Interleukin-2 muteins for the treatment of autoimmune diseases

Abstract

Disclosed are IL-2 agents, including IL-2 variants, and methods, compositions, and uses thereof. The IL-2 agents described herein include point mutations and can be used to treat and / or prevent a variety of disorders and conditions, particularly autoimmune diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 309,293, filed February 11, 2022. The contents of the aforementioned application are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on February 9, 2023, is named P2029-704810_SL.xml, and is 1,534,863 bytes in size. [Background technology]

[0003] Interleukin-2 (IL-2) is a cytokine that regulates the activity of the immune system. It is produced by white blood cells such as T cells, natural killer (NK) cells, dendritic cells, and macrophages in response to antigenic or mitogenic stimuli. IL-2 is important for T cell proliferation, B cell stimulation, and other activities related to immunity and tolerance. It is part of the body's adaptive immune response and distinguishes foreign from host antigens. IL-2 mediates its effects by binding to the IL-2 receptor, which then activates downstream signaling events.

[0004] Human IL-2 is an FDA approved drug for the treatment of diseases such as metastatic renal carcinoma and melanoma. The use of IL-2 in eligible patients can be limited due to the severe toxicity associated with IL-2 therapy, with only a small proportion of eligible patients actually receiving the therapy. Toxicities associated with IL-2 therapy can include severe fever, nausea, vomiting, vascular leakage, and severe hypotension. However, despite these toxicities, IL-2 is typically effective for its approved indications.

[0005] There remains an unmet need for novel IL-2-based agents that are well characterized for the development of safe and effective therapeutics for patients with a variety of diseases and conditions treatable with IL-2. Summary of the Invention

[0006] The disclosure provides, at least in part, IL-2 agents (e.g., IL-2 variants, IL-2 fusion proteins, IL-2 complexes, and IL-2 conjugates) that include one or more amino acid modifications (e.g., substitutions) in IL-2 and that include one or more of the structural or functional properties disclosed herein. In one embodiment, nucleic acid molecules encoding the IL-2 agents, expression vectors, host cells, compositions (e.g., pharmaceutical compositions), kits, containers, and methods for making the IL-2 agents are also provided. The IL-2 agents disclosed herein can be used (alone or in combination with other agents or therapeutic modalities) to treat, prevent, and / or diagnose disorders, such as the disorders and conditions disclosed herein.

[0007] In one aspect, the disclosure features an IL-2 agent (e.g., an IL-2 agent described herein) for use in a method of treating an autoimmune disease in a subject (e.g., a human subject), where the IL-2 agent is administered at a dose of 0.5 μg / kg to 300 μg / kg.

[0008] In one embodiment, the IL-2 agent is an IL-2 variant or an IL-2 fusion protein comprising an IL-2 variant. In one embodiment, the IL-2 variant comprises, relative to human IL-2 (SEQ ID NO: 1031), (i) the amino acid substitutions H16L or H16N, and / or the amino acid substitution I92S, and (ii) the amino acid substitutions V69A, Q74P, and C125S.

[0009] In one embodiment, the IL-2 agent is 0.5 μg / kg to 100 μg / kg, 1 μg / kg to 50 μg / kg, 2 μg / kg to 40 μg / kg, 3 μg / kg to 30 μg / kg, 4 μg / kg to 25 μg / kg. g, 5μg / kg~20μg / kg, 10μg / kg~15μg / kg, 1μg / kg~40μg / kg, 1μg / kg~30μg / kg, 1μg / kg~20μg / kg, 1μg / kg~10μg / kg , 1 μg / kg to 5 μg / kg, 5 μg / kg to 50 μg / kg, 10 μg / kg to 50 μg / kg, 20 μg / kg to 50 μg / kg, 30 μg / kg to 50 μg / kg, 40 μg / kg to 50 μg / kg, 0.5 μg / kg to 2 μg / kg, 3 μg / kg to 5 μg / kg, 6 μg / kg to 10 μg / kg, 12 μg / kg to 20 μg / kg, or 25 μg / kg to 40 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 1 μg / kg, 4 μg / kg, 8 μg / kg, 16 μg / kg, or 32 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 1 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 4 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 8 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 16 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 32 μg / kg. In one embodiment, the IL-2 agent is administered once a week, once every two weeks, or once every four weeks, for example, for at least eight weeks. In one embodiment, the IL-2 agent is administered once every two weeks. In one embodiment, the IL-2 agent is administered subcutaneously.

[0010] In one embodiment, the autoimmune disorder is systemic lupus erythematosus (SLE), autoimmune hepatitis (AIH), focal segmental glomerulosclerosis (FSGS) (e.g., immune-mediated focal segmental glomerulosclerosis (IM-FSGS)), or alopecia areata (AA). In one embodiment, the autoimmune disorder is systemic lupus erythematosus (SLE). In one embodiment, the autoimmune disorder is autoimmune hepatitis (AIH). In one embodiment, the autoimmune disorder is focal segmental glomerulosclerosis (FSGS) (e.g., immune-mediated focal segmental glomerulosclerosis (IM-FSGS)). In one embodiment, the autoimmune disorder is alopecia areata (AA).

[0011] In one embodiment, the IL-2 variant comprises the amino acid substitutions H16L, V69A, Q74P, and C125S. In one embodiment, the IL-2 variant further comprises the amino acid substitution T3A. In one embodiment, the IL-2 variant comprises the amino acid sequence of any of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, an amino acid sequence at least 95% identical thereto or differing therefrom by no more than 1, 2, 3, 4, or 5 amino acids, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises the amino acid sequence of any of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, or a functional fragment thereof.

[0012] In one embodiment, the IL-2 agent comprises an IL-2 fusion protein comprising an IL-2 variant. In one embodiment, the IL-2 fusion protein further comprises an Fc region. In one embodiment, the Fc region comprises an Fc region of IgG1 allotype m3 comprising an N297G substitution according to EU numbering. In one embodiment, the Fc region comprises an amino acid sequence of SEQ ID NO: 1003, or an amino acid sequence at least 95% identical thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or a functional fragment thereof. In one embodiment, the Fc region is fused to the C-terminus of the IL-2 variant.

[0013] In one embodiment, the IL-2 fusion protein further comprises a linker. In one embodiment, the linker is4 S) 4 (SEQ ID NO: 48).

[0014] In one embodiment, the fusion protein comprises any of the amino acid sequences of SEQ ID NOs: 1004, 1005, 1006, 1007, 1008, or 1009, an amino acid sequence that is at least 95% identical thereto or differs therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or a functional fragment thereof. In one embodiment, the fusion protein forms a dimer. In one embodiment, the fusion protein comprises any of the amino acid sequences of SEQ ID NOs: 1004, 1005, 1006, 1007, 1008, or 1009, or a functional fragment thereof.

[0015] In one aspect, the disclosure features a method of treating an autoimmune disease, including administering to a subject (e.g., a human subject) in need of treatment for an autoimmune disease an IL-2 agent (e.g., an IL-2 agent described herein) at a dose of 0.5 μg / kg to 300 μg / kg, thereby treating the autoimmune disease.

[0016] In one embodiment, the IL-2 agent is an IL-2 variant or an IL-2 fusion protein comprising an IL-2 variant. In one embodiment, the IL-2 variant comprises, relative to human IL-2 (SEQ ID NO: 1031), (i) the amino acid substitutions H16L or H16N, and / or the amino acid substitution I92S, and (ii) the amino acid substitutions V69A, Q74P, and C125S.

[0017] In one embodiment, the IL-2 agent is 0.5 μg / kg to 100 μg / kg, 1 μg / kg to 50 μg / kg, 2 μg / kg to 40 μg / kg, 3 μg / kg to 30 μg / kg, 4 μg / kg to 25 μg / kg. g, 5μg / kg~20μg / kg, 10μg / kg~15μg / kg, 1μg / kg~40μg / kg, 1μg / kg~30μg / kg, 1μg / kg~20μg / kg, 1μg / kg~10μg / kg , 1 μg / kg to 5 μg / kg, 5 μg / kg to 50 μg / kg, 10 μg / kg to 50 μg / kg, 20 μg / kg to 50 μg / kg, 30 μg / kg to 50 μg / kg, 40 μg / kg to 50 μg / kg, 0.5 μg / kg to 2 μg / kg, 3 μg / kg to 5 μg / kg, 6 μg / kg to 10 μg / kg, 12 μg / kg to 20 μg / kg, or 25 μg / kg to 40 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 1 μg / kg, 4 μg / kg, 8 μg / kg, 16 μg / kg, or 32 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 1 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 4 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 8 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 16 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 32 μg / kg. In one embodiment, the IL-2 agent is administered once a week, once every two weeks, or once every four weeks, for example, for at least eight weeks. In one embodiment, the IL-2 agent is administered once every two weeks. In one embodiment, the IL-2 agent is administered subcutaneously.

[0018] In one embodiment, the autoimmune disorder is systemic lupus erythematosus (SLE), autoimmune hepatitis (AIH), focal segmental glomerulosclerosis (FSGS) (e.g., immune-mediated focal segmental glomerulosclerosis (IM-FSGS)), or alopecia areata (AA). In one embodiment, the autoimmune disorder is systemic lupus erythematosus (SLE). In one embodiment, the autoimmune disorder is autoimmune hepatitis (AIH). In one embodiment, the autoimmune disorder is focal segmental glomerulosclerosis (FSGS) (e.g., immune-mediated focal segmental glomerulosclerosis (IM-FSGS)). In one embodiment, the autoimmune disorder is alopecia areata (AA).

[0019] In one embodiment, the IL-2 variant comprises the amino acid substitutions H16L, V69A, Q74P, and C125S. In one embodiment, the IL-2 variant further comprises the amino acid substitution T3A. In one embodiment, the IL-2 variant comprises the amino acid sequence of any of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, an amino acid sequence at least 95% identical thereto or differing therefrom by no more than 1, 2, 3, 4, or 5 amino acids, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises the amino acid sequence of any of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, or a functional fragment thereof.

[0020] In one embodiment, the IL-2 agent comprises an IL-2 fusion protein comprising an IL-2 variant. In one embodiment, the IL-2 fusion protein further comprises an Fc region. In one embodiment, the Fc region comprises an Fc region of IgG1 allotype m3 comprising an N297G substitution according to EU numbering. In one embodiment, the Fc region comprises an amino acid sequence of SEQ ID NO: 1003, or an amino acid sequence at least 95% identical thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or a functional fragment thereof. In one embodiment, the Fc region is fused to the C-terminus of the IL-2 variant.

[0021] In one embodiment, the IL-2 fusion protein further comprises a linker. In one embodiment, the linker is4 S) 4 (SEQ ID NO: 48).

[0022] In one embodiment, the fusion protein comprises the amino acid sequence of any of SEQ ID NOs: 1004, 1005, 1006, 1007, 1008, or 1009, an amino acid sequence that is at least 95% identical thereto or differs therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or a functional fragment thereof. In one embodiment, the fusion protein comprises the amino acid sequence of any of SEQ ID NOs: 1004, 1005, 1006, 1007, 1008, or 1009, or a functional fragment thereof. In one embodiment, the fusion protein forms a dimer.

[0023] In one aspect, the disclosure features the use of an IL-2 agent (e.g., an IL-2 agent described herein) in the manufacture of a medicament for the treatment of an autoimmune disease in a subject (e.g., a human subject), where the IL-2 agent is administered at a dose of 0.5 μg / kg to 300 μg / kg.

[0024] In one embodiment, the IL-2 agent is an IL-2 variant or an IL-2 fusion protein comprising an IL-2 variant. In one embodiment, the IL-2 variant comprises, relative to human IL-2 (SEQ ID NO: 1031), (i) the amino acid substitutions H16L or H16N, and / or the amino acid substitution I92S, and (ii) the amino acid substitutions V69A, Q74P, and C125S.

[0025] In one embodiment, the IL-2 agent is 0.5 μg / kg to 100 μg / kg, 1 μg / kg to 50 μg / kg, 2 μg / kg to 40 μg / kg, 3 μg / kg to 30 μg / kg, 4 μg / kg to 25 μg / kg. g, 5μg / kg~20μg / kg, 10μg / kg~15μg / kg, 1μg / kg~40μg / kg, 1μg / kg~30μg / kg, 1μg / kg~20μg / kg, 1μg / kg~10μg / kg , 1 μg / kg to 5 μg / kg, 5 μg / kg to 50 μg / kg, 10 μg / kg to 50 μg / kg, 20 μg / kg to 50 μg / kg, 30 μg / kg to 50 μg / kg, 40 μg / kg to 50 μg / kg, 0.5 μg / kg to 2 μg / kg, 3 μg / kg to 5 μg / kg, 6 μg / kg to 10 μg / kg, 12 μg / kg to 20 μg / kg, or 25 μg / kg to 40 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 1 μg / kg, 4 μg / kg, 8 μg / kg, 16 μg / kg, or 32 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 1 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 4 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 8 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 16 μg / kg. In one embodiment, the IL-2 agent is administered at a dose of 32 μg / kg. In one embodiment, the IL-2 agent is administered once a week, once every two weeks, or once every four weeks, for example, for at least eight weeks. In one embodiment, the IL-2 agent is administered once every two weeks. In one embodiment, the IL-2 agent is administered subcutaneously.

[0026] In one embodiment, the autoimmune disorder is systemic lupus erythematosus (SLE), autoimmune hepatitis (AIH), focal segmental glomerulosclerosis (FSGS) (e.g., immune-mediated focal segmental glomerulosclerosis (IM-FSGS)), or alopecia areata (AA). In one embodiment, the autoimmune disorder is systemic lupus erythematosus (SLE). In one embodiment, the autoimmune disorder is autoimmune hepatitis (AIH). In one embodiment, the autoimmune disorder is focal segmental glomerulosclerosis (FSGS) (e.g., immune-mediated focal segmental glomerulosclerosis (IM-FSGS)). In one embodiment, the autoimmune disorder is alopecia areata (AA).

[0027] In one embodiment, the IL-2 variant comprises the amino acid substitutions H16L, V69A, Q74P, and C125S. In one embodiment, the IL-2 variant further comprises the amino acid substitution T3A. In one embodiment, the IL-2 variant comprises the amino acid sequence of any of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, an amino acid sequence at least 95% identical thereto or differing therefrom by no more than 1, 2, 3, 4, or 5 amino acids, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises the amino acid sequence of any of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, or a functional fragment thereof.

[0028] In one embodiment, the IL-2 agent comprises an IL-2 fusion protein comprising an IL-2 variant. In one embodiment, the IL-2 fusion protein further comprises an Fc region. In one embodiment, the Fc region comprises an Fc region of IgG1 allotype m3 comprising an N297G substitution according to EU numbering. In one embodiment, the Fc region comprises an amino acid sequence of SEQ ID NO: 1003, or an amino acid sequence at least 95% identical thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or a functional fragment thereof. In one embodiment, the Fc region is fused to the C-terminus of the IL-2 variant.

[0029] In one embodiment, the IL-2 fusion protein further comprises a linker. In one embodiment, the linker is4 S) 4 (SEQ ID NO: 48).

[0030] In one embodiment, the fusion protein comprises any of the amino acid sequences of SEQ ID NOs: 1004, 1005, 1006, 1007, 1008, or 1009, an amino acid sequence that is at least 95% identical thereto or differs therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or a functional fragment thereof. In one embodiment, the fusion protein forms a dimer. In one embodiment, the fusion protein comprises any of the amino acid sequences of SEQ ID NOs: 1004, 1005, 1006, 1007, 1008, or 1009, or a functional fragment thereof.

[0031] Additional Embodiments The present disclosure is based, at least in part, on the discovery that a combination of mutations in IL-2 that stabilize the protein, reduce its affinity for CD122 (e.g., CD122 / CD132 heterodimer), and / or reduce or have only a minimal effect on its affinity for CD25 can be used to selectively enhance regulatory T cell (Treg) activity via the IL-2 pathway, thus achieving advantageous therapeutic effects for treating disorders and conditions, such as autoimmune diseases. IL-2 agents containing such mutations are suitable for treating conditions resulting from an abnormal immune response, such as autoimmune diseases.

