RU2804456C1 - Binding molecules specific to il-21 and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions is described, including an antibody or its antigen-binding fragment that specifically binds to IL-21, a conjugate for the treatment or prevention of a disease or disorder associated with IL-21, containing an antibody or antigen-binding fragment, a pharmaceutical composition for the treatment or prevention of a disease or disorder, associated with IL-21, containing an antibody or antigen-binding fragment or conjugate, and a method of treating or preventing a disease or disorder associated with IL-21 in a subject in need thereof. In one embodiment, the antibody comprises a heavy chain variable region (VH) CDR1, CDR2 and CDR3 with the amino acid sequences of the following SEQ ID NO: 29, the following SEQ ID NO: 30 and the following SEQ ID NO: 31, respectively; and CDR1, CDR2 and CDR3 of the light chain variable region (VL) with the amino acid sequences of the following SEQ ID NO: 34, the following SEQ ID NO: 35 and the following SEQ ID NO: 36 respectively.

EFFECT: invention expands the arsenal of agents that specifically bind to IL-21.

15 cl, 40 dwg, 7 tbl, 5 ex

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to United States Patent Application No. 61/976684, filed April 8, 2014, the entire contents of which are incorporated herein by reference.

LINK TO LIST OF SEQUENCES PROVIDED ELECTRONICALLY

[0002] The entire contents of the electronically submitted sequence listing in an ASCII text file (Name: IL21_100P1_seqlist.txt; Size: 55898 bytes; Creation No. and date April 7, 2014) filed with the application are incorporated herein by reference.

BACKGROUND OF THE ART

[0003] This application provides compositions that specifically bind to IL-21 (interleukin-21) and methods of using such compositions, for example, for the treatment or prevention of inflammatory or autoimmune diseases or disorders.

[0004] IL-21 belongs to a family of cytokines that includes IL-2, IL-4, IL-7, IL-9 and IL-15, all of which bind to their own (or common) receptors in complex with common gamma cytokine receptor chain (γc). Most cytokines in this family are extremely important for both the maintenance and function of T lymphocytes, B lymphocytes and NK (natural killer) cells. The receptor for IL-21 is widely distributed on lymphohemapoietic and nonhematopoietic cells, and IL-21 has multiple functions.

[0005] For example, the interaction of IL-21 with IL-21R leads to the activation of several signaling pathways, including the Jak/STAT pathway (Davis, ID, et al., (2007) Clin Cancer Res 13, 3630-3636; Fuqua, CF, et al., (2008) Cytokine 44, 101-107; Habib, T, et al., (2002) Biochemistry 41, 8725-8731). More specifically, IL-21 activates STAT1, STAT3 and STAT5, as indicated by increased phosphorylation of these molecules in the cell (Asao, H, et al., (2001) J Immunol 167, 1-5; Diehl, SA, et al., (2008) J Immunol 180, 4805-4815; Scheeren, FA, et al., (2008) Blood 111, 4706-4715; Zeng, R, et al., (2007) Blood 109, 4135-4142). In particular, STAT3 activation has been shown to play a critical role in regulating B cell responses to IL-21 (Avery, DT, et al., (2008) J Immunol 181, 1767-1779; Avery, DT, et al., J Exp Med 207, 155-171).

[0006] In addition, IL-21 contributes to the maintenance and function of CD8+ memory T lymphocytes and NK cells, promotes the generation of Th17 and T follicular helper (Tfh) cells in mice (and likely humans), and suppresses the generation of regulatory T lymphocytes (Treg). IL-21 has been shown to modulate the activity of NK cells, including effects on their maturation, growth and cytolytic activity (Spolski, R, and Leonard, WJ, (2008) Curr Opin Immunol 20, 295-301). In addition, IL-21 has been shown to promote the production of IFNγ (interferon γ) by both primary NK cells and the human NK cell line NK-92 (Kasaian, MT, et al., (2002) Immunity 16, 559-569 ;Strengell, M, et al., (2003) J Immunol 170, 5464-5469). IL-21 also has a variety of effects on non-hematopoietic cells, such as stromal cells, where it induces inflammation through the release of matrix metalloproteinases (MPMs) (Monteleone et al., (2006) Gut 55, 1774-1780).

[0007] One non-redundant function of IL-21 is to promote the activation, differentiation or death of B lymphocytes during humoral immune responses. The effect of IL-21 on human B lymphocytes has been studied extensively. IL-21 has significant effects on the survival, activation and proliferation of B cells, as well as on the differentiation of B cells into Ig-secreting plasma cells (P) (Avery, DT, et al., (2008) J Immunol 181, 1767-1779 Avery, D.T., et al., J Exp Med 207, 155-171); Bryant, VL, et al., (2007) J Immunol 179, 8180-8190; Ettinger, R, et al., (2005) J Immunol 175, 7867-7879; Parrish-Novak, J, et al., (2000) Nature 408, 57-63; Pene, J, et al., (2004) J Immunol 172, 5154-5157). In addition, increased IL-21 production is characteristic of certain autoimmune diseases and likely contributes to the production of autoantibodies as well as the pathological features of the autoimmune disease. Activation of B cells in vivo can be triggered by interaction with activated T cells that express co-stimulatory molecules such as CD40L and lead to the production of B cell-tropic cytokines such as IL-21.

[0008] Overexpression of IL-21 is a feature of many inflammatory, immune-mediated or autoimmune diseases or disorders, and is likely an important trigger for antibody production as well as pathological features of autoimmune diseases (Nakou et al., (2013) Clin. Exp. Rheumatol. 31, 172-179). The important role of IL-21 in stimulating the humoral immune response makes it an important target for therapeutic interventions in conditions characterized by overproduction of both IL-21 and pathogenic autoantibodies (Dolff et al., (2011) Arthritis Res. Ther. 13, R157; Kang et al., (2011) Arthritis Res. Ther. 13, R179; McGuire et al., (2011) Immunity 34, 602-615; Liu et al., (2012) Arthritis Res. Ther. 14, R255; Terrier et al., (2012) Arthritis Res. Ther. 14, R255 al., (2012) Arthritis Rheum. 64, 2001-2011; Li Q et al., (2013) PLoS One 8, e68145; Li Y et al., (2014) Neurol. Sci. 35, 29-34). Therefore, it is expected that neutralization of IL-21 in the setting of autoimmune reactions should affect several cell populations thought to be involved in the pathogenesis of immune-related diseases. Such diseases or disorders include, but are not limited to, vasculitis, e.g., antineutrophil cytoplasmic antibody (ANCA) or giant cell arteritis (GCA), Sjogren's syndrome, inflammatory bowel disease, pemphigus vulgaris, lupus nephritis, psoriasis, thyroiditis, type 1 diabetes , idiopathic thrombocytopenic purpura (ITP), ankylosing spondylitis, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Crohn's disease, myasthenia gravis, graft-versus-host disease (GVHD).

[0009] The pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterized by large autoantibodies to proteinase 3 (PR3) and myeloperoxidase (MPO). IL-21-producing CD4+ T lymphocytes proliferate in the blood (Abdulahad et al., (2013) Arth Res & Ther 15:R70). Elevated levels of IL-21 are present in the serum of patients with AAV compared with healthy controls, and exogenous IL-21 induces the secretion of ANCA autoantibodies in vitro in peripheral blood mononuclear cells isolated from patients with AAV in vitro. AAV includes granulomatosis with polyangiitis, formerly called Wegener's granulomatosis, GPA, eosinophilic granulomatosis with polyangiitis, known as Churg-Strauss syndrome (CSS), and microscopic polyangiitis (MPA). They are usually grouped together, but epidemiologically the incidence and distribution are different. Current treatment includes corticosteroids, biologics such as rituximab, immunosuppressants, antibiotics, or plasmapheresis, but the prognosis for patients remains poor. There remains a need to find treatments for AAV.

[0010] Sjögren's syndrome (SS) is an autoimmune disease that is characterized by autoantibodies such as rheumatoid factor (RF) as well as autoantibodies to several nuclear antigens such as Ro/La. SS is the second most common autoimmune disease after rheumatoid arthritis. There are at least 1 million patients with primary Sjogren's syndrome in the United States, of whom 90% are women. SS affects the salivary glands, causing, for example, dry mouth and dry eyes. In addition, SS can affect other organs, including the kidneys, blood vessels, lungs, liver, pancreas, peripheral nervous system, and brain, and is associated with other autoimmune diseases such as lupus and rheumatoid arthritis. Significantly elevated serum IL-21 levels correlate with elevated IgG1 levels and the presence of autoantibodies (Kang, 2011). Increased levels of IL-21 and IL-21R can be found in ectopic salivary gland lesions (Kang, 2011). In addition, IL-21 is known to be directly involved in NK activation and cytotoxicity. The salivary glands of patients with primary Sjögren's syndrome show increased numbers of tissue-resident NKs that overexpress the NKp30 receptor and correlate with the lesion score (Rusakiewicz et al., (2013) Sci. Transl. Med. 5, 195ra96). These NKs are involved in pathogenesis through the direct mutual influence of NKs and stromal cells, which leads to tissue damage. IL-21 and its receptor are also expressed in salivary gland infiltrates (Kang, 2011), and IL-21-producing CCR9+ CD4+ memory Tfh cells have been reported to be distributed in the circulation of most patients with SSc (McGuire, 2011). In addition, increased levels of Tfh cells in patients with elevated IL-21 levels are associated with extraglandular manifestations (Szabo et al., (2013) Clinical Immunol 147, 95-104). Preclinical studies in animal models have demonstrated benefit from IL-21 inhibition (Liu et al. (2012) J Oral Pathol Med). There is no cure for SS; Current treatments have limited effectiveness, and none of them can be considered disease-modifying. Mild to moderate illness is treated symptomatically with over-the-counter medications. Severe disease is currently treated with hydroxychloroquine, steroids, methotrexate, azathioprine, or off-label rituximab. There will continue to be a need to find treatments for SS.

[0011] Neutralization of IL-21 has potential clinical utility in several indications, including primary CV disease and vasculitis (Kang, 2011; Bae et al., (2012) Allergy Asthma Immunol Res. 4, 351-356; Terrier, 2012; Abdulahad, 2013; Szabo, 2013).

[0012] The effect of IL-21 on graft-versus-host disease (GVHD) has been extensively studied. When delivered via a hydrodynamic gene system, IL-21 has been shown to significantly accelerate GVHD in humans to mouse xenogeneic grafts, and has also been shown to increase the number of B lymphocytes, plasma cells, and immunoglobulin (Wu et al., (2013) ) Protein Cell 4, 863-871). In contrast, blocking IL-21 in this system has been reported to reduce gastrointestinal injury, Th1 cytokine levels in the spleen, and protect against lethal events (Hippen et al., (2012) Blood 119, 619-628). Additionally, protection from GVHD has been reported to be dependent on the generation of Tregs ( Hippen et al., 2012 ).

[0013] This application provides compositions that specifically bind to IL-21 and methods of using such compositions, for example, for the treatment and prevention of inflammatory, immune-related or autoimmune diseases or disorders.

[0014] According to a specific aspect of the present application, there is provided a method of treating or preventing GHVD using a composition containing an antibody or fragment thereof that specifically binds IL-21. For example, in a mouse xenogeneic graft model of GHVD, an anti-IL-21 formulation was shown to block nascent human IL-21. This composition potentially suppresses GVHD-induced wasting disease and reduces mortality when administered for treatment as well as prophylaxis.

BRIEF DESCRIPTION OF THE INVENTION

[0015] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings and Claims sections of this application. The description in each section of this application should be read in conjunction with the other sections. In addition, the various embodiments disclosed in each section of this application may be combined in various other ways, and all such combinations are intended to fall within the scope of the present invention.

[0016] The present application provides IL-21 binding molecules, such as anti-IL-21 antibodies or antigen binding fragments thereof, such as monoclonal antibodies, capable of inhibiting the interaction of IL-21 and its receptor and inhibiting the activity of IL-21.

[0017] In some embodiments, a single binding molecule or antigen-binding fragment thereof that specifically binds to an IL-21 epitope, wherein the binding molecule specifically binds to the same IL-21 epitope as the antibody or antibody-binding fragment comprises a severe variable region chain (VH) and light chain variable region (VL) 19E3, 9F11, 8B6 or 9H10.

[0018] In some embodiments, a single binding molecule or antigen binding fragment thereof that specifically binds to an epitope of IL-21, competitively inhibits the binding of IL-21 to an antibody or antigen binding fragment thereof, contains VH and VL 19E3, 9F11, 8B6, or 9H10. In some embodiments, VH and VL 19E3 contain SEQ ID Nos: 6 and 11, respectively, VH and VL 9F11 contain SEQ ID Nos: 28 and 33, respectively, VH and VL 8B6 contain SEQ ID Nos: 42 and 47, respectively, a VH and VL 9H10 contain SEQ ID NO: 52 and 57, respectively.

[0019] In some embodiments, a single binding molecule or antigen-binding fragment thereof that specifically binds IL-21 comprises an antibody VL, wherein said VL comprises the amino acid sequences VL-CDR1, VL-CDR2, and VL-CDR3 that are identical or identical to each other except for four, three, two, or one amino acid substitutions in VL-CDRS: SEQ ID No.: 12, 13 and 14, SEQ ID No.: 34, 35 and 36, SEQ ID No.: 48, 49 and 50, or SEQ ID No.: 58, 59 and 60, respectively.

[0020] In some embodiments, a single binding molecule or antigen-binding fragment thereof that specifically binds IL-21 comprises an antibody VJ, wherein said VH comprises the amino acid sequences VH-CDR1, VH-CDR2, and VH-CDR3 that are identical or identical except for four, three, two or one amino acid substitutions in VH-CDRS: SEQ ID No: 7, 8 and 9, SEQ ID No: 29, 30 and 31, SEQ ID No: 43, 44 and 45 or SEQ ID No: 53 , 54 and 55, respectively.

[0021] In some embodiments, a single binding molecule or antigen binding fragment thereof that specifically binds IL-21 comprises an antibody VL, wherein said VL comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical a reference amino acid sequence selected from the group consisting of SEQ ID No.: 11, SEQ ID No.: 17, SEQ ID No.: 21, SEQ ID No.: 25, SEQ ID No.: 33, SEQ ID No.: 39, SEQ ID No.: 40, SEQ ID No: 47 and SEQ ID No: 57.

[0022] In some embodiments, a single binding molecule or antigen binding fragment thereof that specifically binds IL-21 comprises an antibody VH, wherein said VH comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical a reference amino acid sequence selected from the group consisting of SEQ ID No.: 6, SEQ ID No.: 15, SEQ ID No.: 19, SEQ ID No.: 23, SEQ ID No.: 28, SEQ ID No.: 37, SEQ ID No.: 38, SEQ ID No: 42 and SEQ ID No: 52.

[0023] In some embodiments, said binding molecule or fragment thereof comprises an antibody or antigen binding fragment thereof.

[0024] In some embodiments, a single binding molecule or antigen binding fragment thereof that specifically binds IL-21 comprises a VL and a VH comprising the amino acid sequences VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, completely identical or identical except for the substitution of four, three, two or one amino acid in one or more CDRs: SEQ ID No: 12, 13, 14, 7, 8 and 9, SEQ ID No: 34, 35, 36, 29, 30 and 31, SEQ ID Nos: 48, 49, 50, 43, 44 and 45, or SEQ ID Nos: 58, 59, 60, 53, 54 and 55, respectively.

[0025] In some embodiments, a single binding molecule or antigen binding fragment thereof that specifically binds to IL-21, wherein said antibody or antigen binding fragment comprises VH and VL, wherein said VH and VL comprise, respectively, amino acid sequences of at least 85%, 90%, 95% or 100% identical to reference amino acid sequences selected from the group consisting of SEQ ID No: 6 and SEQ ID No: 11, SEQ ID No: 15 and SEQ ID No: 17, SEQ ID No: 19 and SEQ ID No.: 21, SEQ ID No.: 23 and SEQ ID No.: 25, SEQ ID No.: 28 and SEQ ID No.: 33, SEQ ID No.: 37 and SEQ ID No.: 39, SEQ ID No.: 38 and SEQ ID No: 39, SEQ ID No: 37 and 40, SEQ ID No: 42 and SEQ ID No: 47, and SEQ ID No: 52 and SEQ ID No: 57, respectively.

[0026] In some embodiments, said antibody or antigen binding fragment thereof comprises a VH and a VL, wherein said VH comprises the amino acid sequence of SEQ ID No.: 19 and said VL comprises the amino acid sequence of SEQ ID No.: 21. In some embodiments, said antibody or thereof the antigen-binding fragment contains a heavy chain constant region or a fragment thereof. In some embodiments, the heavy chain constant region or fragment thereof is an IgG constant region. In some embodiments, the IgG constant domain contains one or more amino acid substitutions compared to the wild-type IgG constant domain, wherein the modified IgG exhibits an increased half-life compared to the half-life of an IgG containing the wild-type IgG constant domain. In some embodiments, the IgG constant domain comprises one or more amino acid residue substitutions at positions 251-257, 285-290, 308-314, 385-389, and 428-436, wherein the amino acid position numbering corresponds to the EC index as defined by Cabot. In some embodiments, the at least one amino acid substitution in said IgG constant domain is selected from the group consisting of:

(a) replacing the amino acid at Cabot position 252 with tyrosine (Y), phenylalanine (F), tryptophan (W) or threonine (T),

(b) replacing the amino acid at Cabot position 254 with threonine (T),

(c) replacing the amino acid at Cabot position 256 with serine (S), arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D) or threonine (T),

(d) replacing the amino acid at Cabot position 257 with leucine (L),

(e) replacing the amino acid at Cabot position 309 with proline (P),

(f) replacing the amino acid at Cabot position 311 with series (S),

(g) replacing the amino acid at Cabot position 428 with threonine (T), leucine (L), phenylalanine (F) or series (S),

(h) replacing the amino acid at Cabot position 433 with arginine (R), serine (S), isoleucine (I), proline (P) or glutamine (Q),

(i) replacing the amino acid at Cabot position 434 with trypotophan (W), methionine (M), series (S), histidine (H), phenylalanine (F) or tyrosine, and

(j) a combination of two or more of the specified substitutions.

[0027] In some embodiments, the human IgG constant domain contains amino acid substitutions relative to the wild-type human IgG constant domain at Cabot positions 252, 254, and 256, wherein

(a) the amino acid at Cabot position 252 is replaced by tyrosine (Y),

(b) the amino acid at Cabot position 254 is replaced by threonine (T), and

(c) the amino acid at Cabot position 256 is replaced by glutamic acid (E).

[0028] In some embodiments, said constant domain in a human IgG is a human IgG1 constant domain.

[0029] In some embodiments, said heavy chain comprises the amino acid sequence of SEQ ID No.: 16, SEQ ID No.: 20, or SEQ ID No.: 24. In some embodiments, the constant region of said light chain is selected from the group consisting of a human kappa constant region and the human lambda constant region. In some embodiments, the constant region of said light chain is a human kappa constant region. In some embodiments, the heavy and light chain comprise the amino acid sequences of SEQ ID No: 16 and SEQ ID No: 18, SEQ ID No: 20 and SEQ ID No: 22, or SEQ ID No: 24 and SEQ ID No: 26, respectively.

[0030] In some embodiments, the antibody or antigen binding fragment thereof is a mouse antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a multispecific antibody, or an antigen binding fragment thereof. In some embodiments, said antigen binding fragment is Fv, Fab, F(ab')2, Fab', dsFv, scFv, and sc(Fv)2.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof can bind to human IL-21 and cynomolgus IL-21.

[0032] In some embodiments, the antibody or fragment thereof does not bind specifically to human IL-2, IL-4, IL-7, IL-9, or IL-15.

[0033] In some embodiments, the antibody or fragment thereof inhibits the binding of IL-21 to the IL-21 receptor.

[0034] In some embodiments, the antibody or fragment thereof is an antagonist of IL-21 activity.

[0035] In some embodiments, the antibody or fragment thereof can inhibit IL-21-mediated STAT3 phosphorylation in human peripheral blood mononuclear cells (PBMCs).

[0036] In some embodiments, the antibody or fragment thereof can inhibit human and cynomolgus IL-21-mediated phosphorylation of STAT3 in human peripheral blood mononuclear cells (PBMCs).

[0037] In some embodiments, the antibody or fragment thereof can inhibit IL-21-mediated interferon-gamma (IFN-γ) production by NK cells.

[0038] In some embodiments, the antibody or fragment thereof can inhibit human and cynomolgus IL-21-mediated production of interferon gamma (IFN-γ) by NK cells. In some embodiments, said NK cells are NK-92 cells in culture.

[0039] In some embodiments, the antibody or fragment thereof can inhibit IL-21-mediated differentiation of B lymphocytes into plasma cells.

[0040] In some embodiments, the antibody or fragment thereof can inhibit human and cynomolgus IL-21-mediated differentiation of B lymphocytes into plasma cells.

[0041] In some embodiments, the antibody or fragment thereof can inhibit CD4+T cell-activated differentiation of B cells into plasma cells.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human IL-21 with an affinity that is characterized by a dissociation constant (K _D ) of about 100 pM to about 0.1 pM, measured using a kinetic exclusion assay platform ( KinExA) 3000.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to cynomolgus IL-21 with an affinity that is characterized by a dissociation constant (K _D ) of about 100 pM to about 0.1 pM, measured using a kinetic exclusion assay platform ( KinExA) 3000.

[0044] In some embodiments, the antibody or antigen binding fragment thereof has a K _D for human IL-21 of approximately 0.515 pM and a K _D for cynomolgus IL-21 of approximately 0.352 pM.

[0045] In some embodiments, the antibody or fragment thereof is conjugated to an agent selected from the group consisting of an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody, or a fragment thereof, a detectable label, polyethylene glycol (PEG) and a combination of two or more of these agents.

[0046] In some embodiments, the composition contains an antibody or fragment thereof described herein and a carrier. Preferably, said carrier is a pharmaceutically acceptable carrier.

[0047] In some embodiments, the isolated polynucleotide comprises a nucleic acid encoding an antibody VL, wherein the VL comprises the amino acid sequences VL-CDR1, VL-CDR2, and VL-CDR3 that are identical or identical except for four, three, two, or one amino acid substitution in one or more of the specified VL-CDRs, the sequences: SEQ ID Nos: 12, 13 and 14, SEQ ID Nos: 34, 35 and 36, SEQ ID Nos: 48, 49 and 50, or SEQ ID Nos: 58, 59 and 60, respectively.

[0048] In some embodiments, the isolated polynucleotide comprises a nucleic acid encoding an antibody VH, wherein the VH comprises the amino acid sequences VH-CDR1, VH-CDR2, and VH-CDR3 that are identical or identical except for four, three, two, or one amino acid substitution in one or more of the specified VH-CDRs, the sequences: SEQ ID No.: 7, 8 and 9, SEQ ID No.: 29, 30 and 31, SEQ ID No.: 43, 44, and 45, or SEQ ID No.: 53, 54 and 55, respectively.

[0049] In some embodiments, the isolated polynucleotide comprises a nucleic acid encoding an antibody VL, wherein said VL comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID No.: 11, SEQ ID No.: 17, SEQ ID No.: 21, SEQ ID No.: 25, SEQ ID No.: 33, SEQ ID No.: 39, SEQ ID No.: 40, SEQ ID No.: 47 and SEQ ID No: 57. In some embodiments, the isolated polynucleotide comprises a nucleic acid encoding an antibody VH, wherein said VH comprises an amino acid sequence that is at least 85%, 90%, 95%, or 100% identical to a reference amino acid sequence selected from the group consisting of from SEQ ID No.: 6, SEQ ID No.: 15, SEQ ID No.: 19, SEQ ID No.: 23, SEQ ID No.: 28, SEQ ID No.: 37, SEQ ID No.: 38, SEQ ID No.: 42 and SEQ ID No: 52.

[0050] In some embodiments, a polynucleotide described herein comprises a nucleic acid that encodes an antibody or antigen binding fragment thereof comprising VH and VL described herein that can specifically bind to IL-21. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to the same epitope as the antibody or antigen-binding fragment thereof containing the VH and VL of 19E3, 9F11, 8B6, or 9H10.

[0051] In some embodiments, a polynucleotide described herein comprises a nucleic acid that encodes an antibody or antigen binding fragment thereof comprising said VH or said VL that binds to human or cynomolgus IL-21.

[0052] In some embodiments, a vector is provided containing a polynucleotide described herein.

[0053] In some embodiments, a composition is provided comprising a polynucleotide described herein or a vector described herein.

[0054] In some embodiments, a polynucleotide or combination of polynucleotides is provided that encodes a binding molecule or an antigen-binding fragment thereof.

[0055] In some embodiments, a polynucleotide or combination of polynucleotides encoding an antibody or antigen binding fragment thereof described herein is provided.

[0056] In some embodiments, the composition comprises a polynucleotide that contains a nucleic acid encoding a VH, and a polynucleotide that contains a nucleic acid encoding a VL, wherein said VL and VH comprise the amino acid sequences VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, completely identical or identical except for four, three, two or one amino acid substitution in one or more CDRs, sequences: SEQ ID No: 12, 13, 14, 7, 8 and 9 , SEQ ID Nos: 34, 35, 36, 29, 30 and 31, SEQ ID Nos: 48, 49, 50, 43, 44 and 45, or SEQ ID Nos: 58, 59, 60, 53, 54 and 55, respectively.

