Interleukin-2 agents and uses thereof

Abstract

To provide interleukin-2 agents and uses thereof.SOLUTION: IL-2 agents that comprise IL-2 variants are disclosed as well as methods, compositions, and uses thereof. The IL-2 agents described herein can be used to treat and / or prevent various disorders and conditions. Disclosed herein are IL-2 agents (e.g., IL-2 variants, IL-2 fusion proteins, IL-2 complexes, or IL-2 conjugates) that have one or more structural and / or functional properties described herein. Advantageously, several of the IL-2 agents describe herein have one or more improved or desired properties, compared to an IL-2 agent comprising a wild-type IL-2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 879,137, filed July 26, 2019, and U.S. Provisional Application No. 62 / 983,061, filed February 28, 2020, the contents of which are incorporated herein by reference in their entireties.

[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, created on July 20, 2020, is named P2029-7028WO_SL.txt and is 1,728,528 bytes in size. [Background technology]

[0003] Interleukin-2 (IL-2) is a cytokine that regulates the activity of the immune system. Cytokines are 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 between foreign and host antigens. IL-2 mediates its effects by binding to the IL-2 receptor, which also 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 is sometimes limited due to the serious toxicity associated with IL-2 treatment, and only a small proportion of eligible patients will actually receive treatment. Toxicity associated with IL-2 treatment may include severe fever, nausea, vomiting, vascular leakage, and severe hypotension. However, despite these toxicities, IL-2 is usually effective in its approved indications.

[0005] There remains an unmet need for novel IL-2-based agents that are sufficiently characterized for the development of safe and effective therapeutics for patients with a variety of diseases and conditions amenable to treatment with IL-2. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides IL-2 agents (e.g., IL-2 variants, IL-2 fusion proteins, IL-2 complexes, and IL-2 conjugates) that comprise, at least in part, one or more amino acid changes (e.g., substitutions) in IL-2 and that comprise one or more 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 of producing 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] 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., the CD122 / CD132 heterodimer), and / or reduce or minimally affect its affinity for CD25 can be used to selectively enhance regulatory T cell (Treg) activity via the IL-2 pathway, thereby achieving beneficial 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 aberrant immune response, e.g., autoimmune diseases.

[0008] Thus, in certain aspects, the present disclosure provides IL-2 agonists, e.g., IL-2 agonists having 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) is expressed at a higher or increased level in vitro and / or in vivo, e.g., by assay of protein concentration, compared to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, 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 increased, or expressed by 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 increased; b) aggregates at a lower or reduced level in vitro and / or in vivo, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, as determined, for example, by melting temperature analysis (e.g., using fluorescence measurements), dynamic light scattering, and / or size exclusion chromatography, compared to an IL-2 agent comprising a wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant. %, 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 reduced, 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 reduced aggregation; c) enhanced or increased, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, in vitro and / or in vivo, compared to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, for example, by expression in yeast surface display, expression in mammalian cells, chromatography, circular dichroism or related spectroscopic techniques, and / or melting temperature analysis (e.g., using fluorescence measurements); has an increase in stability of 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, 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; d) enhanced or increased in vitro and / or in vivo, 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 an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, for example, by ELISA, flow cytometry and / or mass spectrometry. have a half-life that is increased by about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or that is increased by 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 reduction in IL-2 activity 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%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95 ... have a lower, reduced or decreased rate or level of turnover and / or clearance in vivo that is reduced by 0%, about 95%, about 100% or more, or is reduced by 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; f) a decrease 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%), compared to an IL-2 agent comprising 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) and / or surface plasmon resonance (e.g., Biacore). A decrease or increase of 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, 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 5 times, 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, have a reduced, decreased, or substantially unchanged binding affinity for CD25 (e.g., human CD25) that is about 9-fold, about 9.5-fold, about 10-fold or more decreased, 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 decreased or increased; g) low affinity for CD25 (e.g., human CD25), e.g., as determined by yeast surface display, 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 ~about 490pM, about 20pM to about 480pM, about 30pM to about 470pM, about 40pM to about 460pM, about 50pM to about 450pM, about 6 0pM to about 440pM, about 70pM to about 430pM, about 80pM to about 420pM, about 90pM to about 410pM, about 100pM to about 400pM , about 110pM to about 390pM, about 120pM to about 380pM, about 130pM to about 370pM, about 140pM to about 360pM, about 150p M ~ about 350pM, about 160pM - about 340pM, about 170pM - about 330pM, about 180pM - about 320pM, about 190pM - about 310p M, 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 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 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,Dissociation constants (K, ) of greater than about 400, about 450, or about 500 pM D ) to join; h) low affinity for CD25 (e.g., human CD25), e.g., as determined by, for example, biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore), 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, 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 1 0 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 and a dissociation constant (K) of greater than 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 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. D ) to join; i) e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, compared to an IL-2 agent comprising 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) and / or surface plasmon resonance (e.g., Biacore). 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%, about 5% to about 10%, about 1% to about 40%, about 1% to about 30%, about 1% to about 100%) % 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%), or decreased by about 0.5 times, about 1 time, 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, or about 5 times , 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) decreased, diminished, or reduced binding affinity for a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer); j) low affinity for the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), e.g., as determined by yeast surface display, 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 0.2 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 0.5 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 dissociation constant (K D ) to join; k) low affinity for CD122 / CD132 heterodimers (e.g., human CD122 / CD132 heterodimers), for example, as determined by biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore), for example, about 0.2 to 300 nM, 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 80nM, about 90nM, about 100nM, about 110nM, about 120nM, about 130nM, about 140nM, about 150nM, about 160nM, about 170nM, about 180nM, about 190nM, about 200nM, about 210nM, about 220nM, about 230nM, about 240nM, about 250nM, about 260nM, about 270nM, about 280nM, about 290nM or about 300nM, or for example, about 0.2 to about 280nM, about 0.2 to about 260nM, about 0.2 to about 240nM, about 0.2 to about 220nM, about 0.2 to about 200nM, about 0.2 to about 180nM, about 0.2 to about 160nM, about 0.2 to about 140nM, about 0.2 to about 120nM, about 0.2 to about 100nM, about 0.2 to about 80nM, about 0.2 to about 60nM, about 0.2 to about 40nM, about 0.2 to about 20nM, or for example, about 0.5 to about 300nM, about 1 to about 300nM, about 5 to about 300nM, about 10 to about 300nM, about 20 to about 300nM, about 40 to about 300nM, about 60 to about 300nM, about 80 to about 300nM, about 100 to about 300nM, 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 nM, about 100 to about 120 nM, or for example, about 0.2, about 0.a dissociation constant (K.sub.i) of greater than 5, about 1, about 2, about 5, about 10, about 15, 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 greater than about 300 nM; D ) to join; l) selectively activates IL-2 signaling in vitro and / or in vivo relative to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, 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, e.g., as determined by flow cytometry; 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 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 More than 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, 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 about 20, about 2 to 30, about 2 to 40, about 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 having a ratio; m) selectively activates IL-2 signaling in vitro and / or in vivo relative to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, 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, as determined, e.g., by flow cytometry; 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 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 More than 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, 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 about 20, about 2 to 30, about 2 to 40, about 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 NK cell ECs 50 / Treg EC 50 having a ratio; n) (i) have a potency and / or ability to induce or promote enhanced or increased regulatory T cell activity, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or more, relative to an IL-2 agent comprising 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 regulatory T cell proliferation or expansion assays, and / or T cell suppression assays. %, 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 reduced, 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 EC50 / ... 50 having; (ii) have a potency and / or ability to induce or promote regulatory T cell activity that is reduced or decreased, 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%, or less, as determined, for example, by flow cytometry, in vitro or in vivo regulatory T cell proliferation or expansion assays, and / or T cell suppression assays, relative to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant. %, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more increased, or decreased by, 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 50having; o) modulating (e.g., decreasing (e.g., inhibiting, blocking, or neutralizing) or increasing (e.g., activating, priming, 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 binding specificity, or both, as the IL-2 agents described herein; q) exhibits the same or similar binding affinity or binding 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) changes (e.g., substitutions) described herein; r) exhibits the same or similar binding affinity or binding specificity, or both, as an IL-2 agent comprising an amino acid sequence described herein; s) exhibits the same or similar binding affinity or binding specificity, or both, as an IL-2 agonist comprising an amino acid sequence encoded by a nucleotide sequence described herein; t) inhibiting, e.g., competitively inhibiting, 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) an agent that competes with a second IL-2 agent for binding to an IL-2 receptor, wherein the second IL-2 agent is an IL-2 agent described herein; v) has one or more biological properties of the IL-2 agonists described herein; w) has one or more structural characteristics of an IL-2 agonist described herein; or x) has one or more pharmacokinetic properties of the IL-2 agonists described herein.

[0009] In one embodiment, the IL-2 agent is expressed at a higher or increased level in vitro and / or in vivo, e.g., by protein concentration assay, compared to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, 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, 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. In one embodiment, the IL-2 agent aggregates at a lower or reduced level in vitro and / or in vivo, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, as compared to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, for example, by melting temperature analysis (e.g., using fluorescence measurements), dynamic light scattering, and / or size exclusion chromatography. , 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 reduced, 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 reduced aggregation.

[0010] In one embodiment, the IL-2 agent exhibits enhanced or increased in vitro and / or in vivo activity, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 25%, about 30%, about 45%, about 50%, 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 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 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 690%, about 690%, about 690%, about 690%, about 690%, about 690%, about 690%, about The stability may be increased by 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, or by 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.

[0011] In one embodiment, the IL-2 agent exhibits enhanced or increased in vitro and / or in vivo activity, 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 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 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, 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 750%, about 760%, about 770%, about 780%, about 790%, about 800%, about 810%, The half-life may be increased by 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 by 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.

[0012] 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 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 550%, about 550%, 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 710%, about 720%, about 730%, about 740%, about 750%, about 760%, about 770%, %, about 85%, about 90%, about 95%, about 100% or more reduced, or have a lower, reduced or decreased rate or level of turnover and / or clearance in vivo that is reduced by 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.

[0013] In one embodiment, the IL-2 agent 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% or more (e.g., about 1% to about 20%, about 2% to about 15%, or about 5% to about 10%) decreased activity, or about 1% decreased activity, 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 yeast surface display, biolayer interferometry (e.g., Octet binding), and / or surface plasmon resonance (e.g., Biacore). , decreased or increased by 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 about 50% or less, or by 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, 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, or The antibody has a reduced, decreased, or substantially unchanged binding affinity for CD25 (e.g., human CD25) that is 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more decreased, 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 decreased or increased. In one embodiment, the decrease or reduction in binding affinity for CD25 is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% less than the decrease or reduction in binding affinity for CD25. In one embodiment, the binding affinity for CD25 is not substantially reduced or decreased.

[0014] In one embodiment, the IL-2 agonist has low affinity for CD25 (e.g., human CD25), e.g., as determined by yeast surface display, e.g., between about 5 and 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, 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, 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 410pM, about 100pM to about 400pM, about 110pM to about 390pM, about 120pM to about 380pM, about 130pM to about 370pM, Approximately 140pM to approximately 360pM, approximately 150pM to approximately 350pM, approximately 160pM to approximately 340pM, approximately 170pM to approximately 330pM, approximately 180pM to about 320 pM, about 190 pM to about 310 pM, 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 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 1 0, 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,Dissociation constants (K, ) of greater than about 200, about 250, about 300, about 350, about 400, about 450, or about 500 pM D ) to join them.

[0015] In one embodiment, the IL-2 agent has low affinity for CD25 (e.g., human CD25), 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 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 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 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 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 M, 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 dissociation constant (K D ) to join them.

[0016] In one embodiment, the IL-2 agent exhibits a cytotoxic effect of, 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 20%, 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 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 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 50%, about 2% to about 40%, about 3% to about 30%, about 4% to about 20%, about 5% to about 10%, about 1% to about 40%, about 1 % to about 30%, 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%), or decreased by about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, or about 4.5 times , about 5-fold, 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) decreased, diminished, or reduced binding affinity for CD122 / CD132 heterodimers (e.g., human CD122 / CD132 heterodimers). In one embodiment, the reduction or reduction in binding affinity for CD122 / CD132 heterodimers is at least 1-, 1.5-, 2-, 2.5-, 3-, 3.5-, 4-, 4.5-, or 5-fold greater than the reduction or reduction in binding affinity for CD25. In one embodiment, the binding affinity for CD25 is not substantially reduced or decreased.

[0017] In one embodiment, the IL-2 agonist has low affinity for the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), 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, or about 12 nM, ... , 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 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 10 nM, a dissociation constant (K) of greater than 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 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 D ) to join them.

[0018] In one embodiment, the IL-2 agent has low affinity for CD122 / CD132 heterodimers (e.g., human CD122 / CD132 heterodimers), 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, e.g., as determined by biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore). , 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, 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 1 20 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 nM, about 100 to about 120 nM, or for example, about 0.2, about 0.a dissociation constant (K.sub.i) of greater than 5, about 1, about 2, about 5, about 10, about 15, 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 greater than about 300 nM; D ) to join them.

[0019] In one embodiment, the IL-2 agent selectively activates IL-2 signaling in vitro and / or in vivo relative to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, 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, 0, 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 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 4 100, 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 2-1 0, about 2 to 20, about 2 to 30, about 2 to 40, about 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 50In one embodiment, the T helper cells are CD45+CD3+CD4+Foxp3- cells, as determined, for example, by flow cytometry. In one embodiment, the Tregs are CD45+CD3+CD4+Foxp3+ cells, as determined, for example, by flow cytometry.

[0020] In one embodiment, the IL-2 agent selectively activates IL-2 signaling in vitro and / or in vivo relative to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, 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, 0, 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 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 4 00, 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 to 20, about 2 to 30, about 2 to 40, about 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 NK cell ECs 50 / Treg EC 50In one embodiment, NK cells are CD45+CD3- cells that are CD56+ and / or CD16+, as determined, for example, by flow cytometry. In one embodiment, NK cells are CD45+CD3-CD56+ 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.

[0021] In one embodiment, the IL-2 agent has (i) a potency and / or ability to induce or promote enhanced or increased regulatory T cell activity, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, compared to an IL-2 agent comprising 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 regulatory T cell proliferation or expansion assays, and / or T cell suppression assays. , 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 reduced, 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 It has.

[0022] In one embodiment, the IL-2 agent has a potency and / or ability to induce or promote regulatory T cell activity that is reduced or decreased, 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%, or greater, compared to 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 flow cytometry, in vitro or in vivo regulatory T cell proliferation or expansion assays, and / or T cell suppression assays. EC2 for Tregs that is increased by about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or decreased by, 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. 50 In one embodiment, the IL-2 agent has a reduced or decreased potency and / or ability to induce or promote regulatory T cell activity, e.g., has an EC50 for Tregs that is about 100-fold or greater, and does not activate or does not significantly activate NK cells, compared to an IL-2 agent that comprises wild-type IL-2 or an IL-2 agent that comprises a reference IL-2 variant (e.g., as determined by flow cytometry, in vitro or in vivo regulatory T cell proliferation or expansion assays, and / or T cell suppression assays).

[0023] In one embodiment, the IL-2 agonist modulates (e.g., decreases (e.g., inhibits, blocks, or neutralizes) or increases (e.g., activates, primes, or enhances) one or more biological activities of T cells (e.g., Tregs) in vitro, ex vivo, or in vivo.

[0024] In one embodiment, the IL-2 agonist exhibits the same or similar binding affinity or binding specificity, or both, as an IL-2 agonist described herein.

[0025] In one embodiment, the IL-2 agonist exhibits the same or similar binding affinity or binding specificity, or both, as an IL-2 agonist containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) changes (e.g., substitutions) described herein.

[0026] In one embodiment, the IL-2 agonist exhibits the same or similar binding affinity or binding specificity, or both, as an IL-2 agonist comprising an amino acid sequence described herein.

[0027] In one embodiment, the IL-2 agonist exhibits the same or similar binding affinity or binding specificity, or both, as an IL-2 agonist comprising an amino acid sequence encoded by a nucleotide sequence described herein.

