WO2009132821A1 - Interleukin (il-21) binding proteins and methods of making and using same - Google Patents

Abstract

The present invention provides a family of binding proteins that bind and neutralize the activity of IL-21 in particular human IL-21. The binding proteins can be used as diagnostic and/or therapeutic agents. With regard to their therapeutic activity, the binding proteins can be used to treat certain IL-21 responsive disorders, for example, certain inflammatory bowel diseases or psoriasis.

Description

INTERLEUKIN-21 (IL-21) BINDING PROTEINS AND METHODS OF MAKING AND

USING SAME

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/084,296, filed July 29, 2008, and European Patent Application No. 08425294.9, filed April 28, 2008; the contents of each application is incorporated herein by reference in its entirety.

BACKGROUND

[0002] During the last decade, studies in humans and animal models have advanced the understanding of pathogenesis of immuno-mediated diseases. It has been shown that T lymphocytes play a major pathogenic role in for example, psoriasis, rheumatoid arthritis, bronchial asthma, primitive biliary cirrhosis, celiac disease, Helicobacter pylori (Hp)-associated gastric disease, and multiple sclerosis. Psoriasis, for example, is a chronic skin disorder with no currently available drugs for this disease that offer satisfactory efficacy and safety. Psoriatic lesions appear to stem from hyperproliferation of keratinocytes.

[0003] IL-21 is a cytokine that mediates immunological responses, including inflammation. For example, analysis of the signal transduction cascade induced by interaction of IL-21 with its receptor has shown that this cytokine is able to trigger some intracellular signals and to allow specific immune- inflammatory responses. Several studies carried out in B cells have shown that IL-21 can either promote or inhibit cell death programs in human or murine cells respectively. Similarly, it has been shown that human peripheral blood T lymphocytes produce high levels of interferon-gamma (IFN-γ), indicative of a ThI response, following IL-21 stimulation. Experiments carried out in animal models of carcinogenesis and diabetes have confirmed the ability of IL-21 to modulate T lymphocytes/ natural killer cell activities and cytokine production. Such studies underlined the potential role of IL-21 in the development and perpetuation of immune-inflammatory process.

[0004] Antibodies are multimeric proteins that contain four polypeptide chains. The basic structure common to all antibodies is shown schematically in Figure 1. Two of the polypeptide chains are referred to as heavy or H chains and two of the polypeptide chains typically referred to as light or L chains. The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by a number of interchain disulfide bonds. A light chain is composed of one variable region (V_L in Figure 1) and one constant region (C_L in Figure 1), while the heavy chain is composed of one variable region (V_H in Figure 1) and at least three constant regions (CHj, CH₂ and CH₃ in Figure 1). The variable regions determine the specificity of the antibody and the constant regions have other functions.

[0005] Amino acid and structural information indicate that each variable region comprises three hypervariable regions (also known as complementarity determining regions or CDRs) flanked by four relatively conserved framework regions or FRs. The three CDRs, referred to as CDR₁, CDR₂, and CDR₃, are responsible for the binding specificity of individual antibodies. When antibodies are to be used as diagnostic and therapeutic agents, typically it is desirable to create antibodies that have the highest binding specificity and affinity to the target molecule. It is believed that differences in the variable regions can have profound effects on the specificity and affinity of the antibody

SUMMARY

[0006] The invention disclosed herein is directed, in part, to a family of binding proteins that specifically bind IL-21. The binding proteins are antibody based in so far as they contain antigen (i.e., IL-21) binding sites based on the CDRs of an antibody that specifically binds IL-21. The CDRs confer the binding specificity of the binding proteins to IL-21. The binding proteins can be used as diagnostic and therapeutic agents. When used as a therapeutic agent, the binding proteins may be engineered (e.g., humanized) so as to reduce or eliminate the risk of inducing the immune response against the binding protein when administered to the recipient (e.g., a human).

[0007] In one aspect, an isolated binding protein that binds human interleukin-21 (IL-21) comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises a CDR_H i comprising the sequence SEQ ID NO: 8, a CDR_H2 comprising the sequence SEQ ID NO: 9, and a CDR_H3 comprising the sequence SEQ ID NO: 10, and the immunoglobulin light chain variable region comprises a CDRn comprising the sequence SEQ ID NO: 1 1, a CDR_L2 comprising the sequence SEQ ID NO: 12, and a CDR_L3 comprising the sequence SEQ ID NO: 13.

[0008] In certain embodiments, the CDR sequences of the immunoglobulin heavy chain and the immunoglobulin light chain are interposed between human and humanized framework sequences. In another embodiment, the binding protein is a monoclonal antibody or a antigen binding protein fragment thereof.

[0009] In another aspect, an isolated binding protein that binds human interleukin-21 (IL-21) comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, and the immunoglobulin light chain variable region comprises the amino acid sequence of SEQ ID NO: 7.

[0010] In another aspect, an isolated binding protein that binds human interleukin-21 (IL-21) comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain is SEQ ID NO: 19, and the immunoglobulin light chain is SEQ ID NO: 21.

[0011] In certain embodiments, the isolated binding protein that binds human IL-21 is a monoclonal antibody or an antigen binding fragment thereof.

[0012] Methods of producing the binding proteins described herein are also disclosed.

[0013] In another aspect, the binding proteins neutralize the activity of IL-21 and, therefore, can be used as a therapeutic agent. In certain embodiments, the binding proteins prevent IL-21 from binding its cognate receptor, IL-2 IR, thereby neutralizing the activity of IL-21.

[0014] In one embodiment, a method of treating psoriasis is provided, comprising administering to a subject in need thereof an effective amount of one or more isolated binding proteins that bind interleukin-21. Also contemplated is the use of a binding protein that binds human IL-21 in the manufacture of a medicament for treatment of psoriasis.

[0015] In another embodiment, a method for reducing or inhibiting an IL-21 mediated inflammatory response in a cell is provided, comprising exposing the cell to an effective amount of one or more isolated binding proteins that bind human IL-21 to reduce or inhibit the inflammatory response in the cell. Also contemplated are methods for reducing or inhibiting an IL-21 mediated inflammatory response in a subject in need thereof, comprising administering to the subject an effective amount of one or more isolated binding proteins that bind human IL-21.

[0016] A method of treating an immune-inflammatory disease associated with altered IL-21 expression is also disclosed, comprising administering to a subject in need thereof an effective amount of one or more isolated binding protein that binds human IL-21. In certain embodiments, the immune-inflammatory associated with altered IL-21 expression is selected from the group consisting of chronic inflammatory bowel diseases (IBD) and celiac disease. Chronic inflammatory bowel diseases include Crohn's disease and ulcerative colitis.

[0017] Also contemplated is the use of a binding protein that binds human IL-21 in the manufacture of a medicament for the treatment of inflammatory bowel disease. In certain embodiments, the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0018] A method of treating and/or controlling obesity is also disclosed, comprising administering to a subject in need thereof an effective amount of one or more isolated binding proteins that bind interleukin-21. Also contemplated is the use of a binding protein to human IL- 21 in the manufacture of a medicament for the treatment of obesity.

[0019] In another embodiment, a method of treating Hodgkin's lymphoma is provided, comprising administering to a subject in need thereof an effective amount of one or more isolated binding proteins that bind human IL-21. Also contemplated is the use of the binding protein to human interleukin 21 in the manufacture of a medicament for the treatment of Hodgkin's lymphoma.

[0020] These and other aspects and advantages of the invention will become apparent upon consideration of the following figures, detailed description, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The invention can be more completely understood with reference to the following drawings. [0022] Figure 1 is a schematic representation of a typical antibody.

[0023] Figure 2 provides the nucleic acid sequence (A) and the amino acid sequence (B) for human IL-21.

[0024] Figure 3 is a schematic diagram showing the amino acid sequence defining (A) the complete immunoglobulin heavy chain variable region (SEQ ID NO: 5) and (B) the complete immunoglobulin light chain variable region (SEQ ID NO: 7) of monoclonal antibody 3El 1. The regions defining the complementarity determining regions, CDR₁, CDR₂, and CDR₃, are identified in boxes. The unboxed sequences represent framework (FR) sequences.

[0025] Figure 4 depicts the relative expression of IL-21 RNA in lesional psoriatic skin.

[0026] Figure 5 depicts the percentage of proliferating cells of human keratinocytes with IL- 21 concentration.

[0027] Figure 6 depicts RNA relative expression of various markers.

[0028] Figure 7 depicts the reduction in a human graft psoriatic lesion with anti IL-21 mAb vs. control.

[0029] Figure 8 depicts relative expression of inflammatory molecules with anti-IL-21 mAb administration.

[0030] Figure 9 is the result of fluorescence microscopy on stained human psoriasis lesions.

[0031] Figure 10 is a BIAcore sensorgram of a IL-21 antibody immobilized on a BIAcore sensor chip.

[0032] Figure 11 indicates that IL-21 expression is increased in inflammatory bowel diseases.

[0033] Figure 12 indicates that IL-21 expression is increased in psoriasis. (A) Real time PCR for IL-21 RNA transcripts in skin biopsies taken from both lesional (L) and nonlesional (N) skin of 13 psoriatic patients, 15 normal controls (CTR), 4 patients with lichen planus (LP), 5 patients with contact dermatitis (CD), 5 patients with atopic dematitis (AD), and 3 patients with lupus erythematosus (LES). Levels are normalized to β-actin. Values are mean ± SD of all experiments. *, P=0.01 ; ** P=0.01 ; *** P=0.03; **** P=0.04. (B) Analysis of IL-21 protein in skin biopsies taken from both lesional (L) and nonlesional (N) skin of 9 psoriatic patients, from 15 normal controls (CTR) by ELISA. Values are mean ± SD of all experiments. *, P=0.001. (C) Percentage of CD4+ T cells that expressed IL-21, IFN-γ or IL- 17 A, or co-express IL-21 and IFN-γ, or IL-21 and IL- 17 A, as assessed by flow-cytometry. Data indicate mean ± SD of 5 separate experiments in which cells isolated from the lesional skin of 5 psoriatic patients were evaluated. Right inset shows the percentages of IL-21 producing-CD4+ and -CDl 61+ cells. Data indicate mean ± SD of 3 separate experiments in which cells isolated from the lesional skin of 3 psoriatic patients were evaluated by flow-cytometry. (D) Real time PCR for IL-21 RNA transcripts in human PBMC isolated from 5 psoriatic patients and 5 normal controls. Levels are normalized to β-actin. V alues are mean ± SD of all experiments. *, P=(XOl. (E) Percentage of IL-21 -positive CD4+ T cells purified from human PBMC of 6 psoriatic patients and 5 healthy volunteers as assessed by flow-cytometry. Data indicate mean ± SD of all experiments. *, P=0.02. Right Inset shows representative dot-plots showing the percentages of IL-21 -positive T cells expressing CLA in PBMC samples isolated from one psoriatic patient and one normal control. (F) Percentage of blood CD4+ T cells expressing IL-21, IFN-γ or IL- 17 A, or co- expressing IL-21 and IFN-γ, or IL-21 and IL- 17 A, as assessed by flow-cytometry. Data indicate mean ± SD of 4 separate experiments in which cells isolated from the blood of 5 psoriatic patients were evaluated.