[0057] In some embodiments, the composition comprises a polynucleotide that contains a nucleic acid encoding a VH and a polynucleotide that contains a nucleic acid encoding a VL, wherein VL and VH comprise, respectively, amino acid sequences of at least 85%, 90% , 95% or 100% identical to reference amino acid sequences selected from the group consisting of SEQ ID No: 6 and SEQ ID No: 11, SEQ ID No: 15 and SEQ ID No: 17, SEQ ID No: 19 and SEQ ID No. : 21, SEQ ID No.: 23 and SEQ ID No.: 25, SEQ ID No.: 28 and SEQ ID No.: 33, SEQ ID No.: 37 and SEQ ID No.: 39, SEQ ID No.: 38 and SEQ ID No.: 39 , SEQ ID No.: 37 and 40, SEQ ID No.: 42 and SEQ ID No.: 47, and SEQ ID No.: 52 and SEQ ID No.: 57, respectively.

[0058] In some embodiments, said VH and VL are encoded by nucleotide sequences that are at least 85%, 90%, 95%, or 100% identical to reference nucleotide sequences selected from the group consisting of SEQ ID No: 5 and SEQ ID No.: 10, SEQ ID No.: 27 and SEQ ID No.: 32, SEQ ID No.: 41 and SEQ ID No.: 46, or SEQ ID No.: 51 and SEQ ID No.: 56, respectively.

[0059] In some embodiments, a polynucleotide containing a nucleic acid encoding a VH and a polynucleotide containing a nucleic acid encoding a VL are in the same vector. In some embodiments, a vector is provided as described herein.

[0060] In some embodiments, the polynucleotide containing the VH-encoding nucleic acid and the polynucleotide containing the VL-encoding nucleic acid are in different vectors. Some embodiments provide the vectors described herein.

[0061] In some embodiments, the compositions described herein include an antibody or antigen binding fragment thereof that contains said VH and said VL that can specifically bind to IL-21. In some embodiments, the antibody or antigen binding fragment thereof comprising VH or VL may bind to human or cynomolgus IL-21.

In some embodiments, the antibody or antigen binding fragment thereof may specifically bind to the same epitope as the antibody or antigen binding fragment thereof containing VH or VL 19E3, 9F11, 8B6 or 9H10.

[0062] In some embodiments, host cells are provided containing polynucleotides described in the present invention, compositions described in the present invention, or a vector or vectors described in the present invention.

[0063] In some embodiments, a method of producing an antibody or antigen binding fragment thereof described herein comprises (a) culturing a cell described herein; and (b) isolating the antibody or antigen-binding fragment thereof.

[0064] In some embodiments, a diagnostic agent or kit is provided containing an antibody or antigen binding fragment thereof described herein.

[0065] In some embodiments, a method of reducing IL-21-induced STAT3 phosphorylation in an IL-21R-expressing cell comprises contacting said cell with a binding molecule or antigen-binding fragment thereof described herein, an antibody or antigen-binding fragment thereof described herein application, or the composition described in this application.

[0066] In some embodiments, a method of reducing IL-21-mediated production of IFN-γ by NK cells comprises contacting said NK cell with a binding molecule or antigen binding fragment thereof described herein, an antibody or antigen binding fragment thereof described herein , or the composition described in this application.

[0067] In some embodiments, a method of reducing IL-21-mediated differentiation of naïve or memory B lymphocytes comprises contacting said naïve or memory B lymphocyte with a binding molecule or antigen-binding fragment thereof described herein , an antibody or antigen-binding fragment thereof described herein, or a composition described herein.

[0068] In some embodiments, plasma cell differentiation is activated by CD4+ T lymphocytes.

[0069] In some embodiments, a method of treating an inflammatory, immune-related, or autoimmune disease or disorder in a subject comprises administering to the subject in need of treatment an effective amount of a binding molecule or antigen binding fragment thereof described herein, an antibody or antigen binding fragment thereof described herein in this application, or the composition described in this application.

[0070] In some embodiments, a method of preventing an inflammatory, immune-related, or autoimmune disease or disorder in a subject includes administering to a subject predisposed to such disease or disorder an effective amount of a binding molecule or antigen-binding fragment thereof described herein, an antibody, or an antigen-binding fragment thereof. a fragment described in this application, or a composition described in this application.

[0071] In some embodiments, said autoimmune disease is Sjogren's syndrome (SS), vasculitis (ANCA/GCA), systemic lupus erythematosus or lupus nephritis, rheumatoid arthritis (RA), Crohn's disease, myasthenia gravis, or any combination thereof.

[0072] In some embodiments, said immune-related disease or disorder is GVHD.

[0073] In some embodiments, the method of preventing GVHD includes reducing or eliminating the symptoms of GVHD.

[0074] In a given case, the symptoms of GHVD that are reduced or eliminated are rash, blisters, loss of appetite, vomiting, early feeling of fullness after eating, diarrhea, abdominal discomfort, bloating, blood in stool, jaundice, dark urine, upper discomfort abdominal pain, fluid weight gain (edema), arthritis-like symptoms, pain and stiffness, dry eyes, eye irritation, mouth ulcers, persistent cough, shortness of breath, difficulty breathing.

[0075] In some embodiments, a method of suppressing weight gain due to GHVD includes administering to a subject suffering from GHVD or predisposed to GHVD a binding molecule or an antigen binding fragment thereof described herein, an antibody or an antigen binding fragment thereof described herein, or the composition described in this application.

[0076] In some embodiments, a method of reducing anemia due to GHVD includes administering to a subject suffering from or predisposed to GHVD a binding molecule or antigen binding fragment thereof described herein, an antibody or antigen binding fragment thereof described herein, or a composition described in this application.

[0077] In some embodiments, a method of inhibiting the proliferation of T lymphocytes comprises administering to a subject suffering from or predisposed to GHVD a binding molecule or an antigen binding fragment thereof described herein, an antibody or an antigen binding fragment thereof described herein, or a composition, described in this application.

[0078] In some embodiments, a method of reducing cytokine levels in a subject suffering from or predisposed to GHVD comprises administering to the subject a binding molecule or antigen binding fragment thereof described herein, an antibody or antigen binding fragment thereof described herein, or a composition described in this application.

[0079] In some embodiments, a method of determining the level of IL-21 expression in a sample comprises (a) contacting said sample with a binding molecule or antigen binding fragment thereof as described herein, an antibody or antigen binding fragment thereof as described herein, or the composition described in this application, and (b) determining the binding to IL-21 in the specified sample.

BRIEF DESCRIPTION OF DRAWINGS/FIGURES

[0080] In FIG. 1A shows an alignment of the variable regions of the mouse monoclonal antibody 9F11 (VH: SEQ ID No: 28, VL: SEQ ID No: 33) and three humanized variants (VH-4 and VH-11: SEQ ID No: 41, VH-6: SEQ ID No.: 42, VL-4 and VL-6: SEQ ID No.: 43, VL-11: SEQ ID No.: 44). Residues that differ between human and mouse antibodies in the frame regions are double underlined. Mouse back mutations in frame regions are highlighted in bold, and CDR regions are single underlined. In FIG. 1B shows an alignment of the variable regions of the mouse monoclonal antibody 19E3 (VH: SEQ ID No: 6, VL: SEQ ID No: 11) and three humanized VH variants (SEQ ID Nos. 61, 62 and 63) and two humanized VL variants (SEQ ID No. : 64 and 65). Residues that differ between human and mouse antibodies in the frame regions are double underlined. Mouse back mutations in frame regions are highlighted in bold, and CDR regions are single underlined.

[0081] In FIG. 2A and FIG. 2B shows the results of the KinExA Dual Curve Fit assay for concentrations of 500 fM and 50 pM IgG K44VHa-N56Q (MEDI7169) in liquid phase competition with human (FIG. 2A) and cynomolgus (FIG. 2B) IL-21.

[0082] In FIG. Figure 3A shows the effect of human and cynomolgus IL-21 on STAT3 phosphorylation of human PBMCs. FIGURE 3B shows the suppression of human and cynomolgus IL-21-induced activation of human PBMC STAT3 phosphorylation by anti-IL-21 antibodies K2Ha-N56Q (inverted triangles), K44VHa-N56Q (MEDI7169) (diamonds), and K44VHa6-N56Q (circles).

[0083] In FIG. 4 shows the suppression of IFNγ production by NK-92 cells caused by human (FIG. 4A) and cynomolgus (FIG. 4B) IL-21.

[0084] In FIG. 5 shows the suppression of IL-21-induced differentiation of human B lymphocytes into plasma cells by human IL-21 (FIG 5A) or cynomolgus monkey. The graphs show a decrease in the level of IgD ^- CD38 ^hi PC compared to the control. FIG 5C shows that under control conditions, when stimulated with IL-21, anti-CD40 and anti-IgM antibodies lead to IgG production in the range of 30-40 μg/ml by day 6, and 19E3.1 and 9F11.1 are suppressed production of Ig.

[0085] In FIG. 6A shows that co-cultivation of B lymphocytes with activated CD4+ T lymphocytes leads to the appearance of an IgD ^- CD38 ^hi PC population, with 68.5% of CD19+ cells expressing this genotype of plasma cells by day 7. In FIG. 6B and 6C show suppression of plasma cell differentiation upon addition of 19E3.1 or 9F11.1.

[0086] In FIG. 7 shows that anti-IL21 antibody (19E3.1) blocks IL-21-induced T cell expansion.

[0087] Human T lymphocytes were stimulated with recombinant human IL-21 in combination with anti-CD3. Antibody 19E3.1 was added at the indicated concentrations, and T cell expansion was quantified on day 4. All conditions were tested in duplicate. The experiment was carried out with two unique donors. One representative donor is shown.

In FIG. 8 shows that K44VHa-N56Q (MEDI7169) blocks GVHD-induced wasting disease by limiting the proliferation of CD4+ T lymphocytes. PBMCs from donor 1 (12.71 x 10 ⁶ ) or donor 2 (13.46 x 10 ⁶ ) were transferred to NOD/SCID/γc mice on study day 0. Mice received 200 μg of each control monoclonal antibody (FIG. 8A). or K44VHa-N56Q (FIG. 8B) administered three times a week starting on day 1. Mice were bled every two weeks and flow cytometry was performed to determine the number and phenotype of human CD45+ lymphocytes. In FIG. 8C and FIG. 8D shows the percentage of human CD45+ lymphocytes collected from mice treated with control mAb or K44VHa-N56Q, respectively (day 21, donor 1; n=3 control mAb, n=5 K44VHa-N56Q). In FIG. 8E and FIG. 8F shows the percentage of CD4+ or CD8+ cells in the human CD45+ lymphocyte fraction in mice treated with control mAb or K44VHa-N56Q, respectively (from day 35, donor 2; n=10 control mAb + PBS backbone/n=5 K44VHa-N56Q). For all conditions, 100 μl of blood was collected and flow cytometry was performed for the same time, so that the number of events shown reflects the relative number of cells. Mean ± standard error of absolute numbers of human CD45+ lymphocytes collected from blood on day 21 for donor 1, control monoclonal antibody, n=3 (729111±113899) and K44VHa-N56Q, n=5 (39.104±5.159), P <0.0357. For donor 2, control monoclonal antibody and PBS combined, n=10 (237.294±35.628) and K44VHa-N56Q, n=5, (14.980±2.275) P=0.0007.

[0088] In FIG. Figure 9 shows that K44VHa-N56Q reversibly inhibits the proliferation of human cells in recipient mice. Donor 2 PBMCs (13.46×10 ⁶ ) were transferred into NOD/SCID/γc mice on study day 0. We received 200 μl of PBS base (squares) or 200 μg of K44VHa-N56Q antibody (triangles) or control monoclonal antibody (circles). ) administered three times a week, from day -+1 to day 44. Mice were bled every two weeks and performed flow cytometry to determine the number and phenotype of human CD45+ lymphocytes. The data shows the number of human cells sampled. For all conditions, 100 μl of blood was stained and the same volume was collected on a flow cytometer, so that the number of events shown reflects the relative number of cells.

[0089] In FIG. 10 shows that K44VHa-N56Q protects against GVHD-induced weight loss and mortality. Donor 1 PBMCs (12.71×10 ⁶ ) were transferred into NOD/SCID/γc mice on study day 0, and mice were monitored for weight loss (FIG. 10A) and mortality (FIG. 10B). K44VHa-N56Q antibodies (triangles) or control monoclonal antibody (filled circles) were administered three times per week, from day −+1 to day 31. Naïve animals (open circles) received no human cells or any monoclonal antibodies. Survival percentage indicates either when mice died or when they reached 20% body weight loss and were sacrificed. Shown is one of two independent experiments from two different PBMC donors.

[0090] In FIG. Figure 11 shows the suppression of cell proliferation and cytokine production by K44VHa-N56Q. Donor 2 PBMCs (13.46 x 10 ⁶ ) were transferred into NOD/SCID/γc mice on study day 0 and injected with either 200 μg K44VHa-N56Q or control monoclonal antibody three times per week starting on day 1. In mice Blood was collected and serum cytokines were assessed to determine the presence of cytokines using the MILLIPLEX Human Th17 MAP kit from Millipore. Serum levels (pg/ml) are shown for IL-21 (FIG. 11A), interferon gamma (FIG. 11B), tumor necrosis factor alpha (FIG. 11C), IL-9 (FIG. 11D), granulocyte-macrophage colony -stimulating factor (FIG. 11E), IL-10 (FIG. 11F) and IL-5 (FIG. 11G).

[0091] In FIG. Figure 12 shows that the suppression of cell proliferation and cytokine production by anti-IL-21 is reversible. Donor 1 PBMCs (12.71 x ¹⁰⁶ ) were transferred to NOD/SCID/γc mice on study day 0 and injected with either 200 μg K44VHa-N56Q (filled symbols) or control monoclonal antibody (white symbols) three times per week. , starting on day 1 until day 31, when K44VHa-N56Q administration was stopped. Mice were bled on days 15, 28, 43, and 57, and interferon gamma (FIG. 12A), tumor necrosis factor alpha (FIG. 12B), and IL-10 (FIG. 12C) were quantified. ) in serum at the indicated time points using the MILLIPLEX Human Th17 MAP kit from Millipore. Mice were also bled on days 7, 21, 35, 51 and 57, and the number of human CD45+ lymphocytes was determined (FIG. 12D). All mice treated with the control monoclonal antibody died by day 28 of the study; thus, there was only one cytokine determination time point for these animals.

[0092] In FIG. 13 shows that the therapeutic antibody K44VHa-N56Q protects subjects from GVHD. Donor 1 PBMCs (12.71×10 ⁶ ) were transferred into NOD/SCID/γc mice on study day 0 and their survival was monitored. Mice were treated with control monoclonal antibody from day 7 (circles), K44VHa-N56Q from day 7 (triangles), or K44VHa-N56Q from day 14 (inverted triangles) three times a week at a dose of 200 μg/mouse. Mice were euthanized when body weight had decreased by 20%, or if the mouse was emaciated/fading, or was in pain or distress. Death and humane euthanasia have been used interchangeably for purposes of tracking survival.

[0093] In FIG. 14 shows that K44VHa-N56Q can protect against GVHD-induced anemia. Mice were injected and handled as described for FIG. 13. Mice have not been previously exposed, i.e. received no cells (asterisks), or received human PBMCs followed by control mAb starting on day 7 (circles), K44VHa-N56Q from day 7 (triangles), or K44VHa-N56Q from day 14 ( inverted triangles) three times a week at a dose of 200 μg/mouse. Heparinized whole blood samples collected from the retro-orbital sinus were diluted 1:10 for analysis on an automated hematology analyzer (Sysmex XT-2000iv). The resulting CBC (FIG. 14A) and leukocyte count (FIG. 14C), hematocrit (FIG. 14B) and hemoglobin (FIG. 14D) were multiplied by a dilution factor of ten.

DETAILED DESCRIPTION OF THE INVENTION

[0094] The present invention provides molecules and antigen-binding fragments thereof that bind to IL-21. In some embodiments, such molecules are antibodies and antigen-binding fragments thereof that specifically bind IL-21. Also proposed are related polynucleotides, compositions containing anti-IL-21 antibodies or antigen-binding fragments thereof, and methods for producing anti-IL-21 antibodies and antigen-binding fragments thereof. Additionally provided are methods of using anti-IL-21 antibodies, such as methods of treating inflammatory, immune-related or autoimmune diseases or disorders in a subject and methods of diagnosis.

[0095] To make the present invention more clear, definitions of certain terms are first introduced. Additional definitions are introduced throughout the detailed description.

I. Definitions

[0096] In the present description and the accompanying claims, the singular form includes references to the plural unless the context clearly requires otherwise. The terms “one,” as well as the terms “one or more” and “at least one,” may be used interchangeably in the application.

[0097] In addition, “and/or” should be taken as a specific disclosure of each of the two properties or components described, with or without the second. Thus, the term “and/or” used in a phrase such as “A and/or B” is intended to include “A and B,” “A or B,” “A” (one), and “B.” (one). Likewise, the term “and/or” as used in a phrase such as “A, B and/or C” is intended to include each of the following: A, B and C; A, B or C, A or C; A or B; B or C; A and C; A and B; B and C; A (one); In one); and C (one).

[0098] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as generally accepted by those skilled in the art to which this invention relates. For example, many of the terms used in the present invention can be found in the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press.

[0099] Units, prefixes and symbols are in accordance with the International System of Units (SI). Numeric value areas include values defined by a range. Unless otherwise noted, amino acid sequences are written from left to right in an N-terminal to C-terminal orientation. The headings set forth herein are not intended to be limiting of the various aspects and embodiments, which may be generally understood from the full description. Accordingly, the terms defined below are fully defined by reference to the description in its entirety.

[00100] Where embodiments are described using the language "including", other similar embodiments described in terms of "consisting of" and/or "consisting essentially of" are also provided.

[00101] In this application, amino acids are designated by conventional three-letter symbols or single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Likewise, nucleotides are designated by common one-letter codes.

[00102] The term "IL-21" refers to the cytokine interleukin-21, and/or active fragments thereof. IL-21 binds to the IL-21 receptor on various cells of the immune system, including signal transduction in those cells described elsewhere in this application. IL-21 has been characterized in the most common animal model systems. The cDNA and amino acid sequences of human IL-21 can be found, for example, in GenBank under accession number BC066260.1 (SEQ ID No: 1) and AAH69124.1 (SEQ ID No: 2). The cDNA and amino acid sequences of cynomolgus (Macaca fascicularis) IL-21 can be found, for example, at SEQ ID No.: 3 (SEQ ID No.: 3) and 4 (SEQ ID No.: 4) in US patent application No. 2008-0267910 A1 (incorporated herein by reference to its full version).

[00103] The terms “inhibits,” “blocks,” and “suppresses” are used interchangeably herein and refer to any statistically significant reduction in biological activity, including complete blocking of activity. For example, “inhibition” may refer to a reduction in biological activity by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Accordingly, when the terms “inhibition” or “suppression” are used to describe, for example, an effect on a signal transduction pathway from IL-21, for example, signal transduction in a cell expressing the IL-21 receptor (IL-21R) in the presence of IL-21 ( e.g., STAT3 phosphorylation, interferon gamma (IFN-γ) production in NK, B cell differentiation into plasma cells, or suppression of IL-21-triggered proliferation of naïve human CD4+ T lymphocytes), these terms refer to the ability of the IL-21 binding molecule to for example, an anti-IL-21 antibody or antigen-binding fragment thereof statistically significantly reduces IL-21-induced signal transduction through the IL-21 receptor compared to signal transduction in an untreated (control) cell. The cell that expresses IL-21R may be a naturally occurring cell or cell line (eg, a T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell, or other type of lymphoid lineage cell) or may be produced recombinantly. methods by a cell by introducing a nucleic acid encoding IL-21 R into a host cell. In one embodiment, the IL-21 binding molecule, e.g., an anti-IL-21 antibody or antigen binding fragment thereof, can inhibit IL-21-induced signal transduction in an IL-21R-expressing cell by at least 10%, or by at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80% , or at least 90% or approximately 100%, as determined, for example, by flow cytometry, Western blotting, ELISA (enzyme-linked immunosorbent assay), or other methods as described in the Examples section below.

[00104] As used herein, the terms “antibody” or “immunoglobulin” are used interchangeably and include whole antibodies and any antigen-binding fragment or single chain thereof.

[00105] A typical antibody contains at least two heavy chains (H) and two light chains (L) linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this application) and a light chain constant region. The light chain constant region consists of a single C1 domain. The VH and VL regions can be further subdivided into regions of hypervariability, called hypervariable regions (CDR), separated by regions that are more conserved, called framework regions (FW). Each VH and VL consists of three CDRs and four FWs, arranged from N-terminus to C-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system. Examples of antibodies herein include hybridoma-derived mouse monoclonal antibodies 19E3, 9F11, 8H10, and 8B6, humanized, affinity-optimized, germline and/or other variants thereof, and serum half-life-optimized anti-IL-21-YTE antibodies. (for example, K44VHa-N56Q K44VHa6-N56Q or K2Ha-N56Q).

[00106] The term "humanization" means that amino acids at specific positions in the antibody are reverse mutated back to a germline state.

[00107] The term "antibody" may refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the above, by recognizing at least one site of an antigen in variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes intact polyclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2 and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated by of at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigen recognition portion of the antibody, and any other modified immunoglobulin molecule containing the antigen recognition site, provided that the antibodies exhibit the desired biological activity. An antibody may belong to any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or their subclasses (isotypes) (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), depending on the nature of their constant domains heavy chains called alpha, delta, epsilon, gamma and mu, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[00108] By "blocking" antibody or "antagonistic" antibody is meant an antibody that inhibits or reduces the biological activity of the antigen to which it binds, for example, IL-21. In certain aspects, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. It is desirable that the biological activity is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95% or even 100%.

[00109] The term "IL-21 antibody" or "antibody that binds IL-21" or "anti-IL-21 antibody" refers to an antibody that is capable of binding to IL-21 with sufficient affinity such that the antibody is useful as a therapeutic agent or diagnostic reagent targeting IL-21. The extent of binding of an anti-IL-21 antibody to an unrelated, non-IL-21 protein is less than approximately 10% of the binding of the antibody to IL-21, as measured, for example, by radioimmunoassay (RIA), BIACORE® (using recombinant IL -21 as the analyte and the antibody as the ligand, or vice versa), KINEXA®, or other binding assay known in the art. In certain embodiments, an antibody that binds to IL-21 exhibits a dissociation constant ( _KD ) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤10 pM, ≤1 pM, or ≤0.1 pM.

[00110] The term "antigen binding fragment" refers to a portion of an intact antibody and refers to the hypervariable regions of an intact antibody. The antigen-binding fragment of an antibody may be fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, single chain antibodies (eg, ScFvs), and multispecific antibodies formed from antibody fragments.

[00111] The term "monoclonal antibody" (mAb) refers to a homogeneous population of antibodies involved in highly specific recognition and binding to a single antigenic determinant or epitope. They differ from polyclonal antibodies, which usually include different antibodies against different antigenic determinants. The term "monoclonal antibody" includes both intact and full-length antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins containing antibodies and any other modified molecules immunoglobulins containing an antigen recognition site. In addition, the term “monoclonal antibody” refers to those antibodies that are produced by a variety of methods, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[00112] The term “humanized antibody” refers to an antibody derived from a non-human (eg, murine) immunoglobulin that has been engineered to contain a minimum of non-human (eg, mouse) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the hypervariable region (CDR) are replaced by residues from the CDR of a non-human species (eg, mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and capacity (Jones et al. , 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some embodiments, human immunoglobulin Fv framework (FW) residues are replaced with corresponding residues in an antibody from a non-human species that have the desired specificity, affinity, and capacity.

[00113] The humanized antibody may be further modified by replacing additional residues either in the Fv framework region and/or within the replaced residues from a non-human species to improve and optimize the specificity, affinity and/or capacity of the antibody. Typically, humanized antibodies will consist of substantially at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to an immunoglobulin of a non-human species, while all or substantially all of the regions FRs are regions of the human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically a region from human immunoglobulin. Examples of methods used to generate humanized antibodies are described in US Pat. No. 5,225,539 or 5,639,641.

[00114] The term "variable region" of an antibody refers to a light chain variable region of an antibody or a heavy chain variable region of an antibody, either alone or in combination. Each variable region of the heavy and light chains consists of four framework regions (FW) connected by three hypervariable regions (CDR). The CDRs in each chain are held together in close proximity to each other by the FW regions and, with the CDRs from the other chain, facilitating the formation of the antigen binding site of the antibodies. There are at least two methods for determining CDRs: (1) an approach based on interspecies sequence variability (for example, Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)) ; and (2) an approach based on crystallographic studies of antigen-antibody complexes (A1 - lazikani et al. (1997) J. Molec. Biol. 273:927-948)). In addition, sometimes in technology a combination of these two approaches is used to determine the CDR.