[0028] 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 (wherein the second IL-2 agent is an IL-2 agent described herein).

[0029] In one embodiment, the IL-2 agent is an agent that 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.

[0030] In one embodiment, the IL-2 agonist has one or more biological properties of the IL-2 agonists described herein.

[0031] In one embodiment, the IL-2 agonist has one or more structural characteristics of the IL-2 agonists described herein.

[0032] In one embodiment, the IL-2 agonist has one or more pharmacokinetic properties of the IL-2 agonists described herein.

[0033] In one embodiment, the interleukin-2 (IL-2) agent comprises a human IL-2 variant comprising an amino acid change (e.g., a substitution) 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 T3, H16, 128, K35, R38, F42, E68, V69, Q74, D84, S87, N88, 192, C125, Q126, or a combination thereof, e.g., corresponding to wild-type human IL-2. In another embodiment, the IL-2 agent comprises an amino acid change (e.g., a substitution) at V69, Q74, or a combination thereof. In one embodiment, the IL-2 agent comprises amino acid changes (e.g., substitutions) 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 change (e.g., a substitution) at position H16, I92, D84, or a combination thereof. In one embodiment, the IL-2 agent comprises an amino acid change (e.g., a substitution) at position H16, I92, D84, or a combination thereof. In one embodiment, the IL-2 agent comprises an amino acid change (e.g., a substitution) at position H16, optionally the amino acid substitution is H16N, H16L, or H16D. In one embodiment, the IL-2 agent comprises the amino acid substitution H16N. In one embodiment, the IL-2 agent comprises the amino acid substitution H16L. In one embodiment, the IL-2 agent comprises an amino acid change (e.g., a substitution) at position I92, optionally the amino acid substitution is I92S. In one embodiment, the IL-2 agent comprises an amino acid change (e.g., a substitution) at position D84, optionally the amino acid substitution is D84V. In one embodiment, the IL-2 agonist comprises an amino acid change (e.g., a substitution) at position K35, R38, F42, E68, or a combination thereof. In one embodiment, the IL-2 agonist comprises an amino acid change (e.g., a substitution) at position K35, optionally the amino acid substitution is K35E.In one embodiment, the IL-2 agent comprises an amino acid change (e.g., a substitution) at position R38, optionally 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 change (e.g., a substitution) at position F42, optionally the amino acid substitution is F42K or F42Q. In one embodiment, the IL-2 agent comprises the amino acid substitution F42Q.

[0034] In one embodiment, the IL-2 agonist includes one or more (e.g., two, three, four, or all) amino acid changes of (i)-(v): (i) one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid changes (e.g., substitutions) that reduce, or are identified as reducing, its affinity for CD122 (e.g., a CD122 / CD132 heterodimer), for example, changes (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 changes (e.g., substitutions) that increase or are identified as increasing the stability of the IL-2 agonist, e.g., changes (e.g., substitutions) at positions V69 (e.g., V69A) and / or Q74 (e.g., Q74P); (iii) one or more (e.g., 2, 3, or 4) amino acid changes (e.g., substitutions) that reduce, or are identified as reducing, affinity for CD25, e.g., changes (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 changes (e.g., substitutions) that reduce or are identified as reducing O-glycosylation of the IL-2 agonist, e.g., a change (e.g., substitution) at position T3 (e.g., T3A); or (v) One or more amino acid changes (e.g., substitutions) identified to reduce or decrease incorrect disulfide pairing and / or aggregation of the IL-2 agent (e.g., to improve stability), e.g., a change (e.g., substitution) at position C125 (e.g., C125S).

[0035] In one embodiment, the IL-2 agonist comprises (i). In one embodiment, the IL-2 agonist comprises (ii). In one embodiment, the IL-2 agonist comprises (iii). In one embodiment, the IL-2 agonist comprises (iv). In one embodiment, the IL-2 agonist comprises (v).

[0036] In one embodiment, the IL-2 agonist comprises (i) and (ii). In one embodiment, the IL-2 agonist comprises (i) and (iii). In one embodiment, the IL-2 agonist comprises (i) and (iv). In one embodiment, the IL-2 agonist comprises (i) and (v). In one embodiment, the IL-2 agonist comprises (ii) and (iii). In one embodiment, the IL-2 agonist comprises (ii) and (iv). In one embodiment, the IL-2 agonist comprises (ii) and (v). In one embodiment, the IL-2 agonist comprises (iii) and (iv). In one embodiment, the IL-2 agonist comprises (iii) and (v). In one embodiment, the IL-2 agonist comprises (iv) and (v).

[0037] In one embodiment, the IL-2 agonist includes (i), (ii), and (iii). In one embodiment, the IL-2 agonist includes (i), (ii), and (iv). In one embodiment, the IL-2 agonist includes (i), (ii), and (v). In one embodiment, the IL-2 agonist includes (i), (iii), and (iv). In one embodiment, the IL-2 agonist includes (i), (iii), and (v). In one embodiment, the IL-2 agonist includes (i), (iv), and (v). In one embodiment, the IL-2 agonist includes (ii), (iii), and (iv). In one embodiment, the IL-2 agonist includes (ii), (iii), and (v). In one embodiment, the IL-2 agonist includes (ii), (iii), and (v). In one embodiment, the IL-2 agonist includes (ii), (iv), and (iv). In one embodiment, the IL-2 agonist includes (iii), (iv), and (v).

[0038] In one embodiment, the IL-2 agonist includes (i), (ii), (iii), and (iv). In one embodiment, the IL-2 agonist includes (i), (ii), (iii), and (v). In one embodiment, the IL-2 agonist includes (i), (ii), (iv), and (v). In one embodiment, the IL-2 agonist includes (i), (iii), (iv), and (v). In one embodiment, the IL-2 agonist includes (ii), (iii), (iv), and (v).

[0039] In one embodiment, the IL-2 agonist comprises (i), (ii), (iii), (iv), and (v).

[0040] In one embodiment, the IL-2 agonist does not include (i). In one embodiment, the IL-2 agonist does not include (ii). In one embodiment, the IL-2 agonist does not include (iii). In one embodiment, the IL-2 agonist does not include (iv). In one embodiment, the IL-2 agonist does not include (v).

[0041] In one embodiment, the IL-2 agonist does not include (i) and (ii). In one embodiment, the IL-2 agonist does not include (i) and (iii). In one embodiment, the IL-2 agonist does not include (i) and (iv). In one embodiment, the IL-2 agonist does not include (i) and (v). In one embodiment, the IL-2 agonist does not include (ii) and (iii). In one embodiment, the IL-2 agonist does not include (ii) and (iv). In one embodiment, the IL-2 agonist does not include (ii) and (v). In one embodiment, the IL-2 agonist does not include (iii) and (iv). In one embodiment, the IL-2 agonist does not include (iii) and (v). In one embodiment, the IL-2 agonist does not include (iv) and (v).

[0042] In one embodiment, the IL-2 agonist does not include (i), (ii), and (iii). In one embodiment, the IL-2 agonist does not include (i), (ii), and (iv). In one embodiment, the IL-2 agonist does not include (i), (ii), and (v). In one embodiment, the IL-2 agonist does not include (i), (iii), and (iv). In one embodiment, the IL-2 agonist does not include (i), (iii), and (v). In one embodiment, the IL-2 agonist does not include (i), (iv), and (v). In one embodiment, the IL-2 agonist does not include (ii), (iii), and (iv). In one embodiment, the IL-2 agonist does not include (ii), (iii), and (v). In one embodiment, the IL-2 agonist does not include (ii), (iii), and (v). In one embodiment, the IL-2 agonist does not include (ii), (iii), and (v). In one embodiment, the IL-2 agonist does not include (ii), (iv), and (iv). In one embodiment, the IL-2 agonist does not include (iii), (iv), and (v).

[0043] In one embodiment, the IL-2 agonist does not include (i), (ii), (iii), and (iv). In one embodiment, the IL-2 agonist does not include (i), (ii), (iii), and (v). In one embodiment, the IL-2 agonist does not include (i), (ii), (iv), and (v). In one embodiment, the IL-2 agonist does not include (i), (iii), (iv), and (v). In one embodiment, the IL-2 agonist does not include (ii), (iii), (iv), and (v).

[0044] In one embodiment, the IL-2 agonist does not include (i), (ii), (iii), (iv), and (v).

[0045] In one embodiment, the IL-2 agonist comprises an amino acid alteration (e.g., substitution) (i) positions V69 and Q74, and / or position K35; and (ii) positions H16, I92, or D84; as appropriate; (iii) positions R38, F42, E68, or any combination thereof; Included.

[0046] In one embodiment, the IL-2 agonist comprises an amino acid alteration (e.g., substitution) (i) positions V69 and Q74, and / or position K35; and (ii) positions H16, I92, or D84; (iii) positions R38, F42, E68, or any combination thereof; Included.

[0047] In one embodiment, the IL-2 agonist comprises an amino acid alteration (e.g., substitution) (i) positions V69 and Q74, and / or position K35; and (ii) position H16, I92, or D84; or (iii) positions R38, F42, E68, or any combination thereof; Included.

[0048] In one embodiment, the IL-2 agonist comprises an amino acid alteration (e.g., substitution) (i) positions V69 and Q74, and / or position K35; and (ii) positions H16, I92, D84 or combinations thereof; and (iii) positions R38, F42, E68, or any combination thereof; Included.

[0049] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and H16, optionally with amino acid substitutions V69A, Q74P, and H16N or H16L, respectively, optionally with amino acid substitutions V69A, Q74P, and H16L. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, and H16L.

[0050] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and 192, optionally the amino acid substitutions are V69A, Q74P, and 192S, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, and 192S.

[0051] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and D84, optionally with amino acid substitutions V69A, Q74P, and D84V, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, and D84V.

[0052] In one embodiment, the IL-2 agonist comprises amino acid changes (eg, substitutions) at positions V69, Q74, and R38, optionally the amino acid substitutions are V69A, Q74P, and R38Q, respectively.

[0053] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and F42, optionally with the amino acid substitutions V69A, Q74P, and F42Q, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, and F42Q.

[0054] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and R38, optionally with the amino acid substitutions V69A, Q74P, and R38N, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, and R38N.

[0055] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and R38, optionally with amino acid substitutions V69A, Q74P, and R38E, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, and R38E.

[0056] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions V69, Q74, K35, and H16, optionally with the amino acid substitutions being V69A, Q74P, K35E, and H16N or H16L, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, K35E, and H16N or H16L. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, K35E, and H16N. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, K35E, and H16L.

[0057] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions V69, Q74, K35, H16, and R38, optionally with the amino acid substitutions V69A, Q74P, K35E, H16N, and R38N, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, K35E, H16N, and R38N.

[0058] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions V69, Q74, H16, and R38, optionally with amino acid substitutions V69A, Q74P, H16N or H16L, and R38N or R38Q, respectively, optionally with amino acid substitutions V69A, Q74P, H16N or H16L, and R38Q. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, H16L, and R38Q.

[0059] In one embodiment, the IL-2 agonist comprises an amino acid change (e.g., a substitution) at position 128, E68, S87, N88, Q126, or a combination thereof. In one embodiment, the IL-2 agonist comprises an amino acid change (e.g., a substitution) at position 128, optionally the amino acid substitution is 128T or 128F. In one embodiment, the IL-2 agonist comprises the amino acid substitution 128T. In one embodiment, the IL-2 agonist comprises the amino acid substitution 128F.

[0060] In one embodiment, the IL-2 agent comprises an amino acid change (e.g., a 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.

[0061] In one embodiment, the IL-2 agonist comprises an amino acid change (e.g., a substitution) at position S87, optionally the amino acid substitution is S87R. In one embodiment, the IL-2 agonist comprises the amino acid substitution S87R.

[0062] In one embodiment, the IL-2 agent comprises an amino acid change (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.

[0063] In one embodiment, the IL-2 agonist comprises an amino acid change (e.g., a substitution) at position Q126, optionally the amino acid substitution is Q126T, Q126K, or Q126R. In one embodiment, the IL-2 agonist comprises the amino acid substitution Q126T. In one embodiment, the IL-2 agonist comprises the amino acid substitution Q126K. In one embodiment, the IL-2 agonist comprises the amino acid substitution Q126R.

[0064] In one embodiment, the IL-2 agonist comprises an amino acid change (e.g., a substitution) at position C125, optionally the amino acid substitution is C125S. In one embodiment, the IL-2 agonist comprises the amino acid substitution C125S.

[0065] In one embodiment, the IL-2 agonist comprises an amino acid change (e.g., a substitution) at position T3, optionally the amino acid substitution is T3A. In one embodiment, the IL-2 agonist comprises the amino acid substitution T3A.

[0066] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and C125, optionally with the amino acid substitutions V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions V69A, Q74P, and C125S.

[0067] In one embodiment, the IL-2 agonist comprises an amino acid change (eg, a substitution) at position T3, H16, I92, or a combination thereof, optionally the amino acid substitutions are T3A, H16N, and I92S, respectively.

[0068] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125, optionally with the amino acid substitutions H16N, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions H16N, V69A, Q74P, and C125S.

[0069] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125, optionally with the amino acid substitutions H16L, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 agonist comprises the amino acid substitutions H16L, V69A, Q74P, and C125S. Various technical effects are associated with IL-2 agonists comprising combinations of the aforementioned amino acid changes. 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 may have at least one or more of the following advantageous properties: (i) reduced binding affinity to CD122 and / or CD132, increasing the potency and selectivity of the IL-2 agent for regulatory T cells (Tregs) relative to other T cell types; (ii) significantly stable, e.g., due to the presence of the stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity to (or having no more than a minimal effect on) CD25, improving the longevity of the IL-2 agent; (iv) not substantially promoting 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) reduced disulfide mispairing, improving 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 for 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 agents comprising the amino acid substitutions H16L, V69A, Q74P, and C125S particularly suitable for the treatment of disorders and conditions resulting from an aberrant immune response, such as autoimmune diseases.

[0070] 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., two, three, four, five, six, seven, or all) of the following properties 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 capacity and / or binding affinity to human CD122 in vitro and / 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 (e.g., 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 regulatory T 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.

[0071] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions H16, V69, Q74, 192, and C125, optionally with the amino acid substitutions H16L, V69A, Q74P, 192S, and C125S, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions H16L, V69A, Q74P, 192S, and C125S.

[0072] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions T3, V69, Q74, and C125, optionally with the amino acid substitutions T3A, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions T3A, V69A, Q74P, and C125S.

[0073] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions T3, H16, V69, Q74, and C125, optionally with the amino acid substitutions T3A, H16N or H16L, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions T3A, H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 agonist comprises amino acid substitutions T3A, H16L, V69A, Q74P, and C125S.

[0074] In one embodiment, the IL-2 agonist comprises amino acid changes (e.g., substitutions) at positions T3, V69, Q74, I92, and C125, optionally the amino acid substitutions are T3A, V69A, Q74P, I92S, and C125S, respectively. In one embodiment, the IL-2 agonist comprises amino acid substitutions T3A, V69A, Q74P, I92S, and C125S. In one embodiment, the IL-2 agonist comprises amino acid substitutions T3A, V69A, Q74P, I92S, and C125S.

[0075] In one embodiment, the IL-2 agonist 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: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, 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, SEQ ID NO:55, 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 5, 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 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 thereto.

