Anti-IL2 receptor gamma antigen binding protein

Abstract

The present invention provides antibodies and antigen-binding fragments (e.g., human antibodies) that specifically bind to human IL2 receptor γ (IL2Rγ). Also provided are methods for using the antibodies and fragments to treat or prevent diseases mediated by IL2Rγ (e.g., graft-versus-host disease) and methods for preparing the antibodies and fragments.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 799,851, filed on February 1, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to antibodies that bind to anti-IL2 receptor gamma protein and methods of using the same, such as methods for treating or preventing diseases. Background Art

[0003] The common cytokine receptor gamma chain (γc) was originally identified as the third chain of the interleukin-2 (IL-2) receptor complex and was named IL-2Rγ. The same subunit was identified as part of several other cytokine receptor complexes: IL-4, IL-7, IL-9, IL-15 and IL-21, and can therefore be referred to as γc (common cytokine receptor gamma chain). γc is involved in the signal transduction and ligand binding of these cytokine receptors.

[0004] The binding of cytokines to their receptors activates Janus kinase (JAK) family protein tyrosine kinases JAK1 and JAK3, and triggers JAK1 and JAK3 to transphosphorylate tyrosine.JAK1 associates with the unique α or β chain of the receptor, and JAK3 associates with the γc of the receptor. Phosphorylated JAK in turn can activate signal transduction and transcription activator (signal transducer and activator of transcription, STAT) proteins, which together form the JAK / STAT signaling pathway. STAT phosphorylation leads to dimerization of STAT, which now uses high-affinity DNA binding activity and translocation to the nucleus. Here, it acts as a transcription factor to induce the transcription of target genes.

[0005] The γc gene (IL2RG) is located on chromosome Xq13. IL-2Rγ is mutated in patients with X-linked severe combined immunodeficiency (X-SCID). Patients with this disease exhibit severe immunodeficiency due to the lack of T cells, NK cells, and fully mature B cells.

[0006] IL-7, IL-9, and IL-15 are associated with psoriasis and rheumatoid arthritis (Pathak, The expanding role of IL-7 and thymic stromal lymphopoietin as therapeutic target for rheumatoid arthritis. Expert Opin Ther Targets. 18(5): 581-94 (2014); Hughes-Austin et al., Multiple cytokines and chemokines are associated with rheumatoid arthritis-related autoimmunity in first-degree relatives without rheumatoid arthritis: Studies of the Aetiology of Rheumatoid Arthritis (SERA), Ann Rheum Dis.; 72(6): 901-7 (2013); Dantas et al., Increased Serum Interleukin-9 Levels in Rheumatoid Arthritis and Systemic Lupus Erythematosus: Pathogenic Role or Just an Epiphenomenon?, Dis Markers. 2015; 2015: 519638; Yang et al., Therapeutic potential of IL-15 in rheumatoid arthritis, Hum Immunol. 2015 Nov; 76(11): 812-8; Lesiak et al., Are interleukin-15 and -22 a new pathogenic factor in pustular palmoplantar psoriasis?, Postepy Dermatol Alergol. 33(5): 336-339 (2016); Raeber et al., The role of cytokines in T-cell memory in health and disease, Immunol Rev. 283(1): 176-193 (2018)).

[0007] IL-4 and IL-9 antibodies with antibodies to the antibody (Generoso et al.,Prospects forMonoclonal Antibody Therapy in Pediatric Asthma,Curr Allergy Asthma Rep.18(9):45(2018);Tashkin&Wechsler,Role of eosinophils in airway inflammation ofchronic obstructive pulmonary disease,Int J Chron Obstruct Lung Dis.13:335-349(2018),Buzney et al.,Asthma and Atopic Dermatitis:A Review of TargetedInhibition of Interleukin-4 and Interleukin-13 As Therapy for Atopic Disease,J Drugs Dermatol.15(2):165-71(2016),Lloyd&Harker,Epigenetic Control ofInterleukin-9 in Asthma,N Engl J Med.379(1):87-89(2018),Neurath&Finotto,IL-9 signaling as a key driver of chronic inflammation in mucosal immunity,CytokineGrowth Factor Rev.29:93-9(2016)).

[0008] IL-21 is associated with a variety of inflammatory diseases, including Crohn's disease and rheumatoid arthritis. (Holm et al., Evaluating IL-21 as a Potential Therapeutic Target in Crohn's Disease, Gastroenterol Res Pract. 2018: 5962624 (2018); Dinesh & Rasool Multifaceted role of IL-21 in rheumatoid arthritis: Current understanding and future perspectives, J Cell Physiol. 233 (5): 3918-3928 (2018)). Summary of the invention

[0009] The present invention provides isolated antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof, e.g., which are monospecific or multispecific), characterized by one or more of the following: a kinase activity of about 2.75×10 -9 M is about 3.36×10 -7 M of K D Binds to human IL2Rγ; at 37°C, approximately 6.42×10 -9 M is about 3.53×10 -7 M of K D binds to human IL2Rγ; or at a rate of less than about 3.53×10 -7 M of K D Combined; at 25 ° C, about 3.18 × 10 -9 M is about 2.38×10 -7 M of K D Binds to cynomolgus monkey (Macaca fascicularis) IL-2Rγ; at 37°C, approximately 8.29×10 -9 M is about 3.20×10 -7 M of K D Binds to cynomolgus monkey IL-2Rγ; or less than about 3.20×10 -7 M of K D Combined; at 25°C, about 2.45×10 -9 M is about 1.20×10 -8 M of K D binds to human IL2Rγ; or at a rate of less than about 1.20×10 -8 M of K D Binding; at 37°C, about 1.86×10 -11 M to about 3.00×10-8 M of K D binds to human IL2Rγ; or less than about 3.00×10 -8 M of K D Combined; at 25 ° C, about 1.84 × 10 -8 M, 3.76×10 -9 M, 1.08×10 -7 M, 2.17×10 -8 M, 6.02×10 -9 M or 7.93×10 -8 M of K D Binds to mouse IL2Rγ; or no detectable binding; at 37°C, approximately 5.59×10 -8 M, 6.11×10 -9 M, 3.87×10 -7 M, 5.16×10 -8 M, 8.70×10 -9 M or 2.15×10 -7 M of K D Binds to mouse IL2Rγ; or no detectable binding; at 25°C, approximately 3.32×10 -9 M is about 1.97×10 -7 M of K D Binds to human IL2Rγ domain 1; or no detectable binding; at 37°C at approximately 4.13×10 -9 M is about 2.25×10 -7 M of K D Binds to human IL2Rγ domain 1; or no detectable binding; at 25°C at approximately 2.91×10 -7 M is about 5.35×10 -10 K D Binds to human IL2Rγ domain 2; or no detectable binding; at 37°C at approximately 1.14×10 -8 or about 1.27×10 -8 K DBinding to human IL2Rγ domain 2; or no detectable binding; blocking STAT phosphorylation in T cells induced by IL-2, IL-4, IL7, IL-15 and / or IL-21; blocking STAT phosphorylation in mast cells induced by IL-9; reducing the number of human immune cells injected into mice; reducing the levels of serum human cytokines and / or mouse serum cytokines in mice with human immune cells; no detectable binding to mouse or rat IL2Rγ; protecting mice from weight loss and / or death caused by GvHD in a GvHD mouse model; blocking the binding of a hybrid receptor comprising IL2Rγ complexed with cytokine-specific receptor subunits to IL-2, IL-4, IL-7, IL-9, IL-15 and / or IL-21; and / or reducing the number of CD45+ cells, B cells, T cells and / or NK cells (but optionally, not, for example, neutrophils) in the blood or serum of a subject. The following antibodies and antigen-binding fragments that specifically bind to IL2Rγ form part of the present invention, being variants of any of the antibodies or fragments whose sequences are specifically set forth herein and are characterized by one or more of the features described above.

[0010] The present invention also provides the following isolated antigen-binding protein, for example, an antibody or an antigen-binding fragment thereof, which antigen-binding protein: (i) specifically binds to the same epitope on IL2Rγ as a reference antibody or an antigen-binding fragment thereof; or (ii) competes with a reference antibody or an antigen-binding fragment thereof for binding to an IL2Rγ polypeptide, wherein the reference antibody or an antigen-binding fragment thereof comprises: (a) an immunoglobulin heavy chain or a variable region thereof comprising SEQ ID NO: 2, 18, 22, 38, 42, 58, 62, 77, 81, 97, 101, 115, 119, 134, 138, 152, 156, 170, 174, 186, 190, 198, 200, 208, 210, 216, 218, 234, 238, 254, 258, 272, 276, 284, 286, 294, 296, 311, 315, 331, 335, 343, 345, 357, 361 and / or 376 or a variant thereof, or CDR-H1, CDR-H2 and CDR-H3 of an immunoglobulin heavy chain or variable region thereof; and / or (b) an immunoglobulin light chain or variable region thereof, comprising a CDR-H1 comprising SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 79, 89, 99, 109, 117, 127, 136, 146, 154, 164, 172, 182, 188, 226, 236, 246, 256, 266, 274, 304, 313, 323, 333, 353, 359, 368 and / or 378 or variants thereof, or CDR-L1, CDR-L2 and CDR-L3 of an immunoglobulin light chain or variable region thereof having an amino acid sequence shown in or variants thereof. In one embodiment of the invention, the reference antibody or fragment is pre-bound to the IL2Rg antigen prior to the addition of the antigen binding protein and the binding is evaluated. In one embodiment of the invention, the antigen binding protein is pre-bound to the antigen prior to the addition of the reference antibody or fragment and the binding is evaluated.

[0011] The present invention also provides the following isolated antigen-binding protein (eg, antibody or antigen-binding fragment thereof), comprising: (a) an immunoglobulin heavy chain or variable region thereof, comprising a sequence of SEQ ID NO: 2, 18, 22, 38, 42, 58, 62, 77, 81, 97, 101, 115, 119, 134, 138, 152, 156, 170, 174, 186, 190, 198, 200, 208, 210, 216, 218, 234, 238, 254, 258, 272, 276, 284, 286, 294, 296, 311, 315, 331, 335, 343, 345, 357, 361 and / or 376 or a variant thereof, or CDR-H1, CDR-H2 and CDR-H3 of an immunoglobulin heavy chain or variable region thereof; and / or (b) an immunoglobulin light chain or variable region thereof, comprising a sequence comprising SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 79, 89, 99, 109, 117, 127, 136, 146, 154, 164, 172, 182, 188, 226, 236, 246, 256, 266, 274, 304, 313, 323, 333, 353, 359, CDR-L1, CDR-L2 and CDR-L3 of the immunoglobulin light chain or its variable region whose amino acid sequences are shown in NO: 10, 20, 30, 40, 50, 60, 70, 79, 89, 99, 109, 117, 127, 136, 146, 154, 164, 172, 182, 188, 226, 236, 246, 256, 266, 274, 304, 313, 323, 333, 353, 359, 368 and / or 378 or their variants.

[0012] In one embodiment of the present invention, the invention comprises (a) an immunoglobulin heavy chain or a variable region thereof, which comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 2, 18, 22, 38, 42, 58, 62, 77, 81, 97, 101, 115, 119, 134, 138, 152, 156, 170, 174, 186, 190, 198, 200, 208, 210, 216, 218, 234, 238, 254, 258, 272, 276, 284, 286, 294, 296, 311, 315, 331, 335, 343, 345, 357, 361 and / or 376; and / or (b) an immunoglobulin light chain or a variable region thereof, which comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: NO:10, 20, 30, 40, 50, 60, 70, 79, 89, 99, 109, 117, 127, 136, 146, 154, 164, 172, 182, 188, 226, 236, 246, 256, 266, 274, 304, 313, 323, 333, 353, 359, 368 and / or 378 have an amino acid sequence with at least 90% amino acid sequence identity.For example, in one embodiment of the invention, the antigen binding protein comprises: (a) an immunoglobulin heavy chain or variable region thereof comprising an amino acid sequence as shown in SEQ ID NO: 2, 18, 22, 38, 42, 58, 62, 77, 81, 97, 101, 115, 119, 134, 138, 152, 156, 170, 174, 186, 190, 198, 200, 208, 210, 216, 218, 234, 238, 254, 258, 272, 276, 284, 286, 294, 296, 311, 315, 331, 335, 343, 345, 357, 361 and / or 376 and amino acids corresponding to SEQ ID NO: NO: 2, 18, 22, 38, 42, 58, 62, 77, 81, 97, 101, 115, 119, 134, 138, 152, 156, 170, 174, 186, 190, 198, 200, 208, 210, 216, 218, 234, 238, 254, 258, 272, 276, 284, 286, 294, 296, 311, 315, 331, 335, 343, 345, 357, 361 and / or 376 having at least 90% amino acid sequence identity with the amino acid sequence set forth in the immunoglobulin heavy chain or its variable region CDR-H1, CDR-H2 and CDR-H3; and / or (b) an immunoglobulin light chain or its variable region comprising a CDR-H1 comprising SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 79, 89, 99, 109, 117, 127, 136, 146, 154, 164, 172, 182, 188, 226, 236, 246, 256, 266, 274, 304, 313, 323, 333, 353, 359, the amino acid sequences shown in 368 and / or 378, and the amino acid sequences corresponding to SEQ ID NO:10, 20, 30, 40, 50, 60, 70, 79, 89, 99, 109, 117, 127, 136, 146, 154, 164, 172, 182, 188, 226, 236, 246, 256, 266, 274, 304, 313, 323, 333, 353, 359, CDR-L1, CDR-L2 and CDR-L3 of an immunoglobulin light chain or its variable region having an amino acid sequence with at least 90% amino acid sequence identity as shown in the amino acid sequence shown in NO:10, 20, 30, 40, 50, 60, 70, 79, 89, 99, 109, 117, 127, 136, 146, 154, 164, 172, 182, 188, 226, 236, 246, 256, 266, 274, 304, 313, 323, 333, 353, 359, 368 and / or 378.

[0013] In one embodiment of the invention, the antigen binding protein comprises:

[0014] (i) a heavy chain CDR set: a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 8; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 28; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 46, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 48; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 64, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 66, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 68; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 83, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 84, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 85; NO: 85, and CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 87; and / or CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 103, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 105, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 107; and / or CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 121, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 123, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 125; and / or CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 140, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 142, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 144; and / or CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 158, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 159, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 160. NO: 160, and CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 162, and / or CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 176, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 178, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 180;and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 192, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 194, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 196; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 202, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 204, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 206; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 176, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 214; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 222, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 224; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 22 NO: 240, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 242, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 244; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 260, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 262, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 264; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 278, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 280, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 282; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 288, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 290, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 292; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 298, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 290, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: NO: 298, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 300, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 302; and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 317, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 319, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 321;and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 337, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 339, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 341; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 347, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 349, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 351; and / or a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 363, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 66, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 366; and / or;

[0015] (ii) a light chain CDR set: a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 36; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 56; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 75; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 76 NO:91, a CDR-L2 having the amino acid sequence shown in SEQ ID NO:93, and a CDR-L3 having the amino acid sequence shown in SEQ ID NO:95; and / or a CDR-L1 having the amino acid sequence shown in SEQ ID NO:111, a CDR-L2 having the amino acid sequence shown in SEQ ID NO:54, and a CDR-L3 having the amino acid sequence shown in SEQ ID NO:113; and / or a CDR-L1 having the amino acid sequence shown in SEQ ID NO:129, a CDR-L2 having the amino acid sequence shown in SEQ ID NO:54, and a CDR-L3 having the amino acid sequence shown in SEQ ID NO:132; and / or a CDR-L1 having the amino acid sequence shown in SEQ ID NO:148, a CDR-L2 having the amino acid sequence shown in SEQ ID NO:54, and a CDR-L3 having the amino acid sequence shown in SEQ ID NO:150; and / or a CDR-L1 having the amino acid sequence shown in SEQ ID NO:166, a CDR-L2 having the amino acid sequence shown in SEQ ID NO:54, and a CDR-L3 having the amino acid sequence shown in SEQ ID NO:167. NO:14, and CDR-L2 comprising the amino acid sequence shown in SEQ ID NO:14, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO:168; and / or CDR-L1 comprising the amino acid sequence shown in SEQ ID NO:72, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO:54, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO:184;and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 184; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 184; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 184; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 228, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 230, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 232; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 228, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 230, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 232 NO: 248, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 250, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 252; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 268, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 270; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 184; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 184; and / or a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 306, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: NO: 230, and CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 309; and / or CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 325, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 327, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 329;and / or a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 184; and / or a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 355; and / or a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 370, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 372, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 374. ;

[0016] In one embodiment of the present invention, the antigen binding protein of the present invention comprises the following heavy chain CDR set and light chain CDR set:

[0017] (i) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 8; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16; (ii) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 28; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: NO:36; (iii) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:44, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:46, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:48; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:52, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:56; (iv) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:64, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:66, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:68; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:54, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:73. (v) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 83, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 85, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 87;and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 91, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 93, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 95; (vi) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 103, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 105, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 107; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 111, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 113; (vi) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 121, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 123, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: NO: 125; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 129, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 132; (vii) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 140, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 142, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 144; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 148, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 150; (viii) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 158, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 159, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: NO: 160, and CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 160, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 162; and a light chain variable region comprising CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 166, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 168;(ix) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 176, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 178, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 180; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 184; (x) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 192, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 194, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 196; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: NO: 184; (xi) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 202, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 204, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 206; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 184; (xii) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 176, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 214; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: (xiii) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 220, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 222, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 224;and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 228, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 230, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 232; (xiv) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 240, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 242, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 244; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 248, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 250, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 252; (xv) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 260, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 262, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: NO: 264; and a light chain variable region comprising CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 268, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 270; (xvi) a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 278, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 280, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 282; and a light chain variable region comprising CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 184; (xvii) a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 288, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 280, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 282 NO: 290, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 292; and a light chain variable region comprising CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 184;(xviii) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 298, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 300, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 302; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 306, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 230, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 309; (xix) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 317, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 319, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 321; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 325, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 327, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: NO: 329; (xx) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 337, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 339, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 341; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 184; (xxi) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 347, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 349, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 351; and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: (xxii) a heavy chain variable region comprising a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 363, a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 66, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 366;and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 370, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 372, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 374. ;

[0018] Complexes comprising an antigen binding protein of the invention bound to an IL2Rγ polypeptide or an antigenic fragment thereof are also part of the present invention.

[0019] The present invention also provides methods for preparing an antigen-binding protein (eg, an antibody or an antigen-binding fragment thereof) or an immunoglobulin chain thereof (eg, V H 、V L , HC or LC), comprising: (a) introducing one or more polynucleotides encoding one or more immunoglobulin chains of the antigen binding protein (or a vector comprising such a polynucleotide) into a host cell (e.g., a CHO cell); (b) culturing the host cell under conditions conducive to the expression of the polynucleotide; and (c) optionally, isolating the antigen binding protein or immunoglobulin chain from the host cell and / or the culture medium in which the host cell is cultured. Antigen binding proteins or immunoglobulin chains that are the product of such a method also form part of the present invention.

[0020] The present invention also provides a polypeptide comprising: (a) an immunoglobulin heavy chain or a variable region thereof comprising the amino acid sequence shown in SEQ ID NO: 2, 18, 22, 38, 42, 58, 62, 77, 81, 97, 101, 115, 119, 134, 138, 152, 156, 170, 174, 186, 190, 198, 200, 208, 210, 216, 218, 234, 238, 254, 258, 272, 276, 284, 286, 294, 296, 311, 315, 331, 335, 343, 345, 357, 361 and / or 376 or a variant thereof; and / or (b) a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 2, 18, 22, 38, 42, 58, 62, 77, 81, 97, 101, 115, 119, 134, 138, 152, 156, 170, 174, 186, 190, 198, 200, 208, 210, 216, 218, 234, 238, 254, 258, 272, 276, 284, 286, 294, 296, 311, 315, 331, 335, 343, 345, 357, NO: 10, 20, 30, 40, 50, 60, 70, 79, 89, 99, 109, 117, 127, 136, 146, 154, 164, 172, 182, 188, 226, 236, 246, 256, 266, 274, 304, 313, 323, 333, 353, 359, 368 and / or 378 or variants thereof, or (c) an amino acid sequence or variants thereof selected from a member selected from SEQ ID NO: 1 to 378. The present invention also provides polynucleotides encoding one or more such polypeptides or vectors (e.g., plasmids) comprising such polynucleotides.