[00115] Typically, when specifying a residue in a variable domain, the Cabot numbering system (approximately residues 1-107 of the light chain and 1-113 of the heavy chain) is used (e.g., Kabat et al., Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[00116] Cabot numbering of amino acid positions refers to the numbering system used for heavy chain variable domains or light chain variable domains in antibody formulations in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). According to this numbering system, the actual linear amino acid sequence may contain fewer amino acids or additional amino acids corresponding to a truncation or insertion in the FW or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion (Cabot residue 52a) after H2 residue 52 and inserted residues (e.g., Cabot residues 82a, 82b, and 82c, etc.) after residue 82 in the FW heavy chain. chains.

[00117] Cabot numbering of residues can be determined for a particular antibody by the alignment of regions of homology in the sequence of the specified antibody with a standard Cabot numbering sequence. Chothia numbering, on the other hand, refers to the position of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chotia CDR-H1 loop, when numbered by Cabot, varies from H32 to H34 depending on the length of the loop (this is because the Cabot numbering scheme produces insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). AnM hypervariable regions represent a compromise between Cabot's CDRs and Chotia's structural loops, and are used in Oxford Molecular's AbM modeling software. See Table 1.

[00118] IMGT (ImMunoGeneTics) also proposes a numbering system for immunoglobulin variable regions, including CDRs. See, for example, Lefranc, M.R. et al., Dev. Comp. Immunol. 27: 55-77(2003), which is incorporated herein by reference. The IMGT numbering system was based on over 5000 sequence alignments, structural data, and hypervariable loop characterizations, and allows for easy comparison of variable regions and CDR regions across species. According to the IMGT numbering scheme, VH-CDR1 is in positions 26-35, VH-CDR2 is in positions 51-57, VH-CDR3 is in positions 93-102, VL-CDR1 is in positions 27-32, VL-CDR2 is in positions 50-52, a VL-CDR3 - in positions 89-97.

[00119] In the present description, VH CDR sequences are understood to be arranged according to the classical Cabot numbering, namely Cabot VH-CDR1 is located at positions 31-35, VH-CDR2 at positions 50-65, VH-CDR3 at positions 95-102. VL-CDR1, VL-CDR2 and VL-CDR3 also correspond to the location of the classical Cabot numbering, namely at positions 24-34, 50-56 and 89, 97, respectively.

[00120] The term “human antibody” refers to an antibody produced in a human body, or an antibody with an amino acid sequence corresponding to an antibody produced in a human body, produced by any method known in the art. This definition of human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies containing at least one heavy and/or light chain polypeptide of a human antibody, for example, an antibody containing polypeptides of a light chain from a mouse antibody and a heavy chain from a human antibody.

[00121] The term "chimeric antibodies" refers to antibodies in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from a single mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in the antibodies obtained from another species (usually a human) to preclude an immune response in that species.

[00122] The terms “YTE” or “YTE mutant” refers to a mutation in the IgG1 Fc that results in increased binding to human FcRn and improves the serum half-life of an antibody containing such mutation. The YTE mutant contains a combination of three mutations, M252Y/S254T/T256E (EC numbering, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, U.S. Public Health Service, National Institutes of Health, Washington, D.C.), introduced into the heavy chain of IgG1 . See US Pat. No. 7,658,921, which is incorporated herein by reference. The YTE mutation has been shown to increase the serum half-life of antibodies by approximately fourfold compared with the wild-type variant of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006); Robbie et al., (2013) Antimicrob. Agents Chemother. 57, 6147-6153). Also see US Pat. No. 7,083,784, which is incorporated herein by reference.

[00123] The term “binding affinity” generally refers to the total strength of all non-covalent interactions between a particular binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, “binding affinity” refers to the natural binding affinity that reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of molecule X for molecule Y can usually be represented by the dissociation constant (K _D ). Affinity can be measured by conventional methods known in the art, including the methods disclosed herein. Antibodies with low affinity tend to bind to the antigen slowly and tend to dissociate easily, whereas antibodies with high affinity tend to bind to the antigen more quickly and tend to remain bound to it longer. A number of methods are known in the art for measuring affinity, any of which can be used for the purposes of the present invention.

[00124] “Activity” is typically expressed as an IC50 (half-maximal inhibitory concentration) value, nM or pM, unless otherwise noted. IC ₅₀ is the median inhibitory concentration of an antibody molecule. In functional tests, IC ₅₀ refers to the concentration that reduces the biological response by 50% of its maximum. In ligand binding studies, the IC ₅₀ is the concentration that reduces ligand binding by 50% of the maximum specific binding level. IC ₅₀ can be calculated by any number of methods known in the art.

[00125] The fold increase in activity for antibodies or polypeptides of the present invention compared to a reference antibody can be at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold, at least at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, or at least about 180 times or more.

[00126] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound on Fc receptors (FcRs) is present on certain cytotoxic cells (eg, natural killer (NK) cells, neutrophils and macrophages) and allows said cytotoxic effector cells to specifically bind to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. Specific antibodies - IgG, directed to the surface of target cells, “arm” cytotoxic cells and are absolutely necessary for such destruction. Lysis of target cells occurs extracellularly, requires direct cell-to-cell contact, and does not involve complement. It is assumed that in addition to antibodies, other proteins containing Fc regions, especially Fc-fusion proteins that can bind specifically to the antigen-bearing target cell, can influence cell-mediated cytotoxicity. For simplicity, cell-mediated cytotoxicity resulting from the activity of Fc-fusion proteins is also referred to herein as ADCC activity.

[00127] A polypeptide, antibody, polynucleotide, vector, cell, or composition that is “isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include polypeptides, antibodies, polynucleotides, vectors, cells or compositions that have been purified to such an extent that they are no longer in the form in which they are found in nature. In some embodiments, the antibody, polynucleotide, vector, cell, or composition that is isolated is substantially pure.

[00128] By “subject,” “individual,” “animal,” or “patient” is meant any subject, particularly a mammal, for whom diagnosis, prognosis, or therapy is desired. Mammals include humans, domestic animals, farm animals, sporting animals and zoo animals, including, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc.

[00129] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient to occur and that does not contain additional components that are unacceptably toxic to the subject to whom the composition might be administered. Such a composition may be sterile.

[00130] As used herein, the term “effective amount” refers to an amount sufficient to accomplish a specific intended purpose. The "effective amount" can be determined empirically and in a standardized manner, depending on the intended purpose.

[00131] The term “label” as used herein refers to a analyte or composition that is conjugated directly or indirectly to an antibody to form a “labeled” antibody. The label may itself be detectable (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a chemical transformation of the substrate compound or composition that is detectable.

[00132] Terms such as “treating,” “treating,” or “alleviating,” refer to therapeutic measures that cure, slow, reduce symptoms, and/or arrest the progression of a diagnosed pathological condition or disorder. Thus, those who need treatment include subjects who already suffer from the disorder. In certain embodiments, a subject has been successfully "treated" for an inflammatory, immune-related, or autoimmune disease or disorder according to the methods provided herein if said patient demonstrates, for example, complete, partial, or temporary relief or resolution of symptoms associated with said disease or disorder.

[00133] The terms “prevent” or “prophylaxis” refer to prophylactic or preventative measures that are aimed at preventing and/or slowing the development of the pathological condition or disorder in question. Thus, those in need of prevention include subjects prone to or susceptible to developing the disorder. In certain embodiments, an inflammatory, immune-related, or autoimmune disease or disorder is successfully prevented according to the methods provided herein if the patient develops, for example, temporary or permanent, lesser or less severe symptoms associated with the disease or disorder, or symptoms associated with a given disease or disorder begin later than in a patient who has not undergone the methods of the present invention.

[00134] As used herein, the term "polynucleotide" may include one or more "nucleic acids", "nucleic acid molecules" or "nucleotide sequences" and refers to a polymer of nucleotides of any length, and includes DNA and RNA. Polynucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and analogs thereof. The previous description applies to all polynucleotides mentioned in this application, including RNA and DNA.

[00135] The term “vector” refers to a construct that is capable of delivering and, in some embodiments, expressing one or more of the subject gene(s) or sequence(s) in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes , and certain eukaryotic cells, such as producer cells.

[00136] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length. Said polymer may be linear or branched, it may contain modified amino acids, and they may alternate with non-amino acids. These terms also include a polymer of amino acids that has been modified naturally or by action; for example, formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification such as conjugation with a labeled moiety. The present definition also includes, for example, polypeptides containing one or more amino acid analogues (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It should be understood that since the polypeptides of the present invention are antibody based, in certain embodiments the polypeptides may occur as single chains or associated chains.

[00137] The terms "identical" or percentage of "identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are the same or have a certain percentage of nucleotides or amino acids that are the same when compared and aligned (when by introducing gaps) for maximum matching, counting any conservative amino acid substitutions as part of the sequence identity. Percentage identity can be measured using sequence comparison software or visual scoring algorithms. Various algorithms and programs are known in the art that can be used to obtain amino acid or amino acid alignments. nucleotide sequences.

[00138] One such non-limiting example of a sequence alignment algorithm is the algorithm disclosed in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, and modified from Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and included in the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In certain embodiments, gap BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389–3402. Other publicly available programs that can be used for sequence alignment include BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech) , South San Francisco, California) or Megalign (DNASTAR). In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GGG software package (e.g., using the NWSgapdna.CMP matrix, a gap opening penalty of 40, 50, 60, 70, or 90, and a continuation penalty of 1, 2, 3, 4, 5 or 6). In certain alternative embodiments, the GAP program in the GCG software package, which includes the Needleman and Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)) can be used to determine the percent identity between amino acid sequences (e.g., using BLOSUM 62 matrix or RAM250 matrix, and a penalty for opening a gap 16, 14, 12, 10, 8, 6 or 4 and a penalty for continuing 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the Myers and Miller algorithm (CABIOS, 4:11-17 (1989)). For example, percent identity can be determined using the ALIGN program (version 2.0) and using PAM120 with a table of residues, a gap length penalty of 12, and a gap length penalty of 4. One skilled in the art can determine the appropriate parameters for maximizing alignment using a specific program for alignment. In certain embodiments, default program parameters for alignment are applied.

[00139] In certain embodiments, the percent identity "X" of the first amino acid sequence of the second amino acid sequence is calculated as 100 x (Y/Z), where Y is the number of amino acid residues counted as identical matches in the alignment of the first and second sequences (which are aligned by visual estimation or using a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is greater than the length of the second sequence, the percent identity of the first sequence with respect to the second will be greater than the percent identity of the second sequence with respect to first.

[00140] By “conservative amino acid substitution” is meant a substitution in which one amino acid residue is replaced by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been identified in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncontaminated polar side chains (e.g., asparagine, glutamine, series, threonine, tyrosine, cysteine), non-polar side chains (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). For example, replacing phenylalanine with tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the polypeptide and antibody sequences of the present invention do not abrogate the binding of said polypeptide or antibody containing such amino acid sequence to the antigen(s), i.e. IL-21 to which the specified polypeptide or antibody binds. Methods for identifying succleotide and amino acid conservative substitutions that do not preclude antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12 (10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

II. Anti-IL-21 binding molecules

[00141] The present invention provides IL-21-binding molecules, such as anti-IL-21 antibodies and antigen-binding fragments thereof, that specifically bind IL-21. The full-length amino acid (AA) and nucleotide (NT) sequences of IL-21 are known in the art. See, for example, GenBank Accession No. BC066260.1 (SEQ ID No.: 1) and AAH69124.1 (SEQ ID No.: 2), respectively, for IL-21, or US Patent Application No. 2008-0267910 A1 for IL -21 Cynomolgus Macaque (Masaca fasciculari) SEQ ID No: 3 and 4, respectively). In some embodiments, the IL-21 binding molecules, such as anti-IL-21 antibodies and antigen binding fragments thereof, provided herein are murine antibodies, humanized antibodies, or human antibodies. In certain embodiments, said IL-21 binding molecules are antibodies or antigen binding fragments thereof. In some embodiments, the IL-21 binding molecules, e.g., anti-IL-21 antibodies or antigen binding fragments thereof, comprise a Fab, Fab', F(ab') ₂ , Fd, single chain Fv, or scFv, disulfide-linked Fv, V domain -NAR, IgNar, antibody, IgGΔCH2, mini-antibody, F(ab') ₃ , tetrabody, tribody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb ² , (scFv) ₂ , or scFv-Fc. In some embodiments, the antibody is an antibody of the IgG1 subtype and contains a triple YTE mutant, which is described above in the Definitions section. Specific anti-IL-21 antibodies or fragments thereof are suggested in Table 2 and the Examples section.

[00142] In accordance with certain aspects of the present application, there is provided an IL-21 binding molecule or antigen binding fragment thereof, such as an anti-IL-21 antibody or antigen binding fragment thereof, that can specifically bind to the same epitope of IL-21 as the antibody or antigen binding fragment thereof. antigen-binding fragment containing a heavy chain variable region (VH) and a light chain variable region (VL) 19E3, 9F11, 8B6 or 9H10. Also provided is an IL-21-binding molecule or an antigen-binding fragment thereof, for example, an anti-IL-21 antibody or an antigen-binding fragment thereof, which can competitively inhibit, or can bind to, IL-21 with greater affinity than an antibody or antigen-binding fragment thereof containing VH and VL 19E3, 9F11, 8B6 or 9H10. As proposed herein, VH and VL 19E3 contain SEQ ID Nos: 6 and 11, respectively, VH and VL of 9F11 contain SEQ ID Nos: 28 and 33, respectively, VH and VL 8B6 contain SEQ ID Nos: 42 and 47, respectively, and VH and VL 9H10 contain SEQ ID NO: 52 and 57, respectively.

[00143] Also provided herein is an IL-21 binding molecule or an antigen binding fragment thereof, for example an anti-IL-21 antibody or an antigen binding fragment thereof comprising the VL region of the antibody. In certain aspects, said VL region includes one, two, or three VL-CDRs, such as VL-CDRs that are completely identical or identical except for eight, seven, six, five, four, three, two, or one amino acid substitution, of SEQ ID NO. : 12, SEQ ID No.: 34, SEQ ID No.: 48 or SEQ ID No.: 58, VLCDR2, completely identical or identical except for eight, seven, six, five, four, three, two or one amino acid substitutions, SEQ ID sequence No.: 13, SEQ ID No.: 35, SEQ ID No.: 49 or SEQ ID No.: 59, or VLCDR3, completely identical or identical except for eight, seven, six, five, four, three, two or one amino acid substitution, sequence SEQ ID No.: 14, SEQ ID No.: 36, SEQ ID No.: 50, or SEQ ID No.: 60. In certain aspects, the VL region contains the amino acid sequences VL-CDR1, VL-CDR2, and VL-CDR3 that are identical or identical except eight, seven, six, five, four, three, two or one amino acid substitution of VL-CDR sequences SEQ ID Nos: 12, 13 and 14, SEQ ID Nos: 34, 35 and 36, SEQ ID Nos: 48, 49 and 50 or SEQ ID No.: 58, 59 and 60, respectively. In certain aspects, the VL region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the reference amino acid sequence SEQ ID No: 11, SEQ ID No: 17. SEQ ID No.: 21, SEQ ID No.: 25, SEQ ID No.: 33, SEQ ID No.: 39, SEQ ID No.: 40, SEQ ID No.: 47 or SEQ ID No.: 57.

[00144] Additionally provided is a binding molecule or an antigen-binding fragment thereof that specifically binds to IL-21, for example, an anti-IL-21 antibody or an antigen-binding fragment thereof comprising the VH region of the antibody. In certain aspects, said VH region includes one, two, or three VH-CDRs, such as VH-CDR1, which is identical or identical except for eight, seven, six, five, four, three, two, or one amino acid substitutions to SEQ ID NO. : 7, SEQ ID No: 29, SEQ ID No: 43 or SEQ ID No: 53, VH-CDR2, identical or identical except for eight, seven, six, five, four, three, two or one amino acid substitution, sequence SEQ ID No.: 8, SEQ ID No.: 30, SEQ ID No.: 44 or SEQ ID No.: 54 or VH-CDR3, completely identical or identical except for eight, seven, six, five, four, three, two or one amino acids substitution, sequence SEQ ID No: 9, SEQ ID No: 31, SEQ ID No: 45 or SEQ ID No: 55. In certain aspects, the VH region contains the amino acid sequences VH-CDR1, VH-CDR2 and VH-CDR3, completely identical or identical except for eight, seven, six, five, four, three, two or one amino acid substitution, VH-CDR sequences SEQ ID No: 7, 8 and 9, SEQ ID No: 29, 30 and 31, SEQ ID No: 43 , 44 and 45 or SEQ ID Nos: 53, 54 and 55, respectively. In certain aspects, the VH region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the reference amino acid sequence SEQ ID No.: 6, SEQ ID No.: 15. SEQ ID No.: 19, SEQ ID No.: 23, SEQ ID No.: 28, SEQ ID No.: 37, SEQ ID No.: 38, SEQ ID No.: 42 or SEQ ID No.: 52.

[00145] In specific aspects, the IL-21 binding molecule provided herein includes an anti-IL-21 antibody or an antigen binding fragment thereof. For example, the present application provides an isolated antibody or antigen-binding fragment thereof that specifically binds IL-21, comprising a VL region AND a VH region, wherein VL and VH together contain the amino acid sequences VL-CDR1, VL-CRD2, VL-CDR3, VH- CDR1, VH-CDR2 and VH-CDR3, completely identical or identical except for eight, seven, six, five, four, three, two or one amino acid substitution in one or more CDR sequences: SEQ ID No: 12, 13, 14 , 7, 8 and 9, SEQ ID Nos: 34, 35, 36, 29, 30 and 31, SEQ ID Nos: 48, 49, 50, 43, 44 and 45 or SEQ ID Nos: 58, 59, 60, 53 , 54 and 55, respectively.

[00146] For example, this application provides an isolated antibody or antigen-binding fragment thereof that specifically binds IL-21, comprising VH and VL, wherein said VH and VL contain, respectively, amino acid sequences that are at least 70% identical, 75 %, 80%, 85%, 90%, 95% or 100% of the reference amino acid sequences SEQ ID No: 6 and SEQ ID No: 11, SEQ ID No: 15 and SEQ ID No: 17, SEQ ID No: 19 and SEQ ID No.: 21, SEQ ID No.: 23 and SEQ ID No.: 25, SEQ ID No.: 28 and SEQ ID No.: 33, SEQ ID No.: 37 and SEQ ID No.: 39, SEQ ID No.: 38 and SEQ ID No. : 39, SEQ ID No.: 37 and 40, SEQ ID No.: 42 and SEQ ID No.: 47, or SEQ ID No.: 52 and SEQ ID No.: 57, respectively.

[00147] In one aspect, the present application provides an anti-IL-21 antibody or antigen-binding fragment thereof comprising VH and VLK44VHa-N56Q, VH having the amino acid sequence SEQ ID No: 19, and VL having the amino acid sequence SEQ ID No: 21.

[00148] The anti-IL-21 antibody or antigen binding fragment thereof provided herein, such as those described above, may be, for example, a mouse antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a multispecific antibody or any combination thereof. Said anti-IL-21 antibody antigen binding fragment may be an Fv fragment, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, a dsFv fragment, a scFv fragment, or a sc(Fv)2 fragment.

[00149] In one aspect, the present application provides an anti-IL-21 antibody or antigen-binding fragment thereof that can bind to a variety of types of IL-21 molecules, for example, the antibody or fragment can bind to murine IL-21, IL-21 rabbit, human IL-21 and/or cynomolgus monkey IL-21. For example, the antibody or fragment may bind to human IL-21 and cynomolgus IL-21. In a further example, the antibody or fragment may also bind to mouse IL-21.

[00150] The IL-21 receptor has a gamma chain common to a number of other cytokine receptors, for example, IL-2R, IL-4R, IL-7R, IL-9R and IL-15R. In certain embodiments, an anti-IL-21 antibody or antigen-binding fragment thereof provided herein may specifically bind to IL-21, e.g., human and cynomolgus IL-21, and mouse IL-21, but does not specifically bind to IL-21. Human IL-2, IL-4, IL-7, IL-9 or IL-15.

[00151] An anti-IL-21 antibody or antigen-binding fragment thereof provided herein, such as those described above, may include, in addition to VH and VL, a heavy chain constant region or fragment thereof. In certain embodiments, said heavy chain constant region is a human heavy chain constant region, such as a human IgG constant region, such as a human IgG1 constant region. As described elsewhere herein, in certain aspects, a heavy chain constant region or fragment thereof, such as a human IgG constant region or fragment thereof, may include one or more amino acid substitutions relative to the wild-type IgG constant domain, wherein the modified IgG has an increased half-life compared to the half-life of IgG containing the wild-type IgG constant domain. For example, an IgG constant domain may contain one or more amino acid substitutions at positions 251-257, 285-290, 308-314, 385-389 and 428-436, where the numbering of amino acid positions corresponds to the EC index as specified by Cabot. In certain aspects, an IgG constant domain may contain one or more amino acid substitutions at Cabot position 252 to tyrosine (Y), phenylalanine (F), tryptophan (W), or threonine (T), amino acid substitutions at Cabot position 254 to threonine (T ), amino acid substitutions at Cabot position 256 to series (S), arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D) or threonine (T), amino acid substitutions at Cabot position 257 to leucine (L), amino acid substitutions at Cabot position 309 to proline (P), amino acid substitutions at Cabot position 311 to series (S), amino acid substitutions at Cabot position 428 to threonine (T), leucine (L), phenylalanine (F) or series (S), Cabot 433 amino acid substitutions to arginine (R), series (S), isoleucine (I), proline (P), or glutamine (Q), or Cabot 434 amino acid substitutions to tryptophan (W), methionine (M), serine (S), histidine (H), phenylalanine (F) or tyrosine. More specifically, the IgG constant domain may contain amino acid substitutions relative to the wild-type human IgG constant domain, including amino acid substitutions at Cabot 252 to tyrosine (Y), amino acid substitutions at Cabot 254 to threonine (T), and amino acid substitutions at Cabot position 256 for glutamic acid (E).

[00152] This application provides an anti-IL-21 antibody or antigen binding fragment thereof, wherein the heavy chain is a YTE-mutated IgG1, for example, said heavy chain may comprise a heavy chain amino acid sequence that is at least 70%, 75% , 80%, 85%, 90%, 95% or 100% identical to the reference amino acid sequence SEQ ID No: 16, SEQ ID No: 20 or SEQ ID No: 24.

[00153] An anti-IL-21 antibody or antigen-binding fragment thereof provided herein, such as those described above, may include, in addition to VH and VL and an optional heavy chain constant region or fragment thereof, a light chain constant region or fragment thereof. In certain aspects, said light chain constant region is a kappa or lambda light chain constant region, for example, a human lambda constant region. In one particular aspect, said light chain constant region is a human kappa constant region.

[00154] This application provides an anti-IL-21 antibody or antigen-binding fragment thereof, wherein the light chain comprises a human kappa constant region, for example, the light chain may comprise a light chain with an amino acid sequence that is at least 70%, 75% 80%, 85%, 90%, 95% or 100% identical to the reference amino acid sequence of SEQ ID No: 18, SEQ ID No: 22 or SEQ ID No: 26. In certain aspects, the present application provides an anti-IL-21 antibody or an antigen binding fragment thereof comprising a human IgG1 heavy chain with a YTE mutation and a human kappa light chain, said antibody consisting of the sequences SEQ ID No: 16 and SEQ ID No: 18, SEQ ID No: 20 and SEQ ID No: 22, or SEQ ID No: 24 and SEQ ID No: 26, respectively, or any antigen binding fragment thereof.

[00155] The anti-IL-21 antibody or antigen-binding fragment thereof provided herein may have beneficial properties. For example, the antibody or fragment thereof may inhibit, suppress or block the binding of IL-21 to an IL-21 receptor, for example, expressed on a T lymphocyte, B lymphocyte, natural killer cell, NK T lymphocyte or dendritic cell, or expressed by a recombinant by way of a non-lymphoid germ cell. As a further example, the antibody or fragment thereof may inhibit the binding of human or cynomolgus IL-21 to the human or cynomolgus IL-21 receptor, but not inhibit the binding of mouse IL-21 to the mouse IL-21 receptor. Such an anti-IL-21 antibody or fragment thereof may also inhibit, inhibit or block various activities mediated by IL-21, ie, the antibody or fragment thereof may act as antagonists of IL-21 activity. In certain aspects, an anti-IL-21 antibody or fragment thereof provided herein can inhibit, suppress or block IL-21-mediated phosphorylation in the Jak/STAT pathway, for example, inhibit, suppress or block phosphorylation of STAT3 in IL-21R expressing cells, for example, in human peripheral blood mononuclear cells (PBMCs).

[00156] In certain aspects, an anti-IL-21 antibody or fragment thereof provided herein can inhibit, inhibit, or block IL-21-mediated interferon-gamma (IFN-γ) production by natural killer cells. Such inhibition may be observed for naturally occurring NKs, such as human NK, or for cultured NKs, such as NK-92.

