions shown. There are a total of 63 positions across the six CDRs. The CPE® library contained a total of 1,068 antibody variants (472 variants with mutations in the light chain CDRs and 596 variants with mutations in the heavy chain CDRs). Each antibody variant was verified by sequencing. CPE® variants were transfected into CHO cells in a 96-well format, and supernatants were harvested 48 hours posttransfection. The expression levels of CPE® variants in the supernatants were measured using a quantitative ELISA assay based on commercially available purified human IgG.

[0247] The CPE® library was screened for antibodies capable of binding to both human IL-22 and mouse IL-22 with high affinity. Specifically, CPE® variants were screened for mouse IL-22 (mIL-22) binding affinity by affinity ELISA. hum10 was included on every affinity ELISA plate as a control. CPE® variants with increased binding affinity to mIL-22 compared to hum10 were scored as primary CPE® hits. Primary CPE® hits were re-arrayed, re-transfected, and screened again by affinity ELISA, as well as by titration ELISA with both mIL-22 and human IL-22 (hIL-22) for antibodies that bound to both mIL-22 and hIL-22. Screening identified 10 mutant antibodies, as shown in Table 1. These 10 mutant antibodies were selected based on ELISA data, sequence diversity, and the ability to scale up and purify.

[0248] Example 2: Purified anti-IL-22 antibody The selected anti-IL-22 antibodies were expressed in CHO cells, and the expressed antibodies were purified for further testing. To confirm the purity of the antibodies, they were loaded onto a 10% SDS-PAGE gel. Under non-reducing conditions, the antibodies showed a single band (Figure 4, left half), indicating intact antibodies with paired light and heavy chains. Under reducing conditions, the antibodies separated into light and heavy chains, appearing as two bands in each lane (Figure 4, right half). Lane M shows the molecular weight markers in kDa for each band marked on the gel.

[0249] The purified antibodies were also analyzed by size exclusion chromatography on a Superdex200 5 / 150 GL column at a flow rate of 0.3 ml / min. The wash buffer was 1x PBS buffer. A total of 50 μl of antibody was injected onto the column. SEC analysis yielded a single peak (Figures 5A-5C), indicating the antibody was highly pure. Figure 5A shows the purity of CPS02, and Figure 5B shows the purity of CPS09. A commercially available human IgG standard was used as a control, and the results are shown in Figure 5C. The single peak indicated that the purified antibody was sufficiently pure and did not form aggregates under the test conditions.

[0250] Example 3: Specificity of anti-IL-22 antibodies The specificity of the 10 antibodies in Table 1 was assayed by ELISA using both human and mouse IL-22, as well as related antigens (human IL19, human IL20, human IL24, human IL26, INF alpha A, INF-gamma, INF-lambda 1, INF-2, and a nonspecific antigen). Plates were coated with 100 μl of antigen at a concentration of 1 μg / ml. The plates were exposed to antibody at a concentration of 100 ng / ml. Bound antibody was detected with anti-hIgG-HRP in an ELISA assay.

[0251] The 10 antibodies in Table 1 were found to be able to bind to human and mouse IL-22 with comparable affinity, but they showed very low affinity for related antigens (Figure 6), indicating that the 10 antibodies are specific for human and mouse IL-22 and have little affinity for other related antigens.

[0252] Example 4: Binding affinity of anti-IL-22 antibodies to mIL-22 and hIL-22 The affinities of the 10 antibodies listed in Table 1 were measured by surface plasma resonance (SPR) using a capture assay on an SPR-2 instrument (Sierra Sensors, Hamburg, Germany). The antigens hIL-22 and mIL-22 were immobilized on the chip. The running buffer for the assay contained 10 mM HEPES, pH 7.4, 500 mM NaCl, and 0.05% TWEEN® 20. The buffer flow rate was 25 μl / min throughout the assay. The off-rate was measured for 6 min. Binding constants were calculated using the curve-fitting software Analyzer2 (Sierra Sensors). Sensorgrams were analyzed using the instrument-specific software and BIAevaluation 4.1 software. Binding data were analyzed using a Langmuir 1:1 model (human IL-22) or a heterogeneous ligand model (murine IL-220).