[0021] The present invention also provides host cells (e.g., CHO cells) comprising an antigen binding protein (e.g., an antibody or antigen binding fragment thereof) or an immunoglobulin chain (e.g., V H 、V L , HC or LC) or polypeptide or polynucleotide or vector.

[0022] The present invention also provides a composition or kit comprising one or more antigen binding proteins (e.g., antibodies or antigen binding fragments thereof) described herein, optionally in combination with an additional therapeutic agent (e.g., an anti-inflammatory agent, an anti-TNFα antibody or binding protein, infliximab, adalimumab, etanercept, golimumab, corticoid, prednisolone, methylprednisolone, anti-thymocyte globulin, alemtuzumab, dacluzimab, extracorporeal photophoresis, mycophenolate mofetil, sirolimus, pentostatin, mesenchymal stem cells, inolimomab, denileukin, or basiliximab).

[0023] The present invention also provides a pharmaceutical formulation comprising an antigen binding protein (e.g., an antibody or antigen binding fragment thereof) as described herein and a pharmaceutically acceptable carrier, and optionally, an additional therapeutic agent (e.g., an anti-inflammatory agent, an anti-rNFα antibody or binding protein, infliximab, adalimumab, etanercept, golimumab, adrenocortical hormone, prednisolone, methylprednisolone, anti-thymocyte globulin, alemtuzumab, daclizumab, extracorporeal photophoresis, mycophenolate mofetil, tacrolimus, cyclosporine, sirolimus, pentostatin, mesenchymal stem cells, inomucomab, denileukin or basiliximab).

[0024] The present invention also provides a container or injection device (eg, a vial, a syringe, a prefilled syringe, or an autoinjector) comprising an antigen binding protein or composition (eg, a pharmaceutical preparation) described herein.

[0025] The present invention also provides a method for administering an antigen binding protein or composition as shown herein to a subject (e.g., a human), comprising introducing the antigen binding protein or composition into, for example, injecting (e.g., subcutaneously, intravenously, or intramuscularly) into the subject. The present invention also provides a method for treating or preventing an IL2Rγ-mediated disease or condition (e.g., graft-versus-host disease, organ transplant rejection, skin transplant rejection, heart transplant rejection, lung transplant rejection, kidney transplant rejection, liver transplant rejection, birdshot chorioretinopathy, multiple sclerosis, uveitis, autoimmune disease, type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and / or myasthenia gravis) in a subject in need thereof, comprising administering, for example, injecting an effective amount of an antigen binding protein or composition as shown herein.

[0026] The present invention also provides a method for performing the following in a subject: blocking STAT phosphorylation in peripheral blood mononuclear cells (e.g., T cells) induced by cytokines (e.g., IL-2, IL-4, IL-7, IL-15 and / or IL-21); blocking STAT (e.g., STAT3) phosphorylation in mast cells induced by cytokines (e.g., IL-9); reducing serum levels of interferon-γ, tumor necrosis factor-α, IL-6, IL-8, IL-10 and / or mKC / GRO (e.g., in a transplant recipient); blocking JAK-STAT-mediated (e.g., STAT3) intracellular signaling (e.g., in NK cells) induced by cytokines in the IL2Rγ family (e.g., IL-2, IL-4, IL-7, IL-9, IL-15 and / or IL-21); and / or reducing serum levels of CD45+ immune cells, NK cells, T cells and / or B cells (e.g., excluding neutrophils); the method comprises administering to the subject an effective amount of an anti-IL2Rγ antigen binding protein as described herein, or a composition thereof, or a formulation thereof. In one embodiment of the invention, the subject has an IL2Rγ-mediated disease or condition, such as graft-versus-host disease, organ transplant rejection, b-islet cell transplant rejection, skin transplant rejection, heart transplant rejection, lung transplant rejection, kidney transplant rejection, liver transplant rejection, birdshot chorioretinopathy, multiple sclerosis, uveitis, autoimmune disease, type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, aplastic anemia, atopic dermatitis, asthma, mast cell activation disorder, mast cell activation syndrome (MCAS), systemic mastocytosis (SM) and / or mast cell leukemia (MCL). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 (A to E). Blockade of human (A) IL-2, (B) IL-4, (C) IL7, (D) IL-15 and (E) IL-21-induced STAT phosphorylation in human CD4+ T cells by various concentrations of anti-IL-2Rγ antibodies H4H12857P, H4H12874P, H4H12886P, H4H12889P and H4H12922P2, and antibodies REGN1945 and COMP1499.

[0028] Figure 2Blockade of human IL-9-induced STAT3 phosphorylation in in vitro differentiated human mast cells by anti-IL-2Rγ antibodies H4H12874P, H4H12886P, H4H12889P, H4H12922P2, and antibodies COMP1499 and REGN1945.

[0029] Figure 3 (A to F). Percentage of initial body weight over time of mice with human PBMCs administered with anti-IL2Rγ antibodies (E) H4H12889P and (F) H4H12922P2 and antibody (D) COMP1499. Also shown are control experiments in mice (B) not administered with antibodies, (C) administered with isotype control antibodies, or (A) without human PBMCs. The beginning of antibody injections at day 21 and the end of antibody injections at day 59 are indicated by dotted lines.

[0030] Figure 4 .Survival of mice injected with anti-IL2Rγ antibodies H4H12889P and H4H12922P2, antibody COMP1499, antibody REGN1945, and without antibody injection over time is shown. The no huPBMC group is not shown. Differences in animal survival relative to the isotype control antibody group were analyzed by the Mantel-Cox log-rank test. P values ​​<0.05 were considered statistically significant. **, P value <0.0021; ****, P value <0.0001. The start of antibody injection on day 21 and the end of antibody injection on day 59 are indicated by dotted lines.

[0031] Figure 5 (A to D). Absolute human cell counts ((A) human CD45 cells; (B) human T cells; (C) human CD4 T cells; and (D) human CD8 T cells) in the blood on day 35 after huPBMC injection in mice that were not administered with antibodies (no IgG), administered with REGN1945, COMP1499, or the anti-IL2Rγ antibodies H4H12889P or H4H12922P2. The “no huPBMC” group is not shown; #, significantly different from the “no huPBMC” group; Significantly different from the "huPBMC-no IgG" group; *, significantly different from the "huPBMC-REGN1945" group. Each symbol represents one mouse. For plotting purposes (logarithmic scale), the zero value was arbitrarily changed to a value of 0.01.

[0032] Figure 6(A to D). Blood counts of human (A) CD45+ cells, (B) T cells, (C) CD4+ T cells, and (D) CD8+ T cells over time in mice administered anti-IL2Rγ antibodies H4H12889P or H4H12922P2, or COMP1499 or isotype control antibodies. The start of antibody injection at day 21 and the end of antibody injection at day 59 are indicated by dotted lines.

[0033] Figure 7 (A to I). Serum levels of human and mouse cytokines ((A) human interferon-γ; (B) human TNFα; (C) human IL-6; (D) human IL-8; (E) human IL-10; (F) mouse TNFα; (G) mouse IL-6; (H) mouse KC / GRO; and (I) mouse IL-10) at day 42 after injection of huPBMCs in mice without antibody administration (no IgG), REGN1945, COMP1499, or anti-IL2Rγ antibodies H4H12889P or H4H12922P2, or in mice without human PBMCs. #, significantly different from the “no huPBMC” group; Significantly different from the "huPBMC-no IgG" group; *, significantly different from the "huPBMC-REGN1945" group. Each symbol represents one mouse.

[0034] Figure 8 (A to D). Serum levels of (A) human IFN-γ, (B) human TNFα, (C) mouse TNFα, and (D) mouse IL-6 over time in mice administered anti-IL2Rγ antibodies H4H12889P or H4H12922P2, or COMP1499, or an isotype control antibody.

[0035] Figure 9 (A to E). Levels of total human antibodies or CD45+ immune cells (A), NK cells (B), T cells (C), B cells (D), or neutrophils (E) in the blood of mice treated with various doses of antibodies REGN1945 or H4H12889P.

[0036] Fig.10 . Experimental design for in vivo skin graft rejection experiments.

[0037] Fig.11 . Time to onset of skin graft rejection in mice not administered antibody, administered REGN1945, or H4H12889P.

[0038] Fig.12 . Time to complete skin graft rejection in mice not administered antibody, administered REGN1945, or H4H12889P.

[0039] Fig.13 Total donor-specific IgG antibodies in non-transplanted mice or transplanted mice without antibody administration, REGN1945 or H4H12889P administration. DETAILED DESCRIPTION

[0040] The present invention provides antibodies and antigen-binding fragments thereof, which specifically bind to human and cynomolgus monkey IL2Rγ and show unique biological activities, especially in blocking cytokine-induced STAT phosphorylation in T cells and blocking graft-versus-host disease in an applicable mouse model.

[0041] In accordance with the present invention, conventional molecular biology, microbiology and recombinant DNA techniques within the skill of the art may be employed. Such techniques are fully explained in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (hereinafter "Sambrook, et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover, ed., 1985); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (hereinafter "Sambrook, et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover, ed., 1985); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds. (1985)); Cloning (1984); FM Ausubel, et al. (Editors), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0042] IL-2Rγ

[0043] Interleukin-2 receptor subunit gamma is also known as CD132, common cytokine receptor gamma c chain, IL-2RG, IL-2Rg, IL2R gamma, IL-2Rγ, IMD4, P64:SCIDX, or SCIDX1. IL2Rγ is a subunit shared by several interleukin receptors, including IL-2R, IL-4R, IL-7R, IL-9R, IL-15R, and IL21R.

[0044] In one embodiment of the present invention, human IL2Rγ is encoded by the nucleotide sequence shown in Genbank Accession No. NM_000206. In one embodiment of the present invention, human IL2Rγ comprises the amino acid sequence shown in Genbank Accession No. NP_000197.

[0045] Antigen Binding Protein

[0046] The present invention provides antigen binding proteins that specifically bind to IL2Rγ protein or its antigenic fragment (e.g., the extracellular domain of IL2Rγ), such as antibodies (e.g., human antibodies, monoclonal antibodies and recombinant antibodies) and antigen binding fragments thereof. Antigen binding proteins that bind to the same epitope on IL2Rγ as any antigen binding protein shown herein or compete with any antigen binding protein shown herein for binding to IL2Rγ are also part of the present invention.

[0047] The present invention also provides any polypeptide comprising the amino acid sequence shown below or a variant thereof:

[0048] SEQ ID NO: 2, 4, 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, 6 0, 62, 64, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 1 15, 117, 119, 121, 123, 125, 127, 129, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290 , 292, 294, 296, 298, 300, 302, 304, 306, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 366, 368, 370, 372, 374, 376 and / or 378. Optionally, the polypeptide is fused to one or more additional polypeptides, e.g., human Fc (e.g., human IgG, e.g., IgG1 or IgG4 (e.g., comprising an S108P mutation)).

[0049] As used herein, the term "antibody" refers to an immunoglobulin molecule (i.e., a "complete antibody molecule") comprising four polypeptide chains: two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds (e.g., IgG) - e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2.

[0050] In one embodiment of the invention, each antibody heavy chain (HC) comprises a heavy chain variable region ("HCVR" or "V H ”) (e.g., SEQ ID NO: 2, 22, 42, 62, 81, 101, 119, 138, 156, 174, 190, 200, 210, 218, 238, 258, 276, 286, 296, 315, 335, 345 or 361 or a variant thereof) and a heavy chain constant region (comprising domain C H 1. C H 2 and C H 3); and each antibody light chain (LC) comprises a light chain variable region ("LCVR" or "V L ”) (e.g., SEQ ID NO: 10, 30, 50, 70, 89, 109, 127, 146, 164, 182, 226, 246, 266, 304, 323, 353 or 368 or a variant thereof) and a light chain constant region (CL). V H and V L The regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). VH and VL each contain three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FRs of the antibody (or its antigen-binding fragment) are identical to human germline sequences or are naturally or artificially modified.

[0051] Typically, the variable domains of both immunoglobulin heavy and light chains include three hypervariable regions, also known as complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). Typically, from N-terminus to C-terminus, the variable domains of both light and heavy chains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In one embodiment of the invention, amino acids are assigned to each domain according to the definitions in: Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th Edition; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252: 6609-6616; Chothia, et al., (1987) J Mol. Biol. 196: 901-917 or Chothia, et al., (1989) Nature 342: 878-883. Thus, the invention includes proteins containing V H CDR and V L The CDRs of the antibodies and antigen-binding fragments, the V H and V L Comprising an amino acid sequence as set forth herein (or a variant thereof), wherein the CDRs are defined according to Kabat and / or Chothia.

[0052] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody or antigen-binding protein includes any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments (heavy chain portion of Fab fragment cleaved with papain); (iv) Fv fragments (V H or V L); and (v) single-chain Fv (scFv) molecules; composed of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementary determining region (CDR), such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. Other modified molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, and small modular immunopharmaceuticals (SMIPs), are also included in the expression "antigen-binding fragment" used herein. In one embodiment of the invention, the antigen-binding fragment comprises the following three or more CDRs (e.g., CDR-H1, CDR-H2, and CDR-H3; or CDR-L1, CDR-L2, and CDR-L3):

[0053] or H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2.

[0054] In one embodiment of the present invention, the antigen-binding protein (eg, antibody or antigen-binding fragment thereof) of the present invention comprises: H (e.g., HC) is an immunoglobulin heavy chain, the V H A combination of heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3) shown in Table A below; and / or an immunoglobulin light chain containing a VL (e.g., LC) having a V L Combinations of light chain CDRs (CDR-L1, CDR-L2 and CDR-L3) are included as shown in Table B below.

[0055] Table A. Heavy chain CDRs in the immunoglobulins of the present invention

[0056] CDR-H combination CDR-H1 CDR-H2 CDR-H3 1 4 6 8 2 24 26 28 3 44 46 48 4 64 66 68 5 83 85 87 6 103 105 107 7 121 123 125 8 140 142 144 9 158 160 162 10 176 178 180 11 192 194 196 12 202 204 206 13 176 212 214 14 220 222 224 15 240 242 244 16 260 262 264 17 278 280 282 18 288 290 292 19 298 300 302 20 317 319 321 21 337 339 341 22 347 349 351 23 363 66 366

[0057] *The numbers correspond to the amino acid sequence shown in the SEQ ID NO.

[0058] Table B. Light chain CDRs in the immunoglobulins of the present invention

[0059]

[0060]

[0061] *The numbers correspond to the amino acid sequence shown in the SEQ ID NO.

[0062] In one embodiment of the present invention, the antigen-binding protein (eg, antibody or antigen-binding fragment thereof) of the present invention comprises a V H (e.g., HC) and V L An immunoglobulin heavy and light chain (e.g., LC) comprising the combination of heavy and light chain CDRs shown in Table C below (CDR-H1, CDR-H2, and CDR-H3; and CDR-L1, CDR-L2, and CDR-L3).

[0063] Table C. Heavy and light chain CDRs in the immunoglobulins of the invention

[0064]

[0065]

[0066] *The numbers correspond to the amino acid sequence shown in the SEQ ID NO.

[0067] The present invention includes the following V H and V L An antigen-binding protein (eg, an antibody or antigen-binding fragment thereof) of a polypeptide pair having an amino acid sequence:

[0068] SEQ ID NO: 2 and SEQ ID NO: 10;

[0069] SEQ ID NO: 22 and SEQ ID NO: 30;

[0070] SEQ ID NO:42 and SEQ ID NO:50;

[0071] SEQ ID NO: 62 and SEQ ID NO: 70;

[0072] SEQ ID NO: 81 and SEQ ID NO: 89;

[0073] SEQ ID NO: 101 and SEQ ID NO: 109;

[0074] SEQ ID NO: 119 and SEQ ID NO: 127;

[0075] SEQ ID NO: 138 and SEQ ID NO: 146;

[0076] SEQ ID NO: 156 and SEQ ID NO: 164;

[0077] SEQ ID NO: 174 and SEQ ID NO: 182;

[0078] SEQ ID NO: 190 and SEQ ID NO: 182;

[0079] SEQ ID NO: 200 and SEQ ID NO: 182;

[0080] SEQ ID NO: 210 and SEQ ID NO: 182;

[0081] SEQ ID NO: 218 and SEQ ID NO: 226;

[0082] SEQ ID NO: 238 and SEQ ID NO: 246;

[0083] SEQ ID NO: 258 and SEQ ID NO: 266;

[0084] SEQ ID NO: 276 and SEQ ID NO: 182;

[0085] SEQ ID NO: 286 and SEQ ID NO: 182;

[0086] SEQ ID NO: 296 and SEQ ID NO: 304;

[0087] SEQ ID NO:315 and SEQ ID NO:323;

[0088] SEQ ID NO:335 and SEQ ID NO:182;

[0089] SEQ ID NO: 345 and SEQ ID NO: 353; or

[0090] SEQ ID NO:361 and SEQ ID NO:368.

[0091] The present invention includes antigen binding proteins (eg, antibodies or antigen binding fragments thereof) comprising the following amino acid sequence pair encoding HC and LC:

[0092] SEQ ID NO: 18 and SEQ ID NO: 20;

[0093] SEQ ID NO:38 and SEQ ID NO:40;

[0094] SEQ ID NO:58 and SEQ ID NO:60;

[0095] SEQ ID NO: 77 and SEQ ID NO: 79;

[0096] SEQ ID NO: 97 and SEQ ID NO: 99;

[0097] SEQ ID NO: 115 and SEQ ID NO: 117;

[0098] SEQ ID NO: 134 and SEQ ID NO: 136;

[0099] SEQ ID NO: 152 and SEQ ID NO: 154;

[0100] SEQ ID NO: 170 and SEQ ID NO: 172;

[0101] SEQ ID NO: 186 and SEQ ID NO: 188;

[0102] SEQ ID NO: 198 and SEQ ID NO: 188;

[0103] SEQ ID NO: 208 and SEQ ID NO: 188;

[0104] SEQ ID NO: 216 and SEQ ID NO: 188;

[0105] SEQ ID NO: 234 and SEQ ID NO: 236;

[0106] SEQ ID NO: 254 and SEQ ID NO: 256;

[0107] SEQ ID NO: 272 and SEQ ID NO: 274;

[0108] SEQ ID NO: 284 and SEQ ID NO: 188;

[0109] SEQ ID NO: 294 and SEQ ID NO: 188;

[0110] SEQ ID NO: 311 and SEQ ID NO: 313;

[0111] SEQ ID NO: 331 and SEQ ID NO: 333;

[0112] SEQ ID NO:343 and SEQ ID NO:188;

[0113] SEQ ID NO: 357 and SEQ ID NO: 359; or

[0114] SEQ ID NO: 376 and SEQ ID NO: 378.

[0115] Some embodiments of the present invention further include immunoglobulin V H and V L , or an antigen-binding protein of HC and LC, such as an anti-IL2Rγ antibody and an antigen-binding fragment thereof, wherein the immunoglobulin V H and V L , or HC and LC contain the corresponding V specifically shown in this article H 、V L In some embodiments, the amino acid sequence of the variant antigen-binding protein has 70% or more (e.g., 80%, 85%, 90%, 95%, 97% or 99%) overall amino acid sequence identity or similarity, but wherein the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of such immunoglobulin are not variants and contain the amino acid sequences shown in this article. Therefore, in some such embodiments, the CDRs in the variant antigen-binding protein themselves are not variants.

[0116] The present invention includes monoclonal anti-IL2Rγ antigen binding proteins, such as antibodies and antigen binding fragments thereof, and monoclonal compositions comprising a plurality of isolated monoclonal antigen binding proteins. The term "monoclonal antibody" or "mAb" as used herein refers to a member of a population of substantially homogeneous antibodies, i.e., the antibody molecules contained in the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in small amounts. A "plurality" of such monoclonal antibodies and fragments in a composition refers to the concentration of the same (i.e., identical in amino acid sequence except for possible naturally occurring mutations that may be present in small amounts, as discussed above) antibodies and fragments that are higher than the concentration that normally exists in nature (e.g., in the blood of a host organism (e.g., a mouse or a human)).

[0117] In one embodiment of the invention, the anti-IL2Rγ antigen binding protein (e.g., antibody or antigen binding fragment) comprises a heavy chain constant domain, such as a heavy chain constant domain of the IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 and IgG4 (e.g., comprising S228P and / or S108P mutations)) or IgM type. In one embodiment of the invention, the antigen binding protein (e.g., antibody or antigen binding fragment) comprises a light chain constant domain, such as a light chain constant domain of the κ or λ type. The invention includes antigen binding proteins comprising a variable domain as set forth herein (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12894P) linked to a heavy and / or light chain constant domain (e.g., as set forth above). (4H12890P; H4H12899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2).