[0032] Thus, in one embodiment, the IL-2 agent has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all) of the following properties a)-x): a) e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, e.g., as determined by an assay of protein concentration, compared to an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant , about a 95%, about a 100% or greater increase, or an increase of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or greater; b) a decrease in the IL-2 activity of, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 400%, about 400%, about 45 ... aggregates in vitro and / or in vivo at lower or reduced levels, such as 0%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more reduction, or about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction; c) e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, as compared to, e.g., an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, e.g., by yeast surface display, expression in mammalian cells, chromatography, circular dichroism or related spectroscopic techniques, and / or melting temperature analysis (e.g., using fluorometry). , has enhanced or increased stability in vitro and / or in vivo, such as an increase of about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more, or an increase of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, or more; d) a decrease in, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 95%, about 100%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, about 138%, about 139%, about 140%, about 141%, about 142%, about 143%, about 144%, about 145%, about 146%, about 147%, about 148%, about 149%, about 150%, about 151%, about 152%, about 153%, about 154%, about 155%, about 156%, about 157%, about 158%, about 159%, about 160%, about 161%, about 162%, about 163%, about 164%, about 165%, about 166%, about 167%, have an enhanced or increased half-life in vitro and / or in vivo, such as an 80%, about 85%, about 90%, about 95%, about 100% or more increase, or an increase of about 0.5 fold, about 1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 5.5 fold, about 6 fold, about 6.5 fold, about 7 fold, about 7.5 fold, about 8 fold, about 8.5 fold, about 9 fold, about 9.5 fold, about 10 fold or more; e) a decrease in the expression level of, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 95%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, about 138%, about 139%, about 140%, about 141%, about 142%, about 143%, about 144%, about 145%, about 146%, about 147%, about 148%, about 149%, about 150%, about 151%, about 152%, about 153%, about 154%, about 155%, about 156%, about 157%, about 158%, about 159%, about 160%, have a lower, reduced or decreased rate or level of turnover and / or clearance in vivo, such as a 90%, about 95%, about 100% or more reduction, or a reduction of, for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction; f) a reduction of, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more (e.g., about 1% to about 20%, about 2% to about 15%, or about 5% to about 10%), or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 10%, about 10%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 10%, about 10%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20 ... , about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% or less decrease or increase, or about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more decrease, or about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold or less decrease or increase; g) a dissociation constant (K) of, for example, about 5-500 pM, as determined, for example, by yeast surface display D), for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 pM, or, for example, about 10 pM to about 490 pM, about 20 pM to about 480 pM, about 30 pM to about 470 pM, about 40 pM to about 460 pM, about 50 pM to about 450 pM M, about 60 pM to about 440 pM, about 70 pM to about 430 pM, about 80 pM to about 420 pM, about 90 pM to about 410 pM, about 100 pM to about 400pM, about 110pM to about 390pM, about 120pM to about 380pM, about 130pM to about 370pM, about 140pM to about 360pM, Approximately 150pM to approximately 350pM, approximately 160pM to approximately 340pM, approximately 170pM to approximately 330pM, approximately 180pM to approximately 320pM, approximately 190pM ~Approx. 310pM, approx. 200pM ~ approx. 300pM, approx. 210pM ~ approx. 290pM, approx. 220pM ~ approx. 280pM, approx. 230pM ~ approx. 270p M, about 240 pM to about 260 pM, or for example, about 5 pM to about 450 pM, about 5 pM to about 400 pM, about 5 pM to about 350 pM, about 5 pM to about 300 pM, about 5 pM to about 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM, about 5 pM to about 100 pM, about 5 pM to about 50 pM, or for example, about 10 pM to about 500 pM, about 20 pM to about 500 pM, about 50 pM to about 500 pM, about 100 pM to about 500 pM, about 150 pM to about 500 pM, about 200 pM to about 500 pM, about 250 pM to about 500 pM, about 300 pM to about 500 pM, about binds to CD25 (e.g., human CD25) with low affinity, such as from 50 pM to about 500 pM, from about 400 pM to about 500 pM, from about 450 pM to about 500 pM, or, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or greater than about 500 pM; h) a dissociation constant (K) of, for example, about 0.1-10 nM, as determined, for example, by biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore) D ), for example, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 6, about 7, about 8, about 9, or about 10 nM, or for example, about 0.1 to about 9 nM, about 0.1 to about 8 nM, about 0.1 to about 7 nM, or about 0.1 to about 6 nM, for example, about 0.1 to about about 5 nM, about 0.1 to about 4 nM, about 0.1 to about 3 nM, about 0.1 to about 2 nM, about 0.1 to about 1 nM, or about 0.1 to about 0.5 nM, or for example, about 0.1 to about 10 nM, about 0.5 to about 10 nM, about 1 to about 10 nM, about 1.5 to about 10 nM, about 2 to about 10 nM, about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM , about 5 to about 10 nM, about 5.5 to about 10 nM, about 6 to about 10 nM, about 6.5 to about 10 nM, about 7 to about 10 nM, about 7.5 to about 10 nM, about 8 to about 10 nM, about 8.5 to about 10 nM, about 9 to about 10 nM, or about 9.5 to about 10 nM, or for example, about 0.1 to about 9.5 nM, about 0.5 to about 9 nM, about 1 to about 8.5 nM, about 1.5 to about 8 nM, about 2 to about 7.5 nM, about 2.5 binds to CD25 (e.g., human CD25) with low affinity, such as about 7 nM, about 3 to about 6.5 nM, about 3.5 to about 6 nM, about 4 to about 5.5 nM, or about 4.5 to about 5 nM, or greater than about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nM; i) e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, e.g., compared to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, as determined, e.g., by yeast surface display, biolayer interferometry (e.g., Octet binding), and / or surface plasmon resonance (e.g., Biacore). , about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more (e.g., about 1% to about 50%, about 2% to about 40%, about 3% to about 30%, about 4% to about 20%, or about 5% to about 10%, about 1% to about 40%, about 1% to about 30%, about 1% a decrease of about 0.5 times, about 1 times, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, have a reduced or decreased binding affinity for the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), such as a reduction of about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, or more (e.g., about 0.5-fold to about 5-fold, about 1-fold to about 4-fold, or about 2-fold to about 3-fold); j) a dissociation constant (K) of, for example, about 0.2 to 20 nM, as determined, for example, by yeast surface display; D), for example, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 20 nM, or for example, about 0.2 to about 19 nM, about 0.2 to about 18 nM, about 0.2 to about 17 nM, or about 0.2 to about 16 nM, For example, about 0.2 to about 15 nM, about 0.1 to about 4 nM, about 0.1 to about 3 nM, about 0.1 to about 2 nM, about 0.1 to about 1 nM, or about 0.1 to about 0.5 nM, or for example, about 0.1 to about 10 nM, about 0.5 to about 10 nM, about 1 to about 10 nM, about 1.5 to about 10 nM, about 2 to about 10 nM, about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM, about 5 to about 10 nM, about 5.5 to about 10 nM , about 6 to about 10 nM, about 6.5 to about 10 nM, about 7 to about 10 nM, about 7.5 to about 10 nM, about 8 to about 10 nM, about 8.5 to about 10 nM, about 9 to about 10 nM, or about 9.5 to about 10 nM, or for example, about 0.1 to about 9.5 nM, about 0.5 to about 9 nM, about 1 to about 8.5 nM, about 1.5 to about 8 nM, about 2 to about 7.5 nM, about 2.5 to about 7 nM, about 3 to about 6.5 nM, about 3.5 to about 6 nM, about 4 to about 5.5 nM, or about 4.5 to about 5 nM, or binds to a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with low affinity, such as, for example, greater than about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 20 nM; k) a dissociation constant (K) of, for example, about 0.2 to 300 nM, as determined, for example, by biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore) D), for example, about 0.2 nM, about 0.5 nM, about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, or about 300 nM, or for example, about 0.2 to about 280 nM, about 0.2 to about 260 nM, about 0.2 to about 240 nM, about 0.2 to about 220 nM, about 0.2 to about 200 nM, about 0.2 to about 180 nM, about 0.2 to about 160 nM, about 0.2 to about 140 nM, about 0.2 to about 120 nM, about 0.2 to about 100 nM, About 0.2 to about 80 nM, about 0.2 to about 60 nM, about 0.2 to about 40 nM, about 0.2 to about 20 nM, or, for example, about 0.5 to about 300 nM, about 1 to about 300 nM, about 5 to about 300 nM, about 10 to about 300 nM, about 20 to about 300 nM, about 40 to about 300 nM, about 60 to about 300 nM, about 80 to about 300 nM, about 100 to about 300 nM, about 120 to about 300 nM, about 140 to about 300 nM, about 160 to about 300 nM, about 18 0 to about 300 nM, about 200 to about 300 nM, about 220 to about 300 nM, about 240 to about 300 nM, about 260 to about 300 nM, about 280 to about 300 nM, or for example, about 0.5 to about 280 nM, about 1 to about 260 nM, about 5 to about 240 nM, about 10 to about 220 nM, about 20 to about 200 nM, about 40 to about 180 nM, about 60 to about 160 nM, about 80 to about 140 mM, about 100 to about 120 nM, or for example, about 0.2, about 0.5, about 1, about 2, about 5, about 10, about 15, about 20nM, about 25nM, about 30nM, about 40nM, about 50nM, about 60nM, about 70nM, about 80nM, about 90 nM, approximately 100nM, approximately 110nM, approximately 120nM, approximately 130nM, approximately 140nM, approximately 150nM, approximately 160nM, approximately 170nM, approximately 180nM, approximately 190 binds to a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with low affinity, such as greater than about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, or about 300 nM; l) e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, 2, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, More than about 1000, about 1500, about 2000, about 2500, or about 3000 or more, or, for example, more than 1 and about 1-2, about 2-3, about 3-4, about 4-5, more than 1 and about 1-10, more than 1 and about 1-20, more than 1 and about 1-30, more than 1 and about 1-40, more than 1 and about 1-50, about 2-10, about 2-20, about 2-30, about 2- 40, 2 to 50, about 5 to 10, about 5 to 20, about 5 to 30, about 5 to 40, about 5 to 50, about 10 to 20, about 10 to 30, about 10 to 40, about 10 to 50, about 20 to 40, about 20 to 50, about 50 to 100, about 100 to 200, about 200 to 500, about 500 to 1000, about 1000 to 2000, or about 1000 to 3000 T helper ECs. 50 / Treg EC 50 ratio to selectively activate IL-2 signaling in T regulatory cells in vitro and / or in vivo; m) e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, 2, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, More than about 1000, about 1500, about 2000, about 2500, or about 3000 or more, or, for example, more than 1 and about 1-2, about 2-3, about 3-4, about 4-5, more than 1 and about 1-10, more than 1 and about 1-20, more than 1 and about 1-30, more than 1 and about 1-40, more than 1 and about 1-50, about 2-10, about 2-20, about 2-30, about 2 NK cell ECs such as about 40, 2 to 50, about 5 to 10, about 5 to 20, about 5 to 30, about 5 to 40, about 5 to 50, about 10 to 20, about 10 to 30, about 10 to 40, about 10 to 50, about 20 to 40, about 20 to 50, about 50 to 100, about 100 to 200, about 200 to 500, about 500 to 1000, about 1000 to 2000, or about 1000 to 3000. 50 / Treg EC 50 ratio to selectively activate IL-2 signaling in T regulatory cells in vitro and / or in vivo; n) (i) a reduction in the expression level of, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or more than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or more than about 10%, as compared to an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, for example, by flow cytometry, in vitro or in vivo T regulatory cell proliferation or expansion assays, and / or T cell suppression assays. %, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more lower, or for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduced EC for Tregs. 50 having the potency and / or ability to induce or promote enhanced or increased T regulatory cell activity; (ii) e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80% compared to an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, for example, by flow cytometry, in vitro or in vivo T regulatory cell proliferation or expansion assays, and / or T cell suppression assays. , about 85%, about 90%, about 95%, about 100% or more higher, or, for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 50-fold, about 100-fold, about 200-fold, about 500-fold, about 1000-fold, about 2000-fold, about 5000-fold, about 10,000-fold, about 15,000-fold, about 20,000-fold or more reduced EC 50 having reduced or diminished potency and / or ability to induce or promote T regulatory cell activity; o) modulating (e.g., reducing (inhibiting, blocking, or neutralizing) or increasing (e.g., activating, initiating, or enhancing) one or more biological activities of T cells (e.g., Tregs) in vitro, ex vivo, or in vivo; p) exhibits the same or similar binding affinity or specificity, or both, as the IL-2 agents described herein; q) exhibits the same or similar binding affinity or specificity, or both, as an IL-2 agent containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) modifications (e.g., substitutions) described herein; r) exhibits the same or similar binding affinity or specificity, or both, as an IL-2 agent comprising an amino acid sequence described herein; s) exhibits the same or similar binding affinity or specificity, or both, as an IL-2 agent comprising an amino acid sequence encoded by a nucleotide sequence described herein; t) inhibits, e.g., competitively inhibits, the binding of a second IL-2 agent to an IL-2 receptor, wherein the second IL-2 agent is an IL-2 agent described herein; u) competes for binding to an IL-2 receptor with a second IL-2 agent, the second IL-2 agent being an IL-2 agent described herein; v) has one or more biological properties of the IL-2 agents described herein; w) having one or more structural characteristics of an IL-2 agent described herein; x) has one or more pharmacokinetic properties of the IL-2 agents described herein.

[0033] In one embodiment, the IL-2 agent is, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 87%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 102%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, about 138%, about 139%, about 140%, about 142%, about 143%, about 144%, about 145%, about 146%, about 147%, about 148%, about 149%, about 150%, about 151%, about 152%, about 153%, about 154%, about 155%, about 155%, about 156%, about 157%, about 15 The polypeptides are expressed in vitro and / or in vivo at higher or increased levels, such as a 5%, about 90%, about 95%, about 100% or more increase, or an increase of about 0.5 fold, about 1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 5.5 fold, about 6 fold, about 6.5 fold, about 7 fold, about 7.5 fold, about 8 fold, about 8.5 fold, about 9 fold, about 9.5 fold, about 10 fold or more. In one embodiment, the IL-2 agent has, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 400%, about 45 ... %, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more reduction, or at lower or reduced levels, such as about a 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction.

[0034] In one embodiment, the IL-2 agent exhibits, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% IL-2 activity, as compared to, e.g., an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, e.g., by yeast surface display, expression in mammalian cells, chromatography, circular dichroism or related spectroscopic techniques, and / or melting temperature analysis (e.g., using fluorometry). , about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more increase, or about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more increase.

[0035] In one embodiment, the IL-2 agent exhibits a IL-2 expression level that is, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 9 ... have an enhanced or increased half-life in vitro and / or in vivo, such as an increase of about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or an increase of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more.

[0036] In one embodiment, the IL-2 agent has, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 400%, about 450%, about 400%, about 450%, about 500%, about 550%, about 600%, about 450%, about 400%, about 450%, about 500%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 900%, about 1000%, about 1100%, about 1200%, about 1300%, about 1400%, about 1500%, about 1600%, about 1700%, about 1800%, about 1900%, about 2100%, about 2200%, about 2300%, about 240 In some embodiments, the antibody has a lower, reduced or decreased rate or level of turnover and / or clearance in vivo, such as about 85%, about 90%, about 95%, about 100% or more reduction, or about a 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction.

[0037] In one embodiment, the IL-2 agent exhibits, for example, about a 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more reduction (e.g., about 1% to about 20%, about 2% to about 15%, or about 5% to about 10%) in the expression level of IL-2 compared to, for example, an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, for example, by yeast surface display, biolayer interferometry (e.g., Octet binding), surface plasmon resonance (e.g., Biacore). , about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% or less decrease or increase, or about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold The antibody has a reduced or decreased or substantially unchanged binding affinity for CD25 (e.g., human CD25), such as a reduction of about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, or more, or a reduction or increase of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold or less. In one embodiment, the reduction or decrease in binding affinity for CD25 is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% less than the reduction or decrease in binding affinity for CD25. In one embodiment, the binding affinity for CD25 is not substantially reduced or decreased.

[0038] In one embodiment, the IL-2 agent has a dissociation constant (KD) of, e.g., about 5 to 500 pM, e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 pM, or, e.g., about 10 pM to 500 pM, as determined, e.g., by yeast surface display. Approximately 490pM, approximately 20pM to approximately 480pM, approximately 30pM to approximately 470pM, approximately 40pM to approximately 460pM, approximately 50pM to approximately 450pM, approximately 60 pM ~ about 440pM, about 70pM - about 430pM, about 80pM - about 420pM, about 90pM - about 410pM, about 100pM - about 400pM, Approximately 110pM to approximately 390pM, approximately 120pM to approximately 380pM, approximately 130pM to approximately 370pM, approximately 140pM to approximately 360pM, approximately 150pM ~350pM, 160pM~340pM, 170pM~330pM, 180pM~320pM, 190pM~310pM , about 200 pM to about 300 pM, about 210 pM to about 290 pM, about 220 pM to about 280 pM, about 230 pM to about 270 pM, about 240 pM to about 260 pM, or for example, about 5 pM to about 450 pM, about 5 pM to about 400 pM, about 5 pM to about 350 pM, about 5 pM to about 300 pM, about 5 pM to about 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM, about 5 pM to about 100 pM, about 5 pM to about 50 pM, or for example, about 10 pM to about 500 pM, about 20 pM to about 500 pM, about 50 pM to about 500 pM, about 100 pM to about 500 pM, about 150 pM M to about 500 pM, about 200 pM to about 500 pM, about 250 pM to about 500 pM, about 300 pM to about 500 pM, about 350 pM to about 500 pM, about 400 pM to about 500 pM, about 450 pM to about 500 pM, or, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 3 5, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350,It binds to CD25 (e.g., human CD25) with low affinity, such as greater than about 400, about 450, or about 500 pM.

[0039] In one embodiment, the IL-2 agent has a dissociation constant (KD) of, e.g., about 0.1 to 10 nM, e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 8 about 8, about 9, or about 10 nM, or, for example, about 0.1 to about 9 nM, about 0.1 to about 8 nM, about 0.1 to about 7 nM, or about 0.1 to about 6 nM, for example, about 0.1 to about 5 nM, about 0.1 to about 4 nM, about 0.1 to about 3 nM, about 0.1 to about 2 nM, about 0.1 to about 1 nM, or about 0.1 to about 0.5 nM, or, for example, about 0.1 to about 10 nM, about 0.5 to about 10 nM, about 1 to about 10 nM, about 1.5 to about 10 nM, about 2 to about 10 nM , about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM, about 5 to about 10 nM, about 5.5 to about 10 nM, about 6 to about 10 nM, about 6.5 to about 10 nM, about 7 to about 10 nM, about 7.5 to about 10 nM, about 8 to about 10 nM, about 8.5 to about 10 nM, about 9 to about 10 nM, or about 9.5 to about 10 nM, or for example, about 0.1 to about 9.5 nM, about 0.5 to about 9 nM, about 1 to about 8.5 nM , about 1.5 to about 8 nM, about 2 to about 7.5 nM, about 2.5 to about 7 nM, about 3 to about 6.5 nM, about 3.5 to about 6 nM, about 4 to about 5.5 nM, or about 4.5 to about 5 nM, or a low affinity of, for example, greater than about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nM.

[0040] In one embodiment, the IL-2 agent is, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10% lower than, for example, an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined by, for example, yeast surface display, biolayer interferometry (e.g., Octet binding), and / or surface plasmon resonance (e.g., Biacore). , about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more (e.g., about 1% to about 50%, about 2% to about 40%, about 3% to about 30%, about 4% to about 20%, or about 5% to about 10%, about 1% to about 40%, about 1% to about 3 0%, about 1% to about 20%, about 1% to about 10%, about 40% to about 50%, about 30% to about 50%, about 20% to about 50%, about 10% to about 50%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 10% to about 30%, or 20% to about 40%) decrease, or about 0.5 times, about 1 times, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about The antibody has a reduced or decreased binding affinity to a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer), such as a 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, or more (e.g., about 0.5-fold to about 5-fold, about 1-fold to about 4-fold, or about 2-fold to about 3-fold) reduction. In one embodiment, the reduction or decrease in binding affinity to a CD122 / CD132 heterodimer is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5-fold greater than the reduction or decrease in binding affinity to CD25. In one embodiment, the binding affinity to CD25 is not substantially reduced or decreased.

[0041] In one embodiment, the IL-2 agent has a dissociation constant (KD) of, e.g., about 0.2 to 20 nM, e.g., about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 20 nM, or, e.g., about 0. 2 to about 19 nM, about 0.2 to about 18 nM, about 0.2 to about 17 nM, or about 0.2 to about 16 nM, for example, about 0.2 to about 15 nM, about 0.1 to about 4 nM, about 0.1 to about 3 nM, about 0.1 to about 2 nM, about 0.1 to about 1 nM, or about 0.1 to about 0.5 nM, or for example, about 0.1 to about 10 nM, about 0.5 to about 10 nM, about 1 to about 10 nM, about 1.5 to about 10 nM, about 2 to about 10 nM, about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM, about 5 to about 10 nM, about 5.5 to about 10 nM, about 6 to about 10 nM, about 6.5 to about 10 nM, about 7 to about 10 nM, about 7.5 to about 10 nM, about 8 to about 10 nM, about 8.5 to about 10 nM, about 9 to about 10 nM, or about 9.5 to about 10 nM, or for example, about 0.1 to about 9.5 nM, about 0.5 to about 9 nM, about 1 to about 8.5 nM, about 1.5 to about 8 nM, about 2 to about 7.5 nM, about 2.5 to about 7 nM, about 3 to about 6.5 nM, about 3.5 to about 6 nM, about 4 to about 5.5 nM, or binds to a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with a low affinity of about 4.5 to about 5 nM, or for example, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or greater than about 20 nM.

[0042] In one embodiment, the IL-2 agent has a dissociation constant (KD) of, e.g., about 0.2 to 300 nM, e.g., about 0.2 nM, about 0.5 nM, about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, e.g., about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, about 300 nM, about 310 nM, about 320 nM, about 330 nM, about 340 nM, about 350 nM, about 360 nM, about 370 nM, about 380 nM, about 390 nM, about 40 ...500 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 n nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, or about 300 nM, or for example, about 0.2 to about 280 nM, about 0.2 to about 260 nM, about 0.2 to about 240 nM, about 0.2 to about 220 nM, about 0.2 to about 200 nM, about 0.2 to about 18 0 nM, about 0.2 to about 160 nM, about 0.2 to about 140 nM, about 0.2 to about 120 nM, about 0.2 to about 100 nM, about 0.2 to about 80 nM, about 0.2 to about 60 nM, about 0.2 to about 40 nM, about 0.2 to about 20 nM, or for example, about 0.5 to about 300 nM, about 1 to about 300 nM, about 5 to about 300 nM, about 10 to about 300 nM, about 20 to about 300 nM, about 40 to about 300 nM, about 60 to about 300 nM, about 80 to about 300 nM, about 100 to about 300 nM, about 120 to about 300 nM, about 140 to about 300 nM, about 160 to about 300 nM, about 180 to about 300 nM, about 200 to about 300 nM, about 220 to about 300 nM, about 240 to about 300 nM, about 260 to about 300 nM, about 280 to about 300 nM, or, for example, about 0.5 to about 280 nM, about 1 to about 260 nM, about 5 to about 240 nM, about 10 to about 220 nM, about 20 to about 200 nM, about 40 to about 180 nM, about 60 to about 160 nM, about 80 to about 140 mM, about 100 to about 120 nM, or, for example, about 0.2, about 0.5, about 1, about 2, about 5, about 10, about 15, about 20nM, about 25nM, about 30nM, about 40nM, about 50nM, about 60nM, about 70nM, about 80nM, about 90 nM, approximately 100nM, approximately 110nM, approximately 120nM, approximately 130nM, approximately 140nM, approximately 150nM, approximately 160nM, approximately 170nM, approximately 180nM, approximately 190 The antibody binds to the CD122 / CD132 heterodimer (e.g., the human CD122 / CD132 heterodimer) with low affinity, such as greater than about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, or about 300 nM.

[0043] In one embodiment, the IL-2 agent has, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more percentiles compared to an IL-2 agent that comprises a wild-type IL-2 or an IL-2 agent that comprises a reference IL-2 variant, as determined, e.g., by flow cytometry. , about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900 , about 1000, about 1500, about 2000, about 2500, or about 3000 or more, or, for example, greater than 1 and about 1-2, about 2-3, about 3-4, about 4-5, greater than 1 and about 1-10, greater than 1 and about 1-20, greater than 1 and about 1-30, greater than 1 and about 1-40, greater than 1 and about 1-50, about 2-10, about 2-20, about 2-30, about 2-4 and selectively activate IL-2 signaling in T regulatory cells in vitro and / or in vivo, having a T helper EC50 / Treg EC50 ratio of 0, 2-50, about 5-10, about 5-20, about 5-30, about 5-40, about 5-50, about 10-20, about 10-30, about 10-40, about 10-50, about 20-40, about 20-50, about 50-100, about 100-200, about 200-500, about 500-1000, about 1000-2000, or about 1000-3000, etc. In one embodiment, the T helper cells are CD45+CD3+CD4+Foxp3- cells, as determined, for example, by flow cytometry. In one embodiment, Tregs are CD45+CD3+CD4+Foxp3+ cells, as determined, for example, by flow cytometry.

[0044] In one embodiment, the IL-2 agent has a IL-2 activity greater than or equal to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 1 4, about 25, about 26, about 27, about 28, about 29, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1000, about 1500, about 2000, about 2500, or More than about 3000 or more, or for example, greater than 1 and about 1-2, about 2-3, about 3-4, about 4-5, greater than 1 and about 1-10, greater than 1 and about 1-20, greater than 1 and about 1-30, greater than 1 and about 1-40, greater than 1 and about 1-50, about 2-10, about 2-20, about 2-30, about 2-40, 2-50, about 5-10, about 5-20, about 5-30, about 5-40, about 5-50, about 10-20 , about 10-30, about 10-40, about 10-50, about 20-40, about 20-50, about 50-100, about 100-200, about 200-500, about 500-1000, about 1000-2000, or about 1000-3000, and the like, to selectively activate IL-2 signaling in T regulatory cells in vitro and / or in vivo. In one embodiment, the NK cells are CD45+CD3- cells that are CD56+ and / or CD16+, e.g., as determined by flow cytometry. In one embodiment, the NK cells are CD45+CD3-CD56+ cells, e.g., as determined by flow cytometry. In one embodiment, the Tregs are CD45+CD3+CD4+Foxp3+ cells, e.g., as determined by flow cytometry.

[0045] In one embodiment, the IL-2 agent is, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 100%, about 120%, about 140%, about 160%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 550%, about 560%, about 570%, about 580%, about 590%, about 610%, about 620%, about 630%, about 640%, about 650%, about 660%, about 670%, about 680%, about 690%, about 700%, about 710%, about 720%, about 730%, about 740%, about 750%, about 760%, about 770%, about 780%, about 790%, about 800%, about 820%, about 830%, about 840%, about 850%, about 860%, about 870%, about having an EC50 against Tregs that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more lower, or, for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more decreased, and having a potency and / or ability to induce or promote enhanced or increased T regulatory cell activity.

[0046] In one embodiment, the IL-2 agent exhibits, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 155%, about 15 ... 00% or more higher, or for example, about 0.5 fold, about 1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 5.5 fold, about 6 fold, about 6.5 fold, about 7 fold, about 7.5 fold, about 8 fold, about 8.5 fold, about 9 fold, about 9.5 fold, about 10 fold, about 50 fold, about 100 fold, about 200 fold, about 500 fold, about 1000 fold, about 2000 fold, about 5000 fold, about 10,000 fold, about 15,000 fold, about 20,000 fold or more decreased, and / or have a reduced or diminished potency and / or ability to induce or promote T regulatory cell activity. In one embodiment, the IL-2 agent has a reduced or diminished potency and / or ability to induce or promote T regulatory cell activity, e.g., has an EC50 for Tregs that is about 100-fold or greater higher, and does not activate, or does not substantially activate, NK cells, compared to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant (e.g., as determined by flow cytometry, in vitro or in vivo T regulatory cell proliferation or expansion assays, and / or T cell suppression assays).

[0047] In one embodiment, the IL-2 agent modulates (e.g., reduces (e.g., inhibits, blocks, or neutralizes) or increases (e.g., activates, initiates, or enhances) one or more biological activities of T cells (e.g., Tregs) in vitro, ex vivo, or in vivo.

[0048] In one embodiment, the IL-2 agent exhibits the same or similar binding affinity or specificity, or both, as the IL-2 agents described herein.

[0049] In one embodiment, the IL-2 agent exhibits the same or similar binding affinity or specificity, or both, as an IL-2 agent containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) modifications (e.g., substitutions) described herein.