[0253] Antibodies were tested at four different concentrations of each antibody: 0.33 nM, 0.67 nM, 3.3 nM, and 6.7 nM for hIL-22; and 1, 2, 10, and 20 nM for mIL-22. Antibody CPE-LC-E049K was used as a control. Binding curves were generated and the K, Kd, ​​and K of the antibodies were measured as shown in Table 2. D was used to calculate

[0254] Example 5: Induction of Stat3 phosphorylation 30,000 human HepG2 cells were seeded in a 96-well plate and serum-starved overnight. Three-fold dilutions of anti-IL-22 antibodies and control hum10 antibodies at concentrations of 0.11 μg / mL, 0.33 μg / mL, 1 μg / mL, and 3 μg / mL were incubated with 200 ng / mL hIL-22 or mIL-22 at 37°C for 1 hour before being added to serum-starved human HepG2 cells. HepG2 cells were incubated with the antibody-IL-22 mixture for 20 minutes at 37°C. After incubation, HepG2 cells were washed with PBS and lysed. Half of the cell lysates were used to measure phosphorylated Stat3 levels using Cell Signaling kit #7300 according to the vendor's protocol, and the other half was used to measure total Stat3 levels by Western blot analysis using a Santa Cruz rabbit polyclonal antibody against human Stat3 (Sc-7179).

[0255] Inhibition of Stat3 phosphorylation by anti-IL-22 antibodies via binding to human IL-22 is shown in Figure 7A, and inhibition of Stat3 phosphorylation via binding to murine IL-22 is shown in Figure 7B. A negative control human IgG antibody showed no inhibition of Stat3 phosphorylation. Select anti-IL-22 antibodies showed dose-dependent inhibition of Stat3 phosphorylation (Figures 7A-7B).

[0256] Example 6: Inhibition of CXCL1 production Various concentrations of anti-IL-22 antibody were incubated with HT29 cells, which normally produce the cytokine CXCL1. CXCL1 production by HT29 cells was measured over time, and the results are shown in Figure 8. Inhibition of CXCL1 production by the antibody was dose-dependent, indicating the role of the antibody in inhibiting cytokine production.

[0257] Example 7: Pharmacokinetics of anti-IL-22 antibodies in mice Two anti-IL-22 antibodies, CPS02 and CPS09, were tested in NOD SCID mice. The antibodies were injected intravenously at two doses: 0.3 mg / kg and 10 mg / kg. Three mice were used for each dose group. Blood samples were collected from the mice at 10 minutes, 1 hour, 3 hours, 7 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 240 hours, and 336 hours after intravenous injection. The concentrations of the injected antibodies in the blood samples were measured by solid-phase ELISA.

[0258] The half-lives of the antibodies were long in mice, as shown in Figures 9A-9B for CPS02 and Figures 10A-10B for CPS09. The half-life of CPS02 in mice was approximately 18 hours after a 10 mg / kg injection. The half-life of CPS09 in mice was approximately 16 hours after a 10 mg / kg injection.

[0259] Example 8: Suppression of acute phase responses by IL-22 Five micrograms of mIL-22 was used to stimulate an acute phase response in mice. The acute phase response was measured by serum amyloid (SAA) levels in the mouse blood. Anti-IL-22 antibody was administered at two different doses to inhibit the IL-22-induced acute phase response: 5 μg and 50 μg. Five mice were used in each dose group. SAA levels were measured 24 hours after injection of IL-22 and / or antibody, and the results are shown in Figure 11.