[0034] Figure 13 depicts real-time PCR analysis for IL- 15 (A) and IL-7 (B) RNA transcripts in biopsies taken from lesional skin of 9 psoriatic patients, and from 15 normal controls. Levels are normalized to β-actin. Values are mean ± SD of all experiments. *, P<0.001.

[0035] Figure 14 characterizes IL-21 R in psoriasis. (A) Percentages of CD4+, CD8+, CD20+, CD56+ cells, and primary keratinocytes expressing IL-2 IR. Cells were isolated from lesional skin of 4 psoriatic patients and evaluated by flow-cytometry. Data indicate mean ± SD of all experiments. Right inset shows a representative dot-plot showing IL-2 IR staining in primary keratinocytes. Numbers in quadrants indicate the percentage of IL-21 R-positive cells. Staining with a control IgG is also shown. (B) IL-21 dose-dependently enhances the growth of primary keratinocytes. CFSE-labelled keratinocytes were either left unstimulated (Unst) or stimulated with increasing doses of IL-21 or epidermal growth factor (EGF, 20 ng/ml). After 48 hours, the percentages of proliferating cells in the first, second, and third generation were evaluated by flow cytometry. Data indicate mean ± SD of four separate experiments using cells isolated from four psoriatic patients. (C) Representative Western blots showing both phosphorylated (p) and total ERK 1/2 in primary keratinocytes cultured in media alone (Unst) or stimulated with IL-21 (100 ng/ml) for the indicated time-points. One of four separate experiments in which similar results were obtained is shown. (D) Blockade of ERK 1/2 activation by PD98059 abrogates IL-21 -driven keratinocyte proliferation. Cells were pre- incubated with PD98059 or vehicle (DMSO) for one hour and then stimulated with 100 ng/ml IL-21. After 48 hours, the proliferative index was evaluated by flow cytometry. Data indicate mean ± SD of 4 separate experiments. *, P=0.01. Right right inset shows that in the same cell cultures blockade of ERK 1/2 activation by PD98059 did not modify the fraction of propidium iodide (PΙ)-positive cells as assessed by flow cytometry. Data indicate mean ± SD of four experiments.

[0036] Figure 15 depicts (A) representative photomicrographs (original magnification X 100), of hematoxylin & eosin (H&E)-stained paraffin sections of skin biopsies taken from mice (n=8) injected intradermally on day 0, 1, 2 and 3 with 500 ng IL-21 or PBS in a total volume of 50 μL. On day 4, mice were sacrificed and skin samples were collected for H&E staining. Right inset shows the epidermal thickness measured at day 4. Data indicate mean ± SD of all experiments. *, P=O.001. (B) Immunohistochemical staining for CD3+ T cells in paraffin- embedded sections of skin samples collected from mice treated with IL-21 or PBS (Original magnification x40). In the upper right panel, CD3 staining is shown at higher magnification (x 400), while in lower right panel, the same section was stained with a control isotype antibody. (C) Intradermal injection of IL-21 to mice results in increased expression of ThI and ThI 7- related markers. IFN-γ, IL- 17 A, and IL-21 transcripts were analyzed by real-time PCR, and levels were normalized to β-actin. Values are mean ± SEM of all experiments. *, P<0.01. D-E. IL-21 -induced epidermal hyperplasia is IL-22-independent. (D) Mice were injected as indicated in A. IL-22 transcripts were analyzed by real-time PCR, and levels were normalized to β-actin. Values are mean ± SEM of all experiments. *, P<0.001. Mice were injected with a neutralizing IL-22 (aIL-22) (200 μg) or control (IgG) antibody (200 μg) and the day after treated daily with 500 ng IL-21 or 500 ng IL-23. On day 4, mice were sacrificed and skin samples were collected for H&E staining. The epidermal thickness was measured at day 4. IL-23+IgG vs. IL-23 + anti- IL-22. *, P=0.01. [0037] Figure 16 depicts real-time PCR analyses for keratin (KRT) 6 (A) and KRT 16 (B) in RNA samples prepared from skin biopsies of mice treated with IL-21 or PBS daily for 4 days. Levels are normalized to β-actin. Values are mean ± SD of all experiments. *, PO.001.

[0038] Figure 17 depicts neutralization of IL-21 ameliorates psoriasis-like hyperplasia in a psoriasis skin-SCID mouse xenograft model. (A) SCID mice were engrafted with human psoriatic skin (day 0) and 14 days later injected with autologous anti-CD3+CD28-activated PBMC. On day 21, SCID mice were either left untreated (control=ctr) or treated intra- peritoneally with a neutralizing human IL-21 antibody (anti-IL-21, 1 mg twice weekly) or control IgG for four weeks. Skin grafts were then harvested, embedded in paraffin, and stained with H&E. The tissue samples shown are from the same patient, and are representative of six separate experiments in which psoriatic skin obtained from ten different patients was used to engraft ten mice per each group. (B) Epidermal thickness of mice treated as indicated in (A) was measured at day four. Data indicate mean ± SD of all experiments. *, P=O.001. (C) Paraffin- embedded skin grafts of mice treated with control IgG or anti-IL-21 were stained with Ki-67 nuclear antigen or DAPI. The tissue samples shown are from the same patient, and are representative of four separate experiments in which psoriatic skin obtained from four different patients was used to engraft eight mice per each group. (D) Real-time PCR for IFN-γ, and IL- 17A in RNA samples prepared from skin grafts of mice either left untreated or treated with anti- IL-21 or control IgG as indicated in (A). Levels are normalized to β-actin. Values are mean ± SD of all experiments. **, P<0.03.

DETAILED DESCRIPTION

[0039] The invention is based, in part, upon the discovery of a family of binding proteins that specifically bind, and neutralize the activity of, IL-21 , in particular, human IL-21. The binding proteins can be used in a variety of diagnostic and therapeutic applications. The binding proteins are based upon the antigen binding sites of certain monoclonal antibodies that have been selected for their ability to bind, and neutralize the activity of IL-21. In particular, the binding proteins contain immunoglobulin variable region CDR sequences that together define a binding site for IL-21.

[0040] It is understood that each of the binding proteins discussed herein can be an intact antibody, for example, a monoclonal antibody. Alternatively, the binding protein can be an antigen binding fragment of an antibody, or can be a biosynthetic antibody binding site. Antibody fragments include Fab, Fab', (Fab')₂ or Fv fragments. Techniques for making such antibody fragments are known to those skilled in the art. A number of biosynthetic antibody binding sites are known in the art and include, for example, single Fv or sFv molecules. Other biosynthetic antibody binding sites include bispecific or bifunctional binding proteins, for example, bispecific or bifunctional antibodies, which are antibodies or antibody fragments that bind at least two different antigens. For example, bispecific binding proteins can bind IL-21, for example, human IL-21, and another antigen of interest. Methods for making bispecific antibodies are known in art and, include, for example, by fusing hybridomas or by linking Fab' fragments.

[0041] In another aspect, the invention provides an isolated binding protein that binds human IL-21 with a k_off of 5.OxIO^'3 s^'1 or lower, 4.OxIO^"3 s^"1 or lower, or 3.OxIO^"3 s^"1 or lower. The isolated binding proteins can bind human IL-21 with a k_Ofr from 5.OxIO^"3 s^"1 to O.5xlO^"3 s^"1, or from 4.OxIO^"3 s^"1 to 1.OxIO^"3 s^"1, or from 3.OxIO^"3 s^"1 to 1.5xlO^"3 s^"1. In another aspect, the invention provides an isolated binding protein that binds human IL-21 with a K_D of 10 nM or lower, 7 nM or lower, 5 nM or lower, or 3 nM or lower. The isolated binding proteins can bind human IL-21 with a K_D from 10 nM to 3 nM, or from 7 nM to 3 nM, or from 5 nM to 3 nM. Unless otherwise specified, K_D values are determined by the methods, and under the conditions, described in Example 3.

II - Production of Binding Proteins

[0042] Binding proteins of the invention can be produced in various ways using approaches know in the art. For example, DNA molecules encoding light chain variable regions and heavy chain variable regions can be chemically synthesized, using a commercial synthesizer and sequence information provided herein. Such synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs encoding the desired binding proteins. Production of defined gene constructs is within routine skill in the art. Alternatively, the sequences provided herein can be cloned out of hybridomas by conventional hybridization techniques or PCR techniques, using synthetic nucleic acid probes whose sequences are based on sequence information provided herein or prior art sequence information regarding genes encoding the heavy and light chains of murine antibodies in hybridoma cells. Production and use of such probes is within ordinary skill in the art.

[0043] The nucleic acids encoding the desired binding proteins can be introduced (ligated) into expression vectors, which can be introduced into a host cell via standard transfection or transformation techniques known in the art. Exemplary host cells include, for example, E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce immunoglobulin protein. Transfected host cells can be grown under conditions that permit the host cells to express the genes of interest, for example, the genes that encode the immunoglobulin light or heavy chain variable regions. The resulting expression products can be harvested using techniques known in the art.

[0044] The particular expression and purification conditions will vary depending upon what expression system is employed. For example, if the gene is to be expressed in E. coli, it is first cloned into an expression vector. This is accomplished by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a signal sequence, e.g., a sequence encoding fragment B of protein A (FB). The resulting expressed fusion protein typically accumulates in refractile or inclusion bodies in the cytoplasm of the cells, and may be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the expressed proteins refolded and cleaved by the methods already established for many other recombinant proteins.