[0050] In one embodiment, the IL-2 agent exhibits the same or similar binding affinity or specificity, or both, as an IL-2 agent comprising an amino acid sequence described herein.

[0051] In one embodiment, the IL-2 agent exhibits the same or similar binding affinity or specificity, or both, as an IL-2 agent comprising an amino acid sequence encoded by a nucleotide sequence described herein.

[0052] In one embodiment, the IL-2 agent inhibits, e.g., competitively inhibits, the binding of a second IL-2 agent to an IL-2 receptor, and the second IL-2 agent is an IL-2 agent described herein.

[0053] In one embodiment, the IL-2 agent competes with a second IL-2 agent for binding to the IL-2 receptor, and the second IL-2 agent is an IL-2 agent described herein.

[0054] In one embodiment, the IL-2 agent has one or more biological properties of an IL-2 agent described herein.

[0055] In one embodiment, the IL-2 agent has one or more structural characteristics of an IL-2 agent described herein.

[0056] In one embodiment, the IL-2 agent has one or more pharmacokinetic properties of the IL-2 agents described herein.

[0057] In one embodiment, the interleukin-2 (IL-2) agent comprises a human IL-2 variant comprising an amino acid modification at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) positions selected from the following: T3, H16, I28, K35, R38, F42, E68, V69, Q74, D84, S87, N88, I92, C125, Q126, or a combination thereof, corresponding to wild-type human IL-2. In another embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position V69, Q74, or a combination thereof. In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at positions V69 and Q74. In one embodiment, the IL-2 agent comprises the amino acid substitution V69A. In one embodiment, the IL-2 agent comprises the amino acid substitution Q74P. In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position H16, I92, D84, or a combination thereof. In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position H16, optionally the amino acid substitution is H16N, H16L, or H16D. In one embodiment, the IL-2 agent comprises an amino acid substitution H16N. In one embodiment, the IL-2 agent comprises an amino acid substitution H16L. In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position I92, optionally the amino acid substitution is I92S. In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position D84, optionally the amino acid substitution is D84V. In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position K35, R38, F42, E68, or a combination thereof. In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position K35, optionally wherein the amino acid substitution is K35E. In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position R38, optionally wherein the amino acid substitution is R38E, R38N or R38Q. In one embodiment, the IL-2 agent comprises the amino acid substitution R38N. In one embodiment, the IL-2 agent comprises the amino acid substitution R38Q. In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position F42, optionally wherein the amino acid substitution is F42K or F42Q.In one embodiment, the IL-2 agent comprises the amino acid substitution F42Q.

[0058] In one embodiment, the IL-2 agent includes one or more (e.g., two, three, four, or all) of (i)-(v): (i) one or more (e.g., two, three, four, five, six, or seven) amino acid modifications (e.g., substitutions) that reduce, or are identified as reducing, its affinity for CD122 (e.g., a CD122 / CD132 heterodimer), e.g., modifications (e.g., substitutions) at positions H16 (e.g., H16L, H16N, or H16D), I28 (e.g., I28T or I28F), D84 (e.g., D84V), S87 (e.g., S87R), N88 (e.g., N88S, N88L, or N88D), I92 (e.g., I92S), and / or Q126 (e.g., Q126T, Q126K, or Q126R); (ii) one or more (e.g., two) amino acid modifications (e.g., substitutions) that increase or are identified as increasing the stability of the IL-2 agent, e.g., a modification (e.g., substitution) at positions V69 (e.g., V69A) and / or Q74 (e.g., Q74P); (iii) one or more (e.g., two, three, or four) amino acid modifications (e.g., substitutions) that reduce, or are identified as reducing, its affinity for CD25, e.g., modifications (e.g., substitutions) at positions K35 (e.g., K35E), R38 (e.g., R38E, R38N, or R38Q), F42 (e.g., F42K or F42Q), and / or E68 (e.g., E68Q or E68N); or (iv) one or more amino acid modifications (e.g., substitutions) that reduce, or are identified as reducing, O-glycosylation of the IL-2 agent, e.g., a modification (e.g., substitution) at position T3 (e.g., T3A); or (v) One or more amino acid modifications (e.g., substitutions) that are identified as reducing or reducing incorrect disulfide pairing and / or aggregation of the IL-2 agent (e.g., to improve stability), e.g., a modification (e.g., substitution) at position C125 (e.g., C125S).

[0059] In one embodiment, the IL-2 agent comprises (i). In one embodiment, the IL-2 agent comprises (ii). In one embodiment, the IL-2 agent comprises (iii). In one embodiment, the IL-2 agent comprises (iv). In one embodiment, the IL-2 agent comprises (v).

[0060] In one embodiment, the IL-2 agent comprises (i) and (ii). In one embodiment, the IL-2 agent comprises (i) and (iii). In one embodiment, the IL-2 agent comprises (i) and (iv). In one embodiment, the IL-2 agent comprises (i) and (v). In one embodiment, the IL-2 agent comprises (ii) and (iii). In one embodiment, the IL-2 agent comprises (ii) and (iv). In one embodiment, the IL-2 agent comprises (ii) and (v). In one embodiment, the IL-2 agent comprises (iii) and (iv). In one embodiment, the IL-2 agent comprises (iii) and (v). In one embodiment, the IL-2 agent comprises (iv) and (v).

[0061] In one embodiment, the IL-2 agent comprises (i), (ii), and (iii). In one embodiment, the IL-2 agent comprises (i), (ii), and (iv). In one embodiment, the IL-2 agent comprises (i), (ii), and (v). In one embodiment, the IL-2 agent comprises (i), (iii), and (iv). In one embodiment, the IL-2 agent comprises (i), (iii), and (v). In one embodiment, the IL-2 agent comprises (i), (iv), and (v). In one embodiment, the IL-2 agent comprises (ii), (iii), and (iv). In one embodiment, the IL-2 agent comprises (ii), (iii), and (iv). In one embodiment, the IL-2 agent comprises (ii), (iii), and (v). In one embodiment, the IL-2 agent comprises (ii), (iii), and (v). In one embodiment, the IL-2 agent comprises (ii), (iv), and (iv). In one embodiment, the IL-2 agent comprises (iii), (iv), and (v).

[0062] In one embodiment, the IL-2 agent comprises (i), (ii), (iii), and (iv). In one embodiment, the IL-2 agent comprises (i), (ii), (iii), and (v). In one embodiment, the IL-2 agent comprises (i), (ii), (iv), and (v). In one embodiment, the IL-2 agent comprises (i), (iii), (iv), and (v). In one embodiment, the IL-2 agent comprises (ii), (iii), (iv), and (v). In one embodiment, the IL-2 agent comprises (ii), (iii), (iv), and (v).

[0063] In one embodiment, the IL-2 agent includes (i), (ii), (iii), (iv), and (v).

[0064] In one embodiment, the IL-2 agent does not include (i). In one embodiment, the IL-2 agent does not include (ii). In one embodiment, the IL-2 agent does not include (iii). In one embodiment, the IL-2 agent does not include (iv). In one embodiment, the IL-2 agent does not include (v).

[0065] In one embodiment, the IL-2 agent does not include (i) and (ii). In one embodiment, the IL-2 agent does not include (i) and (iii). In one embodiment, the IL-2 agent does not include (i) and (iv). In one embodiment, the IL-2 agent does not include (i) and (v). In one embodiment, the IL-2 agent does not include (ii) and (iii). In one embodiment, the IL-2 agent does not include (ii) and (iv). In one embodiment, the IL-2 agent does not include (ii) and (v). In one embodiment, the IL-2 agent does not include (iii) and (iv). In one embodiment, the IL-2 agent does not include (iii) and (v). In one embodiment, the IL-2 agent does not include (iii) and (v). In one embodiment, the IL-2 agent does not include (iv) and (v).

[0066] In one embodiment, the IL-2 agent does not include (i), (ii), and (iii). In one embodiment, the IL-2 agent does not include (i), (ii), and (iv). In one embodiment, the IL-2 agent does not include (i), (ii), and (v). In one embodiment, the IL-2 agent does not include (i), (iii), and (iv). In one embodiment, the IL-2 agent does not include (i), (iii), and (v). In one embodiment, the IL-2 agent does not include (i), (iv), and (v). In one embodiment, the IL-2 agent does not include (ii), (iii), and (iv). In one embodiment, the IL-2 agent does not include (ii), (iii), and (v). In one embodiment, the IL-2 agent does not include (ii), (iii), and (v). In one embodiment, the IL-2 agent does not include (ii), (iii), and (v). In one embodiment, the IL-2 agent does not include (ii), (iv), and (iv). In one embodiment, the IL-2 agent does not include (iii), (iv), and (v).

[0067] In one embodiment, the IL-2 agent does not include (i), (ii), (iii), and (iv). In one embodiment, the IL-2 agent does not include (i), (ii), (iii), and (v). In one embodiment, the IL-2 agent does not include (i), (ii), (iv), and (v). In one embodiment, the IL-2 agent does not include (i), (iii), (iv), and (v). In one embodiment, the IL-2 agent does not include (ii), (iii), (iv), and (v).

[0068] In one embodiment, the IL-2 agent does not include (i), (ii), (iii), (iv), and (v).

[0069] In one embodiment, the IL-2 agent contains amino acid modifications (e.g., substitutions) at the following positions: (i) positions V69 and Q74, and / or K35, and (ii) position H16, I92, or D84, and optionally, (iii) positions R38, F42, E68, or any combination thereof;

[0070] In one embodiment, the IL-2 agent contains amino acid modifications (e.g., substitutions) at the following positions: (i) positions V69 and Q74, and / or K35, and (ii) position H16, I92, or D84, and (iii) positions R38, F42, E68, or any combination thereof;

[0071] In one embodiment, the IL-2 agent contains amino acid modifications (e.g., substitutions) at the following positions: (i) positions V69 and Q74, and / or K35, and (ii) position H16, I92, or D84, or (iii) positions R38, F42, E68, or any combination thereof;

[0072] In one embodiment, the IL-2 agent contains amino acid modifications (e.g., substitutions) at the following positions: (i) positions V69 and Q74, and / or K35, and (ii) positions H16, I92, D84, or combinations thereof, and (iii) positions R38, F42, E68, or any combination thereof;

[0073] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and H16, optionally the amino acid substitutions are V69A, Q74P, and H16N or H16L, respectively, optionally the amino acid substitutions are V69A, Q74P, and H16L. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, and H16L.

[0074] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and I92, optionally the amino acid substitutions are V69A, Q74P, and I92S, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, and I92S.

[0075] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and D84, optionally the amino acid substitutions are V69A, Q74P, and D84V, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, and D84V.

[0076] In one embodiment, the IL-2 agent comprises amino acid modifications (eg, substitutions) at positions V69, Q74, and R38, optionally, the amino acid substitutions are V69A, Q74P, and R38Q, respectively.

[0077] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and F42, and optionally, the amino acid substitutions are V69A, Q74P, and F42Q, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, and F42Q.

[0078] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and R38, optionally the amino acid substitutions are V69A, Q74P, and R38N, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, and R38N.

[0079] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and R38, optionally the amino acid substitutions are V69A, Q74P, and R38E, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, and R38E.

[0080] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, K35, and H16, optionally the amino acid substitutions are V69A, Q74P, K35E, and H16N or H16L, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, K35E, and H16N or H16L. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, K35E, and H16N. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, K35E, and H16L.

[0081] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, K35, H16, and R38, and optionally the amino acid substitutions are V69A, Q74P, K35E, H16N, and R38N, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, K35E, H16N, and R38N.

[0082] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, H16, and R38, optionally the amino acid substitutions are V69A, Q74P, H16N or H16L, and R38N or R38Q, respectively, optionally the amino acid substitutions are V69A, Q74P, H16N or H16L, and R38Q. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, H16L, and R38Q.

[0083] In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position I28, E68, S87, N88, Q126, or a combination thereof. In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position I28, optionally the amino acid substitution is I28T or I28F. In one embodiment, the IL-2 agent comprises the amino acid substitution I28T. In one embodiment, the IL-2 agent comprises the amino acid substitution I28F.

[0084] In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position E68, optionally the amino acid substitution is E68Q or E68N. In one embodiment, the IL-2 agent comprises the amino acid substitution E68Q. In one embodiment, the IL-2 agent comprises the amino acid substitution E68N.

[0085] In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., a substitution) at position S87, optionally, the amino acid substitution is S87 R. In one embodiment, the IL-2 agent comprises the amino acid substitution S87R.

[0086] In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position N88, optionally the amino acid substitution is N88R, N88S, N88L, or N88D. In one embodiment, the IL-2 agent comprises the amino acid substitution N88R. In one embodiment, the IL-2 agent comprises the amino acid substitution N88S. In one embodiment, the IL-2 agent comprises the amino acid substitution N88L. In one embodiment, the IL-2 agent comprises the amino acid substitution N88D.

[0087] In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position Q126, optionally the amino acid substitution is Q126T, Q126K or Q126R. In one embodiment, the IL-2 agent comprises the amino acid substitution Q126T. In one embodiment, the IL-2 agent comprises the amino acid substitution Q126K. In one embodiment, the IL-2 agent comprises the amino acid substitution Q126R.

[0088] In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position C125, optionally the amino acid substitution is C125S. In one embodiment, the IL-2 agent comprises the amino acid substitution C125S.

[0089] In one embodiment, the IL-2 agent comprises an amino acid modification (e.g., substitution) at position T3, optionally the amino acid substitution is T3A. In one embodiment, the IL-2 agent comprises the amino acid substitution T3A.

[0090] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and C125, optionally the amino acid substitutions are V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions V69A, Q74P, and C125S.

[0091] In one embodiment, the IL-2 agent comprises an amino acid modification (eg, substitution) at positions T3, H16, I92, or a combination thereof, optionally, the amino acid substitutions are T3A, H16N, and I92S, respectively.

[0092] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions H16, V69, Q74, and C125, and optionally, the amino acid substitutions are H16N, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions H16N, V69A, Q74P, and C125S.

[0093] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions H16, V69, Q74, and C125, optionally the amino acid substitutions are H16L, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions H16L, V69A, Q74P, and C125S. Various technical effects are associated with IL-2 agents that comprise the aforementioned combinations of amino acid modifications. Without wishing to be bound by theory, in one embodiment, it is believed that an IL-2 agent comprising the amino acid substitutions H16L, V69A, Q74P, and C125S can have at least one or more of the following advantageous properties: (i) have reduced binding affinity to CD122 and / or CD132, which increases the potency and selectivity of the IL-2 agent for regulatory T cells (Tregs) compared to other T cell types; (ii) are significantly more stable, e.g., due to the presence of the stabilizing V69A and Q74P mutations; (iii) have reduced or decreased (or have only a minimal effect on) binding ability and / or binding affinity to CD25, which improves the longevity of the IL-2 agent; (iv) do not substantially promote the expansion, activity, survival and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) reduce incorrect disulfide pairing and improve stability, e.g., due to the presence of the C125S mutation. In one embodiment, an IL-2 agent comprising the H16L mutation has reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Tregs over other T cell types, as compared to IL-2 agents comprising other H16 mutations. These properties make IL-2 agents comprising the amino acid substitutions H16L, V69A, Q74P, and C125S particularly suitable for treating disorders and conditions resulting from an aberrant immune response, such as, for example, autoimmune diseases.

[0094] Thus, in one embodiment, an IL-2 agent comprising the amino acid substitutions H16L, V69A, Q74P, and C125S has, inter alia, one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties, in particular as compared to wild-type IL-2 or a reference IL-2 variant that does not comprise the amino acid substitutions: (i) enhanced or increased stability in vitro or in vivo, (ii) reduced or decreased binding ability and / or binding affinity to human CD122 in vitro and / or in vivo, (iii) reduced or decreased binding ability and / or binding affinity to human CD122 in vitro and / or in vivo, (iv) reduced or decreased binding ability and / or binding affinity to human CD122 in vivo, (v) reduced or decreased binding ability and / or affinity to human CD122 in vivo, (vi) reduced or decreased binding ability and / or affinity to human CD122 in vivo, (vii) reduced or decreased binding ability and / or affinity to human CD122 in vivo, (viii ... (iv) reduced or decreased affinity of the IL-2 variant for the heterodimeric IL-2 receptor composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) reduced or decreased (moderately reduced or decreased) binding ability and / or binding affinity for human CD25 in vitro and / or in vivo; (vi) reduced or decreased binding ability and / or binding affinity for regulatory T cells (e.g., Foxp3 + (vii) selective activation of the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0095] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions H16, V69, Q74, I92, and C125, and optionally the amino acid substitutions are H16L, V69A, Q74P, I92S, and C125S, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions H16L, V69A, Q74P, I92S, and C125S.

[0096] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions T3, V69, Q74, and C125, and optionally the amino acid substitutions are T3A, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions T3A, V69A, Q74P, and C125S.

[0097] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions T3, H16, V69, Q74, and C125, and optionally the amino acid substitutions are T3A, H16N or H16L, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions T3A, H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 agent comprises amino acid substitutions T3A, H16L, V69A, Q74P, and C125S.

[0098] In one embodiment, the IL-2 agent comprises amino acid modifications (e.g., substitutions) at positions T3, V69, Q74, I92, and C125, and optionally the amino acid substitutions are T3A, V69A, Q74P, I92S, and C125S, respectively. In one embodiment, the IL-2 agent comprises amino acid substitutions T3A, V69A, Q74P, I92S, and C125S. In one embodiment, the IL-2 agent comprises amino acid substitutions T3A, V69A, Q74P, I92S, and C125S.

[0099] In one embodiment, the IL-2 agent is SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:1000, SEQ ID NO:1001, SEQ ID NO:1002, or a functional fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.

[0100] In one embodiment, the amino acid modification (e.g., substitution) provides an IL-2 agent with at least one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all) of the following properties compared to a reference IL-2 agent that does not include the amino acid modification (e.g., substitution): (i) enhanced or increased expression of IL-2; (ii) inhibited or reduced aggregation of IL-2 agents; (iii) enhanced or increased stability of the IL-2 agent; (iv) an enhanced or increased half-life of the IL-2 agent; (v) inhibition or decreased turnover and / or clearance of IL-2 agents; (vi) inhibited or reduced (e.g., moderately inhibited or reduced) or substantially unchanged binding of the IL-2 agent to human CD25; (vii) inhibiting or decreasing the affinity of the IL-2 agent for human CD122; (viii) inhibited or decreased affinity of the IL-2 agent for human CD132; or (ix) inhibited or reduced affinity of the IL-2 agent for the dimeric IL-2 receptor composed of human CD122 and human CD132; (x) selective binding to regulatory T cells (e.g., Foxp3+ T cells); (xi) selective activation of the IL-2 signaling pathway in Tregs; or (xii) Enhanced or increased, or decreased or diminished, ability to induce or promote expansion, activation, survival, and / or proliferation of Tregs.

[0101] In one embodiment, an IL-2 agent comprises an IL-2 variant that includes one or more amino acid modifications (e.g., substitutions) selected from H16D, H16N, H16L, I28T, K35E, R38Q, R38N, R38E, F42K, F42Q, V69A, Q74P, D84V, S87R, N88L, N88S, I92S, C125S; a polypeptide linker as described herein; and a non-IL-2 moiety as described herein, wherein the amino acid modification (e.g., substitution) provides the IL-2 agent with at least one or more of the following properties relative to a reference IL-2 agent that does not include the amino acid modification (e.g., substitution): (i) enhanced or increased expression of IL-2; (ii) inhibited or reduced aggregation of IL-2 agents; (iii) enhanced or increased stability of the IL-2 agent; (iv) an enhanced or increased half-life of the IL-2 agent; (v) inhibition or decreased turnover and / or clearance of IL-2 agents; (vi) inhibited or reduced (e.g., moderately inhibited or reduced) or substantially unchanged binding of the IL-2 agent to human CD25; (vii) inhibiting or decreasing the affinity of the IL-2 agent for human CD122; (viii) inhibited or decreased affinity of the IL-2 agent for human CD132; (ix) inhibited or reduced affinity of the IL-2 agent for the dimeric IL-2 receptor composed of human CD122 and human CD132; (x) selective binding to regulatory T cells (e.g., Foxp3+ T cells); (xi) selective activation of the IL-2 signaling pathway in Tregs; and / or (xii) Enhanced or increased, or decreased or diminished, ability to induce or promote expansion, activation, survival, and / or proliferation of Tregs.

[0102] In one embodiment, the human IL-2 variant contains the following amino acid modifications (e.g., substitutions): (i) C125S; (ii) V69A, Q74P, and C125S; (iii) H16D, V69A, Q74P, and C125S; (iv) H16N, V69A, Q74P, and C125S; (v) H16L, V69A, Q74P, and C125S; (vi) I28T, V69A, Q74P, and C125S; (vii) V69A, Q74P, D84V, and C125S; (viii) V69A, Q74P, S87R, and C125S; (ix) V69A, Q74P, N88L, and C125S; (x) V69A, Q74P, N88S, and C125S; (xi) V69A, Q74P, I92S, and C125S; (xii) K35E, V69A, Q74P, and C125S; (xii) K35E, H16N, V69A, Q74P, and C125S; (xiv) K35E, H16L, V69A, Q74P, and C125S; (xv) K35E, D84V, V69A, Q74P, and C125S; (xvi) K35E, I92S, V69A, Q74P, and C125S; (xvii) R38Q, V69A, Q74P, and C125S; (xvii) R38Q, H16N, V69A, Q74P, and C125S; (xix) R38Q, H16L, V69A, Q74P, and C125S; (xx)R38Q, D84V, V69A, Q74P, and C125S; (xxi) R38Q, I92S, Q74P, and C125S; (xxii) R38N, V69A, Q74P, and C125S; (xxii) R38N, H16N, V69A, Q74P, and C125S; (xxiv) R38N, H16L, V69A, Q74P, and C125S; (xxv) R38N, D84V, V69A, Q74P, and C125S; (xxvi) R38N, I92S, Q74P, and C125S; (xxvii) R38E, V69A, Q74P, and C125S; (xxviii) F42K, V69A, Q74P, and C125S; (xxix)F42Q, V69A, Q74P, and C125S; (xxx)F42A, Y45A, L72G, N88D, V69A, Q74P, and C125S, (xxxi) R38N, S87R, V69A, Q74P, and C125S; (xxxii) R38E, H16N, V69A, Q74P, and C125S; (xxxii) R38E, D84V, V69A, Q74P, and C125S; (xxxiv) R38E, S87R, V69A, Q74P, and C125S; (xxxv) R38E, I92S, V69A, Q74P, and C125S; (xxxvi) F42Q, H16N, V69A, Q74P, and C125S; (xxxvii) F42Q, I92S, V69A, Q74P, and C125S; or (xxxviii) K35E, R38N, H16N, V69A, Q74P, and C125S.

