HK1157358A - Interleukin-21 receptor binding proteins - Google Patents

Description

Interleukin-21 receptor binding proteins

RELATED APPLICATIONS

This application claims priority to U.S. provisional patent application No.61/055,500, filed on 23/5/2008, the contents of which are hereby incorporated by reference in their entirety.

Technical Field

The present invention relates to binding proteins that bind to interleukin-21 receptor (IL-21R), particularly human IL-21R, and antigen-binding fragments thereof, and their use in modulating IL-21R-associated activity. The binding proteins disclosed herein are useful in the treatment and/or diagnosis of IL-21R associated disorders such as inflammatory disorders, autoimmune diseases, allergies, transplant rejection, hyperproliferative blood disorders, and other immune system disorders.

Background

The antigen elicits an immune response and activates the largest two populations of lymphocytes: t cells and B cells. Upon encountering antigen, T cells proliferate and differentiate into effector cells, while B cells proliferate and differentiate into antibody-secreting plasma cells. These effector cells secrete and/or respond to cytokines, small proteins (less than about 30kDa) secreted by lymphocytes and other cell types.

Human IL-21 is a cytokine that shows sequence homology to IL-2, IL-4 and IL-15 (Parrish-Novak et al (2000) Nature 408: 57-63). Although the sequence homology between interleukin cytokines is low, the cytokines share a common fold, folding into a "four-helix bundle" structure representative of the family. Most cytokines bind to either class I or class II cytokine receptors. Class II Cytokine receptors include receptors for IL-10 and interferons, while class I Cytokine receptors include receptors for IL-2 through IL-7, IL-9, IL-11, IL-12, IL-13, and IL-15, as well as hematopoietic growth factors, leptin, and growth hormone (Cosman (1993) Cytokine 5: 95-106).

Human IL-21R is a class I cytokine receptor. Nucleotide and amino acid sequences encoding human IL-21 and its receptor (IL-21R) are described in International application publication Nos. WO 00/053761 and WO 01/085792; Parrish-Novak et al (2000) supra; and Ozaki et al (2000) Proc.Natl.Acad.Sci.USA 97: 11439-44. IL-21R shares the highest sequence homology with the beta chain of the IL-2 receptor and the alpha chain of the IL-4 receptor (Ozaki et al (2000) supra). Upon ligand binding, IL-21R associates with a common gamma cytokine receptor chain (yc) shared by the receptor complexes of IL-2, IL-3, IL-4, IL-7, IL-9, IL-13, and IL-15 (Ozaki et al (2000) supra; Asao et al (2001) J.Immunol.167: 1-5).

IL-21R is expressed in lymphoid tissues, particularly on T cells, B cells, Natural Killer (NK) cells, Dendritic Cells (DCs) and macrophages (Parrish-Novak et al (2000) supra), which allows these cells to respond to IL-21(Leonard and Spolski (2005) nat. Rev. Immunol.5: 688-98). The broad lymphoid distribution of IL-21R indicates that IL-21 plays an important role in immune regulation. In vitro studies have shown that IL-21 significantly modulates B cell, CD4⁺And CD8⁺T cells, and NK cells (Parrish-Novak et al (2000) supra; Kasaian et al (2002) Immunity 16: 559-69). Recent evidence suggests that IL-21-mediated signaling may have anti-tumor activity (Sivakumar et al (2004) Immunology 112: 177-82), and that IL-21 can prevent antigen-induced elimination in mice (Shang et al (2006) cell. immunol.241: 66-74).

In autoimmunity, disruption of the IL-21 gene and injection of recombinant IL-21 was shown to regulate the development of Experimental Autoimmune Myasthenia Gravis (EAMG) and Experimental Autoimmune Encephalomyelitis (EAE) (King et al (2004) Cell 117: 265-77; Ozaki et al (2004) J.Immunol.173: 5361-71; Vollmer et al (2005) J.Immunol.174: 2696-2701; Liu et al (2006) J.Immunol.176: 5247-54), respectively. In these experimental systems, manipulation of IL-21-mediated signaling was suggested to directly alter CD8⁺Cell, B cell, T helper cell, and NK cell function.

Summary of The Invention

The present invention describes the isolation and characterization of binding proteins, e.g., human antibodies and fragments thereof, that specifically bind to human and murine IL-21R. The binding proteins described herein are derived from antibody 18a5, which is disclosed in U.S. patent No.7,495,085, incorporated herein by reference in its entirety. The binding proteins of the invention have an affinity for human and/or murine IL-21R to a much greater extent than the parent 18a5 antibody.

The present invention provides, at least in part, IL-21R binding agents (such as binding proteins and antigen binding fragments thereof) that bind IL-21R, particularly human IL-21R, with high affinity and specificity. The binding proteins of the invention and antigen binding fragments thereof are also referred to herein as "anti-IL-21R binding proteins" and "fragments thereof," respectively. In one embodiment, the binding protein or fragment thereof reduces, inhibits, or antagonizes IL-21R activity. Such binding proteins are useful for modulating immune responses or IL-21R-associated disorders by antagonizing IL-21R activity. In other embodiments, anti-IL-21R binding proteins can be used for diagnosis, or as targeting binding proteins for delivery of therapeutic or cytotoxic agents to IL-21R expressing cells. Thus, the anti-IL-21R binding proteins of the invention are useful in the diagnosis and treatment of IL-21R associated disorders, such as inflammatory disorders, autoimmune diseases, allergies, transplant rejection, hyperproliferative disorders, and other immune system disorders.

Thus, in one aspect, the binding protein of the invention features an isolated binding protein (e.g., an isolated antibody) or antigen-binding fragment thereof that binds IL-21R, particularly human IL-21R. In certain embodiments, an anti-IL-21R binding protein (e.g., an antibody) may have one or more of the following characteristics: (1) it is a monoclonal or monospecific binding protein; (2) it is a human binding protein; (3) it is a binding protein produced in vitro; (4) it is a binding protein produced in vivo (e.g., transgenic mouse system); (5) it inhibits IL-21 binding to IL-21R; (6) it is IgG 1; (7) it is at least about 10⁵M^-1s^-1Binds to human IL-21R; (8) it is at least about 5x10⁴M^-1s^-1Binds to murine IL-21R; (9) it has a molecular weight of about 10^-3s^-1Or less dissociation constant binds human IL-21R; (10) it has a molecular weight of about 10^-2s^-1Or less dissociation constant binds murine IL-21R; (11) it has an IC of about 1.75nM or less₅₀Inhibiting human IL-21R-mediated proliferation of human IL-21R-expressing BaF3 cells; (12) it has an IC of about 0.5nM or less₅₀Inhibiting murine IL-21R-mediated proliferation of murine IL-21R-expressing BaF3 cells; (13) it is at about 14.0nM orLess IC₅₀Inhibiting human IL-21R-mediated proliferation of human IL-21R-expressing TF1 cells; (14) it has an IC of about 1.9nM or less₅₀Inhibiting IL-21 mediated proliferation of human primary B cells; (15) it has an IC of about 1.5nM or less₅₀Inhibition of IL-21 mediated human primary CD4⁺T cell proliferation; and (16) it has an IC of about 5.0nM or less₅₀Inhibition of IL-21 mediated murine primary CD4⁺T cells proliferate.

Non-limiting exemplary embodiments of the binding proteins of the invention (the term "binding protein" also includes and refers to antigen-binding fragments thereof as appropriate) are referred to herein as AbA-AbZ, and the relationship of these terms to the terms used in U.S. provisional patent application No.61/055,500 is presented in Table 2A. Other exemplary embodiments of the binding proteins of the invention, i.e., the scfvs, are referred to herein as H3-H6, L1-L6, L8-L21, and L23-L25, as detailed in table 2B.

One embodiment of the invention is an isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises at least one amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 165 and 168, 171 and 193, 213 and 229, 240, 242, 244, 246, and 248. The isolated binding protein or antigen binding fragment may be, for example, an antibody, scFv, V_H、V_LOr a CDR.

Another embodiment of the invention is an isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises at least one amino acid sequence encoded by a nucleotide sequence that is at least about 95% identical to a nucleotide sequence selected from the group consisting of seq id nos: SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 239, 241, 243, 245, and 247.

Yet another embodiment of the invention is an isolated binding protein or antigen-binding fragment comprising at least one amino acid sequence selected from the group consisting of seq id no: SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 165 and 168, 171 and 193, 213 and 229, 240, 242, 244, 246, and 248.

Yet another embodiment of the invention is an isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises at least one amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 195, 213, 229, 240, 242, 244, 246, 248 and 248, and wherein, if the binding protein or antigen binding fragment comprises at least one amino acid sequence that is at least about 95% identical to a sequence selected from the group consisting of: SEQ ID NO: 6, 8, 10, 12, 163, 164, 169, 170, 194, and 195, then the binding protein or antigen-binding fragment must further comprise at least one amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 165 and 168, 171 and 193, 213 and 229, 240, 242, 244, 246, and 248.

Yet another embodiment of the invention is an isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises at least one amino acid sequence encoded by a nucleotide sequence that is at least about 95% identical to a nucleotide sequence selected from the group consisting of seq id nos: SEQ ID NO: 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 159, 161, 239, 241, 243, 245, and 247, and wherein, if the binding protein or antigen binding fragment comprises at least one amino acid sequence encoded by a nucleotide sequence that is at least about 95% identical to a sequence selected from the group consisting of: SEQ ID NO: 5,7, 9, and 11, then the binding protein or antigen-binding fragment must further comprise at least one amino acid sequence encoded by a nucleotide sequence that is at least about 95% identical to a nucleotide sequence selected from the group consisting of seq id no: SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 239, 241, 243, 245, and 247.

Yet another embodiment of the invention is an isolated binding protein or antigen-binding fragment comprising at least one amino acid sequence selected from the group consisting of seq id no: SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 195, 213, 229, 240, 242, 244, 246, 248 and 248, wherein, if the binding protein or antigen binding fragment comprises at least one amino acid sequence that is at least about 95% identical to a sequence selected from the group consisting of: SEQ ID NO: 6, 8, 10, 12, 163, 164, 169, 170, 194, and 195, then the binding protein or antigen-binding fragment must further comprise at least one amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 165 and 168, 171 and 193, 213 and 229, 240, 242, 244, 246, and 248.

Another embodiment of the invention is an isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises a light chain and a heavy chain, and wherein the heavy chain comprises a heavy chain toAt least one amino acid sequence selected from the group consisting of: SEQ ID NO: 14, 16, 18, 20, 68, 70, 72, 88, 90, 92, 94, 213, 218, 219, 240, and 242, or a sequence substantially identical thereto (e.g., a sequence substantially identical thereto includes a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical thereto), or a sequence substantially homologous thereto (e.g., a sequence substantially homologous thereto includes a sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical thereto). In yet another embodiment, the binding protein or antigen binding fragment comprises V_LField and V_HDomain, and the V_HThe domain comprises at least one amino acid sequence selected from the group consisting of: SEQ ID NO: 14, 16, 18, and 20, or sequences substantially identical or homologous thereto. Another embodiment of the invention is an isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises a light chain and a heavy chain, and wherein the light chain comprises at least one amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 74, 76, 78, 80, 82, 84, 86, 96, 98, 100, 102, 104, 106, 108, 214 and 217, 220 and 229, 244, 246, and 248, or sequences substantially identical or homologous thereto. In yet another embodiment, the binding protein or antigen binding fragment comprises V_LField and V_HDomain, and the V_LThe domain comprises at least one amino acid sequence selected from the group consisting of: SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 215, 217, 221, 223, 225, 227, and 229, or sequences substantially identical or homologous thereto. In yet another embodiment, the heavy chain comprises an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO: 88, 90, 92, 94, 213, 218, 219, 240, and 242, and the light chain comprises an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 96, 98, 100, 102, 104, 106, 108, 214, 216, 220, 222, 224, 226, 228, 244, 246, and 248, orA sequence substantially identical or homologous thereto.

Binding proteins of the invention, such as antibodies, may be germline or non-germline. They can specifically bind to the same IL-21R epitope or to similar epitopes (e.g., overlapping epitopes) as the epitope bound by AbA-AbZ, H3-H6, L1-L6, L8-L21, or L23-L25. In other embodiments, the binding protein specifically binds to an IL-21R fragment, such as the IL-21R fragment of SEQ ID NO: 2 or 4 or a fragment of at least 10, 20, 50, 75, 100, 150, or 200 contiguous amino acids of a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical thereto.

Another embodiment of the invention is an isolated binding protein or antigen-binding fragment thereof that binds to an epitope of IL-21R recognized by a binding protein selected from the group consisting of: AbA-AbZ, H3-H6, L1-L6, L8-L21, and L23-L25, wherein the binding protein or antigen binding fragment competitively inhibits binding of a binding protein selected from the group consisting of: AbA-AbZ, H3-H6, L1-L6, L8-L21, and L23-L25. In another embodiment, the binding protein or antigen-binding fragment comprises a heavy chain, light chain, or Fv fragment comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 165, 168, 171, 193, 213, 229, 240, 242, 244, 246, and 248, or sequences substantially identical or homologous thereto. In yet another embodiment, the binding protein or antigen-binding fragment comprises a heavy chain, light chain, or Fv fragment comprising an amino acid sequence encoded by a nucleotide sequence selected from the group consisting of seq id nos: SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 239, 241, 243, 245, and 247, or sequences substantially identical or homologous thereto.

In other embodiments, the binding protein comprises these V_HAnd V_LAt least one Complementarity Determining Region (CDR) (e.g., one or more, two or more, three or more, four or more, or five or more contiguous CDRs (e.g., two or more CDRs separated by a Framework Region (FR) or a linker)) or non-contiguous CDRs (e.g., two or more CDRs separated by at least one other CDR and, e.g., a FR)) of a domain. For example, the binding protein may comprise V_HDomain and/or V_LOne, two, three or more CDRs of a domain.

The present disclosure provides V from AbA-AbZ, H3-H6, L1-L6, L8-L21, and L23-L25_HAnd V_LThe nucleic acid sequence of the domain. Also encompassed are nucleic acid sequences comprising at least one CDR from AbA-AbZ, H3-H6, L1-L6, L8-L21, and L23-L25. The disclosure also provides vectors and host cells comprising such nucleic acids.

Binding proteins of the invention can be full-length (e.g., comprising at least one intact heavy chain and at least one intact light chain), or can comprise only antigen-binding fragments (e.g., Fab ', F (ab')₂Fv, single chain Fv fragments, Fd fragments, dAb fragments, CDRs, or other fragments). The binding protein may comprise a constant region or a portion thereof selected from any one of kappa (κ), lambda (λ), alpha (α), gamma (γ), delta (δ), ertron (ε), and paradox (μ) constant region genes. For example, heavy chain constant regions of various isotypes can be used, including: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. The light chain constant region may be selected from kappa or lambda. The binding protein may be an IgG, or it may also be an IgG1 κ or IgG1 λ.

The anti-IL-21R binding proteins described herein may be derivatized or linked to another functional molecule (such as another peptide or protein, e.g., a Fab fragment). For example, a binding protein of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to at least one other molecular entity, such as another binding protein (e.g., a bispecific or multispecific binding protein), a toxin, a radioisotope, a cytotoxic or cytostatic agent, or the like.

In one embodiment of the invention, the binding protein or antigen binding fragment has a binding constant for human IL-21R of at least about 10⁵M^-1s^-1. In another embodiment, the binding protein or antigen binding fragment has an IC of about 1.75nM or less₅₀Inhibits IL-21-mediated proliferation of BaF3 cells, and the BaF3 cells comprise human IL-21R. In another embodiment, the binding protein or antigen binding fragment has an IC of about 14.0nM or less₅₀Inhibits IL-21-mediated proliferation of TF1 cells, and the TF1 cells comprise human IL-21R. In another embodiment, the binding protein or antigen binding fragment has an IC of about 1.9nM or less₅₀Inhibits IL-21-mediated proliferation of primary human B cells, and the B cells comprise human IL-21R. In yet another embodiment, the binding protein or antigen-binding fragment has an IC of about 1.5nM or less₅₀Inhibition of IL-21 mediated Primary human CD4⁺The cells proliferate, and the CD4⁺The cells comprise human IL-21R.

In one embodiment, the invention provides a binding protein or antigen binding fragment that binds to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, or at least about 95% identical to any sequence of at least 100 contiguous amino acids. Another embodiment provides a binding protein or antigen-binding fragment that inhibits IL-21 binding to IL-21R. In at least one embodiment, the binding protein or antigen binding fragment is IgG 1. In at least one embodiment, the binding protein or antigen-binding fragment is human.

In another aspect, the invention features a pharmaceutical composition that includes at least one anti-IL-21R binding protein and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise a combination of at least one anti-IL-21R binding protein and at least one other therapeutic agent (e.g., cytokine and growth factor inhibitors, immunosuppressive agents, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, cytotoxic agents, cytostatic agents, or combinations thereof, as described in more detail herein). Combinations of anti-IL-21R binding proteins and therapeutic agents are also within the scope of the invention. The compositions and combinations of the invention are useful for modulating IL-21R-associated immune disorders, for example, by modulating IL-21R signaling.

In one embodiment, the binding protein of the invention is an antibody. In further embodiments, the antibody is polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted (grafted), generated in vitro, and/or multispecific (e.g., a bispecific antibody formed from at least two intact antibodies).

Other embodiments of the invention include isolated nucleic acids encoding anti-IL-21R binding proteins, expression vectors comprising the nucleic acids, and host cells transformed with the vectors. The host cell may be a bacterium, a mammalian cell, a yeast cell, a plant cell, or an insect cell.

The binding protein may be directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound binding protein. Suitable detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials.

In another aspect, the invention provides methods for delivering or targeting an agent, such as a therapeutic or cytotoxic agent, to an IL-21R expressing cell in vivo. The method comprises administering to the subject an anti-IL-21R binding protein under conditions that allow the binding protein to bind to IL-21R. The binding protein may be coupled to a second therapeutic moiety, such as a toxin.

In another embodiment, the invention provides a diagnostic kit comprising a binding protein or antigen-binding fragment of the invention.

Additional aspects of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The invention is set forth and particularly pointed out in the claims, and this disclosure should not be construed as limiting the scope of the claims. The following detailed description includes exemplary representations of various embodiments of the invention, which are not limiting of the claimed invention. The accompanying drawings are incorporated in and constitute a part of this specification, and together with the description, serve to illustrate embodiments and not to limit the invention.

Brief Description of Drawings

FIG. 1(a-c) depicts neutralization of scFv against human IL-21R-BaF3 cell proliferation. Cells were mixed with the indicated scFv and then incubated with 100pg/ml human IL-21. After 48 hours pass through CELLTITER GLO(Promega Corporation, Madison, Wis.) measure proliferation.

FIG. 2(a-c) depicts neutralization of scFv against human IL-21R-TF1 cell proliferation. Cells were mixed with the indicated scFv and then incubated with 100pg/ml human IL-21. Passing CELLTITER-GLO after 48 hoursProliferation was measured.

FIG. 3(a-c) depicts scFv neutralization of murine IL-21R-BaF3 cell proliferation. Cells were mixed with the indicated scFv and then incubated with 400pg/ml murine IL-21. Passing CELLTITER-GLO after 48 hoursProliferation was measured.

FIG. 4(a-c) depicts scFv competing with the parent antibody 18A5IgG for binding to murine IL-21R. The scFv were mixed with biotinylated murine IL-21R H/F and the mixture was added to antibody 18A5 immobilized on an ELISA plate. mIL-21R capture was detected with HRP-streptavidin and competition for mIL-21R binding was indicated by a decrease in A450 signal.