[0045] If the engineered gene is to be expressed in eukayotic host cells, for example, myeloma cells or CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, immunoglobulin enhancers, and various introns. This expression vector optionally can contain sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed. The gene construct can be transfected into myeloma cells or CHO cells using established transfection protocols. Such transfected cells can express V_L or V_H fragments, V_L-V_H heterodimers, V_H-V_L or V_L-VH single chain polypeptides, complete heavy or light immunoglobulin chains, or portions thereof, each of which may be attached to a protein domain having another function (e.g., cytotoxicity).

Ill - Modifications to the Binding Proteins [0046] It is understood that the binding proteins can be modified to optimize performance depending upon the intended use of the binding proteins. For example, when the binding protein is being used as a therapeutic agent, the binding protein can be modified to reduce its immunogenicity in the intended recipient. Alternatively or in addition, the binding protein can be fused or coupled to another protein or peptide, for example, a growth factor, cytokine, or cytotoxin. Such modifications can be achieved by using routine gene manipulation techniques known in the art.

[0047] Various techniques for reducing the antigenicity of antibodies and antibody fragments are known in the art. These techniques can be used to reduce or eliminate the antigenicity of the binding proteins of the invention. For example, when the binding proteins are to be administered to a human, the binding proteins preferably are engineered to reduce their antigenicity in humans. This process often is referred to as humanization. Preferably, the humanized binding proteins have the same or substantially the same affinity for the antigen as the original non- humanized binding protein it was derived from.

[0048] In one well known humanization approach, chimeric proteins are created in which immunoglobulin constant regions of antibodies from one species, e.g., mouse, are replaced with immunoglobulin constant regions from a second, different species, e.g., a human. In this example, the resulting antibody is a mouse-human chimera, where the human constant region sequences, in principle, are less immunogenic than the counterpart murine sequences.

[0049] In another approach, known as CDR grafting, the CDRs of the light and heavy chain variable regions of an antibody of interest are grafted into frameworks (FRs) from another species. For example, murine CDRs can be grafted into human FR sequences. In some embodiments, the CDRs of the light and heavy chain variable regions of an anti-IL-21 antibody are grafted into human FRs or consensus human FRs. In order to create consensus human FRs, FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence.

[0050] In an approach called "superhumanization," antibodies in which human immunogenicity is reduced or eliminated are created by an alternative form of grafting. In superhumanization, human FR sequences are chosen from a set of human germline genes based on the structural similarity of the human CDRs to those of the mouse antibody to be humanized. [0051] Other approaches to reduce immunogenicity include, techniques are known as "reshaping," "hyperchimerization," or "veneering/resurfacing" to produce humanized antibodies. In the veneering/resurfacing approach, the surface accessible amino acid residues in the murine antibody are replaced by amino acid residues more frequently found at the same positions in a human antibody. One exemplary approach for converting a mouse antibody into a form suitable for medical use in humans is known as ACTIVMAB™ technology (Vaccinex, Inc., Rochester, NY), which involves a vaccinia virus-based vector to express antibodies in mammalian cells. High levels of combinatorial diversity of immunoglobulin heavy and light chains are said to be produced.

[0052] Another exemplary approach for converting a mouse antibody into a form suitable for use in humans is technology practiced commercially by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This technology involves the use of a proprietary human "acceptor" library to produce an "epitope focused" library for antibody selection.

[0053] Another exemplary approach for modifying a mouse antibody into a form suitable for medical use in humans is HUMAN ENGINEERING™ (HE™) technology, which is practiced commercially by XOMA (US) LLC.

[0054] Any suitable approach, including any of the above approaches, can be used to reduce or eliminate human immunogenicity of a binding protein of interest.

[0055] In addition, it is possible to create fully human antibodies in mice. In this approach, human antibodies are prepared using a transgenic mouse in which the mouse's antibody- producing genes have been replaced by a substantial portion of the human antibody producing genes. Such mice may produce, e.g., human immunoglobulin instead of murine immunoglobulin molecules.

[0056] Binding proteins of the invention can be conjugated with other molecules, depending upon their intended use. For example, if the binding protein is to be used as a therapeutic, then the binding protein can be conjugated with another agent, for example, an effector molecule that modulates or otherwise promotes the therapy. To the extent that the effector is non-protein based agent, for example, a small molecule drug, a radiolabel or toxin, then, the agent can be chemically coupled to the binding protein using standard in vitro coupling chemistries. If, on the other hand, the effector molecule is a protein or peptide, for example, an enzyme, receptor, toxin, growth factor, cytokine or other immunomodulator, then the binding protein can either be chemically coupled to the effector using in vitro coupling chemistries or can be coupled to the effector as a fusion protein. Fusion proteins can be constructed and expressed using the techniques similar to those discussed in section II.

IV - Use of Binding Proteins

[0057] The binding proteins described herein can be used as a diagnostic agent or a therapeutic agent.

(1) Therapeutic Applications

[0058] Because the binding proteins of the invention neutralize the activity of IL-21 , the proteins can be used in various therapeutic applications. For example, certain binding proteins of the invention are useful in the prevention or treatment of immune mediated diseases and/or an inflammatory disease associated with altered IL-21 expression, such as chronic inflammatory bowel diseases (IBD) (e.g., Crohn's disease (CD) or ulcerative colitis (UC)) and celiac disease. For example, Figure 11 indicates that the IL-21 expression is increased in such inflammatory bowel diseases.

[0059] This disclosure also provides for methods of treating psoriasis by administering to a patient one or more isolated binding proteins that bind to IL-21 to a patient or subject.

[0060] In addition, the binding protein can be used to inhibit, or slow the course of such diseases in a mammal. In such a method, an effective amount of the binding protein is administered to the mammal so as to inhibit or slow down the course of the disease in the mammal. The method may include administering to the mammal a therapeutically effective amount of the binding protein. The binding protein can be administered alone or in combination with another pharmaceutically active molecule, so as to treat, for example, the inflammatory disease.

[0061] Further, binding proteins of IL-21 are useful in the prevention or treatment of obesity. Also contemplated herein are methods for treating diseases associated with obesity in a patient or subject, for example, hypertension, dyslipidemia, type 2 diabetes, elevated plasma insulin concentrations; insulin resistance; dyslipidemias; hyperlipidemia, arteriosclerosis, coronary heart disease, stroke, gallbladder disease, and osteoarthritis. Such a patient may have, for example, a body mass index greater than or equal to about 30 kg/m², e.g., between about 30 kg/m² and about 60 kg/m² before treatment. Alternatively, a patient may have a body mass index between about 25 kg/m² and about 30 kg/m² before treatment.

[0062] Binding proteins of IL-21 may also be useful for reducing the risk of secondary outcomes of obesity, such as reducing the risk of left ventricular hypertrophy. Methods for treating patients at risk of obesity, such as those patients who are overweight, e.g., with a BMI of between about 25 and 30 kg/m², are also contemplated. Also provided herein is a method of inducing weight loss in a patient by administering a binding protein of IL-21.

[0063] Also contemplated herein are methods for treating Hodgkin's lymphoma using an isolated binding protein that binds human interleukin-21. In some embodiments, a method of treating Hodgkin's lymphoma a patient in need thereof is provided that includes administering an anti-IL-21 antibody to the patient. A characteristic morphological appearance of Hodgkin's lymphoma (HL) is a minority of neoplastic Hodgkin and Reed-Sternberg (HRS) cells surrounded by a vast majority of reactive infiltrating cells, consisting of CD4+ T cells, B cells, eosinophils, neutrophils, plasma cells, histiocytes, and fibroblasts. IL-21 may be a regulator of growth of T cells, B cells, and NK cells.

[0064] In addition, the binding protein can be used to inhibit, or slow the course of such diseases in a mammal. In such a method, an effective amount of the binding protein is administered to the mammal so as to inhibit or slow down the course of the disease, e.g., obesity or Hodgkin's lymphoma in the mammal. The method may include administering to the mammal a therapeutically effective amount of a binding protein of IL-21. The binding protein can be administered alone or in combination with another pharmaceutically active molecule, so as to treat, for example, the inflammatory disease.

[0065] As used herein, "treat, "treating" and "treatment" refer to the treatment of a disease- state in a mammal, particularly in a human, and include: (a) preventing the disease-state from occurring in a mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it; (b) inhibiting the disease-state, i.e., arresting its development; and/or (c) relieving the disease-state, i.e., causing regression of the disease state.

[0066] Generally, a therapeutically effective amount of active component will be in the range of from about 0.1 mg/kg to about 100 mg/kg, optionally from about 1 mg/kg to about 100 mg/kg, optionally from about 1 mg/kg to 10 mg/kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health status of the particular patient, the relative biological efficacy of the binding protein delivered, the formulation of the binding protein, the presence and types of excipients in the formulation, and the route of administration. The initial dosage administered may be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue level, or the initial dosage may be smaller than the optimum and the daily dosage may be progressively increased during the course of treatment depending on the particular situation. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors such as route of administration, dosage amount and the disease condition being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. A preferred route of administration is parenteral, e.g., intravenous infusion. Formulation of monoclonal antibody-based drugs is within ordinary skill in the art. In some embodiments of the invention, the binding protein, e.g., monoclonal antibody, is lyophilized and reconstituted in buffered saline at the time of administration.

[0067] The binding proteins may be administered either alone or in combination with other pharmaceutically active ingredients. The other active ingredients, e.g., immunomodulators, can be administered together with the binding protein, or can be administered before or after the binding protein.

[0068] Formulations containing the binding proteins for therapeutic use, typically include the binding proteins combined with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means buffers, carriers, and excipients, that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The carrier(s) should be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient. Pharmaceutically acceptable carriers, in this regard, are intended to include any and all buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. [0069] The formulations can be conveniently presented in a dosage unit form and can be prepared by any suitable method, including any of the methods well known in the pharmacy art. A pharmaceutical composition of the invention should be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration or non-parenteral administration, for example, intravenous, intradermal, inhalation, transdermal (topical), transmucosal, and rectal administration. Useful solutions for oral or parenteral administration can be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0070] Formulations suitable for oral administration can be in the form of: discrete units such as injectables, capsules, gelatin capsules, sachets, tablets, troches, or lozenges, each containing a predetermined amount of the binding protein; a powder or granular composition; a solution or a suspension in an aqueous liquid or non-aqueous liquid; or an oil-in-water emulsion or a water-in- oil emulsion.

