Interleukin-2 formulations and uses thereof

Abstract

Disclosed are IL-2 formulations comprising IL-2 variants, and methods, compositions, and uses thereof. The IL-2 formulations described herein can be used to treat and / or prevent various diseases or conditions.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefits 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 the above applications are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application contains a sequence list that has been electronically submitted in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy created on July 20, 2020, is named P2029-7028WO_SL.txt and has a size of 1,728,528 bytes. Background Technology

[0005] Interleukin-2 (IL-2) is a cytokine that regulates the activity of the immune system. It is produced by leukocytes (such as T cells, natural killer (NK) cells, dendritic cells, and macrophages) in response to antigens or mitotic 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, distinguishing foreign antigens from host antigens. IL-2 mediates its action by binding to its receptor, thereby activating downstream signal transduction events.

[0006] Human IL-2 is an FDA-approved drug for the treatment of diseases such as metastatic renal cell carcinoma and melanoma. Due to serious toxicities associated with IL-2 treatment, its use in eligible patients is sometimes limited, and only a small percentage of eligible patients 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 generally effective for its approved indications.

[0007] For patients with a variety of diseases and conditions suitable for IL-2 treatment, there remains a need for novel IL-2-based drugs with sufficient characteristics to develop safe and effective therapeutic agents. Summary of the Invention

[0008] The present disclosure provides, at least in part, IL-2 agents (e.g., IL-2 variants, IL-2 fusion proteins, IL-2 complexes, and IL-2 conjugates) comprising one or more amino acid changes (e.g., substitutions) in IL-2 and comprising 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 making the IL-2 agents are also provided. The IL-2 agents disclosed herein are useful (alone or in combination with other agents or therapeutic modalities) in the treatment, prevention, and / or diagnosis of diseases, e.g., the diseases and disorders disclosed herein.

[0009] The present disclosure is based, at least in part, on the discovery that a combination of mutations in IL-2 that stabilize the protein, reduce its affinity for CD122 (e.g., CD122 / CD132 heterodimer), and / or reduce its affinity for CD25 or no more than a minimal effect, can be used to selectively enhance regulatory T cell (Treg) activity through the IL-2 pathway, resulting in a favorable therapeutic effect in the treatment of diseases and disorders, e.g., autoimmune diseases. IL-2 agents comprising such mutations are suitable for treating disorders caused by aberrant immune responses, e.g., autoimmune diseases.

[0010] Accordingly, in some aspects, the present disclosure provides an IL-2 agent, e.g., an IL-2 agent 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):

[0011] a) expresses at a higher or increased level, e.g., by 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 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.g., 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 a protein concentration assay;

[0012] b) aggregates at a lower or reduced level in vitro and / or in vivo, e.g., by 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 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.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by melting temperature analysis (e.g., using a fluorimetric method), dynamic light scattering, and / or size exclusion chromatography;

[0013] c) has enhanced or increased stability in vitro and / or in vivo, e.g., by 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 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.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by expression in yeast surface display, expression in mammalian cells, chromatography, circular dichroism assay or related spectroscopic techniques, and / or melting temperature analysis (e.g., using a fluorimetric method);

[0014] d) has an enhanced or increased half-life in vitro and / or in vivo, e.g., by 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 greater than 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.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by ELISA, flow cytometry, and / or mass spectrometry;

[0015] e) has a lower, reduced, or decreased turnover and / or clearance rate or level in vivo, e.g., by 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 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.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by ELISA, flow cytometry, and / or mass spectrometry;

[0016] f) reduced or decreased or substantially unchanged binding affinity for CD25 (e.g., human CD25), e.g., decreased by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more (e.g., about 1% to about 20%, about 2% to about 15%, or about 5% to about 10%), or decreased or increased by no more than about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%, or decreased 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, or decreased or increased by no more than 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, e.g., relative to an IL-2 preparation comprising wild-type IL-2 or an IL-2 preparation comprising a reference IL-2 variant, e.g., as determined by yeast surface display, bio-layer interferometry (e.g., Octet binding), and / or surface plasmon resonance (e.g., Biacore);

