Anti-IL-2 antibodies, their compositions and uses

Abstract

This invention provides antibodies or antigen-binding moieties thereof that specifically bind to IL-2 and reduce the affinity of IL-2 for binding to IL-2Rα and IL-2Rβ. This invention also provides methods for obtaining such antibodies and nucleic acids encoding said antibodies. This invention further relates to compositions and treatments using these antibodies for the treatment and / or prevention of autoimmune diseases, conditions, or states, as well as for immunosuppression, including but not limited to the administration of a complex comprising an antibody and IL-2.

Description

[0001] Contracting parties to the joint research agreement

[0002] This invention was made by or on behalf of the parties to the joint research agreements listed below. These joint research agreements entered into force on or before the date of completion of this invention, and the invention was completed as a result of activities undertaken within the scope of these joint research agreements. The parties to these joint research agreements are PFIZER INC. and THE REGENTS OF THE UNIVERSITY OF CALIFORNIA.

[0003] Related applications

[0004] This patent application is a divisional application of Chinese Patent Application No. 201680068098.1 and claims the benefit of U.S. Provisional Application No. 62 / 408,360, filed October 14, 2016; and U.S. Provisional Application No. 62 / 245,600, filed October 23, 2015, the contents of which are incorporated herein by reference in their entirety.

[0005] sequence list

[0006] This patent application includes a sequence list, which has been electronically submitted as a text file in ASCII format and is incorporated herein by reference in its entirety. The text file was created on October 21, 2016, named PCFC-993-WO1-SL.txt, and has a size of 106,549 bytes. Technical Field

[0007] This disclosure relates to antibodies, such as full-length antibodies that specifically bind to interleukin-2 (IL-2) and their antigen-binding moieties. This disclosure also relates to compositions comprising antibodies against IL-2, and methods of using said antibodies as pharmaceutical agents. Anti-IL-2 antibodies can be used to treat and prevent autoimmune diseases, symptoms, and conditions, as well as for immunosuppression. Background Technology

[0008] Interleukin-2 (IL-2) plays a crucial role in the immune response and is a potential target for treating diseases, conditions, and illnesses associated with the immune response, such as autoimmune diseases, symptoms, and conditions, as well as for immunosuppression. There is a long-standing unmet need for novel therapies to treat IL-2-mediated diseases, symptoms, and conditions. This disclosure addresses these needs. Summary of the Invention

[0009] The inventors have developed novel and advantageous anti-IL-2 antibodies. In some aspects, this disclosure relates to isolated antibodies or antigen-binding moieties thereof that specifically bind to human IL-2 (hIL-2), wherein said antibody reduces the binding of hIL-2 to the IL-2 receptor chains IL-2Rα and IL-2Rβ, and inhibits CD8. + The activity of T cells is even higher than that of regulatory T (Treg) cells.

[0010] In some aspects, this disclosure relates to isolated antibodies or antigen-binding portions thereof that specifically bind to human IL-2 (hIL-2), wherein said antibody binds to helical A and C and BC loops of hIL-2.

[0011] In some aspects, this disclosure relates to isolated antibodies or antigen-binding portions thereof that compete with antibodies containing the amino acid sequences of SEQ ID NO: 13 and 14 for binding to human IL-2 (hIL-2), or bind to the same hIL-2 epitopes with antibodies containing the amino acid sequences of SEQ ID NO: 13 and 14.

[0012] In some aspects, this disclosure relates to isolated antibodies or antigen-binding moieties thereof that specifically bind to human IL-2 (hIL-2), wherein said antibody reduces the binding affinity of hIL-2 to IL-2Rα by 1 to 199-fold. In some embodiments, said antibody reduces the binding affinity of hIL-2 to IL-2Rα by 10-fold.

[0013] In some aspects, this disclosure relates to isolated antibodies or antigen-binding portions thereof that specifically bind to hIL-2, wherein said antibody or portion reduces the binding of hIL-2 to IL-2Rα and IL-2Rβ, and inhibits CD8. + STAT5 is phosphorylated to a higher degree in T cells than in Treg cells.

[0014] In some aspects, this disclosure relates to isolated antibodies or antigen-binding portions thereof that specifically bind to hIL-2, wherein said antibody or portion reduces the binding of hIL-2 to IL-2Rα and IL-2Rβ, and increases the binding of Treg cells to CD8+ in vivo, as measured in peripheral blood mononuclear cell (PBMC) cultures or remodeling assays. + or CD4 + The ratio of T cells to NK cells.

[0015] In some aspects, this disclosure relates to isolated antibodies or antigen-binding portions thereof that specifically bind to hIL-2, wherein said antibody or portion reduces the binding of hIL-2 to IL-2Rα and IL-2Rβ and increases the expression of one or more of FOXP3, CD25 and Icos in Treg cells.

[0016] In some aspects, this disclosure relates to isolated antibody or antigen-binding moieties, wherein said antibody or moieties have a) equal to or greater than about 4.53 x 10⁻⁶. -4 s -1 a) hIL-2 binding dissociation rate; and / or b) equal to or greater than about 1.14 x 10⁻⁶. -10 The binding affinity of M to hIL-2.

[0017] In some aspects, this disclosure relates to isolated antibody or antigen-binding portions, wherein said antibody or portion comprises:

[0018] (a) HCDR1 containing SEQ ID NO:73 (Kabat), 74 (Chothia), or 75 (extended); HCDR2 containing SEQ ID NO:76 (Kabat) or 77 (Chothia); HCDR3 containing SEQ ID NO:78; LCDR1 containing SEQ ID NO:79; LCDR2 containing SEQ ID NO:80; and LCDR3 containing SEQ ID NO:81;

[0019] (b) HCDR1 containing SEQ ID NO:82 (Kabat), 83 (Chothia), or 84 (extended); HCDR2 containing SEQ ID NO:85 (Kabat) or 86 (Chothia); HCDR3 containing SEQ ID NO:87; LCDR1 containing SEQ ID NO:88; LCDR2 containing SEQ ID NO:89; and LCDR3 containing SEQ ID NO:90;

[0020] (c) HCDR1 containing SEQ ID NO:91 (Kabat), 92 (Chothia), or 93 (extended); HCDR2 containing SEQ ID NO:94 (Kabat) or 95 (Chothia); HCDR3 containing SEQ ID NO:96; LCDR1 containing SEQ ID NO:97; LCDR2 containing SEQ ID NO:98; and LCDR3 containing SEQ ID NO:99;

[0021] (d) HCDR1 containing SEQ ID NO: 100 (Kabat), 101 (Chothia), or 102 (extended); HCDR2 containing SEQ ID NO: 103 (Kabat) or 104 (Chothia); HCDR3 containing SEQ ID NO: 105; LCDR1 containing SEQ ID NO: 106; LCDR2 containing SEQ ID NO: 107; and LCDR3 containing SEQ ID NO: 108;

[0022] (e) HCDR1 containing SEQ ID NO: 109 (Kabat), 110 (Chothia), or 111 (extended); HCDR2 containing SEQ ID NO: 112 (Kabat) or 113 (Chothia); HCDR3 containing SEQ ID NO: 114; LCDR1 containing SEQ ID NO: 115; LCDR2 containing SEQ ID NO: 116; and LCDR3 containing SEQ ID NO: 117;

[0023] (f) HCDR1 containing SEQ ID NO: 118 (Kabat), 119 (Chothia), or 120 (extended); HCDR2 containing SEQ ID NO: 121 (Kabat) or 122 (Chothia); HCDR3 containing SEQ ID NO: 123; LCDR1 containing SEQ ID NO: 124; LCDR2 containing SEQ ID NO: 125; and LCDR3 containing SEQ ID NO: 126;

[0024] (g) HCDR1 containing SEQ ID NO:127 (Kabat), 128 (Chothia), or 129 (extended); HCDR2 containing SEQ ID NO:130 (Kabat) or 131 (Chothia); HCDR3 containing SEQ ID NO:132; LCDR1 containing SEQ ID NO:133; LCDR2 containing SEQ ID NO:134; and LCDR3 containing SEQ ID NO:135;

[0025] (h) HCDR1 containing SEQ ID NO:136 (Kabat), 137 (Chothia), or 138 (extended); HCDR2 containing SEQ ID NO:139 (Kabat) or 140 (Chothia); HCDR3 containing SEQ ID NO:141; LCDR1 containing SEQ ID NO:142; LCDR2 containing SEQ ID NO:143; and LCDR3 containing SEQ ID NO:144;

[0026] (i) HCDR1 containing SEQ ID NO:145 (Kabat), 146 (Chothia), or 147 (extended); HCDR2 containing SEQ ID NO:148 (Kabat) or 149 (Chothia); HCDR3 containing SEQ ID NO:150; LCDR1 containing SEQ ID NO:151; LCDR2 containing SEQ ID NO:152; and LCDR3 containing SEQ ID NO:153;

[0027] (j) HCDR1 containing SEQ ID NO:154 (Kabat), 155 (Chothia), or 156 (extended); HCDR2 containing SEQ ID NO:157 (Kabat) or 158 (Chothia); HCDR3 containing SEQ ID NO:159; LCDR1 containing SEQ ID NO:160; LCDR2 containing SEQ ID NO:161; and LCDR3 containing SEQ ID NO:162;

[0028] (k) HCDR1 containing SEQ ID NO:163 (Kabat), 164 (Chothia), or 165 (extended); HCDR2 containing SEQ ID NO:166 (Kabat) or 167 (Chothia); HCDR3 containing SEQ ID NO:168; LCDR1 containing SEQ ID NO:169; LCDR2 containing SEQ ID NO:170; and LCDR3 containing SEQ ID NO:171;

[0029] (l) HCDR1 containing SEQ ID NO:172 (Kabat), 173 (Chothia) or 174 (extended); HCDR2 containing SEQ ID NO:175 (Kabat) or 176 (Chothia); HCDR3 containing SEQ ID NO:177; LCDR1 containing SEQ ID NO:178; LCDR2 containing SEQ ID NO:179; and LCDR3 containing SEQ ID NO:180;

[0030] (m) HCDR1 containing SEQ ID NO:181 (Kabat), 182 (Chothia), or 183 (extended); HCDR2 containing SEQ ID NO:184 (Kabat) or 185 (Chothia); HCDR3 containing SEQ ID NO:186; LCDR1 containing SEQ ID NO:187; LCDR2 containing SEQ ID NO:188; and LCDR3 containing SEQ ID NO:189;

[0031] (n) HCDR1 containing SEQ ID NO:190 (Kabat), 191 (Chothia), or 192 (extended); HCDR2 containing SEQ ID NO:193 (Kabat) or 194 (Chothia); HCDR3 containing SEQ ID NO:195; LCDR1 containing SEQ ID NO:196; LCDR2 containing SEQ ID NO:197; and LCDR3 containing SEQ ID NO:198;

[0032] (o) HCDR1 containing SEQ ID NO:199 (Kabat), 200 (Chothia), or 201 (extended); HCDR2 containing SEQ ID NO:202 (Kabat) or 203 (Chothia); HCDR3 containing SEQ ID NO:204; LCDR1 containing SEQ ID NO:205; LCDR2 containing SEQ ID NO:206; and LCDR3 containing SEQ ID NO:207;

[0033] (p) HCDR1 containing SEQ ID NO:208 (Kabat), 209 (Chothia), or 210 (extended); HCDR2 containing SEQ ID NO:211 (Kabat) or 212 (Chothia); HCDR3 containing SEQ ID NO:213; LCDR1 containing SEQ ID NO:214; LCDR2 containing SEQ ID NO:215; and LCDR3 containing SEQ ID NO:216; or

[0034] (q) HCDR1 containing SEQ ID NO:217; HCDR2 containing SEQ ID NO:218; HCDR3 containing SEQ ID NO:219; LCDR1 containing SEQ ID NO:220; LCDR2 containing SEQ ID NO:221; and LCDR3 containing SEQ ID NO:222.

[0035] In some embodiments, the antibody or antigen-binding portion includes a heavy chain variable domain (V). H The heavy chain variable structural domain (V) H It includes the following: a) the heavy chain complementarity determination region (HCDR) 3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 71 shown in Table 7; or b) HCDR1-3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 71 shown in Table 7.

[0036] In some embodiments, the antibody or antigen-binding portion of this disclosure includes a light chain variable domain (V). L The light chain variable structural domain (V) L It includes the following: a) the light chain complementarity determination region (LCDR) 3 in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 72 shown in Table 7; or b) LCDR1-3 in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 72 shown in Table 7.

[0037] In some of the above embodiments, the antibody or antigen binding portion comprises: a) HCDR1-3 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 71 shown in Table 7; and / or b) LCDR1-3 of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 72 shown in Table 7.

[0038] In some aspects, this disclosure relates to isolated antibodies or antigen-binding moieties thereof that specifically bind to human interleukin-2 (hIL-2), said isolated antibody or antigen-binding moieties comprising a heavy chain variable domain (V... H The heavy chain variable structural domain (V) H It contains: a) HCDR3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 71 shown in Table 7; or b) HCDR1-3 in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 71 shown in Table 7; or c) the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 71.

[0039] In some aspects, this disclosure relates to isolated antibodies or antigen-binding moieties thereof that specifically bind to hIL-2, said isolated antibody or antigen-binding moieties comprising a light chain variable domain (V... L The light chain variable structural domain (V) L It contains: a) LCDR3 of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 72 shown in Table 7; or b) LCDR1-3 of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 72 shown in Table 7; or c) amino acid sequences of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 72.

[0040] In some embodiments, this disclosure relates to isolated antibody or antigen-binding moieties, whose V HContains the amino acid sequence of a) HCDR3 as shown in Table 7, SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 71; or b) HCDR1-3 as shown in Table 7, SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 71; or c) the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 71; and its V L Contains an amino acid sequence of a) LCDR3 as shown in Table 7, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 72; or b) LCDR1-3 as shown in Table 7, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 72; or c) an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 72.

[0041] In some aspects, this disclosure relates to isolated antibodies or antigen-binding moieties thereof that specifically bind to hIL-2, said antibody comprising the HCDR1-3 and LCDR1-3 amino acid sequences respectively shown in Table 7:

[0042] SEQ ID NO:1 and 2,

[0043] SEQ ID NO:3 and 4,

[0044] SEQ ID NO:5 and 6,

[0045] SEQ ID NO:7 and 8,

[0046] SEQ ID NO:9 and 10,

[0047] SEQ ID NO:11 and 12,

[0048] SEQ ID NO:13 and 14,

[0049] SEQ ID NO:15 and 16,

[0050] SEQ ID NO:17 and 18,

[0051] SEQ ID NO:19 and 20,

[0052] SEQ ID NO:21 and 22,

[0053] SEQ ID NO:23 and 24,

[0054] SEQ ID NO:25 and 26,

[0055] SEQ ID NO:27 and 28,

[0056] SEQ ID NO:29 and 30,

[0057] SEQ ID NO:31 and 32 or

[0058] SEQ ID NO:71 and 72.

[0059] In some respects, this disclosure relates to isolated antibodies or their antigen-binding moieties, whose V H and V L Each contains the following amino acid sequences:

[0060] SEQ ID NO:1 and 2,

[0061] SEQ ID NO:3 and 4,

[0062] SEQ ID NO:5 and 6,

[0063] SEQ ID NO:7 and 8,

[0064] SEQ ID NO:9 and 10,

[0065] SEQ ID NO:11 and 12,

[0066] SEQ ID NO:13 and 14,

[0067] SEQ ID NO:15 and 16,

[0068] SEQ ID NO:17 and 18,

[0069] SEQ ID NO:19 and 20,

[0070] SEQ ID NO:21 and 22,

[0071] SEQ ID NO:23 and 24,

[0072] SEQ ID NO:25 and 26,

[0073] SEQ ID NO:27 and 28,

[0074] SEQ ID NO:29 and 30,

[0075] SEQ ID NO:31 and 32 or

[0076] SEQ ID NO:71 and 72.

[0077] In some embodiments of this disclosure, the isolated antibody is IgG (e.g., IgG1, IgG2, IgG3, or IgG4). The antibody may comprise a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 33, and / or a light chain constant region containing the amino acid sequence of SEQ ID NO: 34 or 35. In some of these embodiments, the C-terminal lysine residue of the heavy chain is absent.

[0078] In some respects, this disclosure relates to isolated antibodies or antigen-binding portions thereof that specifically bind to hIL-2, wherein said antibody binds to the same epitope as any of the aforementioned antibodies, or competes with any of the aforementioned antibodies for binding to IL-2Rα and IL-2Rβ.

[0079] In some respects, this disclosure relates to an antibody or antigen-binding portion, whose V H and V L The amino acid sequences are at least 90% (e.g., 95%, 98%, or 99%) identical to the following amino acid sequences:

[0080] SEQ ID NO:1 and 2,

[0081] SEQ ID NO:3 and 4,

[0082] SEQ ID NO:5 and 6,

[0083] SEQ ID NO:7 and 8,

[0084] SEQ ID NO:9 and 10,

[0085] SEQ ID NO:11 and 12,

[0086] SEQ ID NO:13 and 14,

[0087] SEQ ID NO:15 and 16,

[0088] SEQ ID NO:17 and 18,

[0089] SEQ ID NO:19 and 20,

[0090] SEQ ID NO:21 and 22,

[0091] SEQ ID NO:23 and 24,

[0092] SEQ ID NO:25 and 26,

[0093] SEQ ID NO:27 and 28,

[0094] SEQ ID NO:29 and 30 or

[0095] SEQ ID NO:31 and 32.

[0096] In some of the above embodiments of this disclosure, the antibody is a human antibody.

[0097] This disclosure also provides isolated nucleic acids of the heavy chain, light chain, or both, encoding the antibody or antigen-binding moiety of this disclosure. In some aspects, the isolated nucleic acid comprises: a) a nucleotide sequence of SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, or 66; b) a nucleotide sequence of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, or 67; or c) both a) and b). For example, the isolated nucleic acid may comprise the following nucleotide sequences:

[0098] SEQ ID NO:36 and 37,

[0099] SEQ ID NO:38 and 39,

[0100] SEQ ID NO:40 and 41,

[0101] SEQ ID NO:42 and 43,

[0102] SEQ ID NO:44 and 45,

[0103] SEQ ID NO:46 and 47,

[0104] SEQ ID NO:48 and 49,

[0105] SEQ ID NO:50 and 51,

[0106] SEQ ID NO:52 and 53,

[0107] SEQ ID NO:54 and 55,

[0108] SEQ ID NO:56 and 57,

[0109] SEQ ID NO:58 and 59,

[0110] SEQ ID NO:60 and 61,

[0111] SEQ ID NO:62 and 63,

[0112] SEQ ID NO:64 and 65 or

[0113] SEQ ID NO:66 and 67.

[0114] In some aspects, this disclosure relates to vectors comprising one or more of the isolated nucleic acids described above. In other aspects, this disclosure provides host cells (e.g., mammalian cells, such as NSO cells and CHO cells) comprising isolated nucleic acids or vectors, said isolated nucleic acids or vectors encoding a heavy chain, light chain, or both of the antibody or antigen-binding moiety of this disclosure. This disclosure also provides a method for producing an antibody or antigen-binding moiety specifically binding to hIL-2, said method comprising: a) culturing host cells under conditions allowing expression of said antibody or antigen-binding moiety, said host cells comprising nucleotide sequences encoding heavy and light chains of said antibody or antigen-binding moiety; and b) isolating said antibody or antigen-binding moiety from the culture.

[0115] In some respects, this disclosure relates to pharmaceutical compositions comprising the antibody or antigen-binding portion of this disclosure and a pharmaceutically acceptable carrier or excipient.

[0116] In some aspects, this disclosure relates to methods for treating inflammatory conditions such as autoimmune diseases or inducing immunosuppression in human subjects in need, said methods comprising administering to the subject an effective amount of an antibody or antigen-binding portion of the present disclosure or a pharmaceutical composition of the present disclosure. In some embodiments, the antibody is administered in combination with IL-2. In related aspects, this disclosure provides for the use of an antibody or antigen-binding portion of the present disclosure or a pharmaceutical composition of the present disclosure for treating human subjects suffering from inflammatory conditions such as autoimmune diseases or requiring immunosuppression, wherein said antibody or portion is optionally administered in combination with IL-2; and the use of the antibody or antigen-binding portion of the present disclosure in the preparation of a medicament for treating inflammatory conditions such as autoimmune diseases or inducing immunosuppression, wherein said antibody or portion is optionally administered in combination with IL-2.

[0117] Conditions treatable with the compositions of the present invention (including, but not limited to, the antibodies or antibody / IL-2 complexes described herein) and methods include, but are not limited to: inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); dermatomyositis; systemic scleroderma and sclerosis; conditions associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); colitis; gastritis; respiratory distress syndrome (including adult respiratory distress syndrome and ARDS); dermatitis; meningitis; encephalitis; uveitis; glomerulonephritis; allergic conditions such as eczema and asthma, and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion defects; rheumatoid arthritis; Systemic lupus erythematosus (SLE); diabetes (e.g., type 1 diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; and cytokine- and T-lymphocyte-mediated immune responses associated with acute and delayed hypersensitivity reactions, commonly found in tuberculosis, sarcomatoid diseases, polymyositis, granulomatous diseases, and vasculitis; Wegener's disease; pernicious anemia (Addison's disease); diseases involving leukocyte exudation. Diseases; inflammatory diseases of the central nervous system (CNS); multiple organ injury syndromes; hemolytic anemia (including but not limited to cryoglobulinemia or Kum's positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Langerhans-Ellison myasthenic syndrome; pemphigoid; pemphigus; autoimmune polyendocrine disorders; vitiligo; Reiter's disease; stiff-person syndrome; Behçet's disease; giant cell arteritis; Immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia and autoimmune hemolytic diseases; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveitis; G. Bartholomew's syndrome; Goodpassovudine syndrome; mixed connective tissue disease; autoimmune-related infertility; polyarteritis nodosa; alopecia areata; idiopathic myxedema; graft-versus-host disease; and muscular dystrophy (Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, limb-girdle muscular dystrophy, facial-shoulder-humeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, and Edgner's muscular dystrophy). In some embodiments, the condition that can be treated with the compositions of the present invention (including, but not limited to, the antibodies or antibody / IL-2 complexes described herein) and methods is diabetes (e.g., type 1 diabetes). In some embodiments, the condition that can be treated with the compositions of the present invention (including, but not limited to, the antibodies or antibody / IL-2 complexes described herein) and methods is type 1 diabetes.In some embodiments, the condition that can be treated with the compositions of the present invention (including, but not limited to, the antibodies or antibody / IL-2 complexes described herein) and methods is juvenile diabetes.

[0118] Other features and advantages of this disclosure will become apparent from the following detailed description and exemplary embodiments. Attached Figure Description

[0119] The patent or application documents contain at least one drawing executed in color. A copy of the patent or patent application publication with color drawings is provided by the patent office after the application is filed and the necessary fees are paid.

[0120] Figure 1 Antibody / IL-2 affinity and binding to IL-2Rα and IL-2Rβ were depicted. Responses were reported as binding to IL-2α and IL-2Rβ after 60 seconds, as the percentage of binding to IL-2Rα and IL-2Rβ by two representative clones, d1C7 and 16C3, respectively, which are associated with IL-2 complexation.

[0121] Figure 2 The antibody / IL-2 affinity and the binding of IL-2Rα and IL-2Rβ were depicted. Both the parent and affinity-mature clones showed complete inhibition of IL-2Rβ binding of the antibody / IL-2 complex, and reduced binding to IL-2Rα compared to the d1C7 / IL-2 complex.

[0122] Figure 3 The phenotypes of Treg cells after IL-2:anti-IL-2 mAb treatment were depicted. The Treg population's response to hCD45 was also analyzed. + CD3 + CD4 + Helios + FoxP3 + Cells perform gating.

[0123] Figure 4A -C depicts that 16C3 (25 μg) and F5.1.11.02 (1, 5 and 25 μg) in combination with hIL-2 increased the Treg / CD4, Treg / CD8 and Treg / NK cell ratios. Figure 4D -F depicts how 16C3 (25 μg) and F5.1.11.02 (1 μg, 5 μg, and 25 μg) in combination with hIL-2 increased the Treg / CD4, Treg / CD8, and Treg / NK cell ratios.

[0124] Figure 5A -C depicts that 16C3 (25 μg) and F5.1.11.02 (1, 5, and 25 μg) in combination with hIL-2 increased CD4 in the spleen.+ CD8 + Total number of cells and Tregs. Figure 5D -F depicts the increased CD4+ levels in the spleen by 16C3 (25 μg) in combination with hIL-2 and F5.1.11.02 (1 μg, 5 μg, and 25 μg). + CD8 + Total number of cells and Tregs.

[0125] Figure 6A -C depicts the mean fluorescence intensity (MFI) of CD25, Icos, and FoxP3 on Treg after antibody treatment.

[0126] Figure 7A -V describes the effect on CD8 after antibody treatment. + pSTAT5 signaling in effector T cells and Treg cells. The curves represent treatments with 0.2 ng / mL hIL-2, 10 ng / mL hIL-2, or 500 ng / mL hIL-2.

[0127] Figure 8A -F describes the effect on CD8 after antibody treatment. + pSTAT5 signaling in effector T cells and Treg cells. The curves represent treatments with 0.5 ng / mL hIL-2, 5 ng / mL hIL-2, 50 ng / mL hIL-2, or 500 ng / mL hIL-2.

[0128] Figure 9A -D describes the effect of antibody treatment on CD8. + / CD25 high T cells, CD8 + / CD25 pSTAT5 signaling in low T cells and Treg cells. The x-axis represents nM antibody. The curves represent treatments with 0.8 ng / mL hIL-2, 20 ng / mL hIL-2, or 500 ng / mL hIL-2.

[0129] Figure 10A -B depicts CD8 with different concentrations of IL-2. + / CD25 high T cells, CD8 + pSTAT5 signaling in / CD25-low T cells and Treg cells.

[0130] Figure 11A -H describes the effects of treatment with antibodies F5.1.9, F5.1.9.5, and F5.1.11.04 on CD8. +pSTAT5 signaling in effector T cells and Treg cells. The x-axis represents nM antibody. The curves represent treatments with 0.8 ng / mL hIL-2, 20 ng / mL hIL-2, or 500 ng / mL hIL-2.

[0131] Figure 12A -B describes the production of IL-2 in mouse spleen cells after in vitro stimulation with PMA / ionomycin.

[0132] Figure 13A -B describes the activation of (CD44) in hIL-2Tg, NOD, or NOD mIL-2+ / - mice. + CD62L - CD4 + or CD8 + The percentage of T cells.

[0133] Figure 14 The cell surface expression of CD25 on Treg cells from hIL-2Tg, NOD, or NOD mIL-2+ / - mice was depicted.

[0134] Figure 15 The effects of the F5.1.11.02:IL-2 complex on overall cellularity of the spleen on day 7 were described.

[0135] Figure 16A -I describes the effect of the F5.1.11.02:IL-2 complex on the percentage of Tregs in the spleen, pLN, and pancreas.

[0136] Figure 17A -F describes the effects of F5.1.11.02 (25 μg) in combination with hIL-2 on the Treg / CD4 and Treg / CD8 ratios in the spleen, pLN, and pancreas.

[0137] Figure 18A -C depicts the effect of the F5.1.11.02:IL-2 complex (5 μg and 25 μg F5.1.11.02) on the mean fluorescence intensity (MFI) of CD25 on Tregs in the spleen, pLN and pancreas.

[0138] Figure 19A -B describes the effect of 25 μg of F5.1.11.02 in combination with hIL-2 on the total number of spleen cells.

[0139] Figure 20A -F describes the effect of the F5.1.11.02 antibody:IL-2 complex on the total number of Teff and Treg cells in the spleen. The Treg population's effect on hCD45... + CD3 + CD4+ Helios + FoxP3 + Cells perform gating.

[0140] Figure 21A -D describes the effect of the F5.1.11.02 antibody:IL-2 complex on the Treg / CD4 and Treg / CD8 ratios in the spleen. The Treg population's effect on hCD45... + CD3 + CD4 + Helios + FoxP3 + Cells perform gating.

[0141] Figure 22A -H describes the effect of F5.1.11.02 antibody:IL-2 complex treatment on Treg proliferation.

[0142] Figure 23A -H describes the effect of F5.1.11.02 antibody:IL-2 complex treatment on CD8T cell proliferation.

[0143] Figure 24A -B depicts the increase in mean fluorescence intensity (MFI) of CD25 on Treg and CD8 populations in the spleen induced by treatment with two doses of the F5.1.11.02 antibody:IL-2 complex compared to the isotype control.

[0144] Figure 25A -D depicts a comparison between the effects of F5.1.11.02 and F5.1.11 on Treg and CD8 cell numbers.

[0145] Figure 26A -C depicts a comparison between the effects of F5.1.11.02 and F5.1.11 on the Treg / CD8 ratio.

[0146] Figure 27A -H describes the effect of the antibody on Treg proliferation at various doses.

[0147] Figure 28A -H describes the effect of the antibody on CD8 cell proliferation at various doses.

[0148] Figure 29A -B depicts the equilibrium binding analysis of IL-2Rα with increased concentrations of IL-2(A) and IL-2 / F5.1.11Fab(B).

[0149] Figure 30The interaction of F5.1.11Fab with IL-2 was described via light chain (LC) CDR1 and CDR3 rings and heavy chain (HC) CDR2 and CDR3 rings.

[0150] Figure 31 The F5.1.11Fab / IL-2 complex, covered by the IL-2 receptor quaternary complex, is depicted, showing the overlap of the binding sites of F5.1.11Fab and IL-2Rβ (left panel). Upon binding to F5.1.11Fab, the IL-2 conformation shows a conformational change relative to receptor-bound IL-2, resulting in allosteric regulation of the IL-2Rα binding site (right panel).

[0151] Figure 32 Structure-based library design for cross-reactivity in non-human primates was described. Alignment of the IL-2 amino acid sequences of human (SEQ ID NO: 224; NCBI accession number NP_000577.2) and cynomolgus monkey (SEQ ID NO: 225; predicted from the reference genome NCBI accession number NC_022276.1) showed that the IL-2 variable loop corresponds to the F5.1.11Fab binding interface.