FIG. 5 depicts neutralization of IL-21-dependent proliferation by heavy/light chain pair 21. Antibodies shown in the figure were added to the cells. IL-21 was subsequently added and after 48 hours CELLTITER GLO was usedProliferation was measured. Human IL-21R-BaF3 cells were assayed at 100pg/ml human IL-21 (FIGS. 5a-c), human IL-21R-TF1 cells at 100pg/ml human IL-21 (FIGS. 5d-f), or murine IL-21R-BaF3 cells at 400pg/ml murine IL-21 (FIGS. 5 g-i).

FIG. 6 depicts the binding of 21 anti-IL-21R IgGs to CHO cells transiently expressing human IL-21R (FIGS. 6a-c), rat IL-21R (FIGS. 6d-f), cynomolgus IL-21R (FIGS. 6g-i), and human gamma (γ) common chain (FIGS. 6 j-l). CHO cells were transiently transfected with IL-21R or a control gamma (. gamma.) common strand and binding was measured in a cell-based ELISA using HRP-conjugated anti-human IgG.

FIG. 7(a-c) depicts the binding specificity of specific anti-IL-21R antibodies (FIG. 7a, AbS; FIG. 7b, AbQ, AbT, AbO; FIG. 7c, AbR, AbP, and AbU), as measured by surface plasmon resonance. Capture of anti-IL-21R antibodies on anti-human IgG and BIACORE^TM(GE Healthcare, Piscataway, NJ) instrument was followed to measure binding to either murine IL-21R-H/F, human IL-13-H/F, human IL-2R β, or human soluble IL-4R. FIG. 7d shows that human IL-2R β and human soluble IL-4R are captured by specific anti-IL-2R β and anti-IL-4R antibodies, respectively (control).

FIG. 8(a-d) depicts the binding of anti-IL-21R antibodies to human and murine IL-21R. In BIACORE^TMThe indicated human anti-IL-21R antibody was captured on immobilized anti-human IgG on the chip. Different concentrations of human IL-21R-His/FLAG (FIGS. 8a-b) and murine IL-21R-His/FLAG (FIGS. 8c-d) were flowed through the chip and binding and dissociation monitoredAnd (5) separating.

FIG. 9 depicts the binding of anti-IL-21R antibodies to human and cynomolgus IL-21R. In BIACORE^TMThe immobilized anti-human IgG on the chip captured the human anti-IL-21R antibodies AbS and AbT. Different concentrations of human and cynomolgus IL-21R-His/FLAG were flowed through the chip and binding and dissociation monitored. FIG. 9a shows cynomolgus IL-21R-His/FLAG binding to AbS. FIG. 9b shows binding of human IL-21R-His/FLAG to the AbS. FIG. 9c shows cynomolgus IL-21R-His/FLAG binding to AbT. FIG. 9d shows human IL-21R-His/FLAG binding to AbT.

FIG. 10 depicts epitope evaluation of IL-21R antibodies. In the experiment shown in FIG. 10a (see also the illustration to the left of the Y-axis), the experiment is performed by BIACORE^TMImmobilized anti-IL-21R antibody, AbS on chip, was used to capture murine IL-21R-H/F (His-Flag fusion protein). Additional anti-IL-21R antibodies (AbS, AbT, D5(D5-20, a neutralizing anti-murine IL-21R antibody), and 7C 2(a non-neutralizing anti-murine IL-21R control antibody)) were flowed over the chip and their binding to the captured IL-21R-H/F was monitored. In the experiment shown in FIG. 10b, the results were obtained by BIACORE^TMThe immobilized anti-IL-21R antibody, AbS, on the chip captures human IL-21R-H/F. Additional anti-IL-21R antibodies (AbS, AbT, and 9D 2(a non-neutralizing anti-human IL-21R control antibody)) were flowed over the chip and their binding to captured IL-21R-H/F was monitored.

FIG. 11 depicts the neutralization of proliferation of the indicated antibodies on human IL-21R-BaF3 cells and murine IL-21R-BaF3 cells. Antibodies were added to the cells. IL-21 was subsequently added and after 48 hours CELLTITER GLO was usedProliferation was measured. Human IL-21R-BaF3 cells were assayed at 100pg/ml for human IL-21 (FIG. 11a), 200pg/ml for murine IL-21R-BaF3 cells (FIG. 11b), and 100pg/ml for human IL-21R-TF1 cells (FIG. 11 c).

Fig. 12 depicts neutralization of IL-21-dependent proliferation of human primary B cells. The indicated antibodies were added to primary human B cells along with anti-CD 40 antibodies and human IL-21. Measured after 3 days³Incorporation of H-thymidine. FIG. 12a depicts AbQ, AbR, AbS, AbT, AbUComparison between IL-13 triple mutant, and 18a5 parent antibody; figure 12b depicts a comparison between AbT, AbV, AbW, AbU, and human IgG1 control (hIg 1).

FIG. 13 depicts human primary CD4⁺Neutralization of IL-21-dependent proliferation of T cells. Addition of the indicated antibodies to activated primary human CD4 with human IL-21⁺T cells, and measured after 3 days³Incorporation of H-thymidine.

FIG. 14 depicts the targeting of murine primary CD8⁺Neutralization of IL-21-dependent proliferation of T cells. Addition of the indicated antibodies to activated primary murine CD8 along with human IL-21⁺T cells, and measured after 3 days³Incorporation of H-thymidine.

FIG. 15 depicts measurement of antibody-dependent cellular cytotoxicity (ADCC) induced by anti-IL-21R antibodies. PBMC-dependent killing of CFSE-labeled BJAB cells coated with the indicated anti-IL-21R antibody was measured by propidium iodide incorporation. Including the anti-CD 20 antibody Rituximab (RITUXAN)Genentech, inc., South San Francisco, CA) as a positive control, and an anti-IL-13 antibody as a negative control.

FIG. 16 depicts complement C1q binding of anti-IL-21R antibodies. The anti-IL-21R antibodies shown were immobilized on ELISA plates and after incubation with human serum, C1q binding was measured with chicken anti-human C1q and HRP conjugated anti-chicken IgY antibodies. Including the anti-CD 20 antibody Rituximab (RITUXAN)) As a positive control, and an anti-IL-13 antibody was included as a negative control.

FIG. 17(a-c) depicts the amino acid sequence of AbQ, including V_HAnd V_LDomains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 18(a-c) depicts the amino acid sequence of AbR, including V_HAnd V_LDomains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 19(a-c) depicts the amino acid sequence of AbW, including V_HAnd V_LDomains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 20(a-c) depicts the amino acid sequence of the AbS, including V_HAnd V_LDomains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 21(a-c) depicts the amino acid sequence of AbT, including V_HAnd V_LDomains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 22(a-c) depicts the amino acid sequence of AbO, including V_HAnd V_LDomains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 23(a-c) depicts the amino acid sequence of AbP, including V_HAnd V_LDomains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 24(a-c) depicts the amino acid sequence of AbU, including V_HAnd V_LDomains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

FIG. 25(a-c) depicts the amino acid sequence of AbV, including V_HAnd V_LDomains, CDRs (H1, H2, H3, L1, L2, and L3), and constant regions.

Fig. 26(a-g) depict results from additional studies performed similarly to the studies that produced the results shown in fig. 5, 11, 12, 13, and 14 (described above).

FIG. 27 depicts IL-21 cytokine competes with antibody AbT for binding to murine IL-21R. Either vehicle (vehicle) or increasing amounts of IL-21 were mixed with biotinylated murine IL-21R-His/FLAG and the mixture was added to AbT immobilized on an ELISA plate. mIL-21R capture was detected with HRP-streptavidin and competition for mIL-21R binding was indicated by a decrease in A450 signal.

Detailed Description

The binding proteins of the invention were originally derived from the parent antibody 18a5, but differ from 18a5 in the amino acid sequence of the heavy and/or light chain complementarity determining region 3(CDR3) portion. In addition, the binding proteins of the invention show improved potency in binding and neutralizing both human and murine IL-21R compared to an equivalent form (e.g., scFv or IgG) of 18a 5. High potency neutralization of IL-21R from both species (human and mouse) by a single binding protein has not been previously reported. Binding proteins of the invention with greater neutralizing potency than their parent antibodies may translate to higher efficacy compared to previously documented agents. In addition, V has been changed_HAnd V_LThe amino acid sequence of the framework regions to match the sequence encoded by the human genomic sequence, thereby reducing the potential for human anti-human antibody responses in patients treated with the binding proteins of the invention.

Definition of

In order that the invention may be more readily understood, certain terms are first defined. Other definitions are set forth throughout the detailed description and elsewhere in the specification.

The term "binding protein" as used herein includes any naturally occurring, recombinant, synthetic, or genetically engineered protein, or combination thereof, that binds an antigen, target protein, or peptide, or fragment thereof. The binding proteins of the present invention may comprise an antibody, or be derived from at least one antibody fragment. Binding proteins may include naturally occurring proteins and/or synthetically engineered proteins. Binding proteins of the invention can bind to an antigen or fragment thereof to form a complex and elicit a biological response (e.g., agonize or antagonize a particular biological activity). The binding protein may comprise an isolated antibody fragment. An "Fv" fragment consisting of the variable regions of the heavy and light chains of an antibody, a recombinant single chain polypeptide molecule in which the variable regions of the light and heavy chains are joined by a peptide linker ("scFv protein"), and a minimal recognition unit consisting of amino acid residues that mimic the hypervariable region. Binding protein fragments may also include functional antibody fragments, such as, for example, Fab ', F (ab')₂、Fc、Fd、Fd', Fv, and a single antibody variable domain (dAb). The binding protein antigen is double-or single-stranded and may comprise a single binding domain or multiple binding domains.

Binding proteins may also include binding domain-immunoglobulin fusion proteins in which a binding domain polypeptide is fused or otherwise linked to an immunoglobulin hinge or hinge-acting region polypeptide, which in turn is fused or otherwise linked to a region comprising one or more native or engineered constant regions from an immunoglobulin heavy chain, which constant regions are different from C_H1, C, e.g. IgG and IgA_H2 and C_H3 region or C of IgE_H3 and C_HZone 4 (see, e.g., Ledbetter et al, U.S. patent publication 2005/0136049 for a more complete description). The binding domain-immunoglobulin fusion protein may further include a region comprising a native or engineered immunoglobulin heavy chain C fused or otherwise linked to a hinge region polypeptide_H2 constant region polypeptide (or C)_H3, in the case where the construct is fully or partially derived from IgE) and fused or otherwise linked to C_H2 constant region polypeptide (or C)_H3, in the case of constructs wholly or partially derived from IgE)) of natural or engineered immunoglobulin heavy chain C_H3 constant region Polypeptides (or C)_H4, in the case where the construct is fully or partially derived from IgE). Typically, such binding domain-immunoglobulin fusion proteins have at least one immunological activity selected from the group consisting of: antibody-dependent cell-mediated cytotoxicity, complement fixation, and/or binding to a target (e.g., a target antigen). The binding proteins of the present invention may be derived from any species, including but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, chicken, and cow.

The term "antibody" as used herein refers to an immunoglobulin that is reactive with a specified protein or peptide or fragment thereof. Such antibodies include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, monoclonal antibodies, monospecific antibodies, polyclonal antibodies, multispecific antibodies, non-specific antibodies, bispecific antibodies, multispecific antibodies, humanized antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, graft-coupled antibodies (i.e., antibodies coupled or fused to other proteins, radiolabels, cytotoxins), and antibodies generated in vitro. The antibody can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgG1, IgG2, IgG3, and IgG 4). The antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG 4. The antibody may also have a light chain selected from, for example, kappa (. kappa.) or lambda (. lamda.). The antibodies of the invention may be derived from any species, including but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, chicken, and cow. The constant region of an antibody can be altered, e.g., mutated, to modify a property of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function). Typically, the antibody specifically binds to a predetermined antigen, e.g., an antigen associated with a disorder, e.g., an inflammatory, immune, autoimmune, neurodegenerative, metabolic, and/or malignant disorder.

The term "single domain binding protein" as used herein includes any single domain binding scaffold that binds an antigen, protein, or polypeptide. Single domain binding proteins may include any natural, recombinant, synthetic, or genetically engineered protein scaffold, or combination thereof, that binds to an antigen or fragment thereof to form a complex and elicit a biological response (e.g., agonize or antagonize a particular biological activity). Single domain binding proteins may be derived from naturally occurring proteins or antibodies, or they may be synthetically engineered or generated by recombinant techniques. The single domain binding protein may be any prior art single domain binding protein or any future single domain binding protein and may be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, chicken, and cow. In some embodiments of the invention, the single domain binding protein scaffold may be derived from the variable region of an immunoglobulin found in fish, such as for example from the variable region of an immunoglobulin isotype known as Novel Antigen Receptor (NAR) found in shark serum. Methods for generating single domain binding scaffolds derived from NAR variable regions ("IgNAR") are described in international application publication No. wo 03/014161 and Streltsov (2005) Protein sci.14 (11): 2901-09.

In other embodiments, the single domain binding protein is a naturally occurring single domain binding protein described in the art as a heavy chain antibody without a light chain. Such single domain binding proteins are disclosed, for example, in international application publication No. WO 94/004678. For clarity reasons, the variable domain binding protein derived from a heavy chain antibody naturally free of light chain is referred to herein as VHH or "nanobody" (nanobody) to link it to the V of a conventional four-chain immunoglobulin_HAre distinguished. Such VHH molecules may be derived from antibodies raised in Camelidae (Camelidae) species, for example in camels, llamas, dromedary, alpacas, and guanacos. Other families than camelidae may also be used to produce heavy chain binding proteins which do not naturally contain a light chain. VHH molecules are approximately 10 times smaller than traditional IgG molecules. They are single polypeptides and are very stable and resistant to extreme pH and temperature conditions. Furthermore, they are resistant to the action of proteases, unlike conventional antibodies. Furthermore, expression of VHH in vitro can produce high yields of correctly folded functional VHH. In addition, the binding proteins produced in camelids recognize epitopes other than those recognized by antibody libraries or by antibodies produced in vitro by mammals other than immunized camelids (see, e.g., international application publication nos. WO 97/049805 and WO 94/004678, both incorporated herein by reference).

The terms "antigen binding domain" and "antigen binding fragment" refer to a portion of a binding protein that comprises amino acids responsible for containing the specific binding between the binding protein and an antigen. The portion of the antigen that is specifically recognized and bound by the binding protein is referred to as an "epitope". The antigen binding domain may comprise the light chain variable region (V) of an antibody_L) And heavy chain variable region (V)_H) (ii) a However, it need not contain both. For example, Fd fragment has two V_HAnd often retains the antigen binding function of the intact antigen binding domain. Examples of antigen binding fragments of binding proteinsIncluding but not limited to: (1) fab fragments, i.e. having V_L、V_H、C_LAnd C_H1 domain of a monovalent fragment; (2) f (ab')₂Fragments, i.e. bivalent fragments with two Fab fragments connected by a disulfide bridge at the hinge region; (3) fd fragment having two V_HAnd a C_H1 domain; (4) fv fragment having a V with one arm of an antibody_LAnd V_HA domain; (5) dAb fragments (see, e.g., Ward et al (1989) Nature 341: 544-46) having a V_HA domain; (6) an isolated CDR; and (7) single chain variable fragments (scFv). Although two domains of the Fv fragment (i.e., V)_LAnd V_H) Encoded by separate genes, but which can be joined together using recombinant methods, through synthetic linkers, such that they can be produced as a single protein chain, wherein V_LAnd V_HThe regions pair to form monovalent molecules (called scFv) (see, e.g., Bird et al (1988) Science 242: 423-26; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85: 5879-83). These binding domain fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be functionally evaluated in the same manner as intact binding proteins such as, for example, antibodies.

The term "neutralize" refers to a binding protein or antigen-binding fragment thereof (e.g., an antibody) that reduces or blocks the activity of a signaling pathway or antigen, such as the IL-21/IL-21R signaling pathway or IL-21R antigen.

The term "effective amount" refers to a dose or amount sufficient to modulate IL-21R activity to ameliorate or reduce the severity of clinical symptoms or to achieve a desired biological outcome, e.g., reduce T cell and/or B cell activity, suppress autoimmunity, suppress transplant rejection.

The term "Human binding protein" includes binding Proteins having variable and constant regions that substantially correspond to Human germline immunoglobulin Sequences known in the art, including, for example, those described by Kabat et al (5th ed.1991) Sequences of Proteins of Immunological Interest, U.S. department of Health and Human Services, NIH Publication No. 913242. The human antibodies of the invention may comprise amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, particularly in CDR 3. A human antibody may have at least one, two, three, four, five, or more positions replaced by amino acid residues that are not encoded by human germline immunoglobulin sequences.

The phrases "inhibit," "antagonize," "block," or "neutralize" IL-21R activity and the like refer to at least one activity of IL-21R that is reduced, inhibited, or otherwise reduced by binding to an anti-IL-21R antibody, wherein the reduction is relative to the activity of IL-21R in the absence of the antibody. IL-21R activity can be measured using any technique known in the art. Inhibition or antagonism does not necessarily indicate complete elimination of IL-21R biological activity. The reduction in activity may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

The term "interleukin-21 receptor" or "IL-21R" and the like refers to a receptor of the cytokine family class I that binds IL-21 ligand, also known as MU-1 (see, e.g., U.S. patent application No.09/569,384 and U.S. application publication No. 2004/0265960; 2006/0159655; 2006/0024268; and 2008/0241098), NILR or zalpha11 (see, e.g., International application publication No. WO 01/085792; Parrish Novak et al (2000) supra; Ozaki et al (2000) supra). IL-21R shares the beta chain with IL-2 and IL-15 receptors, and IL-4 alpha homology (Ozaki et al (2000) supra). Upon ligand binding, IL-21R is able to interact with the common gamma cytokine receptor chain (gammac) and induce phosphorylation of STAT1 and STAT3(Asao et al (2001) supra) or STAT5(Ozaki et al (2000) supra). IL-21R shows a broad lymphoid tissue distribution. The term "interleukin-21 receptor" or "IL-21R" and the like also refers to a polypeptide (preferably mammalian-derived, e.g., murine or human IL-21) or a polynucleotide encoding such a polypeptide (where desired) that is capable of interacting with IL-21 (preferably mammalian-derived, e.g., murine or human IL-21) and has at least one of the following characteristics: (1) an amino acid sequence of a naturally occurring mammalian IL-21R polypeptide, or a fragment thereof, such as SEQ ID NO: 2 (human-pair)In GENBANK(U.S. depth. of Health and Human Services, Bethesda, MD) accession No. NP _068570) or SEQ ID NO: 4 (murine-corresponds to GENBANK)Accession number NP _068687) or a fragment thereof; (2) and SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment thereof, e.g., an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99% homologous; (3) consisting of a naturally occurring mammalian IL-21R nucleotide sequence or a fragment thereof (e.g., SEQ ID NO: 1 (human-corresponding to GENBANK)Accession number NM — 021798) or SEQ ID NO: 3 (murine-corresponds to GENBANK)Accession No. NM — 021887) or a fragment thereof); (4) consists of a nucleotide sequence identical to SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof, e.g., an amino acid sequence encoded by a nucleotide sequence that is substantially homologous, e.g., at least 85%, 90%, 95%, 98%, or 99% homologous; (5) consisting of a nucleotide sequence identical to a naturally occurring IL-21R nucleotide sequence or fragment thereof, e.g. SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof; or (6) a nucleotide sequence that hybridizes under stringent conditions (e.g., high stringency conditions) to one of the foregoing nucleotide sequences. In addition, other non-human and non-mammalian IL-21R useful in the disclosed methods are contemplated.