[0076] In one embodiment, the IL-2 agonist of any one of the preceding items, wherein the amino acid alterations (e.g., substitutions) provide the IL-2 agonist with at least one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all) of the following properties relative to a reference IL-2 agonist that does not include the amino acid alterations (e.g., substitutions): (i) enhanced or increased expression of IL-2 agonists; (ii) inhibiting or reducing aggregation of IL-2 agonists; (iii) enhancing or increasing the stability of the IL-2 agonist; (iv) enhancing or increasing the half-life of the IL-2 agonist; (v) inhibition or reduction of IL-2 agonist turnover and / or clearance; (vi) inhibition or reduction (e.g., moderate inhibition or reduction), or substantially no change, in binding of the IL-2 agent to human CD25; (vii) inhibiting or reducing the affinity of the IL-2 agent for human CD122; (viii) inhibiting or reducing the affinity of the IL-2 agent for human CD132; or (ix) inhibiting or reducing the affinity of the IL-2 agonist 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) an enhanced or increased, or a reduced or decreased, ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0077] In one embodiment, the IL-2 agent comprises a human IL-2 variant comprising one or more amino acid changes (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 described herein; and a non-IL-2 moiety described herein, wherein the amino acid changes (e.g., substitutions) provide the IL-2 agent with at least one or more of the following properties compared to a reference IL-2 agent that does not comprise the amino acid changes (e.g., substitutions): (i) enhanced or increased expression of IL-2 agonists; (ii) inhibiting or reducing aggregation of IL-2 agonists; (iii) enhancing or increasing the stability of the IL-2 agonist; (iv) enhancing or increasing the half-life of the IL-2 agonist; (v) inhibition or reduction of IL-2 agonist turnover and / or clearance; (vi) inhibition or reduction (e.g., moderate inhibition or reduction), or substantially no change, in binding of the IL-2 agent to human CD25; (vii) inhibiting or reducing the affinity of the IL-2 agent for human CD122; (viii) inhibiting or reducing the affinity of the IL-2 agonist for human CD132; (ix) inhibiting or reducing the affinity of the IL-2 agonist 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) an enhanced or increased, or a reduced or decreased, ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0078] In one embodiment, the human IL-2 variant is Amino acid changes (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; (xiii) 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; (xviii) 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; (xxiii) 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; (xxxiii) 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; (xxxviii) K35E, R38N, H16N, V69A, Q74P, and C125S; (xxxix) T3A, H16N, V69A, Q74P and C125S; (xl) T3A, H16L, V69A, Q74P, and C125S; or (xli) T3A, V69A, Q74P, I92S and C125S Includes:

[0079] In one embodiment, the IL-2 agonist 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:1002, or a functional fragment thereof or an interleukin-2 (IL-2) agent comprising: a human IL-2 variant comprising an amino acid sequence selected from amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 or fewer amino acids thereto; 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 the human IL-2 polypeptide variant: (i) enhanced or increased expression of IL-2 agonists; (ii) inhibiting or reducing aggregation of IL-2 agonists; (iii) enhancing or increasing the stability of the IL-2 agonist; (iv) enhancing or increasing the half-life of the IL-2 agonist; (v) inhibition or reduction of IL-2 agonist turnover and / or clearance; (vi) inhibition or reduction (e.g., moderate inhibition or reduction), or substantially no change, in binding of the IL-2 agent to human CD25; (vii) inhibiting or reducing the affinity of the IL-2 agent for human CD122; (viii) inhibiting or reducing the affinity of the IL-2 agonist for human CD132; (ix) inhibiting or reducing the affinity of the IL-2 agonist 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) an enhanced or increased, or a reduced or decreased, ability to induce or promote the expansion, activity and / or proliferation of Tregs.

[0080] Various technical effects are associated with IL-2 agonists 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) reduced binding affinity to CD122 and / or CD132, increasing the potency and selectivity of the IL-2 agent for regulatory T cells (Tregs) compared to other T cell types; (ii) significantly stable, e.g., due to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity to (or having no more than a minimal effect on) CD25, improving the longevity of the IL-2 agent; (iv) not substantially promoting 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) reduced disulfide mispairing and improved stability, e.g., due to the presence of a C125S mutation. In one embodiment, an IL-2 agent comprising the H16L mutation has reduced binding affinity for 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 treating disorders and conditions resulting from an aberrant immune response, such as autoimmune diseases.

[0081] Thus, in one embodiment, an IL-2 agent comprising the amino acid sequence of SEQ ID NO: 5 has, inter alia, one or more (e.g., two, three, four, five, six, seven, or all) of the following properties compared to wild-type IL-2 or a reference IL-2 variant that does not contain the amino acid substitution: (i) enhanced or increased stability in vitro or in vivo; (ii) reduced or decreased binding capacity and / or binding affinity to human CD122 in vitro and / 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 (e.g., 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 of the IL-2 variant for regulatory T cells (e.g., Foxp3 + (vii) selective activation of the IL-2 signaling pathway in regulatory T 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.

[0082] 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.

[0083] 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, wherein the linker is a polypeptide linker, optionally a flexible linker, a rigid linker, or a cleavable linker. In one embodiment, the polypeptide linker is a Gly-Ser linker (e.g., (G4S) n linkers, where n=1, 2, 3, 4, 5, 6 or more (SEQ ID NO: 1020)), proline-rich extension linkers (e.g., V1 GPc, V2 GPGc, V3 GcGcP, Cellulase Linker 4, Cellulase Linker 4), rigid linkers (e.g., A(EAAAK) n A, 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 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.

[0084] In one embodiment, the non-IL-2 moiety is an immunoglobulin Fc region, or a fragment or portion (e.g., functional fragment) thereof. 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.

[0085] In one embodiment, the IgG Fc region comprises a mutant IgG1 or mutant IgG4 Fc region, or a fragment or portion thereof. In one embodiment, the IgG Fc region comprises one or more (e.g., 2, 3, 4, or 5) mutations, such as one or more (e.g., 2, 3, 4, or 5) mutations described herein.

[0086] In one embodiment, the IgG Fc region comprises a variant IgG4 Fc region or a fragment or portion thereof, wherein the variant IgG4 Fc region is human.

[0087] In one embodiment, the variant IgG4 Fc region, or fragment or portion thereof, comprises an amino acid change (e.g., substitution) at Ser228 according to EU numbering, optionally wherein the amino acid change (e.g., substitution) at Ser228 is S228P. In one embodiment, the variant IgG4 Fc region comprises the amino acid substitution S228P.

[0088] In one embodiment, the mutant IgG4 Fc region, or fragment or portion thereof, comprises an amino acid change (e.g., substitution) at Arg409 according to EU numbering, optionally wherein the amino acid change (e.g., substitution) at Arg409 is R409K. In one embodiment, the mutant IgG4 Fc region comprises the amino acid substitution R409K.

[0089] In one embodiment, the mutant IgG4 Fc region, or fragment or portion thereof, comprises amino acid changes (e.g., substitutions) at Thr307, Gln311, and Ala378 according to EU numbering, optionally with the amino acid changes (e.g., substitutions) being T307Q, Q311V, and A378V, respectively. In one embodiment, the mutant IgG4 Fc region comprises the amino acid substitutions T307Q, Q311V, and A378V.

[0090] In one embodiment, the variant 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 thereto.

[0091] In one embodiment, the IgG Fc region comprises a mutant IgG1 Fc region or a fragment or portion thereof, and the mutant IgG1 Fc region is human. In one embodiment, the mutant IgG1 Fc region (e.g., comprising an N297G substitution) has the IgG1 m3 allotype.

[0092] In one embodiment, the mutant IgG1 Fc region, or fragment or portion thereof, comprises an amino acid change (e.g., substitution) at Asn297 according to EU numbering, optionally wherein the amino acid change (e.g., substitution) at Asn297 is N297G. In one embodiment, the mutant IgG1 Fc region comprises the amino acid substitution N297G.

[0093] In one embodiment, the variant IgG1 Fc region, or fragment or portion thereof, comprises amino acid changes (e.g., substitutions) at Leu234, Leu235, and Pro329 according to EU numbering, optionally with amino acid changes (e.g., substitutions) at L234A, L235A, and P329G, respectively. In one embodiment, the variant IgG1 Fc region comprises amino acid substitutions L234A, L235A, and P329G.

[0094] In one embodiment, the mutant IgG1 Fc region, or fragment or portion thereof, comprises amino acid changes (e.g., substitutions) at Thr307, Gln311, and Ala378 according to EU numbering, optionally with the amino acid changes (e.g., substitutions) being T307Q, Q311V, and A378V, respectively. In one embodiment, the mutant IgG1 Fc region comprises the amino acid substitutions T307Q, Q311V, and A378V.

[0095] In one embodiment, the variant 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 thereto. In one embodiment, the variant 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 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 thereto.

[0096] In one embodiment, the non-IL-2 moiety inhibits or reduces the ability of the IL-2 agent to induce an Fc receptor-mediated immune effector function.

[0097] In one embodiment, the IL-2 agonist 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, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or an IL-2 variant comprising an amino acid sequence selected from 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 agonist comprises a Gly-Ser linker, optionally wherein the Gly-Ser linker comprises (G4S)4 (SEQ ID NO:48), and 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.

[0098] In one embodiment, the IL-2 agonist 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.

[0099] In one embodiment, the IL-2 agonist 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.

[0100] In one embodiment, the IL-2 agonist 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.

[0101] In one embodiment, the IL-2 agonist 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.

[0102] In one embodiment, the IL-2 agonist 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.

[0103] In one embodiment, the IL-2 agonist 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.

[0104] In one embodiment, the IL-2 agonist 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.

[0105] In one embodiment, the IL-2 agonist 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.

[0106] 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. 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. 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.

[0107] Various technical effects are associated with an IL-2 agonist 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 may have at least one or more of the following advantageous properties: (i) reduced binding affinity to CD122 and / or CD132, increasing the potency and selectivity of the IL-2 agent for regulatory T cells (Tregs) compared to other T cell types; (ii) significantly stable, e.g., due to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity to (or having no more than a minimal effect on) CD25, improving the longevity of the IL-2 agent; (iv) not substantially promoting 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) reduced disulfide mispairing and improved stability, e.g., due to the presence of a C125S mutation. In one embodiment, an IL-2 agent comprising the H16L mutation has reduced binding affinity for 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: 1008 particularly suitable for the treatment of disorders and conditions resulting from an aberrant immune response, such as autoimmune diseases.

[0108] Thus, in one embodiment, an IL-2 agent comprising the amino acid sequence of SEQ ID NO: 1008 has, inter alia, one or more (e.g., two, three, four, five, six, seven, or all) of the following properties compared to wild-type IL-2 or a reference IL-2 variant that does not contain the amino acid substitution: (i) enhanced or increased stability in vitro or in vivo; (ii) reduced or decreased binding capacity and / or binding affinity for human CD122 in vitro and / or in vivo; (iii) reduced or decreased binding capacity and / or binding affinity for 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 (e.g., 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 regulatory T 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.

[0109] In one embodiment, the IL-2 agonist forms a dimer (eg, a homodimer or a heterodimer).

[0110] 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.

[0111] In some aspects, the present disclosure provides pharmaceutical compositions comprising a described IL-2 agent and a pharmaceutically acceptable carrier. In some aspects, the present disclosure provides nucleic acids encoding the IL-2 agents described herein. In some aspects, the present disclosure provides vectors (e.g., expression vectors) comprising nucleic acids encoding the IL-2 agents described herein. In some aspects, the present disclosure provides cells (e.g., isolated cells) comprising nucleic acids encoding the IL-2 agents described herein or vectors (e.g., expression vectors) comprising nucleic acids encoding the IL-2 agents described herein.

[0112] In some aspects, the present disclosure provides a method of producing an IL-2 agent, the method comprising culturing (e.g., maintaining) a cell comprising a nucleic acid encoding an IL-2 agent described herein or a vector (e.g., an expression vector) comprising a nucleic acid encoding an IL-2 agent described herein under conditions that allow expression of the IL-2 agent. In one embodiment, the method further comprises obtaining the IL-2 agent. In one embodiment, the method further comprises purifying the IL-2 agent.

[0113] In some aspects, the present disclosure provides methods for enhancing the expansion, activity, survival, and / or proliferation of regulatory T cells (Tregs), comprising contacting (e.g., in vitro, ex vivo, or in vivo) an effective amount of an IL-2 agent described herein, or a pharmaceutical composition comprising an IL-2 agent, with a Treg cell or a population of Treg cells, or administering to a subject in need thereof. The IL-2 agent can comprise, for example, the amino acid substitutions H16L, V69A, Q74P, and C125S, or the amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 agent comprises the amino acid substitutions H16L, V69A, Q74P, and C125S.

[0114] In some aspects, the present disclosure provides a method of selectively activating the IL-2 signaling pathway in regulatory T cells (Tregs), comprising contacting (e.g., in vitro, ex vivo, or in vivo) an effective amount of an IL-2 agent described herein, or a pharmaceutical composition comprising an IL-2 agent, with a Treg cell or a population of Treg cells, or administering to a subject in need thereof. The IL-2 agent can include, for example, the amino acid substitutions H16L, V69A, Q74P, and C125S, or the amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 agent includes the amino acid substitutions H16L, V69A, Q74P, and C125S.

[0115] In some aspects, the present disclosure provides a method of inducing immune tolerance in a subject in need thereof, comprising contacting or administering (e.g., in vitro, ex vivo, or in vivo) an effective amount of an IL-2 agent described herein, or a pharmaceutical composition comprising an IL-2 agent, to a Treg cell or a population of Treg cells. The IL-2 agent can include, for example, the amino acid substitutions H16L, V69A, Q74P, and C125S, or the amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 agent includes the amino acid substitutions H16L, V69A, Q74P, and C125S.

[0116] In some aspects, the present disclosure provides a method of treating a subject having a disorder (e.g., a disorder described herein, e.g., an autoimmune disease, lupus nephritis, autoimmune hepatitis, nephrotic syndrome, or cancer), the method comprising administering to the subject an effective amount of an IL-2 agonist described herein, or a pharmaceutical composition comprising an IL-2 agonist. The IL-2 agonist can include, for example, the amino acid substitutions H16L, V69A, Q74P, and C125S, or the amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 agonist includes the amino acid substitutions H16L, V69A, Q74P, and C125S.

[0117] In some aspects, the present disclosure provides an IL-2 agent or composition for use in a method for treating a subject having a disorder (e.g., a disorder described herein, e.g., an autoimmune disease, lupus nephritis, autoimmune hepatitis, nephrotic syndrome, or cancer), the method comprising administering to the subject an IL-2 agent or a pharmaceutical composition comprising an IL-2 agent described herein. The IL-2 agent can include, for example, the amino acid substitutions H16L, V69A, Q74P, and C125S, or the amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 agent includes the amino acid substitutions H16L, V69A, Q74P, and C125S.

[0118] In some aspects, the present disclosure provides use of an IL-2 agent or composition in the manufacture of a medicament for treating a subject having a disorder (e.g., a disorder described herein, e.g., an autoimmune disease, lupus nephritis, autoimmune hepatitis, nephrotic syndrome, or cancer), the method comprising administering to the subject an IL-2 agent or a pharmaceutical composition comprising an IL-2 agent described herein. The IL-2 agent can include, for example, amino acid substitutions H16L, V69A, Q74P, and C125S, or amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 agent includes amino acid substitutions H16L, V69A, Q74P, and C125S.

[0119] In some aspects, the present disclosure provides kits comprising an IL-2 agonist described herein, or a pharmaceutical composition comprising an IL-2 agonist, and instructions for use. The IL-2 agonist may comprise, for example, the amino acid substitutions H16L, V69A, Q74P, and C125S, or the amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 agonist comprises the amino acid substitutions H16L, V69A, Q74P, and C125S.