[0152] Figure 33A -B describes the effects of F5.1.11.02 and F5.1.11 on splenocytes and hCD45. + A comparison of the effects of cellularity.

[0153] Figure 34A -B depicts a comparison between the effects of F5.1.11.02 and F5.1.11 on CD25 expression in Treg and CD8 cells.

[0154] Figure 35A -B describes the effect of the F5.1.11.02 antibody:IL-2 complex on the total number of Treg, CD4 and CD8 cells 5 days after treatment.

[0155] Figure 36A -B describes the effect of the F5.1.11.02 antibody:IL-2 complex on the Treg / CD4 and Treg / CD8 cell ratios 5 days after treatment.

[0156] Figure 37A -B describes the effect of treatment with the F5.1.11.02 antibody:IL-2 complex on the mean fluorescence intensity (MFI) of CD25 in the Treg and CD8 populations compared with the isotype control.

[0157] Figure 38The effect of treatment with the F5.1.11.02 antibody:IL-2 complex on the mean fluorescence intensity (MFI) of FoxP3 on Treg cells was described.

[0158] Figure 39A -B describes the use of antibodies to promote differential Treg amplification, said antibodies binding to different epitopes on IL-2, thereby inhibiting binding to different receptor epitopes / binding domains: IL-2Rα blockers (16C3.4), IL-2Rβ blockers (d1C7), and IL-2Rβ blockers that also reduce IL-2 binding to IL-2Rα (F5.1.11.02). Statistical analysis using one-way ANOVA was performed for 16C3.4 versus d1c7 or 16C3.4 versus F5.1.11.02.

[0159] Figure 40 The effects of treatments with 16C3.4, d1C7, and F5.1.11.02 on the mean fluorescence intensity (MFI) of CD25 in Treg and CD8 populations were described compared with the isotype control.

[0160] Figure 41 The study described the remission of diabetes via the F5.1.11.02 antibody:IL-2 complex.

[0161] Figure 42 The effects of the F5.1.11.02 antibody:IL-2 complex on the number and characteristics of Tregs in the pancreas were described. Detailed Implementation

[0162] The inventors have invented novel and advantageous anti-IL-2 antibodies or their antigen-binding moieties that specifically bind to hIL-2 and reduce the binding of hIL-2 to IL-2Rα and IL-2Rβ. These antibodies and moieties can inhibit non-Treg cells (including effector CD8 cells). + Non-Treg CD4 + This antibody increases the proliferation of NK cells (and their inhibitors) to more than suppress the proliferation of Treg cells; increases Treg proliferation compared to allotype control antibodies; and / or increases the Treg / non-Treg cell ratio or maintains Treg markers. This antibody differs from the IL-2Rα blocking antibody described in International Application PCT / US2015 / 011794 (now published as International Publication No. WO 2015 / 109212 on July 23, 2015, which is incorporated herein by reference in its entirety) because this antibody reduces but does not cancel the binding of hIL-2 to IL-2Rα, and blocks the binding of hIL-2 to IL-2Rβ.

[0163] Anti-IL-2 antibodies or their antigen-binding portions may be used to prevent, treat, and / or improve diseases, conditions, or illnesses caused by and / or associated with IL-2 activity. These diseases, conditions, or illnesses include, in particular, type 1 diabetes, autoimmune diseases, graft-versus-host disease, and other immune diseases in which Treg-mediated inflammation is present, as is known to those skilled in the art to whom the teachings disclosed herein are provided.

[0164] General Technology

[0165] Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure should have meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms should include plural terms, and plural terms should include singular terms. Generally, the terms and techniques used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are those well-known and commonly used in the art.

[0166] Unless otherwise stated, the practice of this disclosure will employ conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology within the art. These techniques are well explained in the literature, such as: *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989), ColdSpring Harbor Press; *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Methods in Molecular Biology*, Humana Press; *Cell Biology: A Laboratory Notebook* (edited by J.E.C. Elis, 1998), Academic Press; *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Introduction to Cell and Tissue Culture* (edited by J.M. Pather and P.E. Roberts, 1998), Plenum Press; *Cell and Tissue Culture: Laboratory Procedures* (edited by A. Doyle, J.B. G. R ... Cells (JMMiller and MPCalos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); and The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

[0167] Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The terminology used in conjunction with the analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and pharmaceutical and medicinal chemistry terms described herein, as well as the laboratory procedures and techniques associated with them, are well-known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0168] definition

[0169] Unless otherwise stated, the following terms should be understood to have the following meanings: the term "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide, or antibody or a portion thereof) is a molecule that, due to its origin or derived source, (1) is not bound to its naturally bound components in its native state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) is not present in nature. Thus, a chemically synthesized molecule or expressed in a cellular system different from the cell from which it naturally originates is "isolated" from its naturally bound components. Molecules can also be isolated to the point of being substantially free of naturally bound components using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a variety of methods well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and staining the gel to reveal the polypeptide using techniques well known in the art. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0170] As used herein, “substantially pure” means that the target species is the dominant species present (i.e., more abundant than any other individual species in the composition on a molar basis), and in some embodiments, a substantially purified fraction is a composition in which the target species (e.g., glycoproteins, including antibodies or receptors) comprises at least about 50 percent (on a molar basis) of all present macromolecular species. Generally, a substantially pure composition comprises more than about 80 percent of all present macromolecular species in the composition, and in some embodiments, more than about 85%, 90%, 95%, and 99%. In some embodiments, the target species is purified to substantially homogeneity (contaminant species are not detectable in the composition by conventional detection methods), wherein the composition consists substantially of a single macromolecular species. In some embodiments, the substantially pure material is at least 50% pure (i.e., free of contaminants), in some embodiments at least 90% pure, in some embodiments at least 95% pure, in other embodiments at least 98% pure, and in some embodiments at least 99% pure. These amounts are not intended to be limiting, and increments between the percentages are specifically contemplated as part of this disclosure.

[0171] An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one antigen recognition site located in a variable domain of an immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also (unless otherwise stated) any antigen-binding portion of the antibody that competes for specific binding with the intact antibody, fusion proteins containing an antigen-binding portion, and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site. Antigen-binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs, such as shark and camel antibodies), portions including complementarity-determining regions (CDRs), single-chain variable fragment antibodies (scFvs), macrobodies, minibodies, intracellular antibodies, biantibodies, triantibodies, tetraantibodies, v-NARs, and biscFvs, as well as polypeptides containing at least a portion sufficient to confer specific antigen binding to the polypeptide. Immunoglobulins can be classified into different classes depending on the amino acid sequence of their heavy chain constant regions. There are five main classes (i.e., isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant regions of the heavy chains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

[0172] As used interchangeably herein, the term "antigen-binding portion" or "antigen-binding fragment" (or simply "antibody portion") of an antibody refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., IL-2). The antigen-binding function of an antibody has been shown to be performed by portions of a full-length antibody. Examples of binding portions covered within the term "antigen-binding portion" of an antibody include (i) the Fab portion, which consists of V... L V H C L (ii) the monovalent portion consisting of the CH1 domain; (iii) the F(ab')2 portion, which is a divalent portion containing two Fab portions connected by disulfide bonds in the hinge region; and (iv) the portion consisting of V H (iv) The Fd region composed of the CH1 domain; and the V arm of the antibody. L and V H The Fv part, composed of structural domains, (v) is formed by V. H The dAb region, composed of structural domains (Ward et al., (1989) Nature 341:544-546); and (vi) the separated complementarity-determining region (CDR), the disulfide-linked Fv (dsFv), and the anti-idiotype (anti-Id) antibody and intracellular antibody. Furthermore, although the two structural domains V of the Fv region... L and V H Encoded by separate genes, but they can be linked together using recombination methods via synthetic adapters that allow them to be prepared as a single protein chain, wherein V L and V H The regions pair to form a monovalent molecule (called a single-chain Fv (scFv)); see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such single-chain antibodies are also expected to be covered within the “antigen-binding portion” of the term antibody. Other forms of single-chain antibodies are also covered, such as biantibodies. Biantibodies are bivalent, bispecific antibodies, in which V H and V L The domain is expressed on a single polypeptide chain, but the linker used is too short to allow pairing between two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., 1994, Structure 2:1121-1123).

[0173] The "variable domain" of an antibody refers to the individual or combined antibody light chain (V... L ) variable structural domains or antibody heavy chains (V H The variable domain of the subject variable domain. As is known in the art, the variable domains of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions, and contribute to the formation of the antigen-binding site of the antibody. If a variant of the subject variable domain is desired, particularly substitutions in amino acid residues outside the CDRs (i.e., in the framework regions), appropriate amino acid substitutions, in some embodiments, conserved amino acid substitutions, can be identified by comparing the subject variable domain with variable domains of other antibodies that contain CDR1 and CDR2 sequences in the same canonical class as the subject variable domain (see, for example, Chothia and Lesk, J. Mol. Biol. 196(4):901-917, 1987).

[0174] In some embodiments, the deterministic characterization of the CDR and the identification of residues containing the antibody binding site are accomplished by resolving the structure of the antibody and / or the antibody-ligand complex. In some embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, various analytical methods can be used to identify or approximate the CDR. Examples of these methods include, but are not limited to, Kabat definition, Chothia definition, AbM definition, contact definition, conformation definition, and IMGT definition.

[0175] Kabat definitions are the standard for numbering residues in antibodies and are commonly used to identify CDR regions. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8. Chothia definitions are similar to Kabat definitions, but Chothia definitions take into account the location of certain structural loop regions. See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83. AbM definitions use a computer program integration suite produced by the Oxford Molecular Group to model antibody structures. See, for example, Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbM..." TM“A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. AbM definitions use a combination of knowledge databases and de novo methods to model the tertiary structure of antibodies from basic sequences, methods such as those described by Samudrala et al., 1999, in “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach” in PROTEINS, Structural, Function and Genetics Suppl., 3:194-198. Contact definitions are based on the analysis of available complex crystal structures. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to herein as “conformational definition”, the position of the CDR can be identified as a residue that contributes enthalpy to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Other CDR boundary definitions may not strictly follow one of the methods described above, but still overlap with at least a portion of the Kabat CDR, although they may be shortened or lengthened depending on whether a particular residue or group of residues significantly affects the predicted or experimental results of antigen binding. As used herein, a CDR may refer to a CDR defined by any method (including combinations of methods) known in the art. The methods used herein may utilize a CDR defined according to any of these methods. For any given embodiment containing more than one CDR, the CDR may be defined according to any of the Kabat, Chothia, extended, AbM, contact, and / or conformational definitions. In some embodiments, an extended CDR refers to all amino acid residues identified by the Kabat and Chothia methods.

[0176] As used herein with respect to antibodies or antigens by which they specifically bind, "contact residue" refers to an amino acid residue present on an antibody / antigen containing at least one heavy atom (i.e., non-hydrogen), the heavy atom of which is present on a homologous antibody / antigen. Or less.

[0177] As is known in the art, the “constant region” of an antibody refers to the constant region of the antibody light chain, alone or in combination, or the constant region of the antibody heavy chain.

[0178] As used herein, “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies in the population are identical except for the possibility of naturally occurring mutations present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier “monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to this disclosure can be prepared by a hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by a recombinant DNA method, for example, as described in U.S. Patent No. 4,816,567. For example, monoclonal antibodies can also be isolated from a generated phage library using techniques described in McCafferty et al., 1990, Nature 348:552-554. As used herein, a “humanized” antibody refers to a non-human (e.g., mouse) antibody form that is a chimeric immunoglobulin, an immunoglobulin chain, or a portion thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues of the receptor CDR are replaced with residues of a CDR from a non-human species (donor antibody) such as a mouse, rat, or rabbit, which has the desired specificity, affinity, and capability. Humanized antibodies may contain residues not found in the receptor antibody and the input CDR or framework sequence, but are included to further modify and optimize antibody performance.

[0179] "Human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or has been prepared using any techniques disclosed herein for the preparation of human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0180] The term "chimeric antibody" is intended to refer to an antibody in which the variable domain sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable domain sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody, or vice versa. The term also covers antibodies that contain a V region from one individual of the same species (e.g., the first mouse) and a constant region from another individual of the same species (e.g., the second mouse).

[0181] The term "antigen (Ag)" refers to a vertebrate molecule used for immunization to generate antibodies (Abs) that recognize Ag, or for screening expression libraries (e.g., especially phage, yeast, or ribosome display libraries). In this document, Ag is defined more broadly and is generally expected to include target molecules specifically recognized by Abs, and therefore includes a portion or mimicry of molecules used in immunization procedures to generate Abs or in library screening to select Abs. Thus, for the IL-2-binding antibodies of this disclosure, full-length IL-2 from mammalian species (e.g., human, monkey, mouse, and rat IL-2), including its monomers and multimers, such as dimers, trimers, etc., as well as truncated variants and other variants of IL-2, are referred to as antigens.

[0182] Generally, the term "epitope" refers to a region or area of ​​an antigen to which an antibody specifically binds, i.e., a region or area in physical contact with the antibody. Therefore, the term "epitope" refers to the portion of a molecule that can be recognized and bound by the antibody at one or more sites within the antigen-binding region of the antibody. Typically, an epitope is defined in the context of the molecular interaction between an antibody or its antigen-binding portion (Ab) and its corresponding antigen. Epitopes often consist of surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural features and specific charge characteristics. In some embodiments, an epitope may be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all interaction sites between the protein and the interacting molecule (e.g., the antibody) occur linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" comprises a non-neighboring polypeptide (or amino acid) within an antigenic protein to which the antibody is specific for binding. As used herein, the term "antigen epitope" is defined as a portion of an antigen to which an antibody can specifically bind, as determined by any method well known in the art (e.g., by routine immunoassay). Alternatively, during the discovery process, antibody generation and characterization can elucidate information about the desired epitope. Based on this information, it is then possible to competitively screen for antibodies that bind to the same epitope. This is achieved by conducting competitive and cross-competitive studies to discover antibodies that compete or cross-competitively bind to IL-2, for example, antibodies competing to bind to antigens.

[0183] As used herein, the terms “wild-type amino acid,” “wild-type IgG,” “wild-type antibody,” or “wild-type mAb” refer to an amino acid or nucleic acid sequence that is naturally present in a population (such as humans, mice, rats, cells, etc.).

[0184] As summarized elsewhere in this document, certain positions on an antibody molecule can be altered. As used herein, “position” refers to a location within the protein sequence. Positions can be sequentially numbered or numbered according to established formats, such as the EU index and Kabat index, which can be used to number amino acid residues in antibodies. For example, position 297 is a position in human antibody IgG1. The corresponding position is typically determined by alignment with other parental sequences, as summarized above.

[0185] As used in this article, "residue" refers to a position in a protein and its associated amino acid identity. For example, asparagine 297 (also known as Asn297, or N297) is a residue in human antibody IgG1.

[0186] The term “T regulatory cell” or “Treg” refers to a type of T cell characterized by functional or biological markers known to those skilled in the art (see Schmetterer et al., FASEB Vol. 26 (2012)). In some embodiments, Treg cells express one or more of the following markers: TCR / CD3, CD4, CD25, and demethylated stable FOXP3 based on key genomic elements of the FOXP3 locus.

[0187] The term "Treg-retaining antibody" refers to an antibody that binds to IL-2 and causes Treg:CD8 to... + The ratio of cells can be detectably shifted to antibodies that favor Treg cells. In some embodiments, Treg-retaining antibodies inhibit CD8. + The degree of cell proliferation is greater than its inhibition of Treg proliferation. In some embodiments, the Treg-retaining antibody makes Treg:CD8 + The cell ratio increased at least twice.

[0188] As is known in the art, and as may be used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a nucleotide chain of any length and includes both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerases. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure may be conferred, if present, before or after chain assembly. The sequence of the nucleotide may be interrupted by non-nucleotide components. Polynucleotides may also be modified post-polymerization, for example, by conjugation to labeled components. Other types of modifications include, for example, "capping," replacing one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those with non-electrolyte bonds (e.g., methylphosphonates, triphosphates, aminophosphates, carbamates, etc.) and those with charged bonds (e.g., thiophosphates, dithiophosphates, etc.), those containing a pendant moiety such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those containing an intercalating agent (e.g., acridine, psoralen, etc.), those containing a chelating agent (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing an alkylating agent, those with modified bonds (e.g., α-anomeric nucleic acids, etc.), and unmodified polynucleotides. Furthermore, any hydroxyl group normally present in sugars can be, for example, replaced with a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional linkages with other nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH groups may be phosphorylated or substituted with amines or organic end-capping groups of 1 to 20 carbon atoms. Other hydroxyl groups may also be derived as standard protecting groups. The polynucleotide may also contain similar forms of ribose or deoxyribose known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars (such as arabinose, xylose, or lythose, pyranose, furanose, sedoheptulose), acyclic analogs, and non-basic nucleoside analogs such as methylriboside. One or more phosphodiester bonds may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amid”), P(O)R, P(O)OR', CO, or CH2 (“methylacetal”), wherein each R or R' is independently H, or optionally a substituted or unsubstituted alkyl group (1-20C) containing an ether (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl group. Not all bonds in a polynucleotide need to be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0189] Antibodies that exhibit “preferential binding” or “specific binding” (used interchangeably herein) epitopes are well-known terms in the art, and methods for determining such specificity or preferential binding are also well-known in the art. An antibody is said to exhibit “specific binding” or “preferential binding” if it reacts or binds to a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with a greater affinity than the molecule reacts or binds to alternative cells or substances. An antibody “specifically binds” or “preferentially binds” to a target if it binds to the target with greater affinity, affinity, ease, and / or for a longer duration than the antibody binds to other substances. Furthermore, an antibody “specifically binds” or “preferentially binds” to a target if it binds to a target in a sample with greater affinity, affinity, ease, and / or for a longer duration than the antibody binds to other substances present in the sample. For example, an antibody that specifically or preferentially binds to an IL-2 epitope is one that binds to that epitope with greater affinity, strength, ease, and / or duration of binding compared to its binding to other IL-2 epitopes or non-IL-2 epitopes. It can also be understood from this definition that, for example, an antibody (or part or epitope) that specifically or preferentially binds to a first target may specifically or preferentially bind to a second target or may not specifically or preferentially bind to a second target. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding.

[0190] Various assay formats can be used to select antibodies or peptides that specifically bind to target molecules. Examples include solid-phase ELISA, immunoprecipitation, and Biacore. TM (GE Healthcare, Piscataway, NJ), KinExA, Fluorescence-Activated Cell Sorting (FACS), Octet TM (FortéBio, Inc., Menlo Park, CA) and Western blotting analysis are used in many assays to identify antibodies (which specifically react with antigens or receptors) or their ligand-binding moieties (which specifically bind to homologous ligands or binding partners). Typically, specific or selective responses are at least twice the background signal or noise, more often more than 10 times the background, even more often more than 50 times the background, more often more than 100 times the background, even more often more than 500 times the background, even more often more than 1000 times the background, and even more often more than 10,000 times the background. Furthermore, when the equilibrium dissociation constant (K... D When the concentration is ≤7nM, the antibody is said to "specifically bind" to the antigen.

[0191] The term "binding affinity" is used in this paper as a measure of the strength of the non-covalent interaction between two molecules (e.g., an antibody or a portion thereof with an antigen). The term "binding affinity" is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules can be determined by determining the dissociation constant (K). D ) can be used to quantify this. In turn, K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the binding and dissociation of monovalent complexes are called the binding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D =kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods, and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double-filtered nitrocellulose filter combined with determinations such as those disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of antibodies to target antigens are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry, as well as other assays exemplified elsewhere herein. Antibody binding kinetics and binding affinity can also be determined using standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore. TM The system or KinExA can be used for evaluation.

[0192] In some embodiments, the antibody may be approximately 1.14 x 10⁻⁶. -10 M or larger K D Binds to hIL-2. For example, antibodies can be approximately 9 x 102 -11 M or larger K D Binds to hIL-2. In some embodiments, the antibody may be approximately 8 x 10⁻⁶. -11 M or larger K D Binds to hIL-2. In some embodiments, the antibody may be approximately 7 x 102 -11 M or larger K D Binds to hIL-2. In some embodiments, the antibody may be approximately 6 x 102 -11 M or larger K D Binds to hIL-2. In some embodiments, the antibody may be approximately 5.00 x 10⁻⁶. -11 M or larger K DBinding with hIL-2. These amounts are not intended to be limiting, and the increments between said values ​​are specifically contemplated as part of this disclosure. In some embodiments, as shown in Table 3, the antibody may bind to the K of the antibody. D Approximately the same kd binds to hIL-2.

[0193] In some embodiments, the antibody may be approximately 4.53 x 10⁻⁶. -4 s -1 Or a larger kDa binds to hIL-2. For example, antibodies can be approximately 3 x 10⁻⁶. -4 s -1 Or a larger kDa antibody binds to hIL-2. In some embodiments, the antibody may be approximately 1x10⁻⁶. -4 s -1 Or a larger kDa binds to hIL-2. In some embodiments, the antibody may be approximately 9x10⁻⁶. -5 s -1 Or a larger kDa binds to hIL-2. In some embodiments, the antibody may be approximately 7 x 10⁻⁶. -5 s -1 Or a larger kDa binds to hIL-2. In some embodiments, the antibody may be approximately 5.00 x 10⁻⁶. -5 s -1 Or a larger kd binds to hIL-2. These amounts are not intended to be limiting, and the increments between said values ​​are specifically contemplated as part of this disclosure. In some embodiments, as shown in Table 3, the antibody may bind to hIL-2 with a kd approximately the same as the antibody's kd.

[0194] Competitive binding assays can be performed, in which the binding of an antibody to a target antigen is compared with the binding of the target to another ligand of that target (e.g., another antibody or soluble receptor that binds to the target in a different manner). The concentration at which 50% inhibition occurs is called K. i Under ideal conditions, K i equals K D K i The value is never less than K D Therefore K i The measurement can be conveniently replaced to provide K. D The upper limit.

[0195] Based on the above definition, the K values ​​of various antibody / antigen complexes can be compared. D The binding affinity is used to compare the binding affinity of different molecules associated with their interactions, for example, comparing the binding affinity of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by identifying and comparing the K-value of the target interaction (e.g., the specific interaction between an antibody and an antigen). DThe value of K for non-target interactions (e.g., control antibodies known not to bind IL-2) D The value is evaluated.

[0196] Antibodies that specifically bind to their targets can bind to their targets with high affinity, i.e., exhibiting low K, as discussed above. D Furthermore, antibodies can bind to other non-target molecules with lower affinity. For example, antibodies can bind 1x10... -6 M or higher, in some embodiments, 1x10 -5 M or higher, in some embodiments, 1x10 -4 M or higher, in some embodiments, 1x10 -3 M or higher, in some embodiments, 1x10 -2 M or higher K D Binding to non-target molecules. In some embodiments, the antibody of this disclosure is capable of binding to its target with an affinity at least 2, 10, 50, 100, 200, 500, 1,000, or 10,000 times greater than its affinity for another non-IL-2 molecule. These amounts are not intended to be limiting, and the increments between said values ​​are specifically contemplated as part of this disclosure.

[0197] As used herein, the term "antibody:IL-2 complex" refers to a complex comprising at least one antibody of the present disclosure or its antigen-binding moiety, which specifically binds IL-2 and at least one IL-2 cytokine molecule. The complex comprises an antibody and an IL-2 molecule bound by covalent, non-covalent, or any other force. Preferably, the antibody and IL-2 remain bound as a complex even after administration of the complex. It should be understood that, among other variables, the antibody and IL-2 will form a complex based on the KD value of their binding interaction.

[0198] "Host cell" includes individual cells or cell cultures that may be, or have been, recipients of vectors for incorporating polynucleotide inserts. Host cells include progeny of a single host cell, and progeny may not necessarily be identical to the original parent cell (in morphology or genomic DNA complementation) due to natural, accidental, or intentional mutations. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of this disclosure.

[0199] As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the segment from the amino acid residue at position Cys226 or Pro230 to its C-terminus. The residues in the Fc region are numbered using the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally contains two constant domains, CH2 and CH3. As is known in the art, the Fc region can exist in dimer or monomeric form.

[0200] A “functional Fc region” possesses at least one effector function of a native Fc region. Exemplary effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors). Such effector functions generally require a combination of the Fc region and a binding domain (e.g., an antibody variable domain or its antigen-binding portion) and can be evaluated using various assays known in the art for assessing such antibody effector functions.

[0201] A “natural sequence Fc region” comprises an amino acid sequence identical to that of an Fc region found in nature. A “variant Fc region” comprises an amino acid sequence that differs from that of the natural sequence Fc region due to at least one amino acid modification, but still retains at least one effector function of the natural sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, compared to the natural sequence Fc region or the Fc region of the parent polypeptide, and in some embodiments, about 1 to about 5 amino acid substitutions in the natural sequence Fc region or the Fc region of the parent polypeptide. In some embodiments, the variant Fc region herein has at least about 80% sequence identity with the natural sequence Fc region and / or the Fc region of the parent polypeptide, and in some embodiments, at least about 90% sequence identity, and in some embodiments, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. These amounts are not intended to be limiting, and increments between these values ​​are specifically contemplated as part of this disclosure.

[0202] As used in the art, “Fc receptor” and “FcR” describe receptors that bind to the Fc region of an antibody. In some embodiments, the FcR is a naturally occurring human FcR. Furthermore, in some embodiments, the FcR is an FcR that binds to an IgG antibody (γ receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”) with similar amino acid sequences that differ primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249).

[0203] As used herein, when the presence of a first antibody detectably reduces the binding of a second antibody to an antigen (or an epitope of the second antibody), the first antibody is said to compete with the second antibody for binding to the antigen (or epitope). The opposite, where the binding of the first antibody to the antigen (or its epitope) is also detectably reduced in the presence of the second antibody, may be true but is not necessarily true. However, when each antibody detectably inhibits the binding of another antibody to a common antigen (whether to the same extent or different extent), the antibodies are said to “cross-compete” with each other for binding to that antigen. This disclosure covers both competitive and cross-compete antibodies. Regardless of the mechanism by which such competition or cross-compete occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or a portion thereof), based on the teachings provided herein, those skilled in the art will understand that such competitive and / or cross-compete antibodies are covered and can be used in the methods disclosed herein.

[0204] As used herein, “treatment” is a method for achieving a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: improved survival (reduced mortality), reduction of inflammation, reduction of the amount of tissue fibrosis, improvement in the appearance of disease lesions, localization of pathological damage to the lesion site, reduction in the extent of damage from the disease, reduction in the duration of the disease, and / or reduction in the number, severity, or duration of disease-related symptoms. This term includes the administration of compounds or agents of this disclosure to prevent or delay the onset of symptoms, complications, or biochemical indicators of the disease, to alleviate symptoms, or to prevent or inhibit the further development of the disease, condition, or symptom. Treatment may be preventative (preventing or delaying the onset of the disease, or preventing the manifestation of its clinical or subclinical symptoms) or therapeutic suppression or relief of symptoms after the onset of the disease.

[0205] "Improvement" means a reduction or improvement in one or more symptoms compared to when IL-2 antibodies were not administered. "Improvement" also includes a reduction or shortening of the duration of symptoms.

[0206] As used herein, an “effective dose” or “effective amount” of a drug, compound, or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired outcomes. More specifically, an effective amount prevents, reduces, or improves symptoms of a disease or infection, and / or prolongs the survival of a subject to be treated. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, reducing severity, or delaying the onset of a disease, including the disease, its complications, and biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes presented during disease development. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing one or more symptoms of an IL-2-mediated disease, condition, or status, or an IL-2-deficient disease, condition, or status, reducing the dose of other agents required to treat the disease, enhancing the effect of another agent, and / or delaying the progression of the patient’s disease. An effective dose may be administered in one or more administrations. For the purposes of this disclosure, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in a clinical context, an effective dose of a drug, compound, or pharmaceutical composition may be achieved with or without another drug, compound, or pharmaceutical composition. Therefore, in the context of administering one or more therapeutic agents, an "effective dose" may be considered, and a single agent may be considered to be administered in an effective amount if it achieves or achieves the desired result when combined with one or more other agents.

[0207] As used herein, “vector” means a construct capable of being delivered to a host cell and, in some embodiments, expressing one or more target genes or sequences. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors bound to cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0208] As used herein, "expression control sequence" refers to the nucleic acid sequence that directs nucleic acid transcription. The expression control sequence can be a promoter, such as a constitutive or inducible promoter or enhancer. The expression control sequence is operatively linked to the nucleic acid sequence to be transcribed.

[0209] As used herein, a “pharmaceuticalally acceptable carrier” or “pharmaceuticalally acceptable excipient” includes any material that, when combined with an active ingredient, allows that ingredient to retain its biological activity and does not react with the subject’s immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated using well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, editor, Mack Publishing Co., Easton, PA, 1990; and R. Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0210] This document mentions "about" values ​​or parameters, including (and describing) embodiments of that value or parameter itself. For example, a description of "about X" includes a description of "X". Numerical ranges include the numbers that define the range.

[0211] Although the numerical ranges and parameters described in this disclosure are approximate, the numerical values ​​described in the specific embodiments are reported as precisely as possible. However, any numerical value inherently includes some error that necessarily arises from the standard deviation found in their respective test measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, the range “1 to 10” should be considered to include any and all subranges (including endpoints) between the minimum value of 1 and the maximum value of 10; that is, all subranges that begin with a minimum value of 1 or greater, such as 1 to 6.1, and all subranges that end with a maximum value of 10 or less, such as 5.5 to 10.

[0212] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the definition included herein shall prevail. Throughout this specification and the claims, the word “comprise” or variations such as “comprises” or “comprising” shall be construed as implying inclusion of the stated whole or group of wholes, but not excluding any other whole or group of wholes. Unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Any examples following the terms “such as” or “for example” are not intended to be exhaustive or limiting.