[0071] Formulations suitable for parenteral administration include, for example, the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0072] In general, compositions suitable for injectable use include aqueous solutions (where water soluble) or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.

[0073] Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted prior to or following lyophilization and reconstitution. Once the pharmaceutical composition has been formulated, it can be stored, for example, in vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.

(2) Diagnostic Applications

[0074] Whenever the binding proteins are used for diagnostic purposes, either in vitro or in vivo, the binding proteins typically are labeled either directly or indirectly with a detectable moiety. The detectable moiety can be any moiety which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as ³Hydrogen (³H), ¹⁴Carbon (¹⁴C), ³²Phosphorus (³²P), ³⁵Sulfur (³⁵S), or ¹²⁵Iodine (¹²⁵I); a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase, or horseradish peroxidase; a spin probe, such as a spin label; or a colored particle, for example, a latex or gold particle. It is understood that the binding protein can be conjugated to the detectable moiety using a number of approaches known in the art. The labels may be detected, e.g., visually or with the aid of a spectrophotometer or other detector.

[0075] The binding proteins can be employed in a wide range of immunoassay techniques available in the art. Exemplary immunoassays include, for example, sandwich immunoassays, competitive immunoassays, immunohistochemical procedures.

[0076] In a sandwich immunoassay, two antibodies that bind an analyte or antigen of interest are used, e.g., one immobilized onto a solid support, and one free in solution and labeled with a detectable moiety. When a sample containing the antigen is introduced into this system, the antigen binds to both the immobilized antibody and the labeled antibody, to form a "sandwich" immune complex on the surface of the support. The complex ed protein is detected by washing away non-bound sample components and excess labeled antibody, and measuring the amount of labeled antibody complexed to protein on the support's surface. Alternatively, the antibody free in solution can be detected by a third antibody labeled with a detectable moiety which binds the free antibody.

[0077] It is contemplated that the labeled binding proteins are useful as in vivo imaging agents, whereby the binding proteins can target the imaging agents to particular tissues of interest in the recipient. A preferred remotely detectable moiety for in vivo imaging includes the radioactive atom Technetium^""¹¹¹ (^99mTc), a gamma emitter with a half-life of about six hours. Non-radioactive moieties also useful in in vivo imaging include nitroxide spin labels as well as lanthanide and transition metal ions all of which induce proton relaxation in situ. In addition to immunoimaging, the complexed radioactive moieties may be used in standard radioimmunotherapy protocols to destroy the targeted cell. Preferred nucleotides for high dose radioimmunotherapy include the radioactive atoms ⁹⁰Yttrium (⁹⁰Yt), ¹³¹Iodine (¹³¹I) and 1¹ indium (¹¹¹In). The binding protein can be labeled with ¹³¹I, ¹¹¹In and ^99mχc using coupling techniques known in the imaging arts. Similarly, procedures for preparing and administering the imaging agent as well as capturing and processing images are well known in the imaging art and so are not discussed in detail herein. Similarly, methods for performing antibody-based immunotherapies are well known in the art.

[0078] Throughout the description, where compositions are described as having, including, or comprising specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Except where indicated otherwise, the order of steps or order for performing certain actions are immaterial so long as the invention remains operable. Moreover, unless otherwise noted, two or more steps or actions may be conducted simultaneously.

EXAMPLES

[0079] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

Example 1 - Production of Anti-hIL-21 Monoclonal Antibodies

[0080] This Example describes the production of an anti-hIL-21 monoclonal antibody.

Murine monoclonal antibodies were generated using hybridoma technology as originally described by Kohler and Milstein (1975, Nature 256:495-497) (see also, Brown et al. (1981) J Immunol 127:539-46; Brown et al. (1980) J. Biol Chem 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J Cancer 29:269-75). The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J Biol. Med, 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet., 3:231-36).

[0081] Peptide GM2 comprises the following sequence NVS IKK LKR KPP STN (SEQ ID NO: 1) corresponding to amino acids 97-1 11 of human IL-21 sequence (Figure 2). Different clones directed against GM2 were generated and the monoclonal antibodies were purified by affinity chromatography on Protein-G resin. An anti-GM2 purified clone was designated 3El 1. Upon purification, the antibody was dialyzed against Phosphate Buffer Saline (PBS buffer: 137mM NaCl, 2.7mm KCl, 8.ImM Na₂HPO₄, 1.47mM, KH₂PO₄, pH 7.4). The concentration of the purified 3El 1 antibody was determined by absorbance at 280 nm.

[0082] ELISA studies demonstrated that the isolated anti-GM2 clones bound recombinant human IL-21 (Invitrogen, Catalog No. PHC0214). Each of the monoclonal antibodies recognized only human IL-21 and did not bind mouse IL-21 (m-IL21), which is attributed to the low sequence homology between human and mouse IL-21.

Example 2 - Sequence Analysis of Anti-hIL-21 Monoclonal Antibodies [0083] This Example describes the isotype and the sequence analysis of the anti-human IL- 21 monoclonal antibody, 3El 1.

[0084] The heavy chain type isotype and the light chain isotype for the 3El 1 antibody was determined using methods known in the art. The 3El 1 antibody isotype is IgGl immunoglobulin heavy chain and Kappa immunoglobulin light chain.

[0085] The nucleotide sequences encoding the immunoglobulin heavy and light chain variable regions of the mouse 3El 1 monoclonal antibody were determined by sequencing. Total RNA was extracted from cell pellets of the mouse hybridoma 3El 1-1 using the RNeasy Midiprep kit according to the manufacturer's protocol (Qiagen). First strand cDNA was generated by reverse transcription using Amersham Biosciences First Strand Synthesis kit according to the manufacturer's protocol. [0086] 3El 1-1 cDNA was amplified by PCR in 23 separate reactions. Immunoglobulin kappa chain variable region (VK) cDNA was amplified using 11 VK primers (MKVl-11) in combination with the kappa constant region primer MKC as shown in Table 1.

Table 1

[0087] Immunoglobulin heavy chain variable region cDNA was amplified by PCR using 12 different VH primers (MM71-12) in combination with a mix of the four IgG constant region primers (MHCG l/2a/2b/3) as shown in Table 2.

Table 2

Name Sequence (5'-> 3')

MHVl ATGAAATGCAGCTGGGGCATSTTCTTC (SEQ ID NO: 34)

MHV2 ATGGGATGGAGCTRTATCATSYTCTT (SEQ ID NO: 35)

MHV3 ATGAAGWTGTGGTTAAACTGGGTTTTT (SEQ ID NO: 36)

MHV4 ATGRACTTTGGGYTCAGCTTGRTTT (SEQ ID NO: 37)

MHV5 ATGGACTCCAGGCTCAATTTAGTTTTCCTT (SEQ ID NO: 38)

MHV6 ATGGCTGTCYTRGSGCTRCTCTTCTGC (SEQ ID NO: 39)

MHV7 ATGGRATGGAGCKGGRTCTTTMTCTT (SEQ ID NO: 40)

MHV8 ATGAGAGTGCTGATTCTTTTGTG (SEQ ID NO: 41)

MHV9 ATGGMTTGGGTGTGGAMCTTGCTATTCCTG (SEQ ID NO: 42)

MHVlO ATGGGCAGACTTACATTCTCATTCCTG (SEQ ID NO: 43)

MHVI l ATGGATTTTGGGCTGATTTTTTTTATTG (SEQ ID NO: 44)

MHV 12 ATGATGGTGTTAAGTCTTCTGTACCTG (SEQ ID NO: 45)

MHCGl CAGTGGATAGACAGATGGGGG (SEQ ID NO: 46)

MHCG2a CAGTGGATAGACCGATGGGGC (SEQ ID NO: 47)

MHCG2b CAGTGGATAGACTGATGGGGG (SEQ ID NO: 48)

MHCG3 CAAGGGATAGACAGATGGGGC (SEQ ID NO: 49) [0088] The result of the initial set of kappa light chain PCR reactions was one single amplification product using primers MKV2 and MKC (See Table 1 for primer sequences). Two products were amplified using MHVl (See Table 2 for primer sequences). The products of the PCR reaction primed by the oligonucleotide primers: MKV2 + MMKC were ligated into the pCR2.1-TOPO® vector using the TOPO-TA Cloning® kit according to the manufacturer's instructions. The 450 bp products from the [MKCl + MKC]-primed PCR reaction were gel purified and ligated into the pCR2.1-TOPO® vector using the TOPO-TA Cloning® kit according to the manufacturer's instructions.

[0089] E.coli TOPl 0 bacteria transformed with the ligated vector were cloned on LB/ ampicillin/X-gal agar plates using methods well known in the art, then picked and transferred into LB culture and into PCR mixture. The cloned plasmid inserts were screened by PCR amplification using 1212 (5'-GTTTTCCCAGTCACGAC-S' (SEQ ID NO: 50)) and 1233 primers (5'-AGCGGATAACAATTTCACACAGGA-S' (SEQ ID NO: 51)). The PCR products were gel electrophoresed and clones producing an approximately 700bp PCR amplification product were identified. DNA plasmid minipreps were produced from overnight cultures (4ml) of each clone using the QIAprep Spin Miniprep Kit Protocol according to the manufacturer's instructions.

[0090] Plasmids were sequenced by GATC in each direction using the Ml 3FP (5 '- TGTAAAACGACGGCCAGT-3' (SEQ ID NO: 52)) and Ml 3RP (5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO: 53)) primers. The complete cycle of RT-PCR, cloning, and DNA sequence analysis was repeated to obtain three completely independent sets of sequence information for each immunoglobulin chain.

[0091] The sequences of the MHVl -primed and MHV2-primed products were aligned and the consensus nucleic acid sequence for the 3El 1 heavy chain variable region and the translated amino acid sequence is shown below.

[0092] The sequences of the MKV 1 -primed products were aligned and the consensus nucleic acid sequence for the 3El 1 light chain variable region and the translated protein sequence is shown below. [0093] In the nucleic acid sequence encoding the 3El 1 heavy chain and light chain variable regions, the coding sequence is shown in upper case letters and the signal sequence is underlined.