The term "interleukin-21" or "IL-21" refers to a cytokine that exhibits sequence homology with IL-2, IL-4, and IL-15 (Parrish-Novak et al (2000) supra) and binds IL-21R. Such cytokines share a common fold, folding into a "four-helix bundle" structure characteristic of this family. IL-21 is predominantly on activated CD4⁺Expressed in T cells and reported to have effects on NK, B and T cells (Parrish)Novak et al (2000) supra; kasaian et al (2002) supra). Activation of IL-21R causes, for example, STAT5 or STAT3 signaling when IL-21 binds to IL-21R (Ozaki et al (2000) supra). The term "interleukin-21" or "IL-21" also refers to a polypeptide (preferably mammalian, e.g., murine or human IL-21) or a polynucleotide (where context requires) encoding such a polypeptide that is capable of interacting with IL-21R (preferably mammalian, e.g., murine or human IL-21R) and has at least one of the following characteristics: (1) a naturally occurring amino acid sequence of mammalian IL-21, or a fragment thereof, such as SEQ ID NO: 212 (human), or a fragment thereof; (2) and SEQ ID NO: 212 or a fragment thereof, e.g., an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99% homologous; (3) an amino acid sequence encoded by a naturally occurring mammalian IL-21 nucleotide sequence or fragment thereof (e.g., SEQ ID NO: 211 (human) or fragment thereof); (4) consists of a nucleotide sequence identical to SEQ ID NO: 211 or a fragment thereof, that is substantially homologous, e.g., at least 85%, 90%, 95%, 98%, or 99% homologous; (5) an amino acid sequence encoded by a nucleotide sequence that is degenerate to a naturally-occurring IL-21 nucleotide sequence, or a fragment thereof; or (6) a nucleotide sequence that hybridizes under stringent conditions (e.g., high stringency conditions) to one of the foregoing nucleotide sequences.

The terms "IL-21R activity" and the like (e.g., "IL-21R activity", "IL-21/IL-21R activity") refer to at least one cellular process that is initiated or disrupted by IL-21R binding. IL-21R activity includes but is not limited to: (1) interaction (e.g., binding) with a ligand (e.g., an IL-21 polypeptide); (2) combine or activate signal transduction (also referred to as "signaling," which refers to intracellular cascade that occurs in response to a particular stimulus) and signaling molecules (e.g., gamma chain (yc) and JAK1), and/or stimulate phosphorylation and/or activation of STAT proteins (e.g., STAT5 and/or STAT 3); and (3) modulating proliferation, differentiation, effector cell function, cytolytic activity, cytokine secretion, and/or survival of immune cells, such as T cells, NK cells, B cells, macrophages, regulatory T cells (tregs), and megakaryocytes.

As used herein, "in vitro generated antibody" refers to an antibody in which all or a portion of the variable region (e.g., at least one CDR) is generated in a non-immune cell selection (e.g., in vitro phage display, protein chip, or any other method capable of testing candidate sequences for their ability to bind antigen).

The term "isolated" refers to a molecule that is substantially free of its natural environment. For example, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it was derived. The term also refers to preparations in which the isolated protein is sufficiently pure for the pharmaceutical composition, or at least 70-80% (w/w) pure, at least 80-90% (w/w) pure, at least 90-95% (w/w) pure, or at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.

The phrase "percent identical" or "percent identity" refers to the similarity between at least two different sequences. This percent identity can be determined by standard algorithms, such as the Basic Local Alignment Search Tool (BLAST) described by Altsull et al ((1990) J.mol.biol.215: 403-10); needleman et al ((1970) J.mol.biol.48: 444-53); or Meyers et al ((1988) Compout. appl. biosci.4: 11-17). One set of parameters may be the Blosum 62 scoring matrix and gap penalty 12, gap extension penalty 4, and frameshift gap penalty 5. The percent identity between two amino acid or nucleotide sequences can also be determined using an algorithm of Meyers and Miller ((1989) CABIOS 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Percent identity is typically calculated by comparing sequences of similar length.

The term "repertoire" refers to at least one nucleotide sequence derived, in whole or in part, from at least one sequence encoding at least one immunoglobulin. Sequences can be generated by rearranging V, D, and J segments of the heavy chain and V and J segments of the light chain in vivo. Alternatively, the rearranged cellular sequences may be generated from cells that undergo rearrangement in response to, for example, in vitro stimulation. Alternatively, some or all of the sequence may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, or other methods (see, e.g., U.S. Pat. No.5,565,332). The repertoire may include only one sequence, or may include multiple sequences, including multiple sequences in a genetically diverse repertoire.

The terms "specifically binds" and the like refer to the formation of a complex of two molecules that is relatively stable under physiological conditions. Specific binding is characterized by high affinity and low to moderate capacity, as opposed to non-specific binding, which typically has low affinity and moderate to high capacity. Generally, when the binding constant Ka is greater than about 10⁶M^-1s^-1Binding is considered specific. If necessary, non-specific binding can be reduced without substantially affecting specific binding by changing the binding conditions. The skilled artisan can use routine techniques to modify appropriate binding conditions such as the concentration of binding protein, the ionic strength of the solution, the temperature, the time allowed for binding to occur, the concentration of blocking agent (e.g., serum albumin or milk casein), and the like. Illustrative conditions are set forth herein, but other conditions known to those of ordinary skill in the art are within the scope of the present invention.

As used herein, the terms "stringency," "stringency," and the like describe conditions for hybridization and washing. The isolated polynucleotides of the present invention can be used as hybridization probes and primers to identify and isolate nucleic acids having a sequence that is the same as or similar to the sequence encoding the disclosed polynucleotides. Thus, polynucleotides isolated in this manner can be used to generate binding proteins for IL-21R or to identify cells expressing such binding proteins. Hybridization methods for identifying and isolating nucleic acids include Polymerase Chain Reaction (PCR), Southern hybridization, in situ hybridization, and Northern hybridization, and are well known to those skilled in the art.

Hybridization reactions can be performed under conditions of varying stringency. The stringency of the hybridization reaction includes the difficulty with which any two nucleic acid molecules will hybridize to each other and the conditions under which they will remain hybridized. Preferably, each hybridized polynucleotide hybridizes to its corresponding polynucleotide under low stringency conditions, more preferably stringent conditions, and most preferably high stringency conditions. Stringent conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989) 6.3.1-6.3.6. Both aqueous and nonaqueous methods are described in this document and can be used. An example of stringent hybridization conditions is hybridization in 6 XSSC/sodium citrate (SSC) at about 45 ℃ followed by at least one wash in 0.2 XSSC/0.1% SDS at 50 ℃. Stringent hybridization conditions can also be achieved with washes in, for example, 0.2 XSSC/0.1% SDS at 55 ℃,60 ℃, or 65 ℃. High stringency conditions include, for example, hybridization in 0.5M sodium phosphate/7% SDS at 65 ℃ followed by at least one wash in 0.2 XSSC/1% SDS at 65 ℃. Other examples of stringent conditions are shown in table 1 below: high stringency conditions refer to conditions at least as stringent as, for example, conditions a-F; stringent conditions refer to conditions at least as stringent as, for example, conditions G-L; whereas low stringency conditions refer to conditions at least as stringent as, for example, conditions M-R.

Table 1: conditions of hybridization

¹Hybrid length is the length expected for the hybridizing region of the hybridizing polynucleotide. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be the length of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the regions of optimal sequence complementarity.

²SSPE (1XSSPE is 0.15M NaCl, 10mM NaH)₂PO₄And 1.25mM EDTA, pH 7.4) can be substituted for SSC in the hybridization and wash buffer (1XSSC is 0.15M NaCl and 15mM sodium citrate); washing was performed for 15 minutes after hybridization was complete.

T_B ^*-T_R ^*: the hybridization temperature for hybrids of expected lengths less than 50 base pairs should be higher than the melting temperature (T) of the hybrid_m) 5-10 ℃ lower, where T_mDetermined according to the following equation. For hybrids less than 18 base pairs in length, T_m(° C) 2 (number of a + T bases) +4 (number of G + C bases). For hybrids between 18 and 49 base pairs in length, T_m(℃)＝81.5+16.6(log₁₀Na⁺) +0.41 (% G + C) - (600/N), where N is the number of bases in the hybrid and Na⁺The concentration of sodium ions (Na in 1 XSSC) in the hybridization buffer⁺＝0.165M)。

Further examples of stringency conditions for polynucleotide hybridization are described in Sambrook et al, Molecular Cloning: a Laboratory Manual, chapters 9 and 11, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and Ausubel et al eds., Current Protocols in Molecular Biology, sections 2.10 and 6.3-6.4, John Wiley & Sons, Inc. (1995), incorporated herein by reference.

The isolated polynucleotides of the present invention can be used as hybridization probes and primers to identify and isolate DNA having a sequence that encodes an allelic variant of the disclosed polynucleotides. Allelic variants refer to naturally occurring variants of the disclosed polynucleotides that encode polypeptides that are the same as or have significant similarity to the polypeptides encoded by the disclosed polynucleotides. Preferably, allelic variants have at least about 90% sequence identity (more preferably at least about 95% identity; most preferably at least about 99% identity) to the disclosed polynucleotides. The isolated polynucleotides of the present invention are also useful as hybridization probes and primers to identify and isolate DNA having a sequence encoding a polypeptide homologous to the disclosed polynucleotides. These homologs are polynucleotides and polypeptides that are isolated from a species that is different from or the same as the species of the disclosed polypeptides and polynucleotides, but have significant sequence similarity to the disclosed polynucleotides and polypeptides. Preferably, a polynucleotide homolog has at least about 50% sequence identity (more preferably at least about 75% identity; most preferably at least about 90% identity) to a disclosed polynucleotide, while a polypeptide homolog has at least about 30% sequence identity (more preferably at least about 45% identity; most preferably at least about 60% identity) to a disclosed binding protein/polypeptide. Preferably, homologs of the disclosed polynucleotides and polypeptides are isolated from mammalian species. The isolated polynucleotides of the invention may additionally be used as hybridization probes and primers to identify cells and tissues expressing the binding proteins of the invention and the conditions under which they are expressed.

The phrases "substantially as listed", "substantially identical", and "substantially homologous" mean that the amino acid or nucleotide sequence (e.g., CDR, V) is of interest_HOr V_LDomains) may be identical to or have non-essential differences (e.g., via conservative amino acid substitutions) from the listed sequences. Non-essential differences include minor amino acid changes, such as one or two substitutions in the sequence of five amino acids of a defined region. In the case of an antibody, the second antibody has the same specificity as the first antibody and has at least about 50% of the affinity of the first antibody.

Sequences that are substantially identical or homologous to the sequences disclosed herein are also part of the present application. In some embodiments, the sequence identity may be about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher. Alternatively, substantial identity or homology exists when a nucleic acid segment will hybridize to the complement of the strand under selective hybridization conditions (e.g., high stringency hybridization conditions). The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially purified form.

The term "therapeutic agent" and the like refers to a substance that treats or contributes to the treatment of a medical condition or symptom thereof. Therapeutic agents may include, but are not limited to, substances that modulate immune cells or immune responses in a manner that complements the use of anti-IL-21R binding proteins. In one embodiment of the invention, the therapeutic agent is a therapeutic antibody, such as an anti-IL-21R antibody. In another embodiment of the invention, the therapeutic agent is a therapeutic binding protein, such as an anti-IL-21R antibody. Non-limiting examples and uses of therapeutic agents are described herein.

As used herein, a "therapeutically effective amount" of an anti-IL-21R binding protein (e.g., an antibody) refers to an amount of the binding protein that is effective to treat, prevent, cure, delay, reduce the severity of, and/or ameliorate at least one symptom of a disorder or relapsed disorder or to prolong survival of a subject beyond that expected in the absence of such treatment when administered to a subject, such as a human patient, in a single dose or multiple doses.

anti-IL-21R binding proteins

The disclosure of the present application provides novel anti-IL-21R binding proteins comprising novel antigen binding fragments. Numerous methods known to those skilled in the art can be used to obtain the binding protein or antigen-binding fragment thereof. For example, anti-IL-21R binding proteins, including antibodies, can be generated using recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). Monoclonal antibodies can also be generated by generating hybridomas according to known methods (see, e.g., Kohler and Milstein (1975) Nature 256: 495-99). Standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE) are then used^TM) Hybridomas formed in this manner are analytically screened to identify one or more hybridomas that produce antibodies that specifically bind to a particular antigen. Any form of the defined antigen may be used as an immunogen, e.g., a naturally occurring form, any variant or fragment thereof, and antigenic peptides thereof.

One exemplary method of generating binding proteins, including antibodies, includes screening protein expression libraries, such as phage or ribosome display libraries. Phage display is described, for example, in U.S. Pat. Nos. 5,223,409; smith (1985) Science 228: 1315-17; clackson et al (1991) Nature 352: 624-28; marks et al (1991) J.mol.biol.222: 581-97; and international application publication No. WO 92/018619; WO 91/017271; WO 92/020791; WO 92/015679; WO 93/001288; WO 92/001047; WO 92/009690; and WO 90/002809.

In addition to using display libraries, non-human animals, such as cynomolgus monkeys, chickens, or rodents (e.g., mice, hamsters, or rats) can be immunized with a defined antigen. In one embodiment, the non-human animal includes at least a portion of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig locus. Using hybridoma technology, antigen-specific monoclonal binding proteins (such as antibodies) derived from genes with the desired specificity can be generated and selected (see, e.g., XENOMOUSE)^TM(Amgen Inc., Thious and Oaks, CA); green et al (1994) nat. Genet.7: 1321; U.S. patent nos. 7,064,244; and international application publication nos. WO 96/034096 and WO 96/033735).

In one embodiment of the invention, the binding protein is a monoclonal antibody obtained from a non-human animal and then modified (e.g., humanized, de-immunized, or chimeric) using recombinant DNA techniques known in the art. Various approaches for generating chimeric antibodies have been described (see, e.g., Morrison et al (1985) Proc. Natl. Acad. Sci. USA 81 (21): 6851-55; Takeda et al (1985) Nature 314 (6010): 452-54; U.S. Pat. Nos. 4,816,567 and 4,816,397; European application publication Nos. EP 0171496 and EP 0173494; and British patent No. GB 2177096). Humanized binding proteins can also be generated, for example, using transgenic mice that express human heavy and light chain genes but are incapable of expressing endogenous mouse immunoglobulin heavy and light chain genes. Winter (U.S. Pat. No.5,225,539) describes an exemplary CDR grafting method that can be used to make the humanized binding proteins described herein. All CDRs of a particular human binding protein may be replaced with at least a portion of the non-human CDRs, or only some CDRs may be replaced with non-human CDRs. Only the number of CDRs required for the humanized binding protein to bind to the predetermined antigen must be replaced.

Humanized binding proteins or fragments thereof can be generated by replacing Fv variable domains not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized binding proteins or fragments thereof are provided, for example, in Morrison (1985) Science 229: 1202-07; oi et al (1986) BioTechniques 4: 214; and U.S. Pat. nos. 5,585,089; 5,693,761; 5,693,762; 5,859,205; and 6,407,213. Those methods include isolating, manipulating, and expressing nucleic acid sequences encoding all or part of an immunoglobulin Fv variable domain from at least one of a heavy or light chain. Such nucleic acids can be obtained from hybridomas that produce antibodies to a predetermined target as described above, as well as other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

In certain embodiments, the humanized binding proteins are improved by introducing conservative substitutions, consensus substitutions, germline substitutions and/or back mutations. Such altered immunoglobulin molecules may be generated by any of several techniques known in the art (see, e.g., Teng et al (1983) Proc. Natl. Acad. Sci. USA 80: 7308-73; Kozbor et al (1983) Immunol. today 4: 7279; Olsson et al (1982) meth. enzymol.92: 3-16); international application publication No. WO 92/006193; and european patent No. ep 0239400).

Binding proteins or fragments thereof may also be modified by specific deletion of human T cell epitopes or "deimmunization" by methods such as those disclosed in International application publication Nos. WO 98/052976 and WO 00/034317. Briefly, MHC class II-binding peptides can be analyzed for the heavy and light chain variable domains of binding proteins (such as, for example, binding proteins derived from antibodies); these peptides represent potential T cell epitopes (as defined, for example, in international application publication nos. WO 98/052976 and WO 00/034317). To detect potential T cell epitopes, a computer modeling approach known as "peptide threading" can be applied, and in addition, databases of human MHC class II binding peptides can be searched for V_HAnd V_LMotifs present in the sequence, as described in international application publication nos. WO 98/052976 and WO 00/034317. These motifs bind to any of the 18 major MHC class II DR allotypes and thus constitute potential T cell epitopes. Detected potentialT cell epitopes can be eliminated by substituting a few amino acid residues in the variable domain or by single amino acid substitutions. Typically, conservative substitutions are made. Typically, but not exclusively, amino acids common to a position in the human germline antibody sequence may be used. Human germline sequences are disclosed, for example, in Tomlinson et al (1992) j.mol.biol.227: 776-98; cook et al (1995) immunol. today 16 (5): 237-42; chothia et al (1992) J.mol.biol.227: 799-; and Tomlinson et al (1995) EMBO J.14: 4628-38. The V BASE catalog provides a comprehensive catalog of human immunoglobulin variable region sequences (compiled by Tomlinson et al, MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, such as framework regions and CDRs. Consensus human framework regions may also be used, as described, for example, in U.S. Pat. No.6,300,064.

In certain embodiments, the binding protein may contain an altered immunoglobulin constant or Fc region. For example, a binding protein produced according to the teachings herein can bind more strongly or with greater specificity to effector molecules, such as complement and/or Fc receptors, which can control several immune functions of the binding protein, such as effector cell activity, lysis, complement-mediated activity, binding protein clearance, and binding protein half-life. Typical Fc receptors that bind to the Fc region of binding proteins (e.g., IgG antibodies) include, but are not limited to, receptors of the Fc γ RI, Fc γ RII, and FcRn subclasses, including allelic variants and alternatively spliced forms of these receptors. For reviews of Fc receptors see, for example, ravatch and Kinet (1991) annu. 457-92; capel et al (1994) immunoassays 4: 25-34; and de Haas et al (1995) J.Lab.Clin.Med.126: 330-41. For additional binding protein/Antibody production techniques, see, e.g., antibodies: a Laboratory Manual (1988) Harlow et al eds, Cold Spring Harbor Laboratory. The invention is not necessarily limited to any particular source, method of production, or other particular characteristics of the binding protein or antibody.

Binding proteins, including antibodies (immunoglobulins), are typically tetramerically glycosylated proteins consisting of two light (L) chains (approximately 25kDa each) and two heavy (H) chains (approximately 50kDa each). Two light chain types can be found in antibodies, calledLambda (. lamda.) and kappa (. kappa.). Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes: A. d, E, G, and M, and several of these can be further divided into subclasses (isotypes), such as IgG1, 1gG2, IgG3, IgG4, IgA1, and IgA 2. Each light chain comprises an N-terminal variable (V) domain (V)_L) And a constant (C) field (C)_L). Each heavy chain comprises an N-terminal V domain (V)_H) Three or four C domains (C)_H) And a hinge region. Closest to V_HC of (A)_HThe domain is called C_H1。V_HAnd V_LThe domain consists of four regions of relatively conserved sequence called framework regions (FR1, FR2, FR3, and FR4) and three regions of high sequence height called CDRs, which form the scaffold for the CDRs. The CDRs contain most of the residues responsible for the specific interaction of the antibody with the antigen. The CDRs are referred to as CDR1, CDR2, and CDR 3. The CDR components on the heavy chain are referred to as H1, H2, and H3 (also referred to herein as CDR H1, CDR H2, and CDR H3, respectively), while the CDR components on the light chain are referred to as L1, L2, and L3 (also referred to herein as CDR L1, CDR L2, and CDR L3, respectively).