[0120] In some aspects, the present disclosure provides a container containing an IL-2 agent described herein, or a pharmaceutical composition comprising an IL-2 agent. The IL-2 agent can include, for example, the amino acid substitutions H16L, V69A, Q74P, and C125S, or the amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 agent includes the amino acid substitutions H16L, V69A, Q74P, and C125S. In an embodiment of the present invention, for example, the following items are provided: (Item 1) Corresponding 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 Interleukin-2 (IL-2) variants, including: (Item 2) 2. The IL-2 variant according to item 1, further comprising the amino acid substitution T3A. (Item 3) 3. The IL-2 variant according to item 1 or 2, comprising the amino acid sequence of any of SEQ ID NOs: 4, 5, 11, 1000, 1001 or 1002, an amino acid sequence that is at least 95% identical thereto or that differs therefrom by no more than 1, 2, 3, 4 or 5 amino acids, or a functional fragment thereof. (Item 4) 4. The IL-2 variant of any one of items 1 to 3, which selectively stimulates regulatory T cells (Tregs). (Item 5) 5. An IL-2 fusion protein comprising the IL-2 variant of any one of items 1 to 4. (Item 6) 6. The IL-2 fusion protein of item 5, further comprising an Fc region. (Item 7) 7. The IL-2 fusion protein of item 6, wherein the Fc region comprises the Fc region of IgG1 allotype m3 containing an N297G substitution according to EU numbering. (Item 8) 8. The IL-2 fusion protein of item 6 or 7, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 1003, or an amino acid sequence that is at least 95% identical to said sequence and differs from said sequence by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or a functional fragment thereof. (Item 9) 9. The IL-2 fusion protein of any one of items 6 to 8, wherein the Fc region is fused to the C-terminus of the IL-2 variant. (Item 10) 10. The IL-2 fusion protein of any one of items 6 to 9, further comprising a linker. (Item 11) 11. The IL-2 fusion protein of item 10, wherein the linker comprises (G4S)4 (SEQ ID NO: 48). (Item 12) 12. The IL-2 fusion protein of any one of items 6 to 11, comprising 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 that differs therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or a functional fragment thereof. (Item 13) 13. The IL-2 fusion protein of any one of items 6 to 12, which forms a dimer. (Item 14) 5. An IL-2 complex comprising the IL-2 variant according to any one of items 1 to 4 and an anti-IL-2 antibody molecule. (Item 15) 5. An IL-2 conjugate comprising an IL-2 variant according to any one of items 1 to 4 and a non-IL-2 moiety. (Item 16) 5. A pharmaceutical composition comprising the IL-2 according to any one of items 1 to 4 and a pharmaceutically acceptable carrier. (Item 17) 14. A pharmaceutical composition comprising the IL-2 fusion protein of any one of items 5 to 13 and a pharmaceutically acceptable carrier. (Item 18) 15. A pharmaceutical composition comprising the IL-2 complex according to item 14 and a pharmaceutically acceptable carrier. (Item 19) 16. A pharmaceutical composition comprising the IL-2 conjugate according to item 15 and a pharmaceutically acceptable carrier. (Item 20) A nucleic acid encoding the IL-2 variant of any one of items 1 to 4. (Item 21) 14. A nucleic acid encoding the IL-2 fusion protein of any one of items 5 to 13. (Item 22) 15. A nucleic acid encoding the IL-2 complex of item 14. (Item 23) 16. A nucleic acid encoding the IL-2 conjugate of item 15. (Item 24) A vector comprising the nucleic acid according to item 20. (Item 25) 22. A vector comprising the nucleic acid of item 21. (Item 26) 23. A vector comprising the nucleic acid of item 22. (Item 27) 24. A vector comprising the nucleic acid of item 23. (Item 28) A cell containing the nucleic acid according to item 20. (Item 29) 22. A cell containing the nucleic acid of item 21. (Item 30) A cell containing the nucleic acid according to item 22. (Item 31) A cell containing the nucleic acid according to item 23. (Item 32) 29. A method for producing an IL-2 variant, comprising culturing the cells of item 28 under conditions that allow expression of said IL-2 variant. (Item 33) 30. A method for producing an IL-2 fusion protein, comprising culturing the cells of item 29 under conditions that allow expression of the IL-2 fusion protein. (Item 34) 31. A method for producing an IL-2 complex, comprising culturing the cells of item 30 under conditions that allow expression of the IL-2 complex. (Item 35) 32. A method for producing an IL-2 conjugate, comprising culturing the cells of item 31 under conditions that allow expression of the IL-2 conjugate. (Item 36) 10. A method for enhancing the expansion, activity, survival and / or proliferation of regulatory T cells (Tregs), comprising contacting an effective amount of an IL-2 variant according to any one of items 1 to 4 with a Treg cell or a population of Treg cells in vitro, ex vivo or in vivo, or administering it to a subject in need thereof. (Item 37) 14. A method for enhancing the expansion, activity, survival and / or proliferation of regulatory T cells (Tregs), comprising contacting an effective amount of an IL-2 fusion protein according to any of items 5 to 13 with a Treg cell or a population of Treg cells in vitro, ex vivo or in vivo, or administering to a subject in need thereof. (Item 38) 15. A method for enhancing the expansion, activity, survival and / or proliferation of regulatory T cells (Tregs), comprising contacting an effective amount of the IL-2 complex of item 14 with a Treg cell or a population of Treg cells in vitro, ex vivo or in vivo, or administering to a subject in need thereof. (Item 39) 16. A method for enhancing the expansion, activity, survival and / or proliferation of regulatory T cells (Tregs), comprising contacting an effective amount of the IL-2 conjugate of item 15 with a Treg cell or a population of Treg cells in vitro, ex vivo or in vivo, or administering to a subject in need thereof. (Item 40) 10. A method for selectively activating the IL-2 signaling pathway in regulatory T cells (Tregs), comprising contacting an effective amount of an IL-2 variant according to any one of items 1 to 4 with a Treg cell or a population of Treg cells in vitro, ex vivo or in vivo, or administering it to a subject in need thereof. (Item 41) 14. A method for selectively activating the IL-2 signaling pathway in regulatory T cells (Treg), comprising contacting an effective amount of an IL-2 fusion protein according to any of items 5 to 13 with a Treg cell or a population of Treg cells in vitro, ex vivo or in vivo, or administering to a subject in need thereof. (Item 42) 15. A method for selectively activating the IL-2 signaling pathway in regulatory T cells (Treg), comprising contacting an effective amount of the IL-2 complex of item 14 with a Treg cell or a population of Treg cells in vitro, ex vivo or in vivo, or administering to a subject in need thereof. (Item 43) 16. A method for selectively activating the IL-2 signaling pathway in regulatory T cells (Treg), comprising contacting an effective amount of the IL-2 conjugate of item 15 with a Treg cell or a population of Treg cells in vitro, ex vivo or in vivo, or administering to a subject in need thereof. (Item 44) 10. A method for inducing immune tolerance, comprising administering to a subject in need thereof an effective amount of an IL-2 variant according to any one of items 1 to 4. (Item 45) 14. A method for inducing immune tolerance, comprising administering to a subject in need thereof an effective amount of an IL-2 fusion protein according to any one of items 5 to 13. (Item 46) 15. A method for inducing immune tolerance, comprising administering to a subject in need thereof an effective amount of the IL-2 complex of item 14. (Item 47) 16. A method for inducing immune tolerance, comprising administering to a subject in need thereof an effective amount of the IL-2 conjugate of item 15. (Item 48) 10. A method for treating an autoimmune disease, comprising administering to a subject in need thereof an effective amount of an IL-2 variant according to any one of items 1 to 4. (Item 49) 14. A method for treating an autoimmune disease, comprising administering to a subject in need thereof an effective amount of an IL-2 fusion protein according to any one of items 5 to 13. (Item 50) 15. A method for treating an autoimmune disease, comprising administering to a subject in need thereof an effective amount of the IL-2 complex of item 14. (Item 51) 16. A method for treating an autoimmune disease, comprising administering to a subject in need thereof an effective amount of the IL-2 conjugate of item 15. (Item 52) 10. A method for treating lupus nephritis, comprising administering to a subject in need thereof an effective amount of an IL-2 variant according to any one of items 1 to 4. (Item 53) 14. A method for treating lupus nephritis, comprising administering to a subject in need thereof an effective amount of the IL-2 fusion protein of any one of items 5 to 13. (Item 54) 15. A method for treating lupus nephritis, comprising administering to a subject in need thereof an effective amount of the IL-2 complex of item 14. (Item 55) 16. A method for treating lupus nephritis, comprising administering to a subject in need thereof an effective amount of the IL-2 conjugate of item 15. (Item 56) 10. A method for treating autoimmune hepatitis, comprising administering to a subject in need thereof an effective amount of an IL-2 variant according to any one of items 1 to 4. (Item 57) 14. A method for treating autoimmune hepatitis, comprising administering to a subject in need thereof an effective amount of an IL-2 fusion protein according to any one of items 5 to 13. (Item 58) 15. A method for treating autoimmune hepatitis, comprising administering to a subject in need thereof an effective amount of the IL-2 complex of item 14. (Item 59) 16. A method for treating autoimmune hepatitis, comprising administering to a subject in need thereof an effective amount of the IL-2 conjugate of item 15. (Item 60) 10. A method for treating nephrotic syndrome, comprising administering to a subject in need thereof an effective amount of an IL-2 variant according to any one of items 1 to 4. (Item 61) 14. A method for treating nephrotic syndrome, comprising administering to a subject in need thereof an effective amount of an IL-2 fusion protein according to any one of items 5 to 13. (Item 62) 15. A method for treating nephrotic syndrome, comprising administering to a subject in need thereof an effective amount of the IL-2 complex of item 14. (Item 63) 16. A method for treating nephrotic syndrome, comprising administering to a subject in need thereof an effective amount of the IL-2 conjugate of item 15. (Item 64) A kit comprising an IL-2 variant according to any one of items 1 to 4 and instructions for use. (Item 65) 14. A kit comprising an IL-2 fusion protein according to any one of items 5 to 13 and instructions for use. (Item 66) 15. A kit comprising the IL-2 complex according to item 14 and instructions for use. (Item 67) A kit comprising the IL-2 conjugate according to item 15 and instructions for use. (Item 68) 5. The IL-2 variant of any one of items 1 to 4 for use in a method for inducing immune tolerance in a subject. (Item 69) 14. The IL-2 fusion protein of any one of items 5 to 13 for use in a method for inducing immune tolerance in a subject. (Item 70) 15. An IL-2 complex according to item 14 for use in a method for inducing immune tolerance in a subject. (Item 71) 16. The IL-2 conjugate according to item 15, for use in a method for inducing immune tolerance in a subject. (Item 72) 5. The IL-2 variant of any one of items 1 to 4 for use in a method for treating an autoimmune disease in a subject. (Item 73) 14. The IL-2 fusion protein of any one of items 5 to 13 for use in a method for treating autoimmune hepatitis and autoimmune diseases in a subject. (Item 74) 15. The IL-2 conjugate according to item 14 for use in a method for treating autoimmune hepatitis and autoimmune diseases in a subject. (Item 75) 16. The IL-2 conjugate according to item 15 for use in a method for treating autoimmune hepatitis and autoimmune diseases in a subject. (Item 76) 5. The IL-2 variant of any one of items 1 to 4 for use in a method for treating lupus nephritis in a subject. (Item 77) 14. The IL-2 fusion protein of any one of items 5 to 13 for use in a method for treating lupus nephritis in a subject. (Item 78) 15. The IL-2 conjugate of item 14 for use in a method for treating lupus nephritis in a subject. (Item 79) 16. The IL-2 conjugate according to item 15, for use in a method for treating lupus nephritis in a subject. (Item 80) 5. The IL-2 variant of any one of items 1 to 4 for use in a method for treating autoimmune hepatitis in a subject. (Item 81) 14. The IL-2 fusion protein of any of items 5 to 13 for use in a method for treating autoimmune hepatitis in a subject. (Item 82) 15. The IL-2 conjugate of item 14 for use in a method for treating autoimmune hepatitis in a subject. (Item 83) 16. The IL-2 conjugate according to item 15, for use in a method for treating autoimmune hepatitis in a subject. (Item 84) 5. The IL-2 variant of any one of items 1 to 4 for use in a method for treating nephrotic syndrome in a subject. (Item 85) 14. The IL-2 fusion protein of any of items 5 to 13 for use in a method for treating nephrotic syndrome in a subject. (Item 86) 15. The IL-2 complex according to item 14 for use in a method for treating nephrotic syndrome in a subject. (Item 87) 16. The IL-2 conjugate according to item 15, for use in a method for treating nephrotic syndrome in a subject. [Brief explanation of the drawings]

[0121] [Figure 1]Figure 1A provides a schematic diagram showing the domain structure of an exemplary, non-limiting embodiment of an IL-2 agent provided herein. The IL-2 agent includes an IL-2 portion or variant (also referred to herein as a "mutein"), a peptide linker, an Fc-containing hinge sequence, and antibody CH2 and CH3 domains, as shown. Figure 1B provides a depiction of the amino acid sequence of human IL-2 (SEQ ID NO: 1030) and shows exemplary, non-limiting positions that, when mutated, affect IL-2 receptor binding and IL-2-mediated signaling activity in vitro and in vivo.

[0122] [Figure 2] Figure 2 provides a schematic diagram illustrating a cell-based method for generating a library of IL-2 variants using yeast surface display and selecting stable and active clones from those libraries. Mutations of IL-2 or IL-2 variants expressed by initial clones are generated by DNA synthesis or error-prone PCR and transformed into yeast cells. Yeast cells are stained with an anti-Myc antibody and a fluorescent secondary antibody to determine IL-2 expression (x-axis) and with recombinant CD25, an anti-6xHis antibody ("6xHis" disclosed as SEQ ID NO: 1028), and a fluorescent secondary antibody to measure bound CD25 (y-axis). In some versions of the experiment, an HA tag is used in addition to or instead of the Myc tag. Fluorescence-activated cell sorting is used to enrich for IL-2 variants that exhibit both high expression and high binding activity.

[0123] [Figure 3]Figure 3A provides a graph showing the results of a method for determining the affinity and binding capacity of IL-2 muteins displayed on the surface of yeast using IL-2 receptor titration. Yeast clones expressing the indicated IL-2 muteins were incubated with various concentrations of 6xHis-tagged CD25 extracellular domain ("6xHis" disclosed as SEQ ID NO: 1028). Bound CD25 was measured by staining with an anti-6xHis antibody ("6xHis" disclosed as SEQ ID NO: 1028) and a fluorescent secondary antibody. Several exemplary IL-2 muteins are shown. Curve fitting was used to determine binding affinity (KD) and maximum binding signal (data not shown). Figure 3B provides a graph showing the relative binding capacity (maximum binding signal normalized to IL-2 expression level) for selected IL-2 muteins.

[0124] [Figure 4-1] Figure 4A provides a graph illustrating the thermal denaturation (melting curves) of selected IL-2 agents (IL-2-Fc fusion proteins) as determined by SYPRO Orange fluorescence. The native IL-2-Fc fusion exhibits a maximum signal at low temperatures, indicating the presence of unfolded protein, while the V69A / Q74P mutein exhibits unfolding events as the temperature increases. Figure 4B provides an HPLC size-exclusion chromatogram showing that the majority of the native IL-2-Fc fusion elutes very early from the column (>670 kDa), suggesting unfolded protein aggregation. In contrast, V69A / Q74P IL-2-Fc elutes as a single peak at the expected time for an 84 kDa protein. [Figure 4-2]Figure 4A provides a graph illustrating the thermal denaturation (melting curves) of selected IL-2 agents (IL-2-Fc fusion proteins) as determined by SYPRO Orange fluorescence. The native IL-2-Fc fusion exhibits a maximum signal at low temperatures, indicating the presence of unfolded protein, while the V69A / Q74P mutein exhibits unfolding events as the temperature increases. Figure 4B provides an HPLC size-exclusion chromatogram showing that the majority of the native IL-2-Fc fusion elutes very early from the column (>670 kDa), suggesting unfolded protein aggregation. In contrast, V69A / Q74P IL-2-Fc elutes as a single peak at the expected time for an 84 kDa protein.

[0125] [Figure 5] Figure 5A provides a scatter plot showing the results of a yeast cell sorting procedure used to identify mutations affecting the interaction of CD122 and / or CD132 with the IL-2 receptor. Figure 5B provides a scatter plot showing the results of a yeast cell sorting procedure used to identify mutations affecting the interaction of CD122 and / or CD132 with the IL-2 receptor. Yeast expressing a library of IL-2 variants on their surface were stained with CD122 / CD132 Fc heterodimer at the indicated concentrations, and bound receptor was detected using a fluorescent anti-human Fc secondary antibody. Surface IL-2 expression was detected with an anti-Myc antibody and a fluorescent secondary antibody. Cells within the indicated gates (boxes) were sorted and collected, and the IL-2 muteins enriched in these populations were determined by a combination of Sanger sequencing and next-generation sequencing.