[0260] When mice were injected with mIL-22 alone, they produced high levels of SAA. When mice were injected with anti-IL-22 antibody alone, the SAA concentration in the mice was low due to lack of stimulation (mIL-22, right panel of Figure 11). When mice were injected with both mIL-22 and anti-IL-22 antibody, the SAA concentration in the mice was significantly lower than that in mice injected with mIL-22 alone, indicating that the anti-IL-22 antibody effectively inhibited the acute phase response in the mice (left panel of Figure 11).

[0261] Example 9: Treatment of imiquimod-induced psoriasis in mice Two anti-IL-22 antibodies of the present invention, CPS02 and CPS09, were used to treat imiquimod (IMQ)-induced psoriasis in mice (Figure 12). The mice were male BALB / c mice, an albino, laboratory-bred strain of house mouse. A total of 70 mice weighing 18-20 g were acclimated for 7 days before psoriasis was induced.

[0262] Seventy mice were randomly assigned to seven groups (10 mice per group). Group 1 (Group 1) served as a negative control; mice were not administered imiquimod, but instead, petrolatum was used. Therefore, psoriasis was not induced in this negative control group. The other six groups (Groups 2-7) were administered imiquimod (5% cream) at a dose of 82.5 mg / mouse once daily (qd) from day 0 to day 7 for 8 consecutive days to induce psoriasis (20 mg on the left ear, 62.5 mg on the shaved back). The dorsal skin was divided into 2 x 3 cm sections. 2 The area was shaved to expose the Vaseline control group. Vaseline was applied at a dose of 82.5 mg / mouse once daily (qd) from day 0 to day 7 for 8 consecutive days (20 mg on the left ear, 62.5 mg on the shaved back).

[0263] Only mice in groups 3 to 7 received psoriasis treatment; groups 1 and 2 were untreated, as shown in Table 5. Group 2 was a group of psoriasis-induced mice that served as a control group without treatment. Groups 3 to 7 were treated with various treatment regimens, as shown in Table 5. The administration period completely overlapped with the imiquimod application period (days 0 to 7). For group 2, mice were administered both imiquimod and dexamethasone on each of the 8 days (days 0 to 7). For the group receiving intravenous (IV) injection, imiquimod was applied within 1 hour after IV injection on days 0 and 3. [Table 5]

[0264] Group 3 was treated with the steroid dexamethasone, a current therapeutic treatment used for psoriasis. Dexamethasone was applied topically twice daily (BID) for 8 days. Group 4 was treated with phosphate-buffered saline (PBS) and served as a negative control. Group 5 received an isotype control antibody, which also served as a negative control. Groups 6 and 7 received the anti-IL-22 antibodies CPS02 (A) and CPS09 (B), respectively. Treatment for Groups 4 through 7 was administered via a single intravenous (IV) injection on days 0 and 3. Three mice in Group 6 died on day 0, and two died on day 1. On day 3, the IV injection dose for this group was reduced to 10 mg / kg.

[0265] During the 8-day treatment period, ear thickness was measured on days 0, 3, 5, and 7. Body weight and various skin scores were recorded daily. Photographs of the psoriasis sites were taken on days 0, 3, 5, and 7. On days 1 and 4, approximately 60 μl of blood was collected via retro-orbital bleeding over a 24-hour period and placed into BD Microtainer® tubes coated with dipotassium ethylenediaminetetraacetic acid (K2EDTA). Plasma was then collected (to obtain at least 20 μl of plasma) and stored at -80°C. These samples were further analyzed, and the results are shown in Figure 12.

[0266] Skin scores for erythema, scaling, and thickness were measured daily according to an established grading system. Erythema, scaling, and thickness were scored independently on a scale of 0 to 4: 0 (none), 1 (mild), 2 (moderate), 3 (marked), and 4 (very marked). A cumulative (total) skin score was also calculated.