[0017] g) binds CD25 (e.g., human CD25) with low affinity, e.g., with a dissociation constant (K D), e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 pM, or, e.g., from about 10 pM to about 490 pM, from about 20 pM to about 480 pM, from about 30 pM to about 470 pM, from about 40 pM to about 460 pM, from about 50 pM to about 450 pM, from about 60 pM to about 440 pM, from about 70 pM to about 430 pM, from about 80 pM to about 420 pM, from about 90 pM to about 410 pM, from about 100 pM to about 400 pM, from about 110 pM to about 390 pM, from about 120 pM to about 380 pM, from about 130 pM to about 370 pM, from about 140 pM to about 360 pM, from about 150 pM to about 350 pM, from about 160 pM to about 340 pM, from about 170 pM to about 330 pM, from about 180 pM to about 320 pM, from about 190 pM to about 310 pM, from about 200 pM to about 300 pM, from about 210 pM to about 290 pM, from about 220 pM to about 280 pM, from about 230 pM to about 270 pM, from about 240 pM to about 260 pM, or, e.g., from about 5 pM to about 450 pM, from about 5 pM to about 400 pM, from about 5 pM to about 350 pM, from about 5 pM to about 300 pM, from about 5 pM to about 250 pM, from about 5 pM to about 200 pM, from about 5 pM to about 150 pM, from about 5 pM to about 100 pM, from about 5 pM to about 50 pM, or, e.g., from about 10 pM to about 500 pM, from about 20 pM to about 500 pM, from about 50 pM to about 500 pM, from about 100 pM to about 500 pM, from about 150 pM to about 500 pM, from about 200 pM to about 500 pM, from about 250 pM to about 500 pM, from about 300 pM to about 500 pM, from about 350 pM to about 500 pM, from about 400 pM to about 500 pM, from about 450 pM to about 500 pM, or, e.g., 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, e.g., as determined by yeast surface display;

[0018] h) binds to CD25 (e.g., human CD25) with low affinity, e.g., with a dissociation constant (Kd) of about 0.1-10 nM D , 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 0.1 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, e.g., 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, e.g., about 0.1 to about 10 nM, about 0.5 to about 10 nM, about 1 to about 10 nM, about 1.5 to about 10 nM, about 2 to about 10 nM, about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM, about 5 to about 10 nM, about 5.5 to about 10 nM, about 6 to about 10 nM, about 6.5 to about 10 nM, about 7 to about 10 nM, about 7.5 to about 10 nM, about 8 to about 10 nM, about 8.5 to about 10 nM, about 9 to about 10 nM, or about 9.5 to about 10 nM, or, e.g., 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, e.g., 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, e.g., as determined by bio-layer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore);

[0019] i) has reduced or decreased binding affinity for a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer), e.g., by 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 50%, about 2% to about 40%, about 3% to about 30%, about 4% to about 20%, or about 5% to about 10%, about 1% to about 40%, about 1% to about 30%, about 1% 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 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.g., about 0.5-fold to about 5-fold, about 1-fold to about 4-fold, or about 2-fold to about 3-fold), e.g., relative to an IL-2 formulation comprising a wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by yeast surface display, bio-layer interferometry (e.g., Octet binding), and / or surface plasmon resonance (e.g., Biacore);

[0020] j) binds to a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with low affinity, e.g., a dissociation constant (K D) is about 0.2-20 nM, e.g., about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 20 nM, or, e.g., about 0.2 to about 19 nM, about 0.2 to about 18 nM, about 0.2 to about 17 nM, or about 0.2 to about 16 nM, e.g., 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, e.g., about 0.1 to about 10 nM, about 0.5 to about 10 nM, about 1 to about 10 nM, about 1.5 to about 10 nM, about 2 to about 10 nM, about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM, about 5 to about 10 nM, about 5.5 to about 10 nM, about 6 to about 10 nM, about 6.5 to about 10 nM, about 7 to about 10 nM, about 7.5 to about 10 nM, about 8 to about 10 nM, about 8.5 to about 10 nM, about 9 to about 10 nM, or about 9.5 to about 10 nM, or, e.g., 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, e.g., 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, e.g., as determined by yeast surface display;