CDR3 is generally the largest source of molecular diversity within the antigen binding site. For example, CDR H3 may be as short as two amino acid residues or greater than 26 amino acids. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of antibody structure see, e.g., Harlow et al (1988) supra. One skilled in the art will recognize each subunit structure (e.g., C)_H、V_H、C_L、V_LCDR, and/or FR structures) comprise an active fragment, e.g., V_H、V_LOr CDR subunits bind to the antigen, i.e. antigen binding fragments, or e.g. C_HThe subunits bind and/or activate, for example, Fc receptors and/or portions of complement. CDRs are generally referred to as Kabat CDRs (as described by Kabat et al (1991) supra). Another criterion for characterizing antigen binding sites is the hypervariable loops, as described, for example, by Chothia et al (1992) supra and by Tomlinson et al (1995) supra. A further criterion is the definition of "AbM" used by the AbM antibody modeling software of Oxford Molecular (see, e.g., Protein sequence in general)The sequence and Structure Analysis of Antibody Variable Domains in: antibody Engineering (2001) Duebel and Kontermann eds, Springer-Verlag, Heidelberg). The embodiments described with respect to the Kabat CDRs can be converted into practice using linkages similarly described with respect to Chothia hypervariable loops or AbM defined loops.

Fab fragments are covalently linked by V through disulfide bonds between constant regions_H-C_H1 and V_L-C_LDomain composition. Fv fragments which are small and are linked by non-covalent bonds_HAnd V_LDomain composition. To overcome the propensity for dissociation of non-covalently linked domains, scfvs may be constructed. scFv containing a linker (1) V_HTo the C terminal of_LN terminal of (1), (2) V)_LTo the C terminal of_HThe N-terminal flexible polypeptide of (1). For example, the 15-mer (Gly)₄Ser)₃Peptides may be used as linkers, but other linkers are known in the art.

The sequences of the antibody genes after assembly and somatic mutation are very different, and these differential genes are estimated to encode 10¹⁰Different antibody molecules were generated (Immunoglobulin Genes (2 nd edition, 1995) edited by Jonio et al, Academic Press, San Diego, Calif.).

In certain embodiments of the invention, the binding protein is a single domain binding protein. Single domain binding proteins include those in which the CDRs are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain binding proteins, binding proteins that do not naturally have a light chain, single domain binding proteins derived from conventional four-chain antibodies, engineered binding proteins, and single domain protein scaffolds other than those derived from antibodies. Single domain binding proteins include any single domain binding protein known in the art, as well as any future established or learned.

Single domain binding proteins may be derived from any species, including but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, chicken, and cow. In one aspect of the invention, the single domain binding protein may be derived from a variable region of an immunoglobulin found in fish, such as, for example, from sharkDerived from immunoglobulin isotypes known as neoantigen receptors (NAR) found in fish serum. Methods for generating single domain binding proteins derived from nar (ignar) variable regions are loaded, for example, in international application publication nos. WO 03/014161 and Streltsov (2005) Protein sci.14: 2901-09. Single domain binding proteins also include naturally occurring single domain binding proteins known in the art as heavy chain antibodies without light chains. This variable domain derived from a heavy chain antibody naturally devoid of a light chain is referred to herein as a VHH, or nanobody, to be conjugated to the conventional V of a four-chain immunoglobulin_HAre distinguished. Such VHH molecules can be derived from antibodies raised in Camelidae species, for example in camels, llamas, dromedary, alpacas, and guanacos, and are sometimes referred to as camelids or camelized variable domains (see for example Muydermans (2001) J.Biotechnol.74 (4): 277-302, incorporated herein by reference). Heavy chain binding proteins that are naturally devoid of light chains may also be produced by species other than camelidae. The VHH molecules are about 10 times smaller than the IgG molecules. They are single polypeptides and are very stable and resistant to extreme pH and temperature conditions. Furthermore, they are resistant to the action of proteases, unlike conventional antibodies. Furthermore, expression of VHH in vitro can produce high yields of correctly folded functional VHH. In addition, the binding proteins produced in camelids recognize epitopes other than those recognized by antibody libraries or by antibodies produced in vitro by mammals other than immunized camelids (see, e.g., international application publication nos. WO 97/049805 and WO 94/004678, incorporated herein by reference).

A "bispecific" or "bifunctional" binding protein refers to an artificial hybrid binding protein having two different pairs of heavy/light chains and two different binding sites. Bispecific binding proteins can be generated by a variety of methods, including fusion of hybridomas or ligation of Fab' fragments (see, e.g., Songsivilai and Lachmann (1990) Clin. exp. Immunol.79: 315-21; Kostelny et al (1992) J. Immunol.148: 1547-53). In one embodiment, the bispecific binding protein comprises a first binding domain polypeptide (such as a Fab' fragment) and a second binding domain polypeptide linked via an immunoglobulin constant region.

The other kind isBinding proteins according to the invention may include, for example, binding domain-immunoglobulin fusion proteins in which a binding domain polypeptide is fused or otherwise linked to an immunoglobulin hinge or hinge-acting region polypeptide, which in turn is fused or otherwise linked to a polypeptide comprising one or more heavy chains, C, from an immunoglobulin_H1 (e.g., C for IgG and IgA 1)_H2 and C_H3 region or C of IgE_H3 and C_HZone 4) (see, e.g., U.S. application publication No.2005/0136049 for a more complete description, incorporated herein by reference). The binding domain-immunoglobulin fusion protein may further comprise a region wherein the immunoglobulin heavy chain C is native or engineered_H2 constant region polypeptide (or C)_H3, in the case where the construct is derived in whole or in part from IgE)) fused or otherwise linked to a hinge region polypeptide and a native or engineered immunoglobulin heavy chain C_H3 constant region Polypeptides (or C)_H4, in the case where the construct is fully or partially derived from IgE), fused or otherwise linked to C)_H2 constant region polypeptide (or C)_H3, in the case where the construct is fully or partially derived from IgE). Typically, such binding domain-immunoglobulin fusion proteins are capable of at least one immunological activity selected from the group consisting of: antibody-dependent cell-mediated cytotoxicity (ADCC), complement fixation, and/or binding to a target (e.g., a target antigen, such as human IL-21R).

Binding proteins of the invention may also include peptidomimetics. Peptidomimetics refer to Peptide-containing molecules that mimic the secondary structural elements of a protein (see, e.g., Johnson et al, Peptide Turn Mimets in Biotechnology and Pharmacy (1993) Pezzuto et al, Chapman and Hall, New York, incorporated herein by reference in its entirety). The underlying principle behind the use of peptidomimetics is that the presence of the peptide backbone of a protein is primarily intended to orient the amino acid side chains in such a way as to drive molecular interactions, such as between an antibody and an antigen. Peptidomimetics are expected to allow molecular interactions similar to the native molecule. These principles can be used to engineer second generation molecules with many of the natural characteristics of the targeting peptides disclosed herein, but with altered and possibly improved characteristics.

Other embodiments of binding proteins useful for practicing the invention include fusion proteins. In these molecules, typically the entire or substantial portion of the targeting peptide (e.g., IL-21R or anti-IL-21R antibody) is linked at the N-or C-terminus to the entire or portion of the second polypeptide or protein. For example, fusion proteins may employ leader (or signal) sequences from other species to allow recombinant expression of the protein in a heterologous host. For example, the amino acid sequence or nucleic acid sequence encoding the amino acid sequence of the binding proteins of the invention and antigen-binding fragments thereof comprising a leader (or signal) sequence may be selected from the group consisting of SEQ ID NOs: 87-109 and 239-248. Another useful fusion involves the addition of immunologically active domains, such as binding protein epitopes, to facilitate purification of the fusion protein. The inclusion of a cleavage site at or near the fusion junction will facilitate removal of the foreign polypeptide after purification. Other useful fusions include linking functional domains such as active sites from enzymes, glycosylation domains, cellular targeting signals, or transmembrane regions. Examples of proteins or peptides that can be incorporated into the fusion protein include, but are not limited to, cytostatic proteins, cytocidal proteins, pro-apoptotic agents, anti-angiogenic agents, hormones, cytokines, growth factors, peptide drugs, antibodies, Fab antibody fragments, antigens, receptor proteins, enzymes, lectins, MHC proteins, cell adhesion proteins, and binding proteins. Methods for generating fusion proteins are well known to those skilled in the art. Such proteins may be generated, for example, by chemical attachment using bifunctional crosslinking reagents, by de novo synthesis of the complete fusion protein, or by attaching a DNA sequence encoding a targeting peptide to a DNA sequence encoding a second peptide or protein, followed by expression of the complete fusion protein.

In one embodiment, a targeting peptide (e.g., IL-21R) is fused to an immunoglobulin heavy chain constant region (such as an Fc fragment containing two constant region domains and a hinge region but lacking a variable region) (see, e.g., U.S. patent nos. 6,018,026 and 5,750,375, incorporated herein by reference). The Fc region may be a naturally occurring Fc region, or may be altered to improve certain properties, such as therapeutic properties, circulation time, reduced aggregation. Peptides and proteins fused to the Fc region typically exhibit a longer half-life in vivo than the unfused counterpart. In addition, fusion to the Fc region allows the fusion polypeptide to dimerize/multimerize.

One aspect of the invention includes binding proteins that bind IL-21R and antigen-binding fragments thereof. The present disclosure provides novel CDRs derived from a human immunoglobulin gene library. Typically, the protein structure used to carry the CDRs is an antibody heavy or light chain or portion thereof, wherein the CDRs are located in regions associated with naturally occurring CDRs. The structure and position of the variable domains can be determined as described in Kabat et al ((1991) supra).

Exemplary embodiments of the binding proteins (and antigen binding fragments thereof) of the present invention are identified as AbA-AbZ, H3-H6, L1-L6, L8-L21, and L23-L25. The DNA and amino acid sequences of non-limiting exemplary embodiments of anti-IL-21R binding proteins of the invention are set forth in SEQ ID NO: 5-195, 213-. DNA and amino acid sequences of some exemplary embodiments of the anti-IL-21R binding proteins of the invention, including scFv fragments thereof, V_HAnd V_LThe domains, and CDRs, as well as their present codes and previous names, are listed in FIGS. 17-25 and tables 2A and 2B.

Table 2A: relationship of present antibody code to previous name

Now code	Previous names
		AbA	VHP/VL2
AbB	VHP/VL3
		AbC	VHP/VL11
AbD	VHP/VL13
		AbE	VHP/VL14
AbF	VHP/VL17
		AbG	VHP/VL18
AbH	VHP/VL19
		AbI	VHP/VL24
AbJ	VH3/VLP

AbK	VH3/VL3
		AbL	VH3/VL13
AbM	VH6/VL13
		AbN	VH6/VL24
AbO	VHP/VL16；VHPTM/VL16
		AbP	VHP/VL20；VHPTM/VL20
AbQ	VH3/VL2；VH3DM/VL2
		AbR	VH3/VL18；VH3DM/VL18
AbS	VHP/VL6；VHPTM/VL6；VL6
		AbT	VHP/VL9；VHPTM/VL9；VL9
AbU	VHP/VL25；VHPTM/VL25
		AbV	VH3TM/VL2
AbW	VH3TM/VL18
		AbX	VHPDM/VL9
AbY	VHPg4/VL9
		AbZ	VHPWT/VL9

Table 2B: v of exemplary binding proteins of the invention_HAnd V_LAmino acid and nucleotide sequences of domains, scFv, and CDRs

TABLE 2B (continuation)

CDRL2

AA

NO：195

NO：195

NO：195

NO：195

NO：195

CDRL3

AA

NO：172

NO：173

NO：174

NO：175

NO：176

VH

DNA

NO：5

NO：5

NO：5

NO：5

NO：5

VL

DNA

NO：23

NO：25

NO：27

NO：29

NO：31

scFv

DNA

NO：119

NO：121

NO：123

NO：125

NO：127

TABLE 2B (continuation)

TABLE 2B (continuation)

TABLE 2B (continuation)

TABLE 2B (continuation)

The anti-IL-21R binding proteins of the invention may comprise antibody constant regions or portions thereof. For example, V_LThe domain may be attached at its C-terminus to a light chain constant domain, like ck or C λ. Similarly, V_HThe domains or portions thereof may be attached to all or part of the heavy chain, like IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. Constant regions are known in the art (see, e.g., Kabat et al (1991) supra). Thus, binding proteins within the scope of the invention include V in combination with constant regions known in the art_HAnd V_LA domain or a portion thereof.

Certain embodiments comprise V from Fv fragments of AbA-AbZ, H3-H6, L1-L6, L8-L21, and/or L23-L25_HDomain, V_LA domain, or a combination thereof. Other embodiments include from V_HAnd V_LOne, two, three, four, five or six CDRs of a domain. The CDR sequence is similar to SEQ ID NO: binding proteins in which one or more CDR sequences present within the sequences listed as 5-195, 213-229, and 239-248 are identical or similar (i.e., not substantially different) are encompassed within the scope of the present invention.

In certain embodiments, V_HAnd/or V_LThe domains may be germlined (fermlined) by mutating the FRs of these domains to match those produced by germ line cells using conventional molecular biology techniques. In other embodiments, the FR sequence remains distinct from the consensus germline sequence.

In one embodiment, mutagenesis is used to generate binding proteins that are more similar to one or more germline sequences. This may be desirable when mutations are introduced into the FRs of a binding protein (e.g., an antibody) via somatic mutagenesis or via error-prone PCR. V_HAnd V_LGermline sequences of domains can be identified by performing amino acid and nucleic acid sequence alignments against the VBASE database (MRC Center for Protein Engineering, UK). VBASE is from more than 1000 published sequences (including the latest version of GENBANK)And those in the EMBL data library) are compiled into a comprehensive list of all human germline variable region sequences. In some embodiments, the FR of the scFv is mutated to be consistent with the closest match in the VBASE database, while the CDR portion remains unchanged.

In certain embodiments, the binding proteins of the invention specifically react with the same epitope as that recognized by AbA-AbZ, H3-H6, L1-L6, L8-L21, or L23-L25 such that they competitively inhibit binding of AbA-AbZ, H3-H6, L1-L6, L8-L21, or L23-L25 to human IL-21R. Such binding proteins can be determined in competitive binding assays. In one embodiment, the binding constant (K) of these binding proteins to human IL-21R_A) Is at least 10⁵M^-1s^-1. Binding affinity the binding affinity may be usedKnown techniques to assay, such as ELISA, biosensor techniques (such as biospecific interaction assays), or other techniques, including those described in the present application.

The binding proteins of the invention may bind other proteins such as, for example, recombinant proteins comprising all or part of IL-21R.

One of ordinary skill in the art will recognize that the disclosed binding proteins can be used to detect, measure, and/or inhibit proteins that differ somewhat from IL-21R. For example, these proteins may be homologues of IL-21R. The anti-IL-21R binding protein is expected to bind to a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4, or at least about 60%, 70%, 80%, 90%, 95%, or more of any sequence of at least 100, 80, 60, 40, or 20 contiguous amino acids of the listed sequences.

In addition to sequence homology analysis, Epitope Mapping (see, e.g., Epitope Mapping Protocols (1996) Morris eds., Humana Press) and secondary and tertiary structure analysis can be performed to identify the specific 3D structures adopted by the presently disclosed binding proteins and their complexes with antigens. Such methods include, but are not limited to, x-ray crystallography (Engstom (1974) biochem. exp. biol. 11: 7-13) and Computer Modeling of the actual appearance of Current binding proteins (Fletterick et al (1986) Computer Graphics and Molecular Modeling in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The present disclosure provides a method for obtaining an anti-IL-21R binding protein. The method includes creating a composite having V as disclosed herein_HAnd/or V_LV resulting from sequence changes_HAnd/or V_LA binding protein of sequence. Such binding proteins may be derived by the skilled person using techniques known in the art. For example, amino acid substitutions, deletions, or additions may be introduced in the FR and/or CDR regions. FR alterations are typically designed to improve the stability and immunogenicity of a binding protein, while CDR alterations are typically designed to increase the affinity of a binding protein for its antigen. The affinity may be improved byOne or more CDR sequences are altered and the affinity of the binding protein to its target is measured (see, e.g., Antibody Engineering (2 nd edition, 1995) borreback, ed. by Oxford University Press).

The CDR sequences of which differ substantially from the sequences SEQ ID NO: binding proteins having CDR sequences set forth in or contained in 5-195, 213-229, and 239-248 are encompassed by the present invention. Typically, such insubstantial differences involve the substitution of an amino acid with an amino acid having similar charge, hydrophobicity, or stereochemical characteristics. In contrast to the CDR regions, more drastic substitutions may also be made in the FR regions, provided that they do not adversely affect the binding characteristics of the binding protein (e.g., by more than 50% decrease in affinity compared to the binding protein without substitution). Substitutions may also be made to germline the binding protein or to stabilize its antigen binding site.

Conservative modifications will produce molecules having functional and chemical characteristics similar to those of the molecule undergoing such modification. In contrast, substantial modification of the function and/or characteristics of a molecule can be achieved by selecting amino acid sequence substitutions that differ significantly in their effect on maintaining: (1) the structure of the molecular backbone of the replacement region, such as a sheet or helical conformation; (2) the charge or hydrophobicity of the molecule at the target site; and/or (3) the size of the molecule.

For example, a "conservative amino acid substitution" may involve the replacement of a natural amino acid residue with a standard residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position (see, e.g., MacLennan et al (1998) Acta Physiol. Scand. suppl.643: 55-67; Sasaki et al (1998) adv. Biophys.35: 1-24).

Desired amino acid substitutions (conservative or non-conservative) may be determined by one of skill in the art when such substitutions are desired. For example, amino acid substitutions can be used to identify important residues in the sequence of a molecule, or to increase or decrease the affinity of a molecule described herein. Exemplary amino acid substitutions include, but are not limited to, those listed in table 3.

Table 3: exemplary amino acid substitutions

Initial residue	Example alternatives	More conservative substitutions
			Ala(A)	Val，Leu，Ile	Val
Arg(R)	Lys，Gln，Asn	Lys
			Asn(N)	Gln	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser，Ala	Ser
Gln(Q)	Asn	Asn
			Gly(G)	Pro，Ala	Ala
His(H)	Asn，Gln，Lys，Arg	Arg
			Ile(I)	Leu, Val, Met, Ala, Phe, norleucine	Leu
Leu(L)	Norleucine, Ile, Val, Met, Ala, Phe	Ile
			Lys(K)	Arg, 1, 4-diaminobutyric acid, Gln, Asn	Arg
Met(M)	Leu，Phe，Ile	Leu
			Phe(F)	Leu，Val，Ile，Ala，Tyr	Leu
Pro(P)	Ala，Gly	Gly

Ser(S)	Thr，Ala，Cys	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr，Phe	Tyr
			Tyr(Y)	Trp，Phe，Thr，Ser	Phe
Val(V)	Ile, Met, Leu, Phe, Ala, norleucine	Leu

In certain embodiments, conservative amino acid substitutions also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems.

In one embodiment, for generating variant V_HThe method of the domain includes adding, deleting, or replacing the disclosed V_HAt least one amino acid in the domain, or V disclosed in combination_HDomains and at least one V_LDomain, and for mutation V_HDomain-testing of IL-21R binding orIL-21R/IL-21 activity modulation.

For generating variant V_LSimilar methods for domains include V disclosed in_LAddition, deletion, or substitution of at least one amino acid in a domain, or combination of the disclosed V_LDomains and at least one V_HDomain, and for mutation V_LThe domain tests IL-21R binding or IL-21R activity modulation.

In some alternative embodiments, the anti-IL-21R binding protein may be linked to a protein (e.g., albumin) by chemical cross-linking or recombinant means. Also disclosed in U.S. Pat. Nos. 4,640,835; 4,496,689, respectively; 4,301,144, respectively; 4,670,417, respectively; 4,791,192, respectively; and 4,179,337 to link the disclosed binding proteins to a variety of non-proteinaceous polymers (e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes). Binding proteins may be chemically modified by covalent coupling to polymers, for example in order to extend their half-life in the blood circulation. Exemplary polymers and attachment methods are shown in U.S. Pat. nos. 4,766,106; 4,179,337; 4,495,285, respectively; and 4,609,546.