[0094] (1) Nucleic Acid Sequence Encoding the 3El 1 Heavy Chain Variable Region (SEQ ID NO: 4) atgaaatgcagctggggcatsttcttcctgatggcagtggttataggggtcaactcaGAGGTTCAGCTGC

CATTAAAGACGAATATATGCACTGGGTGAAGCAGAGGCCTGAAAATGGCCTGGAGTGGATTGGATGGATT GATCCTGAGAATGGTGATACTGAATATGCCTCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACAT CCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTACTAC ATGGGATTACTACGGTAGTCCCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA gccaaaacgacacccccatcwgtctatccactg

[0095] (2) Protein Sequence Defining the 3El 1 Heavy Chain Variable Region (SEQ ID NO: 5)

1 EVQLQQSGAE LVRPGASVKL SCTASGFNIK DEYMHWVKQR PENGLEWIGW IDPENGDTEY 61 ASKFQGKATI TADTSSNTAY LQLSSLTSED TAVYYCTTWD YYGSPYAMDY WGQGTSVTVS 121 S

[0096] (3) Nucleic Acid Sequence Encoding the 3El 1 Kappa Chain Variable Region (SEQ ID NO: 6) atgagttgcctgttaggctgttggtgctgatgttctggattcctgcttccagcagtGATGTTGTGATGAC CCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGC

GTTCCGCTCACGTTCGGTGCTGGGACCAAGGTGGAGCTGAAAcgggctgatgctgcaccaactgtatcca tcttcccaccatccag

[0097] (4) Protein Sequence Defining the 3El 1 Kappa Chain Variable Region (SEQ ID NO:

7)

1 DWMTQTPLS LPVSLGDQAS ISCRSSQSLV HSNGETYLHW YLQKPGQSPK LLIYQVSNRF 61 SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP LTFGAGTKVE LK

[0098] Table 3 summarizes the heavy chain CDR sequences (Kabat definition) of monoclonal antibody 3El 1. Table 3

[0099] Table 4 summarizes the light chain CDR sequences (Kabat definition) of monoclonal antibody 3El 1.

Table 4

[00100] In order to create the complete immunoglobulin heavy chain or kappa chain antibody sequences, the variable sequence noted above are combined with their respective constant region sequences (e.g., a complete heavy chain sequence comprises a heavy chain variable sequence followed by the murine IgGl heavy chain constant sequence and a complete kappa chain comprises a kappa variable region followed by the murine kappa light chain constant sequence).

[00101] The following sequences represent the actual or contemplated full length immunoglobulin heavy and light chain sequences (i.e., containing both the variable and constant regions sequences) for the 3El 1 antibody described herein. The immunoglobulin heavy chain and light chain variable regions are shown as uppercase letters and the murine IgGl and kappa constant regions are shown in lower case letters in the full length heavy and light chain sequences.

[00102] It is appreciated, however, that the variable region sequences described herein can be ligated to each of a number of other constant region sequences known to those skilled in the art to produce active full length immunoglobulin heavy and light chains. For example, the heavy chain variable region may be ligated with the human IgGl heavy chain constant region and the light chain variable region may be ligated with the human kappa light chain constant region to create a chimeric antibody. [00103] Nucleic Acid Sequence Encoding the Murine IgGl Heavy Chain Constant Region (SEQIDNO: 14) l gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac

61 tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc

121 tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac

181 ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc

241 acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg

301 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc

361 cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg

421 gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag

481 gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc

541 agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc

601 aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg

661 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc

721 agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg

781 aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct

841 tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc

901 acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac

961 tctcctggta aatga

[00104] Protein Sequence Defining the Murine IgGl Heavy Chain Constant Region (SEQ ID NO: 15)

1 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd

61 lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif

121 ppkpkdvlti tltpkvtcw vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv

181 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv

241 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf

301 tcsvlheglh nhhtekslsh spgk

[00105] Nucleic Acid Sequence Encoding the Murine Kappa Light Chain Constant Region (SEQIDNO: 16)

1 egg gctgatgctg caccaactgt atccatcttc ccaccatcca gtgagcagtt aacatctgga

61 ggtgcctcag tcgtgtgctt cttgaacaac ttctacccca aagacatcaa tgtcaagtgg

121 aagattgatg gcagtgaacg acaaaatggc gtcctgaaca gttggactga tcaggacagc

181 aaagacagca cctacagcat gagcagcacc ctcacgttga ccaaggacga gtatgaacga

241 cataacagct atacctgtga ggccactcac aagacatcaa cttcacccat cgtcaagagc

301 ttcaacagga atgagtgtta g

[00106] Protein Sequence Defining the Murine Kappa Light Chain Constant Region (SEQ ID NO: 17)

1 adaaptvsif ppsseqltsg gaswcflnn fypkdinvkw kidgserqng vlnswtdqds 61 kdstysmsst ltltkdeyer hnsytceath ktstspivks fnrnec [00107] Nucleic Acid Sequence Encoding the Full Length 3El 1 Heavy Chain Sequence (^"3El 1 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 18)

GAGGTTCAGC TGCAGCAGTC TGGGGCTGAG CTTGTGAGGC CAGGGGCCTC AGTCAAGTTG TCCTGCACAG CTTCTGGCTT TAACATTAAA GACGAATATA TGCACTGGGT GAAGCAGAGG CCTGAAAATG GCCTGGAGTG GATTGGATGG ATTGATCCTG AGAATGGTGA TACTGAATAT GCCTCGAAGT TCCAGGGCAA GGCCACTATA ACAGCAGACA CATCCTCCAA CACAGCCTAC CTGCAGCTCA GCAGCCTGAC ATCTGAGGAC ACTGCCGTCT ATTACTGTAC TACATGGGAT TACTACGGTA GTCCCTATGC TATGGACTAC TGGGGTCAAG GAACCTCAGT CACCGTCTCC TCA gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac tctcctggta aatga

[00108] Protein Sequence Defining the Full Length 3El 1 Heavy Chain Sequence (3El 1 Heavy Chain Variable Region and IgGl Constant Region) (SEQ ID NO: 19)

EVQLQQSGAE LVRPGASVKL SCTASGFNIK DEYMHWVKQR PENGLEWIGW IDPENGDTEY ASKFQGKATI TADTSSNTAY LQLSSLTSED TAVYYCTTWD YYGSPYAMDY WGQGTSVTVS Sakttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif ppkpkdvlti tltpkvtcw vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf tcsvlheglh nhhtekslsh spgk

[00109] Nucleic Acid Sequence Encoding the Full Length 3El 1 Light Chain Sequence (3El 1 Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 20)

GATGTTGTGA TGACCCAAAC TCCACTCTCC CTGCCTGTCA GTCTTGGAGA TCAAGCCTCC ATCTCTTGCA GATCTAGTCA GAGCCTTGTA CACAGTAATG GAGAAACCTA TTTACATTGG TACCTGCAGA AGCCAGGCCA GTCTCCAAAG CTCCTGATCT ACCAGGTTTC CAACCGATTT TCTGGGGTCC CAGACAGGTT CAGTGGCAGT GGATCAGGGA CAGATTTCAC ACTCAAGATC AGCAGAGTGG AGGCTGAGGA TCTGGGAGTT TATTTCTGCT CTCAAAGTAC ACATGTTCCG CTCACGTTCG GTGCTGGGAC CAAGGTGGAG CTGAAA gctgatgctg caccaactgt atccatcttc ccaccatcca gtgagcagtt aacatctgga ggtgcctcag tcgtgtgctt cttgaacaac ttctacccca aagacatcaa tgtcaagtgg aagattgatg gcagtgaacg acaaaatggc gtcctgaaca gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc ctcacgttga ccaaggacga gtatgaacga cataacagct atacctgtga ggccactcac aagacatcaa cttcacccat cgtcaagagc ttcaacagga atgagtgtta g

[00110] Protein Sequence Defining the Full Length 3El 1 Light Chain Sequence (3El 1 Kappa Chain Variable Region and Constant Region) (SEQ ID NO: 21)

DWMTQTPLS LPVSLGDQAS ISCRSSQSLV HSNGETYLHW YLQKPGQSPK LLIYQVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP LTFGAGTKVE LKadaaptvsif ppsseqltsg gasvvcf Inn fypkdinvkw kidgserqng vlnswtdqds kdstysmsst ltltkdeyer hnsytceath ktstspivks fnrnec

Example 3-Binding Affinity of the Anti-human IL-21 Monoclonal Antibody

[00111] All the experiments were performed on BIAcore 1000 Upgraded (BIACORE AB, Uppsala, Sweden). The mAb 3El 1 was covalently bound via NH2- groups on sensor chip CM5(GE Healthcare), immobilizing 7400 RUs. IL-21 diluted in HBS-BSA 0.1% (Hepes 0.01M, NaCl 0.15 M, EDTA 0.003M, p20 0.005% BSA 0.1% pH 7.4) at 20OnM, 10OnM, 5OnM, 25nM concentration was injected on the immobilized mAb for 3 minutes at a flow rate of 20 μl/min. Sensor chip was regenerated with glycine 0.010 M pH 2.2. The same experiment was also performed by injecting the antigen on an empty flow cell in order to identify if an a specific binding of the IL-21 antigen on sensor chip's surface occurred.

[00112] Kinetic binding constants were measured using the BIA evaluation 4.1 software (BIAcore). Kinetic parameters for each antibody, Ic_0n (association rate constant), k_off (dissociation rate constant), and K_D (equilibrium dissociation constant were determined. The 3El 1 monoclonal antibody has a Ic_0n of 5.89 e+5 I/Ms, a Ic_0n-Of 3.81 e-3 1/s, and a K_D of 6.47 e-9

M.

[00113] Figure 10 shows the BIAcore sensorgram obtained by injecting four serial dilutions of 11-21 on the anti-IL-21 antibody immobilized on BIAcore sensor chip. Different curves of correspond to different antibody concentrations. Figure 10 also shows the corresponding response curves generated by the BIAevaluation software.