[0213] It should be understood that wherever embodiments are described herein with the language “comprising”, similar embodiments in other respects as described with “consisting of” and / or “substantially consisting of” are also provided.

[0214] When aspects or embodiments of this disclosure are described in accordance with alternatives to the Markush group or other groupings, this disclosure covers not only the entire group listed as a whole, each member of each group, and the individual and all possible subgroups of the main group, but also the main group in which one or more of the group members are absent. This disclosure also contemplates the explicit exclusion of one or more of any group members from the claimed disclosure.

[0215] This document describes exemplary methods and materials, although similar or equivalent methods and materials may also be used in the practice or testing of this disclosure. The materials, methods, and examples are illustrative only and not limiting.

[0216] Overview

[0217] Interleukin-2 (IL-2) is a four-helix type I cytokine that acts as a growth factor for a broad range of leukocytes, including T cells and natural killer (NK) cells. Significant efforts have been devoted to researching IL-2 as a therapeutic target for a wide range of immune diseases, from AIDS to cancer. Recombinant human High doses are used to treat metastatic melanoma and renal cell carcinoma. However, only a small subset of patients (5-10%) experience long-term survival from this treatment. The adverse effects of high-dose IL-2 therapy, ranging from flu-like symptoms to life-threatening vascular leakage syndromes and pulmonary edema, have greatly limited its use. Importantly, IL-2 therapy has unpredictable biological effects.

[0218] IL-2 mediates its effects by binding to a complex receptor consisting of three chains: CD25 (IL-2Rα), CD122 (IL-2Rβ), and a common γ chain (γ). c The quaternary complex is composed of three chains, hence the name IL-2Rαβγ. The three chains are differentially expressed by receptor trimers exhibiting the highest affinity. Individually, each of the three receptor chains in the quaternary complex binds to hIL-2 with low affinity, with hIL-2Rα exhibiting the strongest relative affinity (K2). D ~10 nM). hIL-2Rα expression increases the binding rate of hIL-2 to T cells, and the three receptors are β-blockers. D ~10 pM co-binds hIL-2. After being captured by IL-2Rα, IL-2 is presented to IL-2Rβ, and then γ c It can form a quaternary signaling complex in a pre-formed receptor dimer. Although hIL-2 alone has low affinity (K... D ~150-300 nM) binds to hIL-2Rβ, but the hIL-2 / hIL-2Rα complex binds with a higher affinity (K). D IL-2 (~60 nM) binds to hIL-2Rβ, however, there is no direct contact between IL-2Rα and IL-2Rβ in the quaternary receptor complex. Recent studies suggest that wild-type IL-2 exists in a "dormant" form, which is induced into a structurally altered conformation representing a high-affinity form that binds to IL-2Rα.

[0219] IL-2 has been engineered or modified to enhance its therapeutic potential by altering its ability to selectively target T effector cells (Teff) or T regulator cells (Treg). Interestingly, numerous approaches to more effective and targeted IL-2 exist. In mouse models not directly similar to humans, Boyman et al. have demonstrated that, under certain circumstances, rat anti-mouse IL-2 mAb (JES6-1) can be combined with wild-type IL-2 and preferentially enhance T cells.REG (Boyman et al., 2006, Science 311:1924-1927; and International Patent Publication No. WO 2007 / 095643). Although the mechanistic basis of this effect is not yet elucidated, it is not desirable to be bound by any particular theory. IL-2 complexed with certain antibodies can be "fixed" in a conformation that selectively triggers the recognition signal, leading to individual T... REG or T EFF Selective expansion of cell subsets. Additionally, efforts have focused on developing IL-2 mutant proteins that enhance CD25 binding by increasing the binding of IL-2 to the three-molecule IL-2R complex CD25. + T cell activation and minimization of CD25 - NK cell activation. In this respect, mutant IL-2 prepared by Bayer inhibits IL-2Rβγ. c With significantly lower affinity, treatment with this variant of IL-2 is expected to not activate NK or memory CD8. + T cells. However, neither the IL-2 mutant nor the IL-2 / anti-IL-2 antibody complex approach described above takes into account the potential incompetence of pharmacologically altered endogenous wild-type IL-2, which is produced during inflammatory T cell responses. Furthermore, any treatment with an IL-2 variant that induces endogenous IL-2 (a proven feedback mechanism) can result in an in vivo wild-type IL-2 effect that may not be observed in vitro.

[0220] Therefore, although IL-2 initially enhances the effect of T cells (T cells) EFF IL-2 was developed as an immunostimulant due to its ability to activate NK cells, but it is now believed that the primary function of IL-2 is not immune activation but rather T cells. REG The generation and survival of the T REG It acts to suppress the immune response and prevent autoimmune diseases. There is an increasing understanding using animal models (including mice deficient in IL-2 and its receptor (IL-2R)) that IL-2 plays a role in the inhibition of the immune response and prevention of autoimmune diseases. REG It plays a key role in cell-mediated peripheral immune tolerance. Studies have shown that low-dose IL-2 therapy preferentially activates T cells due to the constitutive expression of high-affinity IL-2R. REG More specifically, increased animal studies have confirmed T REGIt can suppress GVHD and autoimmunity. Furthermore, this potential inhibitory effect has been confirmed in humans in two recent studies, one in GVHD and the other in autoimmune vasculitis, where short-term low-dose therapy led to disease improvement in some individuals. Therefore, there has long been a need for IL-2-based therapeutics to selectively activate tolerance relative to the effector immune response, i.e., designed to make T... REG :T EFF Balance towards T REG Inclined treatment allows for the treatment of a wide variety of diseases. This disclosure addresses this need.

[0221] IL-2 antibody

[0222] This invention relates to antibodies that specifically bind to IL-2, i.e., they bind to IL-2 but cannot detectably bind to other molecules or bind to other molecules with low affinity. In some embodiments, the antibody specifically binds to human IL-2. In some embodiments, the antibody specifically binds to helices A and C and the BC loop of hIL-2. In some embodiments, the antibody specifically binds to helix A of hIL-2. In some embodiments, the antibody specifically binds to helix C of hIL-2. In some embodiments, the antibody specifically binds to helices A and C of hIL-2. In some embodiments, the antibody specifically binds to the BC loop of hIL-2. In some embodiments, the antibody competes with antibodies containing the amino acid sequence SEQ ID NO: 13, 14, 126, 127, 128, 129, 130, 131, 132, 133, and 134 for binding to human IL-2 (hIL-2), or binds to the same hIL-2 epitope with antibodies containing the amino acid sequence SEQ ID NO: 13, 14, 126, 127, 128, 129, 130, 131, 132, 133, and 134. In some embodiments, the antibody specifically binds to human IL-2 (hIL-2) and reduces the binding affinity of hIL-2 to IL-2Rα by about 1 to about 199-fold. In some embodiments, the antibody reduces the binding affinity of hIL-2 to IL-2Rα by about 10-fold. In some embodiments, the antibody reduces the binding affinity of hIL-2 to IL-2Rα by approximately 2, 10, 25, 50, 75, 100, 125, 150, or 175 times.

[0223] In some embodiments, the IL-2 antibody reduces the binding of IL-2 to IL-2Rα and IL-2Rβ without inhibiting the activity of regulatory T (Treg) cells. For example, in some embodiments, the IL-2 antibody blocks the binding of IL-2 to IL-2Rβ and reduces the affinity of hIL-2 for binding to IL-2Rα. In some embodiments, the IL-2 antibody reduces the binding of IL-2 to IL-2Rα by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%. In some embodiments, the IL-2 antibody reduces the binding of IL-2 to IL-2Rβ by at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%. In some embodiments, the IL-2 antibody completely blocks the binding of IL-2 to IL-2Rβ. In particular, this disclosure relates to antibodies that specifically bind to IL-2, and further, to antibodies that inhibit non-Treg cells (including effector CD8 cells). + Non-Treg CD4 + Antibodies that inhibit the proliferation of Treg cells (including NK cells) or increase Treg cell proliferation compared to allotype control antibodies, or increase the Treg cell / non-Treg cell ratio or maintain Treg markers or combinations thereof. In some embodiments, the antibody inhibits CD8. + Non-Treg CD4 + Or, the proliferation of NK cells exceeds the proliferation of Treg cells by at least two times. In some embodiments, the antibody inhibits CD8... + Non-Treg CD4 + The antibody inhibits Treg proliferation by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. In some embodiments, in peripheral blood mononuclear cell (PBMC) cultures or remodeling assays, the antibody reduces the binding of IL-2 to IL-2Rα and IL-2Rβ and increases the binding of T regulatory cells (Tregs) to CD8+. + Non-Treg CD4 + Or the ratio of NK cells. In some embodiments, this ratio is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more. In some embodiments, the antibody induces Treg:CD8 +The cell ratio increased two (2) times or more, and this reflected increased Treg proliferation. In some embodiments, when IL-2 was limited in vitro to, for example, a concentration of less than 1 nM, the antibody enhanced T cell (Treg) proliferation greater than that of the isotype control. In some embodiments, the antibody inhibited CD8 + Cell proliferation. In some embodiments, the antibody reduces the binding of IL-2 to IL-2Rα and IL-2Rβ and maintains the expression of one or more of FOXP3, CD25, and Icos in Treg cells. In some embodiments, the antibody reduces the binding of IL-2 to IL-2Rα and IL-2Rβ and increases the expression of one or more of FOXP3, CD25, and Icos in Treg cells. In some embodiments, the IL-2 antibody of this disclosure has at least one of these features, and in some embodiments, the antibody has two or more of these features. In some embodiments, the antibody has all of these features.

[0224] In some embodiments, antibodies that specifically bind to IL-2 or their antigen-binding moiety reduce the binding of IL-2 to IL-2Rα and IL-2Rβ, and inhibit CD8. + STAT5 is phosphorylated to a higher degree in T cells than in regulatory T (Treg) cells. In some embodiments, the IL-2 antibody or antigen-binding moiety maintains STAT5 phosphorylation in Tregs greater than 50%. In some embodiments, the IL-2 antibody or antigen-binding moiety maintains STAT5 phosphorylation in Tregs greater than 60%. In some embodiments, the IL-2 antibody or antigen-binding moiety maintains STAT5 phosphorylation in Tregs greater than 70%. In some embodiments, the IL-2 antibody or antigen-binding moiety maintains STAT5 phosphorylation in Tregs greater than 80%. In some embodiments, the IL-2 antibody or antigen-binding moiety maintains STAT5 phosphorylation in Tregs greater than 90%. In some embodiments, the antibody specifically binds to hIL-2. These amounts are not intended to be limiting, and the increments between the percentages are specifically contemplated as part of this disclosure.

[0225] This disclosure also relates to compositions comprising such antibodies and the uses of such antibodies, including therapeutic and pharmaceutical uses.

[0226] The term “IL-2” refers to any naturally occurring form of IL-2, whether monomeric or polymeric, including dimers, trimers, etc., derived from any suitable organism. As used herein, “IL-2” refers to mammalian IL-2, such as human, rat, or mouse IL-2, and non-human primate IL-2, bovine, ovine, or swine IL-2. In some embodiments, IL-2 is human (see, for example, Genbank accession number P60568) or “hIL-2”. IL-2 can also be cynomolgus monkey IL-2 (see, for example, Genbank accession number Q29615). The term “IL-2” also encompasses portions, variants, isotypes, and other homologues of such IL-2 molecules. Variant IL-2 molecules are generally characterized by having the same type of activity as naturally occurring IL-2, such as the ability to bind to IL-2 receptors and the ability to induce receptor-mediated activity.

[0227] IL-2 may contain one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, or fifteen or more surface-accessible residues of IL-2. When IL-2 contains the homopolymer form of IL-2, the target may contain one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, or fifteen or more surface-accessible residues of the first subunit of IL-2, and one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, or fifteen or more surface-accessible residues of the second subunit of IL-2.

[0228] The target molecule may contain a known epitope from IL-2. The target molecule may contain a known epitope from hIL-2. In some embodiments, the target may contain a helix A of hIL-2. In some embodiments, the target may contain a helix C of hIL-2. In some embodiments, the target may contain helices A and C of hIL-2. In some embodiments, the target may contain a BC loop of hIL-2. In some embodiments, the target may contain helices A and C of hIL-2 and a BC loop.

[0229] In one embodiment, this disclosure provides, or comprises, any of the following (including pharmaceutical compositions): an antibody having a light chain sequence or a portion thereof derived from any of the following antibodies and a heavy chain or a portion thereof: F4.7.6, F4.7.8, F5.1.11, F5.1.9, F4.7.062, F5.11.1.01, F5.1.11.02, F5.1.11.03, F5.1.11.04, F5.1.11.05, F5.1.11.06, F5.1.11.07, F5.1.11.08, F5.1.11.09, F5.1.9.5, or d1C7. Antibody F5.1.11 is also referred to as antibody F5111, 5.1.11, or 5111. These terms are interchangeable. Variants of the parent antibody can be referred to by other numbers using this nomenclature, such as antibody F5111.2, 5.1.11.2, F5.1.11.02 or 5111.2; or antibody F5.1.11.01, F5111.1, 5.1.11.1 or 5111.1.

[0230] The antibodies used in this disclosure may encompass monoclonal antibodies, polyclonal antibodies, antibody moieties (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugated antibodies, single-chain (ScFv), mutants thereof, fusion proteins containing antibody moieties (e.g., domain antibodies), humanized antibodies, and any other modified conformations of immunoglobulin molecules containing antigen recognition sites with desired specificity, including glycosylated variants of the antibody, amino acid sequence variants of the antibody, and covalently modified antibodies. The antibodies may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the IL-2 antibody is a monoclonal antibody. In some embodiments, the antibody is a human or humanized antibody.

[0231] The IL-2 antibody disclosed herein can be prepared by any method known in the art. General techniques for generating human and mouse antibodies are known in the art and / or described herein.

[0232] IL-2 antibodies can be identified or characterized using methods known in the art, thereby detecting and / or measuring a reduction, improvement, or neutralization of IL-2 activity, such as pAKT and / or pSTAT5. In some embodiments, IL-2 antibodies are identified by incubating a candidate reagent (e.g., IL-2) with an IL-2 receptor and monitoring for a concomitant reduction or inhibition of IL-2 binding and / or biological activity. In some embodiments, hIL-2 antibodies can be identified or characterized using methods known in the art, thereby detecting and / or measuring a reduction, improvement, or neutralization of hIL-2 activity, such as pAKT and / or pSTAT5. In some embodiments, hIL-2 antibodies are identified by incubating a candidate reagent (e.g., hIL-2) with an hIL-2 receptor and monitoring for a concomitant reduction or inhibition of hIL-2 binding and / or biological activity. Binding assays can be performed using, for example, purified IL-2 peptides or cells naturally expressing various receptors or transfected to express IL-2 receptors. In one embodiment, the binding assay is a competitive binding assay in which the ability of a candidate antibody to compete for IL-2 binding with a known IL-2 antibody is evaluated. This assay can be performed in various forms, including ELISA. In some embodiments, IL-2 antibodies are identified by incubating a candidate antibody with IL-2 and monitoring the reaction.

[0233] Following initial identification, the activity of candidate IL-2 antibodies can be further confirmed and purified using bioassays known for testing target biological activity. In some embodiments, in vitro cellular assays are used to further characterize candidate IL-2 antibodies. For example, bioassays can be used for direct screening of candidates. Some methods for identifying and characterizing IL-2 antibodies are described in detail in the examples.

[0234] IL-2 antibodies can be characterized using methods well-known in the art. One approach, for example, is to identify the epitope to which it binds, or “epitope mapping.” Many methods are known in the art for locating and characterizing epitopes on proteins, including resolving the crystal structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In another example, epitope mapping can be used to determine the sequence to which the IL-2 antibody binds. Epitope mapping is commercially available from various sources, such as Pepscan Systems (Edelhertweg 15, 8219PH Lelystad, Netherlands). The epitope can be a linear epitope, i.e., contained in a single segment of amino acids, or a conformational epitope formed through three-dimensional interactions of amino acids that may not necessarily be contained in a single segment. Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombined) and used for binding assays with IL-2 antibodies. In another example, epitopes bound to IL-2 antibodies can be identified in systematic screening by using overlapping peptides derived from the IL-2 sequence and determining binding via antibody. Based on gene fragment expression assays, open reading frames encoding IL-2 can be randomly or fragmented using specific genetic constructs, and the reactivity of the expressed IL-2 fragment with the test antibody can be determined. For example, gene fragments can be generated by PCR, then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. Binding of the antibody to the radiolabeled IL-2 fragment is then determined by immunoprecipitation and gel electrophoresis. Some epitopes can also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) or yeast (yeast display). Alternatively, identified libraries of overlapping peptide fragments can be tested for binding with the test antibody in a simple binding assay. In other examples, antigen mutagenesis, domain exchange assays, and alanine scan mutagenesis can be performed to identify the residues required, sufficient, and / or essential for epitope binding. For instance, an alanine scan mutagenesis assay can be performed using mutant IL-2 in which various residues of the IL-2 peptide have been replaced with alanine. By evaluating the binding of the antibody to the mutant IL-2, the importance of specific IL-2 residues for antibody binding can be assessed.

[0235] Another method that can be used to characterize IL-2 antibodies is a competitive assay using other antibodies that are known to bind to the same antigen (i.e., various fragments on IL-2) to determine whether the IL-2 antibody binds to the same epitope as the other antibody. Competitive assays are well known to those skilled in the art.

[0236] Furthermore, various experimental and computational epitope mapping methods can be used to define and characterize epitopes for a given antibody / antigen binding pair at different levels of detail. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen / deuterium exchange mass spectrometry (H / D-MS), and various competitive binding methods well-known in the art. Because each method relies on unique principles, the description of an epitope is closely linked to the method it has been defined for. Therefore, depending on the epitope mapping method employed, the epitope for a given antibody / antigen pair will be defined differently.

[0237] At its most detailed level, an epitope of the interaction between Ag and Ab can be defined by the spatial coordinates of the atomic contacts present in the Ag-Ab interaction, and information about their relative contributions to the thermodynamics of binding. At a less detailed level, an epitope can be characterized by the spatial coordinates of the atomic contacts between Ag and Ab. At an even less detailed level, an epitope can be characterized by the amino acid residues it contains, as defined by specific criteria, such as the distance between atoms (e.g., heavy, i.e., non-hydrogen atoms) in Ab and Ag. At an even less detailed level, an epitope can be characterized by its function, for example, by competing for binding with other Abs. An epitope can also be defined more generally as containing amino acid residues whose characteristics are altered by substitution of another amino acid (e.g., using an alanine scan).

[0238] Based on the fact that epitope descriptions and limitations are obtained at different levels of detail depending on the epitope mapping method used, subsequent comparisons of epitopes on the same Ag but different Abs can be similarly performed at different levels of detail.

[0239] Epitopes are said to be identical if they contain the same group of amino acid residues, described at the amino acid level, for example, from X-ray structure. Epitopes are said to overlap if at least one amino acid is shared by the epitopes. Epitopes are said to be separate (unique) if no amino acid residues are shared by the epitopes.

[0240] If the binding of corresponding antibodies is mutually exclusive, meaning that the binding of one antibody excludes the simultaneous or sequential binding of another antibody, then epitopes characterized by competitive binding are said to be overlapping. If an antigen can simultaneously accommodate the binding of two corresponding antibodies, then the epitope is said to be separate (unique).

[0241] The term "complementary site" is defined by reversing the definition of "epitope" mentioned above. Therefore, the term "complementary site" refers to a region or area on an antibody that specifically binds to the antigen, i.e., an amino acid residue on the antibody that contacts the antigen (IL-2), as defined elsewhere in this document.

[0242] Epitopes and complementary sites of a given antibody / antigen pair can be identified using conventional methods. For example, the general location of an epitope can be determined by evaluating the ability of an antibody to bind different fragments or variants of the IL-2 peptide. Specific amino acids within IL-2 that contact the antibody (eptopes) and specific amino acids in the antibody that contact IL-2 (complementary sites) can also be determined using conventional methods, such as those described in the examples. For example, antibodies and target molecules can be combined and antibody / antigen complexes can be crystallized. The crystal structure of the complex can be determined and used to identify specific sites of interaction between the antibody and its target.

[0243] The antibodies according to this disclosure can bind to the same IL-2 epitopes or domains as those specifically disclosed herein. For example, other unidentified antibodies of this disclosure can be identified by comparing their binding to IL-2 with any of the following monoclonal antibodies: F4.7.6, F4.7.8, F5.1.11, F5.1.9, F4.7.062, F5.11.1.01, F5.1.11.02, F5.1.11.03, F5.1.11.04, F5.1.11.05, F5.1.11.06, F5.1.11.07, F5.1.11.08, F5.1.11.09, F5.1.9.5 or d1C7 and their variants; and / or by comparing the epitopes / contact residues on IL-2 of the unidentified antibodies with those of the antibodies of this disclosure. Analyses and assays that can be used for this identification purpose include assays evaluating IL-2 binding competition between the target antibody and the IL-2 receptor in bioactivity assays as described in Examples 1-5, and in analysis of antibody crystal structures.

[0244] As described herein, the antibodies disclosed herein may have the ability to compete or cross-compete with another antibody disclosed herein for binding to IL-2. For example, the antibodies disclosed herein may compete or cross-compete with the antibodies described herein for binding to IL-2, or with a suitable fragment or variant of IL-2 bound by the antibodies disclosed herein.

[0245] That is, if a first antibody competes with a second antibody for binding to IL-2, but does not compete when the second antibody binds to IL-2 first, it is considered to "compete" with the second antibody (also known as one-way competition). When an antibody competes with another antibody, it "cross-competes" with the other antibody for binding to IL-2, regardless of which antibody binds to IL-2 first. Such competitive or cross-competitive antibodies can be identified based on their ability to compete / cross-compete with known antibodies of this disclosure in a standard binding assay. For example, SPR, for instance, uses Biacore... TM Systematic, ELISA, or flow cytometry assays can be used to confirm competition / cross-competition. This competition / cross-competition can indicate that two antibodies bind to the same, overlapping, or similar epitopes.

[0246] Therefore, the antibodies of this disclosure can be identified by methods including a binding assay, which evaluates whether the test antibody can compete / cross-competitively bind to a site on a target molecule with reference antibodies of this disclosure (e.g., F4.7.6, F4.7.8, F5.1.11, F5.1.9, F4.7.062, F5.11.1.01, F5.1.11.02, F5.1.11.03, F5.1.11.04, F5.1.11.05, F5.1.11.06, F5.1.11.07, F5.1.11.08, F5.1.11.09, F5.1.9.5, or d1C7). Methods for performing competitive binding assays are disclosed herein and / or are well known in the art. For example, they may involve binding the reference antibody of this disclosure to a target molecule using conditions under which the antibody can bind to the target molecule. The antibody / target complex can then be exposed to the test / second antibody, and the extent to which the test antibody can displace the reference antibody of this disclosure from the antibody / target complex can be evaluated. An alternative approach may involve contacting the test antibody with the target molecule under conditions that allow antibody binding, then adding the reference antibody of this disclosure capable of binding to the target molecule, and evaluating the extent to which the reference antibody of this disclosure can displace the test antibody from the antibody / target complex or simultaneously bind to the target (i.e., a non-competitive antibody).

[0247] The ability of the test antibody to inhibit the binding of the reference antibody of this disclosure to the target confirms that the test antibody can compete with the reference antibody of this disclosure for target binding, and therefore the test antibody binds to the same or substantially the same epitope or region on the IL-2 protein as the reference antibody of this disclosure. The test antibody identified in this method as competing with the reference antibody of this disclosure is also an antibody of this disclosure. The fact that the test antibody can bind IL-2 in the same region as the reference antibody of this disclosure, and can compete with the reference antibody of this disclosure, suggests that the test antibody can act as a ligand at the same binding site as the antibody of this disclosure, and the test antibody can therefore mimic the action of the reference antibody, and is therefore an antibody of this disclosure. This can be confirmed by comparing the IL-2 activity in the presence of the test antibody with the IL-2 activity in the presence of the reference antibody under otherwise identical conditions, using an assay as more fully described elsewhere herein.

[0248] The reference antibody of this disclosure may be an antibody as described herein, such as F4.7.6, F4.7.8, F5.1.11, F5.1.9, F4.7.062, F5.11.1.01, F5.1.11.02, F5.1.11.03, F5.1.11.04, F5.1.11.05, F5.1.11.06, F5.1.11.07, F5.1.11.08, F5.1.11.09, F5.1.9.5, or d1C7, or any variant or portion thereof that retains the ability to bind to IL-2 as described herein. The antibodies of this disclosure may bind to the same epitopes as the reference antibody described herein, or any variant or portion thereof that retains the ability to bind to IL-2 as described herein.

[0249] As previously described elsewhere in this document, specific binding can be evaluated by referring to the binding of the antibody to a non-target molecule. This comparison can be made by comparing the antibody's ability to bind to the target and to another molecule. This comparison can be made as described above at K D or K i The evaluation is conducted. The other molecule used in this comparison can be any molecule that is not the target molecule. In some embodiments, the other molecule is different from the target molecule. In some embodiments, the target molecule is not a fragment of the target molecule.

[0250] Other molecules used to determine specific binding may be structurally or functionally unrelated to the target. For example, these other molecules may be irrelevant or accompanying materials in the environment.

[0251] Another molecule used to determine specific binding can be another molecule that involves the same in vivo pathway as the target molecule (i.e., IL-2). Unwanted in vivo cross-reactivity can be avoided by ensuring that the antibody of this disclosure has more specificity for IL-2 than another such molecule.

[0252] The antibody disclosed herein retains the ability to bind to some molecules associated with the target molecule.

[0253] Alternatively, the antibodies of this disclosure may be specific to a particular target molecule. For example, it may bind to one target molecule as described herein, but may not bind to different target molecules as described herein, or may bind to different target molecules with significantly reduced affinity. For example, full-length mature human IL-2 may be used as a target, but an antibody binding to that target may not bind or may bind with lower affinity to other IL-2 proteins, such as other mammalian IL-2, for example. In some embodiments, the antibody binds to both human and mouse IL-2.

[0254] The peptide or antibody “fragment” or “part” according to this disclosure can be prepared by truncation, for example by removing one or more amino acids from the N and / or C terminus of the peptide. In this way, up to 10, 20, 30, 40 or more amino acids can be removed from the N and / or C terminus. Parts can also be generated by one or more internal deletions.

[0255] The antibody disclosed herein may be a portion of or a variant of any one of antibodies F4.7.6, F4.7.8, F5.1.11, F5.1.9, F4.7.062, F5.11.1.01, F5.1.11.02, F5.1.11.03, F5.1.11.04, F5.1.11.05, F5.1.11.06, F5.1.11.07, F5.1.11.08, F5.1.11.09, F5.1.9.5, or d1C7. The antibody disclosed herein may be or may contain an antigen-binding portion of such antibody or a variant thereof. For example, the antibody disclosed herein may be the Fab portion of the antibody or a variant thereof, or may be a single-chain antibody derived from the antibody or a variant thereof.

[0256] Variant antibodies may contain one, two, three, four, five, up to ten, up to twenty, up to thirty or more amino acid substitutions and / or deletions and / or insertions from specific sequences and portions discussed above. "Deletion" variants may contain the deletion of individual amino acids, a small group of amino acids such as 2, 3, 4, or 5 amino acids, or a larger amino acid region, such as the deletion of a specific amino acid domain or other feature. "Insertion" variants may contain the insertion of individual amino acids, a small group of amino acids such as 2, 3, 4, or 5 amino acids, or an insertion of a larger amino acid region, such as the insertion of a specific amino acid domain or other feature. In some embodiments, "substitution" variants involve replacing one or more amino acids with the same number of amino acids and performing conserved amino acid substitutions. For example, the amino acid may be replaced with an alternative amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. Some properties of the 20 major amino acids that can be used to select suitable substituents are described below.

[0257] Substitutional variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted at its position. The most favorable sites for substitutional mutagenesis include hypervariable domains, but skeletal alterations are also considered. Conservative substitutions are shown under the heading “Conservative Substitutions” in Table 1. If such substitutions result in changes in biological activity, a greater number of alterations, designated as “Exemplary Substitutions,” as shown below or further described below with respect to amino acids, can be introduced, and products screened.

[0258] Table 1: Amino Acid Substitutions

[0259]

[0260]

[0261] The fundamental modification of the biological properties of antibodies is accomplished through selective substitution, which differs significantly in maintaining: (a) the structure of the polypeptide backbone in the substituted region, such as a β-sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the volume of the side chains. Naturally occurring residues are grouped into several groups based on common side chain properties:

[0262] (1) Nonpolar: Leucine, Met, Ala, Val, Leu, Ile;

[0263] (2) Polar and uncharged: Cys, Ser, Thr, Asn, Gln;

[0264] (3) Acidic (negatively charged): Asp, Glu;

[0265] (4) Alkaline (positively charged): Lys, Arg;

[0266] (5) Residues affecting chain orientation: Gly, Pro; and

[0267] (6) Aromatics: Trp, Tyr, Phe, His.

[0268] Non-conservative substitution is achieved by exchanging members of one of these categories for another.

[0269] For example, one type of substitution that can be performed is changing one or more chemically reactive cysteine ​​residues in the antibody to another residue, such as, but not limited to, alanine or serine. For example, non-canonical cysteine ​​substitutions may be present. Substitutions can be performed in the CDR or framework regions or constant regions of the variable structural domains of the antibody. In some embodiments, cysteine ​​is canonical. Any cysteine ​​residue not involved in maintaining the correct conformation of the antibody may also generally be substituted with serine to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bonds can be added to the antibody to improve its stability, particularly when the antibody is an antibody moiety such as the Fv moiety.

[0270] This disclosure also provides methods for generating, selecting, and preparing IL-2 antibodies. The antibodies of this disclosure can be prepared using procedures known in the art. In some embodiments, the antibodies can be recombinantly prepared and expressed using any method known in the art. This disclosure also provides methods for generating, selecting, and preparing hIL-2 antibodies. The antibodies of this disclosure can be prepared using procedures known in the art. In some embodiments, the antibodies can be recombinantly prepared and expressed using any method known in the art.