[0267] At the end of treatment (day 7), the following endpoints were collected: Spleen index (spleen weight / body weight) Blood serum collected and stored at -80℃ Affected skin samples taken from the back and left ear (1) One slide for the back skin and one slide for the left ear for each mouse (2) One representative photograph per group Real-time PCR was performed on dorsal skin samples. Total mRNA was extracted from affected dorsal skin and subjected to real-time PCR to determine the expression of IL-23, IL-22, CCL3, CXCL3, NPG, IL-17A, S100A7, and loricrin (β-actin as a control).

[0268] Treatment effects were statistically analyzed using two-way analysis of variance (ANOVA, Bonferroni posttest) or one-way ANOVA. Comparisons between treatment and control groups are shown in Figures 13-18 with p values ​​less than 0.05 (*), less than 0.01 (**), or less than 0.001 (***).

[0269] It was observed that the anti-IL-22 antibodies CPS02 (A) and CPS09 (B) reduced weight loss in treated mice compared to mice treated with dexamethasone. In fact, the anti-IL-22 antibody CPS09 (B) resulted in weight loss similar to that of the PBS buffer negative control. This is a clear indication that the anti-IL-22 antibodies of the present invention reduced at least one adverse side effect when compared to known treatment with dexamethasone. See Figure 13.

[0270] Psoriasis symptoms are measured by observing skin thickness, erythema, and scaling. Thinner skin thickness is an indicator of more successful treatment. Mice treated with anti-IL-22 antibodies CSP02 (A) and CSP09 (B) had thinner ear skin thickness than mice treated with the negative control, isotype-matched antibody, PBS, and vehicle. The ear thickness of mice treated with dexamethasone was similar to that of mice not receiving imiquimod induction. See Figure 14.

[0271] Skin erythema scores in mice treated with anti-IL22 antibodies CSP02 (A) and CSP09 (B) were also lower than those observed in mice treated with the negative control, isotype-matched antibody, PBS, and vehicle. Again, skin erythema scores in mice treated with dexamethasone were similar to those in mice not treated with imiquimod. See Figure 15.

[0272] The skin scaling scores of mice treated with anti-IL-22 antibodies CSP02 (A) and CSP09 (B) were also lower than those of mice treated with the negative control, isotype-matched antibody, PBS, and vehicle. Again, the skin scaling scores of mice treated with dexamethasone were similar to those of mice not subjected to imiquimod induction. See Figure 16.

[0273] The skin thickness of mice treated with anti-IL-22 antibodies CSP02 (A) and CSP09 (B) was also lower than that of mice treated with the negative control, isotype-matched antibody, PBS, and vehicle. Again, the skin thickness of mice treated with dexamethasone was similar to that of mice not induced with imiquimod. See Figure 17.

[0274] Finally, the overall condition of the skin was evaluated as a total skin score for treated mice. The total skin scores for mice treated with the anti-IL-22 antibodies CSP02 (A) and CSP09 (B) were also lower than those for mice treated with the negative control, isotype-matched antibody, PBS, and vehicle. Mice treated with the anti-IL-22 antibody CSP09 (B) had particularly good total skin scores. Again, the total skin scores for mice treated with dexamethasone were similar to those for mice not challenged with imiquimod. See Figure 18.

[0275] These results demonstrate that anti-IL-22 antibodies are effective in treating psoriasis in a mouse model by improving psoriasis symptoms such as skin thickness, erythema, and scaling. Furthermore, conditionally active anti-IL-22 antibodies exhibited reduced side effects compared to known treatments for psoriasis. The anti-IL-22 antibody CSP09(b) was particularly effective in treating psoriasis, with minimal side effects.

[0276] However, although numerous features and advantages of the present invention have been set forth in the foregoing description, together with details of the structure and function of the invention, it is to be understood that the disclosure is illustrative only, and that changes may be made in details, particularly as to the shape, size, and arrangement of parts, within the principles of the invention to all extents indicated in the broad sense of the terms expressed in the appended claims.