[0118] The term "human" antigen-binding proteins used herein, such as antibodies or antigen-binding fragments, include antibodies and fragments with variable regions and constant regions derived from human germline immunoglobulin sequences, whether in human cells or transplanted into non-human cells (such as mouse cells). See, for example, US8502018, US6596541 or US5789215. In one embodiment of the invention, human antibodies and antigen-binding fragments of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., with mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDR, particularly in CDR3. However, the term "human antibody" used herein is not intended to include mAbs in which the CDR sequences of the germline derived from another mammalian species (e.g., mice) have been grafted to human FR sequences. The term includes antibodies recombinantly produced in cells of non-human mammals or non-human mammals. The term is not intended to include antibodies separated from human subjects or produced in human subjects. The present invention includes human antigen binding proteins (e.g., antibodies or antigen binding fragments thereof, such as H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12 899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2).

[0119] The present invention includes anti-IL2Rγ chimeric antigen binding proteins, such as antibodies and antigen-binding fragments thereof, and methods of use thereof. As used herein, a "chimeric antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first antibody and the second antibody are from different species. (See, e.g., US4816567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851L6855). The present invention includes chimeric antibodies containing the variable domains described herein (e.g., from H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899P; H4H1 chimeric antibodies comprising the variable domains of H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2).

[0120] The term "recombinant" antigen binding protein, such as an antibody or antigen binding fragment thereof, refers to such a molecule produced, expressed, isolated or obtained by a technique or method known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term includes antibodies expressed in a non-human mammal (including a transgenic non-human mammal, such as a transgenic mouse) or a host cell (such as a Chinese hamster ovary (CHO) cell) or a cell expression system, or antibodies isolated from a recombinant combinatorial human antibody library. The present invention includes recombinant antigen binding proteins as described herein (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899 P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2).

[0121] In one embodiment of the invention, the antigen-binding fragment of an antibody comprises at least one variable domain. The variable domain can be of any size or amino acid composition and generally comprises at least one (e.g., 3) CDR adjacent to or within a frame with one or more framework sequences. L Domain-associated V H In the antigen-binding fragment of the structural domain, V H and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise non-covalently bound monomers V H and / or V L Structural domain.

[0122] In certain embodiments, the antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be present in the antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -CH3; (xi)V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) VL -C H 2-C H 3; and (xiv) V L -C L . In any configuration of variable domains and constant domains including any of the exemplary configurations listed above, the variable domains and constant domains may be directly connected to each other or may be connected through a complete or partial hinge region or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragments of the antibodies of the present invention may comprise any of the variable and constant domain configurations listed above, non-covalently associated with each other and / or with one or more monomeric V H or V L The present invention includes antigen-binding fragments of the antigen-binding proteins described herein, for example,

[0123] H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2.

[0124] Antigen binding proteins (e.g., antibodies and antigen binding fragments) can be monospecific or multispecific (e.g., bispecific). Multispecific antigen binding proteins are further discussed herein. The present invention includes monospecific as well as multispecific (e.g., bispecific) antigen binding fragments comprising antigen binding proteins specifically described herein (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; one or more variable domains of the invention.

[0125] The term "specific binding" refers to those proteins that bind to an antigen (e.g., IL2Rγ protein) with a K D The binding affinity expressed is at least about 10 -7 M (e.g., 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M) antigen binding protein (e.g., antibody or antigen binding fragment thereof), as determined by real-time, label-free biolayer interferometry, e.g., at 25° C. or 37° C., e.g. HTX biosensors, or by surface plasmon resonance, such as BIACORE TM , or measured by solution affinity ELISA. The present invention includes antigen binding proteins that specifically bind to IL2Rγ protein. In one embodiment of the present invention, the anti-IL2Rγ antigen binding protein comprises a KD value for binding to human and / or mouse and / or cynomolgus monkey and / or rat IL2Rγ or its domain, which is shown in any one of Tables 3-1 to 3-12. "Anti-IL2Rγ" refers to an antigen binding protein (or other molecule), such as an antibody or an antigen binding fragment thereof, that specifically binds to IL2Rγ.

[0126] "Isolated" antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof), polypeptides, polynucleotides, and vectors are at least partially free of other biomolecules from the cells or cell cultures in which they are produced. Such biomolecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other substances such as cell debris and growth medium. An isolated antigen-binding protein may also be at least partially free of expression system components, such as biomolecules from host cells or their growth medium. In general, the term "isolated" is not intended to mean: the complete absence of such biomolecules (e.g., a small or insignificant amount of impurities may remain); or the absence of water, buffers, or salts; or components of a pharmaceutical preparation comprising an antigen-binding protein (e.g., an antibody or antigen-binding fragment).

[0127] The present invention includes antigen binding proteins of the present invention (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12888P; H4H12889P; H4H12890P; H4H12899P; H4H12900P; H4 or H4H13545P2) an antigen binding protein, such as an antibody or antigen binding fragment, that binds to the same epitope.

[0128] Antigens are molecules, such as peptides (e.g., IL2Rγ or fragments thereof (antigenic fragments)) to which antibodies bind. The specific region on an antigen that an antibody recognizes and binds to is called an epitope. Antigen binding proteins (e.g., antibodies) of the invention that specifically bind to such antigens are part of the invention.

[0129] The term "epitope" refers to an antigenic determinant (e.g., on IL2Rγ) that interacts with a specific antigen binding site of an antigen binding protein, such as a variable region of an antibody molecule called a paratope. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on an antigen and may have different biological effects. The term "epitope" may also refer to a site on an antigen to which B cells and / or T cells respond, and / or to an antigen region bound by an antibody. An epitope may be defined as structural or functional. Functional epitopes are typically subsets of structural epitopes and have those residues that directly contribute to the affinity of the interaction. An epitope may be linear or conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope may include a determinant that is a chemically active surface grouping of a molecule such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments may have specific three-dimensional structural features and / or specific charge characteristics. The epitope to which the antigen binding proteins of the present invention bind may be contained in a fragment of IL2Rγ (eg, human IL2Rγ), such as its extracellular domain, domain 1 or domain 2. Antigen binding proteins of the present invention (eg, antibodies) that bind to such epitopes are part of the present invention.

[0130] Methods for determining the epitope of an antigen binding protein, such as an antibody or fragment or polypeptide, include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can also be used (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify amino acids in a polypeptide that interact with an antigen binding protein (such as an antibody or fragment or polypeptide) is to detect hydrogen / deuterium exchange by mass spectrometry. See, for example, Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

[0131] The invention includes antigen binding proteins that compete for binding to IL2Rγ, e.g., a variant IL2Rγ epitope as discussed herein, with, e.g., the following antigen binding proteins of the invention: H4H12857P; H4H12858P; H4H12859P; H4H128663P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12887P 4H12890P; H4H12899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2. As used herein, the term "competition" refers to an antigen binding protein (e.g., antibody or antigen binding fragment thereof) that binds to an antigen (e.g., IL2Rγ) and inhibits or blocks another antigen binding protein (e.g., antibody or antigen binding fragment thereof) from binding to the antigen. Unless otherwise indicated, the term also includes competition between two antigen binding proteins, such as antibodies, in both directions, i.e., the first antibody binds to the antigen and blocks the binding of the second antibody, and vice versa. Therefore, in one embodiment of the invention, competition occurs in such a direction. In certain embodiments, the first antigen binding protein (e.g., antibody) and the second antigen binding protein (e.g., antibody) may bind to the same epitope. Alternatively, the first and second antigen binding proteins (e.g., antibodies) may bind to different but, for example, overlapping or non-overlapping epitopes, one of which is inhibited or blocked by binding, such as by steric hindrance. Competition between antigen binding proteins (e.g., antibodies) can be measured by methods known in the art, such as by real-time, label-free biolayer interferometry. In addition, the binding competition between anti-IL2Rγ antigen binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using real-time, label-free biolayer interferometry on an Octet RED384 biosensor (Pall ForteBio Corp.).

[0132] Typically, an antibody or antigen-binding fragment of the invention modified in some manner retains the ability to specifically bind to IL2Rγ, for example, retains at least 10% of its IL2Rγ binding activity (when compared to the parent antibody), when the activity is expressed on a molar basis. Preferably, the antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the IL2Rγ binding affinity of the parent antibody. It is also contemplated that the antibodies or antigen-binding fragments of the invention may contain conservative or non-conservative amino acid substitutions that do not substantially change their biological activity (referred to as "conservative variants" or "functional conservative variants" of antibodies).

[0133] polypeptides, such as immunoglobulin chains (e.g., H4H12857P, H4H12858P, H4H12859P, H4H12863P, H4H12874P, H4H12871P, H4H12884P, H4H12886P, H4H12889P, H4H12890P, H4H12899P, H4H12890P, H4H12891P, H4H12892P, H4H12893P, H4H12894P, H4H12895P, H4H12896P, H4H12897P, H4H12898P, H4H12899P, H4H12891P, H4H12894P, H4H12897P 4H12900P, H4H12908P, H4H12913P2, H4H12922P2, H4H12924P2, H4H12926P2, H4H12927P2, H4H12934P2, H4H13538P, H4H13541P, H4H13544P2 or H4H13545P2 V H 、V L8, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 170, 171, 172, 173, 174, 175 2,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236 ,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341 , 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 366, 368, 370, 372, 374, 376 or 378) is at least about 70% to 99.9% (e.g., at least 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%.9%) identical or similar amino acid sequences; when the comparison is performed by the BLAST algorithm, the parameters of the algorithm are selected to give the maximum match between the corresponding sequences over the entire length of the corresponding reference sequences (e.g., expectation threshold: 10; word length: 3; maximum match in query range: 0; BLOSUM 62 matrix; gap penalty: existence 11, extension 1; conditional combination score matrix adjustment). .

[0134] In addition, variants of the polypeptide may include polypeptides such as immunoglobulin chains (e.g., H4H12857P, H4H12858P, H4H12859P, H4H12863P, H4H12874P, H4H12871P, H4H12884P, H4H12886P, H4H12889P, H4H12890P, H4H12891P, 899P, H4H12900P, H4H12908P, H4H12913P2, H4H12922P2, H4H12924P2, H4H12926 P2, H4H12927P2, H4H12934P2, H4H13538P, H4H13541P, H4H13544P2, or H4H13545P2 V H 、V L , HC or LC or its CDR), which may contain the amino acid sequence of the reference polypeptide whose amino acid sequence is specifically shown herein, but there are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations, such as one or more missense mutations (e.g., conservative substitutions), nonsense mutations, deletions or insertions. For example, the present invention includes an anti-IL2Rγ antigen binding protein comprising: an immunoglobulin light chain (or V chain) comprising the amino acid sequence shown in SEQ ID NO: 10 but having one or more such mutations L ) variants, and / or immunoglobulin heavy chains (or V chains) comprising the amino acid sequence shown in SEQ ID NO: 2 but having one or more such mutations H ) variants. In one embodiment of the invention, the anti-IL2Rγ antigen binding protein comprises: an immunoglobulin light chain variant comprising CDR-L1, CDR-L2, and CDR-L3, wherein one or more (e.g., 1 or 2 or 3) of such CDRs have one or more such mutations (e.g., conservative substitutions); and / or an immunoglobulin heavy chain variant comprising CDR-H1, CDR-H2, and CDR-H3, wherein one or more (e.g., 1 or 2 or 3) of such CDRs have one or more such mutations (e.g., conservative substitutions).

[0135] The following references relate to the BLAST algorithm that is frequently used for sequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J. 272(20): 5101-5109; Altschul, SF, et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W., et al., (1993) Nature Genet. 3: 266-272; Madden, TL, et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF, et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J., et al., (1997) Genome Res. 7: 649-656; Wootton, JC, et al. al., (1993) Comput. Chem. 17: 149-163; Hancock, JMet al., (1994) Comput. Appl. Biosci. 10: 67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, MO, et al., "A model of evolutionary change in proteins." Atlas of Protein Sequence andStructure, (1978) Vol.5, suppl.3.MODayhoff (editor), pp.345-352, Natl.Biomed.Res.Found., Washington, DC; Schwartz, RM, et al., "Matrices for detecting distant relationships." Atlas of Protein Sequence and Structure, (1978) vol.5, suppl.3. "MO Dayhoff (editor), pp.353-358, Natl.Biomed.Res.Found., Washington, DC; Altschul, SF, (1991) J.Mol.Biol.219: 555-565; States, DJ, etal., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919; Altschul, SF, et al., (1993) J. Mol. Evol. 36: 290-300; ALIGNMENTSTATISTICS: Karlin, S., et al., (1990) Proc.Natl.Acad.Sci.USA 87: 2264-2268; Karlin, S., et al., (1993) Proc.Natl.Acad.Sci.USA 90: 5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22: 2022-2039; and Altschul, SF "Evaluating the statistical significance of multiple distinct local alignments." Theoretical and Computational Methods in Genome Research (edited by S. Suhai), (1997) pp. 1-14, Plenum, NY. .

[0136] "Conservatively modified variants" or "conservative substitutions" (e.g., of an immunoglobulin chain as described herein) refer to variants in which one or more replacements of amino acids in a polypeptide are present with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.). Such changes can often be made without significantly destroying the biological activity of the antibody or fragment. Those skilled in the art recognize that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed.)). In addition, replacements of amino acids that are structurally or functionally similar are unlikely to significantly destroy biological activity. The present invention includes anti-IL2Rγ antigen binding proteins containing such conservatively modified variant immunoglobulin chains.

[0137] Examples of groups of amino acids with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Alternatively, a conservative substitution is any change disclosed in Gonnet et al. (1992) Science 256: 1443-45 that has a positive value in the PAM250 log-likelihood matrix.

[0138] The anti-IL2Rγ antigen binding proteins disclosed herein, e.g., anti-IL2Rγ antigen binding proteins comprising variant immunoglobulin chains, may exhibit one or more of the following properties:

[0139] At 25°C, about 2.75×10 -9 M is about 3.36×10 -7 M of K D binds to human IL2Rγ (e.g., a fusion thereof, such as a myc-myc-His6 fusion);

[0140] At 37°C, the -9 M is about 3.53×10 -7 The KD of M binds to human IL2Rγ (e.g., a fusion thereof, such as myc-myc-His6 fusion) (or binds to human IL2Rγ with a KD of less than about 3.53×10 -7 M of K D Combined);

[0141] At 25°C, about 3.18×10 -9 M is about 2.38×10 -7 The KD of M binds to cynomolgus IL-2Rγ (e.g., a fusion thereof, such as a myc-myc-His6 fusion);

[0142] At 37°C, the -9 M is about 3.20×10 -7 M of K D Binds to cynomolgus IL-2Rγ (e.g., a fusion thereof, such as a myc-myc-His6 fusion) (or binds to less than about 3.20×10 -7 M of K D Combined);

[0143] At 25°C, about 2.45×10 -9 M is about 1.20×10 -8The KD of M binds to human IL2Rγ (e.g., a fusion thereof, such as a fusion with a C-terminal mouse IgG2a Fc tag) (or binds to human IL2Rγ with a KD of less than about 1.20×10 -8 M of K D Combined);

[0144] At 37°C, the -11 M to about 3.00×10 -8 The KD of M binds to human IL2Rγ (e.g., a fusion thereof, such as a fusion with a C-terminal mouse IgG2a Fc tag) (or binds to human IL2Rγ with a KD of less than about 3.00×10 -8 M of K D Combined);

[0145] At 25°C, the -8 M, 3.76×10 -9 M, 1.08×10 -7 M, 2.17×10 -8 M, 6.02×10 - 9 M or 7.93×10 -8 M of K D binds (or does not bind) to mouse IL2Rγ (e.g., a fusion thereof, such as a myc-myc-His6 fusion);

[0146] At 37°C, the -8 M, 6.11×10 -9 M, 3.87×10 -7 M, 5.16×10 -8 M, 8.70×10 - 9 M or 2.15×10 -7 M of K D binds (or does not bind) to mouse IL2Rγ (e.g., a fusion thereof, such as a myc-myc-His6 fusion);

[0147] At 25°C, about 3.32×10 -9 M is about 1.97×10 -7 M of K D binds (or does not bind) to human IL2Rγ domain 1 (e.g., a fusion thereof, such as a myc-myc-His6 fusion);

[0148] At 37°C, the -9 M is about 2.25×10 -7 M of K Dbinds (or does not bind) to human IL2Rγ domain 1 (e.g., a fusion thereof, such as a myc-myc-His6 fusion);

[0149] At 25°C, the -7 M is about 5.35×10 -10 K D binds (or does not bind) to human IL2Rγ domain 2 (e.g., a fusion thereof, such as a myc-myc-His6 fusion);

[0150] At 37°C, the -8 or about 1.27×10 -8 K D binds (or does not bind) to human IL2Rγ domain 2 (e.g., a fusion thereof, such as a myc-myc-His6 fusion);

[0151] Blockade of T cells (e.g. human CD4 + T cells), e.g., STAT phosphorylation induced by IL-2 (e.g., at about 10 nM), IL-4 (e.g., at about 50 pM), IL7 (e.g., at about 1 pM), IL-15 (e.g., at about 0.5 nM), and / or IL-21 (e.g., at about 50 pM), e.g., at an IC of about 1 nM to about 0.5 nM. 50 ;

[0152] Blocking STAT phosphorylation in mast cells (e.g., differentiated human mast cells), such as STAT phosphorylation induced by IL-9 (e.g., at about 2 nM), such as at about 4×10 -10 IC of M 50 ;

[0153] Reducing the number of human immune cells (e.g., human PBMCs (peripheral blood mononuclear cells), such as human CD45+ cells, human T cells, human CD4+ T cells and / or human CD8+ T cells) in mice (e.g., NOD-scid IL2rγ null (NSG) mice) following injection with human peripheral blood mononuclear cells (PBMCs);

[0154] Reducing the levels of serum human cytokines (e.g., human IFN-γ, human TNFα, human IL-6, human IL-8 and / or human IL-10) and / or mouse cytokines (e.g., mouse TNFα, mouse IL-6, mouse KC / GRO and / or mouse IL-10) in mice (e.g., NOD-scid IL2rγ null (NSG) mice) following injection with human peripheral blood mononuclear cells (PBMCs);

[0155] competes with any one or more of the anti-IL2Rγ antibodies set forth herein for binding to human IL-2Rγ, e.g., human IL-2Rγ on the surface of a cell (e.g., tagged with a C-terminal myc-myc-hexahistidine tag);

[0156] that binds to the same epitope on IL2Rγ as any one or more of the anti-IL2Rγ antibodies set forth herein, e.g., IL2Rγ on the surface of a cell (e.g., tagged with a C-terminal myc-myc-hexahistidine tag);

[0157] Does not detectably bind to mouse or rat IL2Rγ (e.g., as measured by Biacore at 37°C);

[0158] Protected mice from GvHD-induced weight loss and / or death in a GvHD mouse model;

[0159] Blocking the binding of a hybrid receptor comprising IL2Rγ complexed with a cytokine-specific receptor subunit to a cytokine such as IL-2, IL-4, IL-7, IL-9, IL-15 and / or IL-21; and / or

[0160] Inhibition of IL2Rγ intracellular signaling through the JAK-STAT pathway (e.g., in human B lymphocytes or human natural killer cells) induced by, e.g., IL2, IL4, IL7, IL9, IL15 and / or IL21, e.g., as measured by luciferase expression in cells comprising a luciferase gene operably linked to a STAT3 response element.