[0021] k) binds a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with low affinity, e.g., a dissociation constant (K D) is about 0.2-300 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 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, or about 300 nM, or for example, about 0.2 to about 280 nM, about 0.2 to about 260 nM, about 0.2 to about 240 nM, about 0.2 to about 220 nM, about 0.2 to about 200 nM, about 0.2 to about 180 nM, about 0.2 to about 160 nM, about 0.2 to about 140 nM, about 0.2 to about 120 nM, about 0.2 to about 100 nM, about 0.2 to about 80 nM, about 0.2 to about 60 nM, about 0.2 to about 40 nM, about 0.2 to about 20 nM, or for example, about 0.5 to about 300 nM, about 1 to about 300 nM, about 5 to about 300 nM, about 10 to about 300 nM, about 20 to about 300 nM, about 40 to about 300 nM, about 60 to about 300 nM, about 80 to about 300 nM, about 100 to about 300 nM, about 120 to about 300 nM, about 140 to about 300 nM, about 160 to about 300 nM, about 180 to about 300 nM, about 200 to about 300 nM, about 220 to about 300 nM, about 240 to about 300 nM, about 260 to about 300 nM, about 280 to about 300 nM, or for example, about 0.5 to about 280 nM, about 1 to about 260 nM, about 5 to about 240 nM, about 10 to about 220 nM, about 20 to about 200 nM, about 40 to about 180 nM, about 60 to about 160 nM, about 80 to about 140 mM, about 100 to about 120 nM, or for example, greater than about 0.2, about 0.5, 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, e.g., as determined by biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore);

[0022] l) selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, e.g., T helper ECs with 50 / Treg EC 50 Ratio: greater than 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 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1000, about 1500, about 2000, about 2500, or about 3000, or more, or, e.g., greater than 1 and about 1 to 2, about 2 to 3, about 3 to 4, about 4 to 5, greater than 1 and about 1 to 10, greater than 1 and about 1 to 20, greater than 1 and about 1 to 30, greater than 1 and about 1 to 40, greater than 1 and about 1 to 50, about 2 to 10, about 2 to 20, about 2 to 30, about 2 to 40, 2 to 50, about 5 to 10, about 5 to 20, about 5 to 30, about 5 to 40, about 5 to 50, about 10 to 20, about 10 to 30, about 10 to 40 about 10 to 50, about 20 to 40, about 20 to 50, about 50 to 100, about 100 to 200, about 200 to 500, about 500 to 1000, about 1000 to 2000, or about 1000 to 3000, relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by flow cytometry;

[0023] m) selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, e.g., NK cell ECs with50 / Treg EC 50 Ratio: greater than 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 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1000, about 1500, about 2000, about 2500, or about 3000, or more, or for example, greater than 1 and about 1 to 2, about 2 to 3, about 3 to 4, about 4 to 5, greater than 1 and about 1 to 10, greater than 1 and about 1 to 20, greater than 1 and about 1 to 30, greater than 1 and about 1 to 40, greater than 1 and about 1 to 50, about 2 to 10, about 2 to 20, about 2 to 30, about 2 to 40, 2 to 50, about 5 to 10, about 5 to 20, about 5 to 30, about 5 to 40, about 5 to 50, about 10 to 20, about 10 to 30, about 10 to 40 about 10 to 50, about 20 to 40, about 20 to 50, about 50 to 100, about 100 to 200, about 200 to 500, about 500 to 1000, about 1000 to 2000, or about 1000 to 3000, relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, for example, as determined by flow cytometry assay;

[0024] n) (i) enhanced or increased potency and / or ability to induce or promote T regulatory cell activity, for example, EC 50 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 for example, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction, for example, as determined by flow cytometry, in vitro or in vivo T regulatory cell proliferation or expansion assay and / or T cell suppression assay;

[0025] (ii) reduced or decreased efficacy and / or ability to induce or promote T regulatory cell activity, e.g., EC 50 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, 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, 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, e.g., as determined by a flow cytometry, in vitro or in vivo T regulatory cell proliferation or expansion assay, and / or T cell suppression assay;

[0026] o) modulating (e.g., decreasing (e.g., inhibiting, blocking, or neutralizing) or increasing (e.g., activating, initiating, or enhancing)) one or more biological activities of a T cell (e.g., Treg) in vitro, ex vivo, or in vivo;

[0027] p) exhibiting the same or similar binding affinity or specificity, or both, as an IL-2 agent described herein;

[0028] q) exhibiting the same or similar binding affinity or specificity, or both, as an IL-2 agent comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) alterations (e.g., substitutions) described herein;

[0029] r) exhibiting the same or similar binding affinity or specificity, or both, as an IL-2 agent comprising an amino acid sequence described herein;

[0030] s) exhibiting the same or similar binding affinity or specificity, or both, as an IL-2 agent comprising an amino acid sequence encoded by a nucleotide sequence described herein;

[0031] t) inhibiting, e.g., competitively inhibiting, 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,

[0032] u) competing 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;

[0033] v) one or more biological properties of an IL-2 formulation described herein;

[0034] w) one or more structural properties of an IL-2 formulation described herein; or

[0035] x) one or more pharmacokinetic properties of an IL-2 formulation described herein.