[0277] All documents cited herein are incorporated herein by reference in their entirety to provide at least the disclosure upon which they are specifically relied upon. Applicants do not intend to publicly offer any disclosed embodiments, and to the extent that any modifications or variations of the disclosure may not fall literally within the scope of the claims, they are considered part thereof under the doctrine of equivalents.

[0278] JPEG2025172766000006.jpg216170JPEG2025172766000007.jpg216170JPEG2025172766000008.jpg216170JPEG202 5172766000009.jpg216170JPEG2025172766000010.jpg216170JPEG2025172766000011.jpg216170JPEG20251727660 00012.jpg216170JPEG2025172766000013.jpg216170JPEG2025172766000014.jpg216170JPEG2025172766000015.j pg216170JPEG2025172766000016.jpg216170JPEG2025172766000017.jpg216170JPEG2025172766000018.jpg216170

Claims

1. SASSSVSX 1 a light chain CDR1, X having an amino acid sequence selected from 2 TX 3 KLX 4 a light chain variable region comprising a light chain CDR2 having an amino acid sequence selected from the group consisting of: QQWSSNPYIT (SEQ ID NO: 2), and a light chain CDR3 having an amino acid sequence of QQWSSNPYIT (SEQ ID NO: 3); GYIFX 5 a heavy chain CDR1 having an amino acid sequence selected from: SYWIH (SEQ ID NO: 4); 6 TYYNX 7 a heavy chain CDR2 having an amino acid sequence selected from the group consisting of: 8 X 9 SVX 10 Y (SEQ ID NO: 6), X 1 is Y or K, and X 2 is E or K, and X 3 is S or R, and X 4 is A or L, and X 5 is T or R, and X 6 is N or R, and X 7 is E or R, and X 8 is D or M, and X 9 is S or Y, and X 10 is A or G, Anti-IL-22 antibodies or antibody fragments.

2. The anti-IL-22 antibody or antibody fragment of claim 1, wherein the light chain variable region has an amino acid sequence selected from SEQ ID NOs: 7 to 12, and the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs: 13 to 18.

3. 3. The anti-IL-22 antibody or antibody fragment of claim 2, wherein the antibody or antibody fragment is selected from the following: an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 10 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 10 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17; an antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO:8 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:17; and An antibody or antibody fragment comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

18.

4. The anti-IL-22 antibody or antibody fragment of any one of claims 1 to 3, wherein the antibody or antibody fragment binds to human IL-22 and mammalian non-human IL-22.

5. The anti-IL-22 antibody or antibody fragment of claim 4, wherein the mammalian IL-22 is mouse IL-22.

6. 6. The anti-IL-22 antibody or antibody fragment of any one of claims 4-5, wherein the antibody or antibody fragment binds to human IL-22 and mammalian IL-22 with an affinity within ±20%, ±15%, ±10%, or ±5% of each other.

7. The anti-IL-22 antibody or antibody fragment of any one of claims 1 to 6, wherein the antibody or antibody fragment is capable of inhibiting phosphorylation of Stat3.

8. The anti-IL-22 antibody or antibody fragment of any one of claims 1 to 7, wherein the antibody or antibody fragment is capable of inhibiting IL-22-induced cytokine production.

9. The anti-IL-22 antibody or antibody fragment of any one of claims 1 to 8, wherein the antibody or antibody fragment is capable of inhibiting an immune response in at least one animal or human.

10. 10. The anti-IL-22 antibody or antibody fragment of claim 9, wherein at least one animal comprises a mammal.

11. The anti-IL-22 antibody or antibody fragment according to any one of claims 9 to 10, wherein the mammal is a mouse.

12. The anti-IL-22 antibody or antibody fragment of any one of claims 1 to 11, wherein the anti-IL-22 antibody or antibody fragment is humanized.

13. The anti-IL-22 antibody or antibody fragment of any one of claims 1 to 12, wherein the anti-IL-22 antibody or antibody fragment comprises an altered Fc region.