[0103] (xxxix) T3A, H16N, V69A, Q74P, and C125S; (xl) T3A, H16L, V69A, Q74P, and C125S; or (xli) T3A, V69A, Q74P, I92S, and C125S.

[0104] In one embodiment, the IL-2 agent is SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:1000, SEQ ID NO:1001, or SEQ ID NO:100 2, or a functional fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids; a polypeptide linker as described herein; and a non-IL-2 moiety as described herein, wherein the IL-2 agent exhibits at least one or more of the following properties compared to a reference IL-2 agent that does not comprise a human IL-2 polypeptide variant: (i) enhanced or increased expression of IL-2; (ii) inhibited or reduced aggregation of IL-2 agents; (iii) enhanced or increased stability of the IL-2 agent; (iv) an enhanced or increased half-life of the IL-2 agent; (v) inhibition or decreased turnover and / or clearance of IL-2 agents; (vi) inhibited or reduced (e.g., moderately inhibited or reduced) or substantially unchanged binding of the IL-2 agent to human CD25; (vii) inhibiting or decreasing the affinity of the IL-2 agent for human CD122; (viii) inhibited or decreased affinity of the IL-2 agent for human CD132; (ix) inhibited or decreased affinity of the IL-2 agent for the dimeric IL-2 receptor of human CD122 and human CD132; (x) selective binding to regulatory T cells (e.g., Foxp3+ T cells); (xi) selective activation of the IL-2 signaling pathway in Tregs; and / or (xii) Enhanced or increased, or decreased or diminished, ability to induce or promote expansion, activation, and / or proliferation of Tregs.

[0105] Various technical effects are associated with an IL-2 agent comprising the amino acid sequence of SEQ ID NO: 5. Without wishing to be bound by theory, in one embodiment, it is believed that an IL-2 agent comprising the amino acid sequence of SEQ ID NO: 5 may have at least one or more of the following advantageous properties: (i) having reduced binding affinity to CD122 and / or CD132, which increases the potency and selectivity of the IL-2 agent for regulatory T cells (Tregs) compared to other T cell types, (ii) being significantly stable, e.g., due to the presence of the stabilizing V69A and Q74P mutations, (iii) having reduced or diminished (or having only minimal effect on) binding ability and / or binding affinity to CD25, which improves the longevity of the IL-2 agent, (iv) not substantially promoting the expansion, activity, survival and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo, and / or (v) having reduced imprecise disulfide pairing and improved stability, e.g., due to the presence of the C125S mutation. In one embodiment, an IL-2 agent comprising the H16L mutation has reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Tregs over other T cell types, compared to IL-2 agents comprising other H16 mutations. These properties make an IL-2 agent comprising the amino acid sequence of SEQ ID NO:5 particularly suitable for the treatment of disorders and conditions resulting from an aberrant immune response, such as autoimmune diseases.

[0106] Thus, in one embodiment, an IL-2 agent comprising an amino acid substitution of SEQ ID NO:5 has, inter alia, one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties, particularly as compared to wild-type IL-2 or a reference IL-2 variant that does not comprise the amino acid substitution: (i) enhanced or increased stability in vitro or in vivo, (ii) reduced or decreased binding ability and / or binding affinity to human CD122 in vitro and / or in vivo, (iii) reduced or decreased binding ability and / or binding affinity to human CD122 in vitro and / or in vivo, (iv) reduced or decreased binding ability and / or affinity to human CD122 in vitro and / or in vivo, (v) reduced or decreased binding ability and / or affinity to human CD122 in vivo, (vi) reduced or decreased binding ability and / or affinity to human CD122 in vivo, (vii) reduced or decreased binding ability and / or affinity to human CD122 in vivo, (vii) reduced or decreased binding ability and / or affinity to human CD122 in vivo, (vii) reduced or increased ... (iv) reduced or decreased affinity of the IL-2 variant for the heterodimeric IL-2 receptor composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) reduced or decreased (moderately reduced or decreased) binding ability and / or binding affinity for human CD25 in vitro and / or in vivo; (vi) reduced or decreased binding ability and / or binding affinity for regulatory T cells (e.g., Foxp3 + (vii) selective activation of the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0107] In one embodiment, the reference IL-2 agent comprises the amino acid sequence of SEQ ID NO: 1031, SEQ ID NO: 1, or SEQ ID NO: 2, or a functional fragment thereof. In one embodiment, the reference IL-2 agent comprises the amino acid sequence of SEQ ID NO: 1031. In one embodiment, the reference IL-2 agent comprises the amino acid sequence of SEQ ID NO: 1. In one embodiment, the reference IL-2 agent comprises the amino acid sequence of SEQ ID NO: 2.

[0108] In one embodiment, the IL-2 agent comprises a human IL-2 variant described herein fused to a non-IL-2 moiety described herein by a linker, the linker being a polypeptide linker, optionally the polypeptide linker being a flexible linker, a rigid linker, or a cleavable linker. In one embodiment, the polypeptide linker is a Gly-Ser linker (e.g., a (G4S)n linker, where n=1, 2, 3, 4, 5, 6 or more (SEQ ID NO: 1020)), a proline-rich extension linker (e.g., V1 GPc, V2, GPGc, V3 GcGcP, Cellulase Linker 4, Cellulase Linker 4), a rigid linker (e.g., A(EAAAK)nA, where n=2, 3, 4, 5 or more (SEQ ID NO: 1021), REPR_12), a non-GS linker (e.g., (GGGSA)n, where n=1, 2, 3, 4, 5 or more (SEQ ID NO: 1022)), or an immunoglobulin hinge region or portion thereof. In one embodiment, the polypeptide linker is a Gly-Ser linker comprising (G4S)1 (SEQ ID NO:1023), (G4S)2 (SEQ ID NO:1024), (G4S)3 (SEQ ID NO:1025), (G4S)4 (SEQ ID NO:48), (G4S)5 (SEQ ID NO:1026), or (G4S)6 (SEQ ID NO:1027). In one embodiment, the polypeptide linker is a Gly-Ser linker comprising (G4S)4 (SEQ ID NO:48). In one embodiment, the polypeptide linker comprises an amino acid sequence selected from the following: SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, or SEQ ID NO:55. In one embodiment, the polypeptide linker comprises the amino acid sequence of SEQ ID NO:48.

[0109] In one embodiment, the non-IL-2 moiety is an immunoglobulin Fc region, or a fragment or portion thereof (e.g., a functional fragment). In one embodiment, the immunoglobulin Fc region comprises an IgG Fc region, an IgD Fc region, an IgA Fc region, an IgM Fc region, or an IgE Fc region, or a fragment or portion thereof. In one embodiment, the IgG Fc region comprises a wild-type human IgG1 Fc region (e.g., an IgG1 m3 allotype), a wild-type IgG2 Fc region, or a wild-type human IgG4 Fc region, or a fragment or portion thereof.

[0110] In one embodiment, the IgG Fc region comprises a mutated IgG1 or mutated IgG4 Fc region, or a fragment or portion thereof. In one embodiment, the IgG Fc region comprises one or more (e.g., two, three, four, or five) mutations, e.g., one or more (e.g., two, three, four, or five) mutations described herein.

[0111] In one embodiment, the IgG Fc region comprises a mutated IgG4 Fc region, or a fragment or portion thereof, and the mutated IgG4 Fc region is human.

[0112] In one embodiment, the mutated IgG4 Fc region, or a fragment or portion thereof, comprises an amino acid modification (e.g., substitution) at Ser228 according to EU numbering, optionally the amino acid modification (e.g., substitution) at Ser228 is S228P. In one embodiment, the mutated IgG4 Fc region comprises the amino acid substitution S228P.

[0113] In one embodiment, the mutated IgG4 Fc region, or a fragment or portion thereof, comprises an amino acid modification (e.g., substitution) at Arg409 according to EU numbering, optionally wherein the amino acid modification (e.g., substitution) at Arg409 is R409K. In one embodiment, the mutated IgG4 Fc region comprises the amino acid substitution R409K.

[0114] In one embodiment, the mutated IgG4 Fc region, or a fragment or portion thereof, comprises amino acid modifications (e.g., substitutions) at Thr307, Gln311, and Ala378 according to EU numbering, optionally the amino acid modifications (e.g., substitutions) are T307Q, Q311V, and A378V, respectively. In one embodiment, the mutated IgG4 Fc region comprises the amino acid substitutions T307Q, Q311V, and A378V.

[0115] In one embodiment, the mutant IgG4 Fc region comprises an amino acid sequence selected from SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO:47, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0116] In one embodiment, the IgG Fc region comprises a mutated IgG1 Fc region, or a fragment or portion thereof, and the mutated IgG1 Fc region is human. In one embodiment, the mutated IgG1 Fc region (e.g., comprising an N297G substitution) has an IgG1 m3 allotype.

[0117] In one embodiment, the mutated IgG1 Fc region, or a fragment or portion thereof, comprises an amino acid modification (e.g., substitution) at Asn297 according to EU numbering, optionally wherein the amino acid modification (e.g., substitution) at Asn297 is N297G. In one embodiment, the mutated IgG1 Fc region comprises the amino acid substitution N297G.

[0118] In one embodiment, the mutated IgG1 Fc region, or a fragment or portion thereof, comprises amino acid modifications (e.g., substitutions) at Leu234, Leu235, and Pro329 according to EU numbering, optionally the amino acid modifications (e.g., substitutions) are L234A, L235A, and P329G, respectively. In one embodiment, the mutated IgG1 Fc region comprises the amino acid substitutions L234A, L235A, and P329G.

[0119] In one embodiment, the mutated IgG1 Fc region, or a fragment or portion thereof, comprises amino acid modifications (e.g., substitutions) at Thr307, Gln311, and Ala378 according to EU numbering, optionally the amino acid modifications (e.g., substitutions) are T307Q, Q311V, and A378V, respectively. In one embodiment, the mutated IgG1 Fc region comprises the amino acid substitutions T307Q, Q311V, and A378V.

[0120] In one embodiment, the mutated IgG1 Fc region comprises an amino acid sequence selected from SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:1003, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom. In one embodiment, the mutated IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:1003, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids. In one embodiment, the mutated IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:1003.

[0121] In one embodiment, the non-IL-2 moiety inhibits or reduces the ability of the IL-2 agent to elicit an Fc receptor-mediated immune effector function.

[0122] In one embodiment, the IL-2 agent comprises an IL-2 variant comprising an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38, SEQ ID NO:1000, SEQ ID NO:1001, or SEQ ID NO:1002, or a functional fragment thereof, wherein the IL-2 agent comprises a Gly-Ser linker, and optionally the Gly-Ser linker is 4 S) 4 (SEQ ID NO:48), wherein the IL-2 variant is fused by a Gly-Ser linker to an IgG Fc region comprising an amino acid sequence selected from SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:1003.

[0123] In one embodiment, the IL-2 agent comprises an amino acid sequence selected from SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:1004, SEQ ID NO:1005, SEQ ID NO:1006, SEQ ID NO:1007, SEQ ID NO:1008, or SEQ ID NO:1009, or a functional fragment thereof.

[0124] In one embodiment, the IL-2 agent comprises an amino acid sequence selected from SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, or a functional fragment thereof.

[0125] In one embodiment, the IL-2 agent comprises an amino acid sequence selected from SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, or SEQ ID NO:169, or a functional fragment thereof.

[0126] In one embodiment, the IL-2 agent comprises an amino acid sequence selected from SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, or SEQ ID NO:207, or a functional fragment thereof.

[0127] In one embodiment, the IL-2 agent comprises an amino acid sequence selected from SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, or SEQ ID NO:245, or a functional fragment thereof.

[0128] In one embodiment, the IL-2 agent comprises an amino acid sequence selected from SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273, SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, or SEQ ID NO:283, or a functional fragment thereof.

[0129] In one embodiment, the IL-2 agent comprises an amino acid sequence selected from SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:311, SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:319, SEQ ID NO:320, or SEQ ID NO:321, or a functional fragment thereof.

[0130] In one embodiment, the IL-2 agent comprises an amino acid sequence selected from SEQ ID NO:322, SEQ ID NO:323, SEQ ID NO:324, SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327, SEQ ID NO:328, SEQ ID NO:329, SEQ ID NO:330, SEQ ID NO:331, SEQ ID NO:332, SEQ ID NO:333, SEQ ID NO:334, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337, SEQ ID NO:338, SEQ ID NO:339, SEQ ID NO:340, SEQ ID NO:341, SEQ ID NO:342, SEQ ID NO:343, SEQ ID NO:344, SEQ ID NO:345, SEQ ID NO:346, SEQ ID NO:347, SEQ ID NO:348, SEQ ID NO:349, SEQ ID NO:350, SEQ ID NO:351, SEQ ID NO:352, SEQ ID NO:353, SEQ ID NO:354, SEQ ID NO:355, SEQ ID NO:356, SEQ ID NO:357, SEQ ID NO:358, or SEQ ID NO:359, or a functional fragment thereof.

[0131] In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:59, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:97, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:135, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:173, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:211, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:249, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:287, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:325, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:66, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:104, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:142, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO: 180, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO: 218, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO: 256, or a functional fragment thereof.

[0132] In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:294, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:332, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:60, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:98, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:136, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:174, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:212, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:250, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:288, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:326, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:69, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO: 107, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO: 145, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO: 183, or a functional fragment thereof.

[0133] In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:221, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:259, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:297, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:335, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:1004, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:1005, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:1006, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:1007, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:1008, or a functional fragment thereof. In one embodiment, the IL-2 agent comprises the amino acid sequence of SEQ ID NO:1009, or a functional fragment thereof.

[0134] Various technical effects are associated with an IL-2 agent comprising the amino acid sequence of SEQ ID NO:1008. Without wishing to be bound by theory, in one embodiment, it is believed that an IL-2 agent comprising the amino acid sequence of SEQ ID NO: 1008 can have at least one or more of the following advantageous properties: (i) has reduced binding affinity to CD122 and / or CD132, which increases the potency and selectivity of the IL-2 agent for regulatory T cells (Tregs) compared to other T cell types; (ii) is significantly stable, e.g., due to the presence of the stabilizing V69A and Q74P mutations; (iii) has reduced or decreased (or has only a minimal effect on) binding ability and / or binding affinity to CD25, which improves the longevity of the IL-2 agent; (iv) does not substantially promote the expansion, activation, survival and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) has reduced and imprecise disulfide pairing and improved stability, e.g., due to the presence of the C125S mutation. In one embodiment, an IL-2 agent comprising the H16L mutation has reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Tregs over other T cell types, as compared to IL-2 agents comprising other H16 mutations. These properties make the IL-2 variant, comprising the amino acid sequence of SEQ ID NO: 1008, particularly suitable for the treatment of disorders and conditions resulting from an aberrant immune response, such as autoimmune diseases.

[0135] Thus, in one embodiment, an IL-2 agent comprising an amino acid substitution of SEQ ID NO: 1008 has, inter alia, one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties, particularly as compared to wild-type IL-2 or a reference IL-2 variant that does not comprise the amino acid substitution: (i) enhanced or increased stability in vitro or in vivo, (ii) reduced or decreased binding ability and / or binding affinity to human CD122 in vitro and / or in vivo, (iii) reduced or decreased binding ability and / or binding affinity to human CD132 in vitro and / or in vivo. (iv) reduced or decreased affinity of the IL-2 variant for the heterodimeric IL-2 receptor composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) reduced or decreased (moderately reduced or decreased) or substantially unchanged binding ability and / or binding affinity for human CD25 in vitro and / or in vivo; (vi) reduced or decreased binding ability and / or binding affinity for regulatory T cells (e.g., Foxp3 + (vii) selective activation of the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote the expansion, activation, survival and / or proliferation of Tregs.

[0136] In one embodiment, the IL-2 agent forms a dimer (e.g., a homodimer or a heterodimer). In one embodiment, the IL-2 agent comprises an IL-2 fusion protein. In one embodiment, the IL-2 agent comprises an IL-2 agent / anti-IL-2 antibody complex. In one embodiment, the IL-2 agent comprises a conjugate.

[0137] In one embodiment, the IL-2 agent is formulated in a composition (e.g., a pharmaceutical composition) described herein. In one embodiment, the IL-2 agent is encoded by a nucleic acid described herein. In one embodiment, the IL-2 agent is encoded by a nucleic acid present in a vector (e.g., an expression vector) described herein. In one embodiment, the IL-2 agent is encoded by a nucleic acid present in a cell (e.g., an isolated cell). In one embodiment, the IL-2 agent is produced by a method described herein, including, e.g., culturing (e.g., maintaining) a cell comprising a nucleic acid encoding an IL-2 agent described herein, or a vector comprising a nucleic acid encoding an IL-2 agent described herein (e.g., an expression vector), under conditions that allow expression of the IL-2 agent. In one embodiment, the IL-2 agent is isolated or purified. In one embodiment, the IL-2 agent is present in a kit described herein. In one embodiment, the IL-2 agent is present in a container described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0138] Disclosed herein are IL-2 agents (e.g., IL-2 variants, IL-2 fusion proteins, IL-2 complexes, or IL-2 conjugates) having one or more structural and / or functional properties described herein. Advantageously, some of the IL-2 agents described herein have one or more improved or desirable properties compared to IL-2 agents, including wild-type IL-2. Without wishing to be bound by theory, in one embodiment, the IL-2 agents described herein are believed to selectively promote the activity of regulatory T cells (Tregs) through the IL-2 pathway. Nucleic acid molecules encoding the IL-2 agents, expression vectors, host cells, compositions (e.g., pharmaceutical compositions), kits, containers, and methods for making the IL-2 agents are also provided. The IL-2 agents and pharmaceutical compositions disclosed herein can be used (alone or in combination with other agents or therapeutic modalities) to treat, prevent, and / or diagnose disorders and conditions, e.g., disorders and conditions associated with T cell activity, e.g., disorders or conditions described herein (e.g., autoimmune diseases described herein).

[0139] Immune responses are typically controlled by the recognition of specific foreign or self-antigens, communication between innate and adaptive immune pathways, crosstalk between B and T cells, and other factors. Some autoimmune diseases may be characterized by widespread recognition of self-antigens. These diseases can be treated by therapies that broadly enhance the process of protecting self-antigens from attack by the immune system. Tregs are a type of T cell that recognizes self-antigens. In response to antigenic stimulation, they release immunosuppressive cytokines and directly inhibit other T cells through cell-to-cell contact. Impaired Treg activity contributes to a wide range of autoimmune diseases (e.g., too few cells, or cells that are underactive). IL-2 is a cytokine that causes the expansion and activation of many cell types, but Tregs are typically much more sensitive to IL-2 than other cell types. Low dose IL-2 administration has been shown to be associated with preferential and sustained Treg cell expansion in vivo and amelioration of chronic graft-versus-host disease (GVHD) symptoms in a significant proportion of patients (Koreth et al., N Engl J Med. 2011;365(22):2055-2066). In one embodiment, the IL-2 agents described herein provide long-lasting immunomodulatory (e.g., immunosuppressant) agents for a number of disorders (e.g., autoimmune indications).

[0140] The present disclosure is based, at least in part, on the discovery that IL-2 agents comprising human IL-2 polypeptides with a particular combination of amino acid substitutions as described herein may have advantageous technical effects, such as, for example, increasing the stability of the IL-2 agent and / or providing selective activation of regulatory T cells. The IL-2 agents described herein typically require CD25 for efficient signaling through the IL-2 receptor and are highly selective for Tregs. IL-2 signaling promotes Treg suppressive function and drives proliferation. Without wishing to be bound by theory, it is believed that Tregs activated by the IL-2 agents described herein may attenuate autoimmune activity through various mechanisms.

[0141] In one embodiment, the IL-2 agents described herein selectively bind to and activate regulatory T cells, but not other immune cell types (e.g., CD25 high It has been found that the IL-2 receptor has no effect on the expression of IL-2 in T cells and NK cells. Without wishing to be bound by theory, in one embodiment, the amino acid substitutions described herein are believed to promote the ability of the IL-2 agent to maintain an active conformation while modulating the binding affinity of the IL-2 agent to dimeric receptors including IL-2Rβ (CD122) and IL-2Rγ (CD132) and trimeric receptors including IL-2Rα (CD25) with CD122 and CD132. In one embodiment, the IL-2 agents described herein have optimal affinity to selectively bind and activate IL-2 signaling in regulatory T cells, resulting in selective regulatory T cell activation and expansion both in vitro and in vivo. Without wishing to be bound by theory, in one embodiment, the binding of IL-2 to the IL-2 receptor is believed to be the primary pathway for clearance of IL-2 in vivo. For example, IL-2 agents described herein with reduced affinity for dimeric and trimeric IL-2 receptors have demonstrated increased half-life, and reduced affinity for the IL-2 receptor has been shown to reduce clearance of the IL-2 agent in vivo. IL-2 agents described herein, such as those with amino acid substitutions that increase stability and reduce affinity for the IL-2 receptor, can selectively activate regulatory T cells and exhibit increased half-life in vivo. IL-2 agents described herein, such as those with mutations that prevent CD25 binding, can have improved half-life in vivo. In one embodiment, the IL-2 agent does not promote or does not substantially promote the expansion, activity, survival, and / or proliferation of T effector cells and / or NK cells in vitro and / or in vivo. Without wishing to be bound by theory, in one embodiment, it is believed that the IL-2 agents described herein may have a greater therapeutic window than low-dose IL-2.