[00157] In certain aspects, an anti-IL-21 antibody or fragment thereof provided herein can inhibit, suppress, or block IL-21-mediated activation, differentiation, or death of B lymphocytes during a humoral immune response. For example, an anti-IL-21 antibody or fragment thereof provided herein can inhibit, inhibit, or block IL-21-mediated differentiation of stimulated B lymphocytes into plasma cells. Certain antibodies provided herein can inhibit, inhibit, or block human and cynomolgus IL-21-mediated differentiation of stimulated B lymphocytes into plasma cells. In certain aspects, said B lymphocytes may be isolated B lymphocytes to which IL-21 is added exogenously. In other aspects, said B lymphocytes may come into contact, either naturally or through co-culture, with activated CD4+ T lymphocytes that express IL-21. In other aspects, human B lymphocytes may come into contact, either naturally or through co-culture, with activated human CD4+ T lymphocytes that express IL-21.

[00158] In certain aspects, an antibody or antigen binding fragment thereof provided herein can bind IL-21 with an affinity characterized by a dissociation constant (K _D ) from about 100 pM to about 0.1 pM, measured using a kinetic assay platform exceptions (KinExA) 3000.

[00159] In certain aspects, an anti-IL-21 antibody or antigen-binding fragment thereof can bind to IL-21, such as human IL-21 or cynomolgus IL-21, and antigenic fragments thereof with a dissociation constant or K _D of less than 10- 6 M, or less than 10 ^-7 M, or less than 10 ^-8 M, or less than 10 ^-9 M, or less than 10 ^-10 M, or less than 10 ^-11 M, or less than 10 ^-12 M , or less than 10 ^-13 M, or less than 10 ^-14 M, or less than 10 ^-15 M, measured using KINEXA® or BIACORE®. In one aspect, the humanized anti-IL-21 antibody K44VHa-N56Q can bind to human IL-21 with a K _D of approximately 515 fM (515 x 10 -15 M) and cynomolgus IL-21 with a K _D of approximately 352 fM (352 x 10 ^-15 M), measured using KINEXA®.

[00160] In another embodiment, an anti-IL-21 antibody or antigen-binding fragment thereof of the present invention binds to IL-21 and/or antigenic fragments thereof with a _Koff of less than 1×10 ^-3 s ^-1 , or less than 2×10 ^-3 s ^-1 . In other embodiments, an anti-IL-21 antibody or antigen-binding fragment thereof binds to IL-21 and its antigenic fragments with a _{Koff of} less than 10 ^-3 s ^-1 , less than 5×10 ^-3 s ^-1 , less than 10 ^-4 s ^{- 1} , less than 5×10 ^-4 s ^-1 , less than 10 ^-5 s ^-1 , less than 5×10 ^-5 s ^-1 , less than 10 ^-6 s ^-1 , less than 5×10 ^-6 s ^-1 , less less 5×10 ^-7 s ^-1 , less than 10 ^-8 s ^-1 , less than 5×10 ^-8 s ^-1 , less than 10 ^-9 s ^-1 , less than 5×10 ^-9 s ^-1 or less than 10 ^-10 s ^-1 , measured, for example, using KINEXA® or BIACORE®.

[00161] In yet another embodiment, an anti-IL-21 antibody or antigen-binding fragment thereof of the present invention binds to IL-21 and/or antigenic fragments thereof at a constant rate of association or rate of association of at least 10 ⁵ M ^-1 s ^-1 , at least 5×10 ⁵ M ^-1 s ^-1 , at least 10 ⁶ M ^-1 s ^-1 , at least 5×10 ⁶ M ^-1 s ^-1 , at least 10 ⁷ M ^-1 s ^-1 , at least 5×10 ⁷ M ^-1 s ^-1 , or at least 10 ⁸ M ^-1 s ^-1 , or at least 10 ⁹ M ^-1 s ^-1 , measured, for example, using KINEXA ® or BIACORE®.

[00162] As noted above, the amino acid sequence of VH and/or VL may be, for example, 85%, 90%, 95%, 96%, 97%, 98% or 99% similar to the sequence established in the present application, and /or contain 1, 2, 3, 4, 5 or more substitutions, for example conservative substitutions, compared to the sequence established in this application. An anti-IL-21 antibody containing VH and VL regions that exhibit a certain percentage of similarity to the VH region or VL region, or contain one or more substitutions, such as conservative substitutions, can be produced by mutagenesis (eg, site-directed or PCR mediated mutagenesis) against nickelic acid molecules encoding the VH and/or VL regions described herein, and subsequently testing the encoded altered antibody for binding to IL-21 and optionally testing for conservation of function using the functional assay described herein application.

[00163] The affinity or avidity of an antibody for an antigen can be determined experimentally by any suitable method well known in the art, for example, flow cytometry, an enzyme-linked immunosorbent assay (ELISA), or a radioenzyme-linked immunosorbent assay (RIA), or a kinetic assay (for example, the KINEXA® assay or BIACORE™). The assay can be easily applied in the form of a direct binding assay as well as a competitive binding assay. (See, for example, Berzofsky et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, NY (1984); Kuby, Immunology, W. H. Freeman and Company: New York , NY (1992); and the methods described herein). The measured affinity of a particular antigen-antibody interaction may vary if measured under different conditions (eg, salt concentration, pH, temperature). Thus, measurements of affinity and other antigen binding parameters (eg, _KD or Kd, K _on , K _off ) are performed using standardized solutions of antibody and antigen, and a standardized buffer known in the art, such as the buffer described herein.

[00164] Also provided herein is an anti-IL-21 antibody or fragment thereof described herein, wherein said antibody is conjugated to a heterologous agent. In certain aspects, the agent may be an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), or a combination of two or more of the specified agents. Heteroconjugates of anti-IL-21 antibodies are described in more detail elsewhere in this application.

[00165] In certain embodiments, the IL-21 binding molecule is a polypeptide that is not an antibody. A number of methods are known in the art for identifying and producing non-antibody polypeptides that bind with high affinity to the target protein. See, for example, Skerra, Curr. Opin. Biotechnol., 18:295-304 (2007), Hosse et al., Protein Science, 15:14-27 (2006), Gill et al., Curr. Opin. Biotechnol., 17:653-658 (2006), Nygren, FEBS J., 275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008), each of which is included in this application by reference to their full contents. In certain embodiments, phage display technology can be used to identify/produce an IL-21 binding polypeptide. In certain embodiments, the polypeptide comprises a specific type of protein scaffold selected from the group consisting of protein A, lipocalin, fibronectin domain, ankyrin consensus repeat domain, and thioredoxin.

III. Binding molecules that bind to the same epitope as the anti-IL-21 antibodies and antigen binding fragments thereof of the present invention

[00166] In certain embodiments, the present application provides an IL-21 binding molecule, such as an anti-IL-21 antibody or antigen binding fragment thereof, that binds to the same epitope as various anti-IL-21 antibodies described in this application. As used herein, the term “epitope” refers to a target protein determinant capable of binding to an antibody of the present invention. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or carbohydrate side chains, and usually have specific three-dimensional structure characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that binding to the former, but not to the latter, is lost in the presence of denaturing solvents. Such antibodies can be identified by their ability to cross-compete (eg, the ability to competitively inhibit binding in a statistically significant manner) with antibodies such as 19E3α, 9F11, 9H10 or 8B6 in standard IL-21 binding assays. Accordingly, according to one embodiment of the present invention, there are provided anti-IL-21 antibodies and antigen binding fragments thereof, for example, monoclonal antibodies that compete for binding to IL-21 with another anti-IL-21 antibody or antigen binding fragment thereof according to the present invention, such such as mouse monoclonal antibodies 19E3, 9F11, 9H10 or 8B6, or humanized variants disclosed herein. The ability of a test antibody to inhibit binding of, for example, 19E3α, 9F11, 9H10 or 8B6 demonstrates that the test antibody can compete with such antibody for binding to IL-21; such an antibody may, according to non-limiting theory, bind to the same or related (eg, structurally similar or spatially proximally located) epitope on IL-21 as the anti-IL-21 antibody or antigen binding fragment thereof with which it competes. In one embodiment, the anti-IL-21 antibody or antigen-binding fragment thereof binds to the same epitope on IL-21 as, for example, mouse monoclonal antibodies 19E3, 9F11, 9H10, or 8B6.

IV. Activity of IL-21-binding molecules

[00167] In some embodiments, an IL-21 binding molecule, such as an anti-IL-21 antibody or an antigen binding fragment thereof, can inhibit IL-21-mediated signal transduction in IL-21R-expressing cells. For example, an IL-21-binding molecule, such as an anti-IL-21 antibody or an antigen-binding fragment thereof, can inhibit IL-21-mediated phosphorylation of STAT3. In addition, an IL-21-binding molecule, such as an anti-IL-21 antibody or an antigen-binding fragment thereof, can inhibit IFNγ production by NK cells. Moreover, an IL-21-binding molecule, such as an anti-IL-21 antibody or an antigen-binding fragment thereof, can inhibit the differentiation of stimulated B lymphocytes into plasma cells.

[00168] In some embodiments, an IL-21 binding molecule, e.g., an anti-IL-21 antibody or antigen binding fragment thereof, can inhibit IL-21-mediated STAT3 phosphorylation in IL-21R-expressing cells (e.g., PBMCs), as measured by by flow cytometry, with an IC ₅₀ of less than about 500 pM, less than about 350 pM, less than about 250 pM, less than about 150 pM, less than about 100 pM, less than about 75 pM, less than about 60 pM, less less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 15 pM, less than about 10 pM, or less than about 5 pM. In certain aspects, an IL-21 binding molecule, e.g., an anti-IL-21 antibody or an antigen binding fragment thereof, can inhibit IL-21-mediated STAT3 phosphorylation in IL-21R-expressing cells (e.g., PBMCs) as measured by flow cytometry. with an IC ₅₀ of about 22 pM, about 19 pM, about 17 pM, about 14 pM, about 13 pM, about 12 pM, about 6 pM, about 5 pM, or about 3 pM. In certain aspects, an IL-21 binding molecule, e.g., an anti-IL-21 antibody or an antigen binding fragment thereof, can inhibit cynomolgus IL-21-mediated phosphorylation of STAT3 in IL-21R-expressing cells (e.g., PBMCs) as measured by flow assay. cytometry, with an IC ₅₀ of about 52 pM, about 51 pM, about 46 pM, about 19 pM, about 16 pM, about 10 pM, or about 6 pM.

[00169] In some embodiments, an IL-21 binding molecule, such as an anti-IL-21 antibody or an antigen binding fragment thereof, can inhibit IL-21 mediated IFN production by NK cells, as measured by a human IFNγ MSD singlex assay, with IC ₅₀ less than about 100 pM, less than about 400 pM, less than about 100 pM, less than about 75 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 15 pM, less than about 10 pM, or less than about 5 pM. In certain aspects, an IL-21 binding molecule, e.g., an anti-IL-21 antibody or an antigen binding fragment thereof, can inhibit human IL-21-mediated production of IFNγ by the NK-92 cell line, as measured by a human IFNγ singlex MSD assay, with an IC ₅₀ about 99 pM, about 93 pM, about 55 pM, about 41 pM, or about 20 pM. In certain aspects, an IL-21 binding molecule, e.g., an anti-IL-21 antibody or an antigen binding fragment thereof, can inhibit cynomolgus IL-21-mediated IFNγ production by the NK-92 cell line, as measured by a human IFNγ MSD singlex assay with IC ₅₀ about 83 pM, about 81 pM, about 60 pM, about 37 pM, or about 3 pM.

[00170] In some embodiments, an IL-21 binding molecule, such as an anti-IL-21 antibody or an antigen binding fragment thereof, can inhibit IL-21-mediated differentiation of stimulated B lymphocytes, such as naïve B lymphocytes and/or B lymphocytes. memory lymphocytes stimulated with anti-CD40 and anti-IgM F(ab')2 into plasma cells, e.g., IgG-producing plasma cells, as measured by flow cytometry or ELISA, with an IC ₅₀ of less than about 150 nM, less than about 75 nM, less than about 50 nM, less than about 30 nM, less than about 10 nM, less than about 1600 pM, less than about 1400 pM, less than about 1000 pM, less than about 800 pM, less than about 700 pM or less than about 600 pM. In certain aspects, an IL-21 binding molecule, e.g., an anti-IL-21 antibody or an antigen binding fragment thereof, can inhibit human IL-21-mediated differentiation of stimulated B lymphocytes, e.g., naïve B lymphocytes and/or B lymphocytes memory stimulated with anti-CD40 and anti-IgM F(ab')2 into plasma cells, e.g., IgG-producing plasma cells, as measured by cytometry or ELISA with IC ₅₀ of approximately 115 nM, approximately 47 nM, approximately 1.5 nM, approximately 777 pM, approximately 618 pM, or approximately 573 pM. In certain aspects, an IL-21 binding molecule, e.g., an anti-IL-21 antibody or an antigen binding fragment thereof, can inhibit cynomolgus IL-21-mediated differentiation of stimulated B lymphocytes, e.g., naïve B lymphocytes and/or B lymphocytes memory stimulated with anti-CD40 and anti-IgM F(ab')2, into plasma cells, e.g., IgG-producing plasma cells, as measured by cytometry or ELISA with IC ₅₀ of approximately 74 nM, approximately 30 nM, approximately 9 nM, approximately 1, 3 nM, approximately 1.0 nM, approximately 775 pM, or approximately 659 pM. In certain aspects, an IL-21-binding molecule, e.g., an anti-IL-21 antibody or an antigen-binding fragment thereof, can inhibit cynomolgus IL-21-mediated differentiation of stimulated B lymphocytes into plasma cells, but not human IL-21-mediated differentiation of B lymphocytes into plasma cells. Plasmocyte.

[00171] In certain aspects, an IL-21 binding molecule, such as an anti-IL-21 antibody or an antigen binding fragment thereof, can inhibit the differentiation of B lymphocytes into plasma cells driven by CD4+ T lymphocytes that produce co-stimulatory molecules such as CD40L and B-lymphocyte-tropic cytokines such as IL-21. In other aspects, an IL-21 binding molecule, such as an anti-IL-21 antibody or an antigen binding fragment thereof, can inhibit IL-21-triggered proliferation of naïve human CD4+ T lymphocytes.

V. Preparation of anti-IL-21 antibodies and antigen-binding fragments

[00172] Monoclonal anti-IL-21 antibodies can be produced using hybridoma technologies, such as the methods described in Kohler and Milstein (1975) Nature 256:495. When using the hybridoma method, a mouse, hamster or other suitable host animal is immunized as described above to induce the production of lymphocytes and antibodies that will specifically bind to the immunizing antigen. Lymphocytes can also be immunized in vitro. After immunization, the lymphocytes are isolated and hybridized with a cell of a suitable myeloma lineage, for example, using polyethylene glycol, resulting in the formation of hybridoma cells, which can then be selected for screening out unhybridized lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically to a selected antigen, as determined by immunoprecipitation, immunoblotting, or in vitro binding assays (eg, radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)), can then be propagated in in vitro culture or using standard methods (Coding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo in ascites tumors in animals. Monoclonal antibodies can then be purified from culture media or ascites fluid as described above for polyclonal antibodies.

[00173] As an alternative, anti-IL-21 monoclonal antibodies can also be produced using recombinant DNA technologies, which are described in US Patent No. 4,816,567. Polynucleotides encoding a monoclonal antibody are isolated from mature B lymphocytes or hybridomas, for example, by RT-PCR (real-time polymerase chain reaction) using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and using traditional procedures are determined by their sequence. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which, when transfected into host cells, for example E. coli cells, monkey kidney cells, Chinese hamster ovary (CHO) cells or myeloma cells, which otherwise case do not produce immunoglobulin protein, lead to the generation of monoclonal antibodies by the specified host cells. Also, recombinant anti-IL-21 monoclonal antibodies or antigen-binding fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species, as described in (McCafferty et al., 1990, Nature, 348:552-554; Clackson et al. , 1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol., 222:581-597).

[00174] The polynucleotide(s) encoding an anti-IL-21 antibody or an antigen-binding fragment thereof can be further modified in a variety of ways using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be replaced with (1) such regions, for example, from a human antibody, to generate a chimeric antibody, or (2) with a non-immunoglobulin polypeptide to generate a hybrid antibody . In some embodiments, these constant regions are shortened or deleted to generate the desired monoclonal antibody fragment. To optimize the specificity, affinity, etc. of a monoclonal antibody, site-directed mutagenesis or high-density mutagenesis can be used.

[00175] In certain embodiments, the anti-IL-21 antibody or antigen binding fragment thereof is a human antibody or antigen binding fragment thereof. Human antibodies can be obtained directly using various technologies known in the art. It is possible to generate immobilized human B lymphocytes, immunized in vitro or isolated from an immunized individual, that produce an antibody directed to the target antigen (See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 ( 1985); Boemer et al., 1991, J. Immunol., 147 (1):86-95; and US Patent 5750373).

[00176] Also, the specified human anti-IL-21 antibody or antigen binding fragment thereof can be isolated from a phage library if such phage library expresses human antibodies, as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al., 1991, J. Mol. Biol., 222:581). Methods for generating and using phage libraries that generate antibodies are also described in US Patent Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6544731; 6555313; 6582915; 6593081; 6300064; 6653068; 6706484; and 7264963; and also in Rothe et al., 2007, J. Mol. Bio., doi:10.1016/j.jmb.2007.12.018 (each reference is incorporated herein by reference to its entire contents).

[00177] Affinity maturation strategies and chain shuffling strategies are known in the art (Marks et al., 1992, Bio/Technology 10:779-783, incorporated herein by reference in its entirety) and can be used to generate high affinity human antibodies or antigen binding fragments thereof.

[00178] In some embodiments, the anti-IL-21 monoclonal antibody may be a humanized antibody. Methods for constructing, humanizing, or altering the surface properties of antibodies from non-human species or human antibodies can also be used and are well known in the art. A humanized, surface-altered, or similarly engineered antibody may contain one or more amino acid residues from a non-human source, such as, but not limited to, a mouse, rat, rabbit, nonhuman primate, or other mammal. These non-human antibody amino acid residues are replaced with residues that are often referred to as “import” residues, which are typically taken from an “import” variable, constant, or other domain of known sequence characteristic of a human antibody. Such imported sequences can be used to reduce immunogenicity or reduce, increase or modify binding, affinity, rate of association, rate of dissociation, avidity, specificity, half-life or any other relevant characteristics that are known in the art. In general, CDR residues are directly and most significantly involved in influencing IL-21 binding. Therefore, part or all of the CDR sequences from a non-human antibody or from a human antibody are retained, while the non-human variable and constant region sequences can be replaced with amino acids from a human antibody or other amino acids.

[00179] Antibodies may also be optionally humanized, surface-altered, recombinant, or human antibodies engineered to retain high affinity for the IL-21 antigen and other beneficial biological properties. To achieve this goal, humanized (or human antibodies) or recombinant anti-IL-21 antibodies and antibodies with altered surface properties can be obtained optionally by analyzing the parent sequences and various conceptual humanized and recombinant products using three-dimensional models of the parent, recombinant and humanized sequences. Three-dimensional models of immunoglobulins are publicly available and known to those skilled in the art. There are computer programs that illustrate and show probable three-dimensional conformational structures for the sequences of selected immunoglobulin candidates. Viewing such images allows one to analyze the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. analyze how residues affect the ability of a candidate immunoglobulin to bind to its antigen, such as IL-21. Thus, framework (FW) residues can be selected from consensus and import sequences and combined to produce desired antibody characteristics, such as increased affinity for the target antigen(s).

[00180] Humanization, alteration of surface properties, or engineering of anti-IL-21 antibodies or antigen-binding fragments thereof according to the present invention can be accomplished by any known method, without limitation such as those described in Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), US Patent No. 5639641, 5723323; 5976862; 5824514; 5817483; 5814476; 5763192; 5723323; 5766886; 5714352; 6204023; 6180370; 5693762; 5530101; 5585089; 5225539; 4816567, 7557189; 7538195; and 7,342,110; international patent applications PCT/US98/16280; PCT/US96/18978; PCT/US91/09630; PCT/US91/05939; PCT/US94/01234; PCT/GB89/01334; PCT/GB91/01134; PCT/GB92/01755; publications of international patent applications No. WO90/14443; WO90/14424; WO90/14430; and publication of European patent application No. EP 229246; each of which is incorporated herein by reference to its entire contents, including references cited herein.

[00181] Anti-IL-21 antibodies and antigen-binding fragments thereof can also be produced in transgenic mice containing human immunoglobulin loci that are capable of producing the full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. This approach is described in US patents No. 5545807; 5545806; 5569825; 5625126; 5633425; and 5661016.

[00182] In certain embodiments, an anti-IL-21 antibody fragment is provided. Traditionally, these fragments are obtained by proteolytic cleavage of intact antibodies (eg, Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods 24:107-117; Brennan et al., 1985, Science, 229:81). In certain embodiments, anti-IL-21 antibody fragments are produced recombinantly. Fab, Fv and scFv antibody fragments can also be expressed in and secreted from E. coli cells or other host cells, allowing such fragments to be produced in large quantities. Such anti-IL-21 antibody fragments can also be isolated from the antibody phage libraries discussed above. Anti-IL-21 antibody fragments can also be linear antibodies, as described in US Pat. No. 5,641,870. Other methods for producing antibody fragments will be apparent to those skilled in the art.

[00183] According to the present invention, the methods can be adapted to produce single chain antibodies specific for IL-21 (see, for example, US Pat. No. 4,946,778). In addition, the methods can be adapted for the construction of Fab expression libraries (see, e.g., Huse et al., Science 246:1275-1281 (1989)) to allow rapid and efficient identification of monoclonal Fab fragments with the desired IL-21 specificity , or its derivatives, fragments, analogs or homologues. Antibody fragments can be produced using methods known in the art, including, without limitation: (a) an F(ab')2 fragment produced by digestion of the antibody molecule with pepsin; (b) a Fab fragment generated by reducing the disulfide bridges in the F(ab')2 fragment, (c) a Fab fragment generated by treating the antibody molecule with papain and a reducing agent, and (d) Fv fragments.

[00184] In certain aspects, an anti-IL-21 antibody or antigen binding fragment thereof can be modified to increase its half-life from serum. This can be achieved, for example, by incorporating a salvage receptor binding epitope or antibody fragment by mutating the corresponding site in the antibody or antibody fragment, or by incorporating the epitope into a peptide tag that is then combined into the antibody or antibody fragment at either end or in the middle (e.g., by synthesis of DNA or peptides), or by mutation of YTE. Other methods are known in the art to increase the half-life of an antibody or antigen-binding fragment thereof from serum, for example, conjugation with a heterologous molecule such as PEG.

[00185] Heteroconjugates of anti-IL-21 antibodies and antigen-binding fragments thereof are also within the scope of the present invention. Antibody heteroconjugates consist of two covalently linked antibodies. For example, it has been suggested that such antibodies target immune cells to unwanted cells (see, for example, US Pat. No. 4,676,980). It is contemplated that heteroconjugates of anti-IL-21 antibodies and antigen-binding fragments thereof can be prepared in vitro by methods known in the art of protein synthesis chemistry, including methods involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or through the formation of thiofin bonds. Examples of suitable reagents for this purpose include immunothiolate and methyl 4-mercaptobutyrimidate.

[00186] Modified anti-IL-21 antibodies or antigen-binding fragments thereof as provided herein may contain any type of variable region that causes the antibody or polypeptide to associate with IL-21. In this regard, the variable region may contain or be obtained from any type of mammal in which a humoral response is induced and generates immunoglobulins to the desired antigen. The variable region of an anti-IL-21 antibody or an antigen-binding fragment thereof may itself be derived from, for example, an antibody from a human, mouse, lower primate (eg, cynomolgus, macaque, etc.), or wolf. In some embodiments, both the variable and constant regions of the modified anti-IL-21 antibodies or antigen-binding fragments are derived from a human antibody. In other embodiments, variable regions of compatible antibodies (usually derived from a non-human source) can be designed or manufactured specifically to improve the binding properties or to reduce the immunogenicity of the molecule. In this aspect, variable regions useful in the present invention can be humanized or otherwise modified to include imported amino acid sequences.

[00187] In certain embodiments, the variable domains in both the heavy and light chains of an anti-IL-21 antibody or antigen-binding fragment thereof are altered by at least partially replacing one or more CDRs and/or partially replacing framework regions and altering the sequence. Although CDRs may be derived from an antibody of the same class, or even subclass, as the antibody from which the framework regions are derived, said CDRs are contemplated to be derived from an antibody of a different class, and in certain embodiments, from a different species of antibody. It is not necessary to replace all CDRs with the entire CDRs from the donor variable region to transfer antigen binding capacity from one variable domain to another. Rather, it is necessary to transfer those residues that are necessary to maintain the activity of the antigen-binding site. Considering the explanation given in US Pat. Nos. 5,585,089, 5,693,761, and 5,693,762, it is well within the skill of those skilled in the art to obtain a functional antibody with reduced immunogenicity, either through routine experimentation or trial and error.