[0036] In one embodiment, the IL-2 formulation expresses at a higher or increased level in vitro and / or in vivo, e.g., by 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 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.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by protein concentration. In one embodiment, the IL-2 formulation aggregates at a lower or decreased level in vitro and / or in vivo, e.g., by 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 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.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by melting temperature analysis (e.g., using a fluorescence assay), dynamic light scattering, and / or size exclusion chromatography.

[0037] In one embodiment, the IL-2 formulation has enhanced or increased stability in vitro and / or in vivo, e.g., by 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 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.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as measured by expression in yeast surface display, expression in mammalian cells, chromatography, circular dichroism assay or related spectroscopic techniques, and / or melting temperature analysis (e.g., using fluorescence).

[0038] In one embodiment, the IL-2 formulation has enhanced or increased half-life in vitro and / or in vivo, e.g., by 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 by greater than 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.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as measured by ELISA, flow cytometry, and / or mass spectrometry.

[0039] In one embodiment, the IL-2 formulation has a lower, reduced, or decreased turnover and / or clearance rate or level in vivo, e.g., by 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 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.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by ELISA, flow cytometry, and / or mass spectrometry.

[0040] In one embodiment, the IL-2 agent has reduced or decreased or substantially unchanged binding affinity for CD25 (e.g., human CD25), e.g., decreased by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more (e.g., about 1% to about 20%, about 2% to about 15%, or about 5% to about 10%), or decreased or increased by no more than about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%, or decreased 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, or decreased or increased by no more than 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, e.g., 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 yeast surface display, bio-layer interferometry (e.g., Octet binding), and / or surface plasmon resonance (e.g., Biacore). In one embodiment, the decrease or reduction in binding affinity for CD25 is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% lower than the decrease or reduction in binding affinity for CD25. In one embodiment, the binding affinity for CD25 is not significantly decreased or reduced.

[0041] In one embodiment, the IL-2 formulation binds CD25 (e.g., human CD25) with low affinity, e.g., with a dissociation constant (KD) of about 5-500 pM, e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 pM, or, e.g., about 10 pM to 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 410 pM, about 100 pM to about 400 pM, about 110 pM to about 390 pM, about 120 pM to about 380 pM, about 130 pM to about 370 pM, about 140 pM to about 360 pM, about 150 pM to about 350 pM, about 160 pM to about 340 pM, about 170 pM to about 330 pM, about 180 pM 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, e.g., 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, e.g., 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, e.g., 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.For example, it can be determined by yeast surface display.

[0042] In one embodiment, the IL-2 formulation binds to CD25 (e.g., human CD25) with low affinity, for example with a dissociation constant (K2) of about 0.1-10 nM. D For example, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 6, about 7, about 8, about 9, or about 10 nM, or for example, about 0.1 to about 9 nM, about 0.1 to about 8 nM, about 0.1 to about 7 nM, or about 0.1 to about 6 nM, for example, about 0.1 to about 5 nM, about 0.1 to About 4 nM, about 0.1 to about 3 nM, about 0.1 to about 2 nM, about 0.1 to about 1 nM, or about 0.1 to about 0.5 nM, or for example about 0.1 to about 10 nM, about 0.5 to about 10 nM, about 1 to about 10 nM, about 1.5 to about 10 nM, about 2 to about 10 nM, about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM, about 5 to about 10 nM, about 5 0.5 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 Approximately 3.5 to approximately 6 nM, approximately 4 to approximately 5.5 nM, or approximately 4.5 to approximately 5 nM, or, for example, greater than approximately 0.1, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, or approximately 10 nM, as determined, for example, by biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore).

[0043] In one embodiment, the IL-2 agent has reduced or decreased binding affinity for a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer), e.g., by 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 50%, about 2% to about 40%, about 3% to about 30%, about 4% to about 20%, or about 5% to about 10%, about 1% to about 40%, about 1% to about 30%, about 1% 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 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.g., about 0.5-fold to about 5-fold, about 1-fold to about 4-fold, or about 2-fold to about 3-fold), e.g., 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 yeast surface display, bio-layer interferometry (e.g., Octet binding), and / or surface plasmon resonance (e.g., Biacore). In one embodiment, the reduction or decrease in binding affinity for a CD122 / CD132 heterodimer is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5-fold greater than the reduction or decrease in binding affinity for CD25. In one embodiment, the binding affinity for CD25 is not significantly reduced or decreased.