The disclosed binding proteins can be modified to alter their glycosylation; that is, at least one carbohydrate moiety may be deleted or added to the binding protein. Deletion or addition of glycosylation sites can be accomplished by altering the amino acid sequence to delete or create glycosylation consensus sites well known in the art. Another means of adding a carbohydrate moiety is to chemically or enzymatically couple a glycoside to an amino acid residue of a binding protein (e.g., an antibody) (see, e.g., International application publication No. WO 87/05330 and Aplin et al (1981) CRC Crit. Rev. biochem. 22: 259-306). The elimination of carbohydrate modules can also be achieved chemically or enzymatically (see, for example, Hakimuddin et al (1987) Arch. biochem. Biophys.259: 52; Edge et al (1981) anal. biochem.118: 131; and Thotakura et al (1987) meth. enzymol.138: 350). Modifications to the carbohydrate structure may be preferred because amino acid changes in the Fc domain may enhance the immunogenicity of the pharmaceutical composition (see, e.g., international publication No. WO 2008/052030). For immunoglobulin molecules, it has been demonstrated that N-linked carbohydrates are attached to C_HAsn 297 of the 2 domain is essential for ADCC activity. Its elimination enzymatically or via N-linked consensus site mutations results in little to no ADCC activity. In glycoproteins, carbohydrates can be attached to the amide nitrogen atom in the side chain of asparagine in the tripeptide motif Asn-X-Thr/Ser. This type of glycosylation (known as N-linked glycosylation) begins in the Endoplasmic Reticulum (ER) with the addition of multiple monosaccharides to dolichol phosphate to form a 14-residue branched carbohydrate complex. This carbohydrate complex is then transferred to the protein by the oligosaccharyl transferase (OST) complex. Three glucose molecules are eliminated from 14 residue oligosaccharides before the glycoprotein leaves the ER lumen. The enzymes ER glucosidase I, ER glucosidase II and ER mannosidase are involved in ER processing. Subsequently, the polypeptide is transported to the Golgi complex where the N-linked sugar chain is modified in a number of different ways. In the cis and middle compartments of the golgi complex, the first N-linked complex of 14 sugars can be trimmed via elimination of mannose (Man) residues and extended via addition of N-acetylglucosamine (GlcNac) and/or fructose (Fuc) residues. The various forms of N-linked carbohydrates generally have a common pentasaccharide core, consisting of three mannose and two N-acetylglucosamine residues. Finally, in the trans-golgi, additional GlcNac residues may be added, followed by galactose (Gal) and terminal sialic acid (Sial). Carbohydrate processing in the golgi complex is referred to as "terminal glycosylation" to distinguish it from "core glycosylation" occurring in the ER. The final complex carbohydrate unit may take a variety of forms and structures, some of which have two, three, or four branches (referred to as di-, tri-, or tetra-branched). Various enzymes are involved in golgi processing, including golgi mannosidases IA, IB and IC, GlcNAc transferase I, golgi mannosidase II, GlcNAc transferase II, galactosyltransferase and sialyltransferase.

Methods for altering the constant region of a binding protein, such as, for example, the constant region of an antibody, are known in the art. Binding proteins with altered function (e.g., altered affinity for effector ligands such as the FcR on a cell or the C1 component of complement) can be generated by replacing at least one amino acid residue in the constant portion with a different residue (see, e.g., european application publication No. EP 0388151 and U.S. Pat. nos. 5,624,821 and 5,648,260). Similar types of changes that would reduce or eliminate similar function if applied to binding proteins in murine or other species can be described.

For example, it is possible to alter the affinity of the Fc region of a binding protein (e.g., IgG, such as human IgG) for FcR (e.g., Fc gamma R1) or C1 q. Affinity can be altered by replacing at least one defined residue with at least one residue having the appropriate functionality on its side chain or by introducing a charged functional group (such as glutamic acid or aspartic acid) or possibly an aromatic nonpolar residue (such as phenylalanine, tyrosine, tryptophan, or alanine) (see, e.g., U.S. Pat. No.5,624,821).

For example, replacement of residue 297 (asparagine) in the IgG constant region with alanine significantly inhibited recruitment of effector cells while only slightly decreasing (by about 3-fold weaker) the affinity for CIq (see, e.g., U.S. patent No.5,624,821). The numbering of residues in the heavy chain is that of the EU index (see Kabat et al (1991) supra). This alteration disrupts glycosylation sites and the presence of carbohydrates is thought to be required for Fc receptor binding. Any other substitution at this site that disrupts the glycosylation site is believed to cause a similar decrease in lytic activity. Other amino acid substitutions, such as any of residues 318(Glu), 320(Lys), and 322(Lys) to Ala, are known to also eliminate the binding of C1q to the Fc region of IgG antibodies (see, e.g., U.S. Pat. No.5,624,821).

Modified binding proteins having reduced interaction with Fc receptors can be produced. For example, it is shown that in human IgG3, which binds to the human Fc gamma R1 receptor, changing Leu 235 to Glu disrupts its interaction with the receptor. Mutations in the binding protein at adjacent or nearby sites in the hinge region (e.g., replacement of residues 234, 235, and 237 with Ala) can also be used to alter the affinity of the binding protein for the Fc gamma R1 receptor. The numbering of residues in the heavy chain is based on the EU index (see Kabat et al (1991) supra). Thus, in some embodiments of the invention, the Fc region of the binding proteins of the invention contains at least one constant region mutation, such as, for example, Leu 234 to Ala (L234A), Leu 235 to Ala (L235A), and/or Gly 237 to Ala (G237A). In one embodiment, the Fc region of the binding protein contains two constant region mutations, L234A and G237A (i.e., "double mutant" or "DM"). In another embodiment, the Fc region of the binding protein contains three constant region mutations, i.e., L234A, L235A, and G237A (i.e., "triple mutant" or "TM"). For example, a human IgG constant region triple mutant is set forth in SEQ ID NO: 196.

other methods for altering the lytic activity of binding proteins, e.g. by altering C_HAt least one amino acid in the N-terminal region of domain 2 is described in International application publication No. WO 94/029351 and U.S. Pat. No.5,624,821.

The binding proteins of the invention may be labeled with a detectable or functional label. These include radioactive labels (e.g.¹³¹I and⁹⁹tc), enzyme labels (e.g., horseradish peroxidase and alkaline phosphatase), and other chemical modules (e.g., biotin).

The invention also features an isolated binding protein or antigen-binding fragment thereof that binds IL-21R, particularly human IL-21R. In certain embodiments, an anti-IL-21R binding protein may have at least one of the following characteristics: (1) it is a monoclonal or monospecific binding protein; (2) it is a human binding protein; (3) it is a binding protein produced in vitro; (4) it is a binding protein produced in vivo (e.g., transgenic mouse system); (5) it inhibits IL-21 binding to IL-21R; (6) it is IgG 1; (7) it is at least about 10⁵M^-1s^-1Binds to human IL-21R; (8) it is at least about 5x10⁴M^-1s^-1Binds to murine IL-21R; (9) it has a molecular weight of about 10^-3(1/s) or less dissociation constant binds to human IL-21R; (10) it has a molecular weight of about 10^-2(1/s) or less dissociation constant binds murine IL-21R; (11) it has an IC of about 1.75nM or less₅₀Inhibiting human IL-21R-mediated proliferation of human IL-21R-expressing BaF3 cells; (12) it has an IC of about 0.5nM or less₅₀Inhibiting murine IL-21R-mediated proliferation of murine IL-21R-expressing BaF3 cells; (13) it has an IC of about 14.0nM or less₅₀Inhibiting human IL-21R-mediated proliferation of human IL-21R-expressing TF1 cells; (14) it has an IC of about 1.9nM or less₅₀Inhibiting IL-21 mediated proliferation of human primary B cells; (15) it has an IC of about 1.5nM or less₅₀Inhibition of IL-21 mediated human primary CD4⁺T cell proliferation; and (16) its IC at about 5.0nM or less₅₀Inhibition of IL-21 mediated murine primary CD4⁺T cells proliferate.

Those skilled in the art will appreciate that the modifications described above are not exhaustive and that many other modifications will be apparent to the skilled person in light of the teachings of this disclosure.

Nucleic acid, cloning and expression system

The present disclosure provides isolated nucleic acids encoding the disclosed binding proteins. Nucleic acids may comprise DNA or RNA, and they may be synthetic (in whole or in part) or recombinant (in whole or in part). Reference to the nucleotide sequences listed herein encompasses DNA molecules having the specified sequence, but also encompasses RNA molecules having the specified sequence, wherein U replaces T.

Also contemplated are CDRs comprising one, two, or three CDRs as disclosed herein, V_HDomain, V_LA domain, or a combination thereof, or a sequence substantially identical thereto (e.g., a sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical thereto, or a sequence capable of hybridizing to these sequences under stringent conditions).

In one embodiment, the isolated nucleic acid has a nucleotide sequence encoding the heavy and light chain variable regions of an anti-IL-21R binding protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 163-195, or a sequence encoding a CDR that differs from the sequences described herein by one or two or three or four amino acids.

The nucleic acid may encode only the light or heavy chain variable region, or may encode a binding protein light or heavy chain constant region, optionally linked to a corresponding variable region. In one embodiment, the light chain variable region is linked to a constant region selected from the kappa or lambda constant regions. The light chain constant region may also be of the human kappa or lambda type. In another embodiment, the heavy chain variable region is attached to a heavy chain constant region of a binding protein isotype selected from IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA1, IgA2, IgD, and IgE. The heavy chain constant region may be of the IgG (e.g., IgG1) isotype.

Although often a natural sequence (of cDNA or genomic DNA or mixtures thereof) except for modified restriction sites and the like, the nucleic acid compositions of the invention may be mutated according to standard techniques to provide a gene sequence. For coding sequences, these mutations may alter the amino acid sequence as desired. In particular, nucleotide sequences substantially identical to or derived from native V, D, J, invariant, transformed, and other such sequences described herein are contemplated (wherein "derived" indicates that a sequence is identical to or modified from another sequence).

In one embodiment, the nucleic acid differs (e.g., differs by substitution, insertion, or deletion) from the appropriate (e.g., by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10%, or 20% of the nucleotides in the test nucleic acid) of the provided sequence. If necessary for this analysis, the sequences should be aligned for maximum homology. Sequences that are "looped" for deletion or insertion or mismatch are considered differences. The difference may be a nucleotide encoding a non-critical residue, or the difference may be a conservative substitution.

The present disclosure also provides nucleic acid constructs in the form of plasmids, vectors, and transcription or expression cassettes comprising at least one nucleic acid described herein.

The present disclosure further provides a host cell comprising at least one nucleic acid construct as described herein.

Also provided are nucleic acid constructs prepared from nucleic acids comprising sequences described hereinMethods of encoding proteins. The method comprises culturing the host cells under suitable conditions such that they express the protein from the nucleic acid. Following expression and production, V can be isolated and/or purified using any suitable technique_HOr V_LDomain, or specific binding member, and then used as appropriate. The method may further comprise the steps of fusing a nucleic acid encoding the scFv to a nucleic acid encoding the Fc portion of the binding protein and expressing the fused nucleic acid in the cell. The method may further comprise a step of germlining.

Antigen-binding fragment, V_HAnd/or V_LThe domains, and encoding nucleic acid molecules and vectors, may be isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or in the case of nucleic acids, free or substantially free of nucleic acids or genes of origin other than the sequence encoding a polypeptide having the desired function.

Systems for cloning and expressing polypeptides in a variety of host cells are known in the art. Cells suitable for the production of binding proteins are described, for example, in Fernandez et al (1999) Gene Expression Systems, Academic Press. Briefly, suitable host cells include mammalian cells, insect cells, plant cells, yeast cells, or prokaryotic cells, such as E.coli. Mammalian cells useful in the art for heterologous polypeptide expression include lymphocyte cell lines (e.g., NSO), HEK293 cells, Chinese Hamster Ovary (CHO) cells, COS cells, HeLa cells, baby hamster kidney cells, oocytes, and cells from transgenic animals, such as mammary epithelial cells. In other embodiments, the nucleic acid encoding the binding protein of the invention is placed under the control of a tissue-specific promoter (e.g., a mammary-specific promoter), and the binding protein is produced in a transgenic animal. For example, the binding protein is secreted into the milk of a transgenic animal (such as a transgenic cow, pig, horse, sheep, goat, or rodent).

Suitable vectors can be selected or constructed to contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation signals, enhancer sequences, marker genes, and other sequences. The vector may also comprise a plasmid or viral backbone. For details see, e.g., Sambrook et al, Molecular Cloning: a Laboratory Manual (2 nd edition, 1989) Cold Spring Harbor Laboratory Press. Many established techniques for use with vectors, including manipulation, preparation, mutagenesis, sequencing, and transfection of DNA, are described, for example, in Current Protocols in Molecular Biology (2 nd edition, 1992), eds by Ausubel et al, John Wiley & Sons.

Yet another aspect of the disclosure provides a method of introducing a nucleic acid into a host cell. For eukaryotic cells, suitable transfection techniques may include calcium phosphate, DEAE dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia or baculovirus). For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophages. DNA introduction may be followed by selection methods (e.g., drug resistance) to select for cells containing the nucleic acid.

Use of anti-IL-21R binding proteins

An anti-IL-21R binding protein that acts as an IL-21R antagonist can be used to reduce at least one IL-21R-mediated immune response, such as one or more of cell proliferation, cytokine expression or secretion, chemokine secretion, and cytolytic activity of T cells, B cells, NK cells, macrophages, or synoviocytes. Thus, the binding proteins of the invention are useful for inhibiting the activity (e.g., proliferation, differentiation, and/or survival) of immune or hematopoietic cells (e.g., cells of the myeloid, lymphoid, or erythroid lineages, or precursors thereof), and thus are useful for treating a variety of immunological and hematologic hyperproliferative disorders. Examples of immune disorders that can be treated include, but are not limited to, transplant rejection, Graft Versus Host Disease (GVHD), allergies (e.g., atopic allergy), and autoimmune diseases. Autoimmune diseases include diabetes, arthritic conditions (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), spondyloarthropathies, multiple sclerosis, encephalomyelitis, myasthenia gravisAsthenia, systemic lupus erythematosus, cutaneous lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's diseaseSyndrome, IBD (including Crohn's disease and ulcerative colitis), asthma (including intrinsic asthma and allergic asthma), scleroderma and vasculitis.

Combination therapy

In one embodiment, a pharmaceutical composition comprising at least one anti-IL-21R binding protein and at least one therapeutic agent is administered in a combination therapy. The therapy is useful for treating pathological conditions or disorders, such as immune and inflammatory disorders. The term "combination" in this context means that the binding protein composition and the therapeutic agent are administered substantially simultaneously, either simultaneously or sequentially. If administered sequentially, an effective concentration of the first of the two compounds is still detected at the treatment site at the beginning of the administration of the second compound.

For example, combination therapy may include at least one anti-IL-21R binding protein, such as, for example, an anti-IL-21R antibody, co-formulated and/or co-administered with at least one additional therapeutic agent. Additional agents may include at least one cytokine inhibitor, growth factor inhibitor, immunosuppressive agent, anti-inflammatory agent, metabolic inhibitor, enzyme inhibitor, cytotoxic agent, and/or cytostatic agent. Such combination therapies may advantageously utilize lower doses of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with each monotherapy. Furthermore, the therapeutic agents disclosed herein act on pathways different from the IL-21/IL-21R pathway and are therefore expected to enhance and/or synergize the effects of anti-IL-21R binding proteins.

Another aspect of the invention relates to kits for performing combined administration of an anti-IL-21R binding protein and other therapeutic agents. In one embodiment, the kit comprises at least one anti-IL-21R binding protein and at least one therapeutic agent, suitably formulated in one or more separate pharmaceutical formulations, formulated in a pharmaceutical carrier.

Diagnostic use

The binding proteins of the invention are also useful for detecting the presence of IL-21R in a biological sample. By correlating the presence or levels of these proteins with a medical condition, one skilled in the art can diagnose the relevant medical condition. For example, stimulated T cells increase their IL-21R expression, and in general high concentrations of IL-21R expressing T cells in joints may be indicative of joint inflammation and possibly arthritis. Exemplary medical conditions that can be diagnosed by the binding proteins of the invention include, but are not limited to, multiple sclerosis, rheumatoid arthritis, and transplant rejection.

Binding protein-based detection methods, such as those commonly used for antibodies, are well known in the art and include ELISA, radioimmunoassay, immunoblot, Western blot, flow cytometry, immunofluorescence, immunoprecipitation, and other related techniques. Binding proteins can be provided in a diagnostic kit incorporating at least one of these protocols to detect IL-21R. The kit may contain other components, packaging, instructions, reagents, and/or other materials to aid in the detection of the protein and use of the kit.

The binding protein may be modified with a detectable marker, including a ligand group (e.g., biotin), a fluorophore, a chromophore, a radioisotope, an electron-dense reagent, or an enzyme. Enzymes are detected by their activity. For example, horseradish peroxidase is detected by its ability to convert Tetramethylbenzidine (TMB) to a blue pigment, which can be spectrophotometrically quantified. Other suitable binding partners include biotin and avidin, IgG and protein a, and other receptor-ligand pairs known in the art.

The binding protein may also be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to at least one other molecular entity, such as another binding protein (e.g., a bispecific or multispecific binding protein), a toxin, a radioisotope, a cytotoxic or cytostatic agent, or the like. Other variations and possibilities will be apparent to those of ordinary skill in the art and are considered equivalents within the scope of the present invention.

Pharmaceutical compositions and methods of administration

Certain embodiments of the invention include compositions comprising the disclosed binding proteins. The composition may be suitable for pharmaceutical use and administration to a patient. The compositions comprise a binding protein of the invention and a pharmaceutical excipient. As used herein, "pharmaceutical excipients" include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of these agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds to provide supplemental, additional, or enhanced therapeutic functions. The pharmaceutical composition may also be included in a container, pack, or dispenser with instructions for administration.

The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Methods of effecting administration are known to those of ordinary skill in the art. The pharmaceutical composition may be administered topically or orally, or may be capable of traversing a mucosal membrane. Examples of administration of pharmaceutical compositions include oral ingestion or inhalation. Administration can also be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, dermal, or transdermal.

Solutions or suspensions for intradermal or subcutaneous application typically include at least one of the following: a sterile diluent, such as water, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and tonicity agents such as sodium chloride or dextrose. Methods known in the art may be provided for adjusting the pH with an acid or base. Such preparation preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials.

Solutions or suspensions for intravenous administration include carriers such as physiological saline, bacteriostatic water, CREMOPHOR EL(BASF Corp., Ludwigshafen, Germany), ethanol, or a polyol. In all cases, the composition must be sterile and fluid to facilitate injection. Proper fluidity can often be achieved using lecithin or surfactants. The composition must also be stable under the conditions of manufacture and storage. Protection against microorganisms can be achieved with antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents (sugars), polyols (e.g., mannitol and sorbitol), sodium chloride may be included in the composition. Delayed absorption of the composition can be brought about by the addition of agents which delay absorption, such as aluminum monostearate and gelatin.

Oral compositions include an inert diluent or an edible carrier. For the purpose of oral administration, the binding protein may be incorporated with excipients and placed in, for example, tablets, lozenges, capsules, or gelatin. Pharmaceutically compatible binders or adjuvant materials may be included in the composition. The composition may contain (1) a binder such as microcrystalline cellulose, tragacanth or gelatin; (2) excipients, such as starch or lactose; (3) disintegrants, such as alginic acid, Primogel, or corn starch; (4) lubricants, such as magnesium stearate; (5) glidants such as colloidal silicon dioxide; and/or (6) sweetness or aromaticity.