[0142] There are various technical effects associated with the presence of a particular set of mutations described herein, for example, a set of mutations comprising an amino acid substitution at position H16 in combination with amino acid substitutions at positions V69, Q74, and C125 (e.g., H16L, V69A, Q74P, and C125S). Without wishing to be bound by theory, in one embodiment, an IL-2 agent (e.g., an IL-2 variant or IL-2 fusion protein) comprising H16L, V69A, Q74P, and C125S is believed to be significantly more stable, for example, due to the presence of the stabilizing V69A and Q74P mutations. For example, it has been unexpectedly discovered that the V69A and Q74P substitutions do not substantially increase (or essentially decrease) the binding affinity of the IL-2 agent to CD25, but rather stabilize the IL-2 agent in an active conformation sufficient for binding to CD25. Without wishing to be bound by theory, in one embodiment, the IL-2 agents containing the aforementioned mutations have reduced binding affinity to CD122 and / or CD132, which is also believed to increase the potency and selectivity of the IL-2 agents for regulatory T cells (Tregs) compared to other T cell types. Thus, the IL-2 agents containing these mutations are typically stable and selectively activate regulatory T cells (Tregs). Without wishing to be bound by theory, in one embodiment, the IL-2 agents containing the aforementioned mutations have reduced or decreased binding ability and / or binding affinity to CD25, which is further believed to improve the longevity of the IL-2 agents. Without wishing to be bound by theory, in one embodiment, the IL-2 agents containing these mutations are also believed to not substantially promote the expansion, activity, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo. In one embodiment, IL-2 agents containing the H16L mutation have reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Tregs over other T cell types, compared to IL-2 agents containing other H16 mutations. These properties make IL-2 agents containing the aforementioned mutations particularly suitable for the treatment of disorders and conditions resulting from aberrant immune responses, such as autoimmune diseases.

[0143] Thus, in one embodiment, an IL-2 agent (e.g., an IL-2 variant or IL-2 fusion protein) comprising an amino acid substitution at position H16 (e.g., H16L, V69A, Q74P and C125S) in combination with amino acid substitutions at positions V69, Q74 and C125 has one or more (e.g., 2, 3, 4, 5, 6, 7 or all) of the following properties, particularly compared to wild-type IL-2 or a reference IL-2 agent that does not comprise the amino acid substitution: (i) enhanced or increased stability in vitro or in vivo, (ii) binding ability and / or binding to human CD122 in vitro and / or in vivo. (iii) reduced or decreased binding capacity and / or binding affinity to human CD132 in vitro and / or in vivo; (iv) reduced or decreased affinity of the IL-2 agent to the heterodimeric IL-2 receptor composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) reduced or decreased (e.g., moderately reduced or decreased) binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo; (vi) reduced or decreased binding capacity and / or binding affinity to regulatory T cells (e.g., Foxp3 + (vii) selective activation of the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote Treg expansion, activity, survival and / or proliferation.

[0144] definition As used herein, the articles "a" and "an" refer to one or to more than one (eg, to at least one) of the grammatical object of the article.

[0145] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0146] "About" and "approximately" are generally intended to mean an acceptable degree of error for a quantity measured given the nature or precision of the measurement. An exemplary degree of error is within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values. When "about" or "approximately" is present before a series of numbers or ranges, it is understood that the "about" or "approximately" can modify each of the numbers in the series or range. Similarly, when "at least," "more than," "no more than," "less than," "no less than," or "within" is present before a series of numbers or ranges, it is understood that the "at least," "more than," "no more than," "less than," "no less than," or "within" can modify each of the numbers in the series or range. As used herein, a range includes both upper and lower limits.

[0147] The compositions and methods disclosed herein encompass polypeptides and nucleic acids having the identified sequences, or sequences substantially identical or similar thereto, e.g., sequences at least 85%, 90%, 95% identical or more to the identified sequences.

[0148] In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid sequence that contains a sufficient or minimal number of amino acid residues that are i) identical or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences may have a common structural domain and / or a common functional activity, such as an amino acid sequence that contains a common structural domain that has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence, e.g., a sequence provided herein.

[0149] In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or a minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having a common functional activity or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, a nucleotide sequence having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence, e.g., a sequence provided herein.

[0150] The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as a naturally occurring sequence, or is encoded by substantially the same nucleotide sequence, and is capable of one or more activities of the naturally occurring sequence.

[0151] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[0152] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In a typical embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, e.g., at least 40%, 50%, 60%, e.g., at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0153] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0154] Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. In one embodiment, percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated in the GAP program of the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In a particular embodiment, percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package (available at www.gcg.com), using a NWSgapdna.CMP matrix, and a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. One preferred set of parameters (and those to be used unless otherwise specified) is a Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0155] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0156] The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform searches against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acids described herein. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used, as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0157] As used herein, the term "hybridize under low stringency, medium stringency, high stringency, or very high stringency conditions" describes the conditions of hybridization and washing. Guidance for carrying out hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley&Sons, NY (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and non-aqueous methods are described in the references, and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes with 0.2X SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the temperature of the washes can be increased to 55°C); 2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes with 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes with 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) very high stringency hybridization conditions are 0.5 M sodium phosphate with 7% SDS at 65°C, followed by one or more washes with 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions 4) are appropriate conditions and are the conditions that should be used unless otherwise specified.

[0158] It is understood that the molecules described herein may have additional conservative or non-essential amino acid substitutions, which have no substantial effect on their function.

[0159] The term "amino acid" is intended to include all molecules, whether natural or synthetic, that contain both amino and acid functional groups and can be included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives, and congeners; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes both D- or L-optical isomers and peptidomimetics.

[0160] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0161] The terms "polypeptide," "peptide," and "protein" (when single chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses modified amino acid polymers, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides may be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or may be the product of synthetic procedures.

[0162] As will be appreciated by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of the present invention.For example, any protein fragment (meaning a polypeptide sequence that is at least one amino acid residue shorter than the reference polypeptide sequence but otherwise identical) of a reference protein that is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or more than 100 amino acids long is provided herein.In another example, any protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids that is about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100% identical to any sequence described herein can be utilized according to the present invention. In one embodiment, a protein sequence utilized in accordance with this disclosure contains 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations as set forth in any of the sequences provided or referenced herein.

[0163] The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence", or "polynucleotide sequence", and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may be either single-stranded or double-stranded, and if single stranded, may be the coding strand or the non-coding (antisense) strand. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. A nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin that does not occur in nature or is linked to another polynucleotide in a non-natural configuration.

[0164] As used herein, the term "isolated" refers to a material that is removed from its original or natural environment (e.g., the natural environment, if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but an identical polynucleotide or polypeptide that has been separated by human intervention from some or all of the coexisting materials of the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and still be isolated in that it is not part of the environment in which such a vector or composition is found in nature.

[0165] As used herein, the term "treatment" of a disorder, e.g., myeloma, means that a subject (e.g., a human) having a disorder, e.g., myeloma, and / or experiencing symptoms of a disorder, e.g., myeloma, will, in one embodiment, experience less severe symptoms and / or recover more quickly when the antibody molecule is administered than when the antibody molecule is not administered. In one embodiment, when myeloma is treated, a bone marrow biopsy will show fewer clonal plasma cells after effective treatment for myeloma. For example, a diagnostic assay will detect fewer clonal plasma cells in a subject's biological sample after administration of an antibody molecule described herein for effective treatment of myeloma. Other assays, urine tests, or blood tests can also be used to monitor treatment in a patient or detect the presence, e.g., reduced presence (or absence), of symptoms of myeloma after treatment of myeloma in a subject. In one embodiment, when myeloma is treated, the level of β2 microglobulin (β2M) in serum or urine will be reduced after effective treatment for myeloma. Treatment can, for example, partially or completely, alleviate, ameliorate, mitigate, inhibit, or reduce the severity and / or incidence of, and optionally delay the onset of, one or more of the effects or symptoms, characteristics, and / or causes of a disorder, such as, for example, myeloma. In one embodiment, treatment is treatment of a subject who does not show specific signs of a disorder, such as, for example, myeloma, and / or who shows only early signs of a disorder, such as, for example, nephropathy. In one embodiment, treatment is treatment of a subject who shows one or more established signs of a disorder, such as, for example, myeloma. In one embodiment, treatment is treatment of a subject who has been diagnosed with a disorder, such as, for example, myeloma.

[0166] As used herein, the term "preventing" a disorder, such as, for example, myeloma, means that a subject (e.g., a human) is less likely to have a disorder, such as, for example, myeloma, when receiving the antibody molecule.

[0167] Various aspects of the compositions and methods herein are described in further detail below. Additional definitions are set forth throughout the specification.

[0168] IL-2 agents The present disclosure provides IL-2 agents, including, but not limited to, IL-2 variants, IL-2 fusion proteins, IL-2 complexes, and IL-2 conjugates. For example, the IL-2 agents described herein can have one or more structural and / or functional properties described herein. In one embodiment, the IL-2 agent includes an IL-2 variant comprising one or more amino acid modifications (e.g., substitutions) described herein. In one embodiment, the IL-2 agent includes an IL-2 variant comprising one or more amino acid modifications (e.g., substitutions) described in Table 9. In one embodiment, the IL-2 agent includes an IL-2 variant comprising an amino acid sequence described in Table 9, or a portion thereof. In one embodiment, the IL-2 agent, or a portion thereof, is encoded by a nucleic acid comprising a nucleotide sequence described herein, e.g., Table 10. The one or more amino acid modifications (e.g., substitutions), alone or in combination, can confer one or more desired biological properties described herein. In one embodiment, the IL-2 agent can modulate (e.g., increase) the proliferation, survival, activity, and / or function of Tregs. In one embodiment, the modulation is selective or specific for Tregs. For example, an IL-2 agent can regulate activity in Tregs, but has limited or no ability to promote activity in non-regulatory T cells. In one embodiment, the IL-2 agent comprises a polypeptide (also referred to herein as an "IL-2 polypeptide agent").

[0169] IL-2 variants In one embodiment, the IL-2 agent comprises an IL-2 variant, eg, an IL-2 variant described herein.

[0170] In one embodiment, the IL-2 variant comprises an IL-2 polypeptide described herein (e.g., a human IL-2 polypeptide), or a functional fragment thereof. In one embodiment, the IL-2 variant comprises one or more amino acid modifications (e.g., substitutions) described in Table 9. In one embodiment, the IL-2 variant comprises or consists of an amino acid sequence described in Table 9, or a functional fragment thereof. In one embodiment, the IL-2 variant is encoded by a nucleic acid comprising a nucleotide sequence described herein, e.g., in Table 10.

[0171] Without wishing to be bound by theory, in one embodiment, it is believed that IL-2 variants described herein having reduced human CD25 binding affinity and / or reduced human CD122 / CD132 binding affinity compared to wild-type human IL-2 or a reference IL-2 variant may have improved potency and / or selectivity for binding to and activating regulatory T cells (Tregs) over wild-type IL-2 or other IL-2 variants. The IL-2 variants described herein can be identified, for example, by screening a library of mutant IL-2 polypeptides to identify IL-2 variants having binding affinity for human CD25 and / or human CD122 / CD132 in the desired range.

[0172] In one embodiment, the IL-2 variant has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) properties, e.g., different and / or improved properties, described herein, compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid modifications (e.g., substitutions) that provide different and / or improved properties compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all) of the following different and / or improved properties (e.g., as determined by the assays described herein) compared to wild-type IL-2 or a reference IL-2 variant: i) altering (e.g., enhancing or increasing) expression in vitro and / or in vivo; ii) altering (e.g., reducing or decreasing) aggregation in vitro and / or in vivo; iii) altering (e.g., enhancing or increasing) stability in vitro and / or in vivo; iv) modifying (e.g., enhancing or increasing) half-life in vitro and / or in vivo; v) altering (e.g., reducing or decreasing) turnover and / or clearance in vivo; vi) altering (e.g., reducing or decreasing) susceptibility to proteolysis in vitro and / or in vivo; vii) altered (e.g., enhanced or increased) resistance to proteolysis in vitro and / or in vivo; viii) altering (e.g., reducing or decreasing) the binding ability and / or binding affinity to human CD25 in vitro and / or in vivo; ix) alteration (e.g., reduction or decrease) of binding ability and / or binding affinity to human CD132 in vitro and / or in vivo; x) altering (e.g., reducing or decreasing) the binding ability and / or binding affinity to the dimeric IL-2 receptor comprising human CD122 and human CD132 in vitro and / or in vivo; xi) altering (e.g., enhancing, increasing, decreasing, diminishing, and / or selectively) binding to Tregs in vitro and / or in vivo; xii) modifying (e.g., enhancing, increasing, reducing, diminishing, and / or selectively) the activity of the IL-2 signaling pathway in Tregs in vitro and / or in vivo; xiii) Altering (e.g., enhancing, increasing, reducing, diminishing, and / or selectively) the ability to induce or promote expansion, activity, survival, and / or proliferation of Tregs in vitro and / or in vivo.

[0173] In one embodiment, the IL-2 variant has altered (e.g., enhanced or increased) expression in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has enhanced or increased expression (e.g., in bacterial or mammalian cells) compared to wild-type IL-2. In one embodiment, the IL-2 variant has enhanced or increased expression (e.g., in bacterial or mammalian cells) compared to a reference IL-2 variant. In one embodiment, expression of the IL-2 variant is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, expression of the IL-2 variant is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, the IL-2 variant is expressed in vitro and / or in vivo at a higher or increased level, such as, for example, an increase of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, compared to an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, for example, by an assay of protein concentration. In one embodiment, the IL-2 variant is expressed at a higher or increased level, such as, for example, about a 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more increase, compared to an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, for example, by an assay of protein concentration.

[0174] In one embodiment, the IL-2 variant has altered (e.g., reduced or decreased) aggregation in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has reduced or decreased aggregation compared to wild-type IL-2. In one embodiment, the IL-2 variant has reduced or decreased aggregation compared to a reference IL-2 variant. In one embodiment, aggregation of the IL-2 variant is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, aggregation of the IL-2 variant is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, an IL-2 agent comprising an IL-2 variant described herein aggregates in vitro and / or in vivo at a lower or reduced level, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more reduction, as compared to an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, e.g., by melting temperature analysis (e.g., using fluorimetry), dynamic light scattering, and / or size exclusion chromatography. In one embodiment, an IL-2 agent comprising an IL-2 variant described herein aggregates at a lower or reduced level, e.g., about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction, as compared to an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, e.g., by melting temperature analysis (e.g., using fluorimetry), dynamic light scattering, and / or size exclusion chromatography.

[0175] In one embodiment, the IL-2 variant has altered (e.g., enhanced or increased) stability in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has enhanced or increased stability compared to wild-type IL-2. In one embodiment, the IL-2 variant has enhanced or increased stability compared to a reference IL-2 variant. In one embodiment, the stability of the IL-2 variant is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the stability of the IL-2 variant is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, an IL-2 agent comprising an IL-2 variant described herein exhibits, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 1500%, about 2000%, about 3500%, about 4000%, about 5000%, about 10000%, about 20000%, about 3500%, about 4000%, about 5000%, about 6000%, about 7000%, about 8000%, about 9000%, about 10000%, about 15000%, about 20 ...0000%, about and / or have enhanced or increased stability in vitro and / or in vivo, such as an increase of about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or, for example, an increase of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more.

[0176] In one embodiment, the IL-2 variant has an altered (e.g., enhanced or increased) half-life in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has an enhanced or increased half-life compared to wild-type IL-2. In one embodiment, the IL-2 variant has an enhanced or increased half-life compared to a reference IL-2 variant. In one embodiment, the half-life of the IL-2 variant is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the half-life of the IL-2 variant is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, an IL-2 agent comprising an IL-2 variant described herein exhibits, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55% reduction in IL-2 activity, e.g., as determined by ELISA, flow cytometry and / or mass spectrometry, compared to an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant. , about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more increase, or for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more or the like.

[0177] In one embodiment, the IL-2 variant has altered (e.g., reduced or decreased) turnover in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has reduced or decreased turnover compared to wild-type IL-2. In one embodiment, the IL-2 variant has reduced or decreased turnover compared to a reference IL-2 variant. In one embodiment, the turnover of the IL-2 variant is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the turnover of the IL-2 variant is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, an IL-2 agent comprising an IL-2 variant described herein exhibits, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 400%, about 400%, about 45 ... %, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more reduction, or lower, reduced or decreased rate or level of turnover and / or clearance in vivo, such as, for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction.

[0178] In one embodiment, the IL-2 has an altered (e.g., reduced or decreased) susceptibility to proteolysis in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has a reduced or decreased susceptibility to proteolysis compared to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 variant has a reduced or decreased susceptibility to proteolysis compared to a reference IL-2 variant. In one embodiment, the susceptibility of the IL-2 variant to proteolysis is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the susceptibility of the IL-2 variant to proteolysis is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0179] In one embodiment, the IL-2 variant has an altered (e.g., enhanced or increased) resistance to proteolysis in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has an enhanced or increased resistance to proteolysis compared to wild-type IL-2. In one embodiment, the IL-2 variant has an enhanced or increased resistance to proteolysis compared to a reference IL-2 variant. In one embodiment, the resistance of the IL-2 variant to proteolysis is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the resistance of the IL-2 variant to proteolysis is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0180] In one embodiment, the IL-2 variant has an altered (e.g., reduced or decreased) binding ability and / or binding affinity to human CD25 in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has a reduced or decreased binding ability and / or binding affinity to human CD25 compared to wild-type human IL-2. In one embodiment, the IL-2 variant has a reduced or decreased binding ability and / or binding affinity to human CD25 compared to a reference IL-2 variant. In one embodiment, the binding ability and / or binding affinity of the IL-2 variant to human CD25 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the binding ability and / or binding affinity of an IL-2 variant to human CD25 is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, an IL-2 agent comprising an IL-2 variant described herein exhibits a reduced binding ability and / or binding affinity of, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 120%, about 140%, about 160%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 500%, about 500%, about 500%, about 600%, about 700%, about 750%, about 800%, about 900%, about 900%, about 1000%, about 1100%, about 1200%, about 1300%, about 1400%, and have a reduced or decreased binding affinity for CD25 (e.g., human CD25), such as a 5%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more decrease, or for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more decrease.

[0181] In one embodiment, the IL-2 variant has a dissociation constant (K) of, e.g., about 5-500 pM, as determined, e.g., by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding). D ), e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, 250, about 300, about 350, about 400, about 450, or about 500 pM, or, for example, about 10 to about 400 pM, about 20 to about 300 pM, about 50 to about 200 pM, about 100 to about 150 pM, about 5 to about 10 pM, for example, about 10 to about 20 pM, about 20 to about 30 pM, or about 30 to about 40 pM, for example, about 40 to about 50 pM, about 50 to about 60 pM, about 60 to about 70 pM, about 70 to about 80 pM, about 80 to about 90 pM, about 90 to about 100 pM, About 100 to about 110 pM, about 110 to about 120 pM, about 120 to about 130 pM, about 130 to about 140 pM, about 140 to about 150 pM, about 150 to about 200 pM, about 200 to about 250 pM, about 250 to about 300 pM, about 300 to about 350 pM, about 350 to about 400 pM, about 400 to about 500 pM, or, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, It binds to CD25 (e.g., human CD25) with low affinity, such as greater than about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 pM.

[0182] In one embodiment, the IL-2 variant has a dissociation constant (K) of, e.g., about 0.1-10 nM, as determined, e.g., by surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding). D), for example, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 6, about 7, about 8, about 9, or about 10 nM, or for example, about 0.2 to about 5 nM, about 0.5 to about 2 nM, about 1 to 1.5 nM, about 0.1 to about 0.2 nM, for example, about 0.2 to about 0.3 nM, about 0.3 to about 0.4 nM, or about 0.4 to about 0.5 nM, for example, about 0.5 to about 0.6 nM, about 0.6 to about 0.7 nM, about 0.7 to about 0.8 nM, about 0. It binds to CD25 (e.g., human CD25) with low affinity, such as about 8 to about 0.9 nM, about 0.9 to about 1 nM, about 1 to about 1.5 nM, about 1.5 to about 2 nM, about 2.5 to about 3 nM, about 3.5 to about 4 nM, about 4 to about 4.5 nM, about 4.5 to about 5 nM, about 5 to about 6 nM, about 6 to about 7 nM, about 7 to about 8 nM, about 8 to about 9 nM, or about 9 to about 10 nM, or for example, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or more than about 10 nM.

[0183] In one embodiment, the IL-2 variant has an altered (e.g., reduced or decreased) binding ability and / or binding affinity to human CD132 in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has a reduced or decreased binding ability and / or binding affinity to human CD132 compared to wild-type IL-2. In one embodiment, the IL-2 variant has a reduced or decreased binding ability and / or binding affinity to human CD132 compared to a reference IL-2 variant. In one embodiment, the binding ability and / or binding affinity of the IL-2 variant to human CD132 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the binding ability and / or binding affinity of the IL-2 variant to human CD132 is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0184] In one embodiment, the IL-2 variant has an altered (e.g., reduced or decreased) binding capacity and / or binding affinity to the human dimeric IL-2 receptor comprising human CD122 and human CD132 in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has a reduced or decreased binding capacity and / or binding affinity to the human dimeric IL-2 receptor comprising human CD122 and human CD132 compared to wild-type IL-2. In one embodiment, the IL-2 variant has a reduced or decreased binding capacity and / or binding affinity to the human dimeric IL-2 receptor comprising human CD122 and human CD132 compared to a reference IL-2 variant. In one embodiment, the binding ability and / or binding affinity of the IL-2 variant to a human dimeric IL-2 receptor comprising human CD122 and human CD132 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the binding ability and / or binding affinity of the IL-2 variant to a human dimeric IL-2 receptor comprising human CD122 and human CD132 is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0185] In one embodiment, the IL-2 variant exhibits, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 400%, about 45 ... %, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more reduction, or for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction in binding affinity to the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer).

[0186] In one embodiment, the IL-2 variant has a dissociation constant (KD) of, e.g., about 0.2 to 20 nM, e.g., about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 20 nM, or, for example, about 0.5 to about 15 nM, about 1 to about 10 nM, about 2 to about 5 nM, about 0.2 to about 0.3 nM, about 0.3 to about 0.4 nM, about 0.4 to about 0.5 nM, about 0.5 to about 0.6 nM, about 0.6 to about 0.7 nM, about 0.7 to about 0.8 nM, about 0.8 to about 0.9 nM, about 0.9 to about 1 nM, about 1 to about 1.1 nM, about 1.1 to about 1.2 nM, about 1.2 to about 1.3 nM, about 1.3 to about 1.4 nM, about 1. 4 to about 1.5 nM, about 1.5 to about 2 nM, about 2 to about 3 nM, about 3 to about 4 nM, about 4 to about 5 nM, about 5 to about 6 nM, about 6 to about 7 nM, about 7 to about 8 nM, about 8 to about 9 nM, about 9 to about 10 nM, about 10 to about 11 nM, about 11 to about 12 nM, about 12 to about 13 nM, about 13 to about 14 nM, about 14 to about 15 nM, about 15 to about 16 nM, about 16 to about 17 nM, about 17 to about 18 nM, about 18 to about 19 nM, or about 19 to about 20 nM, or for example, It binds to a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with low affinity, such as about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or greater than about 20 nM.