[00188] Those skilled in the art will appreciate that, notwithstanding changes in the variable region, modified anti-IL-21 antibodies or antigen binding fragments thereof of the present invention will include antibodies (e.g., full-length antibodies or antigen binding fragments thereof) in which at least At least a portion or more of the constant region domains have been deleted or otherwise altered so as to confer desired biochemical characteristics, such as enhanced tumor localization or reduced serum half-life, compared to an antibody of approximately the same immunogenicity containing a native or unaltered constant region . In some embodiments, the constant region of the modified antibodies will comprise a constant region from a human antibody. Modifications to the constant region consistent with the present invention include insertions, deletions, or substitutions of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may contain changes or modifications in one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or in the light chain constant domain (CL). In some embodiments, modified constant regions are provided in which one or more domains are partially or completely deleted. In some embodiments, the modified antibodies may contain domain deleted constructs or variants where the entire CH2 domain has been removed (ΔCH2 constructs). In some embodiments, the deleted constant region domain may be replaced with a short amino acid spacer (eg, 10 residues) that imparts some of the molecular flexibility normally compromised by the absence of the constant region.

[00189] It is known in the art that, in addition to configuration, the constant region mediates several effector functions. For example, the binding of complement component C1 to antibodies activates the complement system. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. Also, antibodies bind to cells through the Fc site, with the Fc receptor site on the antibody Fc site, binding to the Fc receptor on the surface of the cell. There are a number of Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mu receptors). Antibody binding to cell surface Fc receptors triggers a number of important and diverse biological responses, including uptake and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells (termed antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control over the production of immunoglobulins.

[00190] In certain embodiments, an anti-IL-21 antibody or antigen-binding fragment thereof is provided to alter effector functions that in turn affect the biological profile of the administered antibody or antigen-binding fragment thereof. For example, deletion or inactivation (by point mutation or other means) of the constant region domain may reduce Fc receptor binding of the circulating modified antibody. In other embodiments, it may be that modifications to the constant regions of the present invention reduce complement fixation and thereby reduce serum half-life and nonspecific association of the conjugated cytotoxin. Several other constant region modifications can be used to eliminate disulfide bonds or oligosaccharide groups, allowing enhanced localization due to increased antigen specificity or antibody flexibility. Likewise, modifications to the constant region of the present invention can be easily made using well-known biochemical or molecular recombinant technologies that are within the competence of those skilled in the art.

[00191] In certain embodiments, the IL-21 binding molecule, which is an antibody or an antigen binding fragment thereof, lacks one or more effector functions. For example, in some embodiments, the antibody or antigen-binding fragment thereof does not have antibody-dependent cellular cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the anti-IL-21 antibody or antigen-binding fragment thereof does not bind to the Fc receptor and/or complement factors. In certain embodiments, the anti-IL-21 antibody or antigen-binding fragment thereof does not have effector function.

[00192] In certain embodiments, an anti-IL-21 antibody or antigen-binding fragment thereof can be constructed by fusing the CH3 domain directly to the hinge region of corresponding modified antibodies or fragments thereof. In other constructs, a peptide spacer may be introduced between the hinge region and the modified CH2 and/or CH3 domains. For example, compatible constructs can be expressed in which the CH2 domain has been removed and the remaining CH3 domain (modified or unmodified) is connected to the hinge region by a spacer of 5-20 amino acids. Such a spacer can be added, for example, to keep the regulatory elements of the constant domain free and accessible, or to keep the hinge region flexible. Amino acid spacers may, in some embodiments, be demonstrated to be immunogenic and induce an undesirable immune response to the construct. Therefore, in certain embodiments, any spacer introduced into the construct may be relatively non-immunogenic, or even omitted altogether, to maintain the desired biochemical properties of the modified antibodies.

[00193] In addition to deleting entire domains of the anti-IL-21 constant region, antibodies or antigen binding fragments thereof provided herein can be modified by partial deletion or substitution of a few or even one amino acid in the constant region. For example, mutations in a single amino acid in select regions of the CH2 domain may be sufficient to significantly reduce Fc binding and thereby increase tumor localization. Likewise, one or more constant region domains that control effector functions (eg, C1Q complement binding) can be partially or completely removed. Such partial deletions of constant regions may improve selected characteristics of the antibody or antigen binding fragment thereof (eg, serum half-life) while maintaining other desired functions associated with the intact domain of the altered constant region. In addition, the constant regions of the disclosed anti-IL-21 antibodies and antigen-binding fragments thereof can be modified by mutation or substitution of one or more amino acids that improve the profile of the resulting construct. In this regard, it is possible to disrupt the activity provided by the conserved binding site (eg, Fc binding) while essentially maintaining the configuration and immunogenicity profile of the modified antibody or antigen-binding fragment thereof. Certain embodiments may include adding one or more amino acids to the constant region to improve desired characteristics, such as reducing or increasing effecton function or allowing more cytotoxin or carbohydrate to be attached. In such embodiments, it may be desirable to introduce or replicate specific sequences derived from selected constant region domains.

[00194] The present invention also includes variants and equivalents that are substantially homologous to mouse anti-IL-21 antibodies, chimeric, humanized anti-IL-21 antibodies or human anti-IL-21 antibodies, or antigen binding fragments thereof, described in this application. For example, they may contain conservative substitution mutations, ie, the replacement of one or more amino acids with a similar amino acid. For example, a conservative substitution refers to the replacement of an amino acid with another amino acid within the same general class, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. Those skilled in the art are well aware of what a conservative amino acid substitution means.

[00195] The anti-IL-21 antibody or antigen-binding fragment thereof can be further modified to contain additional chemical groups that are not normally part of the protein. Such groups may also reduce or eliminate any unwanted side effects of proteins and the like. A review of such groups can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).

VI. Polynucleotides encoding IL-21-binding molecules and their expression

[00196] The present application provides polynucleotides containing nucleotide sequences that encode a polypeptide that specifically binds IL-21 or an antigen-binding fragment thereof. For example, the present invention provides a polynucleotide comprising a nucleotide sequence that encodes an anti-IL-21 antibody or encodes an antigen-binding fragment of such an antibody. The polynucleotides of the present invention may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, it may be a coding strand or a non-coding (antisense) strand.

[00197] In certain embodiments, the polynucleotide may be isolated. In certain embodiments, the polynucleotide may be substantially pure. In certain embodiments, the polynucleotide may be cDNA or may be derived from cDNA. In certain embodiments, the polynucleotide may be produced recombinantly. In certain embodiments, the polynucleotide may comprise a coding sequence for a mature polypeptide fused in the same reading frame with a polynucleotide that assists, for example, in the expression and secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence to control transport polypeptide from the cell). The polypeptide containing the leader sequence is a precursor protein, and the leader sequence can be cleaved from it to form the mature form of the polypeptide. Also, these polynucleotides may encode an IL-21 binding precursor protein, which is the mature protein plus additional amino acid residues at the 5' end.

[00198] According to certain aspects of the present application, there is provided an isolated polynucleotide containing a nucleic acid encoding an antibody VL, wherein said VL contains the amino acid sequences VL-CDR1, VL-CDR2 and VL-CDR3, which are identical or identical except for eight, seven, six, five, four, three, two or one amino acid substitution in one or more of the VL-CDR sequences: SEQ ID Nos: 12, 13 and 14, SEQ ID Nos: 34, 35 and 36, SEQ ID Nos: 48, 49 and 50 or SEQ ID No.: 58, 59 and 60, respectively.

[00199] Also provided in this application is an isolated polynucleotide containing a nucleic acid encoding an antibody VH, wherein said VH contains the amino acid sequences VH-CDR1, VH-CDR2 and VH-CDR3, completely identical or identical except for eight, seven, six, five , four, three, two or one amino acid substitution in one or more of the VH-CDR, sequences: SEQ ID Nos: 7, 8 and 9, SEQ ID Nos: 29, 30 and 31, SEQ ID Nos: 43, 44 and 45 or SEQ ID Nos: 53, 54 and 55, respectively.

[00200] Also provided herein is an isolated polynucleotide containing a nucleic acid encoding an antibody VL, wherein said VL contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100 % identical to a reference amino acid sequence selected from the group consisting of SEQ ID No: 11, SEQ ID No: 17, SEQ ID No: 21, SEQ ID No: 25, SEQ ID No: 33, SEQ ID No: 39, SEQ ID No.: 40, SEQ ID No.: 7 and SEQ ID No.: 57.

[00201] In addition, this application provides an isolated polynucleotide containing a nucleic acid encoding an antibody VH, wherein said VH contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID No.: 6, SEQ ID No.: 15, SEQ ID No.: 19, SEQ ID No.: 23, SEQ ID No.: 28, SEQ ID No.: 40, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 42 and SEQ ID No: 52.

[00202] In certain aspects, an antibody or antigen binding fragment thereof comprising a VH or VL encoded by a polynucleotide described above can specifically bind to IL-21, such as human or cynomolgus IL-21. In certain embodiments, such an antibody or antigen-binding fragment thereof may specifically bind to the same epitope as the antibody or antigen-binding fragment thereof containing the VH and VL of 19E3, 9F11, 8B6, or 9H10. In certain aspects, the present application provides a polynucleotide or combination of polynucleotides encoding a binding molecule, eg, an antibody or an antigen-binding fragment thereof, eg, an antibody or an antigen-binding fragment thereof, that specifically binds to IL-21.

[00203] Additionally provided is a vector containing the polynucleotide described above. Suitable vectors are described elsewhere in this application and are known to those skilled in the art.

[00204] In certain aspects, this application provides a composition, such as a pharmaceutical composition, comprising a polynucleotide or vector as described above, optionally further containing one or more carriers, diluents, excipients or other additives.

[00205] In certain aspects, the present application provides a composition comprising: a polynucleotide that contains a nucleic acid encoding a VH, and a polynucleotide that contains a nucleic acid encoding a VL. In this aspect, VL and VH together may contain amino acid sequences VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, completely identical or identical except for eight, seven, six, five, four , three, two or one amino acid substitution in one or more of the CDR sequences: SEQ ID Nos: 12, 13, 14, 7, 8 and 9, SEQ ID Nos: 34, 35, 36, 29, 30 and 31, SEQ ID Nos: 48, 49, 50, 43, 44 and 45, or SEQ ID Nos: 58, 59, 60, 53, 54 and 55, respectively.

[00206] According to additional aspects, the present application provides a polynucleotide composition comprising: a polynucleotide that contains a nucleic acid encoding a VH, and a polynucleotide that contains a nucleic acid encoding a VL, wherein said VL and VH contain, respectively, amino acid sequences of at least at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the reference amino acid sequences: SEQ ID No: 6 and SEQ ID No: 11, SEQ ID No: 15 and SEQ ID No: 17 , SEQ ID No.: 19 and SEQ ID No.: 21, SEQ ID No.: 23 and SEQ ID No.: 25, SEQ ID No.: 28 and SEQ ID No.: 33, SEQ ID No.: 40 and SEQ ID No.: 39, SEQ ID No.: 37 and SEQ ID No.: 39, SEQ ID No.: 38 and SEQ ID No.: 39, SEQ ID No.: 37 and 40, SEQ ID No.: 42 and SEQ ID No.: 47 or SEQ ID No.: 52 and SEQ ID No: 57, respectively.

[00207] In certain aspects, said VH and VL are encoded by nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the nucleotide sequences of SEQ ID No: 5 and SEQ ID No.: 10, SEQ ID No.: 27 and SEQ ID No.: 32, SEQ ID No.: 41 and SEQ ID No.: 46 or SEQ ID No.: 51 and SEQ ID No.: 56, respectively.

[00208] In the polynucleotide composition disclosed above, the polynucleotide containing the nucleic acid encoding VH and the polynucleotide containing the nucleic acid encoding VL may be in the same vector or may be in separate vectors. Accordingly, the present application provides one or more vectors containing the polynucleotide composition described above.

[00209] In some embodiments, the polynucleotide composition encoding VH and VL described above may encode an antibody or antigen binding fragment thereof that can specifically bind to IL-21, such as human or cynomolgus IL-21. In some aspects, the polynucleotide composition encodes an antibody or antigen binding fragment thereof that can specifically bind to the same epitope as the antibody or antigen binding fragment of an antibody comprising the VH and VL of 19E3, 9F11, 8B6 or 9H10.

[00210] Also provided herein is a host cell comprising a polynucleotide, polynucleotide composition, or vector as set forth above, wherein the host cell may, in some embodiments, express an antibody or antigen binding fragment thereof that specifically binds IL-21. Such a host cell can be used in a method for producing an antibody or an antigen-binding fragment thereof provided herein, the method comprising (a) culturing the host cell and (b) isolating the expressed antibody or antigen-binding fragment thereof from the host cell.

[00211] In certain embodiments, said polynucleotides comprise a coding sequence for a mature IL-21 binding polypeptide, such as an anti-IL-21 antibody or an antigen binding fragment thereof, linked in frame to a marker sequence that allows, for example, purification encoded polypeptide. For example, such a marker sequence may be a hexahistidine tag delivered by the pQE-9 vector to allow purification of the mature polypeptide coupled to the marker in the case of a bacterial host, or the marker sequence may be a hemagglutinin (HA) tag derived from the influenza virus hemagglutinin protein when used a host cell from a mammalian body (eg, COS-7 cells).

[00212] Polynucleotide variants are also provided. Polynucleotide variants may contain changes in coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variants contain changes that result in silent substitutions, insertions, or deletions, but do not result in a change in the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants are generated by silent substitutions due to degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of purposes, for example, to optimize the expression of codons for a particular host (changing codons in human mRNA to those preferred by a bacterial host, such as E. coli). Also provided are vectors and cells containing the polynucleotides described in this application.

[00213] In some embodiments, a DNA sequence encoding an IL-21 binding molecule, such as an anti-IL-21 antibody or an antigen binding fragment thereof, can be constructed by chemical synthesis using an automated DNA synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and the selection of those codons that are preferred in the host cell in which the recombinant polypeptide in question will be produced. To synthesize the isolated polynucleotide sequence encoding the isolated polypeptide in question, standard methods can be used. For example, to construct a gene with a possible sequence reconstructed from a polypeptide, the complete amino acid sequence can be used. It is also possible to synthesize a DNA oligomer containing a nucleotide sequence encoding a specific isolated polypeptide. For example, several small oligonucleotides encoding parts of the desired polypeptide can be synthesized and then cross-linked. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[00214] Once assembled (by synthesis, site-directed mutagenesis, or other method), polynucleotide sequences encoding the particular isolated polypeptide in question can be introduced into an expression vector and operably linked to an expression control sequence suitable for expressing said protein in the desired host. To achieve robust expression of the transfected gene in a host, the gene can be operably linked or associated with transcriptional and translational expression control sequences that are functional in the host selected for expression.

[00215] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding anti-IL-21 antibodies or antigen-binding fragments thereof. Recombinant expression vectors are replicable DNA constructs that contain synthetic or cDNA-derived DNA fragments encoding the polypeptide chain of an anti-IL-21 antibody or its antigen-binding fragment, functionally linked to the corresponding transcriptional or translational regulatory elements derived from the genes of mammals, microorganisms, viruses or insects. Typically, a transcription unit contains a complex of (1) a genetic element or elements that perform a regulatory function in gene expression, such as transcriptional promoters or enhancers, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) the corresponding transcription and translation initiation and termination sequences are described below. Such regulatory elements may include an operator sequence to control transcription. The ability to replicate within a host may be further included, which is typically imparted by an origin of replication. Regions of DNA are functionally linked when they functionally interact with each other. For example, DNA for a signal peptide (secretory leader sequence) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of said polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of said sequence; or a ribosome binding site is operably linked to a coding sequence if it is located such that it allows translation. Structural elements intended for use in expression systems in yeast cells include a leader sequence that allows extracellular secretion of the translated protein by the host cell. Alternatively, when the recombinant protein is expressed without a leader or transport sequence, the protein may include a B-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to form the final product.

[00216] The choice of expression control sequence and expression vector should depend on the choice of host. A wide range of expression host/vector combinations can be used. Useful expression vectors for eukaryotic cells include, for example, vectors containing expression regulatory sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as those from E. coli, including pCR1, pBR322, pMB9 and derivatives thereof, a broader range of host plasmids such as M13 and filamentous single-stranded DNA phages.

[00217] Suitable host cells for expressing an IL-21-binding molecule, eg, an anti-IL-21 antibody or an antigen-binding fragment thereof include prokaryotic, yeast, insect, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-positive or gram-negative organisms such as E. coli or rods. Higher eukaryotic cells include the common mammalian-derived cell lines described below. Translation systems can also be used in a cell-free environment. Additional information on methods for producing proteins, including producing antibodies, can be found, for example, in US Patent Publication No. 2008/0187954, US Patent Nos. 6,413,746 and 6,660,501, and International Patent Application Publication No. WO 04009823, each of which is incorporated herein by reference. for its full content.

[00218] Various mammalian or insect cell culture systems can be advantageously used to express recombinant IL-21 binding molecules, such as anti-IL-21 antibodies or antigen binding fragments thereof. Recombinant proteins can be expressed in mammalian cells because such proteins are typically folded correctly, modified properly, and are fully functional. Examples of suitable mammalian host cell lines include the HEK-293 and HEK-293T lines, the monkey kidney cell line COS-7 described in (Cell 23:175, 1981), and other cell lines including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO) cell line, HeLa and BHK. Expression vectors for mammalian cells may contain non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-transcribed sequences , such as essential chromosome binding sites, polyadenylation site, splice donor and acceptor sites, and transcription termination sequences. Baculovirus-based systems for producing heterologous proteins in insect cells are discussed in Luckow and Summers, BioTechnology 6:47 (1988).

[00219] IL-21 binding molecules, eg anti-IL-21 antibodies or antigen binding fragments thereof, produced using a transformed host, can be purified according to any suitable method. Such standard methods include chromatography (eg, ion exchange, affinity, and molecular sieve column chromatography), centrifugation, differential dissolution, or any other standard technology for protein purification. To facilitate protein purification through an appropriate affinity column, affinity tags such as hexahistidine, maltose binding domain, influenza virus envelope sequence, and glutathione S-transferase can be attached to the protein. Isolated proteins can also be characterized physically using technologies such as proteolysis, nuclear magnetic resonance, and x-ray crystallography.

[00220] For example, supernatants from systems that secrete recombinant protein into a culture medium can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to the appropriate matrix for purification. Alternatively, anion exchange resins can be used, for example a matrix or substrate containing protruding diethylaminoethyl (DEAE) groups. Matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices consisting of sulfopropyl or carboxymethyl groups. In addition, one or more reverse phase high performance liquid chromatography (RP-HPLC) steps can be used to further purify the IL-21 binding molecule using hydrophobic RP-HPLC media, such as silica gel with exposed methyl or other aliphatic groups. Some or all of the above purification steps, in different combinations, can also be used to obtain a homogeneous recombinant protein.

[00221] A recombinant IL-21 binding protein, e.g., an anti-IL-21 antibody or an antigen-binding fragment thereof, produced in bacterial culture, can be isolated, for example, by primary extraction from a cell conglomerate followed by one or more concentration steps, salting-out steps , aqueous ion exchange chromatography or size exclusion chromatography. High performance liquid chromatography (HPLC) can be used as final purification steps. The microbial cells used in the expression of the recombinant protein can be destroyed by any conventional method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of lysing agents.

[00222] Methods known in the art for purifying antibodies and other proteins also include, for example, those described in US Patent Publication No. 2008/0312425, 2008/0177048 and 2009/0187005, each of which is incorporated herein by reference. its full content.

VI. Treatment methods using therapeutic anti-IL-21 antibodies

[00223] Methods are provided for using IL-21-binding molecules, such as anti-IL-21 antibodies, including antigen-binding fragments, variants and derivatives thereof, to treat patients suffering from a disease associated with IL-21 expression or IL-21-secreting cells. By "IL-21-secreting" cells is meant cells that express IL-21, such as activated T helper cells. Methods for determining IL-21 expression are well known in the art and include, but are not limited to, PCR, immunohistochemistry, flow cytometry, Western blot, ELISA and the like.

[00224] The following discussion relates to methods of diagnosing and methods of treating various diseases and disorders using an IL-21 binding molecule, such as an anti-IL-21 antibody or an antigen-binding fragment, variant or derivative thereof, that retains the desired anti-IL-21 properties. 21 antibodies proposed in this application, for example, are capable of specifically binding IL-21 and counteracting the activity of IL-21. In some embodiments, the IL-21 binding molecules are murine, human, or humanized antibodies. In some embodiments, the anti-IL-21 antibody is identical to mouse monoclonal antibodies 19E3, 9F11, 9H10, or 8B6. In some embodiments, the anti-IL-21 antibody is derived from one of the mouse monoclonal antibodies 19E3, 9F11, 9H10, or 8B6. In certain embodiments, the resulting antibody is a humanized antibody. In other embodiments, the IL-21 binding molecule comprises a YTE-mutated human IgG1 constant region. In specific embodiments, said IL-21 binding molecule is K44VHa-N56Q.

[00225] In one embodiment, the treatment comprises applying or administering an IL-21 binding molecule, such as an anti-IL-21 antibody or an antigen binding fragment, variant, or derivative thereof, as provided herein, to a subject or patient, or applying or administering the An IL-21 binding molecule to an isolated tissue or cell line from a subject or patient, wherein said subject or patient has a disease, a symptom of a disease, or is susceptible to a disease. In yet another embodiment, the treatment also includes applying or administering a pharmaceutical composition comprising an IL-21 binding molecule, e.g., an anti-IL-21 antibody or an antigen binding fragment, variant, or derivative thereof, as provided herein, to a subject or patient, or administering or administering a pharmaceutical composition containing an IL-21 binding molecule to an isolated tissue or cell line from a subject or patient that suffers from a disease, a symptom of a disease, or is predisposed to a disease.

[00226] IL-21-binding molecules, for example, anti-IL-21 antibodies, antigen-binding fragments, variants or derivatives thereof, provided herein are useful in the treatment of various inflammatory, immune-related or autoimmune diseases or disorders. Such inflammatory, immune-related or autoimmune diseases or disorders may include B-lymphocyte-driven disease or T-lymphocyte-driven disease. In one embodiment, IL-21-binding molecules, such as anti-IL-21 antibodies, antigen-binding fragments, variants, or derivatives thereof, are provided for use in the treatment or prevention of inflammatory, immune-related, or autoimmune diseases or disorders. Examples of inflammatory, immune-related or autoimmune diseases or disorders include, but are not limited to, vasculitis, e.g., anti-neutrophil cytoplasmic antibody (ANCA) vasculitis, ANCA-associated vasculitis (AAV) or giant cell arteritis (GCA), Sjogren's syndrome, inflammatory bowel disease (IBD), pemphigus vulgaris, lupus nephritis, psoriasis, thyroiditis, type I diabetes, idiopathic thrombocytopenic purpura (ITP), ankylosing spondylitis, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Crohn's disease, myasthenia gravis, neuromyelitis optica (NMO), related with IgG4 disease, systemic sclerosis, insulin-dependent diabetes mellitus (IDDM), ankylosing spondylitis and graft-versus-host disease (GVHD). In one embodiment, IL-21-binding molecules, such as anti-IL-21 antibodies, antigen-binding fragments, variants, or derivatives thereof, are provided for use in the treatment or prevention of B cell-driven diseases or malignancies. Examples of B cell-driven malignancies may include follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma (HL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma, Burkitt's lymphosarcoma, Waldenström's disease, and multiple myeloma ( MM). In one embodiment, IL-21-binding molecules, such as anti-IL-21 antibodies, antigen-binding fragments, variants, or derivatives thereof, are provided for use in the treatment or prevention of T cell-driven diseases or malignancies. An example of T cell-driven malignancies may include angioimmunoblastic T-cell lymphoma, which is a follicular T helper (Tfh) cell-driven malignancy, aplastic large cell lymphoma, adult T-cell leukemia, cutaneous T-cell lymphoma, and Sézary syndrome.

[00227] In accordance with the methods of the present invention, at least one IL-21 binding molecule, for example, an anti-IL-21 antibody or an antigen binding fragment, variant or derivative thereof, as defined in other sections of this application. By “positive therapeutic response” in terms of autoimmune treatment is meant any improvement in pathological conditions associated with the activity of the specified binding molecules, for example, anti-IL-21 antibodies or antigen-binding fragments, variants or derivatives thereof, and/or improvement in symptoms associated with the disease . Thus, for example, improvement of the disease may be characterized by a complete response. A “complete response” is defined as the absence of clinically detectable disease with normalization of any previous laboratory diagnostic test results. Such a response, in some embodiments, may persist for, for example, one month after treatment with the methods of the present invention. Alternatively, disease improvement may be classified as a partial response.

[00228] Clinical response can be assessed using screening modalities such as magnetic resonance imaging (MRI), radiography, computed tomography (CT), flow cytometry or fluorescence activated cell sorting (FACS), histology, gross pathological examination and blood chemistry. including, without limitation, changes detected by ELISA, ELISPOT, RIA, chromatography, etc. In addition to these positive therapeutic responses, a subject treated with an IL-21 binding molecule, for example, an anti-IL-21 antibody or an antigen binding fragment, variant or derivative thereof, may exhibit a beneficial effect of improvement in terms of symptoms associated with the disease.