[0271] In some embodiments, antibodies can be prepared and selected using phage display technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., Annu. Rev. Immunol. 12:433-455, 1994. Alternatively, phage display technology (McCafferty et al., Nature 348:552-553, 1990) can be used to generate human antibodies and antibody moieties in vitro from a gene bank of immunoglobulin variable (V) domains from unimmunized donors. According to this technology, antibody V domain gene frames are cloned into major or minor coat protein genes such as M13 or fd of filamentous phages and displayed as functional antibody moieties on the surface of phage particles. Because filamentous particles contain single-stranded DNA copies of the phage genome, selection based on antibody functional properties also leads to the selection of genes encoding antibodies exhibiting those properties. Therefore, phages mimic some properties of B cells. Phage display can be performed in various forms; for reviews, see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571, 1993. V gene segments from several sources can be used for phage display. Clackson et al., Nature 352:624-628, 1991, isolated a diverse array of anti-oxazolone antibodies from a small, randomly combined library of V genes derived from the spleen of immunized mice. V gene libraries from human donors can be constructed, and antibodies against antigens (including self-antigens) can be isolated by essentially following the methods described by Mark et al., 1991, J. Mol. Biol. 222:581-597 or Griffith et al., 1993, EMBO J. 12:725-734. In the innate immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the introduced changes will confer higher affinity, and B cells displaying high-affinity surface immunoglobulins will preferentially replicate and differentiate during subsequent antigen challenges. This natural process can be simulated using a technique called “chain shuffling” (Marks et al., 1992, Bio / Technol. 10:779-783). In this method, the affinity of “major” human antibodies obtained by phage display is improved by sequentially replacing the heavy and light chain V region genes with naturally occurring variants (repositories) of the V domain genes obtained from unimmunized donors. This technique allows for the production of antibodies and antibody moieties with affinity in the pM-nM range.A strategy for preparing very large phage antibody libraries (also known as “mother-of-all libraries”) has been described by Waterhouse et al., Nucl. Acids Res. 21:2265-2266, 1993. Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibodies have similar affinity and specificity to the starting rodent antibody. According to this method, also known as “epitope imprinting,” a library of heavy or light chain V domain genes of rodent antibodies obtained via phage display technology is replaced with a library of human V domain genes, producing a rodent-human chimera. Selection of the antigen leads to the separation of the human variable domain capable of restoring a functional antigen-binding site, i.e., selection of the epitope-dominated (imprinted) partner. When this process is repeated to replace the remaining rodent V domains, human antibodies are obtained (see PCT Publication No. WO 93 / 06213). Unlike traditional humanization of rodent antibodies via CDR transplantation, this technique provides fully human antibodies that do not contain rodent-derived frameworks or CDR residues.

[0272] In some embodiments, hybridoma technology can be used to prepare antibodies. Any mammalian subject, including humans, or antibody-producing cells derived therefrom, can be considered as a basis for the production of mammalian (including human) hybridoma cell lines. As further described herein, the route and schedule of immunization of the host animal generally conform to established and routine techniques used for antibody stimulation and production. Typically, the host animal is inoculated with a measured amount of immunogen (including, as described herein), via intraperitoneal, intramuscular, oral, subcutaneous, intrapedic, and / or intradermal administration.

[0273] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using general somatic cell hybridization techniques, as described by Kohler, B. and Milstein, C., 1975, Nature 256:495-497, or as modified by Buck, DW et al., In Vitro, 18:377-381, 1982. Available myeloma lines include, but are not limited to, X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA. Generally, this technique involves fusing myeloma cells and lymphoid cells using a fusion agent such as polyethylene glycol, or by electronic means well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium such as hypoxanthine-aminopterin-thymidine (HAT) medium to eliminate unhybridized parental cells. Any culture medium supplemented with or without serum described herein can be used to culture hybridomas that secrete monoclonal antibodies. As an alternative to cell fusion technology, EBV immortalized B cells can be used to generate the IL-2 monoclonal antibody disclosed herein. If necessary, hybridomas or other immortalized B cells are expanded and subcloned, and the antiimmunogen activity of the supernatant is determined by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

[0274] Hybridomas that can be used as antibody sources encompass all derivatives and daughter cells of parental hybridomas that produce monoclonal antibodies specific to IL-2 or a portion thereof.

[0275] Hybridomas that produce such antibodies can be grown in vitro or in vivo using known procedures. If needed, monoclonal antibodies can be isolated from culture media or body fluids using routine immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. If present, unwanted activity can be removed, for example, by running the preparation on an adsorbent made from an immunogen attached to a solid phase and eluting or releasing the desired antibody from the immunogen. Immunizing host animals with an IL-2 peptide or containing a portion of a target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, such as keyhole hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin, using bifunctional or derivatizing agents such as maleimide benzoyl sulfosuccinimide (conjugated via cysteine ​​residues), N-hydroxysuccinimide (conjugated via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R... 1 N = C = NR, where R and R 1 They are different alkyl groups.

[0276] When needed, the target IL-2 antibody (monoclonal or polyclonal) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the target antibody can be maintained in the vector within the host cell, which can then be amplified and frozen for future use. Recombinant monoclonal antibody production in cell culture can be performed by cloning the antibody gene from B cells using methods known in the art. See, for example, Tiller et al., 2008, J. Immunol. Methods 329,112; U.S. Patent No. 7,314,622.

[0277] The plasmids shown in Table 2 are deposited in accordance with the Budapest Treaty at the American Center for Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209. The plasmids have been assigned the following accession numbers:

[0278] Table 2

[0279] Antibody strain naming Preservation date F5.1.11.02VH F5.1.11.02-VH September 8, 2016 F5.1.11.02VL F5.1.11.02-VL September 8, 2016

[0280] In some embodiments, the polynucleotide sequence can be used for genetic manipulation to “humanize” the antibody or improve its affinity or other characteristics. Antibodies can also be customized for, for example, dogs, cats, primates, horses, and cattle.

[0281] In some embodiments, fully human antibodies can be obtained using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce a more desired (e.g., fully human antibody) or a more robust immune response can also be used to generate humanized or human antibodies. An example of this technique is Xenomouse from Abgenix, Inc. (Fremont, CA). TM And from Medarex, Inc. (Princeton, NJ) and TC Mouse TM .

[0282] Antibodies can be recombinantly prepared by first isolating antibodies and antibody-producing cells from a host animal, obtaining the gene sequence, and then recombinantly expressing the antibody in host cells (e.g., CHO cells) using the gene sequence. Another approach is to express the antibody sequence in plants (e.g., tobacco) or transgenic milk. Methods for recombinantly expressing antibodies in plants or milk have been disclosed. See, for example, Peeters et al., Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar, Int. Rev. Immunol 13:65, 1995; and Pollock et al., J Immunol Methods 231:147, 1999. Methods for preparing antibody derivatives such as domains, single chains, etc., are known in the art.

[0283] Immunoassays and flow cytometry sorting techniques, such as fluorescence activated cell sorting (FACS), can also be used to isolate antibodies specific to IL-2.

[0284] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies. In some embodiments, hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector (such as the one disclosed in PCT Publication No. WO 87 / 04462) and then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or otherwise myeloma cells that do not produce immunoglobulin proteins to obtain the synthesis of monoclonal antibodies in recombinant host cells. See, for example, PCT Publication No. WO 87 / 04462. DNA can also be modified, for example, by replacing homologous mouse sequences with coding sequences of human heavy and light chain constant domains (Morrison et al., Proc. Nat. Acad. Sci. 81:6851, 1984), or by covalently linking all or part of the coding sequence of an immunoglobulin-coding sequence to the coding sequence of a non-immunoglobulin polypeptide. In this way, "chimeric" or "hybrid" antibodies with the binding specificity of the IL-2 antibody described in this article are prepared.

[0285] Antibody moieties can be produced by proteolytic digestion or other degradation of antibodies, by recombinant methods as described above (i.e., single or fusion peptides), or by chemical synthesis. Antibody peptides, particularly shorter peptides of up to about 50 amino acids, are conveniently prepared by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, antibodies can be produced using an automated peptide synthesizer employing a solid-phase method. See also U.S. Patents 5,807,715; 4,816,567; and 6,331,415.

[0286] In some embodiments, the polynucleotide comprises a sequence encoding a heavy chain and / or light chain variable domain of an IL-2 antibody disclosed herein. The sequence encoding the target antibody may be maintained in a vector within a host cell, which can then be amplified and the host cell frozen for future use. Vectors (including expression vectors) and host cells are also described herein.

[0287] This disclosure includes embodiments of affinity maturation. For example, affinity-matured antibodies can be produced by procedures known in the art (Marks et al., 1992, Bio / Technology, 10:779-783; Barbas et al., 1994, ProcNat.Acad.Sci, USA 91:3809-3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J.Immunol., 155:1994-2004; Jackson et al., 1995, J.Immunol., 154(7):3310-9; Hawkins et al., 1992, J.Mol.Biol., 226:889-896; and PCT Publication No. WO2004 / 058184).

[0288] The following methods can be used to modulate antibody affinity and characterize CDRs. One way to characterize the CDR of an antibody and / or alter (e.g., improve) the binding affinity of a peptide such as an antibody is called “library scan mutagenesis.” Generally, library scan mutagenesis works as follows: Using art-recognized methods, one or more amino acid positions in the CDR are replaced with two or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). This generates small clonal libraries (in some embodiments, one for each amino acid position analyzed), each library having the complexity of two or more members (if two or more amino acids are substituted at each position). Generally, the library also includes clones containing native (unsubstituted) amino acids. Screening for binding affinity to the target peptide (or other binding targets) is performed on a small number of clones from each library, such as approximately 20-80 clones (depending on the complexity of the library), and candidates exhibiting increased, identical, decreased, or no binding are identified. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using, for example, Biacore, which detects approximately 2-fold or greater differences in binding affinity. TM Surface plasmon resonance analysis Biosensors, scintillation proximity assay, ELISA Immunoassays, fluorescence quenching, fluorescence transfer, and / or yeast display. Appropriate bioassays can also be used to screen for binding affinity. When the starting antibody has a relatively high affinity, such as about 10 nM or lower K, [further action is needed]. D When combined, Biacore TM It is particularly useful.

[0289] In some embodiments, using art-recognized mutagenesis methods (some of which are described herein), each amino acid position in the CDR is replaced (one at a time in some embodiments) with all 20 natural amino acids. This generates small clone libraries (one for each amino acid position analyzed in some embodiments), each library having the complexity of 20 members (if all 20 amino acids are substituted at every position). In some embodiments, using art-recognized mutagenesis methods, each amino acid position in the CDR is replaced (one at a time in some embodiments) with all 20 natural amino acids except cysteine.

[0290] In some embodiments, the library to be screened contains substitutions at two or more positions, which may be in the same CDR or in two or more CDRs. Thus, the library may contain substitutions at two or more positions in one CDR. The library may contain substitutions at two or more positions in two or more CDRs. The library may contain substitutions at 3, 4, 5 or more positions, which are found in 2, 3, 4, 5 or 6 CDRs. Substitutions can be prepared using low-redundancy codons. See, for example, Table 2 of Balint et al., 1993, Gene 137(1):109-18.

[0291] The CDR can be a heavy chain variable domain (VH) CDR3 and / or a light chain variable domain (VL) CDR3. The CDR can be one or more of VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3. The CDR can be a Kabat CDR, Chothia CDR, extension CDR, AbM CDR, contact CDR, or conformational CDR.

[0292] In some embodiments, the library is prepared according to SEQ ID NO:71 and 72.

[0293] Candidates with improved binding can be sequenced to identify CDR substitution mutants that result in improved affinity (also known as "improved" substitutions). Candidates with binding can also be sequenced to identify CDR substitutions that retain binding.

[0294] Multiple rounds of screening can be performed. For example, candidates with improved binding (each containing amino acid substitutions at one or more positions in one or more CDRs) can also be used to design a second library containing at least the original and substituted amino acids at each improved CDR position (i.e., the amino acid position in the CDR where the mutant is substituted to show improved binding). The preparation, screening, or selection of such a library is discussed further below.

[0295] Library scanning mutagenesis also provides a means of characterizing CDRs, and the frequency of clones exhibiting improved binding, identical binding, reduced binding, or no binding also provides information about the importance of each amino acid position for the stability of the antibody-antigen complex. For example, if a CDR position retains binding when changed to all 20 amino acids, that position is identified as unlikely to be needed for antigen binding. Conversely, if a CDR position retains binding only with a small percentage of substitutions, that position is identified as important for CDR function. Thus, library scanning mutagenesis generates information about positions in the CDR that can be changed to many different amino acids (including all 20 amino acids) and positions in the CDR that cannot be changed or can only be changed to a few amino acids.

[0296] Candidates with improved affinity can be combined in a second library comprising the improved amino acid, the original amino acid at that position, and may also include additional substitutions at that position, depending on the desired library complexity or to allow for the use of desired screening or selection methods. Additionally, if desired, adjacent amino acid positions can be randomized to at least two or more amino acids. Randomization of adjacent amino acids allows for additional conformational flexibility in the mutant CDR, which in turn allows or facilitates the introduction of a larger number of improving mutations. The library may also contain substitutions at positions that did not show improved affinity in the first round of screening.

[0297] Use any method known in the art to screen or select library members with improved and / or altered binding affinity in a second library, including methods such as Biacore and Kinexa. TM Biosensor analysis and selection using any method known in the art for selection, including phage display, yeast display, and ribosome display.

[0298] To express the IL-2 antibody of this disclosure, a DNA fragment encoding the VH and VL regions can first be obtained using any of the methods described above. Various modifications, such as mutations, deletions, and / or additions, can also be introduced into the DNA sequence using standard methods known to those skilled in the art. For example, mutagenesis can be performed using standard methods, such as PCR-mediated mutagenesis, in which mutated nucleotides are incorporated into PCR primers such that the PCR product contains the desired mutation or site-directed mutagenesis.

[0299] This disclosure covers modifications to the variable domains and CDRs shown in Table 7. For example, this disclosure includes antibodies comprising functionally equivalent variable domains and CDRs that do not significantly affect their properties, and variants having enhanced or reduced activity and / or affinity. For example, the amino acid sequence may be mutated to obtain an antibody with the desired binding affinity for IL-2. In some embodiments, the amino acid sequence may be mutated to obtain an antibody with the desired binding affinity for IL-2. Examples of modified peptides include peptides with conserved substitutions of amino acid residues, the deletion or addition of one or more amino acids that do not significantly and harmfully alter the functional activity or affinity of the mature (enhancing) peptide for its ligands, or the use of chemical analogs.

[0300] Amino acid sequence insertions include fusions of the N-terminus and / or C-terminus of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue or antibodies fused to an additional epitope. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of an antibody with an enzyme or peptide, which increase the antibody's half-life in blood circulation.

[0301] Antibodies can also be modified, for example, in variable domains of the heavy and / or light chains, to alter the antibody's binding properties. Changes in the variable domains can alter binding affinity and / or specificity. In some embodiments, no more than one to five conserved amino acid substitutions are prepared within the CDR domain. In other embodiments, no more than one to three conserved amino acid substitutions are prepared within the CDR domain. For example, mutations can be prepared in one or more CDR regions to increase or decrease the antibody's K-reactivity against IL-2. D Increase or decrease k off Alternatively, it can alter the binding specificity of the antibody. Site-directed mutagenesis is well-known in the field. See, for example, Sambrook et al. and Ausubel et al., ibid.

[0302] Modifications or mutations can also be prepared in the framework or constant regions to increase the half-life of IL-2 antibodies. See, for example, PCT Publication No. WO 00 / 09560. Mutations can also be prepared in the framework or constant regions to alter the immunogenicity of the antibody, provide a site for covalent or nonvalent binding to another molecule, or alter properties such as complement fixation, FcR binding, and antibody-dependent cell-mediated cytotoxicity. According to this disclosure, a single antibody may have mutations in any one or more CDRs or framework or constant regions of the variable domain.

[0303] Modifications also include glycosylated and non-glycosylated peptides, as well as peptides with other post-translational modifications, such as glycosylation, acetylation, and phosphorylation with different sugars. Antibodies are glycosylated at conserved sites in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32). Oligosaccharide side chains of immunoglobulins influence protein function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:4175-4180) and intramolecular interactions between glycoprotein moieties, which can affect conformation and present the three-dimensional surface of glycoproteins (Jefferis and Lund, ibid.; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416). Based on specific recognition structures, oligosaccharides can also act to target given glycoproteins to certain molecules. Antibody glycosylation has been reported to affect antibody-dependent cytotoxicity (ADCC). In particular, antibodies produced by CHO cells expressed with tetracycline-regulated β(1,4)-N-acetylglucosamine transferase III (GnTIII) (a glycosyltransferase that catalyzes the formation of bipartite GlcNAc) have been reported to have improved ADCC activity (Umana et al., 1999, Nature Biotech. 17: 176-180).

[0304] Antibody glycosylation is typically N-linked or O-linked. N-linking refers to the attachment of the carbohydrate moiety to the asparagine residue side chain. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine ​​(where X is any amino acid except proline) are recognition sequences used for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0305] Adding glycosylation sites to antibodies can be conveniently achieved by altering the amino acid sequence to include one or more of the aforementioned tripeptide sequences (for N-glycosylation sites). This alteration can also be made by adding one or more serine or threonine residues to the original antibody sequence, or by substituting one or more serine or threonine residues (for O-glycosylation sites).

[0306] The glycosylation pattern of an antibody can also be altered without changing the underlying nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Because the cell types used to express recombinant glycoproteins such as antibodies as potential therapeutic agents are rarely native cells, variations in the glycosylation pattern of the antibody can be expected (see, for example, Hse et al., 1997, J. Biol. Chem. 272:9062-9070).

[0307] Besides host cell selection, factors influencing glycosylation during recombinant antibody production include growth pattern, culture medium formulation, culture density, oxygenation, pH, purification protocol, and more. Various methods have been proposed to alter the glycosylation patterns acquired in specific host organisms, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (US Patent Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain types of glycosylation, can be enzymatically removed from glycoproteins, for example, using endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, and endoglycosidase F3. Additionally, recombinant host cells can be genetically modified to be deficient in processing certain types of polysaccharides. These and similar techniques are well known in the art.

[0308] Other modification methods include the use of coupling techniques known in the art, including but not limited to enzymatic methods, oxidative substitution, and chelation. For example, modification can be used to attach tags for immunoassays. Modified peptides are prepared using procedures established in the art and can be screened using standard assays known in the art, some of which are described below and in examples.

[0309] In some embodiments, the antibody comprises a modified constant region having increased or decreased binding affinity for the human Fcγ receptor and being immunologically inert or partially inert, for example, not triggering complement-mediated cleavage, not stimulating antibody-dependent cell-mediated cytotoxicity (ADCC), or not activating microglia; or having reduced activity (compared to an unmodified antibody) in any one or more of the following: triggering complement-mediated cleavage, stimulating ADCC, or activating microglia. Different modifications to the constant region can be used to achieve optimal levels and / or combinations of effector functions. See, for example, Morgan et al., Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-9 157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000; Tao et al., J. Immunology 143:2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:59-76, 1998. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29:2613-2624; PCT application number PCT / GB99 / 01441; and / or UK patent application number 9809951.8.

[0310] In some embodiments, the antibody constant region can be modified to avoid interaction with the Fcγ receptor, as well as with the complement and immune systems. Techniques for preparing such antibodies are described in WO 99 / 58572. For example, if the antibody is to be used in clinical trials and treatments in humans, the constant region can be modified to more closely resemble the human constant region to avoid an immune response. See, for example, U.S. Patent Nos. 5,997,867 and 5,866,692.

[0311] In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29: 2613-2624; PCT application number PCT / GB99 / 01441; and / or UK patent application number 9809951.8. In such embodiments, Fc may be human IgG2 or human IgG4. Fc may contain mutations A330P331 to S330S331 (IgG). 2ΔaThe antibody comprises human IgG2, wherein the amino acid residues are numbered with reference to the wild-type IgG2 sequence. Eur. J. Immunol., 1999, 29: 2613-2624. In some embodiments, the antibody comprises an IgG4 constant region containing the following mutations (Armour et al., 2003, Molecular Immunology 40 585-593): E233F234L235 to P233V234A235 (IgG 4Δc The numbering is based on wild-type IgG4. In another embodiment, Fc is a variant with G236 (IgG) missing. 4Δb The Fc is any human IgG4 E233F234L235 to P233V234A235 containing hinge-stable mutations S228 to P228. In another embodiment, Fc is any human IgG4 Fc (IgG4, IgG...) containing hinge-stable mutations S228 to P228. 4Δb or IgG 4Δc (Aalberse et al., 2002, Immunology 105, 9-19).

[0312] In some embodiments, the antibody includes a human heavy chain IgG2 constant region containing the following mutations: A330P331 to S330S331 (amino acid numbering references the wild-type IgG2 sequence). Eur. J. Immunol., 1999, 29:2613-2624. In other embodiments, the constant region is glycosylated-free for N-glycosylation. In some embodiments, the constant region is glycosylated-free for N-glycosylation by mutating oligosaccharide attachment residues and / or flanking residues (which are part of the N-glycosylation recognition sequence in the constant region). For example, the N-glycosylation site N297 can be mutated to, for example, A, Q, K, or H. See Tao et al., J. Immunology 143:2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:59-76, 1998. In some embodiments, the constant region is glycosylated-free for N-glycosylation. The constant region can be enzymatically deglycosylated (e.g., by removing carbohydrates via the enzyme PNGase) or by being expressed in glycosylation-deficient host cells, thus being glycosylated-free for N-glycosylation.

[0313] Other antibody modifications include antibodies modified as described in PCT Publication No. WO 99 / 58572. In addition to the binding domain against the target molecule, these antibodies also contain an effector domain having an amino acid sequence substantially homologous to all or part of the constant regions of the human immunoglobulin heavy chain. These antibodies are able to bind to the target molecule without triggering significant complement-dependent cleavage or cell-mediated target destruction. In some embodiments, the effector domain is capable of specifically binding to FcRn and / or FcγRIIb. These are typically based on chimeric domains derived from the CH2 domains of two or more individual immunoglobulin heavy chains. Antibodies modified in this manner are particularly suitable for chronic antibody therapy to avoid inflammation and other adverse reactions to conventional antibody therapy.

[0314] This disclosure also provides antibody constant domains that can be further modified. Variants of the known Fc region (e.g., amino acid substitutions, insertions and / or additions and / or deletions) enhance or reduce effector function. See, for example, Presta et al., 2002, Biochem. Soc. Trans. 30:487-490; Strohl, 2009, Curr. Opin. Biotechnol. 20(6):685-691; U.S. Patents 5,624,821, 5,648,260, 5,885,573, 6,737,056, 7,317,091; PCT Publications WO 99 / 58572, WO 00 / 42072, WO 04 / 029207, WO 2006 / 105338, WO 2008 / 022152, WO 2008 / 150494, WO 2010 / 033736; U.S. Patent Application Publications 2004 / 0132101, 2006 / 0024298. 2006 / 0121032, 2006 / 0235208, 2007 / 0148170; Armour et al., 1999, Eur. J. Immunol. 29(8): 2613-2624 (reduced ADCC and CDC); Shields et al., 2001, J. Biol. Chem. 276(9): 6591-6604 (reduced ADCC and CDC); Idusogie et al., 2000, J. Immunol. 164(8): 4178-4184 (increased ADCC and CDC); Steurer et al., 1995, J. Immunol. 155(3): 1165-1174 (reduced ADCC and CDC); Idusogie et al., 2001, J. Immunol. 166(4): 2571-2575 (increased ADCC and CDC); Lazar et al., 2006, Proc. Natl. Acad. Sci. USA 103(11): 4005-4010 (increased ADCC); Ryan et al., 2007, Mol. Cancer. Ther., 6: 3009-3018 (increased ADCC); Richards et al., 2008, Mol. Cancer Ther. 7(8): 2517-2527.

[0315] In some embodiments, the antibody includes a modified constant region that, compared to an unmodified antibody, has increased binding affinity for FcRn and / or an increased serum half-life.

[0316] During a process known as “germlining,” certain amino acids in the VH and VL sequences can be mutated to match those naturally found in germline VH and VL sequences. Specifically, the amino acid sequences of the framework regions in the VH and VL sequences can be mutated to match germline sequences to reduce the risk of immunogenicity during antibody administration. Germline DNA sequences of the human VH and VL genes are known in the art (see, for example, the “Vbase” human germline sequence database; also see Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al., 1992, J. Mol. Biol. 227:776-798; and Cox et al., 1994, Eur. J. Immunol. 24:827-836).

[0317] Another type of amino acid substitution that can be prepared involves removing potential protease-cleaving sites in the antibody. Such sites may be present in the CDR or framework region or constant region of the variable domain of the antibody. Substitution of cysteine ​​residues and removal of protease-cleaving sites can reduce the risk of heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution involves eliminating the asparagine-glycine pair that forms a potential deamidation site by changing one or two residues. In another example, the C-terminal lysine of the heavy chain of the IL-2 antibody of this disclosure may be cleaved or otherwise removed. In various embodiments of this disclosure, the heavy and light chains of the antibody may optionally include a signal sequence.

[0318] Once the DNA fragments encoding the disclosed VH and VL regions are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable domain genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operatively linked" is intended to mean that the two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain within the frame.

[0319] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operatively linking the DNA encoding the VH region to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but in some embodiments, they are IgG1 or IgG2 constant regions. The IgG constant region sequence can be any of the various alleles or allotypes known to occur in different individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypes represent naturally occurring amino acid substitutions in the IgG1 constant region. For Fab fragment heavy chain genes, DNA encoding VH can be operatively linked to another DNA molecule that encodes only the CH1 constant region of the heavy chain. The CH1 heavy chain constant region can be derived from any of the heavy chain genes.

[0320] By operatively linking the DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, the isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a κ or λ constant region. The κ constant region can be any of the various alleles known to occur in different individuals, such as Inv(1), Inv(2), and Inv(3). The λ constant region can originate from any of the three λ genes.

[0321] To generate the scFv gene, DNA fragments encoding VH and VL are operatively linked to another fragment encoding a flexible adapter, allowing the VH and VL sequences to be expressed as adjacent single-chain proteins, with the VL and VH regions connected by a flexible adapter (see, for example, Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554; adapters for other sequences have been designed and used (Bird et al., 1988, ibid.). The adapter can then be further modified for additional functions, such as drug attachment or solid support attachment. If only a single VH and VL are used, the single-chain antibody can be monovalent; if two VH and VL are used, it can be bivalent. Alternatively, if more than two VH and VL are used, it can be multivalent. Bispecific or multivalent antibodies that specifically bind to IL-2 and another molecule can be generated. Single-chain variants can be produced recombinantly or synthetically. For the synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, which can be a eukaryotic cell such as yeast, plant, insect, or mammalian cell, or a prokaryotic cell such as Escherichia coli. The polynucleotide encoding the target scFv can be prepared by conventional procedures such as polynucleotide ligation. The obtained scFv can be isolated using standard protein purification techniques known in the art.

[0322] It also covers other forms of single-chain antibodies, such as biantibodies. Biantibodies are bivalent bispecific antibodies in which VH and VL are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating two antigen-binding sites (see, for example, Holliger, P. et al., 1993, Proc. Natl. Acad Sci. USA 90: 6444-6488; and Poljak, RJ et al., 1994, Structure 2: 1121-1123).

[0323] Heteroconjugated antibodies comprising two covalently linked antibodies are also within the scope of this disclosure. Such antibodies have been used to target unwanted cells with immune system cells (US Patent No. 4,676,980) and to treat HIV infection (PCT Publications WO 91 / 00360 and WO 92 / 200373; and EP 03089). Heteroconjugated antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are described in US Patent No. 4,676,980.

[0324] Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutyrylimides.

[0325] This disclosure also covers fusion proteins comprising one or more portions or regions of antibodies disclosed herein. In some embodiments, a fusion antibody may be prepared comprising all or a portion of the IL-2 antibody of this disclosure linked to another polypeptide. In another embodiment, only the variable domain of the IL-2 antibody is linked to the polypeptide. In another embodiment, the VH domain of the IL-2 antibody is linked to a first polypeptide, and the VL domain of the IL-2 antibody is linked to a second polypeptide, the second polypeptide binding to the first polypeptide in such a way that the VH and VL domains can interact with each other to form an antigen-binding site. In another embodiment, the VH domain is separated from the VL domain by a linker, such that the VH and VL domains can interact with each other. The VH-linker-VL antibody is then linked to the target polypeptide. Alternatively, fusion antibodies may be generated in which two (or more) single-chain antibodies are linked to each other. This is useful if it is desired to generate bivalent or multivalent antibodies on a single polypeptide chain, or if it is desired to generate bispecific antibodies.

[0326] In some embodiments, a fusion polypeptide is provided comprising at least 10 adjacent amino acids of the variable light chain region shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 72, and / or at least 10 amino acids of the variable heavy chain region shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 71. In other embodiments, a fusion polypeptide is provided comprising at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 adjacent amino acids of the variable light chain region, and / or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 adjacent amino acids of the variable heavy chain region. In yet another embodiment, the fusion polypeptide comprises one or more CDRs. In other embodiments, the fusion peptide comprises VH CDR3 and / or VL CDR3. For the purposes of this disclosure, the fusion protein comprises one or more antibodies and another amino acid sequence to which it is not attached in the natural molecule, such as a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to, "tags," such as FLAG tags or 6His tags (SEQ ID NO: 223). Tags are well known in the art.

[0327] Fusion peptides can be generated by methods known in the art, such as synthesis or recombination. Typically, the fusion proteins of this disclosure are prepared by using the recombination methods described herein to prepare and express the polynucleotide encoding it, although they can also be prepared by other means known in the art, including, for example, chemical synthesis.