[0187] In one embodiment, the IL-2 variant has a dissociation constant (KD) of, e.g., about 0.2 to 300 nM, e.g., about 0.2 nM, about 0.5 nM, about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, about 300 nM, about 310 nM, about 320 nM, about 330 nM, about 340 nM, about 350 nM, about 360 nM, about 370 nM, about 380 nM, about 390 nM, about 40 ... nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, or about 300 nM, or for example, about 0.5 to about 15 nM, about 1 to about 10 nM, about 2 to about 5 nM, about 0.2 nM to about 0.5 nM, about 0.5 nM to about 1 nM, about 1 to about 2 nM, about 2 nM to about 5 nM, Approximately 5nM to approximately 10nM, approximately 10nM to approximately 15nM, approximately 15nM to approximately 20nM, approximately 20nM to approximately 25nM, approximately 25 to approximately 30nM, approximately 30nM to approximately 40nM, approximately 40nM to approximately 50nM, approximately 50 to approximately 60nM, approximately 60 to approximately 70nM, approximately 70nM to approximately 8 0nM, approximately 80nM to approximately 90nM, approximately 90nM to approximately 100nM, approximately 100nM to approximately 110nM, approximately 110nM to approximately 120nM, approximately 120nM to approximately 130nM, approximately 130nM to approximately 140nM, approximately 140nM to approximately 150nM, approximately 150nM to approximately 160 nM, about 160 nM to about 170 nM, about 170 nM to about 180 nM, about 180 nM to about 190 nM, about 190 nM to about 200 nM, about 200 nM to about 210 nM, about 210 nM to about 220 nM, about 220 nM to about 230 nM, about 230 nM to about 240 nM, about 240 nM to about 250 nM, about 250 nM to about 260 nM, about 260 nM to about 270 nM, about 270 nM to about 280 nM, about 280 nM to about 290 nM, or about 290 nM to about 300 nM, or for example, greater than about 0.2, about 0.5, about 1, about 2, about 5, about 10, about 15, about 20nM, about 25nM, about 30nM, about 40nM, about 50nM, about 60nM, about 70nM, about 80nM, about 90 nM, approximately 100nM, approximately 110nM, approximately 120nM, approximately 130nM, approximately 140nM, approximately 150nM, approximately 160nM, approximately 170nM, approximately 180nM, approximately 190 The antibody binds to the CD122 / CD132 heterodimer (e.g., the human CD122 / CD132 heterodimer) with low affinity, such as greater than about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, or about 300 nM.

[0188] In one embodiment, the IL-2 variant has altered (e.g., enhanced, increased and / or selective) binding to Tregs in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has enhanced or increased binding to Tregs compared to wild-type IL-2. In one embodiment, the IL-2 variant has selective binding to Tregs compared to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 variant has enhanced or increased binding to Tregs compared to a reference IL-2 variant. In one embodiment, the IL-2 variant has selective binding to Tregs compared to a reference IL-2 variant. In one embodiment, binding to Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, binding to Tregs is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0189] In one embodiment, the IL-2 variant has altered (e.g., enhanced, increased and / or selective) activation of the IL-2 signaling pathway in Tregs in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has enhanced or increased activation of the IL-2 signaling pathway in Tregs compared to wild-type IL-2. In one embodiment, the IL-2 variant has selective activation of the IL-2 signaling pathway in Tregs compared to wild-type IL-2. In one embodiment, the IL-2 variant has enhanced or increased activation of the IL-2 signaling pathway in Tregs compared to a reference IL-2 variant. In one embodiment, the IL-2 variant has selective activation of the IL-2 signaling pathway in Tregs compared to a reference IL-2 variant. In one embodiment, activation of the IL-2 signaling pathway in Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, activation of the IL-2 signaling pathway in Tregs is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0190] In one embodiment, the IL-2 variant has a IL-2 activity greater than or equal to about 1, about 2, about 3, about 4, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or about 3000 or more to selectively activate IL-2 signaling in T regulatory cells in vitro and / or in vivo.

[0191] In one embodiment, the IL-2 variant has a IL-2 activity greater than or equal to about 1, about 2, about 3, about 4, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3500, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 30 ... 500, or about 3000 or more, or for example, greater than 1 and about 1-2, about 2-3, about 3-4, about 4-5, greater than 1 and about 1-10, greater than 1 and about 1-20, greater than 1 and about 1-30, greater than 1 and about 1-40, greater than 1 and about 1-50, about 2-10, about 2-20, about 2-30, about 2-40, 2-50, about 5-10, about 5 and selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, having an NK cell EC50 / Treg EC50 ratio of about 20, about 5-30, about 5-40, about 5-50, about 10-20, about 10-30, about 10-40, about 10-50, about 20-40, about 20-50, about 50-100, about 100-200, about 200-500, about 500-1000, about 1000-2000, or about 1000-3000.

[0192] In one embodiment, the IL-2 variant has an altered (e.g., enhanced, increased and / or selective) ability to induce or promote expansion, activity, survival and / or proliferation of Tregs in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has an enhanced or increased ability to induce or promote expansion, activity, survival and / or proliferation of Tregs compared to wild-type IL-2. In one embodiment, the IL-2 variant has a selective ability to induce or promote expansion, activity, survival and / or proliferation of Tregs compared to wild-type IL-2. In one embodiment, the IL-2 variant has an enhanced or increased ability to induce or promote expansion, activity, survival and / or proliferation of Tregs compared to a reference IL-2 variant. In one embodiment, the IL-2 variant has a selective ability to induce or promote expansion, activity, survival and / or proliferation of Tregs compared to a reference IL-2 variant. In one embodiment, the ability to induce or promote expansion, activity, survival, and / or proliferation of Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the ability to induce or promote expansion, activity, survival, and / or proliferation of Tregs is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0193] In one embodiment, the IL-2 variant exhibits, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7% or about 8% of the IL-2 variant, as compared to, e.g., an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, e.g., by flow cytometry. , about 90%, about 95%, about 100% or more lower, or for example, about 0.5 fold, about 1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 5.5 fold, about 6 fold, about 6.5 fold, about 7 fold, about 7.5 fold, about 8 fold, about 8.5 fold, about 9 fold, about 9.5 fold, about 10 fold or more reduced, and have potency and / or ability to induce or promote enhanced or increased T regulatory cell activity.

[0194] In one embodiment, the IL-2 variant is, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% or more higher, or, e.g., about 0.5 fold, about 1 fold, or more higher, as determined, e.g., by flow cytometry, compared to an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant. fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 50-fold, about 100-fold, about 200-fold, about 500-fold, about 1000-fold, about 2000-fold, about 5000-fold, about 10,000-fold, about 15,000-fold, or about 20,000-fold or more reduction, and have a reduced or diminished potency and / or ability to induce or promote T regulatory cell activity.

[0195] In one embodiment, the T helper cells described herein are CD45+CD3+CD4+Foxp3- cells, e.g., as determined by flow cytometry. In one embodiment, the Tregs described herein are CD45+CD3+CD4+Foxp3+ cells, e.g., as determined by flow cytometry. In one embodiment, the NK cells described herein are CD45+CD3- cells that are CD56+ and / or CD16+, e.g., as determined by flow cytometry. In one embodiment, the NK cells described herein are CD45+CD3-CD56+ cells, e.g., as determined by flow cytometry.

[0196] In one embodiment, an IL-2 variant has one or more of the same, or substantially the same, structural and / or functional properties as wild-type IL-2 or a reference IL-2 variant.

[0197] In one embodiment, the reference IL-2 variant comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an IL-2 variant described herein. In one embodiment, the reference IL-2 variant comprises the amino acid sequence of SEQ ID NO:1 (IL-2 C125S). In one embodiment, the IL-2 variant comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:1 and includes one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid modifications (e.g., substitutions) described herein.

[0198] For purposes of this disclosure, IL-2 variant position numbering begins with the first amino acid following the signal peptide of an exemplary wild-type (WT) human IL-2 polypeptide: [ka] [ka] The corresponding amino acid sequence without the signal peptide is shown in SEQ ID NO:1031.

[0199] In one embodiment, the IL-2 agent contains amino acid modifications (eg, substitutions) at positions corresponding to human IL-2 (eg, contains the amino acid sequence of SEQ ID NO: 1031).

[0200] In one embodiment, the IL-2 variant is A1 / P2 / X3 / S4 / S5 / S6 / T7 / K8 / K9 / T10 / Q11 / L12 / Q13 / L14 / E15 / X16 / L17 / L18 / L19 / D20 / L21 / Q22 / M23 / I24 / L25 / N26 / G27 / X28 / N29 / N30 / Y31 / K32 / N33 / P34 / X35 / L36 / T37 / X38 / M39 / L40 / T41 / X42 / K43 / F44 / Y45 / M46 / P47 / K48 / K49 / A50 / T51 / E52 / L53 / K 54 / H55 / L56 / Q57 / C58 / L59 / E60 / E61 / E62 / L63 / K64 / P65 / L66 / E67 / X68 / X69 / L70 / N71 / L72 / A73 / X74 / S75 / K76 / N77 / F78 / H79 / L80 / R81 / P82 / R83 / X84 / L85 / I86 / X87 / X88 / I89 / N90 / V9 1 / X92 / V93 / L94 / E95 / L96 / K97 / G98 / S99 / E100 / T101 / T102 / F103 / M104 / C105 / E106 / Y107 / A 108 / D109 / E110 / T111 / A112 / T113 / I114 / V115 / E116 / F117 / L118 / N119 / R120 / W121 / I122 / T123 / F124 / X125 / X126 / S127 / I128 / I129 / S130 / T131 / L132 / T133 (SEQ ID NO: 1032), wherein X3 is T or A;16 is H, L or N;X28 is I, T or F;X35 is K or E;X38 is R, E, N or Q;X42 is F, A, K or Q;X68 is E, Q or N;X69 is V or A;X74 is Q or P;X84 is D or V;X87 is S or R;X88 is N, D, L or S;X92 is I or S;X125 is C or S; and X126 is Q, K, R or T, but the IL-2 variant does not comprise the amino acid sequence of SEQ ID NO: 1 or 1031. In one embodiment, the IL-2 variant comprises or consists of an IL-2 variant amino acid sequence described herein.

[0201] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) of the positions described herein. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) of the positions selected from T3, H16, I28, K35, R38, F42, E68, V69, Q74, D84, S87, N88, I92, C125, or Q126.

[0202] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position T3. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position H16. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position I28. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position K35. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position R38. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position F42. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position E68. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position V69. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position Q74. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position D84. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position S87. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position N88. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position I92. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position C125. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position Q126.

[0203] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position V69, Q74, or a combination thereof. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at positions V69 and Q74. In one embodiment, the IL-2 variant comprises the amino acid substitution V69A. In one embodiment, the IL-2 variant comprises the amino acid substitution Q74P.

[0204] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position H16, I92, D84, or a combination thereof. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position H16, optionally the amino acid substitution is H16N, H16L, or H16D. In one embodiment, the IL-2 variant comprises the amino acid substitution H16N. In one embodiment, the IL-2 variant comprises the amino acid substitution H16L. In one embodiment, the IL-2 variant comprises the amino acid substitution H16D.

[0205] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position 192, optionally the amino acid substitution is 192S. In one embodiment, the IL-2 variant comprises the amino acid substitution 192S.

[0206] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position D84, optionally the amino acid substitution is D84V. In one embodiment, the IL-2 variant comprises the amino acid substitution D84V.

[0207] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position K35, R38, F42, E68, or a combination thereof. In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position K35, optionally the amino acid substitution is K35E. In one embodiment, the IL-2 variant comprises the amino acid substitution K35E.

[0208] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position R38, optionally the amino acid substitution is R38E, R38N or R38Q. In one embodiment, the IL-2 variant comprises the amino acid substitution R38N. In one embodiment, the IL-2 variant comprises the amino acid substitution R38Q.

[0209] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position F42, optionally the amino acid substitution is F42K or F42Q. In one embodiment, the IL-2 variant comprises the amino acid substitution F42K. In one embodiment, the IL-2 variant comprises the amino acid substitution F42Q.

[0210] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at one, two, or all of: (i) (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35, and (ii) positions H16, I92, or D84. In one embodiment, the IL-2 variant further comprises an amino acid modification (e.g., substitution) at one, two, or all of positions R38, F42, or E68.

[0211] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at: (i) (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35, and (ii) (a) one, two, or all of positions H16, I92, or D84, or (b) one, two, or all of positions R38, F42, or E68.

[0212] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at: (i) (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35, and (ii) (a) one, two, or all of positions H16, I92, or D84, and (b) one, two, or all of positions R38, F42, or E68.

[0213] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and H16, optionally the amino acid substitutions are V69A, Q74P, and H16N or H16L, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, and H16N or H16L. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, and H16N. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, and H16N. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, and H16L.

[0214] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and I92, optionally the amino acid substitutions are V69A, Q74P, and I92S, respectively. In one embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and I92S.

[0215] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and D84, optionally the amino acid substitutions are V69A, Q74P, and D84V, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, and D84V.

[0216] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and R38, optionally the amino acid substitutions are V69A, Q74P, and R38Q, respectively. In one embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and R38Q.

[0217] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and F42, and optionally the amino acid substitutions are V69A, Q74P, and F42Q, respectively. In one embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and F42Q.

[0218] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and R38, optionally the amino acid substitutions are V69A, Q74P, and R38N, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, and R38N.

[0219] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and R38, optionally the amino acid substitutions are V69A, Q74P, and R38E, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, and R38E.

[0220] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, K35, and H16, optionally the amino acid substitutions are V69A, Q74P, K35E, and H16N, respectively. In one embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, K35E, and H16N.

[0221] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, K35, H16, and R38, and optionally the amino acid substitutions are V69A, Q74P, K35E, H16N, and R38N, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, K35E, H16N, and R38N.

[0222] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, H16, and R38, optionally the amino acid substitutions are V69A, Q74P, H16N, and R38N or R38Q, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, H16N, and R38N or R38Q. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, H16N, and R38N. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, H16N, and R38N. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, H16N, and R38Q.

[0223] In one embodiment, the IL-2 variant comprises an amino acid modification (eg, a substitution) at position I28, E68, S87, N88, Q126, or a combination thereof.

[0224] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position I28, optionally the amino acid substitution is I28T or I28F. In one embodiment, the IL-2 variant comprises the amino acid substitution I28T. In one embodiment, the IL-2 variant comprises the amino acid substitution I28F.

[0225] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position E68, optionally the amino acid substitution is E68Q or E68N. In one embodiment, the IL-2 variant comprises the amino acid substitution E68Q. In one embodiment, the IL-2 variant comprises the amino acid substitution E68N.

[0226] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position S87, optionally the amino acid substitution is S87R. In one embodiment, the IL-2 variant comprises the amino acid substitution S87R.

[0227] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position N88, optionally the amino acid substitution is N88S, N88L, or N88D. In one embodiment, the IL-2 variant comprises the amino acid substitution N88S, N88L, or N88D. In one embodiment, the IL-2 variant comprises the amino acid substitution N88S. In one embodiment, the IL-2 variant comprises the amino acid substitution N88L. In one embodiment, the IL-2 variant comprises the amino acid substitution N88D.

[0228] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position Q126, optionally the amino acid substitution is Q126T, Q126K, or Q126R. In one embodiment, the IL-2 variant comprises the amino acid substitution Q126T, Q126K, or Q126R. In one embodiment, the IL-2 variant comprises the amino acid substitution Q126T, Q126K, or Q126R. In one embodiment, the IL-2 variant comprises the amino acid substitution Q126T. In one embodiment, the IL-2 variant comprises the amino acid substitution Q126K. In one embodiment, the IL-2 variant comprises the amino acid substitution Q126R.

[0229] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position C125, optionally the amino acid substitution is C125S. In one embodiment, the IL-2 variant comprises the amino acid substitution C125S.

[0230] In one embodiment, the IL-2 variant comprises an amino acid modification (e.g., substitution) at position T3, optionally the amino acid substitution is T3A. In one embodiment, the IL-2 variant comprises the amino acid substitution T3A.

[0231] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and C125, optionally the amino acid substitutions are V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and C125S.

[0232] In one embodiment, the IL-2 variant comprises an amino acid modification (eg, substitution) at position T3, H16, I92, or a combination thereof, optionally, the amino acid substitutions are T3A, H16N, and I92S, respectively.

[0233] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions H16, V69, Q74, and C125, optionally the amino acid substitutions are H16N, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions H16N, V69A, Q74P, and C125S.

[0234] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions H16, V69, Q74, and C125, optionally the amino acid substitutions are H16L, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions H16L, V69A, Q74P, and C125S.

[0235] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions H16, V69, Q74, I92, and C125, and optionally the amino acid substitutions are H16L, V69A, Q74P, I92S, and C125S, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions H16L, V69A, Q74P, I92S, and C125S.

[0236] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions T3, V69, Q74, and C125, and optionally the amino acid substitutions are T3A, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 variant comprises the amino acid substitutions T3A, V69A, Q74P, and C125S.

[0237] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions T3, H16, V69, Q74, and C125, and optionally the amino acid substitutions are T3A, H16N or H16L, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions T3A, H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 variant comprises amino acid substitutions T3A, H16L, V69A, Q74P, and C125S.

[0238] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions T3, V69, Q74, I92, and C125, and optionally the amino acid substitutions are T3A, V69A, Q74P, I92S, and C125S, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions T3A, V69A, Q74P, I92S, and C125S. In one embodiment, the IL-2 variant comprises amino acid substitutions T3A, V69A, Q74P, I92S, and C125S.

[0239] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions H16, K35, V69 and Q74, optionally the amino acid substitutions are H16L, K35E, V69A, and Q74P, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions H16L, K35E, V69A, and Q74P.

[0240] In one embodiment, the IL-2 variant comprises amino acid modifications (e.g., substitutions) at positions H16, R38, V69A, and Q74P, and optionally the amino acid substitutions are H16L, R38Q, V69A, and Q74P, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions H16L, R38Q, V69A, and Q74P.

[0241] In one embodiment, the IL-2 variant comprises the amino acid substitutions H16L, V69A, Q74P, and C125S. In one embodiment, the IL-2 variant comprises the amino acid substitutions H16N, V69A, Q74P, and C125S.

[0242] There are various technical effects associated with the presence of a particular set of mutations described herein, for example a set of mutations comprising an amino acid substitution at position H16 combined with amino acid substitutions at positions V69, Q74, and C125 (e.g., H16L, V69A, Q74P, and C125S). Without wishing to be bound by theory, in one embodiment, IL-2 variants comprising the aforementioned mutations have reduced binding affinity to CD122 and / or CD132, which is believed to increase the potency and selectivity of the IL-2 variants for regulatory T cells (Tregs) compared to other T cell types. Without wishing to be bound by theory, in one embodiment, IL-2 variants comprising the aforementioned mutations are also believed to be significantly more stable, for example due to the presence of stabilizing V69A and Q74P mutations. For example, it has been unexpectedly discovered that the V69A and Q74P substitutions do not substantially increase the binding affinity of IL-2 variants to CD25, but rather stabilize the IL-2 variants in an active conformation sufficient for binding to CD25. Thus, IL-2 variants containing these mutations selectively activate regulatory T cells (Tregs) and are significantly stable. Without wishing to be bound by theory, in one embodiment, it is further believed that the IL-2 variants containing the aforementioned mutations have a reduced or decreased binding ability and / or binding affinity to CD25, which improves the lifespan of the IL-2 variants. Without wishing to be bound by theory, in one embodiment, it is believed that the IL-2 variants containing these mutations do not substantially promote the expansion, activity, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo. Without wishing to be bound by theory, in one embodiment, it is further believed that IL-2 variants comprising the aforementioned mutations have reduced imprecise disulfide pairing and improved stability, e.g., due to the presence of the C125S mutation. In one embodiment, IL-2 agents comprising the H16L mutation have reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Tregs over other T cell types, compared to IL-2 agents comprising other H16 mutations.These properties make IL-2 variants containing these mutations particularly suitable for the treatment of disorders and conditions resulting from an aberrant immune response, such as autoimmune diseases.

[0243] Thus, in one embodiment, an IL-2 variant (e.g., an IL-2 variant or IL-2 fusion protein) comprising an amino acid substitution at position H16 (e.g., H16L, V69A, Q74P and C125S) in combination with amino acid substitutions at positions V69, Q74 and C125 has one or more (e.g., 2, 3, 4, 5, 6, 7 or all) of the following properties, particularly compared to a wild-type IL-2 or a reference IL-2 variant that does not comprise the amino acid substitution: (i) enhanced or increased stability in vitro or in vivo, (ii) ability to bind to human CD122 in vitro and / or in vivo. (iii) reduced or decreased binding capacity and / or binding affinity to human CD132 in vitro and / or in vivo; (iv) reduced or decreased affinity of the IL-2 variant to the heterodimeric IL-2 receptor composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) reduced or decreased binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo; (vi) reduced or decreased binding capacity and / or binding affinity to regulatory T cells (e.g., Foxp3 + (vii) selective activation of the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote Treg expansion, activity, survival and / or proliferation.

[0244] In one embodiment, the IL-2 variant is SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or consisting of an amino acid sequence selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:1000, SEQ ID NO:1001, SEQ ID NO:1002, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.

[0245] In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids. In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids. In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids. In one embodiment, the IL-2 variant comprises or consists of an amino acid sequence of SEQ ID NO: 1000, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids. In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 1001, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 1002, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.

[0246] In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of any of SEQ ID NOs: 4, 5, 11, 1000, 1001 or 1002, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 4 or 5, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 5, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 11, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 1000, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 1001, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 1002, or a functional fragment thereof.

[0247] Without wishing to be bound by theory, in one embodiment, an IL-2 variant comprising or consisting of the amino acid sequence of SEQ ID NO:5, or a functional fragment thereof, can have at least one or more of the following advantageous properties: (i) has reduced binding affinity to CD122 and / or CD132, which increases the potency and selectivity of the IL-2 agent for regulatory T cells (Tregs) compared to other T cell types; (ii) is significantly stable, e.g., due to the presence of the stabilizing V69A and Q74P mutations; (iii) has reduced or decreased binding ability and / or binding affinity to CD25, which improves the longevity of the IL-2 agent; (iv) does not substantially promote the expansion, activity, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo, and / or (v) has reduced imprecise disulfide pairing and improved stability, e.g., due to the presence of the C125S mutation. In one embodiment, an IL-2 agent comprising the H16L mutation has reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Tregs over other T cell types, compared to IL-2 agents comprising other H16 mutations. These properties make the IL-2 variant comprising or consisting of the amino acid sequence of SEQ ID NO:5 particularly suitable for the treatment of disorders and conditions resulting from an aberrant immune response, such as autoimmune diseases.