[0161] Unless otherwise indicated, "H4H12857P", "H4H12858P", "H4H12859P", "H4H12863P", "H4H12874P", "H4H12871P", "H4H12884P", "H4H12886P", "H4H12889P", "H4H12890P", "H4H12899P", "H4H12900P" P", "H4H12908P", "H4H12913P2", "H4H12922P2", "H4H12924P2", "H4H12926P2", "H4H12927P2", "H4H12934P2", "H4H13538P", "H4H13541P", "H4H13544P2" or "H4H13545P2" refers to anti-IL2Rγ Antigen binding proteins, such as antibodies and antigen binding fragments thereof (including multispecific antigen binding proteins), respectively comprising: containing the polypeptides described herein for H4H12857P, H4H12858P, H4H12859P, H4H12863P, H4H12874P, H4H12871P, H4H12884P, H4H12886P, H4H12889P, H4H12890P, H4H12891P, 2899P, H4H12900P, H4H12908P, H4H12913P2, H4H12922P2, H4H12924P2, H4H12926P2, H4H12927P2, H4H12934P2, H4H13538P, H4H13541P, H4H13544P2 or H4H13545P2 specifically shown amino acid sequence (e.g., SEQ 376, 384, 396, 408, 412, 424, 436, 442, 458, 461, 472, 484, 492, 508, 512, 520, 530, 531, 542, 550, 561, 572, 576, 581, 592, 608, 612, 620, 630, 640, 658, 661, 672, 684, 686, 692, 708, 713, 714, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 750, 751, 762, 763, 774, 775 H), and / or containing the compounds described herein for H4H12857P, H4H12858P, H4H12859P, H4H12863P, H4H12874P, H4H12871P, H4H12884P, H4H12886P, H4H12889P, H4H12890P, H4H12899P, H4H12900P, H 4H12908P, H4H12913P2, H4H12922P2, H4H12924P2, H4H12926P2, H4H12927P2, H4H12934P2, H4H13538P, H4H13541P, H4H13544P2 or H4H13545P2 specifically shown amino acid sequence (e.g., SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 79, 89, 99, 109, 117, 127, 136, 146, 154, 164, 172, 182, 188, 226, 236, 246, 256, 266, 274, 304, 313, 323, 333, 353, 359, 368 or 378) (or a variant thereof) or its variable region (V L );And / or comprising: a heavy chain or V chain containing its CDRs (CDR-H1 (or its variant), CDR-H2 (or its variant) and CDR-H3 (or its variant)) H , and / or a light chain or V chain containing its CDRs (CDR-L1 (or its variant), CDR-L2 (or its variant) and CDR-L3 (or its variant)) L In one embodiment of the invention, VH is linked to an IgG constant heavy chain domain, e.g., a human IgG constant heavy chain domain (e.g., IgG1 or IgG4 (e.g., comprising S228P and / or S108P mutations)); and / or V L The light chain constant domain, e.g., a human light chain constant domain (e.g., a λ or κ constant light chain domain), is linked to the light chain constant domain. Encoding one or more of any such immunoglobulin chains (e.g., V H 、V L , HC and / or LC) form part of the present invention.

[0162] The present invention includes "neutralizing" or "antagonist" anti-IL2Rγ antigen binding proteins (e.g., antibodies or antigen binding fragments), which include molecules that inhibit IL2Rγ activity to any detectable extent (e.g., inhibit the binding of a hybrid receptor comprising IL2Rγ complexed with cytokine-specific receptor subunits to cytokines such as IL-2, IL-4, IL-7, IL-9, IL-15 and / or IL-21).

[0163] The antibodies and antigen-binding fragments of the invention (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899P; H4H12900P; H4H12908P; H4H129 13P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2) comprises an immunoglobulin chain comprising the amino acid sequence specifically set forth herein (and variants thereof) as well as cellular and in vitro post-translational modifications to the antibody or fragment. For example, the present invention includes antibodies and antigen-binding fragments thereof that specifically bind to IL2Rγ, which comprise the heavy chain and / or light chain amino acid sequences set forth herein; and antibodies and fragments in which one or more asparagine, serine and / or threonine residues are glycosylated, one or more asparagine residues are deamidated, one or more residues (e.g., Met, Trp and / or His) are oxidized, the N-terminal glutamine is pyroglutamic acid (pyroE) and / or the C-terminal lysine or other amino acids are deleted.

[0164] The invention provides a container (e.g., a plastic or glass vial (e.g., with a cap or chromatography column), a hollow needle or a syringe barrel) comprising an anti-IL2Rγ antigen binding protein of the invention, e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H1288 9P; H4H12890P; H4H12899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2.

[0165] The present invention also provides an injection device comprising one or more antigen binding proteins (e.g., antibodies or antigen binding fragments) that specifically bind to IL2Rγ, such as H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12 890P; H4H12899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2, or a pharmaceutical preparation thereof. The injection device may be packaged as a kit. The injection device is a device for introducing a substance into a subject's body by a parenteral route, such as intraocularly, intravitreally, intramuscularly, subcutaneously, or intravenously. For example, the injection device can be a syringe or autoinjector (e.g., pre-filled with a pharmaceutical preparation), which, for example, comprises a barrel or barrel for containing a fluid to be injected (e.g., comprising an antibody or fragment or a pharmaceutical preparation thereof); a needle for piercing the skin, a blood vessel, or other tissue for injecting the fluid; and a stopper for pushing the fluid out of the barrel and through the needle hole and into the body of a subject.

[0166] The present invention also provides methods for administering the anti-IL2Rγ antigen binding protein of the present invention to a subject, such as A method comprising introducing an antigen-binding protein into a subject (e.g., a human), e.g., parenterally. For example, the method comprises piercing the subject's body with a needle of a syringe and injecting the antigen-binding protein into the subject's body, e.g., into a vein, artery, eye, muscle tissue, or subcutaneous tissue of the subject.

[0167] Polynucleotides and preparation methods

[0168] Polynucleotides include DNA and RNA. The present invention includes any polynucleotide of the present invention, for example, encoding H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899P; H4 Immunoglobulin V of H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2 H、V L , CDR-H, CDR-L, HC or LC, optionally, it is operably linked to a promoter or other expression control sequence. For example, the invention provides any polynucleotide (e.g., DNA) comprising the nucleotide sequence shown in the following:

[0169] SEQ ID NO: 1, 3, 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, 6 1, 63, 65, 67, 69, 71, 73, 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, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 20 1,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,2 91, 293, 295, 297, 299, 301, 303, 305, 307, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 365, 367, 369, 371, 373, 375 or 377. In one embodiment of the invention, the polynucleotide of the invention is fused to a secretory signal sequence. Polypeptides encoded by such polynucleotides are also within the scope of the invention.

[0170] In general, a "promoter" or "promoter sequence" is a DNA regulatory region that is capable of binding to RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. The promoter may be operably linked to other expression control sequences including enhancer and repressor sequences and / or to a polynucleotide of the invention. Promoters that can be used to control gene expression include, but are not limited to, the cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist, et al., (1981) Nature 290: 304-310), the promoter contained in the 3' long terminal repeat sequence of Rous sarcoma virus (Yamamoto, et al., (1980) Cell 22: 787-797), the herpes thymidine kinase promoter (Wagner, et al., (1981) Proc. Natl. Acad. Sci. USA 78: 1441-1445), and the regulatory sequence of the metallothionein gene (Brinster, et al., (1982) Nature 296: 39-42); prokaryotic expression vectors, such as the β-lactamase promoter (VIIIa-Komaroff, et al., (1982) Nature 296: 39-42). al., (1978) Proc. Natl. Acad. Sci. USA 75:3727-3731), or tac promoter (DeBoer, et al., (1983) Proc. Natl. Acad. Sci. USA 80:21-25); see also "Useful proteins from recombinant bacteria", Scientific American (1980) 242:74-94; and promoter elements from yeast or other fungi, such as Gal4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or alkaline phosphatase promoter.

[0171] A polynucleotide encoding a polypeptide is "operably linked" to a promoter or other expression control sequence when, in a cell or other expression system, the sequence directs RNA polymerase-mediated transcription of the coding sequence into RNA, preferably mRNA, which may then undergo RNA splicing (if it contains introns) and, optionally, translation into the protein encoded by the coding sequence.

[0172] The present invention includes the following code V H and V L The polynucleotides of the polynucleotide pair:

[0173] SEQ ID NO: 1 and SEQ ID NO: 9;

[0174] SEQ ID NO: 21 and SEQ ID NO: 29;

[0175] SEQ ID NO:41 and SEQ ID NO:49;

[0176] SEQ ID NO: 61 and SEQ ID NO: 69;

[0177] SEQ ID NO: 80 and SEQ ID NO: 88;

[0178] SEQ ID NO: 100 and SEQ ID NO: 108;

[0179] SEQ ID NO: 118 and SEQ ID NO: 126;

[0180] SEQ ID NO: 137 and SEQ ID NO: 145;

[0181] SEQ ID NO: 155 and SEQ ID NO: 163;

[0182] SEQ ID NO: 173 and SEQ ID NO: 181;

[0183] SEQ ID NO: 189 and SEQ ID NO: 181;

[0184] SEQ ID NO: 199 and SEQ ID NO: 181;

[0185] SEQ ID NO: 209 and SEQ ID NO: 181;

[0186] SEQ ID NO: 217 and SEQ ID NO: 225;

[0187] SEQ ID NO: 237 and SEQ ID NO: 245;

[0188] SEQ ID NO: 257 and SEQ ID NO: 265;

[0189] SEQ ID NO: 275 and SEQ ID NO: 181;

[0190] SEQ ID NO: 285 and SEQ ID NO: 181;

[0191] SEQ ID NO: 295 and SEQ ID NO: 303;

[0192] SEQ ID NO: 314 and SEQ ID NO: 322;

[0193] SEQ ID NO:334 and SEQ ID NO:181;

[0194] SEQ ID NO: 344 and SEQ ID NO: 352; or

[0195] SEQ ID NO:360 and SEQ ID NO:367.

[0196] The present invention includes polynucleotides comprising the following polynucleotide sets encoding CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3:

[0197] SEQ ID NOs: 3, 5, 7, 11, 13 and 15;

[0198] SEQ ID NOs: 23, 25, 27, 31, 33 and 35;

[0199] SEQ ID NOs: 43, 45, 47, 51, 53 and 55;

[0200] SEQ ID NOs: 63, 65, 67, 71, 73 and 74;

[0201] SEQ ID NOs: 82, 84, 86, 90, 92 and 94;

[0202] SEQ ID NOs: 102, 104, 106, 110, 73 and 112;

[0203] SEQ ID NOs: 120, 122, 124, 128, 130 and 131;

[0204] SEQ ID NOs: 139, 141, 143, 147, 73 and 149;

[0205] SEQ ID NOs: 157, 159, 161, 165, 13 and 167;

[0206] SEQ ID NOs: 175, 177, 179, 71, 73 and 183;

[0207] SEQ ID NOs: 191, 193, 195, 71, 73 and 183;

[0208] SEQ ID NOs: 201, 203, 205, 71, 73 and 183;

[0209] SEQ ID NOs: 175, 211, 213, 71, 73 and 183;

[0210] SEQ ID NOs: 219, 221, 223, 227, 229 and 231;

[0211] SEQ ID NOs: 239, 241, 243, 247, 249 and 251;

[0212] SEQ ID NOs: 259, 261, 263, 267, 73 and 269;

[0213] SEQ ID NOs: 277, 279, 281, 71, 73 and 183;

[0214] SEQ ID NOs: 287, 289, 291, 71, 73 and 183;

[0215] SEQ ID NOs: 297, 299, 301, 305, 307 and 308;

[0216] SEQ ID NOs: 316, 318, 320, 324, 326 and 328;

[0217] SEQ ID NOs: 336, 338, 340, 71, 73 and 183;

[0218] SEQ ID NOs: 346, 348, 350, 71, 73 and 354; or

[0219] SEQ ID NOs: 362, 364, 365, 369, 371 and 373.

[0220] The present invention includes polynucleotides comprising the following polynucleotide pair encoding HC and LC:

[0221] SEQ ID NO: 17 and SEQ ID NO: 19;

[0222] SEQ ID NO:37 and SEQ ID NO:39;

[0223] SEQ ID NO: 57 and SEQ ID NO: 59;

[0224] SEQ ID NO: 76 and SEQ ID NO: 78;

[0225] SEQ ID NO:96 and SEQ ID NO:98;

[0226] SEQ ID NO: 114 and SEQ ID NO: 116;

[0227] SEQ ID NO: 133 and SEQ ID NO: 135;

[0228] SEQ ID NO: 151 and SEQ ID NO: 153;

[0229] SEQ ID NO: 169 and SEQ ID NO: 171;

[0230] SEQ ID NO: 185 and SEQ ID NO: 187;

[0231] SEQ ID NO: 197 and SEQ ID NO: 187;

[0232] SEQ ID NO: 207 and SEQ ID NO: 187;

[0233] SEQ ID NO: 215 and SEQ ID NO: 187;

[0234] SEQ ID NO: 233 and SEQ ID NO: 235;

[0235] SEQ ID NO: 253 and SEQ ID NO: 255;

[0236] SEQ ID NO: 271 and SEQ ID NO: 273;

[0237] SEQ ID NO: 283 and SEQ ID NO: 187;

[0238] SEQ ID NO: 293 and SEQ ID NO: 187;

[0239] SEQ ID NO: 310 and SEQ ID NO: 312;

[0240] SEQ ID NO: 330 and SEQ ID NO: 332;

[0241] SEQ ID NO:342 and SEQ ID NO:187;

[0242] SEQ ID NO:356 and SEQ ID NO:358; or

[0243] SEQ ID NO:375 and SEQ ID NO:377.

[0244] The present invention includes polynucleotides encoding immunoglobulin polypeptide chains, which are variants of those whose nucleotide sequences are specifically set forth herein. A "variant" of a polynucleotide refers to a polynucleotide comprising a nucleotide sequence that is at least about 70-99.9% (e.g., 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) identical to a reference nucleotide sequence set forth herein; when compared by the BLAST algorithm, wherein the parameters of the algorithm are selected to give maximum matching between each sequence over the entire length of each reference sequence (e.g., expectation threshold: 10; word length: 28; maximum number of matches within the query range: 0; match / mismatch score: 1, -2; gap penalty: linear). In one embodiment of the invention, a variant of the nucleotide sequence specifically shown herein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) point mutations, insertions (e.g., in-frame insertions) or deletions (e.g., in-frame deletions) of one or more nucleotides. In one embodiment of the invention, such mutations may be missense mutations or nonsense mutations. In one embodiment of the invention, such variant polynucleotides encode immunoglobulin polypeptide chains that can be incorporated into an anti-IL2Rγ antigen binding protein, i.e., such that the protein retains specific binding to IL2Rγ.

[0245] Eukaryotic and prokaryotic host cells, including mammalian cells, can be used as hosts for expressing anti-IL2Rγ antigen binding proteins (e.g., antibodies or antigen binding fragments thereof). Such host cells are well known in the art, and many are available from the American Type Culture Collection (ATCC). These host cells include Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (babyhamster kidney, BHK) cells, monkey kidney cells (monkey kidney cell, COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines. Mammalian host cells include cells of humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses, and hamsters. Other cell lines that can be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells, and fungal cells.Fungal cells include cells of yeast and filamentous fungi, including, for example, Pichia, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichialindneri), Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. The present invention includes an isolated host cell (eg, a CHO cell or any type of host cell described above) comprising an antigen binding protein, V. H 、V L , HC, LC or its CDRs (or variants thereof), e.g.

[0246] H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P ;H4H12886P; H4H12889P; H4H12890P; H4H12899P; H4H12900P; H4H12908P; H4H12913 P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2;; and / or a polynucleotide encoding one or more immunoglobulin chains thereof (e.g., as discussed herein).

[0247] The present invention also includes cells expressing IL2Rγ or an antigenic fragment or fusion thereof (e.g., His6, Fc and / or myc) that binds to an antigen binding protein (e.g., an antibody or antigen binding fragment thereof) of the present invention, such as H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12894P; H4H12895P; H4H12896P; H4H12897P; H4H12898P; H4H12899P; H4H12890P; H4H12891P; H4H12894P; H4H12897P; H4H12898P; H4H1289 ...7P; H4H12898P; H4H12899P; H4H12891P; H4H12897P; H4H12898P; H4H12899P; H4H12897P; H4H12898P; H4H12899P; H4H12897P; H4H12898P; H4H12899P; H4H12897P; H4H12897P; H4H12898P; H4H12899P; H4H1 or H4H13545P2., e.g., wherein the cell is in vivo or in vitro in a subject.

[0248] In addition, the present invention also provides a complex comprising an anti-IL2Rγ antigen binding protein, such as an antibody or an antigen binding fragment thereof, as discussed herein, complexed with an IL2Rγ polypeptide or an antigenic fragment thereof or a fusion thereof and / or with a secondary antibody or an antigen binding fragment thereof that specifically binds to an anti-IL2Rγ antibody or fragment (e.g., a detectably labeled secondary antibody). In one embodiment of the present invention, the complex is in vitro (e.g., immobilized on a solid substrate) or in vivo in a subject.

[0249] The recombinant anti-IL2Rγ antigen binding proteins disclosed herein, such as antibodies and antigen binding fragments, can also be produced in an E. coli / T7 expression system. In this embodiment, the recombinant anti-IL2Rγ antibody immunoglobulin molecules (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H1289 HC, LC, V of 9P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2 H and / or V Lor its CDR) can be inserted into a pET-based plasmid and expressed in an E. coli / T7 system. For example, the present invention includes a method for expressing an antibody or antigen-binding fragment thereof or an immunoglobulin chain thereof in a host cell (e.g., a bacterial host cell such as E. coli such as BL21 or BL21DE3), the method comprising expressing a T7 RNA polymerase in the cell, the cell further comprising a polynucleotide encoding an immunoglobulin chain (e.g., comprising a nucleotide sequence of any one or more of the following, or a variant thereof: SEQ ID NO: 1, 3, 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, 6 1, 63, 65, 67, 69, 71, 73, 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, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 20 1,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,2 91, 293, 295, 297, 299, 301, 303, 305, 307, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 365, 367, 369, 371, 373, 375 or 377).For example, in one embodiment of the invention, a bacterial host cell, such as E. coli, comprises a polynucleotide encoding a T7 RNA polymerase gene operably linked to a lac promoter, and expression of the polymerase and the chain is induced by incubating the host cell with IPTG (isopropyl-β-D-thiogalactopyranoside). See US4952496 and US5693489 or Studier & Moffatt, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol. 1986 May 5; 189(1): 113-30.

[0250] Several methods for producing recombinant antibodies are known in the art. US4816567 discloses an example of a method for recombinant production of antibodies.

[0251] Transformation can be carried out by any known method for introducing polynucleotides into host cells. The method for introducing heterologous polynucleotides into mammalian cells is well known in the art, and includes transfection mediated by dextran, calcium phosphate precipitation, transfection mediated by polybrene, protoplast fusion, electroporation, polynucleotides (multiple polynucleotides) are encapsulated in liposomes, biolistic injection and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors. The method for transforming cells is well known in the art. See, for example, No. 4399216, No. 4912040, No. 4740461 and No. 4959455 U.S. Patents.Thus, the present invention includes a recombinant method for preparing an anti-IL2Rγ antigen binding protein, such as an antibody or antigen binding fragment thereof, or an immunoglobulin chain thereof, of the present invention, the method comprising (i) recombining one or more recombinant proteins encoding the antigen binding protein, such as H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12891P; 12899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2 (e.g., comprising any one or more of the following nucleotide sequences, or variants thereof: SEQ ID NO: 1, 9, 17, 19, 21, 29, 37, 39, 41, 49, 57, 59, 61, 69, 76, 78, 80, 88, 96, 98, 100, 108, 114, 116, 118, 126, 133, 135, 137, 145, 151, 153, 155, 1 63, 169, 171, 173, 181, 185, 187, 189, 197, 199, 207, 209, 215, 217, 225, 233, 235, 237, 245, 253, 255, 257, 265, 271, 273, 275, 283, 285, 293 , 295, 303, 310, 312, 314, 322, 330, 332, 334, 342, 344, 352, 356, 358, 360, 367, 375 or 377) into a host cell, for example, wherein the polynucleotide is in a vector; and / or integrated into a host cell chromosome and / or operably linked to a promoter; (ii) culturing the host cell (e.g., CHO or Pichia pastoris or Pichia pastoris) under conditions conducive to expression of the polynucleotide, and (iii) optionally, isolating the antigen binding protein (e.g., antibody or antigen binding fragment) or chains from the host cell and / or the culture medium in which the host cell was cultured. When an antigen binding protein (e.g., antibody or antigen binding fragment) is prepared that comprises more than one immunoglobulin chain, for example, an antibody comprising two immunoglobulin heavy chains and two immunoglobulin light chains, co-expression of these chains in a single host cell results in the association of the chains, e.g., within the cell or on the surface of the cell or outside the cell if such chains are secreted, to form the antigen binding protein (e.g., antibody or antigen binding fragment).The methods of the invention include those in which only immunoglobulin heavy chains or only immunoglobulin light chains or both are expressed in cells (e.g., any of those methods discussed herein including mature fragments and / or variable domains thereof). For example, such a single chain is useful, for example, as an intermediate in expressing an antibody or antigen-binding fragment comprising such a chain. For example, the invention also includes anti-IL2Rγ antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, which are the products of the production methods described herein, and optionally the products of the purification methods described herein.