[00229] Also provided is the use of IL-21 binding molecules, such as anti-IL-21 antibodies or antigen binding fragments, variants or derivatives thereof, for diagnostic monitoring of protein levels (eg, IL-21 levels) in blood or tissue as part of a procedure clinical evaluation, for example to determine the effectiveness of a particular treatment regimen. For example, detection can be facilitated by combining said antibody with the substance to be detected. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythin; an example of a luminescent substance includes luminol; examples of bioluminescent substances include luciferase, luciferin and aquarin; and examples of suitable radioactive substances include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

VII. Pharmaceutical compositions and methods of administration

[00230] Methods for producing and administering IL-21 binding molecules, such as anti-IL-21 antibodies or antigen binding fragments, variants or derivatives thereof, provided herein for a subject in need of such a molecule are well known to those skilled in the art. or are easily determined by them. The route of administration of said IL-21 binding molecule, for example an anti-IL-21 antibody or an antigen binding fragment, variant or derivative thereof, may be, for example, oral, parenteral, inhalation or topical. As used herein, the term “parenteral” includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal routes of administration. While all of these administration forms are clearly intended to fall within the scope of the present invention, another example of an administration form may be an injection solution, particularly for intravenous or intra-arterial injection or drip administration. Typically, a suitable pharmaceutical composition may contain a buffer (eg, acetate, phosphate or citrate buffer), a surfactant (eg, polysorbate), optionally a stabilizing agent (eg, human albumin), etc. According to other methods consistent with the concept described in herein, IL-21-binding molecules, such as anti-IL-21 antibodies or antigen-binding fragments, variants, or derivatives thereof, provided herein can be delivered directly to the site of an unwanted cell population, increasing exposure of the desired tissue to the therapeutic agent. In one embodiment, administration is carried out directly into the respiratory tract, for example, by inhalation or intranasal administration.

[00231] As discussed herein, IL-21 binding molecules, such as anti-IL-21 antibodies or antigen binding fragments, variants or derivatives thereof provided herein, can be administered in a pharmaceutically effective amount for the in vivo treatment of IL -21-related diseases, such as inflammatory immune-related or autoimmune diseases or disorders. In this regard, it should be understood that the binding molecules disclosed herein may be formulated into dosage forms to facilitate administration and promote stability of the active agent. The pharmaceutical compositions of the present invention may contain a pharmaceutically acceptable, non-toxic, sterile carrier such as saline, non-toxic buffers, preservatives and the like. For the purposes of this application, a pharmaceutically effective amount of an IL-21 binding molecule, e.g., an anti-IL-21 antibody or an antigen binding fragment, variant or derivative thereof, conjugated or unconjugated, is understood to be an amount sufficient to achieve effective target binding or benefit. , for example, to relieve symptoms of a disease or condition, or to identify a substance in a cell. Suitable dosage forms for use in the treatment methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).

[00232] Certain pharmaceutical compositions provided herein can be administered orally in a suitable dosage form, such as capsules, tablets, aqueous suspensions or solutions. Certain pharmaceutical compositions can also be administered in the form of a nasal aerosol or inhalation. Such compositions can be formulated in the form of solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability and/or other conventional solvents or disintegrants.

[00233] The amount of IL-21 binding molecule, e.g., anti-IL-21 antibody or fragment, variant or derivative thereof, that can be combined with carrier materials to form a unit dosage form will vary depending on the subject being treated and specific route of administration. The composition can be administered once, repeatedly, or over a predetermined period of time in the form of an infusion. Dosing regimens may also be adjusted to achieve the optimal desired response (eg, therapeutic or prophylactic response).

[00234] In accordance with the scope of the present invention, anti-IL-21 antibodies or antigen binding fragments, variants or derivatives thereof can be administered to a human or other animal in accordance with the above methods of treatment in an amount sufficient to achieve a therapeutic effect. The anti-IL-21 antibodies or antigen binding fragments, variants or derivatives thereof provided herein can be administered to such a human or other animal in a conventional dosage form prepared by combining the antibody or antigen binding fragment, variant or derivative thereof provided herein. , with a conventional pharmaceutically acceptable carrier or solvent according to known technologies. The form and nature of the pharmaceutically acceptable carrier or solvent may be dictated by the amount of active ingredient with which it is combined, the route of administration, and other well known factors. It is also possible to use a mixture containing one or more types of IL-21 binding molecules, for example, anti-IL-21 antibodies, or antigen binding fragments, variants or derivatives thereof according to the present invention.

[00235] By “therapeutically effective dose or amount” or “effective amount” is meant an amount of an IL-21 binding molecule, e.g., an antibody or antigen binding fragment, variant or derivative thereof, which when administered produces a positive therapeutic response with respect to treatment a patient with a disease or condition that needs to be treated.

[00236] Therapeutically effective doses of the compositions of the present invention for the treatment of IL-21-mediated diseases, such as inflammatory, immune-related or autoimmune diseases or disorders, vary depending on a variety of different factors, including the means of administration, the target site, the psychological state of the patient whether the patient is human or animal, other drugs administered, and whether the effect is therapeutic or prophylactic. Typically the patient is a human, but non-human mammals, including transgenic mammals, may also be treated. Treatment doses can be titrated using standard methods known to those skilled in the art to optimize safety and effectiveness.

[00237] The amount of at least one IL-21 binding molecule, such as an antibody or an antigen binding fragment, variant or derivative thereof, to be administered can be readily determined by one of ordinary skill in the art without undue experimentation based on the present disclosure. Factors influencing the mode of administration and the appropriate amount of at least one IL-21-binding molecule, e.g., an antibody or an antigen-binding fragment, variant or derivative thereof, include, but are not limited to, severity of the disease, medical history, and age, height, weight, condition health and physical condition of the individual being treated. Likewise, the amount of IL-21 binding molecule, eg, antibody or antigen binding fragment, variant or derivative thereof, that is required to be administered will depend on the mode of administration and whether the subject will be administered a single dose or multiple doses of the agent.

[00238] Also provided herein is the use of an IL-21 binding molecule, e.g., an antibody or an antigen-binding fragment, variant or derivative thereof, for the treatment of an inflammatory, immune-related or autoimmune disease or disorder, e.g., vasculitis, e.g., ANCA vasculitis or GCA, Sjögren's syndrome, systemic lupus erythematosus and/or lupus nephritis, rheumatoid arthritis, Crohn's disease, myasthenia gravis or GVHD.

[00239] This application also provides the use of an IL-21 binding molecule, for example an antibody or an antigen binding fragment, variant or derivative thereof, for the treatment of the production of a medicament for the treatment of an inflammatory, immune-related or autoimmune disease or disorder, for example, vasculitis, for example, ANCA or GCA vasculitis, Sjögren's syndrome, systemic lupus erythematosus and/or lupus nephritis, rheumatoid arthritis, Crohn's disease, myasthenia gravis or GVHD.

VIII. Diagnostics

[00240] The present application also provides a diagnostic method useful in diagnosing an IL-21-related disease, such as an inflammatory, immune-related or autoimmune disease or disorder, which includes measuring the level of IL-21 protein expression in a tissue or body fluid. , selected from an individual, and comparing the measured level of expression with a standard level of expression of IL-21 in healthy tissue or body fluid, wherein an increase in the level of expression compared to the standard level indicates a disorder that can be treated with an IL-21 binding molecule, for example, an anti-IL-21 antibody or an antigen-binding fragment thereof.

[00241] Anti-IL-21 antibodies and antigen-binding fragments, variants and derivatives thereof provided herein can be used to assess the level of IL-21 protein in a biological sample using classical immunological methods known to those skilled in the art (e.g. see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for determining IL-21 protein expression include an immunoassay such as an enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting.

[00242] By "assessing the expression level of an IL-21 polypeptide" is meant a qualitative or quantitative measurement or assessment of the level of IL-21 polypeptide in a first biological sample, directly (e.g., by determining or estimating the absolute level of the protein) or relatively (e.g., by comparison with the level disease-associated polypeptide in a second biological sample). The level of expression of the IL-21 polypeptide in a first biological sample can be measured or assessed and compared with a standard level of IL-21 polypeptide, wherein the standard is taken from a second biological sample collected from an individual suffering from the disorder or is determined by averaging levels from populations of individuals who do not suffer from the disorder. As is common in the art, if a "standard" level of IL-21 polypeptide is known, it can easily be used as a standard level for comparison.

[00243] By “biological sample” is meant any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing IL-21. Methods for obtaining a biopsy sample of tissue and body fluids from mammals are well known in the art.

IX. Kits containing IL-21 binding molecules

[00244] Also provided herein are kits that contain an IL-21 binding molecule, such as an anti-IL-21 antibody or antigen binding fragment thereof, as described herein, and which are used to implement the methods described herein. In certain embodiments, the kit contains at least one purified antibody or antigen binding fragment thereof in one or more containers. In some embodiments, the kits contain components necessary and/or sufficient to perform the analysis, including control samples, instructions for performing the analysis, and any necessary computer programs for analysis and reporting of results. Those skilled in the art will appreciate that the disclosed IL-21 binding molecules, such as anti-IL-21 antibodies or antigen binding fragments thereof, can be readily included in one of these kit forms, which are well known in the art.

X. Immunological analysis

[00245] IL-21 binding molecules provided herein, such as anti-IL-21 antibodies or antigen binding fragments, variants or derivatives of molecules provided herein, can be assessed for immunospecific binding using any method known in the art . Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using technologies such as Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), ELISPOT, sandwich immunoassay, immunoprecipitation-based assay, reactions with precipitins, diffuse gel precipitation reactions, immunodiffusion-based assays, agglutination-based assays, complement fixation assays, immunoradiometric assays, fluorescence immunoassays, protein A immunoassays, and many others. Such assays are standard and well known in the art (see, for example, Ausubel et al., eds, (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1, which is incorporated herein application by reference to its full contents).

[00246] IL-21 binding molecules, such as anti-IL-21 antibodies or antigen binding fragments, variants or derivatives thereof, provided herein can be used in histological studies such as immunofluorescence, immuno-electron microscopy or non-immunological analysis , upon in situ detection of IL-21, or its conservative variants or peptide fragments. In situ detection can be accomplished by removing a histological sample from a patient and contacting it with a labeled IL-21-binding molecule, for example, an anti-IL-21 antibody or an antigen-binding fragment, variant or derivative thereof, for example, contacting by layering labeled IL-21- binding molecule (eg, antibody or fragment) to a biological sample. Using this procedure, it is possible to determine not only the presence of IL-21, or conserved variants or peptide fragments, but also their distribution in the tissue being examined. Those skilled in the art will readily appreciate that using the present invention, any of a wide variety of histological techniques (such as staining procedures) can be modified to achieve such in situ detection.

[00247] The binding activity of a particular plurality of IL-21 binding molecules, eg, an anti-IL-21 antibody or an antigen binding fragment, variant or derivative thereof, can be determined according to well known methods. Those skilled in the art are able to determine operating and optimal assay conditions for each determination using standard experimentation.

[00248] Methods and reagents suitable for determining the binding characteristics of an isolated IL-21 binding molecule, e.g., an anti-IL-21 antibody or an antigen binding fragment, variant, or altered/mutant derivative thereof, are known in the art and/or are commercially available . Equipment and software designed for such kinetic analysis are commercially available (eg, BIAcore®, BIAevaluation® program, GE Healthcare; KINEXA® program, Sapidyne Instruments).

[00249] In the practice of the present invention, unless otherwise indicated, conventional methods of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which fall within the purview of those skilled in the art, will be employed. Such methods are fully explained in the literature. For example, see Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[00250] General principles for antibody design are established in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). General principles for protein design are established in Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). The general principles of antibody and hapten-antibody binding are established in: Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, N.Y.). In addition, standard methods in immunology, known in the art and not specifically described, generally correspond to the methods in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al., eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co., NY).

[00251] Authoritative references that establish general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett et al., eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyzes (Plenum Press, NY); Campbell (1984) "Monoclonal Antibody Technology" in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al., (Elsevere, Amsterdam); Goldsby et al., eds. (2000) Kuby Immunology (4th ed.; H. Freemand &Co.); Roitt et al. (2001) Immunology (6th ed.; London: Mosby); Abbas et al. (2005) Cellular and Molecular Immunology (5th ed.; Elsevier Health Sciences Division); Kontermann and Dubel (2001) Antibody Engineering (Springer Verlan); Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall 2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

[00252] All sources cited above, as well as sources cited in this application, are incorporated herein by reference in their entirety.

[00253] The following examples are offered for illustration and not limitation.

EXAMPLES

[00254] Embodiments of the present invention can be further characterized by reference to the following non-limiting examples, which detail the preparation of certain antibodies of the present invention and methods of using the antibodies of the present invention. Those skilled in the art will appreciate that many modifications, both materials and methods, can be made without departing from the scope of the present invention.

Example 1: Preparation and Humanization of Anti-IL-21 Mouse Monoclonal Antibody

Immunization of mice and generation of anti-IL-21 hybridoma

[00255] Female Balb/c mice, 6 weeks old, received 8 cycles of intraperitoneal (IP) and metatarsal injections of purified recombinant human IL-21 alternating with cynomolgus IL-21 protein conjugated to BSA (bovine serum albumin) (Kit BSA Imject Maleimide-Activated, "Pierce"). Briefly, starting on day 1, mice were immunized with 10 μg IP and 2.5 μg metatarsal immunogen emulsified in Imject Alum (Thermo Scientific) according to the manufacturer's instructions. Immunization lasted 28 days at 4-day intervals. Blood samples were collected on days 15 and 28 by withdrawing blood from the retro-orbital space into serum separation tubes. Serum was analyzed by ELISA, directly measuring IL-21 binding and competition with IL-2R. Based on neutralization titers in the competitive ELISA, mice were selected to receive a pre-dose of 10 μg of BSA-conjugated human IL-21 and sacrificed on day 30. Splenocytes and lymph node cells were collected from the animals and hybridized with P3-X63-Ag8.653 myeloma cells using PEG to generate hybridomas. Anti-IL-21-specific hybridomas were identified by screening hybridoma supernatants in ELISA to assess direct binding and competition, as described above. Positive hybridomas were also further tested for neutralizing activity in a cell-based assay using negative modulation of STAT3 phosphorylation as a reference point (IL-21 triggers signaling through STAT3). Neutralizing hybridomas were then cloned at limited dilution and propagated to purify the antibody.

[00256] Throughout the hybridoma production cycle, a competitive ELISA was used to identify mice that responded well to IL-21 immunization, then screened in the same manner to neutralize the hybridomas. Briefly, Maxisorp ELISA plates (NUNC) were coated with 2 μg/ml IL-21R-Fc in phosphate-buffered saline (PBS) and left at 4°C overnight. The plates were then blocked with 3% BSA in PBST (PBS + 0.1% Tween) at room temperature for one hour. In parallel, blood samples were serially diluted (to select animals with good responses), or IgG concentrations in the hybridoma supernatant were normalized and preincubated with 2 nM biotinylated IL-21. After preincubation, the mixture was added to each well of the ELISA plate and further incubated at room temperature for half an hour. After washing, a streptavidin-horseradish peroxidase mixture was added to each well, and the level of bound IL-21 was determined as an indicator of inhibition of the IL-21/IL-21R-Fc interaction by the tested blood samples or IgG hybridoma.

Isolation, cloning, sequencing. expression and purification of anti-IL-21 mouse monoclonal antibodies

[00257] Supernatants from hybridoma cultures were analyzed for their ability to inhibit the IL-21/IL-21R-Fc interaction according to the procedure described for the competitive ELISA. Positive samples were subjected to limited dilution cloning (LDC) to ensure clonality. After confirming the functional properties of the subclones, they were subjected to molecular cloning.

[00258] mRNA from hybridomas was isolated using the Dynabeads mRNA Direct Kit according to the manufacturer's instructions. The mRNA was then converted to cDNA by reverse transcriptase polymerase chain reaction using Superscript III reverse transcriptase. To eliminate PCR bias, two sets of cDNA were synthesized. These cDNAs were used as templates for amplification of the variable regions of the light and heavy chains of antibodies (VL and VH). PCR was performed using high-fidelity Taq polymerase from Invitrogen and a mouse Ig primer set from Novagen, which included six sets of primers for VH and seven sets of primers for VL. The amplified VL and VH PCR products were purified and cloned into pCR 2.1 Toro vectors from Invitrogen, which readily ligate DNA sequences to T-A-compatible ends. Transformed E. coli cells were incubated on Petri dishes with X-gal/Carb agar. Bacterial colonies containing PCR inserts turned white. White colonies were randomly selected for PCR to confirm the insertion.

[00259] Forty-eight white colonies from each transformation were selected for sequencing. Colonies were plated into 96-well plates with LB/Carb medium and then imprinted onto Petri dishes with LB/Carb agar. Colonies that grew on agar plates were sequenced using the M13 forward primer. Based on the sequencing results, primers were designed that could allow PCR amplification of VL and VH with the addition of restriction enzyme sites that are compatible with the human IgG expression vector pOE. Restriction enzyme digestion and crosslinking were performed to clone VL and VH into a vector to produce a chimeric antibody construct containing a variable region from a mouse antibody and a constant region from a human antibody. The DNA plasmid was transfected into 293F cells using 293 transfection reagent. The level of IgG expression in cell culture supernatants was assessed using Octet. IgG in the supernatant was purified using protein-A-coated columns. Purified IgG was analyzed on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

[00260] ELISA was used to assess the cross-reactivity of the leader clones. The cytokines tested included IL-2, IL-4, IL-7, IL-9, and IL-15. Maxisorp ELISA plates (NUNC) were coated with 2 μg/ml of different cytokines in PBS and left at 4°C overnight. The plates were then blocked with 3% BSA in PBST (PBS + 0.1% Tween) at room temperature for one hour. Purified IgG leader clones were serially diluted in PBST + 3% BSA, and then the mixture was added to each well of the ELISA plate and further incubated at room temperature for half an hour. After washing, a secondary horseradish peroxidase antibody was added to each well and left for 1 hour. The wells were washed with PBST, and then first PBST was added, and then PBS and TMB solution from Pierce were added to determine the secondary antibody. After 5 min, the reaction was stopped by adding 1N hydrochloric acid solution. Reading was then performed on an ELISA plate reader at 450 nm to determine binding.

Humanization of various anti-IL-21 mouse monoclonal antibodies

[00261] Mouse monoclonal antibody 9F11 was generated and characterized using MedImmune and showed the desired biological activity with a binding KD of 10 nM for IL-21. Humanization work was carried out at Shanghai ChemPartner Co using CDR grafting technology. The properties of the successfully humanized antibody candidates 9F11-4, 9F11-6 and 9F11-11 were tested (results are presented below). The sequences of 9F11 variable regions and humanized 9F11 clones are shown in Figure 1A.

[00262] Humanization of mouse monoclonal antibody 19E3 was accomplished by grafting CDRs from 19E3 into selected human germline antibody scaffolds. The variable heavy chain (VH) and variable light chain (VL) sequences of mouse monoclonal antibody 19E3 were compared with human germline antibody sequences available in the public domain NCBI databases. Human antibody acceptor frameworks were identified based on maximum sequence homology. Several other criteria were used to select the optimal acceptor scaffold, including matching on critical residues (Vernier zone, canonical class residues, and VH/VL surface residues), immunogenicity (germline frequency), stability, and expression. Here, a single germ family, VH1-46, was identified as the VH acceptor framework. The J-Segment genes were compared to the reference sequences at framework region 4, and J-Segments and JH4 were selected for VH. To achieve maximum homology to the VL acceptor sequence, three separate framework regions from three different human germline antibodies were combined to construct an optimal hybrid germline acceptor sequence. The human antibody template selected for the VL chain, referred to as K1, was a combination of O14 (Scaffold1), O18 (Scaffold2), L23 (Scaffold3) and JK1 (Scaffold4). The homology of the framework regions between the mouse sequence and the human antibody template was 71% for VH and 79% for VL.

[00263] Critical residues in the mouse antibody framework that were predicted to influence CDR loops, stabilize variable region structure, and influence VH and VL interchain packing and/or interaction were identified and were selectively introduced back into the germline frameworks from a human antibody to maximize retention of the 19E3 binding epitope and affinity. In this case, residues from the mouse antibody at positions 44V and 87F were backmutated into the VL chain template from the human antibody template as a second acceptor framework template, termed K2. The alignment of the parent VL chain with the indicated acceptor framework regions is shown in Figure 1B.

[00264] For the VH chain, two additional human antibody acceptor framework templates were designed as a way to identify a framework that could influence 19E3 binding activity if the full human antibody (Ha) acceptor template lost binding activity. One template, called Hb, was constructed by combining framework region 1 from a mouse antibody and template VH1-46/JH4 from a human antibody; another template, called Hc, was constructed by combining framework region 3 from a mouse antibody and the VH1-46/JH4 template from a human antibody. The alignment of the parent VH chain with the indicated acceptor framework regions is shown in Figure 1B.

[00265] To generate a humanized antibody, Cabot numbered CDR residues were linked into designed acceptor frameworks for both VH and VL. In total, three humanized VH genes (Ha, Hb and Hc) and two VL genes (K1 and K2) were synthesized using GeneART, then they were cloned into the IgG1 expression vector. A set of humanized variants was generated and characterized in biochemical and biological tests. To evaluate the IL-21 binding activity of these humanized variants, a competition assay for the HTRF epitope was developed. The fully humanized Ha VH template exhibited IL-21 binding activity comparable to that of the mouse 19E3 VH, as well as the activity of the Hb and Hc hybrid variants. The K2 humanized VL template, with two residues from the mouse antibody at positions 44V and 87F, exhibited higher binding activity than the fully humanized K1 variant. The resulting humanized variant was shown to have antigen binding affinity and epitope specificity similar to those of to 19E3. To minimize the content of residues from the mouse antibody in the best-behaved humanized variant (K2Ha), the residue from the mouse antibody at position 87F was replaced by a comparable residue from the human antibody, but not 44V, which was critical to retain IL-21 binding activity. In the 19E3 heavy chain, a high-risk N-terminal glycosylation (NYT) site was identified at position 56 of CDR2, and an N to Q substitution was introduced here. The resulting humanized antibody (K44VHaN56Q), with just one residue from the mouse antibody, exhibited indistinguishable binding activity at compared with mouse 19E3 and demonstrated biological activity 2-3 times higher than that of 19E3 in all biological tests performed.

[00266] Mouse monoclonal antibody 9F11 demonstrated the desired biological activity with an IL-21 binding K-D of 10 nM. Humanization work was carried out at Shanghai ChemPartner Co using CDR grafting technology. The properties of the successfully humanized antibody candidates 9F11-4, 9F11-6 and 9F11-11 were tested (results presented below) and these antibodies were shown to fully retain IL-21 binding and biological activity compared to the parent mouse antibody 9F11 . The sequences of 9F11 variable regions and humanized 9F11 clones are shown in Figure 1.

[00267] The properties of various anti-IL-21 antibodies proposed in this application are shown in Table 2.

Example 2: Binding affinity of K44VHa-N56Q to human and cynomolgus recombinant IL-21

[00268] This example describes the determination of the equilibrium liquid phase binding constant (KD) for the interaction of K44VHa-N56Q with human and cynomolgus IL-21. The equilibrium dissociation constant (KD) for the interaction of K44VHa-N56Q with human (hu) and cynomolgus (cyno) IL-21 was measured using a kinetic exclusion assay (KinExA) 3000 platform (Sapidyne, Boise, ID). 50 mg aliquots of azlactone beads (Thermo Scientific, Rockford, IL) were coated with huIL-21 (obtained from Protein Sciences/ADPE) or cynoIL-21 (obtained from Protein Sciences/ADPE) at concentrations of 75 or 150 μg /ml in accordance with the device manufacturer's instructions. The beads were washed and blocked in 1 M Tris buffer, pH 8 (Invitrogen, Carlsbad, CA) containing 10 mg/ml bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, PA) and stored at 4 °C before use. Lot sizes of K44VHa-N56Q were prepared at 500 fM and 50 pM concentrations (500 fM and 50 pM K44VHa-N56Q batches, respectively) in sample buffer (phosphate-buffered saline, Invitrogen), pH 7.4, 0.1% BSA, 0.02% sodium azide ( _NaN3 , Sigma-Aldrich)); each volume was distributed into 2 separate sets of 13 tubes (1 for each series). Human or cynomolgus IL-21 was added to 1 tube from each series at a concentration of 4 nM (50 pM K44VHa-N56Q) or 100 pM (500 pM K44VHa-N56Q series), then serial dilution was carried out in the remaining 11 tubes, leaving 1 tube for samples in each series as control K44VHa-N56Q, receptor only. Final concentrations of human or cynomolgus IL-21 in individual sample mixtures ranged from 1.95 fM to 100 pM (500 fM K44VHa-N56Q), and from 78.1 fM to 4 nM (50 pM K44VHa-N56Q). These sample mixtures were equilibrated at room temperature for 2-7 days and analyzed on the KinExA platform. Suspensions of IL-21-coated azlkathone beads were diluted to 30 ml with instrumental buffer (PBS, pH 7.4, 0.02% NaN3) in a bead vial and mounted in a KinExA device. To sequentially transfer the azlactone/IL-21 beads into the capillary cuvette, the instrument used a user-defined timing program and individual sample mixtures were applied on top of the IL-21-coated beads. The unbound sample solution was removed by washing the beads with instrumental buffer. A solution of DyLight649-labeled sheep anti-huIgG(H+L) secondary reagent (Thermo Scientific) was passed through the beads to detect the receptor (K44VHa-N56Q) that remained bound to the IL-21-coated beads. The beads were washed again with instrument buffer to remove excess (unbound) label. The level of fluorescence that remains associated with the beads was measured and the signal was converted to the percentage of free receptor (K44VHa-N56Q). At intervals between samples, the bead pack was washed out of the flow cell and replaced with fresh IL-21-coated azlactone beads in preparation for the next sample. When data were collected for all samples in each batch (500 fM and 50 pM K44VHa-N56Q), an isotherm was generated that plotted the amount of free K44VHa-N56Q determined at each IL-21 concentration. The resulting 2 binding curves were then evaluated by dual curve analysis using the evaluation program supplied with the instrument (KinExA Pro software, v. 2.0.1.26, Sapidyne), from which individual _KDs for each interaction were obtained.