Example 4 — IL-21 Expression in Psoriasis

[00114] Figure 4 depicts the relative expression of IL-21 RNA in lesional psoriatic skin psoriasis as compared to non-lesional skin from normal donors. IL-21 also appears to enhance the proliferation of human keratinocytes as depicted in Figure 5. Example 5 — Elevation of Th 17 and ThI -related cytokines

[00115] Figure 6 depicts the enhancement of Th 17 and ThI associated markers (e.g., IL- 17A, IL-22, IFN-γ, ILl 7-F, ROR- γ , and T-bet) in mice that have been subcutaneously administered IL-21 (compared to controls treated with PBS).

Example 6 — Psoriatic Skin Lesion Model

[00116] In brief, symptomless skin from a psoriatic patient is orthotopically transferred onto severe combined immunodeficiency (SCID) mice and autologous peripheral blood mononuclear cells from the same patient, upon activation, are intradermically administered to the mice. Anti- human-IL-21 monoclonal antibody 3El 1 is then administered to the mice. A patient is affected by active psoriasis, but is not under therapy with immunosuppressive drugs or antibodies, is chosen for skin and peripheral blood withdrawal.

[00117] Four severe combined immunodeficiency (SCID) mice, females, 6-8 week-old are used for skin biopsy implantation and antibody administration. On day 1 , symptomless skin is biopsied from the lower back of the patient. Dimension of the biopsied skin: approximately 3 x 2 cm, containing both dermis and epidermis. The skin is placed into a sterile plate containing a physiological solution embedded gauze. The biopsy is cut into 4 fragments (approximately the same dimension), each fragment will be implanted into a different mouse, for a total of four mice.

[00118] The same day, 20ml of peripheral blood are withdrawn from the same patient. Human PBMC are isolated from the collected blood and are then frozen in complete cell culture medium, 5% DMSO, and stored at -80°C until use. Also on the same day the four mice are anesthetized with 2.5% Avertin (300 microL, intra-peritoneally (i.p.)). The back of the mice and the biopsies are treated with betatine. A graft bed (the same size of the human tissue to be implanted) is created in the back of the mouse by removing full thickness skin (containing both dermis and epidermis). The human skin is then placed into the graft bed and is sutured by using a topical skin adhesive (Histoacryl, B Braun, Melsungen)). Mice are then placed into separate cages (i.e., one mouse/cage), checked daily and the transplanted area treated with Betatine daily for 14 days. [00119] On day 11, the autologous Peripheral Blood Mononuclear Cells (PMBC) are thawed and seeded in RPMI medium, 10% FBS, antibiotic mix.

[00120] On day 12, a total 4 x 10⁶ PBMC are transferred in U-bottom multi- wells plates, dispensing 1 x 10⁶PBMC /well and are then activated for 48hr, using T cell activating anti-CD3 and anti-CD28-bound beads (T cell Activation/Expansion Kit, Miltenyi Biotec), according to the manufacturer's instructions. On day 14, 1 x 10⁶ autologous activated T cells are harvested, resuspended in 200 microL of PBS (phosphate buffer saline) and intradermally injected into the xenograft of the mouse. The intradermal injection is performed by using a syringe with a 4 mm long, 3OG needle. The procedure is repeated in four mice.

[00121] On days 21, 24, 27, 30 anti-human-IL-21 purified monoclonal antibody 3El 1 is administered intra-peritoneally (lmg/mouse) into two mice. The remaining two mice (control mice) are administered with commercially available mouse control IgGl.

[00122] On day 32, the mice are sacrificed, skin collected for histological analysis and protein and RNA evaluation.

[00123] Figure 7 shows pictorially the reduction in the human psoriasis lesion of anti-human- IL-21 rnAb 3El 1 administration vs. control. Figure 8 depicts relative expression of inflammatory molecules (e.g., IL- 17 A, IFN-γ, ROR-γT and TNF mRNA) when anti-human-IL- 21 mAb 3El 1 (aIL-21) is administered vs. control (IgG) and indicates that blockade of IL-21 reduces the expression of these inflammatory molecules.

Example 7 — Fluorescence Microscopy

[00124] Figure 9 depicts fluorescence microscopy results obtained by staining the human psoriasis lesion collected from SCID mice, with anti-ki67 (proliferation marker) and DAPI (nuclear marker). The difference between human psoriasis lesion collected from the mice treated with the anti-h- IL-21 3El 1 antibody and the control mice is shown.

Example 8 — Materials and Methods for Examples 9-13 [00125] Patients and Samples

[00126] This study received ethical approval by the local Committee. Biopsy samples were collected from the lesional and non-lesional skin of 13 patients with psoriasis, 15 normal controls, 4 patients with lichen planus, 5 patients with contact dermatitis, 5 patients with atopic dermatitis, and 3 patients with LES, and used to analyze IL-21. Additional lesional and non- lesional skin biopsies were available from 21 psoriatic patients and used either to purify immunocytes and keratinocytes or for the xenograft mouse model. All psoriatic patients had moderate to severe plaque. None of the patients and controls had received any local or systemic treatment during the last 3 months.

[00127] Cell isolation

[00128] All reagents were from Sigma-Aldrich (Milan, Italy) unless specified. Human PBMC were isolated from 17 psoriatic patients and 11 normal controls by Ficoll gradients. PBMC were used to assess cytokines, CLA, CD4, and CD 161, or to purify CD4+, CD8+, CD20+, CD56+ cells, by specific magnetic beads (Miltenyi Biotec, Bologna, Italy). Aliquots of PBMC of psoriatic patients were also resuspended in complete medium consisting of RPMI 1640 supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Life Technologies-GibcoCRL, Milan, Italy), in the presence of activating CD3 and CD28 beads (Miltenyi Biotec) for 48 hours and then used in the human psoriasis-xenograft mouse model.

[00129] Lesional skin biopsies of psoriatic patients were cultured in dispase solution (2 U/ml; Roche, Prodotti Gianni, Milan, Italy) overnight at 4°C to peel off the epidermis. The dermal layer was then minced and resuspended in complete medium for 72 hours at 37°C. The resulting dermal single-cell suspensions were harvested, washed in PBS, and resuspended in complete medium, and seeded in 48-well culture dishes (IxIO⁶ cells/well). Both dermal and circulating CD4+ cells were stimulated with Phorbol 12-myristate 13-acetate (80 pM), ionomycin (1 mg/ml), and monensin (2uM, from eBioscience, San Diego, CA, USA) for 5 hours. At the end, cells were fixed and analyzed by flow-cytometry.

[00130] The epidermis of both lesional and non-lesional skin of psoriatic patients was minced and incubated in the presence of trypsin for 25 minutes at 37°C. Supernatants were then collected, neutralized with FBS, and filtered. The resultant cell preparations were resuspended in Epilife Medium and either cultured according to the manufacturer's instructions (Cascade Biologies, S.I.A.L., Rome, Italy) or used to assess IL-21 R. For functional assays, keratinocytes isolated from non-lesional skin biopsies were seeded in 6-well culture dishes (2x10⁵ cells/well) and stimulated with increasing doses of IL-21 (50-200 ng/ml, Biosource, Camarillo, CA, USA) or epidermal growth factor (EGF), (20 ng/ml, Peprotech, London UK). In parallel, cells were pre-incubated with PD98059 (ERK inhibitor, 50μM, Inalco, Milan, Italy), or DMSO (vehicle) for 1 hour prior to adding IL-21 (100 ng/ml). After 48 hours, cell proliferation and apoptosis were assessed by fiow-cytometry. To assess whether IL-21 induces ERXl /2 activation, primary keratinocytes were stimulated with IL-21 (100 ng/ml) for 10-60 minutes, and cell extracts were then evaluated by Western blotting.

[00131] Flow-cytometry

[00132] Cytokines were evaluated using anti-human IL-21 -PE, IL- 17-APC (both at 1 :50 final dilution, eBioscience), and anti-human IFN-γ-FITC (1:50, final dilution, Becton Dickinson, Milan, Italy). CD4, CDl 61, and CLA were evaluated using anti-human CD4-FITC, CDl 61- APC, and CLA-FITC (1:50, final dilution, all from Becton Dickinson).

[00133] To characterize IL-2 IR expression in freshly isolated PBMC, and purified dermal cells, the following monoclonal anti-human antibodies were used: CD56 APC (1 :50 final dilution, Biolegend, Milan, Italy), CD4-APC, CD8-APC (1 :50, final dilution, Miltenyi Biotec), CD20 FITC (1:50 final dilution, Immunotools, Friesoythe; Germany), IL-21R PE (1 : 10 final dilution, Becton Dickinson, cat. No. FAB991 IP), and isotype control IgGs (Becton Dickinson).

[00134] To track the proliferation, keratinocytes were incubated in 0.2 μM CFSE (Invitrogen, Milan, Italy) at 37°C for 30 minutes and extensively washed before culture, then stimulated as described above. After 48 hours, CFSE fluorescence was evaluated, thus determining the proportion of cells undergoing divisions. The fraction of Annexin V and propidium iodide- positive cells was evaluated using a commercially available kit (Beckmann Coulter, Milan, Italy).

[00135] Real-time PCR

[00136] Complementary DNA was amplified using the following conditions: denaturation 1 minute at 95°C, annealing 30 seconds at 58°C for human IL-21, mouse IL-22, mouse KRT6 and mouse KRT 16, at 60°C for mouse IFN-γ, at 62 °C for mouse IL- 17A, for mouse IL-21, and for human/mouse β-actin, followed by 30 seconds of extension at 72°C. Primers sequence was as follows: human IL-21, FWD: 5 '-GG AGAGG ATTGTC ATCTGTC-3' (SEQ ID NO: 54) and REV: 5 '-CAC AGTTTGTCTCT ACATCTTC-3' (SEQ ID NO: 55); mouse IL-21, FWD: 5'- CCTCCTGATTAGACTTCGTCAC-3' (SEQ ID NO: 56) and REV: 5'- GGTTTGATGGCTTGAGTTTGGC-3' (SEQ ID NO: 57); mouse IFN-γ, FWD: CAATGAACGCTACACACTGC (SEQ ID NO: 58) and REV: CCACATCTATGCCACTTGAG (SEQ ID NO: 59); mouse IL-17A, FWD: CTCAGACTACCTCAACCGTTC (SEQ ID NO: 60) and REV: TTCAGGACCAGGATCTCTTGC (SEQ ID NO: 61); mouse IL-22 FWD: 5'- GACCAAACTCAGCAATC AGC-3' (SEQ ID NO: 62) and REV: 5'- ATCTCTCCACTCTCTCCAAG-3' (SEQ ID NO: 63); mouse KRT 6, FWD: TGAAGGAGTACCAGGAACTC (SEQ ID NO: 64) and REV: CACCACAGAGATGTTGACTG (SEQ ID NO: 65); mouse KRT 16, FWD: AAGACTACAGCCCCTACTTC (SEQ ID NO: 66) and REV: CATTCTCGT ACTTGGTCCTG (SEQ ID NO: 67). Human IL-7 and IL- 15 were evaluated using commercially available TaqMan probes (Applied Biosystems, Foster City, CA, USA), β-actin FWD: 5'- AAGATGACCCAGATC ATGTTTGAGACC-3' (SEQ ID NO: 68) and REV:5'- AGCCAGTCCAGACGCAGGAT-3 (SEQ ID NO: 69) was used as an internal control.