[0328] In other embodiments, other modified antibodies can be prepared using nucleic acid molecules encoding IL-2 antibodies. For example, “κ bodies” (Ill et al., 1997, Protein Eng. 10:949-57), “microantibodies” (Martin et al., 1994, EMBO J. 13:5303-9), “dual antibodies” (Holluger et al., ibid.), or “Janusins” (Traunecker et al., 1991, EMBO J. 10:3655-3659 and Traunecker et al., 1992, Int. J. Cancer (Suppl.) 7:51-52) can be prepared using standard molecular biology techniques following the instructions.

[0329] For example, bispecific antibodies, monoclonal antibodies with binding specificity for at least two different antigens, can be prepared using the antibodies disclosed herein. Methods for preparing bispecific antibodies are known in the art (see, for example, Suresh et al., 1986, Methods in Enzymology 121:210). For example, bispecific antibodies or antigen-binding moieties can be generated by hybridoma fusion or Fab' moieties. See, for example, Songsivilai & Lachmann, 1990, Clin. Exp. Immunol. 79:315-321, and Kostelny et al., 1992, J. Immunol. 148:1547-1553. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, 1983, Nature 305, 537-539). Additionally, bispecific antibodies can be formed as "biantibodies" or "Janusins". In some embodiments, the bispecific antibody binds to two different epitopes of IL-2. In some embodiments, the modified antibody described above is prepared using one or more of the variable domains or CDR regions from the IL-2 antibody provided herein.

[0330] According to a method for preparing bispecific antibodies, an antibody variable domain (antibody-antigen binding site) having the desired binding specificity is fused to an immunoglobulin constant region sequence. In some embodiments, the fusion uses an immunoglobulin heavy chain constant region comprising at least a portion of a hinge region, CH2, and CH3 regions. In some embodiments, a first heavy chain constant region (CH1) containing the desired light chain binding site is present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion and (if needed) the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the relative proportions of the three polypeptide moieties in this example when unequal ratios of the three polypeptide chains used in the construction provide optimal yield. However, when high yields are produced by expressing at least two polypeptide chains at the same ratio, or when the ratios are not particularly significant, it is possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

[0331] In one method, the bispecific antibody comprises: a hybrid immunoglobulin heavy chain having a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair in the other arm (providing a second binding specificity). This asymmetric structure, with the immunoglobulin light chain in only half of the bispecific molecule, facilitates the separation of the desired bispecific compound from the unwanted immunoglobulin chain combination. This method is described in PCT Publication No. WO 94 / 04690.

[0332] This disclosure also provides compositions comprising antibodies conjugated (e.g., linked) to a reagent that facilitates conjugation to a solid support (such as biotin or avidin). For simplicity, the general reference to antibodies implies that these methods are applicable to any IL-2 binding embodiments described herein. Conjugation generally refers to linking these components as described herein. Linking (generally fixing these components in the closest possible combination at least for application) can be achieved in many ways. For example, a direct reaction between the reagent and the antibody is possible when each has a substituent capable of reacting with the other. For example, a nucleophilic group such as an amino or thiosulfate group on one may be capable of reacting with a carbonyl group such as an anhydride or acyl halide, or an alkyl group containing a good leaving group (e.g., a halide) on the other.

[0333] Antibodies can bind to many different carriers. Carriers can be active and / or inert. Well-known examples of carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural and modified cellulose, polyacrylamide, agarose, and magnetite. For the purposes of this disclosure, the carrier can be soluble or insoluble. Those skilled in the art will recognize other suitable carriers for antibody binding, or will be able to determine this using routine experiments.

[0334] The antibodies or peptides disclosed herein can be linked to labeling agents such as fluorescent molecules, radioactive molecules, or any other labels known in the art. Labels that generally provide (direct or indirect) signals are known in the art.

[0335] The amino acid sequences of the light chain variable domain (VL) and heavy chain variable domain (VH) of the IL-2 antibody disclosed in this article are summarized by sequence identifiers in Table 7.

[0336] The antibodies disclosed herein may comprise both of the following:

[0337] a) VH containing the amino acid sequence of SEQ ID NO:1 and VL containing the amino acid sequence of SEQ ID NO:2,

[0338] b) VH containing the amino acid sequence of SEQ ID NO:3, and VL containing the amino acid sequence of SEQ ID NO:4.

[0339] c) VH containing the amino acid sequence of SEQ ID NO:5 and VL containing the amino acid sequence of SEQ ID NO:6.

[0340] d) VH containing the amino acid sequence of SEQ ID NO:7 and VL containing the amino acid sequence of SEQ ID NO:8.

[0341] e) VH containing the amino acid sequence of SEQ ID NO:9 and VL containing the amino acid sequence of SEQ ID NO:10.

[0342] f) VH containing the amino acid sequence of SEQ ID NO:11 and VL containing the amino acid sequence of SEQ ID NO:12.

[0343] g) VH containing the amino acid sequence of SEQ ID NO:13 and VL containing the amino acid sequence of SEQ ID NO:14.

[0344] h) VH containing the amino acid sequence of SEQ ID NO:15 and VL containing the amino acid sequence of SEQ ID NO:16.

[0345] i) VH containing the amino acid sequence of SEQ ID NO:17 and VL containing the amino acid sequence of SEQ ID NO:18

[0346] j) VH containing the amino acid sequence of SEQ ID NO:19 and VL containing the amino acid sequence of SEQ ID NO:20.

[0347] k) VH containing the amino acid sequence of SEQ ID NO:21 and VL containing the amino acid sequence of SEQ ID NO:22.

[0348] 1) VH containing the amino acid sequence of SEQ ID NO:23 and VL containing the amino acid sequence of SEQ ID NO:24.

[0349] m) VH containing the amino acid sequence of SEQ ID NO:25 and VL containing the amino acid sequence of SEQ ID NO:26

[0350] n) VH containing the amino acid sequence of SEQ ID NO:27 and VL containing the amino acid sequence of SEQ ID NO:28

[0351] o) VH containing the amino acid sequence of SEQ ID NO:29 and VL containing the amino acid sequence of SEQ ID NO:30

[0352] p) VH containing the amino acid sequence of SEQ ID NO:31 and VL containing the amino acid sequence of SEQ ID NO:32, or

[0353] q) VH containing the amino acid sequence of SEQ ID NO:71 and VL containing the amino acid sequence of SEQ ID NO:72.

[0354] In another aspect, the antibody comprises variants of these sequences, wherein these variants may include conserved and non-conserved substitutions, deletions and / or additions, and generally include peptides that share at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the specific sequences disclosed herein.

[0355] For example, in one aspect, this disclosure provides an isolated antibody or its antigen-binding portion comprising V as shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32 or SEQ ID NO:72. LThe chain amino acid sequence or a variant thereof. In one aspect, the antibody variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conserved or non-conserved substitutions for SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32 or SEQ ID NO:72, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 additions and / or deletions. In a further aspect, the variant shares at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:72, and wherein the antibody or antigen-binding moiety specifically binds to IL-2. In some embodiments, the antibody or antigen-binding moiety specifically binds to hIL-2.

[0356] In a further aspect, this disclosure provides isolated antibodies or antigen-binding portions thereof, comprising V as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:71. HThe chain amino acid sequence or a variant thereof. In one aspect, the antibody variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conserved or non-conserved substitutions for SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:71, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 additions and / or deletions. In a further aspect, the variant shares at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:71, and wherein the antibody or antigen-binding moiety specifically binds to IL-2. In some embodiments, the antibody or antigen-binding moiety specifically binds to hIL-2.

[0357] The antibodies disclosed herein may include a heavy chain comprising a VH, said VH comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:71, wherein said antibody further comprises a heavy chain constant domain. As more fully described elsewhere herein, the antibody heavy chain constant domain may be selected from the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, but in some embodiments, it is the IgG1 or IgG2 constant region. The IgG constant region sequence can be any of the various alleles or allotypes known to occur in different individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). For Fab fragment heavy chain genes, DNA encoding VH can be operatively linked to another DNA molecule that encodes only the CH1 heavy chain constant region. The CH1 heavy chain constant region can be derived from any of the heavy chain genes.

[0358] In one aspect, the antibody may comprise a heavy chain comprising a VH selected from the following, wherein the VH comprises an amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:71, and further comprises an IgG1 constant domain comprising a triple mutation (hIgG1-3m; SEQ ID NO:2) that reduces or eliminates the function of the Fc effector. In one aspect, the antibody variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conserved or non-conserved substitutions to the full-length heavy chain, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and / or deletions. In a further aspect, the variant shares at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the full-length heavy chain, and wherein the antibody or antigen-binding portion specifically binds to IL-2.

[0359] The antibodies disclosed herein may comprise a light chain containing a VL, said VL comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:72, wherein said antibody further comprises a light chain constant domain. As more fully described elsewhere herein, the antibody light chain constant domain may be selected from a Cκ or Cλ constant region, such as the Cλ constant region of SEQ ID NO:1. In one aspect, the antibody variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conserved or non-conserved substitutions to the full-length light chain, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and / or deletions. In a further aspect, the variant shares at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the full-length light chain, and wherein the antibody or antigen-binding portion specifically binds to IL-2.

[0360] The antibodies disclosed herein may contain a portion of one of the VL or VH amino acid sequences shown in Table 7. For example, the antibody disclosed herein may comprise a portion of at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 20, or at least 25 consecutive amino acids from VH or VL, wherein the VH comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:71, and the VL is derived from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:31, or SEQ ID NO:71. SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:72. Such portions will preferably retain one or more of the functions discussed above, such as the ability to bind to IL-2. In some embodiments, such portions will preferably retain one or more of the functions discussed above, such as the ability to bind to hIL-2.

[0361] In some embodiments, the antibodies of this disclosure comprise VH CDR1, CDR2, and CDR3 according to the Kabat, Chothia, or extended sequences and / or VL CDR1, CDR2, and CDR3 according to the Kabat and / or Chothia amino acid sequences, as shown in Table 7. In some embodiments, the antibodies of this disclosure comprise VH CDR1 according to the following Kabat amino acid sequences: SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:91, SEQ ID NO:100, SEQ ID NO:109, SEQ ID NO:118, SEQ ID NO:127, SEQ ID NO:136, SEQ ID NO:145, SEQ ID NO:154, SEQ ID NO:163, SEQ ID NO:172, SEQ ID NO:181, SEQ ID NO:190, SEQ ID NO:199, or SEQ ID NO:208. In some embodiments, the antibody disclosed herein comprises VH CDR1 according to the following Chothia amino acid sequences: SEQ ID NO:74, SEQ ID NO:83, SEQ ID NO:92, SEQ ID NO:101, SEQ ID NO:110, SEQ ID NO:119, SEQ ID NO:128, SEQ ID NO:137, SEQ ID NO:146, SEQ ID NO:155, SEQ ID NO:164, SEQ ID NO:173, SEQ ID NO:182, SEQ ID NO:191, SEQ ID NO:200, or SEQ ID NO:209. In some embodiments, the antibody disclosed herein comprises VH CDR1 according to the following combined amino acid sequences: SEQ ID NO:75, SEQ ID NO:84, SEQ ID NO:93, SEQ ID NO:102, SEQ ID NO:111, SEQ ID NO:120, SEQ ID NO:129, SEQ ID NO:138, SEQ ID NO:147, SEQ ID NO:156, SEQ ID NO:165, SEQ ID NO:174, SEQ ID NO:183, SEQ ID NO:192, SEQ ID NO:201, or SEQ ID NO:210.In some embodiments, the antibody disclosed herein comprises VH CDR2 according to the following Kabat amino acid sequences: SEQ ID NO:76, SEQ ID NO:85, SEQ ID NO:94, SEQ ID NO:103, SEQ ID NO:112, SEQ ID NO:121, SEQ ID NO:130, SEQ ID NO:139, SEQ ID NO:148, SEQ ID NO:157, SEQ ID NO:166, SEQ ID NO:175, SEQ ID NO:184, SEQ ID NO:193, SEQ ID NO:202, or SEQ ID NO:211. In some embodiments, the antibody disclosed herein comprises VH CDR2 according to the following Chothia amino acid sequences: SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:95, SEQ ID NO:104, SEQ ID NO:113, SEQ ID NO:122, SEQ ID NO:131, SEQ ID NO:140, SEQ ID NO:149, SEQ ID NO:158, SEQ ID NO:167, SEQ ID NO:176, SEQ ID NO:185, SEQ ID NO:194, SEQ ID NO:203, or SEQ ID NO:212. In some embodiments, the antibody disclosed herein comprises VH CDR3 according to the following amino acid sequences: SEQ ID NO:78, SEQ ID NO:87, SEQ ID NO:96, SEQ ID NO:105, SEQ ID NO:114, SEQ ID NO:123, SEQ ID NO:132, SEQ ID NO:141, SEQ ID NO:150, SEQ ID NO:159, SEQ ID NO:168, SEQ ID NO:177, SEQ ID NO:186, SEQ ID NO:195, SEQ ID NO:204, or SEQ ID NO:213.In some embodiments, the antibody disclosed herein comprises VL CDR1 according to the following amino acid sequences: SEQ ID NO:79, SEQ ID NO:88, SEQ ID NO:97, SEQ ID NO:106, SEQ ID NO:115, SEQ ID NO:124, SEQ ID NO:133, SEQ ID NO:142, SEQ ID NO:151, SEQ ID NO:160, SEQ ID NO:169, SEQ ID NO:178, SEQ ID NO:187, SEQ ID NO:196, SEQ ID NO:205, SEQ ID NO:214, or SEQ ID NO:220. In some embodiments, the antibody disclosed herein comprises VL CDR2 according to the following amino acid sequences: SEQ ID NO:80, SEQ ID NO:89, SEQ ID NO:98, SEQ ID NO:107, SEQ ID NO:116, SEQ ID NO:125, SEQ ID NO:134, SEQ ID NO:143, SEQ ID NO:152, SEQ ID NO:161, SEQ ID NO:170, SEQ ID NO:179, SEQ ID NO:188, SEQ ID NO:197, SEQ ID NO:206, SEQ ID NO:215, or SEQ ID NO:221. In some embodiments, the antibody of this disclosure comprises VL CDR3 according to the following amino acid sequences: SEQ ID NO:81, SEQ ID NO:90, SEQ ID NO:99, SEQ ID NO:108, SEQ ID NO:117, SEQ ID NO:126, SEQ ID NO:135, SEQ ID NO:144, SEQ ID NO:153, SEQ ID NO:162, SEQ ID NO:171, SEQ ID NO:180, SEQ ID NO:189, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NO:216, or SEQ ID NO:222. In some embodiments, the antibody of this disclosure comprises VH CDR1 according to SEQ ID NO:217. In some embodiments, the antibody of this disclosure comprises VH CDR2 according to SEQ ID NO:218. In some embodiments, the antibody of this disclosure comprises VH CDR3 according to SEQ ID NO:219.

[0362] In some embodiments, the antibodies of this disclosure comprise VH CDR1, CDR2, and CDR3 based on the Kabat, Chothia, or extended sequences of the antibodies listed below and / or VL CDR1, CDR2, and CDR3 based on the Kabat and / or Chothia amino acid sequences: F4.7.6 VH, F4.7.8 VH, F5.1.11 VH, F5.1.9 VH, F4.7.062 VH, F5.1.11.01 VH, F5.1.11.02 VH, F5.1.11.03 VH, F5.1.11.04 VH, F5.1.11.05 VH, F5.1.11.06 VH, F5.1.11.07 VH, F5.1.11.08 VH, F5.1.11.09 VH, F5.1.9.5 VH, d1C7 VH, F4.7.6 VL, F4.7.8 VL, F5.1.11 VL, F5.1.9 VL, F4.7.062 VL, F5.1.11.01 VL, F5.1.11.02 VL, F5.1.11.03 VL, F5.1.11.04 VL, F5.1.11.05 VL, F5.1.11.06 VL, F5.1.11.07 VL, F5.1.11.08 VL, F5.1.11.09 VL, F5.1.9.5 VL, or d1C7 VL, as shown in Table 7. In some embodiments, the antibodies of this disclosure comprise VH CDR1, CDR2 and CDR3 of antibody F5.1.11.02 according to the Kabat, Chothia or extended sequence and / or VL CDR1, CDR2 and CDR3 according to the Kabat and / or Chothia amino acid sequence, as shown in Table 7.

[0363] Suitable portions or variants of any of these VH or VL sequences will retain the ability to bind to IL-2. In some embodiments, it will preferably retain the ability to specifically bind to IL-2. In some embodiments, it will preferably retain the ability to specifically bind to the same or similar epitopes or regions of the IL-2 molecule from which the antibody is derived. In some embodiments, it will preferably retain one or more additional functions of the antibody from which it is derived, particularly, for example, binding to hIL-2, reducing the binding of hIL-2 to IL-2Rα and IL-2Rβ, and being retained by Treg.

[0364] In some embodiments, a suitable portion or variant of any of these VH or VL sequences will retain the ability to bind to hIL-2. In some embodiments, it will retain the ability to specifically bind to hIL-2. In some embodiments, it will retain the ability to specifically bind to the same or similar epitopes or regions of the hIL-2 molecule from which the antibody is derived. In some embodiments, it will retain one or more additional functions of the antibody from which it is derived, particularly, for example, binding to hIL-2, reducing the binding of hIL-2 to IL-2Rα and IL-2Rβ, and being retained by Treg.

[0365] The antibodies disclosed herein may comprise CDR regions from specific antibodies identified herein, such as CDR regions from SEQ ID NO:1-32. In some embodiments, such antibodies will preferably retain the ability to bind to IL-2 as described herein. In some embodiments, such antibodies will preferably retain the ability to bind to hIL-2 as described herein. For example, the CDR sequences of the antibodies of this disclosure are shown in the sequence listing (Table 7), and the SEQ ID NO is shown in Table 6. In some embodiments, the antibodies of this disclosure comprise 1, 2, 3, 4, 5, or 6 CDRs from the antibodies of this disclosure.

[0366] In one aspect, this disclosure provides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conserved or non-conserved substitutions for one or more CDRs listed above, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and / or deletions. In a further aspect, the variant shares at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one or more CDR sequences listed above, and wherein the antibody or antigen-binding portion specifically binds to IL-2. In some embodiments, the antibody or antigen-binding portion specifically binds to hIL-2.

[0367] Polynucleotides, vectors and host cells

[0368] This disclosure also provides polynucleotides encoding any antibodies, including antibody moieties described herein and modified antibodies, such as antibodies having impaired effector functions. In another aspect, this disclosure provides methods for preparing any of the polynucleotides described herein. Polynucleotides can be prepared and expressed using procedures known in the art. Accordingly, this disclosure provides polynucleotides or compositions, including pharmaceutical compositions, comprising a polynucleotide encoding any one of the following IL-2 antibodies and their antigen-binding moieties: F4.7.6 VH, F4.7.8 VH, F5.1.11 VH, F5.1.9 VH, F4.7.062 VH, F5.1.11.01 VH, F5.1.11.02 VH, F5.1.11.03 VH, F5.1.11.04 VH, F5.1.11.05 VH, F5.1.11.06 VH, F5.1.11.07 VH, F5.1.11.08 VH, F5.1.11.09 VH, F5.1.9.5 VH, d1C7 VH, F4.7.6 VL, F4.7.8 VL, F5.1.11 VL, F5.1.9 VL, F4.7.062 VL, F5.1.11.01 VL, F5.1.11.02 VL, F5.1.11.03 VL, F5.1.11.04 VL, F5.1.11.05 VL, F5.1.11.06 VL, F5.1.11.07 VL, F5.1.11.08 VL, F5.1.11.09 VL, F5.1.9.5 VL, or d1C7 VL, or any part or portion thereof that has the ability to bind IL-2.

[0369] In one embodiment, the VH and VL domains, or their antigen-binding portions, or the full-length HC or LC are encoded by separate polynucleotides. Alternatively, both VH and VL, or their antigen-binding portions, or HC and LC are encoded by a single polynucleotide.

[0370] In another aspect, this disclosure provides polynucleotides encoding IL-2 antibodies and variants thereof, wherein such variant polynucleotides share at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any particular nucleic acid disclosed herein. These amounts are not intended to be limiting, and increments between said percentages are specifically contemplated as part of this disclosure.

[0371] Polynucleotides complementary to any such sequence are also covered by this disclosure. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules containing introns and corresponding one-to-one with DNA molecules, and mRNA molecules without introns. Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of this disclosure, and the polynucleotides may, but need not, be linked to other molecules and / or supporting material.

[0372] The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or a portion thereof) or a variant thereof. Polynucleotide variants contain one or more substitutions, additions, deletions, and / or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the naturally immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be evaluated as described herein. In some embodiments, the variant exhibits at least about 70% identity with the polynucleotide sequence encoding a natural antibody or a portion thereof, in some embodiments at least about 80% identity, in some embodiments at least about 90% identity, and in some embodiments at least about 95% identity. These amounts are not intended to be limiting, and increments between these percentages are specifically contemplated as part of this disclosure.

[0373] Two polynucleotide or polypeptide sequences are said to be “identical” if the nucleotide or amino acid sequences in two sequences are identical when compared for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing sequences within a comparison window to identify and compare local regions of sequence similarity. As used herein, a “comparison window” refers to a segment of at least about 20, typically 30 to about 75, or 40 to about 50 adjacent positions, where, after optimal alignment of two sequences, the sequence can be compared to a reference sequence having the same number of adjacent positions.

[0374] The best alignment of sequences for comparison can be used Bioinformatics software suite ( Inc., Madison, WI) The program was run using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645; Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5: 151-153; Myers, EW and Muller W., 1988, CABIOS 4: 11-17; Robinson, ED, 1971, Comb. Theor. 11: 105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4: 406-425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0375] In some embodiments, the "percentage of sequence identity" is determined by comparing two best-aligned sequences across a comparison window of at least 20 positions, wherein a portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20 percent or less, typically 5 to 15 percent, or 10 to 12 percent of additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions), for the best alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears below it to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to generate the percentage of sequence identity.

[0376] Variants may also, or alternatively, be substantially homologous to the natural gene or a portion thereof or its complement. Such polynucleotide variants are capable of hybridizing with naturally occurring DNA sequences (or complementary sequences) encoding natural antibodies under moderately stringent conditions.

[0377] Suitable “moderately stringent conditions” include prewashing in a solution of 5X SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0); overnight hybridization at 50-65°C with 5X SSC; followed by washing twice at 65°C with 2X, 0.5X, and 0.2X SSC containing 0.1% SDS for a total of 20 minutes.

[0378] As used herein, “highly stringent conditions” or “highly stringent conditions” are as follows: (1) using low ionic strength and high temperature for washing, for example, 0.015M sodium chloride / 0.0015M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) using a denaturing agent, such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50mM sodium phosphate buffer at pH 6.5 with 750mM sodium chloride, 75mM sodium citrate; or (3) using 50% formamide, 5X SSC (0.75 M NaCl, 0.075M sodium citrate) 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X SSC at 42°C. Denhardt's solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate, wherein washing was performed at 42°C in 0.2X SSC (sodium chloride / sodium citrate) and at 55°C in 50% formamide, followed by a high-rigor wash consisting of 0.1X SSC containing EDTA at 55°C. Those skilled in the art will recognize how to adjust the temperature, ionic strength, etc., as needed to accommodate factors such as probe length, etc.

[0379] Those skilled in the art will understand that, due to the degeneracy of the genetic code, there are numerous nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides share minimal homology with the nucleotide sequences of any natural gene. Nevertheless, polynucleotides that differ due to variations in codon usage are specifically considered in this disclosure. Furthermore, alleles of genes containing the polynucleotide sequences provided herein are within the scope of this disclosure. An allele is an endogenous gene altered by one or more mutations in nucleotides (e.g., deletion, addition, and / or substitution). The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification, and / or database sequence comparison).

[0380] The polynucleotides disclosed herein can be obtained using chemical synthesis, recombination methods, or PCR. Methods for the chemical synthesis of polynucleotides are well known in the art and need not be described in detail herein. Those skilled in the art can use the sequences provided herein and commercial DNA synthesizers to generate the desired DNA sequences.

[0381] To prepare polynucleotides using recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, which can then be introduced into a suitable host cell for replication and amplification, as discussed further herein. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing exogenous polynucleotides via direct uptake, endocytosis, transfection, F-conjugation, or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrating vector (e.g., a plasmid) or integrated into the host cell genome. The amplified polynucleotides can then be isolated from the host cell using methods well-known in the art. See, for example, Sambrook et al., 1989.

[0382] Alternatively, PCR allows for the replication of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and in PCR: The Polymerase Chain Reaction, edited by Mullis et al., Birkauswer Press, Boston, 1994.

[0383] RNA can be obtained by using isolated DNA in a suitable vector and inserting it into a suitable host cell. Once the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well-known to those skilled in the art, such as, for example, Sambrook et al., 1989, as described above.

[0384] Suitable cloning vectors can be constructed using standard techniques or selected from a large number of cloning vectors available in the art. While the chosen cloning vector can vary depending on the intended host cell, useful cloning vectors generally possess self-replicating capabilities, may have a single target against a specific restriction endonuclease, and / or carry a gene that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Stragene, and Invitrogen.

[0385] Expression vectors are further provided. Expression vectors are generally replicable polynucleotide constructs containing polynucleotides according to this disclosure. This implies that the expression vector must be able to replicate in the host cell either as an episome or as an integrated part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids disclosed in PCT Publication No. WO 87 / 04462, viral vectors including adenovirus, adeno-associated virus, retrovirus, granules, and expression vectors. Vector components generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; and suitable transcriptional control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational control elements are usually also required, such as ribosome binding sites, translation initiation sites, and stop codons.

[0386] Vectors containing the target polynucleotide and / or the polynucleotide itself can be introduced into host cells by any of a variety of suitable means, including electroporation, transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-glucan or other substances; particle bombardment; lipid transfection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of vector or polynucleotide for introduction often depends on the characteristics of the host cell.

[0387] This disclosure also provides host cells containing any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used to isolate genes encoding a target antibody, peptide, or protein. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (such as Escherichia coli or Bacillus subtilis) and yeasts (such as Saccharomyces cerevisiae, Saccharomyces pombe; or Kluyveromyces lactis). In some embodiments, the host cell expresses cDNA at a level approximately 5-fold higher than the level of the corresponding target endogenous antibody or protein (if present) in the host cell, 10-fold higher in some embodiments, and 20-fold higher in some embodiments. Screening for specific binding of host cells to IL-2 is performed by immunoassay or FACS. Cells overexpressing the target antibody or protein can be identified.

[0388] Expression vectors can be used to direct the expression of IL-2 antibodies. Those skilled in the art are familiar with the administration of expression vectors to achieve the expression of exogenous proteins in vivo. See, for example, U.S. Patent Nos. 6,436,908; 6,413,942; and 6,376,471. Administration of expression vectors includes local or systemic administration, including injection, oral administration, particle gun or catheter administration, and local administration. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglia, or to the coronary arteries, atria, ventricles, or pericardium.

[0389] Targeted delivery of therapeutic compositions containing expression vectors or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described, for example, in: Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer, J.A. Wolff, ed., 1994; Wu et al., J. Biol. Chem., 1988, 263:621; Wu et al., J. Biol. Chem., 1994, 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA, 1990, 87:3655; Wu et al., J. Biol. Chem., 1991, 266:338. Therapeutic compositions containing polynucleotides are administered in the range of about 100 ng to about 200 mg of DNA for local application in gene therapy regimens. In gene therapy protocols, concentrations of DNA ranging from approximately 500 ng to approximately 50 mg, approximately 1 μg to approximately 2 mg, approximately 5 μg to approximately 500 μg, and approximately 20 μg to approximately 100 μg can also be used. Therapeutic polynucleotides and peptides can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (see generally Jolly, Cancer Gene Therapy, 1994, 1:51; Kimura, Human Gene Therapy, 1994, 5:845; Connelly, Human Gene Therapy, 1995, 1:185; and Kaplitt, Nature Genetics, 1994, 6:148). Expression of this coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be constitutive or regulatory.

[0390] Virus-based vectors for delivering desired polynucleotides and expressing them in desired cells are well known in the art. Exemplary virus-based vectors include, but are not limited to, recombinant retroviruses (see, for example, PCT Publications WO90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; US Patents 5,219,740 and 4,777,127; GB Patent 2,200,651; and EP Patent 0 345 242), and alphavirus-based vectors (e.g., Sindbis virus vector, Semliki Forest Virus (ATCC VR-67; ATCC VR-1247), Ross River Virus (ATCC VR-373; ATCC VR-1246), and Venezuelan Equine Encephalitis Virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)). And adeno-associated virus (AAV) vectors (see, for example, PCT Publications WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655). Alternatively, DNA conjugated with killed adenovirus may be used, as described in Curiel, Hum. Gene Ther., 1992, 3:147.

[0391] Non-viral delivery vectors and methods may also be used, including, but not limited to, polycationically condensed DNA linked to or unlinked adenovirus (see, for example, Curiel, Hum. Gene Ther., 1992, 3:147); ligand-linked DNA (see, for example, Wu, J. Biol. Chem., 1989, 264:16985); eukaryotic cell delivery vectors (see, for example, U.S. Patent No. 5,814,482; PCT Publications WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO 97 / 42338); and nucleic acid charge neutralization or fusion with the cell membrane. Naked DNA may also be used. Exemplary methods for introducing naked DNA are described in PCT Publication WO 90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery mediators are described in U.S. Patent No. 5,422,120; PCT Publication Nos. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and EP0524968. Other methods are described in Philip, Mol. Cell Biol., 1994, 14:2411 and Woffendin, Proc. Natl. Acad. Sci., 1994, 91:1581.

[0392] Treatment

[0393] The treatment methods involve administering to a subject in need of treatment a therapeutically effective or “effective” amount of an IL-2 antibody, an antigen-binding moiety, or an antibody:IL-2 complex comprising an IL-2 antibody as described herein, as contemplated by this disclosure. As used herein, a “therapeuticly effective” or “effective” amount means an amount of antibody or a portion thereof sufficient to result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of damage or disability due to disease suffering—either as a single dose or according to a multiple-dose regimen, alone or in combination with other agents. Those skilled in the art will be able to determine such an amount based on factors such as the size of the subject, the severity of the subject’s symptoms, and the particular composition or route of administration chosen. The subject may be a human or a non-human animal (e.g., a rabbit, rat, mouse, monkey, or other lower primate).