[0252] In one embodiment of the invention, the method for preparing an anti-IL2Rγ antigen binding protein (e.g., an antibody or antigen binding fragment thereof) comprises a method for purifying the antigen binding protein, such as by column chromatography, precipitation and / or filtration. As discussed, the products of such a method also form part of the invention.

[0253] Preparation of human antibodies

[0254] The anti-IL2Rγ antibodies of the present invention may be fully human antibodies. Methods for producing monoclonal antibodies (including fully human monoclonal antibodies) are known in the art. Any such known methods may be used in the context of the present invention to prepare human antibodies that specifically bind to human IL2Rγ.

[0255] Use for example VELOCIMMUNE TM The technique or any other similar known method for producing fully human monoclonal antibodies, first isolates a high-affinity chimeric antibody against IL2Rγ having a human variable region and a mouse constant region. As described in the experimental section below, the antibodies are characterized and selected for desired characteristics (including affinity, ligand blocking activity, selectivity, epitope, etc.). If necessary, the mouse constant region is replaced with a desired human constant region, such as a wild-type or modified IgG1 or IgG4, to produce a fully human anti-IL2Rγ antibody. Although the constant region selected may vary depending on the specific use, high-affinity antigen binding and target specificity are characterized by the variable region. In some cases, fully human anti-IL2Rγ antibodies are isolated directly from antigen-positive B cells. For example, see US 6,596,541, Regeneron Pharmaceuticals,

[0256] Anti-IL2Rγ Antibodies Containing Fc Variants

[0257] According to certain embodiments of the present invention, there is provided an anti-IL2Rγ antibody comprising an Fc domain comprising one or more mutations that enhance or weaken the binding of the antibody to the FcRn receptor, for example, at an acidic pH compared to a neutral pH. For example, the present invention comprises a C in the Fc domain. H 2 or C H An anti-IL2Rγ antibody comprising mutations in the 3 region, wherein one or more mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome at a pH range of about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal.

[0258] Non-limiting examples of such Fc modifications include, for example, modifications at the following sites:

[0259] 250 (e.g. E or Q);

[0260] 250 and 428 (e.g. L or F);

[0261] 252 (e.g. L / Y / F / W or T);

[0262] 254 (such as S or T); and / or

[0263] 256 (e.g. S / R / Q / E / D or T);

[0264] and / or modifications at the following sites:

[0265] 428 and / or 433 (e.g. H / L / R / S / P / Q or K), and / or

[0266] 434 (e.g. H / F or Y);

[0267] and / or modifications at the following sites:

[0268] 250 and / or 428;

[0269] and / or modifications at the following sites:

[0270] 307 or 308 (e.g. 308F, V308F), and / or

[0271] ·434.

[0272] In one embodiment of the invention, the modification comprises:

[0273] 428L (e.g., M428L) and 434S (e.g., N434S) modifications;

[0274] 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications;

[0275] 433K (e.g. H433K) and 434 (e.g. 434Y) modifications;

[0276] 252, 254 and 256 (e.g. 252Y, 254T and 256E) modifications;

[0277] 250Q and 428L modifications (e.g. T250Q and M428L); and / or

[0278] · 307 and / or 308 modifications (e.g. 308F or 308P).

[0279] For example, the invention includes an anti-IL2Rγ antibody comprising an Fc domain comprising one or more mutation pairs or groups selected from the group consisting of:

[0280] 250Q and 248L (e.g. T250Q and M248L);

[0281] 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E);

[0282] 428L and 434S (e.g. M428L and N434S); and

[0283] 433K and 434F (e.g. H433K and N434F).

[0284] In one embodiment of the invention, the heavy chain constant domain is γ4 comprising S228P and / or S108P mutations. See Angal et al. A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody, Mol Immunol. 1993 Jan; 30(1): 105-108.

[0285] All possible combinations of the above-described Fc domain mutations and other mutations within the antibody variable domains disclosed herein are contemplated to be within the scope of the present invention.

[0286] The anti-IL2Rγ antibodies of the present invention may comprise a modified Fc domain with reduced effector function. As used herein, a "modified Fc domain with reduced effector function" means any Fc portion of an immunoglobulin that has been modified, mutated, truncated, etc. relative to a wild-type, naturally occurring Fc domain, so that a molecule comprising the modified Fc exhibits a reduction in the severity or extent of at least one effect selected from cell killing (e.g., ADCC and / or CDC), complement activation, phagocytosis, and opsonization relative to a comparison molecule comprising a wild-type, naturally occurring form of the Fc portion. In certain embodiments, a "modified Fc domain with reduced effector function" is an Fc domain with reduced or attenuated binding to an Fc receptor (e.g., FcγR).

[0287] In certain embodiments of the present invention, the modified Fc domain is a variant IgG1 Fc or a variant IgG4 Fc, which comprises a replacement in the hinge region. For example, a modified Fc for use in the context of the present invention may comprise a variant IgG1 Fc, wherein at least one amino acid in the hinge region of IgG1 Fc is replaced by a corresponding amino acid from the hinge region of IgG2 Fc. Alternatively, a modified Fc for use in the context of the present invention may comprise a variant IgG4 Fc, wherein at least one amino acid in the hinge region of IgG4 Fc is replaced by a corresponding amino acid from the hinge region of IgG2 Fc. Non-limiting, exemplary modified Fc regions that can be used in the context of the present invention and any functionally equivalent variants of the modified Fc regions described therein are described in U.S. Patent Application Publication No. 2014 / 0243504, the disclosure of which is hereby incorporated by reference in its entirety.

[0288] Other modified Fc domains and Fc modifications that can be used in the context of the present invention include any modification described in US2014 / 0171623, US8697396, US2014 / 0134162, WO2014 / 043361, the disclosures of which are hereby incorporated by reference in their entirety. Methods for constructing antibodies or other antigen-binding fusion proteins comprising a modified Fc domain as described herein are known in the art.

[0289] Multispecific antigen binding protein

[0290] The present invention includes anti-IL2Rγ antigen binding proteins, such as antibodies and antigen binding fragments thereof, as well as methods of use thereof and methods of making such antigen binding proteins. The term "anti-IL2Rγ" or "anti-IL2R gamma" antigen binding protein, such as an antibody or antigen binding fragment, includes a multispecific (e.g., bispecific or biparatopic) molecule comprising at least one first antigen binding domain that specifically binds to IL2Rγ (e.g., from H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H128 90P; H4H12899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2) and at least one second antigen binding domain that binds to a different antigen or an epitope in IL2Rγ that is different from the epitope of the first antigen binding domain. In one embodiment of the invention, the first and second epitopes overlap. In another embodiment of the invention, the first and second epitopes do not overlap.

[0291] Multispecific binding refers to binding to two or more different epitopes, which can be on the same antigen or on different antigens. Multispecificity includes bispecificity, trispecificity and tetraspecificity.

[0292] "H4H12857P"; "H4H12858P"; "H4H12859P"; "H4H12863P"; "H4H12874P"; "H4H12871P" ;"H4H12884P";"H4H12886P";"H4H12889P";"H4H12890P";"H4H12899P";"H4H12900P "; "H4H12908P"; "H4H12913P2"; "H4H12922P2"; "H4H12924P2"; "H4H12926P2"; "H4H12927P2"; "H4H12934P2"; "H4H13538P"; "H4H13541P"; "H4H13544P2"; or "H4H13545P2"

[0293] Included are multispecific molecules, such as antibodies or antigen-binding fragments, each comprising "H4H12857P"; "H4H12858P"; "H4H12859P"; "H4H12863P"; "H4H12874P"; "H4H12871P"; "H4H12884P"; "H4H12886P"; "H4H12889P"; "H4H12890P"; "H4H12899P"; "H4 HCDR and LCDR, V H and V L , or HC and LC, and one or more antigen binding domains that bind to different epitopes.

[0294] In one embodiment of the present invention, an antigen binding domain that specifically binds to IL2Rγ that may be included in a multispecific molecule comprises: (1)

[0296] (i) Heavy chain variable domain (V H ) sequence comprising CDR-H1, CDR-H2 and CDR-H3 from an immunoglobulin heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 22, 42, 62, 81, 101, 119, 138, 156, 174, 190, 200, 210, 218, 238, 258, 276, 286, 296, 315, 335, 345 and 361 (or variants thereof); and

[0297] (ii) light chain variable domain (V L ) sequence comprising CDR-L1, CDR-L2 and CDR-L3 from an immunoglobulin light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 30, 50, 70, 89, 109, 127, 146, 164, 182, 226, 246, 266, 304, 323, 353 and 368 (or variants thereof);

[0298] or, (2)

[0300] (i) a heavy chain variable domain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 22, 42, 62, 81, 101, 119, 138, 156, 174, 190, 200, 210, 218, 238, 258, 276, 286, 296, 315, 335, 345 and 361 (or variants thereof); and

[0301] (ii) a light chain variable domain (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, 50, 70, 89, 109, 127, 146, 164, 182, 226, 246, 266, 304, 323, 353 and 368 (or variants thereof);

[0302] as well as

[0303] One or more antigen binding domains that bind to different epitopes.

[0304] In one embodiment of the invention, the bispecific antigen-binding fragment comprises a first scFv (e.g., comprising H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; V of H4H12890P; H4H12899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2 H and V L ) and a second scFv with binding specificity for a second, different epitope. For example, in one embodiment of the invention, the first and second scFvs are linked by a linker, such as a peptide linker (e.g., a GS linker, such as (GGGGS) n (SEQ ID NO: 386), wherein n is, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0305] Other bispecific antigen-binding fragments include F(ab)2 of bispecific IgG antibodies comprising H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899P; H4H12900 or H4H13545P2 and the heavy and light chain CDRs of another antibody that binds to a different epitope.

[0306] Immunoconjugates

[0307] The present invention encompasses anti-IL2Rγ antigen binding proteins, such as antibodies or antigen binding fragments, conjugated to another moiety, such as a therapeutic moiety ("immunoconjugates"). In one embodiment of the invention, the anti-IL2Rγ antigen binding protein (e.g., antibody or antigen binding fragment) is conjugated to any other therapeutic agent described herein. As used herein, the term "immunoconjugate" refers to an antigen binding protein, such as an antibody or antigen binding fragment, which is chemically or biologically linked to another antigen binding protein, a drug, a radioactive agent, a reporter moiety, an enzyme, a peptide, a protein, or a therapeutic agent.

[0308] Administration and treatment

[0309] The present invention provides a method for treating or preventing an IL2Rγ-mediated disease or condition in a subject, the method comprising administering to the subject a therapeutically effective dose of an anti-IL2Rg antigen binding protein ( ).

[0310] "IL2Rγ-mediated disease or condition" refers to any disease condition whose symptoms are mediated by the activity of one or more of the cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 and / or receptors that bind such cytokines; for example, autoimmunity and / or inflammation mediated by such cytokines and / or receptors. For example, IL2Rγ-mediated diseases or conditions include graft-versus-host disease (GvHD), organ transplant rejection (e.g., skin transplant (skin graft), b-islet cell transplant, heart transplant, lung transplant, kidney transplant and / or liver transplant), birdshot chorioretinopathy, multiple sclerosis, uveitis, autoimmune diseases (e.g., type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and myasthenia gravis), aplastic anemia; atopic dermatitis; asthma; and mast cell activation disorders (e.g., mast cell activation syndrome (MCAS), systemic mastocytosis (SM), or mast cell leukemia (MCL)).

[0311] The present invention also includes methods for administering to a subject, e.g., a subject suffering from an IL2Rγ-mediated disease or condition, an antigen binding protein (e.g., an antibody or antigen binding fragment thereof) that specifically binds to IL2Rγ, e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; The method comprises introducing the antigen binding protein into the subject, for example by injection.

[0312] GvHD is a disease that may occur after allogeneic transplantation. For example, in GvHD, the donated bone marrow or peripheral blood stem cells can be regarded as foreign to the recipient's body, and the donated cells / bone marrow attack the body. GvHD may occur, for example, in hematopoietic cell transplantation (hematopoietic cell transplantation, HCT; For example, in subjects with acute myeloid leukemia (acute myeloid leukemia, AML) or acute lymphocytic leukemia (acute lymphocyticleukemia, ALL) and / or myelodysplastic syndrome or myeloproliferative tumors), blood transfusion, thymus transplantation, or in patients with thymoma. The types of GvHD include steroid-refractory GvHD, acute graft-versus-host disease (aGvHD) and chronic graft-versus-host disease (cGvHD). Allogeneic transplant recipients may experience aGvHD or cGvHD or both forms, or neither of them. The present invention includes methods for treating or preventing GvHD (of any kind) in a subject, comprising administering to the subject a therapeutically effective dose of an anti-IL2Rγ antigen binding protein.

[0313] Symptoms of aGvHD can include rash or red areas on the skin (signs of cutaneous aGvHD); yellow discoloration of the skin and / or eyes, and abnormal blood test results (signs of liver aGvHD); nausea, vomiting, diarrhea, or abdominal cramping (signs of aGvHD in the digestive tract, or "gut"); and / or increased dryness / irritation of the eyes (signs of ocular GvHD).

[0314] Symptoms of cGvHD may include a rash, raised or discolored areas, thickening or tightness of the skin (signs of cutaneous cGvHD); abdominal distension, yellow discoloration of the skin and / or eyes, and abnormal blood test results (signs of liver cGvHD); dryness of the eyes or vision changes (signs of eye cGvHD); dry mouth, white patches in the mouth, pain, or sensitivity to spicy foods (oral cGvHD—signs of the mouth); shortness of breath or changes seen on a chest x-ray (dry cough pulmonary cGvHD—signs of the lungs); difficulty swallowing, pain when swallowing, or weight loss (signs of digestive tract or "gut" cGvHD); fatigue, muscle weakness or pain (neuromuscular cGvHD—signs of the nerves and muscles); and / or an increased need to urinate (frequent urination), burning or bleeding during urination, vaginal dryness / tightness, or penile dysfunction (signs of genitourinary, bladder, or sex organ cGvHD).

[0315] Organ transplant rejection refers to the rejection of the transplanted organ by the recipient's immune system. Hyperacute rejection occurs within a few minutes after transplantation, acute rejection occurs within a week to 3 months after transplantation, and chronic rejection occurs after many years. Transplanted organs include solid organs such as skin, pancreas, kidney, liver, heart and lung. The present invention includes a method for treating or preventing organ transplantation (of any kind) in a subject, the method comprising administering to the subject a therapeutically effective dose of an anti-IL2Rγ antigen binding protein.

[0316] Shotgun chorioretinopathy is a rare form of posterior uveitis - an inflammation of the uvea, the part of the eye that provides the retina with most of its blood supply. Shotgun chorioretinopathy can be autoimmune. Symptoms of shotgun chorioretinopathy can include night blindness, color vision problems, sensitivity to bright light, seeing flashes of light, visual distortion, pain in the eye, and loss of depth perception and / or peripheral vision. The present invention includes a method for treating or preventing shotgun chorioretinopathy or uveitis in a subject, the method comprising administering to the subject a therapeutically effective dose of an anti-IL2Rγ antigen binding protein, e.g., by intraocular administration, e.g., intravitreal injection.

[0317] The present invention also provides a method for treating or preventing any autoimmune disease or condition by inhibiting IL2Rγ. Blocking the signaling of one or more cytokines in the γc family can be beneficial to patients who suffer from autoimmunity due to the inhibitory effect on the secretion of inflammatory cytokines and the production of autoantibodies. Multiple sclerosis (MS) is a disease of the brain and spinal cord (central nervous system (CNS)), in which the immune system attacks the myelin sheath of nerve fibers and causes communication problems between the brain and other parts of the body. Eventually, the disease can cause the nerves themselves to deteriorate or become permanently damaged. Rheumatoid arthritis (RA) is an autoimmune disease in which the body's immune system attacks the joints. This produces inflammation, which causes the tissue lining the inside of the joint (synovium) to thicken, resulting in swelling and pain in and around the joints. Psoriasis is an autoimmune disease that mainly manifests as affecting the skin. Inflammation can also affect the joints, vascular system and eyes of psoriasis patients. Type 1 diabetes is an autoimmune disease in which the immune system attacks the beta cells that produce insulin in the pancreas and destroys them. The pancreas then produces little or no insulin. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that occurs when the body's immune system attacks its own tissues and organs. Inflammation caused by lupus can affect many different body systems-including joints, skin, kidneys, blood cells, brain, heart and lungs. Myasthenia gravis is an autoimmune disease in which antibodies block the receptors of acetylcholine at the neuromuscular junction, which prevents muscle contraction. In most individuals with myasthenia gravis, this is caused by antibodies to the acetylcholine receptor itself. However, antibodies to other proteins, such as MuSK (muscle-specific kinase) proteins, can also cause impaired transmission at the neuromuscular junction. The present invention includes methods for treating or preventing autoimmune diseases or disorders (e.g., multiple sclerosis or any other central nervous system inflammation, rheumatoid arthritis, psoriasis, type I diabetes, systemic lupus erythematosus and / or myasthenia gravis) in a subject, the method comprising administering to the subject a therapeutically effective dose of an anti-IL2Rγ antigen binding protein.

[0318] An effective dose or therapeutically effective dose of an anti-IL2Rγ antigen binding protein, such as an antibody or antigen binding fragment, for treating or preventing an IL2Rγ-mediated disease or condition refers to an amount of the antigen binding protein sufficient to alleviate one or more signs and / or symptoms of the disease or condition in the treated subject, whether by inducing regression or elimination of these signs and / or symptoms, or by inhibiting the progression of these signs and / or symptoms. In one embodiment of the present invention, the effective dose or therapeutically effective dose of the anti-IL2Rγ antigen binding protein is about 0.05-50 mg / kg body weight. The amount of the dose may vary depending on the age and size of the subject to be administered, the target disease, the condition, the route of administration, etc. In certain embodiments, a second or more subsequent doses of the antigen binding protein may be administered after the initial dose, and the amount of the subsequent dose may be approximately equal to, less than, or greater than the initial dose, wherein the subsequent doses are separated by at least 1 to 3 days, at least one week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0319] As used herein, the term "subject" refers to a mammal (e.g., rat, mouse, cat, dog, cow, sheep, horse, goat, rabbit), preferably a human, who, for example, needs to prevent and / or treat an IL2Rγ-mediated disease. The subject may suffer from an IL2Rγ-mediated disease or be susceptible to such a disease.

[0320] "Preventing" an IL2Rγ-mediated disease or condition as it relates to the use of an anti-IL2Rγ antigen binding protein of the invention refers to administering the disease or condition to a subject before it manifests in the subject, so as to stop such manifestation from occurring.

[0321] Combinations and pharmaceutical preparations

[0322] The present invention provides compositions comprising an anti-IL2Rγ antigen binding protein in combination with one or more ingredients; and methods of use thereof and methods of preparing such compositions. Pharmaceutical formulations comprising an anti-IL2Rγ antigen binding protein and a pharmaceutically acceptable carrier or excipient are part of the present invention.

[0323] To prepare anti-IL2Rγ antigen binding proteins, such as antibodies and antigen binding fragments thereof (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899P; H4H129 00P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2), the antigen binding protein being mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984); Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parental Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.In one embodiment of the invention, the pharmaceutical preparation is sterile. Such compositions are part of the present invention.

[0324] The pharmaceutical preparation of the present invention comprises an anti-IL2Rγ antigen binding protein and a pharmaceutically acceptable carrier, and the carrier includes, for example, water, a buffer, a preservative and / or a detergent.

[0325] The scope of the present invention includes dried, e.g., lyophilized, anti-IL2Rγ antigen binding proteins, e.g., antibodies or antigen binding fragments thereof (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899P ; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2), or a pharmaceutical formulation thereof comprising a pharmaceutically acceptable carrier but substantially lacking water.

[0326] In another embodiment of the present invention, an anti-IL2Rγ antigen binding protein, e.g., an antibody or antigen binding fragment thereof disclosed herein (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; another therapeutic agent that is administered to a subject in combination with the other therapeutic agent that is administered to the subject.

[0327] The mode of administration of the anti-IL2Rγ antigen binding protein or its composition can vary. The routes of administration include parenteral, non-parenteral, oral, rectal, transmucosal, enteral, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intracerebroventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, intraocular, intravitreal, transdermal or intraarterial.