[00269] Human and cynomolgus IL-21 tc binding K44VHa-N56Q was measured by KinExA. K44VHa-N56Q was prepared in 2 concentration batches: 500 fM and 50 pM, and human IL-21 or cynomolgus IL-21 was serially diluted within each batch. When equilibrium was reached, the KinExA instrument was used to measure the concentration of free K44VHa-N56Q remaining in each sample mixture. This was done by recording the level of fluorescence that remained associated with the IL-21-coated beads after samples of each mixture were passed through the beads and then exposed to a fluorescently tagged anti-IgG reagent. The amount of detected free K44VHa-N56Q was plotted against the concentration of huIL-21 or cynoIL-21, and the scoring program included with the KinExA platform was used to fit each data set to a single-site affinity model. The KinExA program generally fits both binding curves (500 fM and 50 pM IgG) in a dual curve analysis (Figures 2A and 2B, respectively), extracting optimal solutions for several parameters simultaneously and calculating the true binding constant in the liquid phase. Under these conditions, the _KD for the binding of K44VHa-N56Q to the huIL-21 protein was calculated to be 515 fM (95% CI for _KD : 279-844 fM), and the binding of K44VHa-N56Q to the cynoIL-21 protein was calculated to be 352 fM ( 95% CI for _KD : 134-685 fM).

[00270] K44VHa-N56Q exhibited weak binding to murine IL-21 with an affinity in the μM region as measured using the KinExA platform.

Example 3 Mechanism of Action Studies for Anti-IL-21 Monoclonal Antibodies

Anti-IL-21 clones block IL-21-induced STAT3 phosphorylation

[00271] The ability of purified anti-IL-21 antibodies to inhibit human and cynomolgus IL-21-induced phosphorylation of STAT3 (pSTAT3) was determined as described below.

[00272] Briefly, all human peripheral blood mononuclear cells (PBMCs) were isolated from a CPT tube (BD Vacutainer) after centrifugation according to the manufacturer's instructions. Anti-IL-21 antibodies were preincubated with cynomolgus IL-21 (20 pM) in 96-well round-bottom plates for 60 minutes at 37 degrees. After this incubation, PBMC (0.4 x 10 ⁶ per well) were quickly added to each well and incubated with IL-21/IgG for 15 minutes at 37 degrees. After stimulation, cells were fixed for 10 minutes (at 37 degrees) with 100 μl/well of prewarmed lysis/fixation buffer (BD Phosflow) and then permeabilized on ice for 30 minutes with 225 μl/well Perm Buffer III (BD Phosflow ). Cells were washed twice (PBS/2% BSA) and stained with anti-STAT3 (pY705, BD Phosflow) for 30 min at room temperature. Cells were washed with staining buffer and analyzed on an LSR II flow pitometer (BD Biosciences) using FACSDiva software.

[00273] Stimulation of human and cynomolgus IL-21 as described caused a 3- to 5-fold increase in pSTAT3 levels in the total human PBMC pool (Figure 3A). Representative graphs demonstrating inhibition of IL-21-induced pSTAT3 inactivation by anti-IL-21 antibodies are shown in Figure 3B. All tested clones achieved 100% inhibition in this assay. The IC50 values of the anti-IL-21 clones in the pSTAT3 assay are summarized in Table 3. These data indicate that the described anti-IL-21 antibodies are capable of inhibiting early signaling events downstream of the IL-21R.

[00274] K44VHa-N56Q has been shown to bind weakly to murine IL-21.

Stimulation of mouse splenocytes with mouse IL-21 caused a 4-fold increase in intracellular levels of phosphorylated STAT3. K44VHa-N56Q could not inhibit murine IL-21-induced STAT3 phosphorylation. These data indicate that K44VHa-N56Q does not neutralize the biological activity of mouse IL-21.

[00275] IL-21 is a member of the _γc receptor family of cytokines that utilize the conventional gamma chain in conjunction with specific receptor(s) to mediate biological activity. Lymphocytes from human blood were stimulated with γ _c chains using the cytokines IL-2, IL-4, IL-7, IL-15 or IL-21 in the presence or absence of K44VHa-N56Q or the human control antibody IgG1-YTE. Signal transduction at subsequent levels was measured by assessing the phosphorylation of STAT3 molecules corresponding to each cytokine. K44VHa-N56Q had no effect on downstream signaling associated with the cytokines IL-2, IL-4, IL-7, or IL-15. These data indicate that K44VHa-N56Q acts specifically to neutralize the IL-21 pathway in human cells.

[00276] Signaling from IL-21 through its own receptor leads to the activation and phosphorylation of STAT3. The data described herein demonstrate that K44VHa-N56Q can inhibit pSTAT3 activation in response to both human and cynomolgus IL-21, but not mouse IL-21. In addition, this inhibition is specific for IL-21, since K44VHa-N56Q does not affect the activation of STAT molecules after activation of cognate _cytokine receptors such as receptors for IL-2, IL-4, IL-7 or IL-15. Therefore, K44VHa-N56Q is a potent and specific inhibitor of IL-21R signaling events.

Blocking IL-21 suppresses IFNγ production by NK-92 cells

[00277] The effect of purified anti-IL-21 antibodies on IL-21-induced IFNγ production by BK-92 cells was assessed as follows.

[00278] NK-92 cells were grown in complete growth medium, which consisted of MEM alpha Glutamax basal medium supplemented with sodium bicarbonate (1.5 g/L), inositol (0.2 M, Sigma Aldrich), 2-mercaptoethanol (0 .1 mM), folic acid (0.02 mM, Sigma Aldrich), recombinant IL-21 (100 U/ml, R&D Systems), horse serum (12.5%), and fetal bovine serum (12.5%). All reagents were purchased from Invitrogen unless otherwise noted. Cells were washed and resuspended in medium that did not contain IL-2 for 16-24 hours before stimulation with IL-21. To evaluate the activity of anti-IL-21 clones, antibodies were preincubated with human or cynomolgus IL-21 (200 pM) in a 96-well flat-bottom plate for 60 minutes at 37 degrees. 0.5 x 105 NK-92 cells were then added to the plate and incubated at 37 degrees for 24 hours. IFNγ production was quantified in the supernatant using the human IFNγ singleplex MSD assay according to the manufacturer's instructions (Meso Scale Discovery).

[00279] The ability of anti-IL-21 clones to inhibit human and cynomolgus IL-21-induced IFNγ production by NK-92 cells is shown in Figure 4. Clones K2Ha-N56Q, K(44V)Ha-N56Q, and K(44V)Ha6-N56Q were able to completely inhibit IFNγ production in these cultures, with similar activity against human and cynomolgus IL-21. Table 4 shows the resulting IC50 values for the clones evaluated in the NK-92 cell assay. These data demonstrate that the studied anti-IL-21 antibodies modulate the activity of NK-92 cells, as demonstrated by complete blocking of IFNγ production after 24 hours.

Plasma cell differentiation induced by exogenous IL-21 is suppressed by anti-IL-21 clones

[00280] To determine the effect of anti-IL-21 antibodies on B cell activity, a plasma cell differentiation assay was performed.

[00281] Human PBMCs were isolated from CPT tubes (BD Vacutainer) after centrifugation according to the manufacturer's instructions. Negative selection for B lymphocytes was performed using MACS cell separation reagents (human B lymphocyte isolation kit II, Miltenyi Biotec). The purity of B lymphocytes using this method was typically >97%. Notably, all B cell preparations isolated using this method contained populations of both naïve and memory B cells, but plasma cells were excluded due to expression of CD43, which is targeted by the selection antibody complex in the MACS kit. The nutrient medium for these experiments was RPMI 1640 medium (Invitrogen) supplemented with 10% FBS, penicillin-streptomycin (100 units/ml penicillin, 100 μg/ml streptomycin), 2-mercaptoethanol (55 μM), L-glutamine (2 mM) and HEPES (5 mM). Anti-IL-21 antibodies were preincubated in 96-well round-bottom plates with a stimulating complex that included human or cynomolgus IL-21 (2 nM), anti-CD40 (0.1 μg/ml, sheep IgG, R&D Systems "), and anti-IgM F(ab')2 (5.0 μg/ml, Jackson ImmunoResearch Laboratories), for 60 minutes at 37 degrees. 0.5-1.0×10 ⁵ B lymphocytes were then added to the plate to a final volume of 150 µl.

[00282] After stimulation for 6 days, cells were stained and analyzed by flow cytometry to quantify the frequency of plasma cells in culture. Cells were washed (PBS/2% FBS) and stained for 30 minutes at 4 degrees with anti-IgD (FITS (fluorescein isothiocyanate) or PE); anti-CD38 (allophycocyanin, clone HB7) (BD Pharmingen), and analyzed on an LSRII flow cytometer using the FACSDiva program. In addition, the supernatant was collected and Ig production was quantified using ELISA. Briefly, 5 μg/ml sheep anti-human IgG diluted in PBS (Bethyl Laboratories) was applied to a 96-well flat-bottom plate and left overnight at 4°C. The plates were washed and blocked with 0.2% BSA in PBS. The supernatant was diluted and incubated in plates overnight. Bound Ig was detected using sheep anti-human IgG-alkaline phosphatase complex (0.2 μg/ml, Bethyl Laboratories) diluted in blocking buffer. The reaction in the plates was accelerated using SigmaFast p-nitrophenyl phosphate tablets (Sigma Aldrich) and specific absorbance was measured at a wavelength of 405 nm using a SpectraMax plate reader (Molecular Devices).

[00283] Stimulation of human B cells as described above with human or cynomolgus IL-21 resulted in the generation of a population of IgD ^- CD38 ^hi plasma cells (Figures 5A and 5B). Several clones, including 19E3.1, 9F11.1 and 9H10.1, were able to completely suppress plasmacytic differentiation under these conditions (Figures 5A and 5B). Clone 18B6.1 suppressed plasma cell differentiation induced by IL-21 in cynomolgus monkeys and not in humans. The resulting IC50 values for the anti-IL-21 clones are shown in Table 5. Additionally, under control conditions, IL-21 stimulation with anti-CD40 and anti-IgM resulted in Ig production in the range of 30-40 μg/ml by day 6 ( Figure 5C). Clones 19E3.1 and 9F11.1 suppress Ig secretion by human B cells in a dose-dependent manner (Figure 5C). Clone 19E3.1 was able to inhibit IgG production in response to both human and cynomolgus IL-21 by >99%, whereas 9F11.1 inhibited IgG production in response to human and cynomolgus IL-21 by 97% and 79%. respectively. Taken together, these data indicate that anti-IL-21 clones are capable of significantly suppressing human plasmacytic differentiation and Ig secretion under the influence of exogenous human and cynomolgus IL-21.

Anti-IL-21 clones suppress plasma cell differentiation induced by activated CD4+ T lymphocytes

[00284] The ability of anti-IL-21 clones to block plasma cell differentiation induced by activated CD4+ T lymphocytes in a co-culture assay as described below was assessed.

[00285] Human PBMCs were isolated from CPT tubes (BD Biosciences) after centrifugation. Negative selection for B lymphocytes and CD4+ T lymphocytes was performed using MACS cell separation reagents (Miltenyi Biotec). The purity of the total population of B lymphocytes and CD4+ T lymphocytes was routinely >97%. CD4+ T lymphocytes were treated with mitomycin-C (30 μg/ml, Sigma Aldrich) for 30 minutes at 37°C, then washed and rested in complete medium at 37°C for another 30 minutes. 1.0 x 10 ⁵ mitomycin C-treated CD4+ T cells and 0.5 x 10 ⁵ purified B cells (per well) were cocultured in a final volume of 200 μl with anti-CD3/anti-CD28 beads (T-ratio lymphocytes and granules 5:1). For all experiments, the culture medium was RPMI 1640 (Invitrogen) supplemented with 10% FBS, penicillin-streptomycin (100 units/ml penicillin, 100 μg/ml streptomycin), 2-mercaptoethanol (55 μM), L-glutamine (2 mM ) and HEPES (5 mM). Anti-IL-21 antibodies were added to cultures at a concentration of 0-200 nM. After 7 days of culture, the number of plasma cells was determined by flow cytometry. Cells were washed (PBS/2% BSA) and stained for 30 minutes at 4 degrees with anti-IgD (FITS or PE); anti-CD38 (allophiocakin, clone HB7) (BD Pharmingen) and analyzed on a flow cytometer using FACSDiva Software. For all conditions, data were collected at the same time, so that the number of events shown reflects the relative number of cells. However, the total cell number was determined using AccuCount Particles (Spherotech).

[00286] Co-culture of B cells with activated CD4+ T cells resulted in a population of IgD ^- CD38 ^hi PC plasma cells, with 68.5% of CD19+ cells expressing this plasma cell phenotype by day 7 (Figure 6A). Addition of 19E3.1 or 9F11.1 resulted in inhibition of plasmacytic differentiation in a dose-dependent manner, with each antibody achieving a maximum inhibition of 80% and 78%, respectively (Figures 6B and 6C). These data indicate that anti-21 clones can significantly block plasma cell differentiation induced by IL-21 produced by newly activated T lymphocytes.

Anti-IL-21 clone suppresses the proliferation of “naive” CD4+ T-lymphocytes

[00287] IL-21 has been shown to support CD4+ T lymphocyte proliferation under suboptimal initiation conditions. Naïve human CD4+ T lymphocytes were purified and stimulated in vitro with anti-CD3 and IL-21 in the presence or absence of the parent antibody 19E3.1. Proliferation was assessed by measuring ATP accumulation after four days in culture. As previously reported, the amount of ATP detected is directly proportional to the number of metabolically active cells in culture. 19E3.1 attenuated IL-21-induced proliferation (mean IC50=61.02 pM for two donors) in a dose-dependent manner. These data show that the parent antibody K44VHa-N56Q (MEDI7169) 19E3.1 suppresses IL-21-triggered proliferation in naïve human CD4+ T lymphocytes (Figure 7).

Pharmacokinetics

[00288] The pharmacokinetics (PK) of K44VHaN56Q, a YTE antibody, was evaluated in three studies using monkeys as an animal model, which allows PK to be predicted with reasonable accuracy (Oitate et al, Drug Metab. Pharmacokinet. 26 (4): 423-430 (2011)). To predict PK in humans, scaled from animal data, the main parameter is clearance; a lower clearance value indicates that the molecule cannot remain in the body. Cl values for K44VHaN56Q in monkeys range from 1.08 to 4.37 ml/d/kg. Typical therapeutic antibodies such as Bevacizumab (Avastin®), Daclizumab (Zenapax®), Infliximab (Remicade®) and Rituximab (Rituxan®) are associated with corresponding Cl values of 5.78, 5.30, 11.5 and 17.0 (ml/d/kg) (Dong et al, Clin Pharmacokinet. 50(2):131-142 (2011). The monkey's body weight is assumed to be 3 kg). The indicated Cl values are greater than the calculated values for K44VHaN56Q. Therefore, K44VHaN56Q is expected to remain in the body longer than a typical therapeutic antibody.

Example 4 Prevention of graft-versus-host disease (GVHD) using anti-IL-21 monoclonal antibodies

Mouse model of xenogeneic GVHD

[00289] GVHD develops when transferred (donor) T lymphocytes recognize foreign antigens presented by MHC/HLA (major histocompatibility complex/human leukocyte antigen) molecules of the host (recipient). NOD/SCID/IL-2 receptor γ-chain-deficient (NSG) mice lack mature T and B lymphocytes, functional NK cells, and macrophages. These mice exhibit decreased cytokine signaling due to loss of the γc receptor.

[00290] In the xenogeneic GVHD model, human PBMCs are transferred into NSG recipient mice. Because mice are immunodeficient, human cells are not rejected by the mouse's immune system. Human cells recognize the environment of the mouse body as foreign, become activated and proliferate. Activated human cells produce a “cytokine storm” that leads to a strong immunoreactive cascade that affects the liver, gastrointestinal tract and skin, ultimately leading to the death of the animal. A large number of cells capable of producing IL-21 can be isolated from the blood of mice with GVHD.

[00291] Thirty-five female NSG mice (NOD.Cg-PrkdcscidIl2rgtmlWjl/SzJ; The Jackson Laboratories, Bar Harbor, Maine) were used in the prophylaxis study. At the beginning of the study, the mice were 5-7 weeks old and weighed 16.8-22.4 grams.

Isolation of human peripheral blood

[00292] Human blood was collected from two healthy donors into cell preparation tubes (CPTs) (Becton Dickinson, San Jose, California) containing sodium heparin. Mononuclear cells were isolated from CPT tubes according to the manufacturer's instructions. Briefly, tubes were centrifuged at room temperature for 25 minutes at 1700 × g. After centrifugation, mononuclear cells were collected. Cells were washed twice in PBS by centrifugation for 10 minutes at a relative centrifugal force of 400 x g. Cells were then counted and resuspended in the appropriate amount of PBS and stored at room temperature until injection (immediately after the last wash).

Antibodies

[00293] In this example, human IgG1κ-YTE antibodies (NIP-228-YTE; MedImmune, Cambridge, UK) were used as control monoclonal antibodies. The control monoclonal antibody was aliquoted and frozen in matrix solution at -80°C. The formulation buffer was a solution of 25 mM histidine, 7% sucrose, 0.02% (wt/vol) polysorbate 80, at pH 6.0. The injection solution was prepared by diluting an aliquot of the matrix solution (60.6 mg/mL) with phosphate-buffered saline (PBS) to a concentration of 1 mg/mL. Working preparations were stored at 4-8°C.

[00294] K44VHa-N56Q (MEDI7169) was stored at -80°C as an aliquoted and frozen matrix solution. The formulation buffer was a solution of 25 mM histidine, 7% sucrose, 0.02% (wt/vol) polysorbate 80, at pH 6.0. The injection solution was prepared by diluting an aliquot of the matrix solution (47.6 mg/mL) with phosphate-buffered saline (PBS) to a concentration of 1 mg/mL. Working preparations were stored at 4-8°C.

Human cell administration, treatment and evaluation of GVHD

[00295] Administration was performed as shown in Table 6. Briefly, 12.71×10 ⁶ purified human PBMCs from donor 2 were administered intraperitoneally to 5-7 week old female NSG mice on day 0. Prior to administration of PBMCs, starting on day 1- On the next day, three times a week, two hundred micrograms (in a volume of 200 μl) of K44VHa-N56Q (MEDI7169), a control monoclonal antibody, or the same volume of PBS alone (base) were injected intraperitoneally.

[00296] Body weight was measured individually throughout the study, at least once a week. Mice were sacrificed when they showed a 20% loss of body weight, or if the mice were emaciated/agonized or in pain or other distress. Death and humane euthanasia have been used interchangeably for purposes of tracking survival.

Assessing the number and phenotype of T lymphocytes by flow cytometry

[00297] Mice were bled from the retro-orbital sinus and placed in microtubes containing heparin. In all experiments, 100 μl of blood was used at all time points. The erythrocyte lysis step was performed using ASA lysis buffer (Life Technologies, Grand Island, New York), the remaining cells were incubated with TruStain Fc blocker (BioLegend, San Diego, California), in a ratio of 1:4, 10 minutes, then stained for 30 min at 4°C with the following antibody complex to visualize CD4 and CD8 T lymphocytes: V500-conjugated anti-human CD45 antibody (clone HI130; BioLegend, San Diego, CA), AF700-conjugated anti-human CD45 antibody Human CD4 (clone RPA-T4; BioLegend, CA) and BV711-conjugated anti-human CD8 antibody (clone RPA-T8; BioLegend, San Diego, CA). Samples were washed, fixed in 2% formaldehyde, and run through a Becton Dickinson LSR II flow cytometer using the FACSDiva computer program. Samples were analyzed using FlowJo software (version 9). Human cells were identified as cells that expressed the human CD45 antigen. Stained blood (100 μl) for all conditions was collected on LSRII in the same volume, so the number of events shown reflected the relative number of cells for different treatments.

Evaluation of human cytokines in serum collected from treated mice using multiplex ELISA

[00298] Fractional serum samples were collected and analyzed using a human Th17 multiplex kit (magnetic beads, premixed) (Merck Millipore, Billerica, Massachusetts) on a Luminex 200 analyzer according to the manufacturer's instructions.

[00299] GraphPad Prism (version 6.0d for Mac OS X) was used. Cytokine results in mice treated with K44VHa-N56Q (MEDI7169) were compared with those in mice treated with a control monoclonal antibody (Donor 1) or a control monoclonal antibody and PBS (backbone) (Donor 2). These data were analyzed using a one-sample t test with Welsh correction if the estimated spread was not significantly different by the F test, and then analysis was performed using Mann-Whitney tests. P<0.05 for all significant differences.

K44VHa-N56Q (MEDI7169) Suppresses Exogenous GVHD-Induced T Cell Depletion and Expansion

[00300] IL-21 has been reported to affect T-lymphocyte function. Thus, the ability of K44VHa-N56Q (MEDI7169) to suppress the development of xenogeneic GVHD, which is mainly mediated by CD4+ T lymphocytes, was studied. Human PBMCs were administered to immunodeficient NSG mice as described above, and by day 21 wasting disease was observed in mice that received control monoclonal antibody (human IgG1 monoclonal antibody) (Figure 8A) or PBS vehicle. Mice that received 200 μg of K44VHa-N56Q (MEDI7169) three times a week starting on day 1 appeared healthy (Figure 8B) and were indistinguishable from control mice that did not receive human cells. Blood analysis revealed that mice that received the control monoclonal antibody (or PBS) had expanded human CD45+ cells (Figure 8C). In comparison, mice that received K44VHa-N56Q (MEDI7169) showed a significantly reduced presence of human CD45+ cells in the blood (Figure 8D). In mice treated with the control monoclonal antibody, this expansion of human CD45+ cells included mainly CD4-positive cells (Figure 8E), while the percentage of CD4-positive cells was significantly reduced in mice treated with K44VHa-N56Q (MEDI7169), as observed in Day 35 (Figure 8F). These results are consistent with the suppression by K44VHa-N56Q (MEDI7169) of the proliferation of CD4+ T-lymphocytes after transplantation of human PBMCs into immunodeficient mice.

[00301] When mice were monitored over time, it was noted that animals treated with PBS vehicle or a control monoclonal antibody showed a consistent increase in the number of CD45+ cells. However, these cells did not expand in mice that received K44VHa-N56Q (MEDI7169) (Figure 9). Importantly, it was shown that approximately 3 weeks after cessation of K44VHa-N56Q (MEDI7169) administration, CD45+ cells began to proliferate, and then decreased again upon resumption of treatment (Figure 9). These data demonstrate that K44VHa-N56Q (MEDI7169) can significantly suppress T cell proliferation, and this effect is reversible when treatment is stopped.

K44VHa-N56Q (MEDI7169) prevents GVHD-induced weight loss and death

[00302] Weight loss and survival were monitored in mice that were not treated with human cells (naive) or that were treated with human PBMC backed with PBS, a control monoclonal antibody, or K44VHa-N56Q (MEDI7169) starting on one day (Day -1) before administration of human PBMCs to mice. It was shown that mice treated with PBS or a control monoclonal antibody rapidly lost weight (Figure 10A) and either died or were sacrificed when body weight had decreased by 20%. All mice (n=16) treated with PBS or control monoclonal antibody in two different studies either died or were sacrificed by study day 37 after administration of human PBMC (Figure 10B). In contrast, all mice (n=10) treated with K44VHa-N56Q (MEDI7169) survived and remained healthy until approximately the cessation of K44VHa-N56Q (MEDI7169) administration (Figure 10B). In one experiment, mice remained healthy for approximately 20 days after cessation of K44VHa-N56Q (MEDI7169) administration and then rapidly deteriorated and died or were sacrificed shortly after day 31 of the study (Figure 10B). In a separate study using PBMC from a second donor, mice remained viable for approximately 40 days after cessation of K44VHa-N56Q (MEDI7169) administration. These data demonstrate that K44VHa-N56Q (MEDI7169) protects against mortality and that this effect is reversible when treatment is stopped.