[0394] Antibodies, antigen-binding portions, or antibodies containing the IL-2 antibody of this disclosure: IL-2 complexes can be co-administered with known agents, and in some cases, the antibody itself can be modified. For example, antibodies can be conjugated to immunotoxins or radioisotopes to potentially further enhance efficacy. Regarding co-administration with other therapeutic agents, such agents may include cytotoxic agents, radiotoxic agents, or immunosuppressants. Antibodies can be linked to agents (as immune complexes) or can be administered separately from agents. In the latter case (separate administration), antibodies can be administered before, after, or simultaneously with agents, or can be co-administered with other known therapies such as anticancer therapies such as radiation. Co-administration of IL-2 antibodies, their antigen-binding portions, or antibodies containing the IL-2 antibody of this disclosure: IL-2 complexes with therapeutic agents provides two agents that, through different mechanisms, can provide therapeutic effects and possible synergistic effects against human diseases.

[0395] Antibodies, antigen-binding portions, and antibody:IL-2 complexes comprising the IL-2 antibody disclosed herein can be used as therapeutic or diagnostic tools in various situations where IL-2 activity is undesirable, such as inflammatory conditions like autoimmune diseases, or situations where immunosuppression is required. In some embodiments, immunosuppressive therapy may be prepared for organ or bone marrow transplantation. Given the involvement of IL-2 in inflammatory pathways and numerous diseases, conditions, and states, many such diseases, conditions, or states are particularly suitable for treatment with antibodies, antigen-binding portions, or antibody:IL-2 complexes comprising the IL-2 antibody disclosed herein. Accordingly, IL-2 antibodies, their antigen-binding portions, or antibody:IL-2 complexes comprising the IL-2 antibody disclosed herein can be used to treat or prevent IL-2-mediated conditions or IL-2 deficiency conditions. Additionally, this disclosure provides the use of IL-2 antibodies, their antigen-binding portions, or antibody:IL-2 complexes comprising the IL-2 antibody disclosed herein in the manufacture of pharmaceutical agents for the treatment or prevention of IL-2-mediated conditions or IL-2 deficiency conditions. In another embodiment, this patent application discloses an IL-2 antibody, its antigen-binding portion, or an antibody:IL-2 complex comprising the IL-2 antibody disclosed herein for treating IL-2-mediated conditions or IL-2 deficiency conditions. In a further embodiment, this patent application discloses a pharmaceutical composition for treating or preventing IL-2-mediated conditions or IL-2 deficiency conditions, comprising an IL-2 antibody, its antigen-binding portion, or an antibody:IL-2 complex comprising the IL-2 antibody disclosed herein. In some embodiments, the antibody specifically binds to hIL-2.

[0396] In some embodiments, these diseases include immune diseases such as graft-versus-host disease. In some embodiments, the disease may be any IL-2-related disease, such as muscular dystrophy and obesity. Exemplary autoimmune diseases and conditions that can be treated with antibodies, their antigen-binding portions, or antibodies containing the IL-2 antibody provided herein include, for example, inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); dermatomyositis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndromes (including adult respiratory distress syndrome and ARDS); dermatitis; meningitis; Encephalitis; uveitis; colitis; gastritis; glomerulonephritis; allergic conditions such as eczema and asthma, and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion defects; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes (e.g., type 1 diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; and cytokine- and T-lymphocyte-mediated immune responses associated with acute and delayed hypersensitivity reactions, commonly found in tuberculosis, sarcomatoid disease, polymyositis, granulomatous disease, and vasculitis; Wegener's disease; pernicious anemia (Addison's disease); diseases involving leukocyte exudation; inflammatory conditions of the central nervous system (CNS); multiple organ injury syndrome; hemolytic anemia (including, but not limited to, cryoglobulinemia or Coombs-positive anemia); Myasthenia gravis; antigen-antibody complex-mediated diseases; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Langerhans-Ellison myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrine disorders; vitiligo; Reiter's disease; stiff-person syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenic purpura and autoimmune hemolytic diseases; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveitis; Graves-Bartholin's syndrome; Goodpassuia syndrome; mixed connective tissue disease; autoimmune-related infertility; polyarteritis nodosa; alopecia areata; and idiopathic myxedema. In some embodiments, the condition that can be treated with the compositions and methods of the present invention is diabetes (e.g., type 1 diabetes). In some embodiments, the disease is type 1 diabetes. In some embodiments, the disease is juvenile diabetes.

[0397] For the treatment of any of the aforementioned conditions, the pharmaceutical compositions used according to this disclosure may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers or excipients, and applied as discussed more fully below.

[0398] The therapeutically effective amount of an antibody, its antigen-binding portion, or an antibody containing an IL-2 antibody of this disclosure, as determined according to this disclosure, depends to a great extent on the specific patient characteristics, route of administration, and nature of the condition to be treated, and is discussed more fully below.

[0399] The administration and delivery of antibodies, their antigen-binding portions, or antibodies containing the IL-2 antibody of this disclosure:IL-2 complexes are discussed more fully elsewhere below.

[0400] Diagnostic methods

[0401] The IL-2 antibody or its antigen-binding moiety disclosed herein can be used for diagnostic testing and imaging. For example, the IL-2 antibody or its antigen-binding moiety can be used in ELISA assays. The antibody or its antigen-binding moiety can also be used as a radiolabeled monoclonal antibody. See, for example, Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging And Therapy, Plenum Press (1988); Chase, "Medical Applications of Radioisotopes," in Remington's Pharmaceutical Sciences, 18th ed., Gennaro et al. (ed.), Mack Publishing Co., pp. 624-652 (1990); and Brown, "Clinical Use of Monoclonal Antibodies," in Biotechnology and Pharmacy, Pezzuto et al. (ed.), Chapman and Hall, pp. 227-249 (1993); Grossman, 1986, Urol. Clin. North Amer. 13: 465-474; Unger et al., 1985, Invest. Radiol. 20: 693-700; and Khaw et al., 1980, Science 209: 295-297. This technique, also known as immunoscintillation imaging, uses a gamma camera to detect the location of gamma-emitting radioactive isotopes conjugated with monoclonal antibodies. Diagnostic imaging can be used to diagnose cancer, autoimmune diseases, infectious diseases, and / or cardiovascular diseases. (See, for example, Brown, ibid.).

[0402] In one embodiment, an IL-2 antibody or its antigen-binding portion can be used to diagnose IL-2-related diseases, conditions, or illnesses, including immune-related diseases. For example, in other uses, the antibody or its antigen-binding portion can be used to detect IL-2 levels in a patient.

[0403] In addition to diagnosis, IL-2 antibodies or their antigen-binding moiety can be used to monitor treatment response, detect disease relapse, and guide subsequent clinical decisions.

[0404] In some embodiments, for diagnostic and monitoring purposes, a radioisotope can be directly or indirectly bound to an antibody via the use of an intermediate functional group. Such intermediate functional groups include, for example, DTPA (diethylenetriaminepentaacetic acid) and EDTA (ethylenediaminetetraacetic acid). The radiation dose delivered to the patient is typically maintained at the lowest possible level. This is achieved by selecting an isotope with an optimal combination of a minimum half-life, a minimum retention time in the body, and a minimum amount of the isotope that allows for detection and accurate measurement. Examples of radioisotopes that can bind to antibodies and are suitable for diagnostic imaging include… 99 mTc and 111 In.

[0405] Studies indicate that the antibody fractions, particularly Fab and Fab', provide a suitable tumor / background ratio. (See, for example, Brown, ibid.)

[0406] IL-2 antibodies or their antigen-binding moieties can also be labeled with paramagnetic ions for in vivo diagnostic purposes. Elements particularly useful for magnetic resonance imaging include Gd, Mn, Dy, and Fe ions.

[0407] IL-2 antibodies or their antigen-binding moieties can also be used to detect the presence of IL-2 in vitro. In such an immunoassay, the antibody or its antigen-binding moieties can be utilized in a liquid phase or bound to a solid-phase support. For example, the intact antibody or its antigen-binding moieties can be attached to a polymer (e.g., glycosaminoglycan) to attach the antibody component to an insoluble support such as polymer-coated beads, plates, or tubes. In some embodiments, the IL-2 used for detection is human IL-2 (hIL-2). In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.1 ng / mL to 1000 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.1 ng / mL to 750 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.5 ng / mL to 500 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.5 ng / mL to 250 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.5 ng / mL to 100 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.5 ng / mL to 50 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.5 ng / mL to 25 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.5 ng / mL to 10 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.5 ng / mL to 5 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.5 ng / mL to 1 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.1 ng / mL to 5 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.1 ng / mL to 1 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.8 ng / mL to 500 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 1 ng / mL to 500 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 1 ng / mL to 250 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 1 ng / mL to 100 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 1 ng / mL to 50 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 1 ng / mL to 25 ng / mL.In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 1 ng / mL to 10 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 50 ng / mL to 500 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 100 ng / mL to 500 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 250 ng / mL to 500 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 400 ng / mL to 500 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 500 ng / mL to 1000 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 750 ng / mL to 1000 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 0.8 ng / mL. In some embodiments, the hIL-2 used for detection is present in vitro at a concentration of 500 ng / mL. These amounts are not intended to be limiting, and increments between these values ​​are specifically envisioned as part of this disclosure.

[0408] Alternatively, IL-2 antibodies or their antigen-binding portions can be used to detect the presence of specific antigens in tissue sections prepared from histological specimens. This in situ detection can be accomplished, for example, by applying a detectable labeled IL-2 antibody or its antigen-binding portion to the tissue section. In situ detection can be used to determine the presence of a specific antigen and the distribution of the antigen in the examined tissue. General techniques for in situ detection are well known to those skilled in the art. (See, for example, Ponder, "Cell Marking Techniques and Their Application," in Mammalian Development: A Practical Approach, Monk (ed.), IRL Press, pp. 115–138 (1987); Coligan et al., ibid.)

[0409] Detectable labels such as enzymes, fluorescent compounds, electron transfer agents, etc., can be linked to carriers using conventional methods well-known in the art. These labeled carriers and antibody conjugates prepared therefrom can be used for in vitro immunoassays and in situ detection, although antibody conjugates can be prepared by direct attachment of the label to the antibody. Loading antibody conjugates with multiple labels can increase the sensitivity of immunoassays or histological procedures where only a low degree of binding of the antibody or antibody fraction to the target antigen is achieved.

[0410] Composition

[0411] This disclosure also provides pharmaceutical compositions comprising an effective amount of the IL-2 antibody described herein. Examples of such compositions and how to formulate them are also described herein. In some embodiments, the composition comprises one or more IL-2 antibodies. In other embodiments, the IL-2 antibody recognizes IL-2. In other embodiments, the IL-2 antibody is a human antibody. In other embodiments, the IL-2 antibody is a humanized antibody. In some embodiments, the IL-2 antibody comprises a constant region capable of triggering a desired immune response (e.g., antibody-mediated cleavage or ADCC). In other embodiments, the IL-2 antibody comprises a constant region that does not trigger an unwanted or undesirable immune response (e.g., antibody-mediated cleavage or ADCC). In other embodiments, the IL-2 antibody comprises one or more CDRs of the antibody (e.g., one, two, three, four, five, or in some embodiments, all six CDRs).

[0412] It should be understood that the composition may contain more than one IL-2 antibody (e.g., a mixture of IL-2 antibodies that recognize different epitopes of IL-2). Other exemplary compositions contain more than one IL-2 antibody that recognizes the same epitope, or different kinds of IL-2 antibodies that bind to different epitopes of IL-2. In some embodiments, the composition contains a mixture of IL-2 antibodies that recognize different variants of IL-2.

[0413] The compositions used in this disclosure may also contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington: The Science and Practice of Pharmacy, 20th edition, 2000, Lippincott Williams and Wilkins, edited by KEHoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the receptor at the specified dose and concentration and may contain buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, etc. Trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are also described in this article.

[0414] IL-2 antibodies and their compositions can also be used in combination with other reagents that enhance and / or supplement the effectiveness of reagents.

[0415] In some embodiments, the IL-2 antibody is conjugated with IL-2 prior to administration. In other embodiments, the IL-2 antibody is not conjugated with IL-2 prior to administration.

[0416] This disclosure also provides compositions comprising any polynucleotide of this disclosure, including pharmaceutical compositions. In some embodiments, the composition comprises an expression vector that encodes a polynucleotide encoding an antibody as described herein. In other embodiments, the composition comprises an expression vector that encodes a polynucleotide encoding any antibody as described herein. In other embodiments, the composition comprises any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:1 and SEQ ID NO:2, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:3 and SEQ ID NO:4, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:5 and SEQ ID NO:6, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:7 and SEQ ID NO:8, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:9 and SEQ ID NO:10, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:11 and SEQ ID NO:12, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:13 and SEQ ID NO:14, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:15 and SEQ ID NO:16, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:17 and SEQ ID NO:18, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:19 and SEQ ID NO:20, and any or both of the polynucleotides encoding SEQ ID NO:21 and SEQ ID NO:14. Any or both of the polynucleotides encoding the sequences shown in NO:22, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:25 and SEQ ID NO:26, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:27 and SEQ ID NO:28, any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:29 and SEQ ID NO:30, or any or both of the polynucleotides encoding the sequences shown in SEQ ID NO:31 and SEQ ID NO:32.In other embodiments, the composition comprises any or both of the polynucleotides comprising the sequences shown in SEQ ID NO:36 and SEQ ID NO:37, any or both of the polynucleotides comprising the sequences shown in SEQ ID NO:38 and SEQ ID NO:39, any or both of the polynucleotides comprising the sequences shown in SEQ ID NO:40 and SEQ ID NO:41, any or both of the polynucleotides comprising the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, any or both of the polynucleotides comprising the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, any or both of the polynucleotides comprising the sequences shown in SEQ ID NO:46 and SEQ ID NO:47, any or both of the polynucleotides comprising the sequences shown in SEQ ID NO:48 and SEQ ID NO:49, any or both of the polynucleotides comprising the sequences shown in SEQ ID NO:50 and SEQ ID NO:51, any or both of the polynucleotides comprising the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, any or both of the polynucleotides comprising the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, and any or both of the polynucleotides comprising SEQ ID NO:36 and SEQ ID NO:37. Any or both of the polynucleotides containing the sequences shown in NO:56 and SEQ ID NO:57, any or both of the polynucleotides containing the sequences shown in SEQ ID NO:58 and SEQ ID NO:59, any or both of the polynucleotides containing the sequences shown in SEQ ID NO:60 and SEQ ID NO:61, any or both of the polynucleotides containing the sequences shown in SEQ ID NO:62 and SEQ ID NO:63, any or both of the polynucleotides containing the sequences shown in SEQ ID NO:64 and SEQ ID NO:65, or any or both of the polynucleotides containing the sequences shown in SEQ ID NO:66 and SEQ ID NO:67.

[0417] In another aspect, the polynucleotide may encode the VH, VL, and / or both VH and VL of the antibody disclosed herein. That is, the composition comprises a single polynucleotide or more than one polynucleotide encoding the antibody disclosed herein or its antigen-binding moiety.

[0418] The pharmaceutical compositions disclosed herein can also be administered in combination therapy, such as in combination with other agents. For example, combination therapy may include the IL-2 antibody of this disclosure or its antigen-binding portion in combination with at least one other therapy, wherein said therapy may be surgical, immunotherapy, or pharmaceutical therapy.

[0419] The pharmaceutical compounds disclosed herein may include one or more pharmaceutically acceptable salts. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, etc., and non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanes, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., and non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucosamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

[0420] The pharmaceutical compositions disclosed herein may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as palmitic acid ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0421] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. For example, by using a coating material such as lecithin, the desired particle size can be maintained in the case of a dispersion, and by using a surfactant, appropriate flowability can be maintained.

[0422] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. The presence of microorganisms can be prevented through sterilization procedures and by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc., in the composition. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0423] Pharmaceutical compositions must generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, by using a coating such as lecithin, the desired particle size can be maintained in the case of a dispersion, and by using a surfactant, appropriate flowability can be maintained. In many cases, including an isotonic agent in the composition, such as sugar, polyols such as mannitol, sorbitol, or sodium chloride, would be suitable. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as monostearate and gelatin, in the composition.

[0424] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound with one or a combination of the ingredients listed above (as needed) into a suitable solvent, followed by sterile microfiltration.

[0425] Generally, dispersions are prepared by incorporating an active compound into a sterile medium containing a base dispersion medium and other desired components from those listed above. In the case of sterile powders for preparing sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying (lyophilization), which yields a powder containing the active ingredient plus any other desired components from its prior sterile filtered solution.

[0426] The pharmaceutical compositions disclosed herein are suitable for the preparation, packaging, or marketing of formulations for ocular application. For example, such formulations may be in the form of eye drops, comprising, for example, a 0.1%-1.0% (w / w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may also contain the buffering agents, salts, or one or more other components described herein. Other useful ocularly applicable formulations include those containing the active ingredient in microcrystalline form or in liposomal formulations.

[0427] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binders; lubricants; sweeteners; flavorings; colorants; preservatives; physiologically degradable compositions such as gelatin; aqueous media and solvents; oily media and solvents; suspending agents; dispersants or wetting agents; emulsifiers; modifiers; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungals; stabilizers; and pharmaceutically acceptable polymers or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions disclosed herein are known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Genaro, edited, Mack Publishing Co., Easton, PA (1985), references which are incorporated herein by reference.

[0428] In one embodiment, the IL-2 antibody or its antigen-binding portion is administered as an intravenous formulation as a sterile aqueous solution containing 5 mg / mL of antibody, or in some embodiments, about 10 mg / mL, or in some embodiments, about 15 mg / mL, or in some embodiments, about 20 mg / mL of sodium acetate, polysorbate 80, and sodium chloride, at a pH in the range of about 5 to 6. In some embodiments, the intravenous formulation is a sterile aqueous solution containing 5 or 10 mg / mL of antibody, and 20 mM sodium acetate, 0.2 mg / mL polysorbate 80, and 140 mM sodium chloride at pH 5.5. Furthermore, among many other compounds, the solution containing the antibody or its antigen-binding portion may contain histidine, mannitol, sucrose, trehalose, glycine, polyethylene glycol, EDTA, methionine, and any combination thereof, as well as many other compounds known in the art.

[0429] In one embodiment, the pharmaceutical composition of this disclosure comprises the following components: 100 mg of the disclosed IL-2 antibody or antigen-binding moiety, 10 mM histidine, 5% sucrose, and 0.01% polysorbate 80, at pH 5.8. The composition can be provided as a lyophilized powder. When the powder is reconstituted at full volume, the composition maintains the same formulation. Alternatively, the powder can be reconstituted at half volume, in which case the composition comprises 100 mg of the disclosed IL-2 antibody or its antigen-binding moiety, 20 mM histidine, 10% sucrose, and 0.02% polysorbate 80, at pH 5.8.

[0430] In one embodiment, a portion of the dose is administered via intravenous bolus injection, and the remainder is administered via infusion of the antibody preparation. For example, an intravenous injection of 0.01 mg / kg IL-2 antibody or its antigen-binding portion may be given as a bolus injection, and the remainder of the antibody dose may be administered via intravenous injection. The predetermined dose of the IL-2 antibody or its antigen-binding portion may be administered, for example, over a period of one and a half hours to two to five hours.

[0431] Regarding therapeutic agents, when the agent is, for example, a small molecule, it can be in the form of a physiologically acceptable ester or salt, for example, in combination with a physiologically acceptable cation or anion in a pharmaceutical composition, as is well known in the art.

[0432] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or subsequently developed in the field of pharmacology. Generally, such preparation methods include the following steps: combining the active ingredient with a carrier or one or more other auxiliary ingredients, and then, if desired or desired, shaping or packaging the product into the desired single-dose or multi-dose units.

[0433] In one embodiment, the composition disclosed herein is a pyrogen-free formulation substantially free of endotoxins and / or associated pyrogens. Endotoxins include toxins confined within microorganisms and released when the microorganisms decompose or die. Pyrogens also include fever-inducing heat-stable substances (glycoproteins) from bacterial outer membranes and other microorganisms. Both of these substances can cause fever, hypotension, and shock if administered to humans. Due to the potential adverse effects, it is advantageous to remove even trace amounts of endotoxins from intravenously administered drug solutions. The U.S. Food and Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EUs) / dose / kg body weight for intravenous drug administration in a single one-hour period (The United States Pharmacopeial Convention, Pharmacopeial Forum 26(1):223(2000)). Removal of even trace amounts of endotoxins is also advantageous when therapeutic proteins are administered in doses of hundreds or thousands of mg / kg body weight. In one embodiment, the endotoxin and pyrogen levels in the composition are less than 10 EU / mg, or less than 5 EU / mg, or less than 1 EU / mg, or less than 0.1 EU / mg, or less than 0.01 EU / mg, or less than 0.001 EU / mg. In another embodiment, the endotoxin and pyrogen levels in the composition are less than about 10 EU / mg, or less than about 5 EU / mg, or less than about 1 EU / mg, or less than about 0.1 EU / mg, or less than about 0.01 EU / mg, or less than about 0.001 EU / mg.

[0434] In one embodiment, this disclosure includes the administration of a composition, wherein the administration is via oral, parenteral, intramuscular, intranasal, vaginal, rectal, tongue, sublingual, buccal, intrabuccal, intravenous, skin, subcutaneous, or transdermal route.

[0435] In another embodiment, this disclosure also includes the administration of the composition in combination with other therapies such as surgery, chemotherapy, hormone therapy, biotherapy, immunotherapy, or radiotherapy.

[0436] Dosage / Administration

[0437] To prepare a pharmaceutical or sterile composition comprising the IL-2 antibody or its antigen-binding portion thereof disclosed herein, the antibody is mixed with a pharmaceutically acceptable carrier or excipient. Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers, for example, in the form of lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, for example, 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. (ed.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al. (ed.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (ed.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0438] The choice of administration regimen for treatment depends on several factors, including the serum or tissue turnover rate of the entity, symptom level, immunogenicity of the entity, and accessibility of target cells in the biological matrix. In some embodiments, the administration regimen maximizes the therapeutic dose delivered to the patient in accordance with acceptable levels of side effects. Accordingly, the amount of biologic delivered depends in part on the specific entity and the severity of the condition being treated. Guidelines for selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, for example, Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.), 1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al., 2003, New Engl. J. Med. 348: 601-608; Milgrom et al., 1999, New Engl. J. Med. 341: 1966-1973; Slamon et al., 2001, New...). Engl. J. Med. 344:783-792; Beniaminovitz et al., 2000, New Engl. J. Med. 342:613-619; Ghosh et al., 2003, New Engl. J. Med. 348:24-32; Lipsky et al., 2000, New Engl. J. Med. 343:1594-1602).

[0439] The determination of the appropriate dosage is made by a clinician, for example, using parameters or factors known or suspected in the art to affect or predicted to affect treatment. Generally, the dosage begins at a level slightly below the optimal dose and is subsequently increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include, for example, symptoms of inflammation or levels of inflammatory cytokines.

[0440] The actual dose level of the active ingredient in the pharmaceutical compositions of this disclosure can be varied to obtain an amount of active ingredient that is non-toxic to the patient and effectively achieves the desired therapeutic response for a particular patient, composition, and administration mode. The selected dose level will depend on various pharmacokinetic factors, including the activity of the particular composition of this disclosure used, or its esters, salts, or amides, route of administration, time of administration, excretion rate of the particular compound used, duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and medical history of the patient to be treated, and similar factors well known in the medical field.

[0441] Compositions containing the IL-2 antibody or its antigen-binding portion of this disclosure may be administered by continuous infusion or at intervals such as daily, weekly, or weekly (1-7 times). Dosage may be administered intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. Specific dosing regimens are those involving the maximum dose or frequency to avoid significant undesirable side effects. The total weekly dose may be at least 0.05 μg / kg body weight, at least 0.2 μg / kg, at least 0.5 μg / kg, at least 1 μg / kg, at least 10 μg / kg, at least 100 μg / kg, at least 0.2 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, or at least 50 mg / kg (see, for example, Yang et al., 2003, New Engl. J. Med. 349:427-434; Herold et al., 2002, New Engl. J. Med. 346:1692-1698; Liu et al., 1999, J. Neurol. Neurosurg. Psych. 67:451-456; Portielji et al., 2003, Cancer. Immunol. Immunother. 52:133-144). The dosage may be at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, or at least 100 μg. The dosage administered to the subject may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more doses.

[0442] For the IL-2 antibody or its antigen-binding portion disclosed herein, the dose administered to a patient may be from 0.0001 mg / kg to 100 mg / kg of patient body weight. The dose may be from 0.0001 mg / kg to 20 mg / kg, 0.0001 mg / kg to 10 mg / kg, 0.0001 mg / kg to 5 mg / kg, 0.0001 to 2 mg / kg, 0.0001 to 1 mg / kg, 0.0001 mg / kg to 0.75 mg / kg, 0.0001 mg / kg to 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.10 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.25 mg / kg, or 0.01 to 0.10 mg / kg of patient body weight.

[0443] The dose of IL-2 antibody or its antigen-binding moiety can be calculated by multiplying the patient's weight in kilograms (kg) by the dose administered in mg / kg. The dosage of the antibody disclosed herein may be 150 μg / kg or less, 125 μg / kg or less, 100 μg / kg or less, 95 μg / kg or less, 90 μg / kg or less, 85 μg / kg or less, 80 μg / kg or less, 75 μg / kg or less, 70 μg / kg or less, 65 μg / kg or less, 60 μg / kg or less, 55 μg / kg or less, 50 μg / kg or less, 45 μg / kg or less, 40 μg / kg or less, 35 μg / kg or less, 30 μg / kg or less, 25 μg / kg or less, 20 μg / kg or less, 15 μg / kg or less, 10 μg / kg or less, 5 μg / kg or less, 2.5 μg / kg Patient weight of 2 μg / kg or less, 1.5 μg / kg or less, 1 μg / kg or less, 0.5 μg / kg or less, or 0.1 μg / kg or less.

[0444] The unit dose of the IL-2 antibody or its antigen-binding portion disclosed herein may be 0.1 mg to 200 mg, 0.1 mg to 175 mg, 0.1 mg to 150 mg, 0.1 mg to 125 mg, 0.1 mg to 100 mg, 0.1 mg to 75 mg, 0.1 mg to 50 mg, 0.1 mg to 30 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 mg to 2.5 mg, 0.25 mg to 20 mg, 0.25 mg to 15 mg, 0.25 mg to 12 mg, 0.25 mg to 10 mg, 0.25 mg to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0445] The dose of the IL-2 antibody or its antigen-binding portion disclosed herein can achieve serum titers in subjects of at least 0.1 μg / mL, at least 0.5 μg / mL, at least 1 μg / mL, at least 2 μg / mL, at least 5 μg / mL, at least 6 μg / mL, at least 10 μg / mL, at least 15 μg / mL, at least 20 μg / mL, at least 25 μg / mL, at least 50 μg / mL, at least 100 μg / mL, at least 125 μg / mL, at least 150 μg / mL, at least 175 μg / mL, at least 200 μg / mL, at least 225 μg / mL, at least 250 μg / mL, at least 275 μg / mL, at least 300 μg / mL, at least 325 μg / mL, at least 350 μg / mL, at least 375 μg / mL, or at least 400 μg / mL. Alternatively, the antibody of this disclosure may be administered in subjects at a serum titer of at least 0.1 μg / mL, at least 0.5 μg / mL, at least 1 μg / mL, at least 2 μg / mL, at least 5 μg / mL, at least 6 μg / mL, at least 10 μg / mL, at least 15 μg / mL, at least 20 μg / mL, at least 25 μg / mL, at least 50 μg / mL, at least 100 μg / mL, at least 125 μg / mL, at least 150 μg / mL, at least 175 μg / mL, at least 200 μg / mL, at least 225 μg / mL, at least 250 μg / mL, at least 275 μg / mL, at least 300 μg / mL, at least 325 μg / mL, at least 350 μg / mL, at least 375 μg / mL, or at least 400 μg / mL.

[0446] The dosage of the IL-2 antibody or its antigen-binding portion disclosed herein can be repeated, and the administration can be separated for at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or at least 6 months.

[0447] The effective dose for a particular patient can vary depending on factors such as the condition to be treated, the patient’s overall health, the route and dosage of administration, and the severity of side effects (see, for example, Maynard et al., 1996, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, FIa.; Dent, 2001, Good Laboratory and Good Clinical Practice, Urch Publ, London, UK).

[0448] The route of administration can be, for example, local or dermal application, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional injection or infusion, or via a continuous release system or implant (see, for example, Sidman et al., 1983, Biopolymers 22:547-556; Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277; Langer, 1982, Chem. Tech. 12:98-105; Epstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030-4034; U.S. Patent Nos. 6,350466 and 6,316,024). If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Alternatively, pulmonary administration may be used, for example by using an inhaler or nebulizer and formulated with an aerosolizer. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publications WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference in its entirety. In one embodiment, Alkermes AIR is used. TMLung drug delivery technology (Alkermes, Inc., Cambridge, Mass.) administers the IL-2 antibody or its antigen-binding portion or composition disclosed herein.

[0449] The compositions of this disclosure can also be administered via one or more of a variety of methods known in the art, through one or more routes of administration. As those skilled in the art will understand, the route of administration and / or mode of administration varies depending on the desired outcome. Routes of administration selected for the antibodies of this disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. Parenteral administration can refer to administration modes other than enteral and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Alternatively, the compositions of this disclosure can be administered via non-parenteral routes, such as local, epidermal, or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration.

[0450] If the IL-2 antibody or its antigen-binding portion of this disclosure is administered in a controlled release or sustained release system, the pump can be used to achieve controlled release or sustained release (see Langer, ibid.; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:501; Saudek et al., 1989, N. Engl. J. Med. 321:514).