[0126] [Figure 6]Figure 6A provides a graph showing the results of a method to determine the saturation of yeast expressing the indicated IL-2 muteins on their surface after titration with CD122 / CD132 Fc heterodimer at the indicated concentrations. All muteins shown contain V69A / Q74P in addition to the indicated mutations. Bound CD122 / CD132 was labeled using an anti-human Fc fluorescent secondary and measured using an Accuri C6 flow cytometer. Saturation was calculated by fitting each curve to a four-parameter dose-response to estimate the maximum binding signal for each curve, then normalizing so that the estimated maximum was defined as 1. Figure 6B provides a graph showing the results of the same method as Figure 6A, except that selected muteins were incubated with 6xHis-tagged ("6xHis" disclosed as SEQ ID NO: 1028) recombinant CD25 extracellular domain, and bound CD25 was detected with an anti-6xHis antibody ("6xHis" disclosed as SEQ ID NO: 1028).

[0127] [Figure 7-1] Figure 7 provides a series of graphs showing the affinity of IL-2-Fc fusion proteins, including different IL-2 variants as indicated, for the extracellular domain of CD122 / CD132 Fc heterodimer and CD25, as measured with an Octet Biolayer Interferometer. The IL-2 variants contain V69A / Q74P plus the indicated mutations. The IL-2-Fc fusion proteins were immobilized on an anti-human Fc capture tip and then incubated with the indicated range of IL-2 receptor concentrations. Association and dissociation phase kinetics were used to estimate binding affinity. An excess of irrelevant antibody was used to prevent nonspecific binding or capture of the CD122 / CD132 Fc protein by the tip. [Figure 7-2]Figure 7 provides a series of graphs showing the affinity of IL-2-Fc fusion proteins, including different IL-2 variants as indicated, for the extracellular domain of CD122 / CD132 Fc heterodimer and CD25, as measured with an Octet Biolayer Interferometer. The IL-2 variants contain V69A / Q74P plus the indicated mutations. The IL-2-Fc fusion proteins were immobilized on an anti-human Fc capture tip and then incubated with the indicated range of IL-2 receptor concentrations. Association and dissociation phase kinetics were used to estimate binding affinity. An excess of irrelevant antibody was used to prevent nonspecific binding or capture of the CD122 / CD132 Fc protein by the tip.

[0128] [Figure 8-1] Figure 8 provides a schematic diagram showing the gating strategy and corresponding flow cytometry data for identifying IL-2-sensitive cell populations from human PBMCs. Singlet lymphocytes are identified based on forward and side scatter. Populations are defined as regulatory T cells (CD4+CD25highFoxp3+), CD25high T helper cells (CD4+CD25highFoxp3-), and natural killer cells (CD3-CD56+). [Figure 8-2] Figure 8 provides a schematic diagram showing the gating strategy and corresponding flow cytometry data for identifying IL-2-sensitive cell populations from human PBMCs. Singlet lymphocytes are identified based on forward and side scatter. Populations are defined as regulatory T cells (CD4+CD25highFoxp3+), CD25high T helper cells (CD4+CD25highFoxp3-), and natural killer cells (CD3-CD56+).

[0129] [Figure 9-1]Figure 9A provides graphs showing the IL-2 signaling response in an IL-2-sensitive cell population (Tregs) within human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce affinity for CD122 / CD132 dimers, as determined by the degree of STAT5 phosphorylation. Figure 9B provides graphs showing the IL-2 signaling response in an IL-2-sensitive cell population (CD25+(high) T helper cells) within human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce affinity for CD122 / CD132 dimers, as determined by the degree of STAT5 phosphorylation. Figure 9C provides graphs showing the IL-2 signaling response in an IL-2-sensitive cell population (NK cells) within human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce affinity for CD122 / CD132 dimers, as determined by the degree of STAT5 phosphorylation. Figure 9D provides a graph showing the IL-2 signaling response in IL-2-sensitive cell populations (CD8+ cytotoxic T cells) within human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce affinity for the CD122 / CD132 dimer, as determined by the degree of STAT5 phosphorylation. Cells were treated for 30 minutes at the indicated concentrations with IL-2-Fc fusion proteins containing the V69A / Q74 mutation plus the indicated mutations, or with the IL-2 N88D mutein (C-terminal N88D) fused to the C-terminus of a nonbinding antibody. The inactive IL-2-Fc fusion protein contains several mutations that reduce its IL-2 signaling activity (F42A, Y45A, L72G, N88D, V69A, Q74P). After treatment, cells were fixed with formaldehyde, permeabilized with cold methanol, and stained for surface markers and the STAT5 transcription factor (pSTAT5) phosphorylated at Tyr694. Each population is identified based on the gating described in Figure 8. Selected muteins were also evaluated for signaling activity on CD8+ cytotoxic T cells. The cells were gated as in Figure 8, except that the CD8 surface marker was used instead of CD4. Median pSTAT5 levels (median fluorescence intensity (MFI)) are shown for each concentration of IL-2-Fc fusion protein tested in each cell population.Curve fitting performed using GraphPad Prism v5.03 with a four-parameter fit to log(agonist) versus response. [Figure 9-2]Figure 9A provides graphs showing the IL-2 signaling response in an IL-2-sensitive cell population (Tregs) within human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce affinity for CD122 / CD132 dimers, as determined by the degree of STAT5 phosphorylation. Figure 9B provides graphs showing the IL-2 signaling response in an IL-2-sensitive cell population (CD25+(high) T helper cells) within human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce affinity for CD122 / CD132 dimers, as determined by the degree of STAT5 phosphorylation. Figure 9C provides graphs showing the IL-2 signaling response in an IL-2-sensitive cell population (NK cells) within human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce affinity for CD122 / CD132 dimers, as determined by the degree of STAT5 phosphorylation. Figure 9D provides a graph showing the IL-2 signaling response in IL-2-sensitive cell populations (CD8+ cytotoxic T cells) within human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce affinity for the CD122 / CD132 dimer, as determined by the degree of STAT5 phosphorylation. Cells were treated for 30 minutes at the indicated concentrations with IL-2-Fc fusion proteins containing the V69A / Q74 mutation plus the indicated mutations, or with the IL-2 N88D mutein (C-terminal N88D) fused to the C-terminus of a nonbinding antibody. The inactive IL-2-Fc fusion protein contains several mutations that reduce its IL-2 signaling activity (F42A, Y45A, L72G, N88D, V69A, Q74P). After treatment, cells were fixed with formaldehyde, permeabilized with cold methanol, and stained for surface markers and the STAT5 transcription factor (pSTAT5) phosphorylated at Tyr694. Each population is identified based on the gating described in Figure 8. Selected muteins were also evaluated for signaling activity on CD8+ cytotoxic T cells. The cells were gated as in Figure 8, except that the CD8 surface marker was used instead of CD4. Median pSTAT5 levels (median fluorescence intensity (MFI)) are shown for each concentration of IL-2-Fc fusion protein tested in each cell population.Curve fitting performed using GraphPad Prism v5.03 with a four-parameter fit to log(agonist) versus response.

[0130] [Figure 10] Figure 10A provides graphs showing the IL-2 signaling response in an IL-2-sensitive cell population (Tregs) within human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce affinity for CD25. Figure 10B provides graphs showing the IL-2 signaling response in an IL-2-sensitive cell population (CD25+(high) T helper cells) within human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce affinity for CD25. Figure 10C provides graphs showing the IL-2 signaling response in an IL-2-sensitive cell population (NK cells) within human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce affinity for CD25. Human PBMCs were treated and analyzed as in Figure 9. Median pSTAT5 levels (MFI) are shown for each treatment in each population. To highlight the effect on EC50, signaling within each mutein was normalized to 0 to 1 across the concentration range of IL-2-Fc treatments.

[0131] [Figure 11-1]Figure 11A provides graphs showing IL-2 signaling responses in IL-2-sensitive cell populations (Tregs) within human PBMCs after treatment with IL-2-Fc fusions containing paired mutations that reduce affinity for CD25 and CD122 / CD132. Figure 11B provides graphs showing IL-2 signaling responses in IL-2-sensitive cell populations (CD25+(high) T helper cells) within human PBMCs after treatment with IL-2-Fc fusions containing paired mutations that reduce affinity for CD25 and CD122 / CD132. Figure 11C provides graphs showing IL-2 signaling responses in IL-2-sensitive cell populations (NK cells) within human PBMCs after treatment with IL-2-Fc fusions containing paired mutations that reduce affinity for CD25 and CD122 / CD132. Human PBMCs were treated and analyzed as in Figure 9. IL-2-Fc fusion proteins containing various IL-2 muteins are separated into upper and lower panels for clarity, as indicated. Median pSTAT5 levels (MFI) are shown for each treatment in each population. [Figure 11-2] Same as above. [Figure 11-3] Same as above.

[0132] [Figure 12] Figure 12A provides a graph showing in vivo Treg expansion, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with IL-2-Fc H16N. Figure 12B provides a graph showing in vivo Treg expansion, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with C-terminal N88D. Homozygous Tg32 mice were administered the indicated amounts of each IL-2Fc fusion protein (dose levels were approximately equimolar) via tail vein injection. At the indicated time points, lymphocyte populations were profiled, and Tregs were defined as CD45+CD3+CD4+CD25highCD127- cells. Data in Figures 12A and 12B are the average of three mice per treatment group. Figure 12C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested.

[0133] [Figure 13-1] Figure 13A provides a graph showing the change in CD4+ T helper cell levels, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with IL-2-Fc H16N. Figure 13B provides a graph showing the change in CD4+ T helper cell levels, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with C-terminal N88D. Mice were dosed as in Figure 12. CD4+ T helper cells were defined as CD45+ CD3+ CD4+ cells that were not CD25highCD127-. Data in Figures 13A and 13B are the average of three mice per treatment group. Figure 13C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested. [Figure 13-2] Figure 13A provides a graph showing the change in CD4+ T helper cell levels, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with IL-2-Fc H16N. Figure 13B provides a graph showing the change in CD4+ T helper cell levels, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with C-terminal N88D. Mice were dosed as in Figure 12. CD4+ T helper cells were defined as CD45+ CD3+ CD4+ cells that were not CD25highCD127-. Data in Figures 13A and 13B are the average of three mice per treatment group. Figure 13C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested.

[0134] [Figure 14-1]Figure 14A provides a graph showing the change in the level of CD8+ cytotoxic T cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with IL-2-Fc H16N. Figure 14B provides a graph showing the change in the level of CD8+ cytotoxic T cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with C-terminal N88D. Mice were dosed as in Figure 12. Cytotoxic T cells were defined as CD45+CD3+CD8+ cells. Data in Figures 14A and 14B are the average of three mice per treatment group. Figure 14C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested. [Figure 14-2] Figure 14A provides a graph showing the change in the level of CD8+ cytotoxic T cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with IL-2-Fc H16N. Figure 14B provides a graph showing the change in the level of CD8+ cytotoxic T cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with C-terminal N88D. Mice were dosed as in Figure 12. Cytotoxic T cells were defined as CD45+CD3+CD8+ cells. Data in Figures 14A and 14B are the average of three mice per treatment group. Figure 14C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested.

[0135] [Figure 15-1]Figure 15A provides a graph showing the change in NK cell levels, measured as a percentage of total CD45+ lymphocytes, in Tg32 mice treated with IL-2-Fc H16N. Figure 15B provides a graph showing the change in NK cell levels, measured as a percentage of total CD45+ lymphocytes, in Tg32 mice treated with C-terminal N88D. Mice were dosed as in Figure 12. NK cells were defined as CD45+CD3-CD56+ cells. Data in Figures 15A and 15B are the average of three mice per treatment group. In each case, the percentage of NK cells is normalized within each mouse so that the pre-treatment value is 1. Figure 15C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested. [Figure 15-2] Figure 15A provides a graph showing the change in NK cell levels, measured as a percentage of total CD45+ lymphocytes, in Tg32 mice treated with IL-2-Fc H16N. Figure 15B provides a graph showing the change in NK cell levels, measured as a percentage of total CD45+ lymphocytes, in Tg32 mice treated with C-terminal N88D. Mice were dosed as in Figure 12. NK cells were defined as CD45+CD3-CD56+ cells. Data in Figures 15A and 15B are the average of three mice per treatment group. In each case, the percentage of NK cells is normalized within each mouse so that the pre-treatment value is 1. Figure 15C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested.

[0136] [Figure 16-1]Figure 16A provides graphs showing the binding kinetics of the CD122 / CD132 Fc heterodimer or CD25 extracellular domain over a range of concentrations to an IL-2-Fc fusion protein (wild-type) containing only the V69A / Q74P mutations anchored at the tip of the anti-human Fc Octet. Figure 16B provides graphs showing the binding kinetics of the CD122 / CD132 Fc heterodimer or CD25 extracellular domain over a range of concentrations to an IL-2-Fc fusion protein (42A, Y45A, L72G, N88D, V69A, Q74P; inactive) containing only the inactivating mutations anchored at the tip of the anti-human Fc Octet. The binding kinetics were used to estimate the KD of each interaction. [Figure 16-2] Figure 16A provides graphs showing the binding kinetics of the CD122 / CD132 Fc heterodimer or CD25 extracellular domain over a range of concentrations to an IL-2-Fc fusion protein (wild-type) containing only the V69A / Q74P mutations anchored at the tip of the anti-human Fc Octet. Figure 16B provides graphs showing the binding kinetics of the CD122 / CD132 Fc heterodimer or CD25 extracellular domain over a range of concentrations to an IL-2-Fc fusion protein (42A, Y45A, L72G, N88D, V69A, Q74P; inactive) containing only the inactivating mutations anchored at the tip of the anti-human Fc Octet. The binding kinetics were used to estimate the KD of each interaction.

[0137] [Figure 17-1]Figure 17A provides a graph illustrating the clearance kinetics of IL-2Fc fusion proteins in mice. Figure 17B provides a graph illustrating the clearance kinetics of IL-2Fc fusion proteins in mice. Figure 17C provides a graph illustrating the clearance kinetics of IL-2Fc fusion proteins in mice. Figure 17D provides a graph illustrating the clearance kinetics of IL-2Fc fusion proteins in mice. Plasma was collected from mice treated as in Figure 12 with various doses of IL-2-Fc fusion proteins containing V69A / Q74P / H16N mutations or C-terminal N88D (Figures 17A-17B) or IL-2-Fc fusion proteins containing inactivating mutations (42A, Y45A, L72G, N88D, V69A, Q74P; inactive; Figures 17C-17D), as indicated. The amount of IL-2-Fc or C-terminal N88D present at each time point was measured using an ELISA assay with an anti-IL-2 capture antibody (R&D Systems, AF-202) and an anti-human Fc secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch 109-035-008). 100% of the starting material was defined as the amount detectable in plasma 1 hour after injection. Note that the x-axis is categorical and not scaled with time. [Figure 17-2]Figure 17A provides a graph illustrating the clearance kinetics of IL-2Fc fusion proteins in mice. Figure 17B provides a graph illustrating the clearance kinetics of IL-2Fc fusion proteins in mice. Figure 17C provides a graph illustrating the clearance kinetics of IL-2Fc fusion proteins in mice. Figure 17D provides a graph illustrating the clearance kinetics of IL-2Fc fusion proteins in mice. Plasma was collected from mice treated as in Figure 12 with various doses of IL-2-Fc fusion proteins containing V69A / Q74P / H16N mutations or C-terminal N88D (Figures 17A-17B) or IL-2-Fc fusion proteins containing inactivating mutations (42A, Y45A, L72G, N88D, V69A, Q74P; inactive; Figures 17C-17D), as indicated. The amount of IL-2-Fc or C-terminal N88D present at each time point was measured using an ELISA assay with an anti-IL-2 capture antibody (R&D Systems, AF-202) and an anti-human Fc secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch 109-035-008). 100% of the starting material was defined as the amount detectable in plasma 1 hour after injection. Note that the x-axis is categorical and not scaled with time.