K44VHa-N56Q (MEDI7169) inhibits the production of several cytokines. and this action is reversible

[00303] Mice receiving cells from Donor 1 (Figure 11) and Donor 1 (Figure 12) had serum collected at different time points and analyzed for the expression of 25 cytokines using the Millipore MILLIPLEX MAP system. More than half of the cytokines studied were below the detection level. For detectable cytokines, K44VHa-N56Q (MEDI7169) caused significant reductions in GM-CSF (granulocyte-macrophage-colony-stimulating factor), IL-10, and TNF (tumor necrosis factor) in two separate experiments. IFNγ was significantly reduced in one experiment and showed a downward trend in the second experiment. It was shown that IL-17A and IL-9 were significantly reduced in one of the two experiments. Both experiments showed a significant increase in IL-5 levels (Figure 11). Clinical blood count (CBC) was performed using a Sysmex XT-2000iv hematology analyzer, and it is important to note that mice with elevated IL-5 levels did not have increased eosinophil counts. It was shown that IL-+21 was increased in both experiments. The increase in IL-21 levels is consistent with results obtained in a toxicity study in cynomolgus monkeys, in which increased IL-21 levels reflected the amount of circulating drug that bound to and neutralized the cytokine.

[00304] As shown in Figure 9, K44VHa-N56Q (MEDI7169) suppressed the expansion of CD4+ T lymphocytes, and this effect was reversible when the drug was stopped. This was confirmed in the second study (Figure 12). Notably, several cytokines were also increased following cell proliferation, including TNF, IFNγ, and IL-10 (Figure 12). These data indicate that the anti-IL-21 antibody, namely K44VHa-N56Q (MEDI7169), suppresses T-lymphocyte expansion and cytokine production, and both effects are reversible when administration of K44VHa-N56Q (MEDI7169) is stopped.

Example 5 Treatment of graft-versus-host disease (GVHD) using anti-IL-21 monoclonal antibodies

Experimental design

[00305] Twenty female NSG mice (NOD.Cg-Prkdcscid Il2rgtmlWjl/SzJ; The Jackson Laboratories, Bar Harbor, Maine) participated in the therapeutic effect study. At the beginning of the study, the mice were 8 weeks old and weighed 18.00-22.3 grams.

[00306] Human blood was collected and prepared as described above in Example 4.

[00307] Control and test monoclonal antibodies were prepared, processed and stored as described above in Example 4.

[00308] Administration was performed as shown in Table 7. Briefly, 12 x 10 ⁶ purified human PBMCs from donor 1 were administered intraperitoneally to 8-week-old female NSG mice on day 0. Beginning on day 7, three times per week i.p. two hundred micrograms (in a 200 µl volume) of K44VHa-N56Q (MEDI7169) or control monoclonal antibody. The other group was administered K44VHa-N56Q (MEDI7169) (200 μg in 200 μl) intravenously three times a week starting on day 14.

[00309] Assessment of GVHD, T cell count and phenotype, and human serum cytokines was performed as described above in Example 4.

Automatic evaluation of clinical blood test

[00310] A Sysmex XT-2000iv automated hematology analyzer (Sysmex America, Mandeley, IL) was used to evaluate total white blood cell count, as well as red blood cell count, hematocrit, and hemoglobin. On days 7, 14, and 21, mice were bled from the retroorbital sinus into a microtube containing heparin. A 1:10 dilution was made (20 μl of each whole blood sample was diluted in 180 μl of PBS), then the samples were injected into the analyzer, and the data obtained were multiplied by a dilution factor of ten.

Therapeutic intervention of K44VHa-N560 (MEDI7169) protects mice from GVHD

[00311] To examine whether the antibody K44VHa-N56Q (MEDI7169), administered for therapeutic purposes, was able to protect mice from GVHD, human PBMCs were administered as described above, and K44VHa-N56Q (MEDI7169) was administered 7 or 14 days after cell injection person. As shown in Figure 13, mice that received the control monoclonal antibody began to die in the first two weeks after administration of human cells, and all died by the 26th day. Mice that received K44VHa-N56Q (MEDI7169) on day 7 were completely protected from GVHD-induced death. Mice that received K44VHa-N56Q (MEDI7169) on day 14 (after control mice began to die) were partially protected; 2 of 5 mice in the day 14 group survived after day 26, when all control mice had already died. Mice treated with human cells and a control monoclonal antibody on day 7 developed severe GVHD-induced anemia. Mice that received K44VHa-N56Q (MEDI7169) from day 7 were able to maintain higher normal red blood cell counts, hematocrit and hemoglobin, which were closer to those of intact animals that did not receive cells (Figure 14).

[00312] These data demonstrate that the anti-IL-21 antibody, namely K44VHa-N56Q, protected mice from GVHD by reducing or eliminating symptoms associated with the disease.

[00313] The foregoing description of specific embodiments is intended to fully demonstrate the general nature of the invention, which others can, by applying knowledge within the scope of those skilled in the art, readily modify and/or adapt to different applications, such as specific embodiments, without unnecessary experiments without deviating from the general concept of the present invention. Therefore, such adaptations and modifications are intended to fall within the meaning and range of equivalents of the disclosed embodiments based on the teachings and guidance presented herein. It should be understood that the language and terminology used herein are intended to be descriptive and not limiting, so that the terminology and language used herein should be interpreted by those skilled in the art in light of the teachings and guidance. The present invention is further described in the following claims.

--->

LIST OF SEQUENCES

<110> MEDIMMUNE, LLC

<120> BINDING MOLECULES SPECIFIC TO IL-21 AND AREAS OF THEIR APPLICATION

<130>IL21-100P1

<160> 65

<170> PatentIn, ver 3.5

<210> 1

<211> 564

<212> DNA

<213> Homo sapiens

<400> 1

ctgaagtgaa aacgagacca aggtccagct ctactgttgg tacttatgag atccagtcct 60

ggcaacatgg agaggattgt catctgtctg atggtcatct tcttggggac actggtccac 120

aaatcaagct cccaaggtca agatcgccac atgattagaa tgcgtcaact tatagatatt 180

gttgatcagc tgaaaaatta tgtgaatgac ttggtccctg aatttctgcc agctccagaa 240

gatgtagaga caaactgtga gtggtcagct ttttcctgtt ttcagaaggc ccaactaaag 300

tcagcaaata caggaaacaa tgaaaggata atcaatgtat caattaaaaa gctgaagagg 360

aaaccacctt ccacaaatgc agggagaaga cagaaacaca gactaacatg cccttcatgt 420

gattcttatg agaaaaaacc acccaaagaa ttcctagaaa gattcaaatc acttctccaa 480

aagatgattc atcagcatct gtcctctaga acacacggaa gtgaagattc ctgaggatct 540

aacttgcagt tggacattgt taca 564

<210> 2

<211> 161

<212> Protein

<213> Homo sapiens

<400> 2

Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met

1 5 10 15

Val Ile Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln

20 25 30

Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln

35 40 45

Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro

50 55 60

Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln

65 70 75 80

Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile

85 90 95

Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala

100 105 110

Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Glu

115 120 125

Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln

130 135 140

Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp

145 150 155 160

Ser

<210> 3

<211> 489

<212> DNA

<213> Macaca fascicularis

<400> 3

atgagatcca gtcctggcaa catggagagg atagtcatct gtctgatggt catcttcttg 60

gggacactgg tccacaaatc aagctcccaa ggtcaagatc gccacatgat tagaatgcgt 120

caacttatag atattgttga tcagctgaaa aattatgtga atgacttgga ccctgaattt 180

ctgccagctc cagaagatgt agagacaaac tgtgagtggt cagctatttc ctgttttcag 240

aaggcccaac taaagtcagc aaatacagga aacaatgaaa ggataatcaa tttatcaatt 300

aaaaagctga agaggaaatc accttccaca ggtgcagaga gaagacagaa acacagacta 360

acatgccctt catgtgattc ttatgagaaa aaaccaccca aagaattcct agaaagattc 420

aaatcacttc tccaaaagat gattcatcag catctgtcct ctagaacaca tggaagtgaa 480

gattcctga 489

<210> 4

<211> 162

<212> Protein

<213> Macaca fascicularis

<400> 4

Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met

1 5 10 15

Val Ile Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln

20 25 30

Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln

35 40 45

Leu Lys Asn Tyr Val Asn Asp Leu Asp Pro Glu Phe Leu Pro Ala Pro

50 55 60

Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Ile Ser Cys Phe Gln

65 70 75 80

Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile

85 90 95

Asn Leu Ser Ile Lys Lys Leu Lys Arg Lys Ser Pro Ser Thr Gly Ala

100 105 110

Glu Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr

115 120 125

Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu

130 135 140

Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu

145 150 155 160

Asp Ser

<210> 5

<211> 357

<212> DNA

<213> Mus sp.

<400> 5

gaagtggagc ttgtggagtc tggaggaggc ttggtgcaac ctggaggatc catgaaactc 60

tcttgtgctg cctctggatt cacttttagt gacgcctgga tggactgggt ccgccagtct 120

ccagagaagg ggcttgagtg ggttgctgaa attagaagcg aagctgataa tcatccaaca 180

tactatgctg agtctgtgaa agggaggttc accatctcaa gagatgattc caaaagtagt 240

gtctacctgc aaatgaacag cttaagagct gaagacagtg gcatttatta ctgtactgaa 300

tatgattacg aggggtttgt acactggggc caggggacgc tggtcactgt ctctgca 357

<210> 6

<211> 116

<212> Protein

<213> Mus sp.

<400> 6

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Thr Ile Asp Pro Ser Asp Asn Tyr Thr Ile Tyr Ser Gln Asn Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 7

<211> 5

<212> Protein

<213> Mus sp.

<400> 7

Asp Tyr Trp Met His

15

<210> 8

<211> 17

<212> Protein

<213> Mus sp.

<400> 8

Thr Ile Asp Pro Ser Asp Asn Tyr Thr Ile Tyr Ser Gln Asn Phe Lys

1 5 10 15

Gly

<210> 9

<211> 7

<212> Protein

<213> Mus sp.

<400> 9

Tyr Gly Phe Ala Met Asp Tyr

15

<210> 10

<211> 321

<212> DNA

<213> Mus sp.

<400> 10

gatatccagc tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagc aattttttaa actggtatca gcagacacca 120

gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtcatacgc ttcctcggac gttcggtgga 300

ggcaccaagc tggaaatcaa a 321

<210> 11

<211> 107

<212> Protein

<213> Mus sp.

<400> 11

Asp Ile Gln Leu Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Thr Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly His Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 11

<212> Protein

<213> Mus sp.

<400> 12

Arg Ala Ser Gln Asp Ile Ser Asn Phe Leu Asn

1 5 10

<210> 13

<211> 7

<212> Protein

<213> Mus sp.

<400> 13

Tyr Thr Ser Arg Leu His Ser

15

<210> 14

<211> 9

<212> Protein

<213> Mus sp.

<400> 14

Gln Gln Gly His Thr Leu Pro Arg Thr

15

<210> 15

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic sequence description: K2Ha-N56Q, synthetic

humanized VH region sequence

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Thr Ile Asp Pro Ser Asp Gln Tyr Thr Ile Tyr Ser Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 16

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic sequence description: K2Ha-N56Q, synthetic

humanized VH+ human CH region sequence

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Thr Ile Asp Pro Ser Asp Gln Tyr Thr Ile Tyr Ser Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

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

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 17

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic sequence description: K2Ha-N56Q, synthetic

humanized VH region sequence

<400> 17

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly His Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 18

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic sequence description: K2Ha-N56Q, synthetic

humanized VL sequence + human CL region

<400> 18

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly His Thr Leu Pro Arg

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 19

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic sequence description: K44VHa-N56Q, synthetic

humanized VH region sequence

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Thr Ile Asp Pro Ser Asp Gln Tyr Thr Ile Tyr Ser Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 20

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic sequence description: K44VHa-N56Q, synthetic

sequence of the humanized VH human CH region

<400> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Thr Ile Asp Pro Ser Asp Gln Tyr Thr Ile Tyr Ser Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

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

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 21

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic sequence description: K44VHa-N56Q, synthetic

humanized VL region sequence

<400> 21

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 22

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic sequence description: K44VHa-N56Q, synthetic

humanized VL + human CL region sequence

<400> 22

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Arg

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 23

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: K44VHa-6N56Q, synthetic

humanized VH region sequence

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Thr Ile Asp Pro Ser Asp Gln Tyr Thr Ile Tyr Ser Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Val Asp Glu Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 24

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: K44VHa-6N56Q, synthetic

sequence region humanized VH + human CH

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Thr Ile Asp Pro Ser Asp Gln Tyr Thr Ile Tyr Ser Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Val Asp Glu Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

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

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 25

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: K44VHa-6N56Q, synthetic

humanized VL region sequence

<400> 25

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 26

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: K44VHa-6N56Q, synthetic

sequence humanized VL + human CL region

<400> 26

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Arg

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 27

<211> 357

<212> DNA

<213> Mus sp.

<400> 27

gaagtggagc ttgtggagtc tggaggaggc ttggtgcaac ctggaggatc catgaaactc 60

tcttgtgctg cctctggatt cacttttagt gacgcctgga tggactgggt ccgccagtct 120

ccagagaagg ggcttgagtg ggttgctgaa attagaagcg aagctgataa tcatccaaca 180

tactatgctg agtctgtgaa agggaggttc accatctcaa gagatgattc caaaagtagt 240

gtctacctgc aaatgaacag cttaagagct gaagacagtg gcatttatta ctgtactgaa 300

tatgattacg aggggtttgt acactggggc caggggacgc tggtcactgt ctctgca 357

<210> 28

<211> 119

<212> Protein

<213> Mus sp.

<400> 28

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

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Glu Ala Asp Asn His Pro Thr Tyr Tyr Ala Glu

50 55 60

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

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ser Gly Ile Tyr

85 90 95

Tyr Cys Thr Glu Tyr Asp Tyr Glu Gly Phe Val His Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 29

<211> 5

<212> Protein

<213> Mus sp.

<400> 29

Asp Ala Trp Met Asp

15

<210> 30

<211> 19

<212> Protein

<213> Mus sp.

<400> 30

Glu Ile Arg Ser Glu Ala Asp Asn His Pro Thr Tyr Tyr Ala Glu Ser

1 5 10 15

Val Lys Gly

<210> 31

<211> 8

<212> Protein

<213> Mus sp.

<400> 31

Tyr Asp Tyr Glu Gly Phe Val His

15

<210> 32

<211> 333

<212> DNA

<213> Mus sp.

<400> 32

gacattgtgc tgacacagtc tcctgcttca ctagctgtat ctctggggca gagggccacc 60

atctcatgca gggccagcaa aagtgtcagt acatatggct atagttatat gcactggtac 120

caacagaaac caggacagcc acccaaactc ctcatctatc ttgcatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcaac acagtaggga gcttccgctc 300

acgttcggtg ctgggaccaa gctggagctg aag 333

<210> 33

<211> 111

<212> Protein

<213> Mus sp.

<400> 33

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

1 5 10 15

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

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 34

<211> 15

<212> Protein

<213> Mus sp.

<400> 34

Arg Ala Ser Lys Ser Val Ser Thr Tyr Gly Tyr Ser Tyr Met His

1 5 10 15

<210> 35

<211> 7

<212> Protein

<213> Mus sp.

<400> 35

Leu Ala Ser Asn Leu Glu Ser

15

<210> 36

<211> 9

<212> Protein

<213> Mus sp.

<400> 36

Gln His Ser Arg Glu Leu Pro Leu Thr

15

<210> 37

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic sequence description: Hu-9F11 VH-4, VH-11, synthetic

humanized VH region sequence

<400> 37

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Ser Glu Ala Asp Asn His Pro Thr Tyr Tyr Ala Glu

50 55 60

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

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Glu Tyr Asp Tyr Glu Gly Phe Val His Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 38

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Hu-9F11 VH-6, synthetic

humanized VH region sequence

<400> 38

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Ser Glu Ala Asp Asn His Pro Thr Tyr Tyr Ala Glu

50 55 60

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

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Glu Tyr Asp Tyr Glu Gly Phe Val His Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 39

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic sequence description: Hu-9F11 VL, VL-4, VL-6, synthetic

humanized VL region sequence

<400> 39

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Tyr

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 40

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic sequence description: Hu-9F11 VL-11, synthetic

humanized VL region sequence

<400> 40

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Tyr

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 41

<211> 363

<212> DNA

<213> Mus sp.

<400> 41

gaagtgcagc tggtggagtc tgggggaggc tcagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggact cactttcagt cactattaca tgtattgggt tcgccagacg 120

ccggaaaaga ggctggagtg ggtcgcaatc attagtgatg gtggtgatta cacccactac 180

tcagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caacctgtac 240

ctacaaatga gcagtctgca gtctgaggac acagccatgt attactgtgc aggagattac 300

ggatatggtt acgaggggtg gtttgcttac tggggccaag ggactctggt cactgtctct 360

aca 363

<210> 42

<211> 121

<212> Protein

<213> Mus sp.

<400> 42

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ile Ile Ser Asp Gly Gly Asp Tyr Thr His Tyr Ser Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Ser Ser Leu Gln Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Gly Asp Tyr Gly Tyr Gly Tyr Glu Gly Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Thr

115 120

<210> 43

<211> 5

<212> Protein

<213> Mus sp.

<400> 43

His Tyr Tyr Met Tyr

15

<210> 44

<211> 17

<212> Protein

<213> Mus sp.

<400> 44

Ile Ile Ser Asp Gly Gly Asp Tyr Thr His Tyr Ser Asp Ser Val Lys

1 5 10 15

Gly

<210> 45

<211> 12

<212> Protein

<213> Mus sp.

<400> 45

Asp Tyr Gly Tyr Gly Tyr Glu Gly Trp Phe Ala Tyr

1 5 10

<210> 46

<211> 321

<212> DNA

<213> Mus sp.

<400> 46

gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc 60

gtcacctgca aggccagtca gaatgtggat acttatgtag cctggtatca acagaaacca 120

gggcaatctc ctaaagcact gatttattcg acatcctatc ggtacagtgg agtccctgat 180

cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagtaa tgtgcagtct 240

gaagacttgg cagagtattt ctgtcagcaa tataacagct atccgtggac gttcggtgga 300

ggcactaagc tggaaatcaa a 321

<210> 47

<211> 107

<212> Protein

<213> Mus sp.

<400> 47

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 48

<211> 11

<212> Protein

<213> Mus sp.

<400> 48

Lys Ala Ser Gln Asn Val Asp Thr Tyr Val Ala

1 5 10

<210> 49

<211> 7

<212> Protein

<213> Mus sp.

<400> 49

Ser Thr Ser Tyr Arg Tyr Ser

15

<210> 50

<211> 9

<212> Protein

<213> Mus sp.

<400> 50

Gln Gln Tyr Asn Ser Tyr Pro Trp Thr

15

<210> 51

<211> 360

<212> DNA

<213> Mus sp.

<400> 51

gaggtccagc tgcaacagtt tggagctgag gtggtgaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggcta cacattcact gactacaaca tgaactgggt gaagcagagc 120

catgaaaaga gccttgagtg gattggagat attcatccta agtttcatag tactaactac 180

aaccagaaat tcaagggaaa ggccacattg actatagaca cgtcctccag cacagcctac 240

atggagctcc gcaacctgac atctgaggac actgcagtct attactgtgc acgagttggt 300

aactacgttt actttgttat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360

<210> 52

<211> 120

<212> Protein

<213> Mus sp.

<400> 52

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

1 5 10 15

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

20 25 30

Asn Met Asn Trp Val Lys Gln Ser His Glu Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asp Ile His Pro Lys Phe His Ser Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ile Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Val Gly Asn Tyr Val Tyr Phe Val Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 53

<211> 5

<212> Protein

<213> Mus sp.

<400> 53

Asp Tyr Asn Met Asn

15

<210> 54

<211> 10

<212> Protein

<213> Mus sp.

<400> 54

Asp Tyr Asn Met Asn Asp Tyr Asn Met Asn

1 5 10

<210> 55

<211> 11

<212> Protein

<213> Mus sp.

<400> 55

Val Gly Asn Tyr Val Tyr Phe Val Met Asp Tyr

1 5 10

<210> 56

<211> 321

<212> DNA

<213> Mus sp.

<400> 56

agtattgtga tgacccagac tcccaaattc ctgcttgtat cagcaggaga cagggttact 60

ataacctgca aggccagtca gagtgtgagt aatgatgtag cttggtacca acagaagcca 120

gggcagtctc ctaaactcct gatatactat gcatccaatc gctacactgg agtccctgat 180

cgcttcagtg gcagtggata tgggacggat ttcactttca ccatcagcac tgtgcaggct 240

gaagacctgg cagtttattt ctgtcagcag gattatacct ctccgtggac gttcggtgga 300

ggcaccaagc tggaaatcaa a 321

<210> 57

<211> 107

<212> Protein

<213> Mus sp.

<400> 57

Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 58

<211> 11

<212> Protein

<213> Mus sp.

<400> 58

Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala

1 5 10

<210> 59

<211> 7

<212> Protein

<213> Mus sp.

<400> 59

Tyr Ala Ser Asn Arg Tyr Thr

15

<210> 60

<211> 9

<212> Protein

<213> Mus sp.

<400> 60

Gln Gln Asp Tyr Thr Ser Pro Trp Thr

15

<210> 61

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

synthetic humanized variant of the VH 19E3 sequence

<400> 61

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Thr Ile Asp Pro Ser Asp Asn Tyr Thr Ile Tyr Ser Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 62

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

sequence of the synthetic humanized variant VH 19E3

<400> 62

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Thr Ile Asp Pro Ser Asp Asn Tyr Thr Ile Tyr Ser Gln Asn Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 63

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

sequence of the synthetic humanized variant of VH 19E3

<400> 63

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Thr Ile Asp Pro Ser Asp Asn Tyr Thr Ile Tyr Ser Gln Asn Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 64

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

sequence of the synthetic humanized variant VL 19E3

<400> 64

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 65

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

sequence of the synthetic humanized variant VL 19E3

<400> 65

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Phe

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly His Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<---

Claims (22)

1. An antibody or antigen-binding fragment thereof that specifically binds to IL-21, containing a heavy chain variable region (VH) and a light chain variable region (VL), where: (a) CDR1, CDR2 and CDR3 of the specified heavy chain variable region (VH) contain SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively; And (b) CDR1, CDR2 and CDR3 of the light chain variable region (VL) contain SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, respectively. 2. Antibody or antigen-binding fragment thereof according to claim 1, characterized in that (a) VH contains the amino acid sequence of SEQ ID NO: 37; And (b) VL contains the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40. 3. Antibody or antigen-binding fragment thereof according to claim 1, characterized in that (a) VH contains the amino acid sequence of SEQ ID NO: 38; And (b) VL contains the amino acid sequence of SEQ ID NO: 39. 4. Antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-3, characterized in that said antibody or antigen-binding fragment contains an IgG constant domain. 5. Antibody or antigen-binding fragment thereof according to claim 4, characterized in that an IgG constant domain contains one or more amino acid substitutions that increase the half-life of an antibody or antigen-binding fragment compared to the half-life of an antibody or antigen-binding fragment containing a wild-type IgG constant domain. 6. The antibody or antigen-binding fragment thereof according to claim 4 or 5, characterized in that the IgG constant domain is human IgG1. 7. Antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-6, characterized in that said antibody is a mouse antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, or a recombinant antibody. 8. Antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-7, characterized in that said antigen-binding fragment is Fv, Fab, F(ab')2, Fab', dsFv, scFv and sc(Fv)2. 9. A conjugate for the treatment or prevention of a disease or disorder associated with IL-21, containing an antibody or antigen-binding fragment according to any one of paragraphs. 1-8, conjugated with an antimicrobial agent, therapeutic agent, prodrug, peptide, protein, enzyme, lipid, biological response modifier, pharmaceutical agent, lymphokine, antibody or fragment thereof, detectable label, polyethylene glycol (PEG). 10. The conjugate according to claim 9, wherein said conjugate inhibits the differentiation of CD4+ T lymphocyte-activated B lymphocytes into plasma cells. 11. A pharmaceutical composition for the treatment or prevention of a disease or disorder associated with IL-21, containing an antibody or antigen-binding fragment according to any one of paragraphs. 1-8 or the conjugate of claim 9, and one or more pharmaceutically acceptable carrier or diluent. 12. A method of treating or preventing a disease or disorder associated with IL-21 in a subject in need thereof, comprising administering to said subject an effective amount of an antibody or antigen binding fragment according to any one of claims. 1-8, the conjugate of claim 9, or the pharmaceutical composition of claim 10, wherein said IL-21-associated disease or disorder is an autoimmune disease or graft-versus-host disease (GVHD) associated with IL-21 . 13. The method of claim 12, wherein said IL-21-associated disease or disorder is an autoimmune disease selected from the group consisting of Sjogren's syndrome (SS), systemic sclerosis, ANCA-associated vasculitis (AAV) , giant cell arteritis (GCA) vasculitis, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis (RA), Crohn's disease, myasthenia gravis, pemphigus vulgaris, idiopathic thrombocytopenic purpura (ITP), type I diabetes, IgG4-related disease or any of these combinations. 14. The method according to claim 13, characterized in that said autoimmune disease is systemic lupus erythematosus. 15. The method of claim 12, wherein said IL-21-associated disease or disorder is IL-21-associated graft-versus-host disease (GVHD).