[0248] Thus, in one embodiment, an IL-2 variant comprising or consisting of an amino acid sequence of SEQ ID NO:5 or a functional fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids, has one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties, particularly compared to wild-type IL-2 or a reference IL-2 variant that does not contain amino acid substitutions: (i) enhanced or increased stability in vitro or in vivo; (ii) enhanced or increased stability in vitro or in vivo; (iii) reduced or decreased binding capacity and / or binding affinity to human CD122 in vitro and / or in vivo; (iv) reduced or decreased affinity of the IL-2 variant for the heterodimeric IL-2 receptor composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) reduced or decreased or substantially unchanged binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo; (vi) reduced or decreased or substantially unchanged binding capacity and / or binding affinity to regulatory T cells (e.g., Foxp3 + (vii) selective activation of the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) the ability to induce or promote enhanced or increased Treg expansion, activity, survival and / or proliferation.

[0249] As further described herein, the present disclosure provides IL-2 fusion proteins, IL-2 complexes, and IL-2 conjugates comprising the IL-2 variants described herein. In one embodiment, one or more distinct and / or improved properties of the IL-2 variants described herein are maintained, transferred, or imparted to the IL-2 fusion protein, IL-2 complex, or IL-2. For purposes of this disclosure, the terms "IL-2 variant" and "IL-2 mutein" may be used interchangeably herein.

[0250] In one embodiment, the IL-2 variant comprises a polypeptide (also referred to herein as an "IL-2 variant polypeptide"). The present disclosure provides isolated nucleic acid molecules encoding the IL-2 variants described herein, as well as vectors and host cells thereof. Nucleic acid molecules include, but are not limited to, RNA, genomic DNA, and cDNA.

[0251] IL-2 fusion proteins In one embodiment, the IL-2 agent comprises an IL-2 fusion protein, eg, an IL-2 fusion protein described herein.

[0252] In one embodiment, the IL-2 fusion protein comprises an IL-2 variant, e.g., an IL-2 variant described herein. In one embodiment, the IL-2 fusion protein comprises one or more amino acid modifications (e.g., substitutions) described in Table 9. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence described in Table 9, or a functional fragment thereof. In one embodiment, the IL-2 variant is encoded by a nucleic acid comprising a nucleotide sequence described herein, e.g., in Table 10.

[0253] Without wishing to be bound by theory, in one embodiment, it is believed that the IL-2 fusion proteins described herein have reduced human CD25 binding affinity and / or reduced human CD122 / CD132 binding affinity compared to IL-2 fusion proteins comprising wild-type human IL-2 or a reference IL-2 fusion protein, and may have improved potency and / or selectivity for binding to and activating regulatory T cells (Tregs) over IL-2 fusion proteins comprising wild-type human IL-2 or other IL-2 fusion proteins.

[0254] In one embodiment, the IL-2 fusion protein has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) properties described herein, e.g., different and / or improved properties, as compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid modifications (e.g., substitutions) that provide different and / or improved properties as compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all) properties described herein, e.g., the following different and / or improved properties (e.g., as determined by an assay described herein), as compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein: i) altering (e.g., enhancing or increasing) expression in vitro and / or in vivo; ii) altering (e.g., reducing or decreasing) aggregation in vitro and / or in vivo; iii) altering (e.g., enhancing or increasing) stability in vitro and / or in vivo; iv) modifying (e.g., enhancing or increasing) half-life in vitro and / or in vivo; v) altering (e.g., reducing or decreasing) turnover and / or clearance in vivo; vi) altering (e.g., reducing or decreasing) susceptibility to proteolysis in vitro and / or in vivo; vii) altered (e.g., enhanced or increased) resistance to proteolysis in vitro and / or in vivo; viii) altering (e.g., reducing or decreasing) the binding ability and / or binding affinity to human CD25 in vitro and / or in vivo; ix) alteration (e.g., reduction or decrease) of binding ability and / or binding affinity to human CD132 in vitro and / or in vivo; x) altering (e.g., reducing or decreasing) the binding ability and / or binding affinity to the dimeric IL-2 receptor comprising human CD122 and human CD132 in vitro and / or in vivo; xi) altering (e.g., enhancing, increasing, decreasing, diminishing, and / or selectively) binding to Tregs in vitro and / or in vivo; xii) modifying (e.g., enhancing, increasing, reducing, diminishing, and / or selectively) activation of the IL-2 signaling pathway in Tregs in vitro and / or in vivo; or xiii) Altering (e.g., enhancing, increasing, reducing, diminishing, and / or selectively) the ability to induce or promote expansion, activity, survival, and / or proliferation of Tregs in vitro and / or in vivo.

[0255] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced or increased) expression in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has enhanced or increased expression (e.g., in bacterial or mammalian cells) compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has enhanced or increased expression (e.g., in bacterial or mammalian cells) compared to a reference IL-2 fusion protein. In one embodiment, expression of the IL-2 fusion protein is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, expression of the IL-2 fusion protein is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, the IL-2 fusion protein is expressed in vitro and / or in vivo at a higher or increased level, such as, for example, an increase of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, compared to, for example, an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, as determined, for example, by an assay of protein concentration. In one embodiment, the IL-2 fusion protein is expressed at a higher or increased level, such as, for example, about a 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more increase, compared to, for example, an IL-2 fusion protein comprising a wild-type IL-2 or a reference IL-2 fusion protein, as determined, for example, by an assay of protein concentration.

[0256] In one embodiment, the IL-2 fusion protein has altered (e.g., reduced or decreased) aggregation in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has reduced or decreased aggregation compared to wild-type IL-2. In one embodiment, the IL-2 fusion protein has reduced or decreased aggregation compared to a reference IL-2 fusion protein. In one embodiment, aggregation of the IL-2 fusion protein is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, aggregation of the IL-2 fusion protein is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, the IL-2 fusion protein aggregates in vitro and / or in vivo at a lower or reduced level, such as, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more reduction, e.g., compared to an IL-2 fusion protein comprising a wild-type IL-2 or a reference IL-2 fusion protein, as determined, for example, by melting temperature analysis (e.g., using fluorimetry), dynamic light scattering, and / or size exclusion chromatography. In one embodiment, the IL-2 fusion protein aggregates at a lower or reduced level, such as, for example, about a 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction, compared to, for example, an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, as determined by, for example, melting temperature analysis (e.g., using fluorimetry), dynamic light scattering, and / or size exclusion chromatography.

[0257] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced or increased) stability in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has enhanced or increased stability compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has enhanced or increased stability compared to a reference IL-2 fusion protein. In one embodiment, the stability of the IL-2 fusion protein is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the stability of the IL-2 fusion protein is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, the IL-2 fusion protein exhibits, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, about 590%, about 610%, about 620%, about 630%, about 640%, about 650%, about 660%, about 670%, about 680%, about 690%, about 700%, about 710%, about 720%, about 730%, about 740%, about 7 and / or have enhanced or increased stability in vitro and / or in vivo, such as an increase of 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or, for example, an increase of about 0.5 fold, about 1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 5.5 fold, about 6 fold, about 6.5 fold, about 7 fold, about 7.5 fold, about 8 fold, about 8.5 fold, about 9 fold, about 9.5 fold, about 10 fold or more.

[0258] In one embodiment, the IL-2 fusion protein has an altered (e.g., enhanced or increased) half-life in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has an enhanced or increased half-life compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has an enhanced or increased half-life compared to a reference IL-2 fusion protein. In one embodiment, the half-life of the IL-2 fusion protein is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the half-life of the IL-2 fusion protein is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, the IL-2 fusion protein has, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 400%, about 45 ... An enhanced or increased half-life in vitro and / or in vivo of greater than 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or, for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more.

[0259] In one embodiment, the IL-2 fusion protein has altered (e.g., reduced or decreased) turnover in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has reduced or decreased turnover compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has reduced or decreased turnover compared to a reference IL-2 fusion protein. In one embodiment, the turnover of the IL-2 fusion protein is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the turnover of the IL-2 fusion protein is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, the IL-2 fusion protein exhibits a IL-2 fusion protein that is, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 9 ... have a lower, reduced or decreased rate or level of turnover and / or clearance in vivo, such as about a 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more reduction, or for example, about a 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction.

[0260] In one embodiment, the IL-2 fusion protein provided by the present disclosure comprises the property of altered (e.g., reduced or decreased) susceptibility to proteolysis in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has reduced or decreased susceptibility to proteolysis compared to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 fusion protein has reduced or decreased susceptibility to proteolysis compared to a reference IL-2 fusion protein. In one embodiment, the susceptibility of the IL-2 fusion protein to proteolysis is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the susceptibility of the IL-2 fusion protein to proteolysis is decreased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0261] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced or increased) resistance to proteolysis in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has enhanced or increased resistance to proteolysis compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has enhanced or increased resistance to proteolysis compared to a reference IL-2 fusion protein. In one embodiment, the resistance of the IL-2 fusion protein to proteolysis is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the resistance of the IL-2 fusion protein to proteolysis is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0262] In one embodiment, the IL-2 fusion protein has an altered (e.g., reduced or decreased) binding ability and / or binding affinity to human CD25 in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has a reduced or decreased binding ability and / or binding affinity to human CD25 compared to wild-type human IL-2). In one embodiment, the IL-2 fusion protein has a reduced or decreased binding ability and / or binding affinity to human CD25 compared to a reference IL-2 fusion protein. In one embodiment, the binding ability and / or binding affinity of the IL-2 fusion protein to human CD25 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the binding ability and / or binding affinity of the IL-2 fusion protein to human CD25 is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, the IL-2 fusion protein is reduced by, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, e.g., compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, as determined, e.g., by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding). , about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more reduction, or for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction.

[0263] In one embodiment, the IL-2 fusion protein has a dissociation constant (K) of, e.g., about 5-500 pM, as determined, e.g., by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding). D ), e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, 250, about 300, about 350, about 400, about 450, or about 500 pM, or, for example, about 10 to about 400 pM, about 20 to about 300 pM, about 50 to about 200 pM, about 100 to about 150 pM, about 5 to about 10 pM, for example, about 10 to about 20 pM, about 20 to about 30 pM, or about 30 to about 40 pM, for example, about 40 to about 50 pM, about 50 to about 60 pM, about 60 to about 70 pM, about 70 to about 80 pM, about 80 to about 90 pM, about 90 to about 100 pM, About 100 to about 110 pM, about 110 to about 120 pM, about 120 to about 130 pM, about 130 to about 140 pM, about 140 to about 150 pM, about 150 to about 200 pM, about 200 to about 250 pM, about 250 to about 300 pM, about 300 to about 350 pM, about 350 to about 400 pM, about 400 to about 500 pM, or, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, It binds to CD25 (e.g., human CD25) with low affinity, such as greater than about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 pM.

[0264] In one embodiment, the IL-2 fusion protein has a dissociation constant (K) of, e.g., about 0.1-10 nM, as determined, e.g., by surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding). D), for example, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 6, about 7, about 8, about 9, or about 10 nM, or for example, about 0.2 to about 5 nM, about 0.5 to about 2 nM, about 1 to 1.5 nM, about 0.1 to about 0.2 nM, for example, about 0.2 to about 0.3 nM, about 0.3 to about 0.4 nM, or about 0.4 to about 0.5 nM, for example, about 0.5 to about 0.6 nM, about 0.6 to about 0.7 nM, about 0.7 to about 0.8 nM, about 0. It binds to CD25 (e.g., human CD25) with low affinity, such as greater than about 8 to about 0.9 nM, about 0.9 to about 1 nM, about 1 to about 1.5 nM, about 1.5 to about 2 nM, about 2.5 to about 3 nM, about 3.5 to about 4 nM, about 4 to about 4.5 nM, about 4.5 to about 5 nM, about 5 to about 6 nM, about 6 to about 7 nM, about 7 to about 8 nM, about 8 to about 9 nM, or about 9 to about 10 nM, or for example, greater than about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nM.

[0265] In one embodiment, the IL-2 fusion protein has an altered (e.g., reduced or decreased) binding ability and / or binding affinity to human CD132 in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has a reduced or decreased binding ability and / or binding affinity to human CD132 compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has a reduced or decreased binding ability and / or binding affinity to human CD132 compared to a reference IL-2 fusion protein. In one embodiment, the binding ability and / or binding affinity of the IL-2 fusion protein to human CD132 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the binding ability and / or binding affinity of the IL-2 fusion protein to human CD132 is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0266] In one embodiment, the IL-2 fusion protein has an altered (e.g., reduced or decreased) binding ability and / or binding affinity to a human dimeric IL-2 receptor comprising human CD122 and human CD132 in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has a reduced or decreased binding ability and / or binding affinity to a human dimeric IL-2 receptor comprising human CD122 and human CD132 compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has a reduced or decreased binding ability and / or binding affinity to a human dimeric IL-2 receptor comprising human CD122 and human CD132 compared to a reference IL-2 fusion protein. In one embodiment, the binding ability and / or binding affinity of the IL-2 fusion protein to a human dimeric IL-2 receptor comprising human CD122 and human CD132 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the binding ability and / or binding affinity of the IL-2 fusion protein to a human dimeric IL-2 receptor comprising human CD122 and human CD132 is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0267] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced, increased, and / or selective) binding to Tregs in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has enhanced or increased binding to Tregs compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has selective binding to Tregs compared to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 fusion protein has enhanced or increased binding to Tregs compared to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has selective binding to Tregs compared to a reference IL-2 fusion protein. In one embodiment, binding to Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more, hi one embodiment, binding to Tregs is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0268] In one embodiment, the IL-2 fusion protein exhibits, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 102%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 109%, about 102%, about 105%, about 106%, about 107%, about 108%, about 109%, about 109%, about 109%, about 109%, about 109%, about 102%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109 ... has a reduced or decreased binding affinity for a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer), such as a 5%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more decrease, or for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more decrease.

[0269] In one embodiment, the IL-2 fusion protein has a dissociation constant (K) of, e.g., about 0.2 to 20 nM, as determined, e.g., by yeast surface display. D), for example, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 20 nM, or, for example, about 0.5 to about 15 nM, about 1 to about 10 nM, about 2 to about 5 nM, M, about 0.2 to about 0.3nM, about 0.3 to about 0.4nM, about 0.4 to about 0.5nM, about 0.5 to about 0.6nM, about 0.6 to about 0.7nM, about 0.7 to about 0.8nM, about 0.8 to about 0.9nM, about 0. 9 to about 1nM, about 1 to about 1.1nM, about 1.1 to about 1.2nM, about 1.2 to about 1.3nM, about 1.3 to about 1.4nM, about 1.4 to about 1.5nM, about 1.5 to about 2nM, about 2 to about 3nM, about 3 to about 4n M, about 4 to about 5 nM, about 5 to about 6 nM, about 6 to about 7 nM, about 7 to about 8 nM, about 8 to about 9 nM, about 9 to about 10 nM, about 10 to about 11 nM, about 11 to about 12 nM, about 12 to about 13 nM, about 13 to about 14 nM, about 14 to about 15 nM, about 15 to about 16 nM, about 16 to about 17 nM, about 17 to about 18 nM, about 18 to about 19 nM, or about 19 to about 20 nM, or for example, about 0.2, about 0.3, about 0.4, about It binds to a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with low affinity, such as greater than 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 20 nM.

[0270] In one embodiment, the IL-2 fusion protein has a dissociation constant (K) of, e.g., about 0.2 to 300 nM, as determined, e.g., by surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding). D), for example, about 0.2 nM, about 0.5 nM, about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, or about 300 nM, or for example, about 0.5 to about 15 nM, about 1 to about 10 nM, about 2 to about 5 nM, about 0.2 nM to about 0.5 nM, about 0.5 nM to about 1 nM, about 1 to about 2 nM, about 2 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 15 nM, about 15 nM to about 20 nM, about 20 nM to about 25 nM, about 25 to about 3 0nM, about 30nM to about 40nM, about 40nM to about 50nM, about 50 to about 60nM, about 60 to about 70nM, about 70nM to about 80nM, about 80nM to about 90nM, about 90nM to about 100nM, about 100nM to about 110 nM, about 110nM to about 120nM, about 120nM to about 130nM, about 130nM to about 140nM, about 140nM to about 150nM, about 150nM to about 160nM, about 160nM to about 170nM, about 170nM to about 1 80 nM, about 180 nM to about 190 nM, about 190 nM to about 200 nM, about 200 nM to about 210 nM, about 210 nM to about 220 nM, about 220 nM to about 230 nM, about 230 nM to about 240 nM, about 240 nM to about 250 nM, about 250 nM to about 260 nM, about 260 nM to about 270 nM, about 270 nM to about 280 nM, about 280 nM to about 290 nM, or about 290 nM to about 300 nM, or for example, greater than about 0.2, about 0.5, about 1, about 2, about 5, about 10, about 15, about 20nM, about 25nM, about 30nM, about 40nM, about 50nM, about 60nM, about 70nM, about 80nM, about 90 nM, approximately 100nM, approximately 110nM, approximately 120nM, approximately 130nM, approximately 140nM, approximately 150nM, approximately 160nM, approximately 170nM, approximately 180nM, approximately 190 The antibody binds to the CD122 / CD132 heterodimer (e.g., the human CD122 / CD132 heterodimer) with low affinity, such as greater than about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, or about 300 nM.

[0271] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced, increased, and / or selective) binding to Tregs in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has enhanced or increased binding to Tregs compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has selective binding to Tregs compared to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 fusion protein has enhanced or increased binding to Tregs compared to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has selective binding to Tregs compared to a reference IL-2 fusion protein. In one embodiment, binding to Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more, hi one embodiment, binding to Tregs is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0272] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced, increased, and / or selective) activation of the IL-2 signaling pathway in Tregs in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has enhanced or increased activation of the IL-2 signaling pathway in Tregs compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has selective activation of the IL-2 signaling pathway in Tregs compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has enhanced or increased activation of the IL-2 signaling pathway in Tregs compared to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has selective activation of the IL-2 signaling pathway in Tregs compared to a reference IL-2 fusion protein. In one embodiment, activation of the IL-2 signaling pathway in Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, activation of the IL-2 signaling pathway in Tregs is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0273] In one embodiment, the IL-2 fusion protein has a IL-2 fusion protein activity that is, for example, about 1, about 2, about 3, about 4, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, or greater than about 3000 or more to selectively activate IL-2 signaling in T regulatory cells in vitro and / or in vivo.

[0274] In one embodiment, the IL-2 fusion protein has a IL-2 fusion protein activity greater than or equal to about 1, about 2, about 3, about 4, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 20 ... 2500, or greater than about 3000 or more, or, for example, greater than 1 and about 1-2, about 2-3, about 3-4, about 4-5, greater than 1 and about 1-10, greater than 1 and about 1-20, greater than 1 and about 1-30, greater than 1 and about 1-40, greater than 1 and about 1-50, about 2-10, about 2-20, about 2-30, about 2-40, 2-50, about 5-10, about 5 and selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, having an NK cell EC50 / Treg EC50 ratio of about 20, about 5-30, about 5-40, about 5-50, about 10-20, about 10-30, about 10-40, about 10-50, about 20-40, about 20-50, about 50-100, about 100-200, about 200-500, about 500-1000, about 1000-2000, or about 1000-3000.

[0275] In one embodiment, the IL-2 fusion protein has an altered (e.g., enhanced, increased, and / or selective) ability to induce or promote expansion, activity, survival, and / or proliferation of Tregs in vitro and / or in vivo compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has an enhanced or increased ability to induce or promote expansion, activity, survival, and / or proliferation of Tregs compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has a selective ability to induce or promote expansion, activity, survival, and / or proliferation of Tregs compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has an enhanced or increased ability to induce or promote expansion, activity, survival, and / or proliferation of Tregs compared to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has a selective ability to induce or promote expansion, activity, survival, and / or proliferation of Tregs compared to a reference IL-2 fusion protein. In one embodiment, the ability to induce or promote expansion, activity, survival, and / or proliferation of Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the ability to induce or promote expansion, activity, survival, and / or proliferation of Tregs is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0276] In one embodiment, the IL-2 fusion protein has, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 400%, about 45 ... %, about 90%, about 95%, about 100% or more lower, or for example, about 0.5 fold, about 1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 5.5 fold, about 6 fold, about 6.5 fold, about 7 fold, about 7.5 fold, about 8 fold, about 8.5 fold, about 9 fold, about 9.5 fold, about 10 fold or more reduced, and / or have potency and / or ability to induce or promote enhanced or increased T regulatory cell activity.

[0277] In one embodiment, the IL-2 fusion protein is, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% or more higher, or, e.g., about 0.5 fold, as determined, e.g., by flow cytometry, compared to, e.g., an IL-2 fusion protein comprising a wild-type IL-2 or a reference IL-2 fusion protein. and have an EC50 against Tregs of about 1 fold, about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, about 5 fold, about 5.5 fold, about 6 fold, about 6.5 fold, about 7 fold, about 7.5 fold, about 8 fold, about 8.5 fold, about 9 fold, about 9.5 fold, about 10 fold, about 50 fold, about 100 fold, about 200 fold, about 500 fold, about 1000 fold, about 2000 fold, about 5000 fold, about 10,000 fold, about 15,000 fold, or about 20,000 fold or more reduction, and have a reduced or diminished potency and / or ability to induce or promote T regulatory cell activity.

[0278] In one embodiment, the T helper cells described herein are CD45+CD3+CD4+Foxp3- cells, e.g., as determined by flow cytometry. In one embodiment, the Tregs described herein are CD45+CD3+CD4+Foxp3+ cells, e.g., as determined by flow cytometry. In one embodiment, the NK cells described herein are CD45+CD3- cells that are CD56+ and / or CD16+, e.g., as determined by flow cytometry. In one embodiment, the NK cells described herein are CD45+CD3-CD56+ cells, e.g., as determined by flow cytometry.

[0279] In one embodiment, the IL-2 fusion protein has one or more structural and / or functional properties that are the same or substantially the same as an IL-2 fusion protein that comprises wild-type IL-2 or a reference IL-2 fusion protein.