[00137] ELISA

[00138] IL-21 was evaluated using a commercially available ELISA kit (eBioscience), and IL- 21 values were expressed as pg/100 μg of total proteins.

[00139] Western blotting

[00140] Cell extracts were prepared as previously described (Caruso, R. et al., J. Immunol. 178, 5957-5965 (2007)). The membranes were blocked with Tris-buffered saline containing 0.05% Tween 20 and 5% non-fat dry milk and then incubated with anti-p-ERKl/2 (1:500 final dilution, Santa Cruz Biotechnology, Santa Cruz, CA). An appropriate horse-radish peroxidase- conjugated secondary antibody (Dako, Milan, Italy) was then used, and bound antibodies visualized using enhanced chemiluminescence (Pierce, S.I.A.L., Rome, Italy). After detection, blots were stripped and incubated with a total ERK1/2 antibody (Santa Cruz Biotechnology).

[00141] Injection of IL-21 into the skin of mice

[00142] All animal studies received approval from the Ethical Local Committee. Hair was removed from the back of the Balb/c mice with electric clipper. Three days later, mice were injected intradermally with IL-21 (500 ng/ 50 μl saline), vehicle control (sterile saline), or IL-23 (500 ng/50 μl saline; R&D Systems, Minneapolis, MN) using a 30-gauge needle. Injections were performed daily, and mice were killed at day 4. The skin was then collected for histological examination and RNA analysis. In parallel experiments, mice were treated intra- peritoneally with a neutralizing IL-22 (200 μg/400 μl saline, R&D Systems ) or control IgG (200 μg/200 μl saline, R&D Systems) 1 day prior the first IL-21 or IL-23 injection.

[00143] Human psoriasis-xenograft mouse model

[00144] The transplantation procedure was conducted as described elsewhere with minor modifications (Wrone-Smith, T. & Nickoloff, B.J., J. Clin. Invest. 98, 1878-1887 (1996)). Briefly, keratome biopsies (6 X 2 X 0.04 cm) were taken from clinically symptomless skin on the lower back of psoriatic patients after informed consent was obtained. Skin xenografts were orthotopically transplanted onto the back of 7-8 — week-old SCID mice, followed 2 weeks later by intradermal injection of autologous anti-CD3+CD28-activated PBMC (3 X 10⁶ cells/mouse). One week later, mice were randomly allocated into 3 groups and either left untreated, or treated intra-peritoneally with a neutralizing human IL-21 or control IgG (1 mg twice weekly intra- peritoneally) for 4 weeks.

[00145] Skin grafts were then harvested, and either embedded in paraffin, or used for RNA extraction.

[00146] Histopathological analysis

[00147] Tissues were processed into paraffin tissue blocks using routine methods, cut in sections, and stained with H&E. Epidermal thickness was measured as previously described (Chan, J.R. et al., J. Exp. Med. 203, 2577-2587 (2006)). For immunohistochemistry, paraffin- embedded sections were dewaxed in xylene and ethanol, rehydrated, and microwaved in 0.01 M citrate buffer, pH 6.0, to optimize antigen retrieval. Sections were then stained with antibodies specific for CD3 (1:10 final dilution, Santa Cruz Biotechnology), or control IgG followed by incubation in a dilution of a secondary antibody using a highly sensitive alkaline phosphatase technique (DBA, Milan, Italy). Skin grafts were also stained Ki-67 nuclear antigen (1 :1000 final dilution, Kovance, S.I.A.L, Rome, Italy) followed by a fluorescent-labeled secondary antibody or DAPI (1 :2000 final dilution, Invitrogen). [00148] Data analysis

[00149] Difference between groups may be compared using either the Mann-Whitney U test or the Student's t test.

Example 9 — IL-21 is Highly Produced in Lesional Psoriatic Skin

[00150] To investigate whether IL-21 expression is enhanced in the psoriatic plaque, skin biopsies were taken from patients with psoriasis and controls, and analyzed for the content of IL- 21 RNA transcripts by real-time PCR. Increased IL-21 RNA expression was seen in all samples taken from lesional psoriatic skin (L) compared to samples taken from non-lesional skin (N) of the same patients and normal controls (Figure 12A). IL-21 RNA expression was also slightly enhanced in biopsies taken from patients with atopic dermatitis (AD) or lupus erythematosus (LES) in comparison to normal controls (CTR). However, this increase was markedly less pronounced than in lesional psoriatic skin. IL-21 transcripts were not enhanced in skin biopsies from patients with lichen planus (LP) or patients with contact dermatitis (CD) (Figure 12A). Among other γ-chain related cytokines, IL- 15 but not IL-7 RNA was increased in lesional psoriatic skin (Figure 13). IL-21 was further analyzed in total extracts prepared by skin biopsies of patients with psoriasis and controls by ELISA. A more pronounced expression of IL-21 protein was observed in lesional psoriatic skin compared to samples taken from non-lesional skin of the same patients and normal controls (Figure 12B).

[00151] Previous studies have shown that IL-21 can be made by both activated CD4+ T and NKT cells (Parrish-Novak, J. et al, Nature 408, 57-63 (2000); Coquet, J.M. et al., J. Immunol. 178, 2827-2834 (2007)). Thus, the cell source of IL-21 in lesional psoriatic skin was evaluated. IL-21 was mostly produced by CD4+ T cells, even though 35 ± 15% of IL-21 -producing cells express the NKT cell marker, CD161 (Figure 12C, right inset). Flow-cytometry analysis of CD4+ T cells infiltrating the psoriatic skin revealed that 8 ± 2.5% of these cells expressed IL-21 alone, while the fraction co-expressing IL-21 and interferon (IFN)-γ or IL-21 and IL- 17A were 2.6 ± 2% and 2.2 ± 2.1% respectively (Figure 12C).

[00152] IL-21 transcripts were also increased in peripheral blood mononuclear cells (PBMC) of psoriatic patients (Figure 12D). IL-21 -expressing circulating CD4+ T cells were more frequent in patients with psoriasis than in controls (Figure 12E). Of the IL-21 -expressing CD4+ T cells from psoriatic patients, 45 ± 4% were positive for the skin homing antigen CLA compared to 27 ±2 % in controls (Figure 12E, right inset). Less than 2% of the circulating IL- 21 -producing CD4+ T cells co-expressed IFN-γ or IL- 17A (Figure 12F).

[00153] Example 10 — IL-21 Induces Keratinocyte Proliferation

[00154] The expression of IL-21 R was also assessed in the cells in the lesional skin of psoriatic patients. T cells (both CD4+ and CD8+), B cells, and NK cells were positive for IL- 21R (Figure 14A), thus confirming results obtained in other systems (Mehta, D. S., Wurster, A.L. & Grusby, M.J., Immunol. Rev. 202, 84-95 (2004); Paπϊsh-Novak, J., Foster, D.C., Holly, R.D. & Clegg, C.H., J. Leukocyte Biol. 72, 856-863 (2002)). IL-21 R was also expressed by primary keratinocytes isolated from both the lesional and non-lesional skin of psoriatic patients (Figure 14 A, and right insets) indicating that these cells are functionally capable of responding to IL-21. To address this issue, keratinocytes isolated from non-lesional skin of psoriatic patients were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and cultured in the presence or absence of increasing doses of IL-21. IL-21 enhanced the proliferation of these cells in a dose-dependent manner (Figure 14B), without affecting the rate of apoptosis.

[00155] Keratinocytes of psoriatic patients over-express active ERK1/2 mitogen-activated protein (Map) kinases (Takahashi, H. et al., J. Dermatol. Sci. 30, 94-99 (2002)). Since these kinases promote keratinocyte proliferation (Hobbs, R.M., Silva- Vargas, V., Groves, R. & Watt, F.M., J. Invest. Dermatol. 123, 503-515 (2004)), the dependence of IL-21 driven keratinocyte growth on ERK 1/2 activity was tested. Western blot analysis initially showed that stimulation of keratinocytes with IL-21 resulted in a time-dependent activation of ERK 1/2 (Figure 14C). Preincubation of cells with an ERK inhibitor completely prevented IL-21 -driven keratinocyte proliferation without affecting the rate of cell death (Figure 14D).

Example 11 — Intradermal injection of IL-21 into mice causes epidermal hyperplasia

[00156] IL-21 was intradermally injected into Balb/c mice daily for four days. Mice treated with IL-21, but not PBS, developed visually apparent erythema. Histological evaluation of IL- 21 -treated skin showed marked epidermal hyperplasia accompanied by few rete pegs and infiltration of the dermis with inflammatory cells (Figure 15A). IL-21 -induced epidermal thickening (Figure 15 A, right inset) was associated with increased expression of keratin-6 and keratin- 16, two proliferation-associated cytokeratins (Figure 16). Immunohistochemical staining of skin sections also demonstrated a marked infiltrate of CD3+ T cells in IL-21 -treated but not in PBS-treated mice (Figure 15B). Real-time PCR analysis showed that IL-21 injection into mouse skin caused a significant increase in transcripts for ThI (IFN -γ) and Th 17 (IL-17A)-related genes (Figure 15C). IL-21 enhanced also its own RNA expression (Figure 15C), thus confirming the existence of an autocrine regulation of IL-21 production (Caprioli, F. et al., J. Immunol. 180, 1800-1807 (2008)).