[0328] The present invention provides methods for administering an anti-IL2Rγ antigen binding protein, such as an antibody or an antigen binding fragment thereof (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899P; H4H12890P) to a subject. 12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2), the method comprising introducing the protein or a pharmaceutical preparation thereof into the subject. For example, in one embodiment of the invention, the method comprises piercing, such as with a needle of a syringe, the body of the subject and injecting the antigen binding protein or a pharmaceutical preparation thereof into the subject, such as into the eye, into a vein, into an artery, into muscle tissue or under the skin of the subject.

[0329] The present invention provides a container (e.g., a plastic bottle or a glass bottle, e.g., with a cap or a chromatography column, a hollow needle or a syringe barrel) comprising any anti-IL2Rγ antigen binding protein, e.g., an antibody or an antigen binding fragment thereof (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2), or a pharmaceutical preparation thereof comprising a pharmaceutically acceptable carrier.

[0330] The present invention includes combinations comprising an anti-IL2Rγ antigen binding protein, e.g., antibody, or antigen binding fragment thereof (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P) of the present invention in combination with one or more additional therapeutic agents. ; H4H12890P; H4H12899P; H4H12900P; H4H12908P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2). The anti-IL2Rγ antigen binding protein and the additional therapeutic agent can be in a single composition or in separate compositions. For example, in one embodiment of the invention, the additional therapeutic agent is an immunosuppressive drug. In one embodiment of the invention, the additional therapeutic agent is an anti-TNFα antibody or binding protein (e.g., infliximab, adalimumab, etanercept or golimumab), tacrolimus, cyclosporine, corticosteroids, prednisolone, methylprednisolone, anti-thymocyte globulin, alemtuzumab, daclizumab, extracorporeal photophoresis, mycophenolate mofetil, sirolimus, pentostatin, mesenchymal stem cells, inomucomab, denileukin, a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof that binds to BCMA (B cell maturation antigen) and CD3, and / or basiliximab.

[0331] For use in combination with another therapeutic agent, an anti-IL2Rγ antigen binding protein (e.g., H4H12857P; H4H12858P; H4H12859P; H4H12863P; H4H12874P; H4H12871P; H4H12884P; H4H12886P; H4H12889P; H4H12890P; H4H12899P; H4H12900P; H4H1290 8P; H4H12913P2; H4H12922P2; H4H12924P2; H4H12926P2; H4H12927P2; H4H12934P2; H4H13538P; H4H13541P; H4H13544P2; or H4H13545P2) to perform said treatment or prevention in a subject in need thereof is part of the invention.

[0332] The term "in combination with..." refers to a component: an anti-IL2Rγ antigen binding protein (e.g., an antibody or antigen binding fragment thereof) of the present invention and another agent (e.g., methotrexate) can be formulated into a single composition, for example, for simultaneous delivery, or separately formulated into two or more compositions (e.g., a kit containing each component). The components administered in combination with each other can be administered to the subject at a different time than when the other component is administered; for example, each administration can be given non-simultaneously (e.g., separately or sequentially) at intervals within a given time period. The separate components administered in combination with each other can also be administered sequentially but substantially simultaneously during the same administration period. In addition, the separate components administered in combination with each other can be administered to the subject by the same or different routes.

[0333] Example

[0334] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0335] Embodiment 1: Identification and isolation of anti-IL2Rγ antibodies

[0336] Immunization with an IL2Rγ protein immunogen containing the IL2Rγ extracellular sequence (ectodomain) Anti-IL2Rγ antibodies were obtained by using mice (ie, mice engineered to contain DNA encoding human immunoglobulin heavy and kappa light chain variable regions).

[0337] Specifically, the immunogen human IL2Rg extracellular-mmh comprises:

[0338] Amino acids (1-240): human IL2Rg extracellular (L23-A262 of NP_000197.1), and

[0339] Amino acids (241-268): Myc-Myc-hexahistidine tag (underlined);

[0340] It contains the amino acid sequence:

[0341]

[0342] *Expressed with mROR signal sequence

[0343] The antibody immune response was monitored using an IL2Rγ-specific immunoassay. A fully human anti-IL2Rγ antibody was isolated and purified.

[0344] Table 1-1. Summary of Anti-IL2Rγ VH, VK and CDR Amino Acid Sequences*

[0345]

[0346] *Numbers refer to SEQ ID NOs corresponding to the indicated sequences.

[0347] Table 1-2. Summary of amino acid sequences of anti-IL2Rγ heavy and light chains*

[0348]

[0349] *Numbers refer to SEQ ID NOs corresponding to the indicated sequences.

[0350] The amino acid sequences of the anti-IL2Rγ antibody immunoglobulin heavy and light chains are shown below (CDRs are underlined; variable regions are in bold).

[0351] H4H12857P

[0352] Heavy chain (SEQ ID NO: 311)

[0353]

[0354] Light chain (SEQ ID NO: 313)

[0355]

[0356] H4H12858P

[0357] Heavy chain (SEQ ID NO: 331)

[0358]

[0359] Light chain (SEQ ID NO: 333)

[0360]

[0361] H4H12859P

[0362] Heavy chain (SEQ ID NO: 18)

[0363]

[0364] Light chain (SEQ ID NO: 20)

[0365]

[0366] H4H12863P

[0367] Heavy chain (SEQ ID NO: 38)

[0368]

[0369] Light chain (SEQ ID NO: 40)

[0370]

[0371] H4H12874P

[0372] Heavy chain (SEQ ID NO: 58)

[0373]

[0374] Light chain (SEQ ID NO: 60)

[0375]

[0376] H4H12871P

[0377] Heavy chain (SEQ ID NO: 376)

[0378]

[0379] Light chain (SEQ ID NO: 378)

[0380]

[0381] H4H12884P

[0382] Heavy chain (SEQ ID NO: 77)

[0383]

[0384] Light chain (SEQ ID NO: 79)

[0385]

[0386] H4H12886P

[0387] Heavy chain (SEQ ID NO: 97)

[0388]

[0389] Light chain (SEQ ID NO: 99)

[0390]

[0391] H4H12889P

[0392] Heavy chain (SEQ ID NO: 357)

[0393]

[0394] Light chain (SEQ ID NO: 359)

[0395]

[0396] H4H12890P

[0397] Heavy chain (SEQ ID NO: 115)

[0398]

[0399] Light chain (SEQ ID NO: 117)

[0400]

[0401] H4H12899P

[0402] Heavy chain (SEQ ID NO: 134)

[0403]

[0404] Light chain (SEQ ID NO: 136)

[0405]

[0406] H4H12900P

[0407] Heavy chain (SEQ ID NO: 152)

[0408]

[0409] Light chain (SEQ ID NO: 154)

[0410]

[0411] H4H12908P

[0412] Heavy chain (SEQ ID NO: 170)

[0413]

[0414] Light chain (SEQ ID NO: 172)

[0415]

[0416] H4H12913P2

[0417] Heavy chain (SEQ ID NO: 186)

[0418]

[0419] Light chain (SEQ ID NO: 188)

[0420]

[0421] H4H12922P2

[0422] Heavy chain (SEQ ID NO: 343)

[0423]

[0424] Light chain (SEQ ID NO: 188)

[0425]

[0426] H4H12924P2

[0427] Heavy chain (SEQ ID NO: 198)

[0428]

[0429] Light chain (SEQ ID NO: 188)

[0430]

[0431] H4H12926P2

[0432] Heavy chain (SEQ ID NO: 208)

[0433]

[0434] Light chain (SEQ ID NO: 188)

[0435]

[0436] H4H12927P2

[0437] Heavy chain (SEQ ID NO: 216)

[0438]

[0439] Light chain (SEQ ID NO: 188)

[0440]

[0441] H4H12934P2

[0442] Heavy chain (SEQ ID NO: 234)

[0443]

[0444] Light chain (SEQ ID NO: 236)

[0445]

[0446] H4H13538P

[0447] Heavy chain (SEQ ID NO: 254)

[0448]

[0449] Light chain (SEQ ID NO: 256)

[0450]

[0451] H4H13541P

[0452] Heavy chain (SEQ ID NO: 272)

[0453]

[0454] Light chain (SEQ ID NO: 274)

[0455]

[0456] H4H13544P2

[0457] Heavy chain (SEQ ID NO: 284)

[0458]

[0459] Light chain (SEQ ID NO: 188)

[0460]

[0461] H4H13545P2

[0462] Heavy chain (SEQ ID NO: 294)

[0463]

[0464] Light chain (SEQ ID NO: 188)

[0465]

[0466] *The antibodies referred to in these Examples are antibodies having immunoglobulin chains containing the amino acid sequences specifically shown in Example 1.

[0467] Example 2 : Surface plasmon resonance binding assay

[0468] The dissociation rate constant (k) of IL-2Rγ reagent binding to purified anti-IL2Rγ monoclonal antibody was determined using the real-time surface plasmon resonance-based Biacore 4000 biosensor platform. dAll binding studies were performed at 25°C and 37°C using two running buffers: (i) 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 2.7 mM KCl, 137 mM NaCl, 0.03% NaN3, 0.05% v / v surfactant Tween-20, pH 7.4 (PBS-T-pH 7.4) and (ii) 8.8 mM NaH2PO4, 1.2 mM Na2HPO4, 2.7 mM KCl, 137 mM NaCl, 0.03% NaN3, 0.05% v / v surfactant Tween-20, pH 6.0 (PBS-T-pH 6.0). Anti-IL2Rγ monoclonal antibody expressing human IgG4 Fc was captured using an amine-derivatized CM5 Biacore sensor surface coupled to a monoclonal mouse anti-human Fc antibody (GE, catalog # BR-1008-39). All IL2Rγ reagents were expressed with a C-terminal myc-myc-hexahistidine tag (subsequently indicated by the -MMH suffix). Different concentrations of human IL2Rγ extracellular domain expressing a C-terminal myc-myc-hexahistidine tag (hIL-2Rg-MMH; SEQ ID NO: 379) or cynomolgus monkey IL2Rγ extracellular domain expressing a C-terminal myc-myc-hexahistidine tag (mfIL-2Rg-MMH; SEQ ID NO: 380) were prepared in PBS-T-pH7.4 running buffer (100 nM-11.11 nM, 3-fold serial dilutions) and injected at a flow rate of 30 μL / min for 4 minutes. Dissociation of bound IL-2Rg-MMH was performed in PBS-T-pH7.4 or PBS-T-pH6.0 running buffer for 6 minutes.

[0469] The dissociation rate constants (k) in both running buffers were determined by fitting the real-time binding sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. d The dissociation rate values ​​of anti-hemojuvelin mAb binding to hIL-2RG-MMH and mfIL-2RG-MMH at 25°C and 37°C in PBS-T-pH7.4 and PBS-T-pH6.0 are shown in Tables 2-1 to 2-8.

[0470] Table 2-1. Dissociation rate constants of anti-IL-2Rγ mAb binding to hIL-2Rg-MMH at 25°C in PBS-T-pH7.4

[0471]

[0472] Table 2-2. Dissociation rate constants of anti-IL-2Rg mAb binding to hIL-2Rg-MMH at 25°C in PBS-T-pH 6.0

[0473]

[0474] Table 2-3. Dissociation rate constants of anti-IL-2Rγ mAb binding to hIL-2Rg-MMH at 37°C in PBS-T-pH7.4

[0475]

[0476] Table 2-4. Dissociation rate constants of anti-IL-2Rγ mAb binding to hIL-2Rg-MMH at 37°C in PBS-T-pH 6.0

[0477]

[0478] Table 2-5. Dissociation rate constants of anti-IL-2Rγ mAb binding to mfIL-2Rg-MMH at 25°C in PBS-T-pH7.4

[0479]

[0480] Table 2-6. Dissociation rate constants of anti-IL-2Rγ mAb binding to mfIL-2Rg-MMH at 25°C in PBS-T-pH 6.0

[0481]

[0482] Table 2-7. Dissociation rate constants of anti-IL-2Rγ mAb binding to mfIL-2Rg-MMH at 37°C in PBS-T-pH7.4

[0483]

[0484] Table 2-8. Dissociation rate constants of anti-IL-2Rγ mAb binding to mfIL-2Rg-MMH at 37°C in PBS-T-pH 6.0

[0485]

[0486] Example 3 : Binding kinetics

[0487] The equilibrium dissociation constant (K) for IL-2Rγ binding to purified anti-IL-2Rγ monoclonal antibodies was determined using a Biacore 4000 instrument equipped with a real-time surface plasmon resonance biosensor. DAll binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-ET) running buffer at 25°C and 37°C. The Biacore sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture the anti-IL2Rγ monoclonal antibody.

[0488] Binding studies were performed on the following IL-2Rγ reagents:

[0489] Expression of a human IL2Rγ extracellular domain with a C-terminal myc-myc-hexahistidine tag (hIL-2Rg-MMH; SEQ ID NO: 379), comprising:

[0490] Amino acids (1-240): human IL2Rg extracellular (L23-A262 of NP_000197.1)

[0491] Amino acids (241-268): Myc-Myc-hexahistidine tag (underlined)

[0492] It contains the amino acid sequence:

[0493]

[0494] *Expressed with mROR signal sequence

[0495] Expression of the cynomolgus monkey IL2Rγ extracellular domain with a C-terminal myc-myc-hexahistidine tag (mfIL-2Rg-MMH; SEQ ID NO: 380), comprising:

[0496] Amino acids (1-240): Cynomolgus monkey IL2Rg extracellular (L23-A262 of XP_005593949.1)

[0497] Amino acids (241-268): Myc-Myc-hexahistidine tag (underlined)

[0498] It contains the amino acid sequence:

[0499]

[0500] Expression of a human IL2Rγ extracellular domain with a C-terminal mouse IgG2a Fc tag (hIL-2Rg-mFc; SEQ ID NO: 381), comprising:

[0501] Amino acids (1-240): human IL2Rg extracellular (L23-A262 of NP_000197.1)

[0502] Amino acids (241-473): mouse IgG2a Fc tag (underlined)

[0503] It contains the amino acid sequence:

[0504]

[0505] *Expressed with mROR signal sequence

[0506] Expression of the D1 domain of the human IL-2Rγ extracellular domain with a C-terminal myc-myc-hexahistidine tag (hIL-2Rg_D1-MMH; SEQ ID NO: 382), comprising:

[0507] Amino acids (1-131): human IL2Rg domain 1 (L23-I153 of NP_000197.1)

[0508] Amino acids (132-159): Myc-Myc-hexahistidine tag (underlined)

[0509] It contains the amino acid sequence:

[0510]

[0511] *Expressed with mROR signal sequence

[0512] Expression of the D2 domain of the human IL2Rγ extracellular domain with a C-terminal myc-myc-hexahistidine tag (hIL-2Rg_D2-MMH; SEQ ID NO: 383), comprising:

[0513] Amino acids (1-88): human IL2Rg domain 2 (P154-S241 of NP_000197.1)

[0514] Amino acids (89-116): Myc-Myc-hexahistidine tag (underlined)

[0515] It contains the amino acid sequence:

[0516]

[0517] *Expressed with mROR signal sequence

[0518] Expression of mouse IL2Rγ extracellular domain with a C-terminal myc-myc-hexahistidine tag (mIL-2Rg-MMH; SEQ ID NO: 384), comprising:

[0519] Amino acids (1-241): mouse IL2Rg extracellular (W23-A263 of NP_038591.1)

[0520] Amino acids (242-269): Myc-Myc-hexahistidine tag (underlined)

[0521] It contains the amino acid sequence:

[0522]

[0523] Expression of rat IL2Rγ extracellular domain with a C-terminal myc-myc-hexahistidine tag (rIL-2Rg-MMH; SEQ ID NO: 385), comprising:

[0524] Amino acids (1-240): rat IL2Rg extracellular (W23-A262 of NP_543165.1)

[0525] Amino acids (241-268): Myc-Myc-hexahistidine tag (underlined)

[0526] It contains the amino acid sequence:

[0527]

[0528] *Expressed with mROR signal sequence

[0529] Different concentrations of IL2Rγ reagent were prepared in HBS-ET running buffer (100 nM-6.25 nM; 4-fold serial dilution or 50 nM-3.125 nM; 4-fold serial dilution for hIL-2Rg-mFc) and injected over the anti-human Fc captured anti-IL2Rγ mAb surface at a flow rate of 30 μL / min for 4 min. Dissociation of mAb-bound IL2Rγ reagent was monitored in HBS-ET running buffer for 8 to 10 min. Kinetic association (k a ) and dissociation (k d ) rate constants were determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. The binding-dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 ) was calculated from the kinetic rate constant as follows:

[0530] and

[0531] The kinetic parameters for the binding of various IL-2Rγ reagents to different IL2Rγ monoclonal antibodies at 25°C and 37°C are shown in Tables 3-1 to 3-14.

[0532] Table 3-1. Binding kinetic parameters of hIL-2Rg-MMH binding to IL-2Rγ monoclonal antibody at 25°C

[0533]

[0534] Table 3-2. Binding kinetic parameters of hIL-2Rg-MMH binding to IL-2Rγ monoclonal antibody at 37°C

[0535]

[0536] *NB indicates no binding was observed under the present experimental conditions.

[0537] Table 3-3. Binding kinetic parameters of mfIL-2Rg-MMH binding to IL-2Rγ monoclonal antibody at 25°C

[0538]

[0539] Table 3-4. Binding kinetic parameters of mfIL-2Rg-MMH binding to IL-2Rγ monoclonal antibody at 37°C

[0540]

[0541] *NB indicates no binding was observed under the present experimental conditions.

[0542] Table 3-5. Binding kinetic parameters of hIL-2Rg-mFc binding to IL-2Rγ monoclonal antibody at 25°C

[0543]

[0544] *NB indicates no binding was observed under the present experimental conditions.

[0545] # indicates that no dissociation was observed under the current experimental conditions, and k d The value is artificially fixed at 1.00E-05s -1 .

[0546] Table 3-6. Binding kinetic parameters of hIL-2Rg-mFc binding to IL-2Rγ monoclonal antibody at 37°C

[0547]

[0548] *NB indicates no binding was observed under the present experimental conditions.

[0549] # indicates that no dissociation was observed under the current experimental conditions, and kd The value is artificially fixed at 1.00E-05s -1 .

[0550] $ The binding data are important for generating reliable binding kinetics and K values. D is uncertain.

[0551] Table 3-7. Binding kinetic parameters of mIL-2Rg-MMH binding to IL-2Rγ monoclonal antibody at 25°C

[0552]

[0553] *NB indicates no binding was observed under the present experimental conditions.

[0554] Table 3-8. Binding kinetic parameters of mIL-2Rg-MMH binding to IL-2Rγ monoclonal antibody at 37°C

[0555]

[0556] *NB indicates no binding was observed under the present experimental conditions.

[0557] Table 3-9. Binding kinetic parameters of rat IL-2Rg-MMH binding to IL-2Rγ monoclonal antibody at 25°C

[0558]

[0559] *NB indicates no binding was observed under the present experimental conditions.

[0560] Table 3-10. Binding kinetic parameters of rat IL-2Rg-MMH binding to IL-2Rγ monoclonal antibody at 37°C

[0561]

[0562] *NB indicates no binding was observed under the present experimental conditions.

[0563] Table 3-11. Binding kinetic parameters of hIL-2Rg_D1-MMH binding to IL-2Rγ monoclonal antibody at 25°C

[0564]

[0565] *NB indicates no binding was observed under the present experimental conditions.

[0566] Table 3-12. Binding kinetic parameters of hIL-2Rg_D1-MMH binding to IL-2Rγ monoclonal antibody at 37°C

[0567]

[0568] *NB indicates no binding was observed under the present experimental conditions.

[0569] Table 3-13. Binding kinetic parameters of hIL-2Rg_D2-MMH binding to IL-2Rγ monoclonal antibody at 25°C

[0570]

[0571] *NB indicates no binding was observed under the present experimental conditions.

[0572] Table 3-14. Binding kinetic parameters of hIL-2Rg_D2-MMH binding to IL-2Rγ monoclonal antibody at 37°C

[0573]

[0574] *NB indicates no binding was observed under the present experimental conditions.