[0138] [Figure 18-1]Figure 18A shows in vivo immune cell expansion in humanized mice after administration of an exemplary IL-2Fc fusion protein. Figure 18B shows in vivo immune cell expansion in humanized mice after administration of an exemplary IL-2Fc fusion protein. Figure 18C shows in vivo immune cell expansion in humanized mice after administration of an exemplary IL-2Fc fusion protein. Figure 18D shows in vivo immune cell expansion in humanized mice after administration of an exemplary IL-2Fc fusion protein. Figure 18A shows a schematic diagram of the experimental design, showing various time points at which blood was collected from humanized mice administered an IL-2Fc fusion polypeptide and a control polypeptide. Flow cytometry was used to measure various lymphocyte populations at each indicated time point. Figure 18B shows the expansion rate of regulatory T cells for each IL-2Fc fusion polypeptide on the Y-axis and its corresponding dose (low or high) on the X-axis. Figure 18C shows the T helper cell proliferation rate on the Y-axis for each IL-2Fc fusion polypeptide and its corresponding dose (low or high) shown on the X-axis. Figure 18D shows the NK cell proliferation rate on the Y-axis for each IL-2Fc fusion polypeptide and its corresponding dose (low or high) shown on the X-axis. As shown from left to right on the X-axis of Figures 18B-18D, the IL-2Fc fusion polypeptides examined were as follows: control monoclonal antibody (motavizumab), inactive IL-2, an IL-2 mutein containing the N88D mutation, wild-type IL-2, an IL-2 mutein containing the mutations H16N / V69A / Q74P / C125S (SEQ ID NO: 1007), and an IL-2 mutein containing the mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008). [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 18-4] Same as above.

[0139] [Figure 19]Figure 19A shows the residual amount of an exemplary IL-2 fusion protein in Tg32 mice. Figure 19B shows the effective half-life of an exemplary IL-2 fusion protein in Tg32 mice. Figure 19A shows the concentration of IL-2 fusion proteins with the indicated combinations of mutations in the blood of mice on the Y-axis, with the number of days sampled after administration represented on the X-axis. Figure 19B shows a comparison of the half-life of IL-2 fusion proteins with the indicated combinations of mutations in the IL-2 moiety, with and without additional mutations in the Fc region. The concentration of the indicated IL-2 fusion protein in the blood is represented on the Y-axis, and the number of days after administration is represented on the X-axis.

[0140] [Figure 20] Figure 20 shows the pharmacokinetic profile of an exemplary IL-2-Fc fusion protein (IL2-118 fused to IgG1 Fc N297G allotype m3) containing the mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) in cynomolgus monkeys. Four monkeys (numbers 3501, 3502, 3503, and 3504) were injected with 100 μg / kg of IL-2-Fc fusion protein four times per week, and serum levels of IL-2-Fc fusion protein were measured over time.

[0141] [Figure 21]Figure 21A shows the effect of an exemplary IL-2-Fc fusion protein (containing mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on the expansion and proliferation of regulatory T cells in cynomolgus monkeys. Figure 21B shows the effect of an exemplary IL-2-Fc fusion protein (containing mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on the expansion and proliferation of regulatory T cells in cynomolgus monkeys. Figure 21A shows the expansion of regulatory T cells over time, expressed as fold change over baseline (baseline = pre-dose), after four weekly injections of 100 μg / kg of IL-2-Fc fusion protein. Figure 21B shows the percentage of Ki67+ regulatory T cells normalized to total regulatory T cells (an indicator of proliferating regulatory T cells) over time after four weekly injections of 100 μg / kg IL-2-Fc fusion protein.

[0142] [Figure 22-1]Figure 22A shows the effect of an exemplary IL-2-Fc fusion protein (containing mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after four weekly injections of 100 μg / kg of IL-2-Fc fusion protein. Figure 22B shows the effect of an exemplary IL-2-Fc fusion protein (containing mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after four weekly injections of 100 μg / kg of IL-2-Fc fusion protein. Figure 22C shows the effect of an exemplary IL-2-Fc fusion protein (containing mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after four weekly injections of 100 μg / kg of IL-2-Fc fusion protein. Figure 22D shows the effect of an exemplary IL-2-Fc fusion protein (containing mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after four weekly injections of 100 μg / kg of IL-2-Fc fusion protein. Figure 22A shows the effect of IL-2-Fc fusion protein on the number of NK cells over time, Figure 22B shows the effect on cytotoxic T cells over time, Figure 22C shows the effect on T helper cells over time, and Figure 22D shows the effect on total T cells over time. Data are presented as fold change over baseline (baseline = pre-dose) for each cell type. [Figure 22-2]Figure 22A shows the effect of an exemplary IL-2-Fc fusion protein (containing mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after four weekly injections of 100 μg / kg of IL-2-Fc fusion protein. Figure 22B shows the effect of an exemplary IL-2-Fc fusion protein (containing mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after four weekly injections of 100 μg / kg of IL-2-Fc fusion protein. Figure 22C shows the effect of an exemplary IL-2-Fc fusion protein (containing mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after four weekly injections of 100 μg / kg of IL-2-Fc fusion protein. Figure 22D shows the effect of an exemplary IL-2-Fc fusion protein (containing mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after four weekly injections of 100 μg / kg of IL-2-Fc fusion protein. Figure 22A shows the effect of IL-2-Fc fusion protein on the number of NK cells over time, Figure 22B shows the effect on cytotoxic T cells over time, Figure 22C shows the effect on T helper cells over time, and Figure 22D shows the effect on total T cells over time. Data are presented as fold change over baseline (baseline = pre-dose) for each cell type.

[0143] [Figure 23-1]Figure 23A shows the effect of an exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with kidney lesions similar to lupus nephritis. Figure 23B shows the effect of an exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with kidney lesions similar to lupus nephritis. Figure 23C shows the effect of an exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with kidney lesions similar to lupus nephritis. Figure 23A shows the proteinuria scores measured weekly in mice after treatment with 40 μg / kg of an exemplary IL-2-Fc fusion protein or PBS vehicle control administered every three days from 3 to 18 weeks of age. Proteinuria scores are shown on the Y-axis, and the ages of the mice in weeks are shown on the X-axis. Figure 23B shows a series of graphs depicting proteinuria scores on the Y-axis in individual mice treated with a vehicle control or an exemplary IL-2-Fc fusion protein, as indicated on the X-axis. From left to right, the first panel shows proteinuria scores at 11 weeks of age, the middle panel shows scores at 12 weeks of age, and the last panel shows scores at 13 weeks of age. Figure 23C shows glomerular pathology quantified on the Y-axis at the end of the study (when mice reached 18 weeks of age) in individual mice treated with a vehicle control or an exemplary IL-2-Fc fusion protein, as indicated on the X-axis. [Figure 23-2]Figure 23A shows the effect of an exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with kidney lesions similar to lupus nephritis. Figure 23B shows the effect of an exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with kidney lesions similar to lupus nephritis. Figure 23C shows the effect of an exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with kidney lesions similar to lupus nephritis. Figure 23A shows the proteinuria scores measured weekly in mice after treatment with 40 μg / kg of an exemplary IL-2-Fc fusion protein or PBS vehicle control administered every three days from 3 to 18 weeks of age. Proteinuria scores are shown on the Y-axis, and the ages of the mice in weeks are shown on the X-axis. Figure 23B shows a series of graphs depicting proteinuria scores on the Y-axis in individual mice treated with a vehicle control or an exemplary IL-2-Fc fusion protein, as indicated on the X-axis. From left to right, the first panel shows proteinuria scores at 11 weeks of age, the middle panel shows scores at 12 weeks of age, and the last panel shows scores at 13 weeks of age. Figure 23C shows glomerular pathology quantified on the Y-axis at the end of the study (when mice reached 18 weeks of age) in individual mice treated with a vehicle control or an exemplary IL-2-Fc fusion protein, as indicated on the X-axis. DETAILED DESCRIPTION OF THE INVENTION

[0144] 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 enhance regulatory T cell (Treg) activity via the IL-2 pathway. Also provided are nucleic acid molecules encoding the IL-2 agents, expression vectors, host cells, compositions (e.g., pharmaceutical compositions), kits, containers, and methods for producing the IL-2 agents. The IL-2 agents and pharmaceutical compositions disclosed herein can be used (alone or in combination with other agents or treatment modalities) to treat, prevent, and / or diagnose disorders and conditions, e.g., disorders and conditions associated with T cell activity, e.g., a disorder or condition described herein (e.g., an autoimmune disorder described herein).

[0145] Immune responses are typically controlled by the recognition of specific foreign or self-antigens, communication between innate and adaptive immune pathways, crosstalk between B cells and T cells, and other factors. Some autoimmune diseases can be characterized by widespread recognition of self-antigens. These diseases can be treated with therapies that broadly enhance the process that protects self-antigens from immune attack. Tregs are a type of T cell that recognizes self-antigens. In response to antigen stimulation, Tregs release immunosuppressive cytokines and directly inhibit other T cells through cell-to-cell contact. Impaired Treg activity can lead to a wide range of autoimmune disorders (e.g., too few or underactive cells). IL-2 is a cytokine that triggers the expansion and activation of many cell types, but Tregs are typically much more sensitive to IL-2 than other cell types. Administration of low doses of IL-2 has been shown to preferentially and sustainably expand Treg cells in vivo, accompanied by 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-lived immunomodulatory (e.g., immunosuppressant) agents for a number of disorders (e.g., autoimmune indications).

[0146] The present disclosure is based, at least in part, on the discovery that IL-2 agents comprising human IL-2 polypeptides having specific combinations of amino acid substitutions described herein can have advantageous technical effects, such as 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, resulting in high selectivity for Tregs. IL-2 signaling promotes Treg suppressor function and promotes proliferation. Without wishing to be bound by theory, it is believed that Tregs activated by the IL-2 agents described herein can attenuate autoimmune activity through various mechanisms.

[0147] In one embodiment, the IL-2 agents described herein selectively bind to and activate regulatory T cells and inhibit other immune cell types (e.g., CD25 high It was found that the effects on regulatory T cells (T cells and NK cells) were simultaneously lacking. 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 agonist to maintain an active conformation and modulate the binding affinity of the IL-2 agonist to dimeric receptors comprising IL-2Rβ (CD122) and IL-2Rγ (CD132), and trimeric receptors comprising IL-2Rα (CD25) together with CD122 and CD132. In one embodiment, the IL-2 agonist described herein has optimal affinity to selectively bind and activate IL-2 signaling in regulatory T cells, selectively resulting in the activation and expansion of regulatory T cells 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 clearance pathway for IL-2 in vivo. For example, IL-2 agents described herein with reduced affinity for dimeric and trimeric IL-2 receptors have demonstrated extended half-lives, indicating that reduced affinity for the IL-2 receptor reduces clearance of the IL-2 agent in vivo. IL-2 agents described herein, for example, those with amino acid substitutions that increase stability and decrease affinity for the IL-2 receptor, can selectively activate regulatory T cells and exhibit increased half-life in vivo. IL-2 agents described herein, for example, 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, activation, 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 larger therapeutic window than low-dose IL-2.

[0148] There are various technical effects associated with the presence of the particular set of mutations described herein, e.g., 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 stable, e.g., 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 for 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, IL-2 agents containing the aforementioned mutations are believed to have reduced binding affinity to CD122 and / or CD132, increasing the potency and selectivity of the IL-2 agent for regulatory T cells (Tregs) compared to other T cell types. Accordingly, 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, IL-2 agents containing the aforementioned mutations are believed to have reduced or diminished binding ability and / or binding affinity to CD25, thereby improving the longevity of the IL-2 agent. Without wishing to be bound by theory, in one embodiment, IL-2 agents containing these mutations are believed to 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. 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 agents containing the aforementioned mutations particularly suitable for the treatment of disorders and conditions resulting from an aberrant immune response, such as autoimmune diseases.

[0149] Thus, in one embodiment, an IL-2 agonist (e.g., an IL-2 variant or IL-2 fusion protein) 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) has, inter alia, one or more (e.g., two, three, four, five, six, seven, or all) of the following properties compared to a wild-type IL-2 or reference IL-2 agonist that does not comprise the amino acid substitution: (i) enhanced or increased stability in vitro or in vivo; (ii) reduced or decreased binding capacity and / or binding affinity 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 agonist 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 (e.g., moderately reduced or decreased) binding ability and / or binding affinity to human CD25 in vitro and / or in vivo; (vi) regulatory T cells (e.g. Foxp3 + selective binding to T cells; (vii) selective activation of the IL-2 signaling pathway in regulatory T 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.

[0150] definition As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0151] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly dictates otherwise.

[0152] "About" and "approximately" generally refer to an acceptable degree of error for the quantity being measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values. When "about" or "approximately" precedes a series of numbers or ranges, it is understood that "about" or "approximately" may modify each number in the series or range. Similarly, when "at least," "greater than," "less than," "more than," or "within" precedes a series of numbers or ranges, it is understood that "at least," "greater than," "less than," "more than," or "within" may modify each number in the series or range. As used herein, a range includes both upper and lower limits.

[0153] The compositions and methods disclosed herein encompass polypeptides and nucleic acids having the designated sequences or sequences substantially identical or similar thereto, for example, sequences at least 85%, 90%, 95% or more identical to the designated sequences.

[0154] 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 minimum number of amino acid residues that are i) identical to or ii) conservative substitutions for 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. For example, an amino acid sequence containing 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.

[0155] 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 minimum number of identical nucleotides to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode polypeptides having a common functional activity, or encode a common structural polypeptide domain or 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.

[0156] 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 that can possess one or more activities of the naturally occurring sequence.

[0157] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[0158] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, for optimal alignment, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison purposes). In a typical embodiment, the length of the reference sequence that is aligned for comparison purposes is at least 30%, for example, at least 40%, 50%, 60%, for example, at least 70%, 80%, 90%, 100% of the length of the reference sequence. Then, the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are 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 this position.

[0159] 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.

[0160] The comparison of sequences and determination of percent identity between two sequences can be achieved using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is determined using the algorithm by Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) incorporated into the GAP program of the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, 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 specific embodiment, the 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, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. One suitable parameter set (which should be used unless otherwise specified) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4 and a frameshift gap penalty of 5.

[0161] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm by E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) 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.

[0162] The nucleic acid and protein sequences described herein can be used as a "query sequence" to search public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST program and the XBLAST program (version 2.0) by Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain nucleotide sequences homologous to the nucleic acids described herein, BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3. To obtain gapped alignments for comparison purposes, gapped BLAST, as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, can be used. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov.

[0163] As used herein, the term "hybridize under low stringency conditions, medium stringency conditions, high stringency conditions or very high stringency conditions" describes the conditions for hybridization and washing.Guidelines 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 this reference, and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by two washes in 0.2× SSC, 0.1% SDS at at least 50° C. (low stringency conditions allow the wash temperature to be increased to 55° C.); 2) 6× SSC at about 45° C., followed by one wash in 0.2× SSC, 0.1% SDS at 60° C. 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one additional wash in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) ultra-high stringency hybridization conditions in 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one additional wash in 0.2×SSC, 1% SDS at 65° C. Ultra-high stringency condition 4) is the appropriate condition and should be used unless otherwise specified.

[0164] It is understood that the molecules described herein may have additional conservative or non-essential amino acid substitutions that do not have a substantial effect on their function.

[0165] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both amino and acid functional groups and can be included in naturally occurring amino acid polymers. 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 above. As used herein, the term "amino acid" includes both D- and L-optical isomers and peptidomimetics.

[0166] 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 with 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).

[0167] The terms "polypeptide," "peptide," and "protein" (when single chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides can be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or can be the product of synthetic procedures.

[0168] As will be recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered within the scope of the present invention. For example, any protein fragment of a reference protein (meaning a polypeptide sequence that is at least one amino acid residue shorter than the reference polypeptide sequence but otherwise identical) having a length of more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 100 amino acids is provided herein. In another example, any protein comprising a stretch of about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 95, about 98%, or about 100% identical to any of the sequences described herein can be utilized in accordance with the present invention. In one embodiment, a protein sequence utilized in accordance with the present disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations set forth in any of the sequences provided or referenced herein.

[0169] 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 their analogs. A polynucleotide can be either single-stranded or double-stranded, and, if single-stranded, can be the coding strand or non-coding (antisense) strand. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, for example, by conjugation with a labeling component. A nucleic acid can be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin, which does not occur in nature or is linked to another polynucleotide in a non-natural configuration.