The compositions may also be administered by transmucosal or transdermal routes. For example, a binding protein (e.g., an antibody) comprising an F region may be capable of passing through mucosa (via Fc receptors) in the intestine, mouth, or lung. Transmucosal administration can be accomplished through lozenges, nasal sprays, inhalants, or suppositories. Transdermal administration may be accomplished using ointments, salves, gels, or creams containing the compositions as known in the art. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used. For administration by inhalation, the binding protein may be delivered as an aerosol spray from a pressurized container or dispenser containing a propellant (e.g., liquid or gas), or a nebulizer.

In certain embodiments, the binding proteins of the invention are prepared with a carrier to protect the binding protein from rapid clearance from the body. Biodegradable polymers (e.g., ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid) are often used. Methods for preparing such formulations are known to those skilled in the art. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. Liposomes can be prepared according to established methods known in the art (see, e.g., U.S. Pat. No.4,522,811).

The binding protein or binding protein composition of the invention is administered in a therapeutically effective amount as described. The therapeutically effective amount may vary with the age, condition, sex, and severity of the pharmaceutical condition of the subject. The physician can determine the appropriate dosage based on clinical indications. The binding protein or composition may be administered as a bolus dose (bolus dose) to maximize circulating levels of binding protein for the longest period of time. Continuous infusion may also be used.

As used herein, the term "subject" is intended to include both human and non-human animals. The subject may include a human patient having a disorder characterized by cells that express an IL-21R (e.g., cancer cells or immune cells). The term "non-human animal" in the present invention includes all vertebrates such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

Examples of dosage ranges that can be administered to a subject may be selected from: 1 to 20mg/kg, 1 to 10mg/kg, 1 to 1mg/kg, 10 to 100 μ g/kg, 100 to 1mg/kg, 250 to 2mg/kg, 250 to 1mg/kg, 500 to 2mg/kg, 500 to 1mg/kg, 1 to 2mg/kg, 1 to 5mg/kg, 5 to 10mg/kg, 10 to 20mg/kg, 15 to 20mg/kg, 10 to 25mg/kg, 15 to 25mg/kg, 20 to 25mg/kg, and 20mg/kg to 30mg/kg (or higher). These doses may be administered once daily, once weekly, once biweekly, once monthly, or less frequently, e.g., twice annually, depending on the dose, method of administration, condition or symptom to be treated, and the characteristics of the individual subject.

In certain instances, it may be advantageous to formulate the compositions in dosage unit form for convenient administration and consistent dosage. As used herein, dosage unit form refers to physically discrete units suitable for use in a patient. Each dosage unit contains a predetermined amount of binding protein in combination with a carrier that, on a calculated basis, produces a therapeutic effect. The dosage unit will depend on the characteristics of the binding protein and the particular therapeutic effect to be achieved.

Toxicity and therapeutic efficacy of the compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD₅₀(dose lethal to 50% of the population) and ED₅₀(a therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects, i.e. the therapeutic index, and it can be expressed as the ratio LD₅₀/ED₅₀. Binding proteins exhibiting a large therapeutic index may be low in toxicity and/or high in therapeutic efficacy.

Data obtained from cell culture assays and animal studies can be used to elucidate the dose range in humans. The dose of these compounds may lie within the range of circulating binding protein concentrations in the blood, which includes ED with little or no toxicity₅₀. The dosage may vary within this range depending upon the dosage composition form employed and the route of administration. For any binding protein used in the present invention, a therapeutically effective dose can be initially estimated using cell culture assays. The inclusion of IC can be demonstrated in animal models₅₀(i.e., the concentration of binding protein that achieves half-maximal inhibition of the symptom) of circulating plasma concentration range. The effect of any particular dose can be monitored by a suitable bioassay. Examples of suitable bioassays include DNA replication assays, transcription-based assays, gene expression assays, IL-21/IL-21R binding assays, and other immunological assays.

All references, patent applications, and patents cited throughout this application are hereby incorporated by reference in their entirety.

Examples

The invention is further illustrated in the following non-limiting examples. These examples are set forth to aid in understanding the invention and are not intended to, and should not be construed to, limit its scope in any way. The examples do not include detailed descriptions of conventional methods that would be well known to one of ordinary skill in the art.

Example 1: generation of binding proteins by phage display

The scFv parental clone 18A5 described in U.S. Pat. No.7,495,085 (incorporated herein by reference) was obtained from CS human scFv libraries by standard phage display using BaF3 cells expressing human IL-21R as targets in rounds 1 and 3 and biotinylated IL-21R-Fc fusion protein as targets in round 2.

Example 2: library construction

The phage display library was based on the parental 18a5scFv using the pCANTAB6 vector, where the scFv was fused at its 3' end to the complete gene III. Various CDR3 sequences were derived using techniques well known in the art.

At V_HAnd V_LRandomizing two overlapping blocks of six consecutive codons in CDR3 of (a), generating a total of four libraries: H3B1, H3B2, L3B1, and L3B 2. The nucleotide and amino acid sequences are identified below, respectively: IL-21R: 18A5V_HCDR3[ SEQ ID NO: 199 and 200](ii) a H3B1 (library Capacity 1.40X 10)⁹) [ SEQ ID NO: 201 and 202](ii) a H3B2 (library Capacity 1.00X 10)⁹) [ SEQ ID NO: 203 and 204]；IL-21R：18A5 V_LCDR3[ SEQ ID NO: 205 and 206, respectively](ii) a L3B1 (library Capacity 9.00X 10)⁹) [ SEQ ID NO: 207 and 208](ii) a L3B2 (library Capacity 6.40X 10)⁹) [ SEQ ID NO: 209 and 210]。

Example 3: phage selection

Isolating all derivatives of 18a5 from the scFv library above by selecting phages capable of binding in solution phase to biotinylated human IL-21R ectodomain His-Flag fusion protein ("biotin-hIL-21R-H/F") and biotinylated murine IL-21R ectodomain His-Flag fusion protein ("biotin-mIL-21R-H/F"); all procedures and techniques involving selection are well known to those skilled in the art. A total of 27 anti-IL-21R scFv were isolated by phage selection protocol.

Example 4: library screening

The resulting binding proteins in scFv format were selected based on their ability to compete with the parent 18A5 in human IgG1 format for binding to biotin-hIL-21R-H/F and biotin-mIL-21R-H/F to prevent hIL-21-dependent proliferation of genetically engineered cell lines expressing human IL-21R and mIL-21-dependent proliferation of genetically engineered cell lines expressing murine IL-21R.

Example 4.1: preparation of crude periplasmic material ("periplasmic preparation") for use in screening assays

Depending on the growth conditions used, the scFv can be expressed in solution in the bacterial periplasmic space. To induce scFv release into the periplasm, a 96-well deep-well plate containing 990. mu.l of 2XTY medium containing 0.1% glucose/100. mu.g/ml ampicillin was inoculated with the thawed glycerol stock (one clone per well) using a QPix2 colony harvester (Genetix, New Milton, England) and incubated at 37 ℃ (999rpm) for about 4 hours. Cultures were induced with IPTG at a final concentration of 0.02mM and cultured overnight at 30 deg.C (999 rpm). The contents of the bacterial periplasm (periplasmic preparation) are released by osmotic shock. Briefly, plates were centrifuged and pellets were resuspended in 150. mu.l TES periplasmic buffer (50mM Tris/HCl (pH 8.0)/1mM EDTA (pH 8.0)/20% sucrose), followed by addition of 150. mu.l 1: 5 TES: water and incubation on ice for 30 min. Plates were centrifuged and supernatants containing scFv were harvested.

Example 4.2: epitope competition assays for library screening

Those scfvs that compete with the parent 18a5 antibody for binding to human or murine IL-21R are fluorescence resolved by homogeneous time-resolved fluorescence (HTRF)) The assay is identified from a selected phage. According to HTRFInstructions in the Cryptate labeling kit (Cisbio, Bedford, MA) purified parental 18a5 antibody was covalently modified with Cryptate (a derivative of europium). Periplasmic preparations of scFv were prepared as described above and incubated in PBS/0.4M Potassium fluoride/0.1% BSA (HTRF)Buffer) to 0.25%; mu.l of the mixture was then transferred to wells of a black 384-well shallow well plate (Nunc, Rochester, NY). Mu.l of Cryptate-conjugated 18A5 antibody was then added to each well, followed by a 1: 800 dilution of streptavidin-XL 665 conjugate (Cisbio) in 5. mu.l mixture with either 4.8nM biotin-hIL-21R-H/F or 40nM biotin-mIL-21R-H/F. The mixture was incubated at room temperature for 2 hours and time resolved fluorescence measurements (340nm excitation, 615nm and 665nm emission) were performed. Competition with the 18a5 antibody is indicated by a decrease in the background-corrected ratio of emission at 665nm to emission at 615 nm.

At HTRFA total of 8280 independently isolated scFv were screened using human IL-21R-H/F in the assay, and 376 clones that were able to compete with the parent 18A5 antibody for binding to biotin-hIL-21R-H/F were selected for further analysis.

Examples5: DNA sequence analysis of library-derived scFv-PCR amplification of scFv regions for sequencing For analysis

The sequence of 287 18A5 derived scFv variants with improved IL-21R binding compared to the parent 18A5 was determined and the amino acid frequency at each position was determined. At V_HOf the clones, only two (1.7%) were derived from mutations such as SEQ ID NO: 169 last six amino acids (at V)_HC-terminal of CDR3), while the remainder are derived from, for example, SEQ ID NO: 169 library of the first six amino acids. At V_LOf the clones, only one clone (0.6%) was derived from a mutation such as SEQ ID NO: 170 (at V)_LC-terminal of CDR3), while most are derived from e.g. SEQ ID NO: 170 (at V)_LN-terminal of CDR 3).

Using VENTPCR amplification of scFv was performed with DNA polymerase (New England Biolabs, Ispwich, MA) in HN buffer (Epicentre Biotechnologies, Madison, Wis.) according to the manufacturer's instructions. Mu.l of a 1: 10 diluted stationary phase bacterial culture was used as template for a final reaction volume of 20. mu.l. The cycling conditions used were 94 ℃ 2 min hot start, 30 cycles of 94 ℃ denaturation (1 min), 55 ℃ primer annealing (2 min) and 72 ℃ extension (1 min), followed by 72 ℃ final extension (5 min). The PCR products were checked by agarose gel electrophoresis and cleaned with ExoI/SAP (shrimp alkaline phosphatase) before sequencing with M13rev primer.

The SEQ ID NOs of the CDR3 sequences of the 27 scfvs are listed in table 4. These scfvs were selected for further analysis based on the assay described in example 6.

Table 4: CDR3 SEQ ID NO of improved 18A5 derived scFv

scFv	Heavy CDR3	Light CDR3
			H3	165	170
H4	166	170
			H5	167	170
H6	168	170
			L1	169	171

L2	169	172
			L3	169	173
L4	169	174
			L5	169	175
L6	169	176
			L8	169	177
L9	169	178
			L10	169	179
L11	169	180
			L12	169	181
L13	169	182
			L14	169	183
L15	169	184
			L16	169	185
L17	169	186
			L18	169	187
L19	169	188
			L20	169	189
L21	169	190
			L23	169	191
L24	169	192
			L25	169	193

Example 6: characterization of library-derived scFv

Example 6.1: preparation of purified scFv for quantitative analysis

In PHYTIPEach scFv clone was purified on a small scale by Ni-NTA purification on a column (PhyNexus, inc., San Jose, CA). A single colony was cultured in a 50ml conical tube to mid-log phase in 20ml of 2XTY medium containing 0.1% glucose/100. mu.g/ml ampicillin with shaking at 250rpm at 37 ℃. scFv expression was induced with IPTG at a final concentration of 0.02mM, and the cultures were incubated overnight at 30 ℃. Cells were harvested by centrifugation and resuspended in 1ml TES periplasmic buffer, followed by addition of 1ml 1: 5 TES: water and incubation on ice for 30 minutes. The lysate was centrifuged at 3200rpm for 10 min at 4 ℃ and the supernatant was brought to 2mM MgCl₂. In Ni-NTA PHYTIPs(PhyNexus) by passing the supernatant repeatedly through a Perkin Elmer (Waltham, MA) MINITRAK^TMPHYTIPs on IX liquid handling robotTo capture scFv, followed by washing in IMAC wash buffer and elution with 200mM imidazole, 50mM Tris, 300mM NaCl (pH 8.0). Buffer exchange to PBS was performed by three 1: 10 dilutions into PBS followed by 10,000 molecular weight cut-off filter plates (Millipore MultiTectoren)ULTRACEL^TM96 well ultra filter plates, Millipore, Billerica, MA). Use of Micro BCA^TMKit (Thermo Fisher Scientific inc., Rockford, IL) samples were quantified using a manufacturer's bovine serum albumin standard.

Example 6.2: assay for IL-21 dependent proliferation of cells overexpressing human or murine IL-21R Method of

Inhibition assays were performed with 18A 5-derived binding proteins (scFv and IgG) to measure their blocking of IL-21-dependent proliferation of cell lines transfected with human or murine IL-21R. BaF3 cells (a murine pre-B cell line) and TF1 cells (a human red blood cell line) were transduced with IL-21R and Green Fluorescent Protein (GFP) via retroviruses. Cells were routinely cultured in RPMI 1640 containing 10% FBS, 2mM L-glutamine, 100U/ml penicillin, 100. mu.g/ml streptomycin, and 0.00036% β -mercaptoethanol. Human IL-21R-BaF3 cell cultures were supplemented with 50ng/ml human IL-21; murine IL-21R-BaF3 cell cultures were supplemented with 10U/ml IL-3; TF1 cell cultures were supplemented with 50ng/ml GM-CSF. Prior to assay, cells were washed 3 times in assay medium lacking supplemental growth factors, resuspended in assay medium, and resuspended at 37 ℃/5% CO₂Incubate for 6 hours. To prepare the assay plates, 5000 cells were added to the central 60 wells of a 96-well flat-bottom white tissue culture plate (Thermo Scientific, Waltham, MA) in a volume of 55 μ Ι/well. Test scFv or IgG samples were prepared by dilution of stock samples in assay medium and serial three-fold dilutions. A25. mu.l sample of binding protein was added to the cells and incubated at 37 ℃/5% CO₂Incubate for 30 minutes. Mu.l of assay medium containing 100-400pg/ml human or murine IL-21 was added to each well and the fine particlesThe cells were incubated for a further 48 hours. Proliferation was measured by adding 15. mu.l/well of CELLTITER-GLO, even when the plates were brought to room temperatureIncubate with room temperature for 10 min and use Perkin Elmer ENVISION^TMThe plate reader measures luminescence. After purification with PhyNexus IMAC tips, 108 scFv were tested for neutralization of IL-21 dependent proliferation on all three cell lines. All showed neutralization of human IL-21R-BaF3 cells, where IC₅₀Less than or equal to the parent 18A5 scFv. A subset showed strong neutralization of proliferation of murine IL-21R-BaF3 cells and human IL-21R-TF1 cells. Data from the 27 most potent clones are shown in FIGS. 1-3 and are summarized in Table 5.

FIGS. 1-3 show neutralization of scFv proliferation for human IL-21R-BaF3 cells (FIGS. 1a-c), human IL-21R-TF1 cells (FIGS. 2a-c), and murine IL-21R-BaF3 cells (FIGS. 3 a-c). Cells were mixed with the indicated scFv and incubated with either 100pg/ml (FIGS. 1-2) or 400pg/ml (FIG. 3) human IL-21.

Example 6.3: quantitative epitope competition assay

The purified scfvs were quantified for their ability to compete with the parent 18a5 antibody for binding to murine IL-21R in an enzyme-linked immunosorbent assay (ELISA). The parent 18A5 antibody was applied to a 96 well Nunc MAXISORP in PBS at a concentration of 0.75. mu.g/mlPlates were coated overnight at 4 ℃. Plates were washed 3 times with PBS and then blocked in PBS/1% BSA/0.05% Tween 20 for 3 hours at room temperature. The scFv was mixed with 36nM biotinylated mIL-21R-H/F and incubated for 10 min at room temperature. The blocked plates were washed 3 times with PBS and 50. mu.l/well scFv/IL-21R mix was transferred to the appropriate plate and incubated for 1 hour at room temperature. The plate was washed 5 times with PBS, after which a 1: 6000 diluted secondary horseradish peroxidase-conjugated streptavidin (Southern Biotech, Birmingham, AL) antibody was added to detect bound biotinylated mIL-21R-H/F. The plates were then incubated at room temperature for 1 hour,and washed 7 times with PBS. Signal development Using 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB), with H₂SO₄Terminating the reaction and reacting in an invader^TMAbsorbance was read at 450nm on a plate reader (Perkin Elmer). 108 scFv purified by PhyNexus IMAC tips were tested in this assay and most competed with the parental 18A5 antibody for binding to biotinylated murine IL-21R-H/F, with its IC₅₀Lower than the parent 18A5 scFv. Epitope competition data for the 27 clones with the highest potency in the cell-based neutralization assay are shown in FIGS. 4a-c and are summarized in Table 5.

Table 5: neutralization of human and murine IL-21R and competition with 18A5 antibody for murine IL-21R binding in cell-based assays

L15	3.3	30.3	53.49	14
					L16	3.7	67.4	4.71	6
L17	1.6	60.3	2.66	12
					L18	3.7	54.4	8.34	8

L19	4.5	35.3	13.59	15
					L20	3.1	57.5	15.39	5
L21	9.4	100 (estimated)	162.27	28
					L23	1.5	15.3	nd	12
L24	2.4	18.7	3.73	6
					L25	3.7	33.1	15.55	9

Example 7: conversion of parent 18A5IgG to germline sequence

Selecting the following 15 species with improved V_LscFv of a region, along with the germline parent 18A5V_L(see below) for conversion to full-length human IgG lambda: l2, L3, L6, L9, L11, L13, L14, L16, L17, L18, L19, L20, L23, L24, and L25. Selection of 4 species with improved V_HscFv of regions (H3, H4, H5, and H6), along with the germlined parent 18A5V_H(see below) for conversion to full-length human IgG 1.