14. 14. A modified anti-IL-22 antibody or antibody fragment comprising the anti-IL-22 antibody or antibody fragment of any one of claims 1 to 13 and at least one moiety selected from an oligosaccharide, a non-proteinaceous moiety, a therapeutic agent, a prophylactic agent, and a diagnostic agent.

15. 15. The modified anti-IL-22 antibody or antibody fragment of claim 14, wherein at least one moiety is an oligosaccharide.

16. 15. The modified anti-IL-22 antibody or antibody fragment of claim 14, wherein at least one moiety is at least one non-proteinaceous moiety.

17. 16. The modified anti-IL-22 antibody or antibody fragment of claim 15, wherein at least one non-proteinaceous moiety is selected from a water-soluble polymer.

18. 15. The modified anti-IL-22 antibody or antibody fragment of claim 14, wherein at least one moiety is selected from a therapeutic agent, a prophylactic agent, and a diagnostic agent.

19. 19. The modified anti-IL-22 antibody or antibody fragment of claim 18, wherein a therapeutic, prophylactic, or diagnostic agent is conjugated to the anti-IL-22 antibody or antibody fragment and is selected from a chemotherapeutic agent, a radioactive atom, a detectable label, a prodrug-activating enzyme, a cytostatic agent, and a cytotoxic agent.

20. The modified anti-IL-22 antibody or antibody fragment of any one of claims 18 to 19, comprising two agents selected from a therapeutic agent, a prophylactic agent, and a diagnostic agent.

21. The modified anti-IL-22 antibody or antibody fragment of any one of claims 18 to 20, wherein the antibody or antibody fragment and the therapeutic, prophylactic, or diagnostic agent are covalently attached to a linker molecule.

22. A pharmaceutical composition comprising the antibody or antibody fragment of any one of claims 1 to 13, or the modified anti-IL-22 antibody or antibody fragment of any one of claims 14 to 21; and a pharmaceutically acceptable carrier.

23. 23. The pharmaceutical composition of claim 22, further comprising at least one additional excipient.

24. The pharmaceutical composition of any one of claims 22-23, further comprising at least one additional therapeutic agent.

25. 22. A method for treating an immune-related disease or cancer, comprising administering to a subject the antibody or antibody fragment of any one of claims 1 to 13, the modified anti-IL-22 antibody or antibody fragment of any one of claims 14 to 21, or the pharmaceutical composition of any one of claims 22 to 24.

26. A diagnostic or therapeutic kit comprising: an antibody or antibody fragment according to any one of claims 1 to 13, a modified anti-IL-22 antibody or antibody fragment according to any one of claims 14 to 21, or a pharmaceutical composition according to any one of claims 22 to 24; and instructions for using the antibody or antibody fragment, the modified antibody or antibody fragment, or the pharmaceutical composition for diagnosis or treatment.

27. a light chain variable region having an amino acid sequence having 90% or more sequence identity with one of the amino acid sequences of SEQ ID NOs: 7 to 12; A heavy chain variable region having an amino acid sequence having 90% or more sequence identity with one of the amino acid sequences of SEQ ID NOs: 13 to 18. An antibody or antibody fragment comprising:

28. 28. The antibody or antibody fragment of claim 27, wherein the light chain variable region has three complementarity determining regions identical to the complementarity determining regions of a light chain variable region having an amino acid sequence selected from SEQ ID NOs: 7-12.

29. The antibody or antibody fragment of any one of claims 27 to 28, wherein the heavy chain variable region has three complementarity determining regions that are identical to the complementarity determining regions of a heavy chain variable region having an amino acid sequence selected from SEQ ID NOs: 13 to 18.

30. 30. The antibody or antibody fragment of any one of claims 27 to 29, wherein the light and heavy chains have at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity.

31. The pharmaceutical composition according to claims 22 to 24, further comprising a pharmaceutically acceptable preservative.

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