[00157] Example 12 — IL-22 is not Required for IL-21-Induced Epidermal Hyperplasia

[00158] IL-22 is over-produced in psoriatic skin lesions, where it is supposed to contribute to the thickening of the epidermis (Boniface, K. et al, Clin. Exp. Immunol. 150, 407-415 (2007); Zheng, Y. et al, Nature 445, 648-651 (2007)). Since IL-22 can be made by Thl7 cells (Zheng, Y. et al, Nature 445, 648-651 (2007)), and IL-21 has been involved in the differentiation of ThI 7 cells (Korn, T. et al, Nature 448, 484-487 (2007)), it was assessed whether IL-21 -induced epidermal hyperplasia was mediated by IL-22. IL-22 mRNA expression was increased in IL-21- treated mouse skin at day 4 (Figure 15D). A blocking monoclonal antibody to IL-22 was used to determine whether IL-22 is required for IL-21 -dependent epidermal thickening. Anti-IL-22 did not inhibit IL-21 -stimulated skin thickening (Figure 15E). By contrast, anti-IL-22 treatment inhibited IL-23-stimulated epidermal hyperplasia (Figure 15F), which has previously been shown to be IL-22 regulated (Zheng, Y. et al, Nature 445, 648-651 (2007)).

[00159] Overall these data indicate that intradermal injection of IL-21 in mice is sufficient to trigger epidermal hyperplasia.

Example 13 — Administration of Neutralizing IL-21 Antibody Reduces Skin Thickening and Inflammation in a Human Psoriasis SCID Mouse Xenograft Model

[00160] To examine further the role of IL-21 in the epidermal hyperplasia, the efficacy of a neutralizing human IL-21 antibody to ameliorate damage in human psoriasis skin grafts transplanted onto severe combined immunodeficient (SCID) mice was tested. This model has previously been validated, and it has been shown that human psoriatic grafts can be successfully transplanted to SCID mice, retaining some histological and immunological psoriatic skin characteristic (Wrone-Smith, T. & Nickoloff, B.J., J. CHn. Invest. 98, 1878-1887 (1996)). Full- thickness human skin from non-lesional areas of psoriatic patients was orthotopically transplanted onto the back of 7-8 — week-old SCID mice, followed 2 weeks later (day 14) by intradermal injection of autologous activated PBMC. Conversion of the uninvolved skin into psoriatic plaque skin was seen 1 week after PBMC injection. At this stage, mice were randomly allocated into 3 groups and either left untreated, or treated with a neutralizing human IL-21 or control IgG (1 mg twice weekly intra-peritoneally) for 4 weeks. Histological evaluation of xenografts from mice either left untreated showed a marked epidermal thickening with hyperkeratosis, elongated rete ridges, and aggregates of mononuclear cells (Figure 17 A-B). Notably, xenografts from mice treated with the anti-IL-21 exhibited a decrease in epidermal thickness and reduced numbers of inflammatory cells (Figure 17A-B). In contrast, injection of mice with an isotype control antibody used at the same concentration as the anti-IL-21 did not reduce the skin thickening (Figure 17 A-B). Consistently, abundant proliferating keratinocytes (Ki67+) were seen in biopsies from IgG-treated but not anti-IL-21 -treated mice (Figure 17C). Anti-IL-21 but not control IgG treatment also reduced transcripts of both IFN-γ and IL- 17A (Figure 17D).

[00161] These results indicate that treatment with a neutralizing IL-21 antibody reduces skin thickening and inflammation associated with psoriasis.

INCORPORATION BY REFERENCE

[00162] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[00163] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

We claim:

1. An isolated binding protein that binds human interleukin-21 (IL-21) comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises a CDR_H i comprising the sequence SEQ ID NO: 8, a CDR_H2 comprising the sequence SEQ ID NO: 9, and a CDR_H3 comprising the sequence SEQ ID NO: 10; and wherein the immunoglobulin light chain variable region comprises a CDR_LI comprising the sequence SEQ ID NO: 11, a CDR_L2 comprising the sequence SEQ ID NO: 12, and a CDR_L3 comprising the sequence SEQ ID NO: 13.

2. The binding protein of claim 1, wherein the CDR sequences are interposed between human and humanized framework sequences.

3. The binding protein of claim 2, wherein the binding protein is a monoclonal antibody or antigen binding protein fragment thereof.

4. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable region of claim 1.

5. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin light chain variable region of claim 1.

6. An expression vector containing the nucleic acid of claim 4.

7. An expression vector containing the nucleic acid of claim 5.

8. A host cell comprising at least one expression vector that expresses a protein of claim 1.

9. A method of producing a binding protein, the method comprising:

(a) growing the host cell of claim 8 under conditions so that the host cell expresses the immunoglobulin heavy chain variable region; and

(b) harvesting the immunoglobulin heavy chain variable region.

10. The method of claim 9, wherein, after step (b), the immunoglobulin heavy chain variable region is covalently linked to an immunoglobulin light chain variable region, so that the heavy and light chain variable regions together bind human IL-21.

11. A method of producing a binding protein, the method comprising:

(a) growing the host cell of claim 8 under conditions so that the host cell expresses the immunoglobulin light chain variable region; and

(b) harvesting the immunoglobulin light chain variable region.

12. The method of claim 11, wherein, after step (b), the immunoglobulin light chain variable region is covalently linked to an immunoglobulin heavy chain variable region, so that the light and heavy chain variable regions together bind human IL-21.

13. An isolated binding protein that binds human interleukin-21 (IL-21 ) comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, and wherein the immunoglobulin light chain variable region comprises the amino acid sequence of SEQ ID NO: 7.

14. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable region of claim 13.

15. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin light chain variable region of claim 13.

16. An expression vector containing the nucleic acid of claim 14.

17. An expression vector containing the nucleic acid of claim 15.

18. A host cell comprising at least one expression vector that expresses a protein of claim 13.

19. A method of producing a binding protein, the method comprising:

(a) growing the host cell of claim 18 under conditions so that the host cell expresses the immunoglobulin heavy chain variable region; and

(b) harvesting the immunoglobulin heavy chain variable region.

20. The method of claim 19, wherein, after step (b), the immunoglobulin heavy chain variable region is covalently linked to an immunoglobulin light chain variable region, so that the heavy and light chain variable regions together bind human IL-21.

21. A method of producing a binding protein, the method comprising:

(a) growing the host cell of claim 18 under conditions so that the host cell expresses the immunoglobulin light chain variable region; and

(b) harvesting the immunoglobulin light chain variable region.

22. The method of claim 21, wherein, after step (b), the immunoglobulin light chain variable region is covalently linked to an immunoglobulin heavy chain variable region, so that the light and heavy chain variable regions together bind human IL-21.

23. An isolated binding protein that binds human interleukin-21 (IL-21) comprising an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain is SEQ ID NO: 19, and the immunoglobulin light chain is SEQ ID NO: 21.

24. The binding protein of claim 13 or 23, wherein the binding protein is a monoclonal antibody or an antigen binding fragment thereof.

25. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain of claim 23.

26. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin light chain of claim 23.

27. An expression vector containing the nucleic acid of claim 25.

28. An expression vector containing the nucleic acid of claim 26.

29. A host cell comprising at least one expression vector that expresses a protein of claim 23.

30. A method of producing a binding protein, the method comprising:

(a) growing a host cell of claim 29 comprising one expression vector that expresses an immunoglobulin heavy chain and a second expression vector that expresses an immunoglobulin light chain under conditions so that the host cell expresses both immunoglobulin heavy chain and immunoglobulin light chain, and

(b) harvesting an antibody comprising an immunoglobulin heavy chain and an immunoglobulin light chain capable of binding human IL-21.

31. The binding protein of claim 1 or 13, wherein the binding protein has a K_D of 7 nM or lower.

32. A pharmaceutical composition comprising the binding protein of any one of claims 1-3, 13, and 23-24, and a pharmaceutically acceptable carrier.

33. A method of treating psoriasis in a subject in need thereof, comprising administering an effective amount of the binding protein of claim 1, 13, or 23.

34. A method for reducing or inhibiting an IL-21 mediated inflammatory response in a cell, the method comprising exposing the cell to an effective amount of the binding protein of claim 1, 13, or 23 to reduce or inhibit the inflammatory response in the cell.

35. A method for reducing or inhibiting an IL-21 mediated inflammatory response in a subject in need thereof, the method comprising administering an effective amount of the binding protein of claim 1, 13, or 23.

36. A method of treating an immune-inflammatory disease associated with altered IL-21 expression in a subject in need thereof, the method comprising administering an effective amount of the binding protein of claim 1, 13, or 23.

37. The method of claim 36, wherein the immune-inflammatory disease associated with altered IL-21 expression is selected from the group consisting of chronic inflammatory bowel diseases (IBD), and celiac disease.

38. The method of claim 37, wherein the chronic inflammatory bowel disease is Crohn's disease.

39. The method of claim 37, wherein the chronic inflammatory bowel disease is ulcerative colitis.

40. A method of treating psoriasis in a subject in need thereof, the method comprising administering an effective amount of an isolated binding protein that binds interleukin-21.

41. The method of claim 40, wherein the isolated binding protein that binds interleukin-21 is the binding protein of claim 1, 13, or 23.

42. A method of treating inflammatory bowel disease in a subject in need thereof, the method comprising administering an effective amount of the binding protein of claim 1, 13, or 23.

43. A method of treating Crohn's disease in a subject in need thereof, the method comprising administering an effective amount of the binding protein of claim 1, 13, or 23.

44. The use of the binding protein of claim 1, 13, or 23 in the manufacture of a medicament for treatment of inflammatory bowel disease.

45. The use of claim 44, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

46. The use of the binding protein of claim 1 , 13, or 23 in the manufacture of a medicament for treatment of psoriasis.

47. A method of treating and/or controlling obesity in a subject in need thereof, the method comprising administering an effective amount of an isolated binding protein that binds interleukin-21.

48. A method of treating Hodgkin's lymphoma in a subject in need thereof, the method comprising administering an effective amount of an isolated binding protein that binds interleukin-21.

49. The use of the binding protein to human interleukin 21 in the manufacture of a medicament for the treatment of obesity.

50. The use of the binding protein to human interleukin 21 in the manufacture of a medicament for the treatment of Hodgkin's lymphoma.