[0170] As used herein, the term "isolated" refers to material that has been removed from its original or native 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 the same polynucleotide or polypeptide that has been separated from some or all of the coexisting materials in the natural system by human intervention is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, but such a vector or composition is still isolated in that it is not part of the environment in which it is found in nature.

[0171] As used herein, the term "treating" a disorder, e.g., myeloma, means that a subject (e.g., a human) having a disorder, e.g., myeloma, and / or experiencing symptoms of the disorder, e.g., myeloma, in one embodiment, suffers from less severe symptoms and / or recovers more quickly when the antibody molecule is administered than when the antibody molecule is not administered. In one embodiment, if the myeloma is treated, a bone marrow biopsy will show fewer clonal plasma cells after effective treatment of the myeloma. For example, a diagnostic assay will detect fewer clonal plasma cells in a biological sample of a subject after administration of an antibody molecule described herein for effective treatment of myeloma. Other assays, such as urine tests or blood tests, can also be used to monitor treatment in a patient or to detect the presence, e.g., a decreased presence (or absence), of symptoms of myeloma after treatment of myeloma in a subject. In one embodiment, if the myeloma is treated, the level of β2 microglobulin (β2M) in the serum or urine will decrease after effective treatment of the myeloma. Treatment may, for example, partially or completely alleviate, improve, alleviate, inhibit, or reduce the severity of one or more of the effects or symptoms, characteristics, and / or causes of a disorder, such as myeloma, and / or reduce the incidence, and optionally delay its onset. In one embodiment, treatment is for subjects who do not show specific signs of a disorder, such as myeloma, and / or who show only early signs of a disorder, such as nephropathy. In one embodiment, treatment is for subjects who show one or more established signs of a disorder, such as myeloma. In one embodiment, treatment is for subjects who have been diagnosed with a disorder, such as myeloma.

[0172] As used herein, the term "preventing" a disorder, e.g., myeloma, means that a subject (e.g., a human) is less likely to have the disorder, e.g., myeloma, when the subject is administered the antibody molecule.

[0173] Various aspects of the compositions and methods herein are described in further detail below. Additional definitions are set forth throughout the specification.

[0174] IL-2 agonist 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 comprises an IL-2 variant comprising one or more amino acid changes (e.g., substitutions) described herein. In one embodiment, the IL-2 agent comprises an IL-2 variant comprising one or more amino acid changes (e.g., substitutions) set forth in Table 9. In one embodiment, the IL-2 agent comprises an IL-2 variant comprising an amino acid sequence set forth 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 set forth herein, e.g., in Table 10. One or more amino acid changes (e.g., substitutions), alone or in combination, can confer one or more desired biological properties described herein. In one embodiment, the IL-2 agonist can modulate (e.g., increase) the expansion, survival, activation, and / or function of Tregs. In one embodiment, the modulation is selective or specific for Tregs. For example, the IL-2 agonist can modulate activity in Tregs, but has limited or no ability to promote activity in non-regulatory T cells. In one embodiment, the IL-2 agonist comprises a polypeptide (sometimes referred to herein as an "IL-2 polypeptide agonist").

[0175] IL-2 variants In one embodiment, the IL-2 agent comprises an IL-2 variant, eg, an IL-2 variant described herein.

[0176] In one embodiment, the IL-2 variant comprises an IL-2 polypeptide (e.g., a human IL-2 polypeptide) described herein, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises one or more amino acid changes (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.

[0177] Without wishing to be bound by theory, in one embodiment, it is believed that IL-2 variants described herein having reduced human CD25 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) relative to 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 mutated IL-2 polypeptides to identify IL-2 variants having binding affinities for human CD25 and / or human CD122 / CD132 in the desired range.

[0178] 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 changes (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) altered (e.g., enhanced or increased) expression in vitro and / or in vivo; ii) altered (e.g., reduced or decreased) aggregation in vitro and / or in vivo; iii) altered (e.g., enhanced or increased) stability in vitro and / or in vivo; iv) altered (e.g., enhanced or increased) half-life in vitro and / or in vivo; v) altered (e.g., slowed or decreased) turnover and / or clearance in vivo; vi) altered (e.g., reduced or decreased) 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) altered (e.g., reduced or decreased) binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo; ix) altered (e.g., reduced or decreased) binding capacity and / or binding affinity of human CD132 in vitro and / or in vivo; x) altered (e.g., reduced or decreased) binding capacity and / or binding affinity to the dimeric IL-2 receptor comprising human CD122 and human CD132 in vitro and / or in vivo; xi) altered (e.g., enhanced, increased, decreased, diminished, and / or selective) binding to Tregs in vitro and / or in vivo; xii) altered (e.g., enhanced, increased, decreased, diminished, and / or selective) activation of the IL-2 signaling pathway in Tregs in vitro and / or in vivo; xiii) An altered (e.g., enhanced, increased, decreased, diminished, and / or selective) ability to induce or promote the expansion, activity, survival, and / or proliferation of Tregs in vitro and / or in vivo.

[0179] 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 bacteria or mammalian cells) compared to wild-type IL-2. In one embodiment, the IL-2 variant has enhanced or increased expression (e.g., in bacteria 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 at a higher or increased level in vitro and / or in vivo, 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, compared to 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 assaying protein concentration. In one embodiment, the IL-2 variant is expressed at a higher or increased 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, compared to 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 assaying protein concentration.

[0180] 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 at a lower or reduced level in vitro and / or in vivo, 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 reduced aggregation, 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 melting temperature analysis (e.g., using fluorescence measurements), 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 reduced aggregation, 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 melting temperature analysis (e.g., using fluorescence measurements), dynamic light scattering, and / or size exclusion chromatography.

[0181] 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-variant described herein exhibits enhanced or increased stability in vitro and / or in vivo, 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 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%, 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 130%, about 131%, about 132%, about 132, about 133%, about 134%, about The stability may be increased by 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, or by, 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.

[0182] 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 has an enhanced or increased half-life in vitro and / or in vivo, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, compared to 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 ELISA, flow cytometry, and / or mass spectrometry. , 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 increased, 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 increased half-life.

[0183] 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 a lower, decreased or reduced rate or level of turnover and / or clearance in vivo, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, or more, as determined, for example, by ELISA, flow cytometry, and / or mass spectrometry, compared to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant. %, 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, or has a rate or level of 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.

[0184] In one embodiment, the IL-2 has 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 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 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.

[0185] In one embodiment, the IL-2 variant has 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 enhanced or increased resistance to proteolysis compared to wild-type IL-2. In one embodiment, the IL-2 variant has 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.

[0186] In one embodiment, the IL-2 variant has 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 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 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 the 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 has a reduced or decreased binding ability and / or binding affinity, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 5 ... or have a binding ability and / or affinity that is reduced by 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, or that is reduced by, 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.

[0187] In one embodiment, the IL-2 variant has low affinity for CD25 (e.g., human CD25), 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 ... 5, 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 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, a dissociation constant (K) of about 400 to about 500 pM, or, for example, greater than 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 D ) to join them.

[0188] In one embodiment, the IL-2 variant has low affinity for CD25 (e.g., human CD25), e.g., as determined by surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding), 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, or about 10 nM, or e.g., about 0.2 to about 5 nM, about 0.5 to about 2 nM, about 1 to about 1.5 nM, about 0.1 to about 0.2 nM, e.g., about 0.2 to about 0 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 0.8 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 a dissociation constant (K) of greater than about 0.5 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, 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 D ) to join them.

[0189] In one embodiment, the IL-2 variant has altered (e.g., reduced or decreased) binding ability and / or binding affinity for 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 reduced or decreased binding ability and / or binding affinity for human CD132 compared to wild-type IL-2. In one embodiment, the IL-2 variant has reduced or decreased binding ability and / or binding affinity for human CD132 compared to a reference IL-2 variant. In one embodiment, the binding ability and / or binding affinity of the IL-2 variant for 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.

[0190] In one embodiment, the IL-2 variant has an altered (e.g., reduced or decreased) binding ability 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 ability 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 ability 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.

[0191] In one embodiment, the IL-2 variant has a reduced or decreased binding affinity, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, to a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) compared to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, as determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding). , 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 decreased, or have a binding ability and / or binding affinity that is reduced by, 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.

[0192] In one embodiment, the IL-2 variant has low affinity for the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), e.g., as determined by yeast surface display, e.g., between about 0.2 and 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, 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 69, about 70, about 71, 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 M, 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 greater than, 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.

[0193] In one embodiment, the IL-2 variant has low affinity for CD122 / CD132 heterodimers (e.g., human CD122 / CD132 heterodimers), 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, or about 40 nM, as determined, for example, by surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding). 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 to about 0.5 nM, about 0.5 to about 1 nM, about 1 to about 2 nM, about 2 to about 5 nM, about 5 to about 10 nM, about 10 to about 15 nM, about 15 to about 20 nM, about 20 to about 25 nM, about 25 to about 30 nM, about 30 to about 40 nM M, about 40 to about 50 nM, about 50 to about 60 nM, about 60 to about 70 nM, about 70 to about 80 nM, about 80 to about 90 nM, about 90 to about 100 nM, about 100 to about 110 nM, about 110 to about 120 nM, about 120 to about 130 nM, about 130 to about 14 0 nM, about 140 to about 150 nM, about 150 to about 160 nM, about 160 to about 170 nM, about 170 to about 180 nM, about 180 to about 190 nM, about 190 to about 200 nM, about 200 to about 210 nM, about 210 to about 220 nM, about 220 to about 230 nM, about 230 to about 240 nM, about 240 to about 250 nM, about 250 to about 260 nM, about 260 to about 270 nM, about 270 to about 280 nM, about 280 to about 290 nM, or about 290 to about 300 nM, or for example, about 0.2, about 0.and binds with a dissociation constant (KD) of greater than 5, about 1, about 2, about 5, about 10, about 15, 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 greater than about 300 nM.

[0194] 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.

[0195] 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, hi 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.

[0196] In one embodiment, the IL-2 variant selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo relative 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, and is at least 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, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 10 or about 3000.

[0197] In one embodiment, the IL-2 variant selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo relative to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, e.g., 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, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7500, 8000, 8500, 9000, 9500, 10 ... 00, 800, 900, 1000, 1500, 2000, 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 about 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.

[0198] In one embodiment, the IL-2 variant has an altered (e.g., enhanced, increased, and / or selective) ability to induce or promote the 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 the 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 the 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 the 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 the 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, hi 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.

[0199] In one embodiment, the IL-2 variant has enhanced or increased potency and / or ability to induce or promote regulatory T cell activity, 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 determined, e.g., by flow cytometry, compared to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant. , about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more reduced, or has an EC50 for Tregs that is reduced by, 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.

[0200] In one embodiment, the IL-2 variant has a reduced or decreased potency and / or ability to induce or promote regulatory T cell activity, as determined by flow cytometry, compared to an IL-2 agent comprising wild-type IL-2 or an IL-2 agent comprising a reference IL-2 variant, 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 ... 0%, about 95%, about 100% or more increased, or have an EC50 for Tregs that is decreased by, 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.

[0201] 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.

[0202] In one embodiment, the IL-2 variant has one or more structural and / or functional properties that are the same or substantially the same as wild-type IL-2 or a reference IL-2 variant.

[0203] 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 that contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid changes (e.g., substitutions) described herein.

[0204] For purposes of this disclosure, position numbering for IL-2 variants begins with the first amino acid after the signal peptide of an exemplary wild-type (WT) human IL-2 polypeptide: [ka] The corresponding amino acid sequence without the signal peptide is shown as SEQ ID NO:1031.

[0205] In one embodiment, the IL-2 agonist comprises amino acid alterations (eg, substitutions) at positions corresponding to human IL-2 (eg, comprising the amino acid sequence of SEQ ID NO: 1031).

[0206] In one embodiment, the IL-2 variant is [ka] It contains the amino acid sequence of wherein X3 is T or A, X16 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, with the proviso that 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.

[0207] In one embodiment, the IL-2 variant comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) amino acid changes (e.g., substitutions) at a position described herein. In one embodiment, the IL-2 variant comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) amino acid changes (e.g., substitutions) at a position selected from T3, H16, 128, K35, R38, F42, E68, V69, Q74, D84, S87, N88, 192, C125, or Q126.

[0208] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position T3. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position H16. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position I28. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position K35. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position R38. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position F42. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position E68. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position V69. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position Q74. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position D84. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position S87. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position N88. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position I92. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position C125. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position Q126.

[0209] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at positions V69, Q74, or a combination thereof. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a 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.

[0210] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position H16, I92, D84, or a combination thereof. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a 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.

[0211] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position 192, optionally the amino acid substitution is 192S. In one embodiment, the IL-2 variant comprises the amino acid substitution 192S.

[0212] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position D84, optionally the amino acid substitution is D84V. In one embodiment, the IL-2 variant comprises the amino acid substitution D84V.

[0213] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position K35, R38, F42, E68, or a combination thereof. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position K35, optionally the amino acid substitution is K35E. In one embodiment, the IL-2 variant comprises the amino acid substitution K35E.

[0214] In one embodiment, the IL-2 variant comprises an amino acid change (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.

[0215] In one embodiment, the IL-2 variant comprises an amino acid change (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.

[0216] In one embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) 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 amino acid changes (e.g., substitutions) at one, two, or all of positions R38, F42, or E68.

[0217] In one embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at (i) (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35, and (ii) one, two, or all of (a) positions H16, I92, or D84; or (b) one, two, or all of positions R38, F42, or E68.

[0218] In one embodiment, the IL-2 variant contains amino acid changes (e.g., substitutions) at: (i) (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35, and (ii) one, two, or all of (a) positions H16, I92, or D84; and (b) one, two, or all of positions R38, F42, or E68.

[0219] In one embodiment, the IL-2 variant comprises amino acid changes (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 H16L.

[0220] In one embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and 192, optionally the amino acid substitutions are V69A, Q74P, and 192S, respectively. In one embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and 192S.

[0221] In one embodiment, the IL-2 variant comprises amino acid changes (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.

[0222] In one embodiment, the IL-2 variant comprises amino acid changes (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 amino acid substitutions V69A, Q74P, and R38Q.

[0223] In one embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and F42, optionally the amino acid substitutions are V69A, Q74P, and F42Q, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions V69A, Q74P, and F42Q.

[0224] In one embodiment, the IL-2 variant comprises amino acid changes (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.

[0225] In one embodiment, the IL-2 variant comprises amino acid changes (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.

[0226] In one embodiment, the IL-2 variant comprises amino acid changes (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 amino acid substitutions V69A, Q74P, K35E, and H16N.

[0227] In one embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, K35, H16, and R38, 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.

[0228] In one embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, H16, and R38, optionally with the amino acid substitutions 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 R38Q.

[0229] In one embodiment, the IL-2 variant comprises an amino acid change (eg, a substitution) at position I28, E68, S87, N88, Q126, or a combination thereof.

[0230] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position 128, optionally the amino acid substitution is 128T or 128F. In one embodiment, the IL-2 variant comprises the amino acid substitution 128T. In one embodiment, the IL-2 variant comprises the amino acid substitution 128F.

[0231] In one embodiment, the IL-2 variant comprises an amino acid change (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.

[0232] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position S87, optionally the amino acid substitution is S87 R. In one embodiment, the IL-2 variant comprises the amino acid substitution S87R.

[0233] In one embodiment, the IL-2 variant comprises an amino acid change (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.

[0234] In one embodiment, the IL-2 variant comprises an amino acid change (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. 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.

[0235] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position C125, optionally the amino acid substitution is C125S. In one embodiment, the IL-2 variant comprises the amino acid substitution C125S.

[0236] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., a substitution) at position T3, optionally the amino acid substitution is T3A. In one embodiment, the IL-2 variant comprises the amino acid substitution T3A.

[0237] In one embodiment, the IL-2 variant comprises amino acid changes (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 amino acid substitutions V69A, Q74P, and C125S.