Modifying V of parent 18A5 antibody_HAnd V_LAmino acid sequence such that sequences outside the CDR regions match the closest human germline sequences: v_HDP67/VH4B + (VBASE _ A) in casesA: WAP00CEAZ _1) and JH1/JH4/JH5, and V_LDPL16/VL3.1 in this case (VBASE _ AA: WAP00CEMI _ 1). The modification was performed by a combination of gene synthesis at GENEART (Regensburg, Germany) and site-directed changes introduced by PCR. In addition, sequences were codon optimized for expression in mammalian cells by GENEART using their proprietary methods. The alignment of the parent 18a5 sequence and germline amended 18a5 sequence is shown below:

18A5 heavy chain comparison

Parent 18A5V _H

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGACTTCGGAGACCCTGTCCCTCACCTGCGCT

GTCTCTGGTTACTCCATCAGCAGTGGTTACTACTGGGGCTGGATCCGGCAGCCCCCAGGGAAGGGGTTG

GAGTGGATTGGGAGTATCTCTCATACTGGGAACACCTACTACAACCCGCCCCTCAAGAGTCGCGTCACC

ATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAACTGAGCTCTGTGACCGCCGCAGACACGGCC

GTGTATTACTGTGCGCGAGGTGGGGGAATTAGCAGGCCGGAGTACTGGGGCAAAGGCACCCTGGTCACC

GTCTCGAGT(SEQ ID NO：5)

Germlining 18A5V _H

CAGGTGCAGCTGCAGGAGTCTGGCCCTGGCCTGGTGAAGCCTTCCGAGACCCTGTCTCTGACCTGTGCC

GTGTCCGGCTACTCCATCTCCTCCGGCTACTACTGGGGCTGGATCAGACAGCCTCCTGGCAAGGGCCTG

GAGTGGATCGGCTCCATCTCTCACACCGGCAACACCTACTACAACCCCCCTCTGAAGTCCAGAGTGACC

ATCTCCGTGGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCTGTGACCGCTGCCGATACCGCC

GTGTACTACTGTGCCAGAGGCGGCGGAATCTCCAGACCTGAGTACTGGGGCCAGGGCACCCTGGTGACC

GTGTCCTCT(SEQ ID NO：7)

Germlining 18A5V _H x parent 18A5V _H

18A5 light chain comparison

Parent 18A5V _L

TCTTCTGAGCTGACTCAGGACCCTCCTGTGTCTGTGGCCTTGGGACAGACAGTCACGCTCACATGCCAA

GGAGACAGCCTCAGAACCTATTATGCAAGCTGGTACCAGCAGAAGTCAGGACAGGCCCCTATACTTCTC

CTCTATGGTAAACACAAACGGCCCTCAGGGATCCCAGACCGCTTCTCTGGCTCCACCTCAGGAGACACA

GCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGACGAGGCTGACTATTACTGTAACTCCCGGGACTCC

AGTGGCAACCCCCATGTTCTGTTCGGCGGAGGGACCCAGCTCACCGTTTTA(SEQ ID NO：9)

Germlining 18A5V _L

TCCTCTGAGCTGACCCAGGATCCTGCTGTGTCTGTGGCCCTGGGCCAGACCGTCAGGATCACCTGCCAG

GGCGATAGCCTGAGAACCTACTACGCCTCCTGGTATCAGCAGAAGCCTGGACAGGCCCCTGTGCTGGTG

ATCTACGGCAAGCACAAGAGGCCATCCGGCATCCCTGACAGATTCTCCGGCTCCTCCTCTGGCAATACC

GCCTCCCTGACCATCACCGGCGCTCAGGCCGAGGACGAGGCCGACTACTACTGTAACTCCCGGGACTCT

TCCGGCAACCCTCACGTGCTGTTTGGCGGCGGAACCCAGCTGACCGTGCTA(SEQ ID NO：11)

Germlining 18A5V _L x parent 18A5V _L

Germline corrected V_HSequence (changes relative to the parent sequence are bold and underlined):

parent (SEQ ID NO: 6) QVQLQESGPGLVKTSETLSLTCAVSGYSISSGYYWGWIRQPPGKG

Germlined (SEQ ID NO: 8) QVQLQESGPGLVKSETLSLTCAVSGYSISSGYYWGWIRQPPGKG

LEWIGSISHTGNTYYNPPLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGGISRP of parent

Germlined LEWIGSISHTGNTYYNPPLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGGISRP

EYWGKGTLVTVSS of parent

Germlined EYWGGTLVTVSS

Germline corrected V_LSequence (changes relative to the parent sequence are bold and underlined):

parent (SEQ ID NO: 10) SSELTQDPPVSVALGQTVTLTCQGDSLRTYYASWYQQKSGQAPIL

Germlined (SEQ ID NO: 12) SSELTQDPVSVALGQTVTCQGDSLRTYYASWYQQKGQAPL

LLYGKHKRPSGIPDRFSGSTSGDTASLTITGAQAEDEADYYCNSRDSSGNPHVLFGGGTQ of parent

GermlinedYGKHKRPSGIPDRFSGSSGTASLTITGAQAEDEADYYCNSRDSSGNPHVLFGGGTQ

LTVL of parent

Germlined LTVL

Example 8: conversion of library-derived scFv to IgG

V modification of 18A5scFv derivatives by PCR amplification_LAnd V_H(ii) the CDR3 region and subcloned into the germline amended parent 18A5V by the following method_LAnd V_HA frame. By using primer BssHII _ II _ V_H_F

(5’-GCTTGGCGCGCACTCTCAGGTGCAGCTGCAGGAG-3’)[SEQ ID NO：230]And GV_H_R_for_BssHII(5’-TCAGGGAGAACTGGTTCTTGG-3’)[SEQ ID NO：231]Amplification ofPlasmid pSMED2_ OP18A5G _ huIgG1 Generation encompasses germlining 18A5V_HPCR fragments of the 5' portion of the gene. With primer G _ V_H_F_for_SalI(5’-TCCAAGAACCAGTTCTCCCTG-3’)[SEQ ID NO：232]And scFv _ SalI _ V_H_R(5’-GCGACGTCGACAGGACTCACCACTCGAGACGGTGACCAGGGTGCC-3’)[SEQ ID NO：233]Amplification of V covering from modified scFv clone VH3_HPCR fragment of 3' part of gene. The fragments were gel purified, then mixed and used as external primer set BssHII _ G _ V_HF and SalI _ V_HR amplification to generate complete V_HA gene fragment. This was digested with bshii and SalI and ligated into a vector containing the human IgG1 constant region and the triple mutant hinge region. With BssHII _ II _ V_HF and New primer (Sal _ V)_H_R_RJ(5’-GCGACGTCGACAGGACTCACCACTCGAGACGG-3’))[SEQ ID NO：234]Reamplifying inserts to alter V_HThe coding sequence for the J segment was in accordance with JH1 germline sequence and ligated into the human IgG 1-triple mutant constant region vector.

Subcloning of V from modified scFv by similar methods_LA gene. By using primer BssHII _ II _ V_L_F(5’-GCTTGGCGCGCACTCTTCCTCTGAGCTGACCCAG-3’)[SEQ ID NO：235]And scFv _ V_L_R_for_BssHII(5’-GCCTGAGCCCCAGTGATGGTCA-3’)[SEQ ID NO：236]Amplification plasmid pSMEN2_ OP18A5G _ hu Lambda Generation covering 18A5V_LPCR fragments of the 5' portion of the gene. With primer GV_L_F_for_XbaI(5’-ACCGCCTCCCTGACCATCAC-3’)[SEQ ID NO：237]And scFv _ XbaI _ V_L_R(5’-GCGCCGTCTAGAGTTATTCTACTCACCTAAAACGGTGAGCTGGGTCCCTC-3’)[SEQ ID NO：238]Amplification of V encompassing clones from modified scFv_LPCR fragment of 3' part of gene. The fragments were gel purified, then mixed with fragments corresponding to the 5 'and 3' portions of each gene, and used with the outer primer set BssHII _ II _ V_LF and scFv _ XbaI _ V_LR amplification to generate complete V_LA gene fragment. These were digested with BssHII and XbaI and ligated into a vector containing the constant region of the human lambda gene.

Example 9: in vitro characterization of IgG

Example 9.1: transient small scale expression of binding proteins

Clones were tested for function after transient expression of the complete IgG format in cos7 cells. 16 test sequences (germlined parental 18A 5V)_LAnd each light chain in the set of L2, L3, L6, L9, L11, L13, L14, L16, L17, L18, L19, L20, L23, L24, and L25) was combined with 5 test sequences (H3, H4, H5, and H6 along with V25)_HP, germlined parent 18A5V_HDomains) are paired with each heavy chain in the collection. Each plasmid (1.4. mu.g) was paired with TRANSITTransfection reagents (Mirus, Madison, WI) were combined according to the manufacturer's instructions and the DNA: TRANSITThe reagent complexes were added to monolayers of cos7 cells grown in Dulbecco's Modified Eagle's Medium (DMEM)/10% heat-inactivated fetal bovine serum/penicillin/streptomycin/2 mM L-glutamine in 6-well tissue culture plates. After 24 hours, the culture medium was replaced with serum-free medium (R1CD1), and then collected after 48 hours. The binding proteins (now including the full length antibody) were quantified by anti-human IgG ELISA.

Example 9.2: activity of anti-IL-21R IgG in cell proliferation and neutralization

The activity in three cell lines in an IL-21 dependent proliferation assay was tested for 80 transiently expressed iggs in serum-free conditioned medium as described above: (1) human IL-21R-BaF3 cells, (2) murine IL-21R-BaF3 cells, and (3) human IL-21R-TF1 cells. All 80 pairs showed neutralization of proliferation of human IL-21R expressing BaF3 cells, and all pairs except those involving VH4 showed neutralization of human IL-21R expressing TF1 cells (data not shown). All 80 pairs also showed neutralization of proliferation of murine IL-21R expressing BaF3 cells, with the strongest neutralization generally associated with the light chain pairing with the parental heavy chain and the weakest neutralization oneTypically associated with the VH4 heavy chain (data not shown). Neutralization data from the most potent 21 IgG combinations (AbA-AbU) are shown in FIG. 5, and IC₅₀The data are summarized in Table 6.

Human IL-21R-BaF3 cells were assayed at 100pg/ml human IL-21 (FIGS. 5a-c), human IL-21R-TF1 cells at 100pg/ml human IL-21 (FIGS. 5d-f), or murine IL-21R-BaF3 cells at 400pg/ml murine IL-21 (FIGS. 5 g-i). Adding IL-21 to the cells after the indicated antibody; after 48 hours CELLTITER-GLO was usedProliferation was measured. FIGS. 26a-c show additional studies demonstrating similar inhibition in the same three cell lines.

Example 9.3: anti-IL-21R IgG binding transiently expressed rat and cynomolgus IL-21R

A subset of the binding proteins were tested for binding to rat, cynomolgus, human IL-21R, or human IL-2R-gamma common subunit transiently expressed on the surface of CHO PA Dukx cells. Cells were transfected 48 hours prior to the assay. On the day of assay, cells were plated on an automated plate washer (Titertek, Huntsville, AL) containing 0.9mM CaCl₂And 0.45mM MgCl₂The cells were washed 5 times with PBS (PBS/CaMg) and blocked in PBS/CaMg/5% skim milk powder for 1 hour at room temperature. Conditioned medium from transient expression of anti-IL-21 RIgG was serially diluted in blocking buffer and added to the cells in the blocked plates for 1 hour at room temperature. Cells were washed 5 times with PBS/CaMg and then incubated with horseradish peroxidase-conjugated anti-human IgG for 1 hour at room temperature. Cells were then washed 10 times in PBS/CaMg and all wash buffer was removed. Cells were incubated with 100. mu.l TMB until the color reaction reached saturation, and 100. mu.l 0.18M H was used₂SO₄Terminated and treated in a Perkin Elmer ENVISION^TMRead on plate reader at a 450.

All 21 IgGs bind to CHO cells transiently expressing human (FIGS. 6a-c), rat (FIGS. 6d-f), or cynomolgus (FIGS. 6g-i) IL-21R. Most did not show binding above background to control proteins transiently expressed on CHO cells (human gamma (γ) common chain), but binding of a subset of iggs (AbD, AbE, AbF, AbH, AbL, and AbM) was 13nM or greater above background (fig. 6 j-l). The data are summarized in Table 6.

Table 6: summary of neutralization of human and murine IL-21R activity in cell proliferation assays and binding of human, rat, and cynomolgus IL-21R expressed on CHO cells

AbB	1.14	3.34	0.421	1.147	1.09	1.333	0.107
								AbC	0.82	3.36	0.03	1.218	0.999	1.277	0.137
AbD	0.91	2.67	0.01	1.247	0.874	1.375	0.197
								AbE	0.56	2.28	0.04	1.257	1.111	1.423	0.223
AbF	0.54	2.41	0.304	1.347	1.001	1.458	0.433
								AbG	0.77	3.84	0.07	1.35	1.112	1.304	0.108

AbH	0.94	3.64	0.327	1.35	1.097	1.324	0.152
								AbI	1.00	3.80	0.224	1.237	1.088	1.209	0.107
AbJ	0.65	4.60	0.4	1.217	1.261	1.273	0.126
								AbK	0.98	4.00	0.079	1.364	1.175	1.338	0.108
AbL	0.68	4.25	0.227	1.454	1.257	1.514	0.219
								AbM	1.08	4.22	0.125	1.197	0.78	1.45	0.224
AbN	0.50	1.59	0.435	1.214	0.702	1.497	0.136
								AbO	0.52	2.91	0.065	1.107	1.101	1.358	0.108
AbP	0.75	3.48	0.03	1.308	1.03	1.313	0.112
								AbQ	0.68	4.62	0.153	1.255	1.161	1.31	0.125
AbR	0.87	3.94	0.302	1.334	1.108	1.35	0.109
								AbS	1.53	5.00	0.04	1.017	1.166	1.224	0.118
AbT	0.67	3.26	0.093	1.078	0.994	1.219	0.102
								AbU	0.73	3.13	0.184	1.289	0.927	1.314	0.104

Example 9.4: selective BIACORE of anti-IL-21R IgG binding to human IL-21R ^TM Analysis of

In BIACORE^TMA subset of transiently expressed anti-IL-21R binding proteins (here antibodies) were tested for binding specificity on a 2000 surface plasmon resonance instrument. Anti-human IgG, anti-mouse immunoglobulin antibody, and mouse IL-21R-H/F were immobilized onto a research-grade carboxymethyl dextran chip (CM5) using standard amine coupling. The sensor chip surface was activated with EDC/NHS for 7 min at a flow rate of 20. mu.l/min. Bulk refractive index (bulk refractive index), matrix effect (matrix effect), and non-specific binding were corrected using the first flow cell as a reference surface. Capture antibodies (anti-human Fc antibody of 7,150 Resonance Units (RU) on flow cell 2 (Invitrogen Corporation, Carlsbad, CA) and anti-mouse Fc antibody of 7,500 RU on flow cell 3) were diluted to 10 μ g/ml in sodium acetate buffer (pH 5.0) and injected onto the activated surface. The remaining activated groups were blocked with 1.0M ethanolamine (pH 8.0). Both anti-human and anti-mouse IgG have a molecular weight of 150kD, while IL-21R monomer has a molecular weight of 27 kD.

Containing anti-IL-21R antibody and antibody control (mouse anti-human IL-2R beta and mouse anti-human IL-4R (R)&D Systems, Minneapolis, MN); human anti-human IL-13(Wyeth, Cambridge, MA)) conditioned medium was diluted in HBS/EP buffer supplemented with 0.2% bovine serum and injected into BIACORE^TMOn all four flow cells of the chip, 500-700(RU) of the antibody was captured on the species appropriate capture antibody. After a5 second wash period, one positive control protein (murine IL-21R-H/F), two human proteins related to IL-21R (human IL-2R β and human sIL-4R (R)&D Systems)), or a 50nM solution of an unrelated His/FLAG-tagged protein (human IL-13-H/F) was injected onto the captured antibody on the chip. The binding and dissociation phases were monitored for 120 and 180 seconds, respectively, followed by two injections of 5 μ l glycine (pH 1.5) to regenerate a fully active capture surface. All binding experiments were performed in HBS/EP buffer at 25 ℃. Subtraction of blank and buffer effects for each sensorgram Using Dual referenceShould be used.

All tested anti-IL-21R antibodies (18A5 antibody and AbA-AbU) showed clear binding to murine IL-21R, but no binding to the IL-21R-related protein human IL-2R β and human soluble IL-4R or to the unrelated His/FLAG tagged protein human IL-13-His/FLAG (FIGS. 7 a-c). Controls indicated that IL-2R β and human soluble IL-4R were captured by specific anti-IL-2R β and anti-IL-4R antibodies (FIG. 7 d).

Example 9.5: purification of transiently expressed antibodies

7 antibodies (human IgG1 triple mutant versions: AbS, AbT, AbO, AbP, and AbU; and double mutant versions: AbQ and AbR) were transiently expressed in cos7 cells and purified for further analysis. In addition, three AbT versions with human IgG tails (wild-type IgG1, IgG4, and IgG1 double mutants) were also prepared that were expected to have different levels of Fc receptor binding. Following TRANSIT as described aboveProtocol except that 25. mu.g of each plasmid was used to transfect cells in each of 8T 175 flasks. After the first harvest of conditioned medium, fresh R1CD1 was added and then collected after another 72 hours. The conditioned media were combined and filtered on a 0.22 μm filter. The antibody was loaded onto protein A resin, eluted with 20mM citric acid/150 mM sodium chloride (pH 2.5), neutralized with Tris (pH 8.5), and dialyzed into PBS.

Example 9.6: BIACORE OF ANTIBODY BIACHIBITING HUMAN AND MOUSE IL-21R ^TM Analysis of

In BIACORE^TMThe kinetics of anti-IL-21R antibody binding to human and murine IL-21R-H/F were tested on a surface plasmon resonance apparatus. Anti-human IgG antibodies (Invitrogen Corporation) were immobilized onto research-grade carboxymethyl dextran chips (CM5) using standard amine coupling. The surface was activated with EDC/NHS for 7 min at a flow rate of 20. mu.l/min. Correcting refractive index of a body using a first flow cell as a reference surfaceMatrix effect, and non-specific binding. Anti-human Fc antibody was diluted to 20. mu.g/ml in 10mM sodium acetate buffer (pH 5.0) and 2950-3405 Resonance Units (RU) were captured on each of the four flow chambers. The remaining activated groups were blocked with 1.0M ethanolamine HCl (pH 8.5).

anti-IL-21R antibody was diluted to 0.1-0.2. mu.g/ml in HBS/EP buffer supplemented with 0.2% bovine serum albumin and loaded into BIACORE^TMOn the chip. After a brief wash period, a solution of 0-100nM human IL-21R-H/F or 10-500nM murine IL-21R-H/F was injected onto the chip at a flow rate of 50. mu.l/min. The binding phase was run for 3 min for human and murine IL-21R kinetics, and the dissociation phase was monitored for hIL-21R for 15 min, for mIL-21R for 5 min, followed by two injections of 10. mu.l and one injection of 30. mu.l glycine (pH 1.5) to regenerate a fully active capture surface. All binding experiments were performed in HBS/EP buffer at 25 ℃ and the sample rack (sample rack) was kept at 15 ℃. The blank and buffer effects were subtracted for each sensorgram using a double reference. Sensorgrams are shown in FIGS. 8a-b (human IL-21R-His/FLAG) and 8c-d (murine IL-21R-His/FLAG). Binding kinetic parameters are shown in table 7A, and additional kinetic data from one replicate experiment are shown in table 7B.

In addition, AbS and AbT were tested for binding kinetics to cynomolgus IL-21R-His/FLAG by the protocol described above. The binding properties of human and cynomolgus IL-21R-H/F were similar for both AbS and AbT (FIG. 9). FIG. 9 shows cynomolgus IL-21R-His/FLAG binding to the AbS (9 a); and AbT (9 c); and human IL-21R-His/FLAG binding AbS (9 b); and AbT (9 d).

Table 7A: kinetic parameters for anti-IL-21R antibody binding to human and murine IL-21R-His/FLAG

Table 7B: kinetic parameters of anti-IL-21R antibody binding to human IL-21R-His/FLAG

Example 9.7: BIACORE ^TM Epitope competition assay

Direct immobilization of antibodies AbS and AbT and parent antibody 18a5 to CM5 BIACORE^TMOn the chip. Murine IL-21R-H/F (100nM) was allowed to flow through the chip for 300 seconds, followed by washing (100sec), and then a 5. mu.g/ml solution of either the AbS, AbT, D5, or a non-neutralizing anti-mIL-21R antibody (7C2) was allowed to flow over the surface. No additional binding was observed for AbS, AbT, and D5, indicating that their binding sites on mIL-21R-H/F were blocked by concurrent binding of the AbS, AbT, or 18a5 antibodies (fig. 10 a). In contrast, the neutralizing control anti-IL-21R antibody 7C2 was able to bind mIL-21R-H/F captured on the AbS, AbT, or 18A5 antibodies, indicating that this control antibody binds at an epitope different from the epitope bound by the capture antibody.

Similarly, AbS and AbT do not bind to CM5 BIACORE^TMOn-chip immobilized AbS or AbT captured human IL-21R-H/F, while control anti-human IL-21R antibody (9D2) was able to bind to human IL-21R-H/F captured by AbS or AbT (fig. 10 b). This observation suggests that the binding site of AbS is blocked by parallel binding of AbT and vice versa.