[0280] In one embodiment, the reference IL-2 fusion protein comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an IL-2 fusion protein described herein. In one embodiment, the reference IL-2 fusion protein comprises an IL-2 variant comprising the amino acid sequence of SEQ ID NO: 57. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 57 and contains one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid modifications (e.g., substitutions) described herein.

[0281] In one embodiment, the IL-2 fusion protein comprises an IL-2 polypeptide (e.g., a human IL-2 polypeptide) described herein. In one embodiment, the IL-2 fusion protein is encoded by a nucleic acid comprising a nucleotide sequence described herein.

[0282] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) positions in IL-2 as described herein. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) positions in IL-2 selected from T3, H16, I28, K35, R38, F42, E68, V69, Q74, D84, S87, N88, I92, C125, or Q126.

[0283] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position T3 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position H16 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position I28 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position K35 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position R38 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position F42 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position E68 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position V69 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position Q74 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position D84 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position S87 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position N88 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position I92 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position C125 of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position Q126 of IL-2.

[0284] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at positions V69, Q74, or both in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at positions V69 and Q74 in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution V69A in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution Q74P in IL-2.

[0285] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position H16, I92, D84, or a combination thereof, of IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position H16, optionally the amino acid substitution is H16N, H16L, or H16D of IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution H16N in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution H16L in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution H16D in IL-2.

[0286] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position 192, optionally the amino acid substitution is 192S in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution 192S in IL-2.

[0287] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position D84, optionally the amino acid substitution is D84V in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution D84V in IL-2.

[0288] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position K35, R38, F42, E68, or a combination thereof, in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position K35, optionally the amino acid substitution is K35E in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution K35E in IL-2.

[0289] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position R38, optionally the amino acid substitution is R38E, R38N or R38Q of IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution R38N in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution R38Q in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position F42, optionally the amino acid substitution is F42K or F42Q of IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution F42K in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution F42Q in IL-2.

[0290] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at one, two, or all of: (i) (a) positions V69 and Q74, (b) K35, or (c) positions V69, Q74, and K35, and (ii) positions H16, I92, or D84 of IL-2. In one embodiment, the IL-2 fusion protein further comprises an amino acid modification (e.g., substitution) at one, two, or all of positions R38, F42, or E68 of IL-2.

[0291] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at (i)(a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35, and (ii)(a) one, two, or all of positions H16, I92, or D84, or (b) one, two, or all of positions R38, F42, or E68 of IL-2.

[0292] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at (i)(a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35, and (ii)(a) one, two, or all of positions H16, I92, or D84, and (b) one, two, or all of positions R38, F42, or E68 of IL-2.

[0293] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and H16, optionally the amino acid substitutions are V69A, Q74P, and H16N or H16L, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, and H16N or H16L in IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, and H16N in IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, and H16N in IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, and H16L in IL-2.

[0294] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and I92, optionally the amino acid substitutions are V69A, Q74P, and I92S, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, and I92S in IL-2.

[0295] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and D84, optionally the amino acid substitutions are V69A, Q74P, and D84V, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, and D84V in IL-2.

[0296] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and R38, optionally the amino acid substitutions are V69A, Q74P, and R38Q, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, and R38Q in IL-2.

[0297] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and F42, optionally the amino acid substitutions are V69A, Q74P, and F42Q, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, and F42Q in IL-2.

[0298] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and R38, optionally the amino acid substitutions are V69A, Q74P, and R38N, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, and R38N in IL-2.

[0299] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and R38, optionally the amino acid substitutions are V69A, Q74P, and R38E, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, and R38E in IL-2.

[0300] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, K35, and H16, optionally the amino acid substitutions are V69A, Q74P, K35E, and H16N, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, K35E, and H16N in IL-2.

[0301] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, K35, H16, and R38, and optionally the amino acid substitutions are V69A, Q74P, K35E, H16N, and R38N, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, K35E, H16N, and R38N in IL-2.

[0302] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, H16, and R38, and optionally the amino acid substitutions are V69A, Q74P, H16N, and R38N or R38Q, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, H16N, and R38N or R38Q in IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, H16N, and R38N in IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, H16N, and R38N in IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, H16N, and R38Q in IL-2.

[0303] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (eg, a substitution) at position I28, E68, S87, N88, Q126, or a combination thereof in IL-2.

[0304] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position I28, optionally the amino acid substitution is I28T or I28F in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution I28T in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution I28F in IL-2.

[0305] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position E68, optionally the amino acid substitution is E68Q or E68N in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution E68Q in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution E68N in IL-2.

[0306] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position S87, optionally the amino acid substitution is S87R in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution S87R in IL-2.

[0307] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position N88, optionally the amino acid substitution is N88S, N88L, or N88D in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution N88S, N88L, or N88D in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution N88S in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution N88L in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution N88D in IL-2.

[0308] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position Q126, optionally the amino acid substitution is Q126T, Q126K, or Q126R in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution Q126T, Q126K, or Q126R in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution Q126T, Q126K, or Q126R in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution Q126T in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution Q126K in IL-2. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution Q126R in IL-2.

[0309] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position C125 in IL-2, optionally the amino acid substitution is C125S. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution C125S in IL-2.

[0310] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at position T3 of IL-2, optionally the amino acid substitution is T3A. In one embodiment, the IL-2 fusion protein comprises the amino acid substitution T3A in IL-2.

[0311] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions V69, Q74, and C125 in IL-2, optionally the amino acid substitutions are V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions V69A, Q74P, and C125S in IL-2.

[0312] In one embodiment, the IL-2 fusion protein comprises an amino acid modification (e.g., substitution) at positions T3, H16, I92, or a combination thereof, of IL-2, optionally the amino acid substitutions are T3A, H16N, and I92S, respectively, of IL-2.

[0313] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions H16, V69, Q74, and C125 of IL-2, optionally the amino acid substitutions are H16N, V69A, Q74P, and C125S, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions H16N, V69A, Q74P, and C125S in IL-2.

[0314] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions H16, V69, Q74, and C125 of IL-2, optionally the amino acid substitutions are H16L, V69A, Q74P, and C125S, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions H16L, V69A, Q74P, and C125S in IL-2.

[0315] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions H16, V69, Q74, I92, and C125 of IL-2, and optionally the amino acid substitutions are H16L, V69A, Q74P, I92S, and C125S, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions H16L, V69A, Q74P, I92S, and C125S in IL-2.

[0316] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions T3, V69, Q74, and C125 of IL-2, and optionally the amino acid substitutions are T3A, V69A, Q74P, and C125S, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions T3A, V69A, Q74P, and C125S in IL-2.

[0317] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions T3, H16, V69, Q74, and C125 of IL-2, and optionally the amino acid substitutions are T3A, H16N or H16L, V69A, Q74P, and C125S, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions T3A, H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions T3A, H16L, V69A, Q74P, and C125S in IL-2.

[0318] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions T3, V69, Q74, I92, and C125 of IL-2, and optionally the amino acid substitutions are T3A, V69A, Q74P, I92S, and C125S, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions T3A, V69A, Q74P, I92S, and C125S in IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions T3A, V69A, Q74P, I92S, and C125S in IL-2.

[0319] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions H16, K35, V69 and Q74, optionally the amino acid substitutions are H16L, K35E, V69A, and Q74P, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions H16L, K35E, V69A, and Q74P in IL-2.

[0320] In one embodiment, the IL-2 fusion protein comprises amino acid modifications (e.g., substitutions) at positions H16, R38, V69A, and Q74P, and optionally the amino acid substitutions are H16L, R38Q, V69A, and Q74P, respectively, of IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions H16L, R38Q, V69A, and Q74P in IL-2.

[0321] In one embodiment, the IL-2 fusion protein contains the amino acid substitutions H16L, V69A, Q74P, and C125S in IL-2.

[0322] Without wishing to be bound by theory, in one embodiment, an IL-2 fusion protein comprising the amino acid substitutions H16L, V69A, Q74P, and C125S can have at least one or more of the following advantageous properties: (i) has reduced binding affinity to CD122 and / or CD132, which increases the potency and selectivity of the IL-2 agent for regulatory T cells (Tregs) compared to other T cell types; (ii) is significantly more stable, e.g., due to the presence of the stabilizing V69A and Q74P mutations; (iii) has reduced or decreased binding capacity and / or binding affinity to CD25, which improves the longevity of the IL-2 agent; (iv) does not substantially promote the expansion, activity, survival and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) has reduced imprecise disulfide pairing and improved stability, e.g., due to the presence of the C125S mutation. In one embodiment, an IL-2 agent comprising the H16L mutation has reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Tregs over other T cell types, compared to IL-2 agents comprising other H16 mutations. These properties make IL-2 variants comprising the amino acid substitutions H16L, V69A, Q74P, and C125S particularly suitable for treating disorders and conditions resulting from aberrant immune responses, such as, for example, autoimmune diseases.

[0323] Thus, in one embodiment, an IL-2 fusion protein comprising the amino acid substitutions H16L, V69A, Q74P, and C125S has one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties, particularly when compared to wild-type IL-2 or a reference IL-2 variant that does not comprise the amino acid substitutions: (i) enhanced or increased stability in vitro or in vivo, (ii) reduced or decreased binding ability and / or binding affinity to human CD122 in vitro and / or in vivo, (iii) reduced or decreased binding ability and / or binding affinity to human CD122 in vitro and / or in vivo, (iv) reduced or increased binding affinity to human CD122 in vivo and / or in vivo, (v) reduced or increased binding affinity to human CD122 in vivo and / or in vivo, (vi) reduced or increased binding affinity to human CD122 in vivo and / or in vivo, (vii ... (iv) reduced or decreased affinity of the IL-2 variant for the heterodimeric IL-2 receptor composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) reduced or decreased or substantially unchanged binding ability and / or binding affinity for human CD25 in vitro and / or in vivo; (vi) reduced or decreased binding ability and / or binding affinity for regulatory T cells (e.g., Foxp3 + (vii) selective activation of the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0324] In one embodiment, the IL-2 fusion protein is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and IL-2 variants comprising an amino acid sequence selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:1000, SEQ ID NO:1001, SEQ ID NO:1002, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.

[0325] In one embodiment, the IL-2 fusion protein comprises an IL-2 variant comprising an amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids. In one embodiment, the IL-2 fusion protein comprises an IL-2 variant comprising an amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO:11, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1000, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1001, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.In one embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1002, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.

[0326] In one embodiment, the IL-2 fusion protein comprises the amino acid sequence of any of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, or a functional fragment thereof. In one embodiment, the IL-2 fusion protein comprises the amino acid sequence of SEQ ID NO: 4 or 5, or a functional fragment thereof. In one embodiment, the IL-2 fusion protein comprises the amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof. In one embodiment, the IL-2 fusion protein comprises the amino acid sequence of SEQ ID NO: 5, or a functional fragment thereof. In one embodiment, the IL-2 fusion protein comprises the amino acid sequence of SEQ ID NO: 11, or a functional fragment thereof. In one embodiment, the IL-2 fusion protein comprises the amino acid sequence of SEQ ID NO: 1000, or a functional fragment thereof. In one embodiment, the IL-2 fusion protein comprises the amino acid sequence of SEQ ID NO: 1001, or a functional fragment thereof. In one embodiment, the IL-2 fusion protein comprises the amino acid sequence of SEQ ID NO: 1002, or a functional fragment thereof.

[0327] Without wishing to be bound by theory, in one embodiment, an IL-2 fusion protein comprising the amino acid sequence of SEQ ID NO:5 or a functional fragment thereof can have at least one or more of the following advantageous properties: (i) has reduced binding affinity to CD122 and / or CD132, which increases the potency and selectivity of the IL-2 agent for regulatory T cells (Tregs) compared to other T cell types; (ii) is significantly stable, e.g., due to the presence of the stabilizing V69A and Q74P mutations; (iii) has reduced or decreased binding ability and / or binding affinity to CD25, which improves the longevity of the IL-2 agent; (iv) does not substantially promote the expansion, activity, survival and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) has reduced imprecise disulfide pairing and improved stability, e.g., due to the presence of the C125S mutation. In one embodiment, an IL-2 agent comprising the H16L mutation has reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Tregs over other T cell types, compared to IL-2 agents comprising other H16 mutations. These properties make the IL-2 fusion protein comprising the amino acid sequence of SEQ ID NO:5 particularly suitable for the treatment of disorders and conditions resulting from an aberrant immune response, such as autoimmune diseases.

[0328] Thus, in one embodiment, an IL-2 fusion protein comprising the amino acid sequence of SEQ ID NO:5, or a functional fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 or fewer amino acids, has one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties, particularly as compared to wild-type IL-2 or a reference IL-2 fusion protein that does not contain amino acid substitutions: (i) enhanced or increased in vitro or in vivo stability, (ii) increased IL-2 activity, (iii) increased IL-2 activity, (iv) increased IL-2 activity, (v) increased IL-2 activity, (vi) increased IL-2 activity, (vii) increased IL-2 activity, (v ...v) increased IL-2 activity, (vii) increased IL-2 activity, (v) increased IL-2 activity, (v) increased IL-2 activity, (v) increased IL-2 activity, (v) increased IL-2 activity, (v) increased IL-2 activity, (v) increased IL-2 activity, (v) increased (iii) reduced or decreased binding ability and / or binding affinity to human CD132 in vitro and / or in vivo; (iv) reduced or decreased affinity of the IL-2 fusion protein for the heterodimeric IL-2 receptor composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) reduced or decreased or substantially unchanged binding ability and / or binding affinity to human CD25 in vitro and / or in vivo; (vi) reduced or decreased binding ability and / or binding affinity to regulatory T cells (e.g., Foxp3 + (vii) selective activation of the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0329] In one embodiment, an IL-2 fusion protein described herein comprises an Fc region, e.g., an Fc region having one or more mutations described herein and / or having one or more structural or functional properties described herein. Without wishing to be bound by theory, it is believed that in one embodiment, the Fc region described herein can reduce (e.g., prevent) renal clearance and / or extend the half-life of the IL-2 agent (e.g., via FcRn).

[0330] As used herein, the term "fusion protein" refers to a protein that includes two or more protein or peptide components. The two or more protein or peptide components may be obtained from different sources or may be encoded by different genes. A fusion protein may also be referred to as a chimeric protein. An Fc fusion protein (also known as an Fc chimeric fusion protein, Fc-Ig, Ig-based chimeric fusion protein, or Fc tag protein) may include an Fc region of an immunoglobulin (e.g., an Fc region described herein) linked (e.g., fused) to a protein or peptide. The Fc region can be linked (e.g., genetically fused) to the protein or peptide directly or indirectly, e.g., via a linker. In one embodiment, the Fc region is derived from an Fc region of an IgG, such as, for example, human IgG, e.g., IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc region is derived from an IgG1 Fc region, e.g., human IgG1.

[0331] An IL-2 fusion protein may comprise an IL-2 variant (e.g., an IL-2 variant described herein) or a functional fragment thereof linked (e.g., fused) to a protein or peptide. In one embodiment, the IL-2 fusion protein is an IL-2-Fc fusion protein, e.g., further comprising an Fc region of an immunoglobulin (e.g., an Fc region described herein) linked (e.g., fused) to an IL-2 polypeptide (e.g., an IL-2 variant described herein) or a functional fragment thereof. In one embodiment, the IL-2 fusion protein is not an IL-2-Fc fusion protein, e.g., an IL-2 fusion variant described herein or a functional fragment thereof, but is linked (e.g., fused) to a protein or peptide other than the Fc region of an IgG, e.g., human IgG, e.g., IgG1, IgG2, IgG3, or IgG4.

[0332] In one embodiment, the IL-2 fusion protein is selected from the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO: 85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.

[0333] In one embodiment, the IL-2 fusion protein is SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, 22, an amino acid sequence selected from SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, or SEQ ID NO:131, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.

[0334] In one embodiment, the IL-2 fusion protein is selected from the group consisting of SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, The present invention also includes an amino acid sequence selected from SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, or SEQ ID NO:169, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0335] In one embodiment, the IL-2 fusion protein is selected from the group consisting of SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, The present invention also includes an amino acid sequence selected from SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, or SEQ ID NO:207, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0336] In one embodiment, the IL-2 fusion protein is selected from the group consisting of SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, The present invention also includes an amino acid sequence selected from SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, or SEQ ID NO:245, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0337] In one embodiment, the IL-2 fusion protein is selected from the group consisting of SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273, The present invention also includes an amino acid sequence selected from SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, or SEQ ID NO:283, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0338] In one embodiment, the IL-2 fusion protein is selected from the group consisting of SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:311, The present invention also includes an amino acid sequence selected from SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:319, SEQ ID NO:320, or SEQ ID NO:321, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0339] In one embodiment, the IL-2 fusion protein is selected from the group consisting of SEQ ID NO:322, SEQ ID NO:323, SEQ ID NO:324, SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327, SEQ ID NO:328, SEQ ID NO:329, SEQ ID NO:330, SEQ ID NO:331, SEQ ID NO:332, SEQ ID NO:333, SEQ ID NO:334, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337, SEQ ID NO:338, SEQ ID NO:339, SEQ ID NO:340, SEQ ID NO:341, SEQ ID NO:342, SEQ ID NO:343, SEQ ID NO:344, SEQ ID NO:345, SEQ ID NO:346, SEQ ID NO:347, SEQ ID NO:348, SEQ ID NO:349, The present invention includes an amino acid sequence selected from SEQ ID NO:350, SEQ ID NO:351, SEQ ID NO:352, SEQ ID NO:353, SEQ ID NO:354, SEQ ID NO:355, SEQ ID NO:356, SEQ ID NO:357, SEQ ID NO:358, or SEQ ID NO:359, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0340] In one embodiment, the IL-2 fusion protein comprises an amino acid sequence selected from SEQ ID NO:1004, SEQ ID NO:1005, SEQ ID NO:1006, SEQ ID NO:1007, SEQ ID NO:1008, SEQ ID NO:1009, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1004, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1005, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1006, or an amino acid s...

Claims

1. 1. A pharmaceutical composition for treating an autoimmune disease in a human subject, comprising an IL-2 agent, wherein the IL-2 agent is administered at a dose of 0.5 μg / kg to 300 μg / kg, the IL-2 agent being an IL-2 variant or an IL-2 fusion protein comprising the IL-2 variant, and the IL-2 variant has a specific activity relative to human IL-2 (SEQ ID NO: 1031): (i) the amino acid substitution H16L or H16N, and / or the amino acid substitution I92S, and (ii) amino acid substitutions V69A, Q74P, and C125S; A pharmaceutical composition comprising:

2. The IL-2 agent is 0.5 μg / kg to 100 μg / kg, 1 μg / kg to 50 μg / kg, 2 μg / kg to 40 μg / kg, 3 μg / kg to 30 μg / kg, 4 μg / kg to 25 μg / kg, 5 μg / kg to 2 0 μg / kg, 10 μg / kg to 15 μg / kg, 1 μg / kg to 40 μg / kg, 1 μg / kg to 30 μg / kg, 1 μg / kg to 20 μg / kg, 1 μg / kg to 10 μg / kg, 1 μg / kg to 5 μg / kg , 5 μg / kg to 50 μg / kg, 10 μg / kg to 50 μg / kg, 20 μg / kg to 50 μg / kg, 30 μg / kg to 50 μg / kg, 40 μg / kg to 50 μg / kg, 0.5 μg / kg to 2 μg / kg, 3 μg / kg to 5 μg / kg, 6 μg / kg to 10 μg / kg, 12 μg / kg to 20 μg / kg, or 25 μg / kg to 40 μg / kg.

3. 3. The pharmaceutical composition of claim 2, wherein the IL-2 agent is administered at a dose of 1 μg / kg, 4 μg / kg, 8 μg / kg, 16 μg / kg, or 32 μg / kg.

4. 10. The pharmaceutical composition of claim 1, wherein the IL-2 agent is administered once a week, once every two weeks, or once every four weeks.

5. 2. The pharmaceutical composition of claim 1, wherein the IL-2 agent is administered subcutaneously.

6. 2. The pharmaceutical composition of claim 1, wherein the autoimmune disorder is systemic lupus erythematosus (SLE), autoimmune hepatitis (AIH), immune-mediated focal segmental glomerulosclerosis (FSGS), or alopecia areata (AA).

7. 2. The pharmaceutical composition of claim 1, wherein the IL-2 variant further comprises the amino acid substitution T3A.

8. 2. The pharmaceutical composition of claim 1, wherein the IL-2 variant comprises the amino acid sequence of any of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, or an amino acid sequence at least 95% identical thereto.

9. 2. The pharmaceutical composition of claim 1, wherein the IL-2 agent comprises an IL-2 fusion protein comprising the IL-2 variant.

10. The pharmaceutical composition of claim 9, wherein the IL-2 fusion protein further comprises an Fc region.

11. The pharmaceutical composition of claim 10, wherein the Fc region comprises an Fc region of IgG1 allotype m3 containing an N297G substitution according to EU numbering.

12. 11. The pharmaceutical composition of claim 10, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 1003, or an amino acid sequence at least 95% identical thereto.

13. The pharmaceutical composition of claim 10, wherein the Fc region is fused to the C-terminus of the IL-2 variant.

14. The pharmaceutical composition of claim 10, wherein the IL-2 fusion protein further comprises a linker.

15. The linker is ４ S) ４ 15. The pharmaceutical composition of claim 14, comprising (SEQ ID NO: 48).

16. 11. The pharmaceutical composition of claim 10, wherein the fusion protein comprises the amino acid sequence of any of SEQ ID NOs: 1004, 1005, 1006, 1007, 1008, or 1009, or an amino acid sequence at least 95% identical thereto.

17. The pharmaceutical composition of claim 10 , wherein the fusion protein forms a dimer.

18. Use of an IL-2 agent for the manufacture of a medicament for treating an autoimmune disease in a human subject, wherein the IL-2 agent is administered at a dose of 0.5 μg / kg to 300 μg / kg, the IL-2 agent is an IL-2 variant or an IL-2 fusion protein containing the IL-2 variant, and the IL-2 variant has a specific activity relative to human IL-2 (SEQ ID NO: 1031): (i) the amino acid substitution H16L or H16N, and / or the amino acid substitution I92S, and (ii) amino acid substitutions V69A, Q74P, and C125S; Including, use.