[0451] Polymer materials can be used to achieve controlled or sustained release of the therapies of this disclosure (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIA. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol.ScL Rev. Macromol.Chem. 23:61; see also Levy et al., 1985, Science 11 225:190; During et al., 19Z9, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105); U.S. Patent No. 5,679,377; U.S. Patent Nos. 5,916,597, 5,912,015, 5,989,463, 5,128,326, PCT Publication No. WO 99 / 15154, and PCT Publication No. WO 99 / 20253. Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolic acid (PLG), polyanhydride, poly(N-vinylpyrrolidone), polyvinyl alcohol, polyacrylamide, polyethylene glycol, polylactide (PLA), poly(glycolic acid-lactide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained-release formulation is inert, free of leaching impurities, stable during storage, sterile, and biodegradable. Controlled-release or sustained-release systems can be placed near the preventive or therapeutic target, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115-138 (1984)).

[0452] Controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533. Any technique known to those skilled in the art can be used to produce sustained-release formulations comprising one or more antibodies or conjugates thereof disclosed herein. See, for example, U.S. Patent No. 4,526,938, International Patent Publication Nos. WO 91 / 05548, WO 96 / 20698, Ning et al., 1996, "Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy and Oncology 59:179-189; Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science and Technology 50:372-397; Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Pro.Ml. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,"Proc.Ml.Symp.Control Rel.Bioact.Mater. 24:759-160, each of which is incorporated herein by reference in its entirety.

[0453] If the IL-2 antibody or its antigen-binding portion of this disclosure is applied topically, it can be formulated as an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other forms well known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th edition, Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, they are typically in viscous to semi-solid or solid form, containing a carrier or one or more excipients compatible with the topical application, and in some cases having a dynamic viscosity greater than that of water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, ointments, etc., which, when necessary, are sterilized or mixed with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) to influence various properties, such as osmotic pressure. Other suitable topical dosage forms include sprayable aerosol formulations, in which, in some cases, the active ingredient, combined with a solid or liquid inert carrier, is packaged in a mixture or squeeze bottle containing a pressurized volatile (e.g., a gaseous propellant, such as Freon). Wetting agents or humectants may also be added to the pharmaceutical composition and dosage form if desired. Examples of such additives are well known in the art.

[0454] If a composition containing an IL-2 antibody or its antigen-binding portion is administered intranasally, it can be formulated in aerosol, spray, mist, or droplet form. In particular, the prophylactic or therapeutic agent used according to the invention can be conveniently delivered in aerosol spray form from a pressurized pack or nebulizer, using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver the measured amount. Capsules and cartridges (composed of, for example, gelatin) for inhalers or blowpipes can be formulated as powder mixtures containing compounds and suitable powder matrices such as lactose or starch.

[0455] Methods of co-administering or treating with a second therapeutic agent (e.g., cytokines, steroids, chemotherapy agents, antibiotics, or radiation) are well known in the art (see, for example, Hardman et al. (edited) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (edited) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams and Wilkins, Phila., Pa.; Chabner and Longo (edited) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams and Wilkins, Phila., Pa.). An effective amount of the therapeutic agent can reduce symptoms by at least 10 percent; at least 20 percent; at least about 30 percent; at least 40 percent, or at least 50 percent.

[0456] Additional therapies (e.g., prophylactic or therapeutic agents) that can be administered in combination with the IL-2 antibody or antigen binding portion of this disclosure may be administered at intervals of less than 5 minutes, less than 30 minutes, 1 hour, about 1 hour, about 1 to about 2 hours, about 2 to about 3 hours, about 3 to about 4 hours, about 4 to about 5 hours, about 5 to about 6 hours, about 6 to about 7 hours, about 7 to about 8 hours, about 8 to about 9 hours, about 9 to about 10 hours, about 10 to about 11 hours, about 11 to about 12 hours, about 12 to 18 hours, 18 to 24 hours, 24 to 36 hours, 36 to 48 hours, 48 ​​to 52 hours, 52 to 60 hours, or 60 to 72 hours. The intervals are 72 to 84 hours, 84 to 96 hours, or 96 to 120 hours. Two or more treatments may be administered within the same patient's visit.

[0457] The IL-2 antibody or its antigen-binding portion disclosed herein can be cyclically administered along with other therapies. Cyclic therapy involves administering a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by administering a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, optionally followed by administering a third therapy (e.g., a prophylactic or therapeutic agent) for a period of time, and so on, and repeating this sequential administration, i.e., cyclic, in order to reduce the development of tolerance to one of the therapies, avoid or reduce the side effects of one of the therapies, and / or improve the efficacy of the therapies.

[0458] In one embodiment, the IL-2 antibody of this disclosure may be co-administered with compositions for treating autoimmune diseases and conditions, including but not limited to doxorubicin, azathioprine, busulfan, cyclophosphamide, cyclosporine A, cytoxan, fludarabine, 5-fluorouracil, methotrexate, mycophenolate mofetil, 6-mercaptopurine, corticosteroids, nonsteroidal anti-inflammatory drugs, sirolimus (rapamycin), and tacrolimus (FK-506). In alternative embodiments, the immunomodulator or immunosuppressant is an antibody selected from: moromuzumab-CD3, alenzusumab. Baliximab, Dacizumab, Morotumab Rituximab, anti-thymocyte globulin, and IVIg, among others, are known to those skilled in the art.

[0459] In one embodiment, the IL-2 antibody disclosed herein may be co-administered with a composition for treating diabetes, said composition including but not limited to biguanides (e.g., buformin, metformin, and phenformin), hormones and their analogues (amyloidin, insulin, insulin aspart, insulin detemir, insulin glargine, insulin glutathione, insulin lispro, liraglutide, and pramlintide), sulfonylurea derivatives (acetylhexylurea, amiloride, chlorpropamide, glibenclamide, gliclazide, glimepiride, glipizide, gliquidone, glimepiride, glibenclamide, glithiazide, glibenclamide, glibenclamide, glibenclamide, glibenclamide, glibenclamide, glimepiride, tolazoline, tolbutamide, and sulfonylurea), thiazolidinediones (pioglitazone, rosiglitazone, and troglitazone), acarbose, exenatide, miglitol, miglitol, mogliflozin, nateglinide, repaglinide, sitagliptin, ticaglipizide, etc. Vidalistin and Voglibose.

[0460] In some embodiments, the IL-2 antibody or its antigen-binding portion of this disclosure may be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of this disclosure cross the BBB (if desired), they may be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548; and 5,399,331. Liposomes may contain one or more portions that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery (see, for example, VVRanade, 1989, J. Clin. Pharmacol. 29:685). Exemplary targeting components include folic acid or biotin (see, for example, U.S. Patent 5,416,016); mannosides (Umezawa et al., Biochem. Biophys. Res. Commun. 153:1038); antibodies (PGBloeman et al., 1995, FEBS Lett. 357:140; M. Owais et al., 1995, Antimicrob. Agents Chemother. 39:180); surfactant protein A receptors (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen, 1994, FEBS Lett. 346:123; Killion; Fidler, 1994; Immunomethods 4:273.

[0461] This disclosure provides a method for administering a pharmaceutical composition, alone or in combination with other therapies, to a subject in need, said pharmaceutical composition comprising the IL-2 antibody of this disclosure or its antigen-binding moiety. The combination therapies of this disclosure (e.g., prophylactic or therapeutic agents) may be administered simultaneously or sequentially to the subject. The combination therapies of this disclosure (e.g., prophylactic or therapeutic agents) may also be cyclically administered. Cyclic therapy involves administering a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by administering a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, and repeating this sequential administration, i.e., cyclic, to reduce the development of tolerance to one of the therapies (e.g., agents), avoid or reduce the side effects of one of the therapies (e.g., agents), and / or improve the efficacy of the therapy.

[0462] The combination therapies of this disclosure (e.g., prophylactic or therapeutic agents) can be administered simultaneously to a subject. The term "simultaneously" is not limited to administering the therapeutic agent (e.g., prophylactic or therapeutic agent) at exactly the same time, but rather means that a pharmaceutical composition comprising the IL-2 antibody of this disclosure or its antigen-binding moiety is administered to a subject in a sequence and at time intervals, such that the antibody or its conjugate can work together with other therapies to provide increased benefit than if they were administered otherwise. For example, each therapy may be administered sequentially to the subject at the same time or at different time points in any order; however, if not at the same time, they should be administered sufficiently close in time to provide the desired therapeutic or preventative effect. Each therapy may be administered to the subject, individually in any suitable form and via any suitable route. In various embodiments, the therapy (e.g., a preventative or therapeutic agent) is administered to the subject in less than 15 minutes, less than 30 minutes, less than 1 hour apart, about 1 hour apart, about 1 hour to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 7 hours apart, about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart. In other embodiments, two or more therapies (e.g., preventative or therapeutic agents) are administered within the same patient visit.

[0463] The prophylactic or therapeutic agent of a combination therapy may be administered to the subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agent of a combination therapy may be administered to the subject simultaneously in separate pharmaceutical compositions. The prophylactic or therapeutic agent may be administered to the subject via the same or different routes of administration.

[0464] Reagent test kit

[0465] This disclosure also provides kits containing any or all of the antibodies described herein. Kits of this disclosure include one or more containers containing the IL-2 antibody described herein and instructions for use according to any method of this disclosure as described herein. Generally, these instructions include a description of antibody administration for the therapeutic treatment described above. In some embodiments, kits are provided for generating a single-dose administration unit. In some embodiments, the kit may contain both a first container having a dried protein and a second container having an aqueous formulation. In some embodiments, kits include applicators, such as single-compartment and multi-compartment pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0466] Instructions for use of IL-2 antibodies generally include information on the dosage, administration regimen, and route of administration for the intended treatment. Containers may be unit dose, bulk packaging (e.g., multi-dose packaging), or subunit dose. Instructions provided with kits disclosed herein are typically written instructions on a label or packaging insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0467] The kit disclosed herein is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bag), etc. Packaging for use with specific devices (e.g., inhalers, nasal delivery devices (e.g., nebulizers)) or infusion devices (e.g., micropumps) is also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is the IL-2 antibody of this disclosure. The container may also contain a second pharmaceutical active agent.

[0468] The kit may optionally include additional components, such as buffer solutions and explanatory information. Typically, the kit includes a container and a label or packaging insert on or associated with the container.

[0469] This disclosure also provides diagnostic kits comprising any or all of the antibodies described herein. The diagnostic kits can be used, for example, to detect the presence of IL-2 in a sample. In some embodiments, the diagnostic kits can be used to identify individuals with a latent disease, condition, or condition that may put them at risk of developing an IL-2-mediated disease, condition, or condition, or an IL-2 deficiency disease, condition, or condition. In some embodiments, the diagnostic kits can be used to detect the presence and / or level of IL-2 in individuals suspected of having an IL-2-mediated disease or an IL-2 deficiency disease, condition, or condition.

[0470] The diagnostic kits disclosed herein include one or more containers containing the IL-2 antibody described herein and instructions for use according to any method of this disclosure described herein. Generally, these instructions include a description of the use of the IL-2 antibody to detect the presence of IL-2 in an individual who is at risk of having, or is suspected of having, an IL-2-mediated disease or IL-2 deficiency disease, condition, or condition. In some embodiments, exemplary diagnostic kits may be configured to contain reagents such as an IL-2 antibody, a negative control sample, a positive control sample, and instructions for using the kit.

[0471] Equivalent scheme

[0472] The foregoing description and the following examples have detailed certain specific embodiments of this disclosure and described the best mode considered by the inventors. However, it should be understood that this disclosure can be practiced in many ways, however detailed the foregoing may be in the text, and this disclosure should be interpreted in accordance with the appended claims and any equivalents thereof.

[0473] Although the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it should be understood that various changes and modifications can be made without departing from the teachings herein and the disclosures claimed below. The examples provided below are intended to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. Although the teachings have been described with reference to these exemplary embodiments, those skilled in the art will readily understand that numerous variations and modifications of these exemplary embodiments are possible without excessive experimentation. All such variations and modifications are within the scope of the teachings.

[0474] All references cited herein, including patents, patent applications, papers, textbooks, etc., and references cited therein, are incorporated herein by reference in their entirety to the extent that they have not already been cited. In the event that one or more of the cited references and similar materials differ from or contradict this patent application, including but not limited to defined terminology, use of terminology, described techniques, etc., this patent application shall prevail.

[0475] Exemplary embodiments

[0476] This disclosure is further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, this disclosure should in no way be construed as limited to the following examples, but rather as encompassing any and all variations that become apparent from the teachings provided herein.

[0477] Example

[0478] Example 1. IL-2 antibody binding kinetics and affinity

[0479] The affinity of antibodies for IL-2 was determined by surface plasmon resonance using a Biacore T200 instrument (GE Healthcare, Piscataway, NJ). Anti-IL-2 IgG was captured on a CM5 sensor chip using anti-human IgG prepared with the Biacore Human Antibody Capture Kit, following manufacturer's instructions (GE Healthcare). Mouse anti-human IL-2 clone 5344 (clone 5344.111, BD Biosciences) was captured on the surface using anti-mouse IgG prepared with the Biacore Mouse Antibody Capture Kit, following manufacturer's instructions (GE Healthcare). Experiments were performed at 25°C using a flow rate of 30 μL / min in 0.01 M HEPES pH 7.4, 0.15 M NaCl, and 0.005% v / v surfactant P20 (HBS-P) buffer. After each cycle, the chip surface was regenerated with 3 M MgCl2 and new antibodies were captured. Recombinant IL-2 (Humanzyme, Chicago, IL) was injected onto the surface for 3 minutes, and binding was monitored again for 20 minutes. Data were analyzed using BiacoreT200 Evaluation software. Background subtraction was performed on signals from adjacent control flow cells containing only the captured antibody, along with buffer-only injections for each antibody. A 1:1 Lammill binding model was used to fit all binding curves.

[0480] result

[0481] The tested human antibodies bound IL-2 with an affinity ranging from 536 pM to 13.4 nM (Table 3). Antibodies identified as having weaker affinity for IL-2, compared to comparative clones 16C3 and d1C7, exhibited a lower affinity for IL-2 in terms of their ability to inhibit IL-2Rβ binding relative to the d1C7 / IL-2 complex and reduce binding to IL-2Rα. Antibody 5344 was found to have a very stable dissociation rate exceeding the detection limit of the Biacore instrument, with calculated affinity assumed to be 50 pM or lower, and a significantly slower dissociation rate compared to the tested human antibody clones. This significantly slower dissociation rate of 5344 contributes to the antibody not exhibiting the desired Treg retention properties shown by other hIL-2 antibodies described below (Example 5) in this patent application.

[0482] Table 3

[0483] clone ka(M -1 s -1 )]]> kd(s -1 )]]> K D (M) F4.7.062 3.77E+06 5.80E-03 1.54E-09 F4.7.6 2.44E+06 7.38E-03 3.03E-09 F5.1.11 2.21E+06 6.88E-03 3.12E-09 F4.7.8 2.31E+05 3.10E-03 1.34E-08 F5.1.9 2.48E+05 3.28E-03 1.33E-08 16C3 9.89E+05 6.19E-04 6.37E-10 d1C7 1.13E+06 6.07E-04 5.36E-10 5344 1.02E+06 <＝5.00E-05* <＝5.00E-11

[0484] *The dissociation rate exceeds the instrument specifications; a dissociation rate of 5.0E-05 or lower has been assumed.

[0485] Studies using antibodies F5.1.11, d1C7, 13A10, and 16C3 to bind to mIL-2 and hIL-2 indicated that F5.1.11 and d1C7 do not bind to mIL-2, while 13A10 and 16C3 bind to mIL-2 with reduced affinity compared to hIL-2 (data not shown).

[0486] Example 2. IL-2 / antibody complex receptor binding

[0487] The ability of antibody-modified IL-2 to bind to the IL-2 receptors IL-2Rα and IL-2Rβ was evaluated using a Biacore T200 instrument (GE Healthcare, Piscataway, NJ). Recombinant IL-2α and IL-2Rβ proteins were biotin-labeled and captured on a Biacore Streptavidin chip (GE Healthcare). Experiments were performed at 25°C using a flow rate of 10 μL / min in 10 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% v / v surfactant P20 buffer. IL-2 (333 nM) was pre-incubated with IgG (1000 nM) for at least 20 minutes. The IL-2 / antibody complex was injected onto the receptor-conjugated chip surface for 60 seconds and allowed to dissociate for 20 minutes. Data were background subtracted using adjacent control flow units and buffer-only injections. The response was reported as the binding percentage of IL-2α and IL-2Rβ after 60 seconds, representing the binding percentages of two representative IL-2-reactive clones, d1C7 and 16C3, to IL-2Rα and IL-2Rβ, respectively.

[0488] result

[0489] The clonal 16C3 / IL-2 complex binds to IL-2Rβ but not IL-2Rα, while the d1C7 / IL-2 complex binds to IL-2Rα but not IL-2Rβ. These two antibody / IL-2 complexes were used as representative clones to normalize IL-2Rα and IL-2Rβ binding. The tested antibody / IL-2 complexes were reported individually for their respective relative binding to the receptor. Clones 4.7.062, F5.1.11, F4.7.6, F4.7.8, F5.1.9, and the commercially available 5344 (clone 5344.111, BD Biosciences) all inhibited IL-2 binding to IL-2Rβ, similar to the d1C7 / IL-2 complex, and showed reduced binding to IL-2Rα compared to the d1C7 / IL-2 complex. Therefore, the ability of these antibodies to block IL-2 binding to IL-2Rβ and reduce the rate of binding to IL-2Rα was identified. Figure 1).

[0490] Example 3. Antibody / IL-2 affinity and receptor binding on affinity maturation clones

[0491] The binding kinetics and receptor binding profile of the affinity-matured F5.1.11 antibody / IL-2 complex were measured using surface plasmon resonance, as described for the parent antibody (Example 1).

[0492] result

[0493] Affinity maturation activity increased the apparent binding affinity of clone F5.1.11 for IL-2 by up to 16-fold. Three affinity-matured variants, F5.1.11.02, F5.1.11.04, and F5.1.11.08, were measured to have binding energies of 114, 624, and 205 pM for IL-2, respectively. Characterization of the antibody / IL-2 complexes bound to IL-2Rα and IL-2Rβ revealed that the receptor binding profile of the higher affinity variants remained unchanged compared to the parent molecule. Both the parent and affinity-matured clones showed complete inhibition of the antibody / IL-2 complex bound to IL-2Rβ and reduced binding to IL-2Rα compared to the clonal d1C7 / IL-2 complex. Some variations in the binding of parental F5.1.11 / IL-2 to IL-2Rα were observed experimentally, indicated by the dashed lines in the figure to describe the potential range. The three highly affinity variants, F5.1.11.02, F5.1.11.04, and F5.1.11.08, all fall within this range (Table 4 and...). Figure 2 ).

[0494] Table 4

[0495]

[0496]

[0497] Example 4. IL-2: Phenotype of Tregs after treatment with anti-IL-2 mAb

[0498] IL-2-anti-IL-2 complex treatment

[0499] PBMCs were isolated from healthy donors using Ficoll and activated overnight with 12.5 ng / mL anti-CD3 and 25 ng / mL anti-CD28. After overnight incubation, cells were harvested and thoroughly washed with PBS and incubated at 30 x 10⁻⁶ cells / mL. 6The concentration of each cell was resuspended in 200 μL PBS. PBMCs were administered intravenously to NSG receptors. NSG mice injected with human PBMCs received intraperitoneal injections of 8,000 U hIL-2 (Proleukin) in combination with 25 μg isoform, 25 μg 16C3, 1 μg, 5 μg, or 25 μg Ab F5.1.11.02 daily for 5 days at 37°C for 30 minutes.

[0500] Isolation and staining of spleen cells

[0501] Mice were sacrificed with CO2, and spleen cells were harvested and stained extracellularly with anti-human CD45 Pacific Orange, CD4PercPCy 5.5, CD3 PeCy7 / Pacific Blue / PercP, CD25 APC-Cy7 / Pe, CD238 Pe, CD39 PeCy7, CD8 APC-H7, Ki67 Pe, NKG2D PeCy7, and CD44 V450, and intracellularly stained with anti-human Helios FITC and FoxP3 APC. Labeled antibodies were purchased from BD PharMingen or Ebioscience. Stained single-cell suspensions were analyzed using a Fortess flow cytometer running FACSDiva (BD Biosciences), and the analysis was performed using FLOWJO software, resulting in FSC 3.0 files.

[0502] result

[0503] Antibody 16C3, which conjugates with hIL-2, increases the percentage of Tregs in the spleen. An incremental increase in the percentage of Tregs was also observed in response to treatment with two different doses (5 μg and 25 μg) of antibody F5.1.11.02. The Treg population responds to hCD45. + CD3 + CD4 + Helios + FoxP3 + Cell gating (Figure 3). Both 16C3 and F5.1.11.02, when combined with hIL-2, increased the Treg / Teff and Treg / NK cell ratios.

[0504] 16C3 (25 μg) in combination with hIL-2 increased the Treg / CD4, Treg / CD8, and Treg / NK cell ratios. In the first experiment, treatment with F5.1.11.02 at the lowest dose (1 μg) did not show an increase in these ratios. Conversely, significant increases in the Treg / CD4, Treg / CD8, and Treg / NK cell ratios were observed in response to 5 μg and 25 μg of F5.1.11.02 in combination with hIL-2. Figure 4A -C). In the second experiment, the same results were observed for 16C3 as well as for 5 μg and 25 μg F5.1.11.02, and additionally, the increase in ratio was also evident for the low dose of F5.1.11.02 (1 μg) (Fig. 4D-F). Both 16C3 and F5.1.11.02 combined with hIL-2 increased the total number of Teff and Treg cells. In both experiments, 16C3 (25 μg) combined with hIL-2 increased CD4+ in the spleen. + CD8 + Total number of cells and Tregs.

[0505] F5.1.11.02, combined with hIL-2, also showed CD4. + CD8 + An increase in the number of cells and total Treg cells. In the first experiment, the effect was more pronounced at doses of 5 μg and 25 μg F5.1.11.02. Figure 5A -C). The second experiment showed a dose-dependent response to F5.1.11.02 in CD4. + CD8 + Increase in the total number of cells and Tregs ( Figure 5D Treatment with F5.1.11.02 also induced an increase in the mean fluorescence intensity (MFI) of CD25, Icos, and FoxP3 on Treg, which was not observed in the presence of 16C3. Figure 6A -C).

[0506] Example 5. Anti-IL-2 antibody inhibits pSTAT5

[0507] Spleen cells from C57B16 cells expressing GFP under the control of the Foxp3 promoter were harvested, processed into single-cell suspensions, and resuspended in RPMI 0.1% BSA. Cells were incubated at 37°C in a tissue culture incubator until the assay time (1-2 hours).

[0508] PBMCs were purified from human Trima remnants (Blood Centers of the Pacific) and resuspended in RPMI 0.1% BSA. Cells were incubated at 37°C in a tissue incubator until assay time (1–2 hours).

[0509] IL-2-induced pSTAT5 assay

[0510] Place PBMCs or spleen cells in 96-well V-bottom plates, ensuring 1 million cells per well in 50 μL (RPMI, 0.1% BSA). Return the plates to a 37°C incubator to maintain the temperature. Titrate the antibody (JES6-1 (JES6-1A12 eBioscience) or anti-human IL-2) and test for IL-2 at four concentrations: 500 ng / mL, 50 ng / mL, 5 ng / mL, and 0.5 ng / mL.

[0511] Commercially available mouse IL-2 monoclonal antibody JES6-1 blocks both the IL-2 IL2Rα and IL2Rβ interfaces (León et al., 2013, "Mathematical models of the impact of IL2 modulation therapies on T cell dynamics," Frontiers in Immunology, 4:439, cited in full). JES6-1 binds to mIL-2 but not hIL-2 because the epitope is not conserved in hIL-2. Key amino acid residues for JES6-1 binding to IL-2 include mIL-2 residues Q36 and E37, corresponding to hIL-2 residues Q22 and M23, as cited in Spangler et al., 2015, "Antibodies to Interleukin-2 Elicit Selective T Cell Subset Production through Distinct Conformational Mechanisms," Immunity, 42:815, cited in full. Figure 3Details are provided in sections 4, S2, S3, and S4. The kDa of mIL-2 / mIL-2Rβ was reported by Spangler et al. as >7 μM, and for mIL-2 / mIL-2Rα, the kDa was 2-4 times weaker than that of hIL-2. A comparison of the amino acid sequences of mouse and human IL-2 is available in Figure 6 of Yokota et al., 1985, “Use of a cDNA expression vector for isolation of mouse interleukin 2 cDNA clones: Expression of T-cell growth-factor activity after transfection of monkey cells,” Proc. Natl. Acad. Sci. USA, 82:68, which is incorporated herein by reference in its entirety.

[0512] An equal volume (50 μL) of 2x IL2:antibody complex (prepared at 37°C for 1 hour) was added to each well, and the cells were incubated at 37°C for 40 minutes. Cells were fixed by adding 100 μL of IC fixation buffer (eBioscience). After 15 minutes of fixation, the cells were washed and stored in FAC buffer (PBS + 0.2% BSA) until staining.

[0513] The assay plate was centrifuged to form cell clumps, and the cells were resuspended in permeation buffer III (BD Biosciences) and then incubated on ice for 30 minutes. Cells were washed twice in FAC buffer and stained with the following Abs: anti-human CD3 APC e780, CD4 Percp e710, and CD127 (PE) from eBiosciences. CD8 FITC, CD25 (PeCy7), FoxP3 (e660), and pSTAT5 (Pacific blue) were purchased from BD Biosciences. For spleen cells, the following anti-mouse antibodies were used: CD4 e660 and CD8a PeCy7 from eBiosciences. pSTAT5 Pacific Blue (BD Biosciences) was used for both mouse and human cells.

[0514] Data were collected on an LSR Fortessa and analyzed using FlowJo software. Data were plotted as background-subtracted MFI, normalized to the maximum signal for each cell type (IL2 500 ng / mL + isotype). Background was defined as unstimulated but stained pSTAT5. Human Tregs were defined as CD3+.+ CD8 - CD4 + CD25hiCD127lo. Mouse Tregs are defined as CD8... - CD4 + Foxp3.GFP + c cells.

[0515] result

[0516] Inhibition of pSTAT5 in PBMCs: F5.1.11 affinity variant

[0517] Affinity variants of F5.1.11, such as F5.1.11.02 and F5.1.11.08, have been shown to have increased affinity for IL-2 in inhibiting CD8+. + Effector T cells and non-Treg CD4 + pSTAT5 signaling is more efficient in T cells. At IL-2 concentrations of 5–500 ng / mL, pSTAT5 levels in Tregs remained greater than 50%. At the lowest tested IL-2 concentration (0.5 ng / mL), pSTAT5 levels in Tregs were suppressed to below 50% of the maximum signal; however, pSTAT5 remained detectable across all Ab concentrations. Figure 7A -V). The 5344 antibody was also tested in a pSTAT5 signaling assay, and in Treg, pSTAT5 was undetectable at low concentrations of IL-2 (data not shown).

[0518] Comparison of JES6-1 and antibody F5.1.11.02

[0519] Compared with the same type ( Figure 8A -F) Similarly, the mouse IL-2 antibody JES6-1 preserved Treg pSTAT5 signaling at most of the IL-2 concentrations tested. In contrast, IL-2-induced pSTAT5 signaling was absent in mouse CD8 at all IL-2 concentrations tested. + JES6-1 strongly inhibited T cell growth. This data is consistent with published observations that JES6-1, in combination with IL-2, promotes the growth of Treg cells in vivo.

[0520] At most IL-2 concentrations tested, the anti-human IL-2 antibody F5.1.11.02 largely preserved human Treg pSTAT5 signaling. At all IL-2 concentrations tested, CD8... +T cell pSTAT5 was inhibited by IL-2 antibody F5.1.11.02. In summary, the pSTAT5 inhibition pattern of IL-2 antibody F5.1.11.02 in CD8 and Treg cells is similar to that observed with mouse IL-2 antibody JES6-1.

[0521] In Table 5, from Figure 8A The pSTAT 5%max value of -F is used to generate the area under the curve (AUC) values ​​for Treg or CD8 cells treated with allotype or anti-IL-2 antibodies. The Treg / CD8 AUC ratios are listed for each IL-2 concentration. In allotype-treated samples, decreasing amounts of IL-2 lead to an increased AUC ratio, reflecting the difference in AUC ratios between Treg and CD8 cells. + Effector cells showed increased pSTAT5 signaling. Although the absolute numbers differed, this observation was consistent between mouse and human cells.

[0522] At higher concentrations of IL-2, JES6-1 treatment shifted this ratio to favor Tregs. A similar change in the AUC ratio occurred in human cells treated with the F5.1.11.02 antibody.

[0523] In summary, F5.1.11.02 appears to have a similar pSTAT5 signal conduction profile to JES6-1.

[0524] Table 5

[0525]

[0526] Antibody epitopes against CD25hi and CD8 + Effect of pSTAT5 inhibition on T cells

[0527] Some peripheral CD8 + T cells express CD25 (IL-2Rα). For example... Figure 10A As shown in -B, if CD25-gated cells are used, CD25hi CD8 cells are more sensitive to low levels of IL-2. Therefore, IL-2 antibodies may be needed to completely or partially block CD25 binding to inhibit CD25hi CD8 cells. + pSTAT5 signaling in T cells.