[0238] In one embodiment, the IL-2 variant comprises an amino acid change (eg, a substitution) at position T3, H16, I92, or a combination thereof, optionally the amino acid substitutions are T3A, H16N, and I92S, respectively.

[0239] In one embodiment, the IL-2 variant comprises amino acid changes (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.

[0240] In one embodiment, the IL-2 variant comprises amino acid changes (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.

[0241] In one embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions H16, V69, Q74, 192, and C125, optionally the amino acid substitutions are H16L, V69A, Q74P, 192S, and C125S, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions H16L, V69A, Q74P, 192S, and C125S.

[0242] In one embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions T3, V69, Q74, and C125, optionally the amino acid substitutions are T3A, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 variant comprises amino acid substitutions T3A, V69A, Q74P, and C125S.

[0243] In one embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions T3, H16, V69, Q74, and C125, 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.

[0244] In one embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions T3, V69, Q74, I92, and C125, 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.

[0245] In one embodiment, the IL-2 variant comprises amino acid changes (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.

[0246] In one embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions H16, R38, V69A, and Q74P, 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.

[0247] 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.

[0248] There are various technical effects associated with the presence of the particular set of mutations described herein, e.g., 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, it is believed that IL-2 variants comprising the aforementioned mutations also have reduced binding affinity to CD122 and / or CD132, increasing the potency and selectivity of the IL-2 variant for regulatory T cells (Tregs) compared to other T cell types. Without wishing to be bound by theory, it is also believed that in one embodiment, IL-2 variants comprising the aforementioned mutations are significantly more stable, e.g., 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 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 more stable. Without wishing to be bound by theory, in one embodiment, it is further believed that IL-2 variants containing the aforementioned mutations have reduced or diminished binding ability and / or binding affinity to CD25, thereby improving the longevity of the IL-2 variants. Without wishing to be bound by theory, it is also believed that in one embodiment, IL-2 variants containing these mutations do 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. Without wishing to be bound by theory, in one embodiment, IL-2 variants comprising the aforementioned mutations are further believed to have reduced disulfide mispairing and improved stability due to the presence of, for example, the C125S mutation.In one embodiment, an IL-2 agent comprising the H16L mutation has reduced binding affinity for 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 these mutations particularly suitable for the treatment of disorders and conditions resulting from aberrant immune responses, such as autoimmune diseases.

[0249] 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 in combination with amino acid substitutions at positions V69, Q74, and C125 (e.g., H16L, V69A, Q74P, and C125S) has, inter alia, one or more (e.g., two, three, four, five, six, seven, or all) of the following properties 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) enhanced or increased stability in vitro and / or in vivo in human C16; (iii) reduced or decreased binding capacity and / or affinity for 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 binding capacity and / or affinity for human CD25 in vitro and / or in vivo; (vi) reduced or decreased binding capacity and / or affinity for regulatory T cells (e.g., Foxp3 + (vii) selective activation of the IL-2 signaling pathway in regulatory T 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.

[0250] In one embodiment, the IL-2 variant 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, SEQ ID NO: 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 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 thereto.

[0251] 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 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 thereto. 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 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 thereto. 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 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 thereto. In one embodiment, the IL-2 variant comprises or consists of the 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 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 thereto.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 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 thereto. 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 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 thereto.

[0252] 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.

[0253] Without wishing to be bound by theory, in one embodiment, it is believed that an IL-2 variant comprising or consisting of the amino acid sequence of SEQ ID NO: 5, or a functional fragment thereof, may have at least one or more of the following advantageous properties: (i) having reduced binding affinity to CD122 and / or CD132, increasing 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 decreased binding capacity and / or binding affinity to CD25, improving the longevity of the IL-2 agent; (iv) not substantially promoting 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) having reduced disulfide mispairing 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 for 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 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.

[0254] Thus, in one embodiment, an IL-2 variant comprising or consisting of the amino acid sequence 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 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 or fewer amino acids thereto, has, inter alia, one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties compared to wild-type IL-2 or a reference IL-2 variant that does not contain the amino acid substitutions: (i) exhibits a specific activity in vitro or in vivo. (ii) reduced or decreased binding capacity and / or binding affinity 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, 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 regulatory T 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.

[0255] As further described herein, the present disclosure provides IL-2 fusion proteins, IL-2 complexes, and IL-2 conjugates that include the IL-2 variants described herein. In one embodiment, one or more different and / or improved properties ascribed to the IL-2 variants described herein are maintained, transferred, or conferred 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.

[0256] In one embodiment, the IL-2 variant comprises a polypeptide (sometimes 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.

[0257] IL-2 fusion protein In one embodiment, the IL-2 agent comprises an IL-2 fusion protein, such as an IL-2 fusion protein described herein.

[0258] 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 changes (e.g., substitutions) set forth in Table 9. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence set forth 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 set forth herein, e.g., in Table 10.

[0259] Without wishing to be bound by theory, in one embodiment, it is believed that IL-2 fusion proteins described herein that have reduced human CD25 and / or reduced human CD122 / CD132 binding affinity compared to IL-2 fusion proteins comprising wild-type human IL-2 or reference IL-2 fusion proteins may have improved potency and / or selectivity for binding to and activating regulatory T cells (Tregs) over IL-2 fusion proteins comprising wild-type IL-2 or other IL-2 fusion proteins.

[0260] 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, e.g., different and / or improved properties, described herein, 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 changes (e.g., substitutions) that provide different and / or improved properties 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) of the following different and / or improved properties (e.g., as determined by the assays described herein) compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein: i) altered (e.g., enhanced or increased) expression in vitro and / or in vivo; ii) altered (e.g., reduced or decreased) aggregation in vitro and / or in vivo; iii) altered (e.g., enhanced or increased) stability in vitro and / or in vivo; iv) altered (e.g., enhanced or increased) half-life in vitro and / or in vivo; v) altered (e.g., slowed or decreased) turnover and / or clearance in vivo; vi) altered (e.g., reduced or decreased) 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) altered (e.g., reduced or decreased) binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo; ix) altered (e.g., reduced or decreased) binding capacity and / or binding affinity of human CD132 in vitro and / or in vivo; x) altered (e.g., reduced or decreased) binding capacity and / or binding affinity to the dimeric IL-2 receptor comprising human CD122 and human CD132 in vitro and / or in vivo; xi) altered (e.g., enhanced, increased, decreased, diminished, and / or selective) binding to Tregs in vitro and / or in vivo; xii) altered (e.g., enhanced, increased, decreased, diminished, and / or selective) activation of the IL-2 signaling pathway in Tregs in vitro and / or in vivo; xiii) An altered (e.g., enhanced, increased, decreased, diminished, and / or selective) ability to induce or promote the expansion, activity, survival, and / or proliferation of Tregs in vitro and / or in vivo.

[0261] 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 bacteria 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 bacteria 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 at a higher or increased level in vitro and / or in vivo, 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, compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, as determined, e.g., by assaying protein concentration. In one embodiment, the IL-2 fusion protein is expressed at a higher or increased 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, compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, as determined, e.g., by an assay of protein concentration.

[0262] In one embodiment, the IL-2 fusion protein has altered (e.g., lowered 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 lowered or decreased aggregation compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has lowered 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 at a lower or reduced level in vitro and / or in vivo, 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 reduced aggregation, compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, as determined, for example, by melting temperature analysis (e.g., using fluorescence measurements), dynamic light scattering, and / or size exclusion chromatography. In one embodiment, the IL-2 fusion protein 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 reduced aggregation, compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, as determined, for example, by melting temperature analysis (e.g., using fluorescence measurements), dynamic light scattering and / or size exclusion chromatography.

[0263] 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 has enhanced or increased stability in vitro and / or in vivo, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 95%, about 100%, about 105%, 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 1 0%, 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 increased stability, 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 increased stability.

[0264] 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 an enhanced or increased half-life in vitro and / or in vivo, 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 5 ... The half-life may be increased by 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, or by, 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.

[0265] In one embodiment, the IL-2 fusion protein has altered (e.g., lowered or decreased) metabolic 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 lowered or decreased metabolic turnover compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has lowered or decreased metabolic turnover compared to a reference IL-2 fusion protein. In one embodiment, the metabolic 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 metabolic 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 lower, decreased or reduced rate or level of turnover and / or clearance in vivo, 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 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%, 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 A decrease of 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, or having a rate or level of decrease 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.

[0266] In one embodiment, the IL-2 fusion proteins provided by the present disclosure comprise the property of having 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 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 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.

[0268] In one embodiment, the IL-2 fusion protein has altered (e.g., reduced or decreased) binding ability and / or binding affinity for 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 reduced or decreased binding ability and / or binding affinity for human CD25 compared to wild-type human IL-2. In one embodiment, the IL-2 fusion protein has reduced or decreased binding ability and / or binding affinity for 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 for 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 has a reduced or decreased binding ability and / or binding affinity, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, to CD25 (e.g., human CD25) compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, as determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding). , 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 decreased, or have a binding ability and / or binding affinity that is reduced by, 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.

[0269] In one embodiment, the IL-2 fusion protein has low affinity for CD25 (e.g., human CD25), 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 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 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, 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 700, about 710, about 720, about 730, about 740, about 750, about 760, about 770, about 780, about 7 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 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 greater than, 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. D ) to join them.

[0270] In one embodiment, the IL-2 fusion protein has low affinity for CD25 (e.g., human CD25), e.g., as determined by surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding), for example, 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, or about 10 nM, or for example, about 0.2 to about 5 nM, about 0.5 to about 2 nM, about 1 to about 1.5 nM, about 0.1 to about 0.2 nM, e.g., 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 0.8 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 a dissociation constant (K) of 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, about 9 to about 10 nM, or greater than, 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 about 10 nM D ) to join them.

[0271] In one embodiment, the IL-2 fusion protein has altered (e.g., reduced or decreased) binding ability and / or binding affinity for 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 reduced or decreased binding ability and / or binding affinity for human CD132 compared to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has reduced or decreased binding ability and / or binding affinity for 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 for 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.

[0272] 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.

[0273] 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. 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.

[0274] In one embodiment, the IL-2 fusion protein has a reduced or decreased binding affinity, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 35%, about 40%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 115%, 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 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 750%, or have a binding ability and / or affinity that is reduced by 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, or that is reduced by, 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.

[0275] In one embodiment, the IL-2 fusion protein has low affinity for the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), 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 7nM, about 7 to about 8nM, about 8 to about 9nM, about 9 to about 10nM, about 10 to about 11nM, about 11 to about 12nM, about 12 to about 13nM, about 13 to about 14nM, about 14 to about 15nM, about 15 to about 16nM, about 16 to about 17nM , about 17 to about 18 nM, about 18 to about 19 nM, or about 19 to about 20 nM, or greater than, 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.

[0276] In one embodiment, the IL-2 fusion protein has low affinity for the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), 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, or about 40 nM, as determined, for example, by surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding). 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 1 0nM, about 2 to about 5nM, about 0.2 to about 0.5nM, about 0.5 to about 1nM, about 1 to about 2nM, about 2 to about 5nM, about 5 to about 10nM, about 10 to about 15nM, about 15 to about 20nM, about 20 to about 25nM, about 25 to about 30nM, about 30 to about 40n M, about 40 to about 50 nM, about 50 to about 60 nM, about 60 to about 70 nM, about 70 to about 80 nM, about 80 to about 90 nM, about 90 to about 100 nM, about 100 to about 110 nM, about 110 to about 120 nM, about 120 to about 130 nM, about 130 to about 14 0 nM, about 140 to about 150 nM, about 150 to about 160 nM, about 160 to about 170 nM, about 170 to about 180 nM, about 180 to about 190 nM, about 190 to about 200 nM, about 200 to about 210 nM, about 210 to about 220 nM, about 220 to about 230 nM, about 230 to about 240 nM, about 240 to about 250 nM, about 250 to about 260 nM, about 260 to about 270 nM, about 270 to about 280 nM, about 280 to about 290 nM, or about 290 to about 300 nM, or for example, about 0.2, about 0.and binds with a dissociation constant (KD) of greater than 5, about 1, about 2, about 5, about 10, about 15, 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 greater than about 300 nM.

[0277] 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. 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.

[0278] 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, hi 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.

[0279] In one embodiment, the IL-2 fusion protein selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by flow cytometry, e.g., 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 or about 3000.

[0280] In one embodiment, the IL-2 fusion protein selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by flow cytometry, e.g., 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 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, about 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.

[0281] In one embodiment, the IL-2 fusion protein has an altered (e.g., enhanced, increased, and / or selective) ability to induce or promote the 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 the 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 the 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 the 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 the 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, hi 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.

[0282] In one embodiment, the IL-2 fusion protein has enhanced or increased potency and / or ability to induce or promote regulatory T cell activity compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by flow cytometry, 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 or has an EC50 for Tregs that is reduced by 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or that is reduced by, 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.

[0283] In one embodiment, the IL-2 fusion protein has a reduced or decreased potency and / or ability to induce or promote regulatory T cell activity compared to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, as determined by flow cytometry, 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 increased, or has an EC50 for Tregs that is decreased by, 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.

[0284] 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.

[0285] In one embodiment, the IL-2 fusion protein has one or more of the same or substantially the same structural and / or functional properties as an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein.

[0286] 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 that contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid changes (e.g., substitutions) described herein.

[0287] 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.

[0288] In one embodiment, the IL-2 fusion protein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) amino acid changes (e.g., substitutions) at a position in IL-2 described herein. In one embodiment, the IL-2 fusion protein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) amino acid changes (e.g., substitutions) at a position selected from T3, H16, 128, K35, R38, F42, E68, V69, Q74, D84, S87, N88, 192, C125, or Q126.

[0289] In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position T3 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position H16 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position I28 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position K35 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position R38 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position F42 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position E68 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position V69 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position Q74 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position D84 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position S87 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position N88 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position I92 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position C125 in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position Q126 in IL-2.

[0290] In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at positions V69, Q74, or both in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a 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.

[0291] In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position H16, I92, D84, or both in IL-2. In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position H16, optionally the amino acid substitution is H16N, H16L, or H16D in 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.

[0292] In one embodiment, the IL-2 fusion protein comprises an amino acid change (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.

[0293] In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a 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.

[0294] In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a 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 change (e.g., a 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.

[0295] In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position R38, optionally the amino acid substitution is R38E, R38N, or R38Q in 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.

[0296] In one embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., a substitution) at position F42, optionally the amino acid substitution is F42K or F42Q in 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 f...

Claims

1. An IL-2 variant comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, and amino acid substitutions H16L, V69A and Q74P.

2. An IL-2 fusion protein comprising an IL-2 variant and an Fc region, An IL-2 fusion protein, wherein said IL-2 variant comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:1, and the amino acid substitutions H16L, V69A and Q74P.

3. The IL-2 fusion protein described in claim 2, wherein the Fc region includes an IgG1 Fc region.

4. The IL-2 fusion protein of claim 3, wherein the Fc region of IgG1 comprises the amino acid substitution N297G according to EU numbering.

5. An Il-2 fusion protein described in any one of claims 2 to 4, wherein the Fc region includes an Fc region of human IgG1 allotype m3.

6. An IL-2 fusion protein described in any one of claims 2 to 5, wherein the Fc region comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1003.

7. The IL-2 fusion protein of claim 6, wherein the Fc region comprises an amino acid sequence as shown in SEQ ID NO:1003.

8. An IL-2 fusion protein according to any one of claims 2 to 7, wherein the IL-2 conjugate is a fusion protein.

9. The IL-2 fusion protein of claim 8, wherein the Fc region is fused to the C-terminus of the IL-2 variant.

10. The IL-2 fusion protein of claim 8 or claim 9, wherein the fusion protein comprises a linker between the IL-2 variant and the Fc region.

11. The IL-2 fusion protein of claim 10, wherein the linker comprises the amino acid sequence shown in SEQ ID NO:

48.

12. An IL-2 fusion protein described in any one of claims 8 to 11, wherein the fusion protein forms a dimer.

13. The IL-2 fusion protein of claim 12, wherein the fusion protein forms a homodimer.

Images