[0575] Example 4 : Octet cross-competition between different anti-IL-2Rγ monoclonal antibodies

[0576] Binding competition between a panel of anti-IL2Rγ monoclonal antibodies was determined using real-time, label-free biolayer interferometry on an Octet HTX biosensor platform (Pall ForteBio). The entire experiment was performed at 25°C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, 1 mg / mL BSA, pH 7.4 (HBS-EBT) buffer using a plate shaker speed of 1000 rpm. To evaluate whether the two antibodies compete with each other for binding to their respective epitopes on a human IL2Rγ extracellular domain expressing a C-terminal myc-myc-hexa-histidine tag (hIL-2Rg-MMH; SEQ ID: 379), an anti-pentahistidine antibody-coated Octet biosensor tip (Fortebio Inc, #18-5122) was used to capture approximately 0.27 nM hIL-2Rg-MMH by immersing the biosensor tip in a well containing 10 μg / mL hIL-2Rg-MMH for 3 minutes. The antigen-capturing biosensor tip was then saturated with the first anti-IL2Rγ monoclonal antibody (subsequently referred to as mAb-1) by immersing in a well containing 50 μg / mL mAb-1 for 300 seconds. The biosensor tip was then subsequently immersed in a well containing 50 μg / mL of the second anti-IL2Rγ monoclonal antibody (subsequently referred to as mAb-2) for 240 seconds. Between each step of the experiment, the biosensor tip was washed in HBS-ETB buffer. Real-time binding responses were monitored throughout the experiment and recorded at the end of each step. The binding responses of mAb-2 and hIL-2Rg-MMH pre-complexed with mAb-1 were compared and the competitive / non-competitive behavior of different anti-IL2Rγ monoclonal antibodies was determined, as shown in Table 4-1.

[0577] Table 4-1. Cross-competition between anti-IL-2Rg monoclonal antibodies

[0578]

[0579]

[0580]

[0581]

[0582] Example 5 :Flow cytometric analysis of STAT phosphorylation in human CD4+ T cells (human PBMC)

[0583] To evaluate the in vitro properties of the IL2Rγ antibodies of the present invention, flow cytometry (BD Biosciences) was used to analyze the TMPhosflow assay) measured their ability to block CD4 + T cell activation capacity. TM Phosflow allows the simultaneous analysis of intracellular phosphoproteins (e.g. STAT proteins) and cell surface markers to analyze cell signaling in discrete cell subsets. The technology was used to analyze STAT phosphorylation in human CD4+ T cells following stimulation with cytokines from the γc family.

[0584] Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood (Bioreclammation IVT) by density gradient centrifugation. K2 EDTA whole blood was centrifuged in X-VIVO TM 15 culture medium (Lonza) was diluted 1:1, added to SepMate tubes (StemCell) containing FicollPaquePLUS (Healthcare) and centrifuged to separate PBMCs. The upper layer containing PBMCs was transferred to a new tube and washed twice with DPBS (Life Technologies). PBMCs were then concentrated to a concentration of approximately 5.0×10 6 Cells / mL resuspended in X-VIVO TM 15 μL of culture medium, plated in 96-well plates (50 μL cells / well; approximately 250,000 cells / well), and incubated at 37°C for 2 hours before adding cytokines and antibodies.

[0585] In the preheating X-VIVO TM Serial dilutions of antibodies (1:5) were prepared in 15% culture medium and added to cells (50 μL), with final antibody concentrations starting at 400 nM. TM Fixed cytokine concentrations were prepared in 15% culture medium and added to the cells (100 μL): the final concentration of IL-7 (R&D Systems) was 1 pM, the final concentrations of IL-4 (R&D Systems) and IL-21 (eBioscience) were 50 pM, the final concentration of IL-15 (R&D Systems) was 0.5 nM, and the final concentration of IL-2 (R&D Systems) was 10 nM; the final volume in each well was 200 μL.

[0586] For cytokine dose response, serial dilutions (1:5) of each cytokine were also added to the pre-warmed X-VIVO TM15 culture medium, with final cytokine concentrations starting at 5 nM for IL-4, IL-7, and IL-21, or 50 nM for IL-2 and IL-15. First, add 50 μL of X-VIVO TM 15 culture medium, followed by the addition of 100 μL of serial dilutions of cytokines, for a total volume of 200 μL per well. After the addition of cytokines and antibodies to the cells, they were incubated at 37 ° C for 15 minutes to allow PBMC activation (STAT phosphorylation). The stimulation was then stopped by adding 200 μL of warm Cytofix (BD) to each well and the cells were incubated at 37 ° C for 10 minutes (fixation step). The cells were then washed twice with staining buffer (BD) and kept at 4 ° C overnight. The next day, the cells were centrifuged and permeabilized by slowly adding 100 μL of cold permeabilization buffer III (BD) to the pellet. The cells were incubated at 4 ° C for 30 minutes and then washed twice with staining buffer. In order to be able to analyze the CD4 + T cell population, cells were stained with a mixture of human FcR binding inhibitor (eBioscience; 1 / 10), anti-CD33-PE (BD; 1 / 200), anti-CD4-PacificBlue (BD; 1 / 200), anti-CD3-PECy7 (BD; 1 / 200), and relevant anti-phospho-STAT-AlexaFluor647 (BD) prepared in staining buffer:

[0587] - Anti-phospho-STAT3 (1 / 10): for cells stimulated by IL-21,

[0588] - Anti-phospho-STAT5 (1 / 20): for cells stimulated by IL-2, IL-7 and IL-15,

[0589] - Anti-phospho-STAT6 (1 / 10): for cells stimulated by IL-4.

[0590] The samples were kept in the dark at room temperature for 1 hour. The cells were then centrifuged and washed twice with staining buffer. Sample data were acquired on an LSR Fortessa X-20 cell analyzer using the HTS accessory (BD). Data analysis was performed using FlowJo X software (Tree Star, OR). + T cells are limited to intact cells, singlets, CD33 - 、CD3 + 、CD4 + ; and STAT phosphorylation was analyzed in this cell population (MFI = mean fluorescence intensity).

[0591] Both H4H12889P and H4H12922P2 similarly and effectively blocked STAT phosphorylation induced by all cytokines tested in this assay (IL-2, IL-4, IL-7, IL-15 and IL-21), while H4H12874P, H4H12886P, H4H12857P and the comparative antibody COMP1499 (anti-IL2Rγ antibody CP.B8, see US2002 / 0028202) only partially blocked or did not block cytokine-induced STAT phosphorylation.

[0592] Table 5-1. Anti-IL-2Rγ antibodies H4H12889P and H4H12922P2 block human CD4 + STAT phosphorylation induced by human IL-2, IL-4, IL7, IL-15, and IL-21 in T cells

[0593] IC50[M] IL-2 IL-4 IL-7 IL-15 IL-21 Constant 10nM 50pM 1pM 0.5nM 50pM H4H12889P 2.06E-09 1.10E-09 8.92E-10 2.55E-09 2.28E-09 H4H12922P2 1.87E-09 8.54E-10 5.80E-10 2.46E-09 2.21E-09

[0594] *IC50 values ​​measured for both antibodies are shown at the indicated concentrations under different interleukins.

[0595] See also Figure 1 (AE), where the level of STAT phosphorylation was determined at each antibody concentration tested.

[0596] Example 6 : Flow cytometric analysis of STAT3 phosphorylation in in vitro differentiated human mast cells

[0597] In order to evaluate the in vitro properties of the anti-IL2Rγ antibodies of the present invention, flow cytometry (BD Biosciences) was used to analyze the TM Phosfiow assay) measured their ability to block IL-9-induced human mast cell activation. We used this technique to observe STAT3 phosphorylation in in vitro differentiated human mast cells following human IL-9 stimulation.

[0598] Briefly, human mast cells were cultured for 6 weeks in StemSpan serum-free medium supplemented with human SCF, IL-6, and IL-3. + Progenitor cells generated in vitro.

[0599] Human mast cells were isolated at a concentration of approximately 4.0 x 10 6 Cells / mL in X-VIVO TM The cells were resuspended in 15% culture medium, plated in 96-well plates (50 μL cells / well; approximately 200,000 cells / well), and incubated at 37°C for 2 hours before adding cytokines and antibodies.

[0600] In the preheating X-VIVO TMSerial dilutions of antibodies (1:5) were prepared in 15% culture medium and added to cells (50 μL), with final antibody concentrations starting at 400 nM. TM A fixed concentration of IL-9 (R&D) was prepared in 15% culture medium and added to the cells (100 μL) to a final concentration of 2 nM; the final volume in each well was 200 μL.

[0601] For cytokine dose response, serial dilutions of IL-9 (1:5) were also added to the pre-warmed X-VIVO TM 15% culture medium, the final cytokine concentration starts at 100nM. First, add 50μL X-VIVO TM 15 μL of culture medium and then 100 μL of serial dilutions of cytokines were added to a total volume of 200 μL per well.

[0602] After adding cytokines and antibodies to the cells, they were incubated at 37°C for 15 minutes to allow mast cell activation (as measured by STAT3 phosphorylation). The stimulation was then stopped by adding 200 μL of warm Cytofix (BD) to each well, and the cells were incubated at 37°C for 10 minutes (fixation step). The cells were then washed twice with staining buffer (BD) and kept overnight at 4°C. The next day, the cells were centrifuged and permeabilized by slowly adding 100 μL of cold permeation buffer III (BD) to the pellet. The cells were incubated at 4°C for 30 minutes and then washed twice with staining buffer. Mast cells were then stained with a mixture of human FcR binding inhibitors (eBioscience; 1 / 10), anti-c-Kit-PE (BD; 1 / 100) and anti-phosphoSTAT3-AlexaFluor647 (BD; 1 / 10) prepared in staining buffer.

[0603] The samples were kept in the dark at room temperature for 1 hour. The cells were then centrifuged and washed twice with staining buffer. Sample data were acquired on an LSR Fortessa X-20 cell analyzer using an HTS accessory (BD). Data analysis was performed using FlowJo X software (Tree Star, OR). Mast cells were defined as intact cells, singlet, c-Kit+; and STAT3 phosphorylation (MFI = mean fluorescence intensity) was analyzed in this cell population.

[0604] Both H4H12889P and H4H12922P2 similarly and effectively blocked STAT3 phosphorylation induced by IL-9.

[0605] Table 6-1. Anti-IL-2Rγ antibodies H4H12889P and H4H12922P2 block STAT3 phosphorylation induced by human IL-9 in human mast cells differentiated in vitro

[0606] IC50[M] IL-9 Constant 2nM H4H12889P 4.41E-10 H4H12922P2 4.16E-10

[0607] *Shows the IC50 values ​​measured for both antibodies when the IL-9 concentration was 2 nM.

[0608] See also Figure 2 , where the level of IL-9-induced STAT phosphorylation was determined at each antibody concentration tested.

[0609] Example 7 In vivo model to evaluate the blocking activity of IL-2Rγ antibodies as therapeutic treatment: testing of monoclonal antibodies in a xenogeneic acute graft-versus-host disease model

[0610] To determine the effects of our anti-IL2Rγ antibodies H4H12889P and H4H12922P2 and the comparator IL-2Rγ antibody COMP1499 in a relevant in vivo model, xenogeneic acute graft-versus-host disease (GvHD) studies were performed. Briefly, to induce GvHD in mice, human peripheral blood mononuclear cells (huPBMCs) were injected into NOD-scid IL2rγ null (NSG) mice (Jackson Laboratory). After transplantation, human immune cells recognize the mouse host as xenogeneic and mount a strong immune response against its tissues.

[0611] In this experiment, NSG mice (Jackson Lab) were retro-orbitally injected with 10 million huPBMCs (ReachBio) resuspended in DPBS (10 million cells / 100 μL; 5 groups of 10 mice each). Briefly, on the day of injection, human PBMCs were thawed in IMDM medium (Irvine Scientific) supplemented with 10% FBS (Seradigm) and incubated in this supplemented medium for 2 hours at 37°C. The cells were then washed in DPBS (Life Technologies) and resuspended at 10 million cells / 100 μL for injection. The control group (10 mice) was retro-orbitally injected with 100 μL of PBS. Four groups of huPBMC-transplanted NSG mice were subcutaneously injected with 25 mg / kg H4H12889P, H4H12922P2, COMP1499 or isotype control antibody (REGN1945; human anti-domestic cat (Felis domesticus) Fel d1 antibody (IgG4 (S108P) / κ)) starting 3 weeks after huPBMC injection, and then twice a week for 6 weeks. The experiment was terminated by killing the remaining mice on day 161 after huPBMC transplantation. The experimental dosing and treatment regimens of each group of mice are shown in Table 7-1.

[0612] Table 7-1. Experimental drug administration and treatment scheme for each group of mice

[0613] Group NSG mice huPBMC injection Antibody 1 10 none none 2 10 10 million none 3 10 10 million Isotype control antibody (REGN1945) 4 10 10 million IL-2Rγ Antibody (COMP1499) 5 10 10 million IL-2Rγ Antibody (H4H12889P) 6 10 10 million IL-2Rγ Antibody (H4H12922P2)

[0614] Throughout the experiment, mice were monitored twice weekly for weight loss and death (to assess the effect of therapeutic antibodies on survival). Human cell engraftment in the blood and serum mouse and human cytokine levels were assessed at different time points, as shown in Table 7-2.

[0615] Table 7-2. Blood / serum collection dates and readouts

[0616]

[0617] The mice were monitored for weight loss twice a week throughout the experiment ( Figure 3 (AF); % initial body weight on the day of huPBMC transplantation) and mortality ( Figure 4 ; to assess the effect of therapeutic antibodies on survival). Animals showing a weight loss of 20% of their initial body weight were euthanized.

[0618] At different time points after huPBMC injection, blood samples from mice were collected into Microtainer tubes (BD, Cat#3659740) and human cell engraftment was assessed by observing the absolute number of human cells in the blood by flow cytometry. Briefly, 50 μL of each blood sample was incubated in ACK lysis buffer (Gibco) at room temperature for 5 minutes to lyse red blood cells. The cells were then washed in DPBS, stained with LIVE / DEAD fixable dead cell stain (Invitrogen), washed in MACS buffer (Miltenyi Biotec), and stained with a flow cytometer for identification of human CD45 + Cells, T cells, CD4 + T cells and CD8 + T cells were labeled with a cocktail of antibodies (anti-human CD45, anti-human CD3, anti-human CD4, and anti-human CD8 [BD] diluted 1 / 50 in brilliant staining buffer [BD], and human and mouse Fc inhibitor antibodies [eBioscience and BD, respectively]). Finally, samples were washed in MACS buffer, fixed in BD CytoFix (BD), and resuspended in MACS buffer containing CountBright beads (Life Technologies) to calculate the absolute cell number in each sample. Sample data were acquired on an LSR Fortessa X-20 cell analyzer using the HTS accessory (BD). Data analysis was performed using FlowJo X software (Tree Star, OR). Human CD45 + T cells were defined as live, singlet, CD45 + In this group, CD4 + T cells and CD8 + T cells were further classified into CD3 + , CD4 + and CD3 + , CD8 + .

[0619] Table 7-3. Blood human immune cells on days 35 and 56 after huPBMC injection (mean ± SD, in cells / μL blood)

[0620]

[0621] Notice:Statistical significance determined by Kruskal-Wallis one-way ANOVA and Dunn's multiple comparison post hoc test is shown (*=p<0.05, **=p<0.01, ***=p<0.001 compared to Group 3: huPBMC-isotype control antibody). n=number of mice analyzed.

[0622] As an example, Figure 5 (AD) shows the absolute human cell counts in the blood on day 35 after huPBMC injection. Blood counts of human CD45+ cells, T cells, CD4+ T cells, and CD8+ T cells over time are shown in Figure 6 (AD) in.

[0623] Serum from mice was collected on different days after huPBMC injection, and serum levels of mouse and human cytokines were evaluated. In short, whole blood was collected in Microtainer tubes (BD, Cat#365967) and allowed to clot by leaving it undisturbed at room temperature for at least 30 minutes. The clotted blood and cells were pelleted by centrifugation at 15,000 x g for 10 minutes at 4 ° C. The resulting supernatant (called serum) was transferred to a clean plate, and two pro-inflammatory (mouse and human) multiple immunoassay kits (Meso Scale Discovery) were used to measure the cytokine concentration in serum according to the manufacturer's instructions. The plate was washed with PBS containing 0.05% (w / v) Tween-20 (Life Technologies). Electrochemiluminescence was read immediately on the MSD Spector instrument. Data analysis was performed using FlowJo X software (Tree Star, OR).

[0624] Table 7-4. Serum human cytokine concentrations on days 42 and 62 after huPBMC injection (mean ± SD, in pg / mL)

[0625]

[0626] Notice: Statistical significance determined by Kruskal-Wallis one-way ANOVA and Dunn's multiple comparison post hoc test is shown (*=p<0.05, **=p<0.01, ***=p<0.001 compared to Group 3: huPBMC-isotype control antibody). n=number of mice analyzed.

[0627] Table 7-5. Serum mouse cytokine concentrations on days 42 and 62 after huPBMC injection (mean ± SD, in pg / mL)

[0628]

[0629] Notice: Statistical significance determined by Kruskal-Wallis one-way ANOVA and Dunn's multiple comparison post hoc test is shown (*=p<0.05, **=p<0.01, ***=p<0.001 compared to Group 3: huPBMC-isotype control antibody). n=number of mice analyzed.

[0630] In addition, as an example, serum human and mouse cytokine levels on day 42 after huPBMC injection are shown in Figure 7 (AI). Figure 8 (AD) Serum levels of human IFN-γ, human TNFα, mouse TNFα, and mouse IL-6 over time are shown.

[0631] This in vivo study demonstrates the efficacy of anti-IL2Rγ antibodies H4H12889P and H4H12922P2 when administered therapeutically in a graft-versus-host disease model. Both H4H12889P and H4H12922P2 effectively blocked the development of GvHD in mice, whereas COMP1499 did not. Mice treated therapeutically with either of these two antibodies were protected from weight loss and death, and this was associated with a dramatic reduction in mouse and human serum cytokine levels and the number of human T cells in the blood. See Tables 7-3, 7-4, and 7-5.

[0632] Example 8 : Bioassays using NK92 / hIL7R / STAT3-Luc and Ramos.2G6.4C10 / STAT3-Luc cells

[0633] IL2Rγ cytokine family: IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 signal through the JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway (Rochman et al., New insights into the regulation of T cells by gamma (c) family cytokines. Nat Rev Immunol. 2009 Jul; 9 (7): 480-90). In order to evaluate the inhibition of cytokine signaling by anti-IL2Rγ antibodies, a bioassay was developed using NK-92 cells (human natural killer cell line, ATCC) stably expressing a luciferase reporter (STAT3-Luc; SABiosciences, #CLS-6028L). NK-92 endogenously expresses IL2Rγ and ligand-selective receptors that mediate IL-2, IL-9, IL-15 and IL-21 signaling. To also evaluate the regulation of IL-7 signaling, NK-92 cells were transduced with a lentivirus containing human IL-7R, and stably expressing cells were selected and maintained in G418. The resulting cell line is referred to as NK-92 / hIL7R / STAT3-Luc hereinafter. To test the regulation of IL-4-mediated signaling, Ramos.2G6.4C10 (human B lymphocyte line, ATCC) cells endogenously expressing IL2Rγ and IL-4R receptors were transduced with a STAT3-luc reporter, and the resulting cell line was referred to as Ramos.2G6.4C10 / STAT3-Luc.

[0634] The anti-IL2γ antibodies of the invention were tested for inhibition of human IL-2 (hIL-2), human IL-7 (hIL-7), human IL-9 (hIL-9), human IL-15 (hIL-15), or human IL-21 (hIL-21) signaling by plating 20,000 NK-92 / hIL7R / STAT3-Luc cells per well in a 96-well plate in growth medium (prepared according to ATCC instructions, but without IL-2) and incubating overnight at 37° C. in 5% CO 2. The next day, the anti-IL2Rγ antibody or isotype control was serially diluted from 500 to 0.008 nM in assay buffer (plus samples containing only buffer without the test molecule), added to the cells and incubated for 30 minutes. After incubation, ligands were added to the cells at the following final concentrations: 30 pM hIL-2, 50 pM hIL-7, 20 pM hIL-9, 60 pM or 100 pM hIL-15, or 5 pM or 3 pM hIL-21. Serial dilutions of ligand (from 10 nM to 0.2 pM) (plus samples containing only buffer without ligand) were added to the cells to determine dose-dependent activation. After incubation for 5 hours at 37°C in 5% CO2, the cells were incubated with OneGlo TM Reagents (Promega, #E6031) and Victor TM Luciferase activity was measured using a X multilabel plate reader (PerkinElmer).