[0528] As shown in Figure 9, IL-2 antibodies (such as 13A10 or F5.1.11.02) that block the binding of IL-2Rα to IL-2 optimally inhibited pSTAT5 induced by a low dose of IL-2 (0.8 ng / mL). Conversely, high concentrations of IL-2 (500 ng / mL) (presumably not requiring the presence of IL-2Rα for signal transduction) were optimally inhibited by antibodies (such as d1C7 or F5.1.11.02) that block the binding of IL-2 to IL-2Rβ. Across a range of IL-2 concentrations, antibody F5.1.11.02 optimally inhibited CD8. + pSTAT5 signaling in effector T cells

[0529] Inhibition of pSTAT5 by IL-2 antibody F5.1.9

[0530] Evaluation of the inhibition of pSTAT5 by IL-2 antibody F5.1.9 and variant F5.1.9.5 ( Figure 11A -H). The affinity variant of F5.1.9, F5.1.9.5, effectively inhibits CD8. + The extent to which pSTAT5 signaling is inhibited in effector T cells is greater than its inhibition of pSTAT5 signaling in Treg cells. F5.1.9.5ka (M -1 s -1 The value is 1.23E+06, kd(s) -1 The value is 8.18E-04, and K D (M) is 6.63E-10.

[0531] Example 6. Generation of NOD mIL-2- / -hIL-2+ / - mice

[0532] A mouse bacterial artificial chromosome (BAC) containing IL-2 (clone RP23-290D8) was engineered to express the human IL-2 coding sequence. The following modifications were made to the BAC: the mouse IL-2 promoter, 5' and 3' UTRs, and introns remained intact; the mouse signal peptide was replaced with a human signal peptide; half of exon 1 downstream of the signal peptide, and exons 2 and 3 were replaced with human sequences; half of exon 4 was replaced with a human coding sequence.

[0533] The modified BAC was then transferred to zygotes of FVB / NJ (Friend Virus B) female mice (Jackson) via pronuclear injection. These zygotes were then implanted into FVB female mice to generate FVB mIL-2+ / +hIL-2BAC+ mice. To generate mice lacking mIL2 expression in a NOD background, FVB mIL-2+ / +hIL-2BAC+ mice were backcrossed with NOD mIL-2- / - mice. NOD mIL-2- / -hIL-2BAC+ / - mice generated from this initial breed were backcrossed with NOD mIL-2- / - mice for eight generations to generate mice with type 1 diabetes. The resulting mice were used for experiments.

[0534] method

[0535] After ACK lysis, whole blood (100 μL) was stimulated overnight with PMA / ionomycin, and the supernatant (125 μL) was collected and ELISA (ELISA: ebioscience) was performed for mIL-2 (50 μL) and hIL-2 (50 μL).

[0536] Female NOD mIL2- / -hIL2+ / - mice were injected at 14 weeks of age and received 8,000 IU of hIL-2 (Proleukin) daily for 5 days, either 25 μg of the same type or 5 μg or 25 μg of F5.1.11.02. IL-2:antibody complexes were formed by incubating IL-2 and antibody together at 37°C for 30 minutes.

[0537] Isolation and cell staining of different organs

[0538] Mice were euthanized with CO2 and immediately perfused with PBS through the left ventricle until the effluent became clear. Spleen cells and pancreatic lymph nodes (pLNs) were harvested and subjected to extracellular staining with CD25 PeCy7, CD4 BV605, CD8 PercP-Cy5.5, CD44 Pe, and CD62L Al647, and intracellular staining with FoxP3 FITC.

[0539] Whole pancreas was prepared by digestion with 0.8 mg / mL collagenase P and 20 μg / mL DNase (Roche) at 37°C for 30 min, followed by cleavage. After digestion, the homogenate was filtered twice through a 40 μm cell filter and subjected to extracellular staining with CD45PercP Cy5.5, CD25 Pe, CD4 PeCy7, CD8 Al647, Thy1.2 BV605 and intracellular staining with FoxP3FITC. Labeled antibodies were purchased from BD Pharmaceuticals or Ebioscience. The stained single-cell suspensions were analyzed and visualized using FLOWJO software.

[0540] result

[0541] Phenotype of human IL-2 transgenic (hIL-2Tg) mice

[0542] Mouse spleen cells were stimulated in vitro with PMA / ionomycin. Figure 12A -B) confirmed the production of human IL-2 from transgenic mice, although the level of human IL-2 produced was lower than that of mouse IL-2 from wild-type mice. hIL-2Tg / mIL-2- / - mice appeared healthy and energetic. However, compared to NOD or NOD mIL-2+ / - mice, hIL-2Tg mice had an increased percentage of activated (CD44) cells. + CD62L-)CD4 + or CD8 + T cells ( Figure 13A -B). Although Treg frequencies are similar to those of NOD or NODmIL-2+ / - mice, Tregs from hIL-2Tg mice have reduced CD25 expression on the cell surface ( Figure 14 ).

[0543] hIL-2Tg mice were treated with F5.1.11.02:hIL-2 complex.

[0544] As described above (methods), hIL-2Tg mice were treated with the F5.1.11.02:IL-2 complex. Treatment on day 7 did not increase the overall cellularity of the spleen, and a slight decrease in total spleen cells was observed in the 5 μg treatment group. Figure 15 Compared with the isotype control, treatment with two doses of F5.1.11.02 in combination with hIL-2 resulted in an increased percentage of Tregs in all organs except pLN, with a more significant increase in the pancreas. Figure 16A -I). In the spleen and pLN, the Treg population is resistant to CD4. + CD25 + FoxP3 +Cells are gated, and in the pancreas, through the inhibition of CD45... + Thy1.2 + CD4 + CD25 + FoxP3 + Cell gating was used to identify Treg cells.

[0545] Following treatment with the complex, a decrease in the total number of CD4, CD8, and Treg cells was observed in the spleen and pLN. However, in the pancreas, treatment with the F5.1.11.02 complex at a dose of 25 μg induced a slight increase in the total number of Treg cells. No difference in the total number of CD4 and CD8 cells was observed in the pancreas (data not shown).

[0546] Treatment of hIL-2Tg mice with F5.1.11.02 (25 μg) in combination with hIL-2 induced a significant increase in the Treg / CD4 and Treg / CD8 cell ratios in the spleen. Figure 17A -F). Similar results were observed in the pancreas, with a significant increase in the Treg / CD4 and Treg / CD8 ratios observed with 5 μg and 25 μg F5.1.11.02. F5.1.11.02: IL2 complex treatment had little effect on cell ratios in pancreatic lymph nodes.

[0547] Finally, treatment of huIL2 Tg mice with the F5.1.11.02:IL-2 complex (5 μg and 25 μg F5.1.11.02) induced an increase in the mean fluorescence intensity (MFI) of CD25 on Tregs in all organs (Fig. 18A-C). This increase was particularly significant in the pancreas, where the Treg CD25 MFI was very low before treatment.

[0548] NSG experiment using CTV

[0549] PBMCs were isolated from healthy donors using Ficoll and activated overnight with 12.5 ng / mL anti-CD3 and 25 ng / mL anti-CD28. After overnight activation, cells were harvested and used with 1 μL CTV / 10x10 6 Cell Trace Violet (Life Technology) labeling at cell concentrations. After staining, cells were thoroughly washed with PBS and then... 6The concentration of cells was resuspended in 200 μL PBS. PBMCs were injected intravenously into the NSG receptor. NSG mice injected with human PBMCs received 8,000 U hIL-2 (Proleukin) in combination with an allotype or antibody via intraperitoneal injection for 5 days at the indicated dose, at 37°C for 30 minutes.

[0550] Isolation and staining of spleen cells

[0551] Mice were sacrificed with CO2 on days 3 and 5, and spleen cells were harvested. Extracellular staining was performed with anti-human CD45 PacificOrange, CD4 PercPCy5.5, CD25Pe, CD8 APC-H7, and NKG2D PeCy7, while intracellular staining was performed with anti-human HeliosFITC and FoxP3 APC. Labeled antibodies were purchased from BD PharMingen or Ebioscience. Stained single-cell suspensions were analyzed using a Fortess flow cytometer running FACSDiva (BD Biosciences), and the analysis was performed using FLOWJO software, resulting in FSC 3.0 files.

[0552] result

[0553] Treatment with F5.1.11.02 in combination with hIL-2 at day 5, instead of day 3, induced an increase in the total number of splenocytes. Figure 19A -B).

[0554] The F5.1.11.02 antibody:IL-2 complex can also increase the total number of Teff and Treg cells in the spleen on day 5. Figure 20A -F) and the ratios of Treg / CD4 and Treg / CD8 ( Figure 21A -D), which was not observed on day 3. Treg populations against hCD45 + CD3 + CD4 + Helios + FoxP3 + Cells perform gating.

[0555] By including CTV markers prior to transfer and treatment, we observed that treatment with the F5.1.11.02 antibody:IL-2 complex induced Treg proliferation compared to the isotype, with an effect already evident on day 3 and resulting in a low number of undivided cells by day 5. Figure 22A-H). Treatment with the F5.1.11.02 antibody:IL-2 complex also increased CD8 T cell proliferation; however, this was only significant on day 5, and a greater number of undivided cells were maintained than in the Treg population. Figure 23A In summary, the CTV data support increased Treg proliferation, as a potential shift was observed in the Treg / CD8 ratio.

[0556] Finally, in the spleen, treatment with both doses of the complex induced an increase in the mean fluorescence intensity (MFI) of CD25 in both Treg and CD8 populations compared to the isotype control. Figure 24A -B). However, the absolute expression of CD25 on Treg cells is many times higher than that on CD8 cells.

[0557] In subsequent experiments, we compared F5.1.11.02 with the lower-affinity 'parental' antibody F5.1.11. We extended the dose range to 125 μg of the antibody complexed with hIL2 to evaluate whether higher doses of the lower-affinity antibody had comparable effects in vivo. At 125 μg, F5.1.11 had comparable effects on Treg and CD8 cell numbers, increased Treg / CD8 ratio, and cellularity, equivalent to 25 μg of F5.1.11.02. Figure 25A -D, 26A-C, and 33A-B). As observed in CTV dilutions, this is again supported by increased Treg proliferation ( Figure 27A -H, 28A-H and 38). We predict that in vitro, F5.1.11.02 will similarly increase the levels of Foxp3 and CD25 proteins on human Tregs.

[0558] Example 7. IL-2 antibody binding affinity for cynomolgus monkey IL-2

[0559] Recombinant cynomolgus macaque (Macaca fascicularis) IL-2 was expressed in mammalian cells and purified using immobilized metal ion affinity chromatography (IMAC). The affinity of the antibody for cynomolgus macaque IL-2 (1–100 nM) was measured using surface plasmon resonance as described for human IL-2 (Example 1). A 1:1 Lammill binding model was used to fit the binding curves or to calculate the steady-state affinity constant from the concentration-response curves. Clones where the observed concentration-response relationship for 1–100 nM IL-2 was insufficient to calculate the apparent binding affinity were designated as “very weak.”

[0560] result

[0561] The tested human antibody clones bound to cynomolgus monkey IL-2 with an affinity ranging from 516 pM to very weak binding that could not be quantified in this assay (Table 6). When selected based on their ability to inhibit IL-2Rβ binding, most clones were tested (Examples 2 and 3), and these clones showed a significant loss of affinity for cynomolgus monkey IL-2 compared to human IL-2. The 16C3 clone (Example 2), which inhibits IL-2Rα binding, was observed to show equivalent binding to both human and cynomolgus monkey IL-2. These results are consistent with our structural analysis, which revealed that the divergent region between human and cynomolgus monkey IL-2 falls within both the IL-2Rβ binding site and the epitope of the F5.1.11 antibody clone (Example 9).

[0562] Table 6

[0563] clone Human IL-2KD(M) cynomolgus monkey IL-2KD(M) F5.1.11 3.12E-09 Very weak F4.7.8 1.34E-08 9.24E-08 F5.1.9 1.33E-08 Very weak 16C3 6.37E-10 5.16E-10 d1C7 5.36E-10 >＝1.08E-07 5344 <＝5.00E-11* >＝5.0E-08 F5.1.11.02 1.14E-10 ～2.11E-07

[0564] *The dissociation rate exceeds the instrument specifications; a dissociation rate of 5.0E-05 or lower has been assumed.

[0565] Example 8. Receptor binding affinity of the IL-2 / Fab complex

[0566] The affinity of IL-2Rα for IL-2 or the IL-2 / F5.1.11Fab complex was measured using a Biacore T200 instrument (GE Healthcare, Piscataway, NJ). Recombinant IL-2α was biotin-labeled and captured on a Biacore Streptavidin chip (GE Healthcare). Experiments were performed at 25°C using a flow rate of 10 μL / min in 10 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% v / v surfactant P20 buffer. IL-2 (1–50 nM) or IL-2 / F5.1.11Fab (pre-complexed 1–500 nM IL-2 with excess Fab) was injected at 30 μL / min onto the receptor-conjugated chip surface for 120 seconds, allowing dissociation for 5 minutes. Data were background subtracted using adjacent control flow units and buffer-only injections. The affinity of the interaction was calculated from the concentration-response relationship at equilibrium.

[0567] result

[0568] Compared to ligandless IL-2, the IL-2 / F5.1.11Fab complex binds to IL-2Rα with a lower affinity. Equilibrium binding analysis of the complex and ligandless IL-2 revealed that IL-2 bound to F5.1.11Fab has approximately 7 times weaker affinity for IL-2Rα (9.97 nM compared to 70.1 nM). Figure 29A -B). This result is consistent with the reduced binding observed in the IL-2 / antibody complex receptor binding assay (Example 2) and the analysis of the IL-2 / F5.1.11 Fab crystal structure (Example 9).

[0569] Example 9. Crystal structure of F5.1.11 Fab bound to human IL-2

[0570] Interleukin-2 expression and purification

[0571] Full-length IL-2 (residues 1-133) was purified as described in (Rickert et al., 2004). In short, the gene was in-frame cloned into the pAcgp67A vector along with the gp67A signal sequence, followed by a C-terminal hexahistine tag (SEQ ID NO: 223). Fall armyworm (Spodoptera frugiperda) (Sf9) cells were used to generate high-titer recombinant virus. Trichopulsia ni (High-Five) cells grown in Insect Xpress medium (Lonza) were infected with the virus and allowed to express the protein at 28°C for 48 hours. The protein was purified by Ni-NTA and digested overnight at 4°C with carboxypeptidase A and B, then purified on a Superdex 200 gel filter column (GE Healthcare).

[0572] Crystallization of the 5.1.11Fab complex with interleukin-2

[0573] F5.1.11Fab was mixed with a 1.5-fold excess of IL-2, and the complex was purified by FPLC on a Superdex 200 column (GE Healthcare). For example, using 1.49 mL*mg... -1 *cm -1 The extinction coefficient was measured at 280 nm. The purified Fab / IL-2 complex was concentrated to 10 mg / mL. The complex was mixed with an equal volume of precipitant solution (100 mM sodium citrate, pH 5.0; 20% polyethylene glycol 6000) and crystallized on a reservoir of precipitant solution via seated drop vapor diffusion. Prismatic crystals formed within one week. The crystals were harvested by cryogenic protection with the addition of glycerol to 30% (v / v) and rapidly cooled by immersion in liquid nitrogen.

[0574] Crystallographic data collection and improvement

[0575] Diffraction data were collected using Advanced Light Source Beamline 8.2.1 (Berkeley, CA). The crystal is indexed in space group P21, with a unit cell size of [missing information]. β = 95.38°. Maximum resolution. For structural resolution and refinement, the structure was resolved using molecular substitutions via the procedure Phaser (McCoy et al., 2007). Search models included the heavy chain constant domain from PDB entry 3U1S, the heavy chain variable domain from 4NPY, the light chain variable domain from 3N9G, the light chain variable domain from 4HP0, and interleukin-2 from 2B5I (Wang et al., 2005; McLellan et al., 2011; Ogata et al., 2013; Sok et al., 2013; Kaufmann et al.). Four copies of the Fab / IL-2 complex were modeled in asymmetric units. The structure was constructed using manual reconstruction and improved iterative loops via the procedures Coot (Emsley et al., 2010) and PHENIX (Adams et al., 2010). Visual inspection in Coot and analysis of protein-protein interactions using PISA (Krissinel and Henrick, 2005; 2007) were performed. Using human apo-IL-2 (PDB ID 1M47 (Arkin et al., 2003) as a template, a homology model of cynomolgus monkey (crab-eating macaque) IL-2 was prepared using I-TASSER (Roy et al., 2010; Yang et al., 2015).

[0576] result

[0577] F5.1.11 interacts with IL-2 via light chain CDR1 and CDR3 rings and heavy chain CDR2 and CDR3 rings at helical A and C and BC rings. Figure 30 Comparison with the IL-2 / IL-2Rβ / γc / CD25 quaternary structure (PDB ID 2B5I) reveals that F5.1.11 blocks the IL-2Rβ binding site of IL-2, rather than CD25 or γc. c Binding sites. The overall structure of IL-2 is similar to the apo structure (RMSD on 95 Cα atoms). When F5.1.11 is combined with the BC ring of IL-2, a significant perturbation occurs; this perturbation propagates to the adjacent IL-2A-B rings. ...

Claims

1. An isolated antibody or its antigen-binding moiety that specifically binds to human IL-2, said antibody comprising: (i) (a) HCDR1 as shown in SEQ ID NO: 73, encoded according to Kabat number; HCDR2 as shown in SEQ ID NO: 76, encoded according to Kabat number; HCDR3 as shown in SEQ ID NO: 78, encoded according to extended number; LCDR1 as shown in SEQ ID NO: 79, encoded according to extended number; LCDR2 as shown in SEQ ID NO: 80, encoded according to extended number; and LCDR3 as shown in SEQ ID NO: 81, encoded according to extended number; (b) HCDR1 as shown in SEQ ID NO: 74, encoded according to Chothia number; HCDR2 as shown in SEQ ID NO: 77, encoded according to Chothia number; HCDR3 as shown in SEQ ID NO: 78, encoded according to extended number; LCDR1 as shown in SEQ ID NO: 79, encoded according to extended number; LCDR2 as shown in SEQ ID NO: 80, encoded according to extended number; and LCDR3 as shown in SEQ ID NO: 81, encoded according to extended number; (c) HCDR1 as shown in SEQ ID NO: 75, encoded according to the extended number; HCDR2 as shown in SEQ ID NO: 76, encoded according to the Chothia number; HCDR3 as shown in SEQ ID NO: 78, encoded according to the extended number; LCDR1 as shown in SEQ ID NO: 79, encoded according to the extended number; LCDR2 as shown in SEQ ID NO: 80, encoded according to the extended number; and LCDR3 as shown in SEQ ID NO: 81, encoded according to the extended number; (d) HCDR1 as shown in SEQ ID NO: 82, encoded according to the Kabat number; HCDR2 as shown in SEQ ID NO: 85, encoded according to the Kabat number; HCDR3 as shown in SEQ ID NO: 87, encoded according to the extended number; LCDR1 as shown in SEQ ID NO: 88, encoded according to the extended number; LCDR2 as shown in SEQ ID NO: 89, encoded according to the extended number; and LCDR3 as shown in SEQ ID NO: 90, encoded according to the extended number; (e) HCDR1 as shown in SEQ ID NO: 83, encoded according to Chothia number; HCDR2 as shown in SEQ ID NO: 86, encoded according to Chothia number; HCDR3 as shown in SEQ ID NO: 87, encoded according to extended number; LCDR1 as shown in SEQ ID NO: 88, encoded according to extended number; LCDR2 as shown in SEQ ID NO: 89, encoded according to extended number; and LCDR3 as shown in SEQ ID NO: 90, encoded according to extended number; (f) HCDR1 as shown in SEQ ID NO: 84, encoded according to the extended number; HCDR2 as shown in SEQ ID NO: 85, encoded according to the Kabat number; HCDR3 as shown in SEQ ID NO: 87, encoded according to the extended number; LCDR1 as shown in SEQ ID NO: 88, encoded according to the extended number; LCDR2 as shown in SEQ ID NO: 89, encoded according to the extended number, comprising; and LCDR3 as shown in SEQ ID NO: 90, encoded according to the extended number; (g) HCDR1 as shown in SEQ ID NO: 91, encoded according to the Kabat number; HCDR2 as shown in SEQ ID NO: 94, encoded according to the Kabat number; HCDR3 as shown in SEQ ID NO: 96, encoded according to the extended number; LCDR1 as shown in SEQ ID NO: 97, encoded according to the extended number; LCDR2 as shown in SEQ ID NO: 98, encoded according to the extended number; and LCDR3 as shown in SEQ ID NO: 99, encoded according to the extended number; (h) HCDR1 as shown in SEQ ID NO: 92, encoded according to Chothia number; HCDR2 as shown in SEQ ID NO: 95, encoded according to Chothia number; HCDR3 as shown in SEQ ID NO: 96, encoded according to extended number; LCDR1 as shown in SEQ ID NO: 97, encoded according to extended number; LCDR2 as shown in SEQ ID NO: 98, encoded according to extended number; and LCDR3 as shown in SEQ ID NO: 99, encoded according to extended number; (i) HCDR1 as shown in SEQ ID NO: 93, encoded according to the extended number; HCDR2 as shown in SEQ ID NO: 94, encoded according to the Kabat number; HCDR3 as shown in SEQ ID NO: 96, encoded according to the extended number; LCDR1 as shown in SEQ ID NO: 97, encoded according to the extended number; LCDR2 as shown in SEQ ID NO: 98, encoded according to the extended number; and LCDR3 as shown in SEQ ID NO: 99, encoded according to the extended number; (j) HCDR1 as shown in SEQ ID NO: 100 according to the Kabat number; HCDR2 as shown in SEQ ID NO: 103 according to the Kabat number; HCDR3 as shown in SEQ ID NO: 105 according to the extended number; LCDR1 as shown in SEQ ID NO: 106 according to the extended number; LCDR2 as shown in SEQ ID NO: 107 according to the extended number; and LCDR3 as shown in SEQ ID NO: 108 according to the extended number; (k) HCDR1 as shown in SEQ ID NO: 101 according to Chothia number; HCDR2 as shown in SEQ ID NO: 104 according to Chothia number; HCDR3 as shown in SEQ ID NO: 105 according to extended number; LCDR1 as shown in SEQ ID NO: 106 according to extended number; LCDR2 as shown in SEQ ID NO: 107 according to extended number; and LCDR3 as shown in SEQ ID NO: 108 according to extended number; (l) HCDR1 as shown in SEQ ID NO: 102 according to the extended numbering; HCDR2 as shown in SEQ ID NO: 103 according to the Kabat numbering; HCDR3 as shown in SEQ ID NO: 105 according to the extended numbering; LCDR1 as shown in SEQ ID NO: 106 according to the extended numbering; LCDR2 as shown in SEQ ID NO: 107 according to the extended numbering; and LCDR3 as shown in SEQ ID NO: 108 according to the extended numbering; (m) HCDR1 as shown in SEQ ID NO: 109, encoded according to the Kabat number; HCDR2 as shown in SEQ ID NO: 112, encoded according to the Kabat number; HCDR3 as shown in SEQ ID NO: 114, encoded according to the extended number; LCDR1 as shown in SEQ ID NO: 115, encoded according to the extended number; LCDR2 as shown in SEQ ID NO: 116, encoded according to the extended number; and LCDR3 as shown in SEQ ID NO: 117, encoded according to the extended number; (n) HCDR1 as shown in SEQ ID NO: 110, encoded according to Chothia number; HCDR2 as shown in SEQ ID NO: 113, encoded according to Chothia number; HCDR3 as shown in SEQ ID NO: 114, encoded according to extended number; LCDR1 as shown in SEQ ID NO: 115, encoded according to extended number; LCDR2 as shown in SEQ ID NO: 116, encoded according to extended number; and LCDR3 as shown in SEQ ID NO: 117, encoded according to extended number; (o) HCDR1 as shown in SEQ ID NO: 111 according to the extended numbering; HCDR2 as shown in SEQ ID NO: 112 according to the Kabat numbering; HCDR3 as shown in SEQ ID NO: 114 according to the extended numbering; LCDR1 as shown in SEQ ID NO: 115 according to the extended numbering; LCDR2 as shown in SEQ ID NO: 116 according to the extended numbering; and LCDR3 as shown in SEQ ID NO: 117 according to the extended numbering; (p) HCDR1 as shown in SEQ ID NO: 145, encoded according to the Kabat number; HCDR2 as shown in SEQ ID NO: 148, encoded according to the Kabat number; HCDR3 as shown in SEQ ID NO: 150, encoded according to the extended number; LCDR1 as shown in SEQ ID NO: 151, encoded according to the extended number; LCDR2 as shown in SEQ ID NO: 152, encoded according to the extended number; and LCDR3 as shown in SEQ ID NO: 153, encoded according to the extended number; (q) HCDR1 as shown in SEQ ID NO: 146, encoded according to Chothia number; HCDR2 as shown in SEQ ID NO: 149, encoded according to Chothia number; HCDR3 as shown in SEQ ID NO: 150, encoded according to extended number; LCDR1 as shown in SEQ ID NO: 151, encoded according to extended number; LCDR2 as shown in SEQ ID NO: 152, encoded according to extended number; and LCDR3 as shown in SEQ ID NO: 153, encoded according to extended number; (r) HCDR1 as shown in SEQ ID NO:147, encoded according to the extended number; HCDR2 as shown in SEQ ID NO:148, encoded according to the Kabat number; HCDR3 as shown in SEQ ID NO:150, encoded according to the extended number; LCDR1 as shown in SEQ ID NO:151, encoded according to the extended number; LCDR2 as shown in SEQ ID NO:152, encoded according to the extended number; and LCDR3 as shown in SEQ ID NO:153, encoded according to the extended number; (s) HCDR1 as shown in SEQ ID NO:181, encoded according to the Kabat number; HCDR2 as shown in SEQ ID NO:184, encoded according to the Kabat number; HCDR3 as shown in SEQ ID NO:186, encoded according to the extended number; LCDR1 as shown in SEQ ID NO:187, encoded according to the extended number; LCDR2 as shown in SEQ ID NO:188, encoded according to the extended number; and LCDR3 as shown in SEQ ID NO:189, encoded according to the extended number; (t) HCDR1 as shown in SEQ ID NO: 182, encoded according to Chothia number; HCDR2 as shown in SEQ ID NO: 185, encoded according to Chothia number; HCDR3 as shown in SEQ ID NO: 186, encoded according to extended number; LCDR1 as shown in SEQ ID NO: 187, encoded according to extended number; LCDR2 as shown in SEQ ID NO: 188, encoded according to extended number; and LCDR3 as shown in SEQ ID NO: 189, encoded according to extended number; (u) HCDR1 as shown in SEQ ID NO:183, encoded according to the extended number; HCDR2 as shown in SEQ ID NO:184, encoded according to the Kabat number; HCDR3 as shown in SEQ ID NO:186, encoded according to the extended number; LCDR1 as shown in SEQ ID NO:187, encoded according to the extended number; LCDR2 as shown in SEQ ID NO:188, encoded according to the extended number; and LCDR3 as shown in SEQ ID NO:189, encoded according to the extended number; (v) HCDR1 as shown in SEQ ID NO: 199, encoded according to the Kabat number; HCDR2 as shown in SEQ ID NO: 202, encoded according to the Kabat number; HCDR3 as shown in SEQ ID NO: 204, encoded according to the extended number; LCDR1 as shown in SEQ ID NO: 205, encoded according to the extended number; LCDR2 as shown in SEQ ID NO: 206, encoded according to the extended number; and LCDR3 as shown in SEQ ID NO: 207, encoded according to the extended number; (w) HCDR1 as shown in SEQ ID NO: 200, encoded according to Chothia number; HCDR2 as shown in SEQ ID NO: 203, encoded according to Chothia number; HCDR3 as shown in SEQ ID NO: 204, encoded according to extended number; LCDR1 as shown in SEQ ID NO: 205, encoded according to extended number; LCDR2 as shown in SEQ ID NO: 206, encoded according to extended number; and LCDR3 as shown in SEQ ID NO: 207, encoded according to extended number; or (x) HCDR1 as shown in SEQ ID NO: 201 according to the extended numbering; HCDR2 as shown in SEQ ID NO: 202 according to the Kabat numbering; HCDR3 as shown in SEQ ID NO: 204 according to the extended numbering; LCDR1 as shown in SEQ ID NO: 205 according to the extended numbering; LCDR2 as shown in SEQ ID NO: 206 according to the extended numbering; and LCDR3 as shown in SEQ ID NO: 207 according to the extended numbering; or (ii) The amino acid sequences of the heavy chain variable domain VH and the light chain variable domain VL shown in the following sequences: a) These are SEQ ID NO: 1 and 2, respectively; b) are SEQ ID NO: 3 and 4 respectively; c) These are SEQ ID NO: 5 and 6, respectively; d) These are SEQ ID NO: 7 and 8, respectively; e) are SEQ ID NO: 9 and 10 respectively; f) are SEQ ID NO: 17 and 18 respectively; g) are SEQ ID NO: 25 and 26, respectively; h) are SEQ ID NO: 29 and 30, respectively; i) respectively SEQ ID NO: 31 and 32; or j) are SEQ ID NO: 71 and 72, respectively.

2. The isolated nucleic acid, said isolated nucleic acid encoding the heavy and light chains of the antibody or antigen-binding moiety of claim 1.

3. An isolated nucleic acid, said isolated nucleic acid encoding a heavy chain and a light chain of an antibody or antigen-binding moiety specifically binding to human interleukin-2 (hIL-2), wherein said nucleic acid comprises: One of the following nucleotide sequence pairs: SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, SEQ ID NO: 42 and 43, SEQ ID NO: 44 and 45, SEQ ID NO: 52 and 53, SEQ ID NO: 60 and 61, SEQ ID NO: 64 and 65, or SEQ ID NO: 66 and 67.

4. A vector comprising the nucleic acid of claim 3.

5. A host cell comprising the nucleic acid of claim 4.

6. The host cell according to claim 5, wherein the cell is a mammalian cell.

7. A method for producing an antibody or antigen-binding moiety thereof that specifically binds to human interleukin-2 (hIL-2), said method comprising: a) Culturing the host cells of claim 6 under conditions that allow expression of the antibody or antigen-binding moiety, wherein the host cells comprise nucleotide sequences encoding the heavy and light chains of the antibody or antigen-binding moiety, and b) Isolate the antibody or antigen-binding portion from the culture.

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