Example 9.8: cell-based proliferation assay

Purified IgG was tested for activity in IL-21 dependent proliferation in three cell lines as described above: human IL-21R-BaF3 cells, murine IL-21R-BaF3 cells, and human IL-21R-TF-1 cells. All showed strong inhibition of both human and murine IL-21R dependent proliferation with greater potency than the parent 18a5IgG (fig. 11, table 8). Human IL-21R-BaF3 cells were assayed at 100pg/ml for human IL-21 (FIG. 11a), 200pg/ml for murine IL-21R-BaF3 cells (FIG. 11b), and 100pg/ml for human IL-21R-TF-1 cells (FIG. 11 c). FIG. 26d depicts the results of another study on the effect of these antibodies on human IL-21R-BaF3 cells.

Table 8: neutralization of proliferation of human IL-21R-BaF3 cells, murine IL-21R-BaF3 cells, and human IL-21R-TF-1 cells

Example 9.9: primary human B cell proliferation assay

anti-IL-21R antibodies were tested for their ability to inhibit IL-21-dependent proliferation of primary human B cells. Buffy coat cells from healthy human donors were obtained from Massachusetts General Hospital (Boston, MA). Contacting the cells with ROSETTESEP^TMThe B cell enrichment mixture (StemCell Technologies, Vancouver, Canada) was incubated together and B cells were isolated according to the manufacturer's instructions. The resulting population (60-80% CD 19)⁺B cells) at 1x10 in 96-well flat-bottom plates⁵Perwell were cultured in RPMI containing 10% FBS, 50U/ml penicillin, 50. mu.g/ml streptomycin, and 2mM L-glutamine. B cells were adjusted to 5% CO with serially diluted anti-human IL-21R antibody₂At 37 ℃ for 30 minutes. The treated B cells were then adjusted to 5% CO with 0.5. mu.g/ml anti-CD 40 monoclonal antibody (BD Biosciences, San Jose, Calif.) and 10ng/ml IL-21 cytokine₂At 37 ℃ for 3 days. On day 3, cultures were plated with 0.5. mu. Ci/well³H-thymidine (Perkin Elmer (NEN)) was pulsed and harvested 5 hours later onto glass fiber filter pads. Determination by liquid scintillation counting³Incorporation of H-thymidine. All of the improved antibodies neutralized IL-21-dependent proliferation with greater potency than the parental 18A5 antibody (FIGS. 12a-b, Table 9; see also FIG. 26 e).

Table 9: neutralization of human primary B cell proliferation

Example 9.10: primary human T cell proliferation assay

anti-IL-21R antibodies tested they inhibit primary human CD4⁺The ability of T cells to proliferate in an IL-21 dependent manner. Buffy coat cells from healthy human donors were obtained from Massachusetts General Hospital. Using ROSETTESEP^TM CD4⁺T cell enrichment mixtures (StemCell Technologies) isolate CD4 by negative selection according to manufacturer's instructions⁺T cells. The resulting population was about 80-90% CD4⁺/CD3⁺T cells. Enriching human CD4⁺T cells are regulated to 5% CO₂Was activated with anti-CD 3/anti-CD 28 coated microspheres in RPMI containing 10% FBS, 100U/ml penicillin, 100. mu.g/ml streptomycin, 2mM L-glutamine, and HEPES for 3 days in an incubator at 37 ℃. After activation, the microspheres were removed, the cells were washed, and placed approximately 1 × 10 in culture medium⁶The cells/ml were allowed to rest overnight. The resting cells were then washed again before addition to the assay plate. Serial dilutions of anti-human IL-21 receptor antibody were prepared in culture medium in flat bottom 96-well plates, followed by sequential addition of human IL-21(20ng/ml final concentration) and activated and resting CD4⁺T cell (10)⁵Individual cells/well). The plate was then incubated for another 3 days and 1. mu. Ci/well was used during the last 6 hours of the assay³Pulses of H-thymidine (Perkin Elmer (NEN)). Cells were harvested onto glass fiber filter pads and assayed by liquid scintillation counting³Incorporation of H-thymidine. All of the improved antibodies neutralized IL-21-dependent proliferation with greater potency than the parental 18a5 antibody (fig. 13, table 10A; see also fig. 26 f).

Table 10A: neutralization of human primary T cell proliferation

Example 9.11: primary murine T cell proliferation assayMethod of

anti-IL-21R antibodies tested they inhibit primary murine CD8⁺The ability of T cells to proliferate in an IL-21 dependent manner. Popliteal, axillary, brachial, and inguinal lymph nodes and spleen were collected from 12 week old female BALB/C mice. 0.16M NH in 0.017M Tris (pH 7.4) was used₄Single cell suspensions of Cl on splenocytes depleted red blood cells. Spleen and lymph node cells were pooled and murine T cell CD8 subcolumn kit (R) was used&D Systems) enriched CD8⁺A cell. Mouse CD8⁺Cell (3X 10)⁴(ii) a Suspended in DMEM containing 10% fetal bovine serum supplemented with 0.05mM beta-mercaptoethanol, 2mM L-glutamine, 0.1mM non-essential amino acids, 1mM sodium pyruvate, 100U/ml penicillin, 100. mu.g/ml streptomycin and 50. mu.g/ml gentamicin) into 96-well anti-mCD 3 activation plates (BD Biosciences); mIL-21(50ng/ml) was added to all wells. The test antibody was titrated in triplicate, starting with 20 μ g/ml. Cells were incubated at 37 ℃/10% CO₂Incubate in incubator for 3 days. During the last 5 hours of culture, 0.5. mu. Ci of methyl-³Cells were labeled with H-thymidine/well (GE Healthcare). Cells were harvested using a Mach III cell harvester (TomTec, Hamden, CT) and counted using a Trilux microbeta counter (Perkin Elmer). With the exception of AbP, all of the improved antibodies neutralized IL-21-dependent proliferation with greater potency than the parent 18A5 antibody (FIG. 14, Table 10B; see also FIG. 26 g).

Table 10B: neutralization of murine Primary T cell proliferation

Antibodies	Neutralization of T cell proliferation IC50(nM)
		AbO	4.92
AbP	Without inhibition
		AbQ	0.85
AbR	0.13
		AbS	0.02
AbT	0.61
		AbU	1.79
18A5 antibody	＞85

Example 9.12: ADCC assay

anti-IL-21R antibodies were tested for their ability to induce antibody-dependent cellular cytotoxicity (ADCC) upon binding to target cells. On the day before the experiment, PBMC were isolated from the buffy coat by diluting the buffy coat 1: 1 in PBS and isolating it in FICOLL(GE Healthcare) was layered and centrifuged at 1200g for 20 min. self-FICOLLTop side of layerPBMCs were removed, washed, and treated with 10ng/ml IL-2 and 10ng/ml IL-12 (R)&D Systems) stimulation overnight. On the day of the experiment, stimulated PBMCs were collected by centrifugation and cultured at 1x10 in medium⁸Resuspend individual cells/ml. The BJAB cells were treated with 0.5. mu.M CFSE (MOLECULAR PROBES)Invitrogen Corporation) was labeled at 37 ℃ for 10 minutes, and then washed once with fetal bovine serum and twice with PBS. Cells were then plated at 2 × 10 in 100 μ l medium⁵Individual cells/well were dispensed into a 96-well flat bottom plate. Add 50. mu.l of 4 Xantibody to BJAB cells, followed by 5X10 in 50. mu.l⁶PBMC, giving a final 1: 25 target: effector cell ratio. Cells were incubated at 37 ℃ for 6 hours and stained with Propidium Iodide (PI) to label dead and dying cells. By applying in FACSCALIBUR^TMFlow cytometry (BD Biosciences) is measuring PI staining to assess target cell (CFSE +) killing. Only one anti-IL-21R antibody, AbZ, with a wild-type human IgG1 constant region, showed ADCC above background levels exhibited by control anti-IL-13 antibodies that did not bind to target cells. All antibodies with the same variable domains as AbZ, including antibodies with each form of human IgG4 (AbY), and double mutant (AbX) and triple mutant (AbT) forms of human IgG1, showed only background levels of ADCC (figure 15). All other anti-IL-21R antibodies tested contained triple mutant forms of human IgG1 and showed background ADCC. A positive control antibody, rituximab (rituximab, RITUXAN)) ADCC was induced in all experiments.

Example 9.13: c1q ELISA

To determine whether cell surface binding of anti-IL-21R antibodies is likely to result in Complement Dependent Cytotoxicity (CDC), the antibodies were tested for their ability to bind complement component C1q in an ELISA. Mixing IL-21R antibody and Rituximab (RITUXAN)) Diluted to 5. mu.g/ml in PBS. Diluted antibody (100. mu.l) was coated onto COSTARHigh binding ELISA plates (Corning Life Sciences, Lowell, Mass.) were incubated overnight at 4 ℃. The plate was washed 3 times with PBS/Tween 20 and with 200. mu.l blocking buffer (0.1M NaPO)₄0.1M NaCl, 0.1% gelatin, 0.01% Tween) was blocked at room temperature for 1 hour. Human serum previously identified as containing C1q (Quidel, San Diego, Calif.) was diluted 1: 50 in PBS. After 1 hour of blocking, plates were washed, 100 μ l of diluted serum was added to each well and incubated for 2 hours at room temperature on a shaker. After washing 3 times, 100. mu.l of 0.1. mu.g/ml chicken polyclonal anti-human C1q antibody (Abcam, Cambridge, MA) was added to each well and incubated for 1 hour at room temperature. The plates were washed again and incubated with 100. mu.l of rabbit anti-chicken Ig-Y polyclonal antibody-HRP (Abcam) diluted 1: 4000 for 1 hour at room temperature. Plates were washed and developed with TMB for 5 minutes followed by 50. mu.l of 1M H2SO₄The reaction was stopped and then read at 450 nm. Only one anti-IL-21R antibody, AbZ, with a wild-type human IgG1 constant region, showed C1q binding above background levels exhibited by a control antibody with a triple mutant human IgG1 constant region and previously shown to lack C1q binding. All antibodies with the same variable domains as AbZ, including antibodies with each form of human IgG4 (AbY), and double mutant (AbX) and triple mutant (AbT) forms of human IgG1, showed only background levels of C1q binding (fig. 16). All other anti-IL-21R antibodies tested contained triple mutant forms of human IgG1 and showed background C1q binding.

Example 9.14: cytokine competition assay

To demonstrate that antibody AbT binds murine IL-21R in a manner that competes with IL-21 cytokine, a cytokine competition assay was performed. Antibody AbT was coated onto ELISA plates at 1. mu.g/ml and then blocked with 1% BSA in PBS/. 05% Tween. Biotinylated murine IL-21R-His/FLAG (1.5ng/ml) was added to each well, either alone or in the presence of increasing concentrations of murine IL-21, and binding of the receptor to the immobilized antibody was detected with HRP-coupled streptavidin followed by incubation with TMB detection reagent. Mouse IL-21 was able to nearly completely block mIL-21R binding AbT above 4ng/ml, indicating that the antibody and the cytokine compete for binding to mouse IL-21R (FIG. 27).

Equivalent scheme

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The appended claims are intended to cover such equivalents.

Claims (49)

1. An isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises at least one amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 165 and 168, 171 and 193, 213 and 229, 240, 242, 244, 246, and 248.

2. The isolated binding protein or antigen-binding fragment of claim 1, wherein the binding protein or antigen-binding fragment is an antibody.

3. The isolated binding protein or antigen-binding fragment of claim 1, wherein the binding protein or antigen-binding fragment is an scFv.

4. The isolated binding protein or antigen-binding fragment of claim 1, wherein the binding protein or antigen-binding fragment is V_H。

5. The isolated binding protein or antigen-binding fragment of claim 1, wherein the binding protein or antigen-binding fragment is V_L。

6. The isolated binding protein or antigen-binding fragment of claim 1, wherein the binding protein or antigen-binding fragment is a CDR.

7. An isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises at least one amino acid sequence encoded by a nucleotide sequence that is at least about 95% identical to a nucleotide sequence selected from the group consisting of seq id nos: SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 239, 241, 243, 245, and 247.

8. The isolated binding protein or antigen-binding fragment of claim 7, wherein the binding protein or antigen-binding fragment is an antibody.

9. The isolated binding protein or antigen-binding fragment of claim 7, wherein the binding protein or antigen-binding fragment is an scFv.

10. The isolated binding protein or antigen-binding fragment of claim 7, wherein the binding protein or antigen-binding fragment is V_H。

11. The isolated binding protein or antigen-binding fragment of claim 7, wherein the binding protein or antigen-binding fragment is V_L。

12. The isolated binding protein or antigen-binding fragment of claim 7, wherein the binding protein or antigen-binding fragment is a CDR.

13. The binding protein or antigen-binding fragment of claim 1, comprising at least one amino acid sequence selected from the group consisting of: SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 165 and 168, 171 and 193, 213 and 229, 240, 242, 244, 246, and 248.

14. The isolated binding protein or antigen-binding fragment of claim 13, wherein the binding protein or antigen-binding fragment is an antibody.

15. The isolated binding protein or antigen-binding fragment of claim 13, wherein the binding protein or antigen-binding fragment is an scFv.

16. The isolated binding protein or antigen-binding fragment of claim 13, wherein the binding protein or antigen-binding fragment is V_H。

17. The isolated binding protein or antigen-binding fragment of claim 13, wherein the binding protein or antigen-binding fragment is V_L。

18. The isolated binding protein or antigen-binding fragment of claim 13, wherein the binding protein or antigen-binding fragment is a CDR.

19. An isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises at least one amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 195, 213, 229, 240, 242, 244, 246, 248, and

wherein, if the binding protein or antigen-binding fragment comprises at least one amino acid sequence that is at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 6, 8, 10, 12, 163, 164, 169, 170, 194, and 195, then the binding protein or antigen-binding fragment must further comprise at least one amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 165 and 168, 171 and 193, 213 and 229, 240, 242, 244, 246, and 248.

20. An isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises at least one amino acid sequence encoded by a nucleotide sequence that is at least about 95% identical to a nucleotide sequence selected from the group consisting of seq id nos: SEQ ID NO: 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 159, 161, 239, 241, 243, 245, and 247, and

wherein, if the binding protein or antigen-binding fragment comprises at least one amino acid sequence encoded by a nucleotide sequence that is at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 5,7, 9, and 11, then the binding protein or antigen-binding fragment must further comprise at least one amino acid sequence encoded by a nucleotide sequence that is at least about 95% identical to a nucleotide sequence selected from the group consisting of seq id no: SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 239, 241, 243, 245, and 247.

21. The binding protein or antigen-binding fragment of claim 19, comprising at least one amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 195, 213, 229, 240, 242, 244, 246, 248, and 248,

wherein, if the binding protein or antigen-binding fragment comprises at least one amino acid sequence that is at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 6, 8, 10, 12, 163, 164, 169, 170, 194, and 195, then the binding protein or antigen-binding fragment must further comprise at least one amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 165 and 168, 171 and 193, 213 and 229, 240, 242, 244, 246, and 248.

22. An isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises a light chain and a heavy chain, and wherein the heavy chain comprises at least one amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 14, 16, 18, 20, 68, 70, 72, 88, 90, 92, 94, 213, 218, 219, 240, and 242.

23. An isolated binding protein or antigen-binding fragment thereof that binds IL-21R, wherein the binding protein or antigen-binding fragment thereof comprises a light chain and a heavy chain, and wherein the light chain comprises at least one amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 74, 76, 78, 80, 82, 84, 86, 96, 98, 100, 102, 104, 106, 108, 214 and 217, 220 and 229, 244, 246 and 248.

24. The binding protein or antigen-binding fragment of claim 22, wherein the binding protein or antigen-binding fragment comprises V_LField and V_HDomain, and wherein the V_HThe domain comprises at least one amino acid sequence selected from the group consisting of: SEQ ID NO: 14, 16, 18, and 20.

25. The binding protein or antigen-binding fragment of claim 23, wherein the binding protein or antigen-binding fragment comprises V_LField and V_HDomain, and wherein the V_LThe domain comprises at least one amino acid sequence selected from the group consisting of: SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 215, 217, 221, 223, 225, 227, and 229.

26. The binding protein or antigen-binding fragment of claim 22 or 23, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 88, 90, 92, 94, 213, 218, 219, 240, and 242, and the light chain comprises an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 96, 98, 100, 102, 104, 106, 108, 214, 216, 220, 222, 224, 226, 228, 244, 246, and 248.

27. An isolated binding protein or antigen-binding fragment thereof that binds to an epitope of IL-21R recognized by a binding protein selected from the group consisting of: AbA-AbW, H3-H6, L1-L6, L8-L21, and L23-L25, wherein the binding protein or antigen binding fragment competitively inhibits binding of a binding protein selected from the group consisting of: AbA-AbW, H3-H6, L1-L6, L8-L21, and L23-L25.

28. The binding protein or antigen-binding fragment of claim 27, comprising a heavy chain, light chain, or Fv fragment comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 165 and 168, 171 and 193, 213 and 229, 240, 242, 244, 246, and 248.

29. The binding protein or antigen-binding fragment of claim 27, comprising a heavy chain, light chain, or Fv fragment comprising an amino acid sequence encoded by a nucleotide sequence selected from the group consisting of seq id nos: SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 239, 241, 243, 245, and 247.

30. The binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20, wherein the binding protein or antigen-binding fragment has a binding constant for human IL-21R of at least about 10⁵M^-1s^-1。

31. The binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20, wherein the binding protein or antigen-binding fragment has an IC of about 1.75nM or less₅₀Inhibits IL-21-mediated proliferation of BaF3 cells, and wherein the BaF3 cells comprise human IL-21R.

32. The binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20, wherein the binding protein or antigen-binding fragment has an IC of about 14.0nM or less₅₀Inhibits IL-21-mediated proliferation of TF1 cells, and wherein the TF1 cells comprise human IL-21R.

33. The binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20, wherein the binding protein or antigen-binding fragment has an IC of about 1.9nM or less₅₀Inhibits IL-21-mediated proliferation of primary human B cells, and wherein the B cells comprise human IL-21R.

34. The binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20, wherein the binding protein or antigen-binding fragment has an IC of about 1.5nM or less₅₀Inhibition of IL-21 mediated Primary human CD4⁺The cells proliferate, and wherein said CD4⁺The cells comprise human IL-21R.

35. The binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20, wherein the binding protein or antigen-binding fragment specifically binds to the amino acid sequence of SEQ ID NO: 2, or any sequence of at least 100 contiguous amino acids that is at least about 95% identical.

36. The binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20, wherein the binding protein or antigen-binding fragment inhibits IL-21 from binding to IL-21R.

37. The binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20, wherein the binding protein or antigen-binding fragment is IgG 1.

38. The binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20, wherein the binding protein or antigen-binding fragment is a human binding protein or antigen-binding fragment.

39. A pharmaceutical composition comprising the binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20.

40. An isolated nucleic acid encoding the binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20.

41. An expression vector comprising the nucleic acid of claim 40.

42. A host cell transformed with the vector of claim 41.

43. The host cell of claim 42, wherein said host cell is a bacterium, a mammalian cell, a yeast cell, a plant cell, or an insect cell.

44. A diagnostic kit comprising the binding protein or antigen-binding fragment of any one of claims 1, 2, 19, or 20.

45. The isolated binding protein or antigen-binding fragment of any one of claims 19-38, wherein the binding protein or antigen-binding fragment is an antibody.

46. The isolated binding protein or antigen-binding fragment of any one of claims 19-38, wherein the binding protein or antigen-binding fragment is an scFv.

47. The isolated binding protein or antigen-binding fragment of any one of claims 19-38, wherein the binding protein or antigen-binding fragment is V_H。

48. The isolated binding protein or antigen-binding fragment of any one of claims 19-38, wherein the binding protein or antigen-binding fragment is V_L。

49. The isolated binding protein or antigen-binding fragment of any one of claims 19-38, wherein the binding protein or antigen-binding fragment is a CDR.