[0635] To test the inhibition of human IL-4 (hIL-4) signaling by the anti-IL2γ antibodies of the present invention, Ramos.2G6.4C10 / STAT3-Luc cells were plated in growth medium (prepared according to ATCC instructions) in 96-well plates at a density of 100,000 cells per well. Anti-IL2Rγ antibodies or isotype controls were serially diluted from 500 to 0.008 nM in assay buffer (plus samples containing only buffer without the test molecule), added to the cells and incubated for 20 minutes. After incubation, hIL-4 was added to the cells at a final concentration of 250 pM or 200 pM. Dose-dependent activation was determined using serial dilutions of hIL-4 (from 10 nM to 0.2 pM) (plus samples containing only buffer without ligand) added to the cells. After overnight incubation at 37°C in 5% CO2, the cells were assayed using OneGlo TM Reagents (Promega, #E6031) and Victor TM Luciferase activity was measured using a X multilabel plate reader (Perkin Elmer).

[0636] The results were analyzed using Prism 5 software (GraphPad) using nonlinear regression (4-parameter logistic) to obtain EC 50and IC 50 The percentage of inhibition was calculated using the RLU values ​​using the following equation:

[0637]

[0638] In this equation, “RLU 基线 ” is the luminescence value from cells treated with a constant amount of ligand in the absence of antibody, “RLU 抑制 " is the minimum luminescence value from cells treated with a dose response of a specific antibody at a specific ligand concentration, "RLU 背景 ” is the luminescence value from cells not treated with any ligand or antibody.

[0639] Table 8-1. Inhibition of IL2Rγ signaling by 19 anti-IL2γ antibodies in bioassays using NK-92 / hIL7R / STAT3-Luc and Ramos.2G6.4C10 / STAT3-Luc cells

[0640]

[0641] NB: No blocking

[0642] WB: Weak blocking

[0643] Table 8-2. Inhibition of IL2Rγ signaling by four anti-IL2γ antibodies in bioassays using NK-92 / hIL7R / STAT3-Luc and Ramos.2G6.4C10 / STAT3-Luc cells

[0644]

[0645] The ability of 23 anti-IL2γ antibodies of the present invention to inhibit the signal transduction of IL2Rγ family cytokines was tested using a bioassay. As shown in Table 8-1, 19 of the 23 anti-IL2γ antibodies inhibited the activation of IL2Rγ to varying degrees, and as shown in Table 8-2, four of the 23 anti-IL2γ antibodies showed no inhibition of IL2Rγ activation of the ligand.

[0646] Example 9 : Cell binding analysis of NK-92, Jurkat, NIH / 3T3, MC / 9, and HEK293 cells by flow cytometry

[0647] To evaluate the binding of anti-IL2Rγ antibodies to human and mouse IL-2Rγ expressed on cells, flow cytometry analysis was performed on cell lines that endogenously express IL-2Rγ: NK-92 (human natural killer cell line), Jurkat (human T lymphocyte line), and MC / 9 (mouse mast cell line). NIH / 3T3 (mouse fibroblasts) and HEK293 (human embryonic kidney) cell lines were included as negative controls.

[0648] For flow cytometry analysis, cells were pre-incubated with mouse IgG at 100 μg / ml for 15 minutes at room temperature (RT) to block antibody binding to Fc receptors. Anti-IL2Rγ antibodies of the invention and isotype control antibodies were used at 10 μg / ml with 0.5 to 1 x 10 cells per cell type. 6 Cells / well were incubated in PBS (without calcium and magnesium) containing 1% FBS (for Jurkat, NIH / 3T3, and HEK293 cells) or in growth medium (prepared according to ATCC instructions) (for NK-92 and MC / 9) for 30 to 45 minutes at room temperature. Cells were washed and incubated with anti-human antibody conjugated to allophycocyanin (APC) (Jackson ImmunoResearch, #109-136-170) on ice for 30 minutes. Cells were washed and fixed with BD CytoFix TM (BD biosciences, #554655) and fixed in Analyze on a flow cytometer or Accuri flow cytometer (BD). For all cell lines, unstained and secondary antibody alone controls were also included. The results were analyzed by the software to determine the geometric mean (MFI) of the fluorescence of the viable cells. The binding ratio was calculated by normalizing the MFI of the tested samples with the MFI of the unstained samples.

[0649] As shown in Table 9-1, 19 of the 23 anti-IL2Rγ antibodies of the present invention tested at 10 μg / ml showed binding to Jurkat and NK-92 cells, with binding ratios of 1-19 and 1-94, respectively. Anti-IL2Rγ antibodies showed binding to NIH / 3T3 and MC / 9 cells, with binding ratios of 1-13 and 1, respectively. Human isotype control antibody REGN1945 and secondary antibody-only control conditions showed binding ratios of 1-13 for all tested cell lines.

[0650] As shown in Table 9-2, 4 of the 23 anti-IL2Rγ antibodies of the present invention tested at 10 μg / ml showed binding to NK-92 cells with a binding ratio of 1 to 37, and binding to HEK293 cells with a binding ratio of 1 to 3. The human isotype control antibody REGN1945 and the secondary antibody-only control condition showed a binding ratio of 1 to 2 for NK-92 and HEK293 cells.

[0651] Table 9-1. Flow cytometric analysis of binding of 19 of the 23 anti-IL2Rγ antibodies to NIH / 3T3, MC / 9, Jurkat, and NK-92 cells

[0652]

[0653] Table 9-2. Flow cytometric analysis of binding of four of the 23 anti-IL2Rγ antibodies to HEK293 and NK-92 cells

[0654]

[0655] Example 10 : In vivo immunosuppression experiments to evaluate the effects of anti-IL2Rγ antibody H4H12889P on immune cell populations in the blood

[0656] Experimental procedures. Propagation of colonies genetically modified to replace the endogenous IL2RG extracellular domain with the corresponding human sequence (VG) background mice (C57BL / 6NTac(75%) / 129S6SvEvTac(25%)) were administered isotype control (REGN1945) or H4H12889P subcutaneously at a dose of 10 mg / kg or 25 mg / kg twice per week for 3 weeks (6 doses in total), or not administered.

[0657] Table 10-1. Experimental drug administration and treatment scheme for each group of mice

[0658] Group Recipient strain n mAb treatment A <![CDATA[II2rg hu / hu ]]> 8 No rnAb B <![CDATA[II2rg hu / hu ]]> 8 REGN1945 (isoform), 10 mg / kg C <![CDATA[II2rg hu / hu ]]> 8 REGN1945 (isoform), 25 mg / kg D <![CDATA[II2rg hu / hu ]]> 8 H4H12889P (anti-hIL2RG), 10 mg / kg E <![CDATA[II2rg hu / hu ]]> 8 H4H12889P (anti-hIL2RG), 25 mg / kg

[0659] Analysis of immune cell populations in the blood over a period of time by flow cytometry. Total immune cell, B cell, T cell, NK cell and neutrophil counts in peripheral blood at different time points (once a week) were analyzed by flow cytometry to assess the effect of H4H12889P on the absolute number of these cell types. In brief, at each time point, blood samples from mice were collected into Microtainer tubes with K2EDTA [BD#365974], and 30-75 μL of each blood sample was incubated in red blood cell lysis buffer [Sigma#R7757] at room temperature for 5 minutes to lyse red blood cells. If necessary, a second round of lysis was performed. The cells were then washed in DPBS [Gibco#14190-144] and washed with LIVE / DEAD diluted 1:500 in DPBS. TM Fixable near-infrared dead cell stain [Invitrogen #L34962] was stained for 20 minutes, washed again in DPBS, and then blocked with purified anti-mouse CD16 / CD32 (Fc shield) [Tonbo Biosciences, #70-0161-M001] diluted 1:50 in MACS buffer [autoMACS running buffer; Miltenyi Biotec, #130-091-221]. Subsequently, cells were stained for cell surface markers to identify CD45+ cells, T cells, B cells, NK cells, and neutrophils by adding a cocktail of fluorescently labeled antibodies (described in Table 2) diluted in BD horizon brilliant staining buffer [BD #566349]. Finally, samples were washed in MACS buffer, fixed in BD CytoFix [BD #554655] diluted 1:4 in DPBS, and then washed and resuspended in MACS buffer before collection. Sample data were acquired on a FACSymphony A5 analyzer using the HTS accessory [BD]. A fixed volume of each sample was run. Data analysis was performed using FlowJov10 software [Tree Star, OR]. CD45 + Immune cells are limited to singlet, live cells, CD45 + ; Within this population, T cells were further defined as CD3 + , B cells were further defined as CD3 - CD19 + NK cells are further defined as CD3 - CD19 - NKp46 + , neutrophils were further defined as F4 / 80 - 6G +The absolute number of each cell type run through the analyzer, the sample volume run, and the volume of blood initially stained were used to calculate a cells / μL blood count for each sample.

[0660] Table 10-2. Antibodies used in flow cytometry analysis

[0661] Antibody Fluorescent dyes Manufacturer Final dilution NKp46 FITC eBioscience 1∶200 6G BB700 BD 1∶100 F4 / 80 PE BD 1∶500 CD3 PE-Cy7 BD 1∶200 CD4 BV786 BD 1∶200 CD8a BUV395 BD 1∶200 CD19 BUV737 BD 1∶200 CD45 Alexa Fluor 700 BioLegend 1∶200

[0662] Analysis of serum therapeutic antibody levels over time by antigen capture ELISA. Serum levels of IL2Rγ antibody or isotype control antibody were measured weekly by human total IgG platinum ELISA kit. Each antibody was serially diluted in 0.5% BSA in PBS to generate standard curves from 1.56 to 100 ng / mL for H4H12889P and REGN1945. Absorbance at 450 nm was measured on a SpectraMax M5 plate reader [Molecular Devices]. Data analysis was performed using Prism 8.1.2 [GraphPad].

[0663] Results Overview and Conclusions. Treatment with H4H12889P (10 mg / kg and 25 mg / kg) resulted in a significant decrease in the number of total CD45+ immune cells (Figure 9(A)), NK cells (Figure 9(B)), T cells (Figure 9(C)), and B cells (Figure 9(D)) in the blood, while neutrophil counts (Figure 9(E)) were not affected. After the 3-week dosing period, serum concentrations of H4H12889P decreased over time. This decrease in H4H12889P concentrations was associated with a sustained increase in the number of total CD45+ immune cells (Figure 9(A)), NK cells (Figure 9(B)), T cells (Figure 9(C)), and B cells (Figure 9(D)). By the end of the study, all of these groups had returned to similar levels observed before treatment, and to levels observed in mice that were untreated or treated with REGN1945 (isotype control).

[0664] Embodiment 11 : An in vivo skin allograft rejection model for evaluating the blocking activity of the IL2Rγ antibody H4H12889P

[0665] Experimental Procedures BALB / cJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and used as skin graft donors. MHC-mismatched mice were obtained from Regeneron. Propagation of colonies genetically modified to replace the endogenous IL2RG extracellular domain with the corresponding human sequence (VG) background mice (C57BL / 6NTac (75%) / 129S6SvEvTac (25%)) were used as recipients. Skin grafts were taken from the tail of donor mice. The skin was peeled off with forceps and punched with a biopsy punch with a diameter of 10 mm. VG mice (humanized for IL2Rγ) used as graft recipients were subcutaneously administered with an isotype control (REGN1945) or H4H12889P at a dose of 25 mg / kg, twice a week, starting 3 weeks before transplantation and continuing until rejection, or no administration. The recipients with shaved surgical sites were anesthetized with isoflurane through a nose cone and an analgesic (buprenorphine sustained release formulation) (ZooPharm) was administered. The shaved dorsal area was wiped by applying povidone iodine and alcohol. The graft bed was created laterally in the middle between the dorsal and ventral sides of the mouse by grasping the skin with forceps and then performing skin excision using a sterile 10 mm diameter biopsy skin punch. The graft was then placed on the graft bed and covered with an adhesive bandage that was secured to the skin with two sterile surgical staples. Aseptic technique was used throughout the process. After 5 days, the bandage and staples were removed and monitoring continued.

[0666] Table 11-1. Experimental drug administration and treatment scheme for each group of mice

[0667] Group Recipient strain n Donor strain Donor tissue mAb treatment A <![CDATA[II2rg hu / hu ]]> 10 BALB / cJ Tail Skin No mAb B <![CDATA[II2rg hu / hu ]]> 10 BALB / cJ Tail Skin REGN1945 (isotype) C <![CDATA[II2rg hu / hu ]]> 10 BALB / cJ Tail Skin H4H12889P (anti-hIL2Rγ)

[0668] The experimental design is shown in Fig.10 middle.

[0669] Monitoring of skin graft rejection. Monitoring of skin grafts included the following criteria: (1) Skin grafts that failed to vascularize properly were considered technical failures and were excluded from analysis. These grafts exhibited scabbing and shrinkage several hours after bandage removal. (2) Late “scabbing” and shrinkage of the graft were used as indicators of graft rejection. The time point for complete rejection was recorded as the first day (day 1) when 100% of the graft tissue was necrotic. Fig.12 The rejection episode was recorded as the first day of rejection signs (ie, redness) ( Fig.11 ). Significance was determined by log-rank test (Mantel-Cox) with Bonferroni correction (adjusted p value 0.005, K=9).

[0670] Donor-specific antibody detection by flow cytometry. Blood was sampled at the 56th day after transplantation to assess the formation of donor-specific antibodies ( Fig.13 ).

[0671] CT26.WT Cells were cultured to 80% confluence in tissue culture bottles. The cells were washed with 1X DPBS and dissociated with TrypLE Express reagent (Gibco) by incubating at room temperature for 5 minutes and washing the flask with complete RPMI1640 culture medium. The cells were then centrifuged (500 g, 10 minutes) and resuspended at room temperature for 15 minutes with 1X DPBS with 4 μg / ml Fc blocking agent (Tonbo) diluted 1:50 at 5 million cells / ml. The suspension was plated in 384-well V-bottom plates with 250,000 cells / well (50 μL).

[0672] 50 μL of serially diluted sample sera from transplanted mice and non-transplanted wild-type VG mice (C57BL / 6NTac (75%) / 129S6SvEvTac (25%)) and wild-type BALB / cJ mice obtained from The Jackson Laboratory were added to their respective wells and incubated at 37°C for 45 minutes. After washing twice with MACS buffer (500 g, 4 minutes), the cells were resuspended in 50 μL of LIVE / DEAD diluted 1:500 in 1X DPBS. TM Fixable blue dead cell staining kit (Invitrogen), total volume per well 50 μL, and incubate at room temperature for 15 minutes. After centrifugation at 500g for 4 minutes, discard the supernatant, resuspend the cells in 25 μL Fc blocking agent (Tonbo), and incubate at 4°C for 15 minutes. Then add 25 μL 2X antibody mixture (Table 11-2) and incubate at 4°C for 25 minutes. After centrifugation (500g, 4 minutes) by adding 100 μL MACS TM Wash the cells in MACS buffer. Resuspend the cells in 100 μL of Cytofix diluted 1:4 in 1X DPBS. TM Fixation buffer (BD) was used to fix the cells and incubated at 4°C for 15 minutes. Then, after centrifugation and discarding the fixative, the samples were resuspended in MACS buffer. Cells were acquired on a BD Fortessa X-20. The acquired events were analyzed with FlowJo (BD). MFI was derived from cells that were double-differentiated (FSC-H, FSC-A) and then Live / Dead dye negative. The results plotted are median fluorescence intensity values ​​at a 1 / 512 dilution of sample serum.

[0673] Table 11-2. Antibodies used in flow cytometry staining mixture

[0674] antigen Conjugate clone supplier Dilution (1 / ) CD45 BV421 30-F11 BioLegend 200 IgG APC Poly4053 BioLegend 200 B220 BUV395 RA3-6B2 BD 200 IgG1 PE-Cy7 RMG1-1 BioLegend 200 IgM APC-Cy7 RMM-1 BioLegend 200 IgG2a FITC R19-15 BD 200 IgG2c FITC Goat polyclonal IgG Bio-Rad 200

[0675] Summary of Results and Conclusions. In a skin transplantation model (BALB / cJ to VG mice), H4H12889P (anti-IL2RγAb) treatment delayed the onset of skin graft rejection and improved overall survival of skin grafts. H4H12889P treatment also prevented the generation of donor-specific antibodies in this transplantation model.

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to interleukin-2 receptor γ (IL2Rγ), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region comprises three heavy chain complementary determining regions CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region comprises three light chain complementary determining regions CDR-L1, CDR-L2 and CDR-L3, wherein CDR-H1 consists of the amino acid sequence of SEQ ID NO: 347, CDR-H2 consists of the amino acid sequence of SEQ ID NO: 349, CDR-H3 consists of the amino acid sequence of SEQ ID NO: 351, CDR-L1 consists of the amino acid sequence of SEQ ID NO: 72, CDR-L2 consists of the amino acid sequence of SEQ ID NO: 54, and CDR-L3 consists of the amino acid sequence of SEQ ID NO:

355.

2. The antibody or antigen-binding fragment thereof according to claim 1, which is an antibody.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody is a human antibody. The antibody or antigen-binding fragment thereof according to claim 1 , which is an antigen-binding fragment of an antibody.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 345 and / or the LCVR comprises the amino acid sequence of SEQ ID NO:

353.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 345 and the LCVR comprises the amino acid sequence of SEQ ID NO:

353.

7. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 357 and / or a light chain comprising the amino acid sequence of SEQ ID NO:

359.

8. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 357, and the light chain comprises the amino acid sequence of SEQ ID NO:

359.

9. The antibody or antigen-binding fragment thereof of claim 1, wherein the HCVR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 345; and / or the LCVR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

353.

10. The antibody or antigen-binding fragment thereof of claim 1, comprising: a heavy chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 357; and / or a light chain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

359.

11. The antibody or antigen-binding fragment thereof of claim 1, which is multispecific. 12 . A complex comprising the antibody or antigen-binding fragment thereof according to claim 1 , bound to an IL2Rγ polypeptide or an antigen-binding fragment thereof.

13. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising: (a) introducing one or more polynucleotides encoding the HCVR and LCVR of the antibody or antigen-binding fragment thereof into a host cell; (b) culturing the host cell under conditions conducive to expression of the polynucleotide; and (c) isolating the antibody or antigen-binding fragment thereof from the host cell and / or the medium in which the host cell is cultured.

14. The method of claim 13, wherein the host cell is a Chinese hamster ovary cell.

15. An antibody or antigen-binding fragment thereof which is the product of the method of any one of claims 13 to 14.

16. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

17. A vector comprising the polynucleotide of claim 16.

18. A host cell comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the polynucleotide according to claim 16, or the vector according to claim 17.

19. A composition or kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and 15.

20. The composition or kit of claim 19, further comprising an additional therapeutic agent.

21. A pharmaceutical preparation comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and 15 and a pharmaceutically acceptable carrier or excipient.

22. The pharmaceutical formulation of claim 21 further comprising an additional therapeutic agent.

23. The composition or kit of claim 20 or formulation of claim 22, wherein the additional therapeutic agent is an anti-inflammatory agent.

24. The composition or kit of claim 20 or the formulation of claim 22, wherein the additional therapeutic agent is one or more members selected from the group consisting of infliximab, adalimumab, etanercept, golimumab, prednisolone, methylprednisolone, antithymocyte globulin, alemtuzumab, daclizumab, tacrolimus, cyclosporine, mycophenolate mofetil, sirolimus, pentostatin, mesenchymal stem cells, inomucomab, denileukin, and basiliximab.

25. The composition or kit of claim 20 or formulation of claim 22, wherein the additional therapeutic agent is one or more members selected from the group consisting of an anti-TNFα antibody or binding protein and a cortical hormone.

26. A container or injection device comprising the antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 11 and 15, or the composition as defined in any one of claims 19, 20 and 23-25, or the formulation as defined in any one of claims 21 to 25.

27. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 and 15 in the preparation of a medicament for treating or preventing an IL2Rγ-mediated disease or condition in a subject in need thereof, wherein the IL2Rγ-mediated disease or condition is graft-versus-host disease or an autoimmune disease.

28. The use of claim 27, wherein the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus or type I diabetes.

29. The use of claim 27, wherein the antibody or antigen-binding fragment thereof is for administration to the subject by subcutaneous, intravenous or intramuscular injection.

30. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 and 15 in the preparation of a medicament for treating or preventing an IL2Rγ-mediated disease or condition in a subject in need thereof, wherein the IL2Rγ-mediated disease or condition is b-islet cell transplant rejection, skin transplant rejection, heart transplant rejection, lung transplant rejection, kidney transplant rejection or liver transplant rejection.

31. The use of claim 30, wherein the antibody or antigen-binding fragment thereof is for administration by subcutaneous, intravenous or intramuscular injection into the subject.

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