[0044] In one embodiment, the IL-2 agent binds to a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with low affinity, e.g., a dissociation constant (K D) is about 0.2-20 nM, e.g., about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 20 nM, or, e.g., about 0.2 to about 19 nM, about 0.2 to about 18 nM, about 0.2 to about 17 nM, or about 0.2 to about 16 nM, e.g., 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, e.g., about 0.1 to about 10 nM, about 0.5 to about 10 nM, about 1 to about 10 nM, about 1.5 to about 10 nM, about 2 to about 10 nM, about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM, about 5 to about 10 nM, about 5.5 to about 10 nM, about 6 to about 10 nM, about 6.5 to about 10 nM, about 7 to about 10 nM, about 7.5 to about 10 nM, about 8 to about 10 nM, about 8.5 to about 10 nM, about 9 to about 10 nM, or about 9.5 to about 10 nM, or, e.g., 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, e.g., 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, e.g., as determined by yeast surface display.

[0045] In one embodiment, the IL-2 formulation binds a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with a low affinity, e.g., a dissociation constant (K D) is about 0.2-300 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 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, or about 300 nM, or for example, about 0.2 to about 280 nM, about 0.2 to about 260 nM, about 0.2 to about 240 nM, about 0.2 to about 220 nM, about 0.2 to about 200 nM, about 0.2 to about 180 nM, about 0.2 to about 160 nM, about 0.2 to about 140 nM, about 0.2 to about 120 nM, about 0.2 to about 100 nM, about 0.2 to about 80 nM, about 0.2 to about 60 nM, about 0.2 to about 40 nM, about 0.2 to about 20 nM, or for example, about 0.5 to about 300 nM, about 1 to about 300 nM, about 5 to about 300 nM, about 10 to about 300 nM, about 20 to about 300 nM, about 40 to about 300 nM, about 60 to about 300 nM, about 80 to about 300 nM, about 100 to about 300 nM, about 120 to about 300 nM, about 140 to about 300 nM, about 160 to about 300 nM, about 180 to about 300 nM, about 200 to about 300 nM, about 220 to about 300 nM, about 240 to about 300 nM, about 260 to about 300 nM, about 280 to about 300 nM, or for example, about 0.5 to about 280 nM, about 1 to about 260 nM, about 5 to about 240 nM, about 10 to about 220 nM, about 20 to about 200 nM, about 40 to about 180 nM, about 60 to about 160 nM, about 80 to about 140 mM, about 100 to about 120 nM, or for example, greater than about 0.2, about 0.5, 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, for example, as determined by biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore).

[0046] In one embodiment, the IL-2 formulation selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, for example, with a T helper EC 50 / Treg EC 50Ratio: greater than 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 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1000, about 1500, about 2000, about 2500, or about 3000, or more, or, for example, greater than 1 and about 1 to 2, about 2 to 3, about 3 to 4, about 4 to 5, greater than 1 and about 1 to 10, greater than 1 and about 1 to 20, greater than 1 and about 1 to 30, greater than 1 and about 1 to 40, greater than 1 and about 1 to 50, about 2 to 10, about 2 to 20, about 2 to 30, about 2 to 40, 2 to 50, about 5 to 10, about 5 to 20, about 5 to 30, about 5 to 40, about 5 to 50, about 10 to 20, about 10 to 30, about 10 to 40 about 10 to 50, about 20 to 40, about 20 to 50, about 50 to 100, about 100 to 200, about 200 to 500, about 500 to 1000, about 1000 to 2000, or about 1000 to 3000, relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by flow cytometry. In an embodiment, the T helper cell is a CD45+CD3+CD4+Foxp3- cell, e.g., as determined by flow cytometry. In an embodiment, the Treg is a CD45+CD3+CD4+Foxp3+ cell, e.g., as determined by flow cytometry.

[0047] In an embodiment, the IL-2 formulation selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, e.g., has an NK cell EC 50 / Treg EC 50Ratio: greater than 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 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1000, about 1500, about 2000, about 2500, or about 3000, or more, or, for example, greater than 1 and about 1 to 2, about 2 to 3, about 3 to 4, about 4 to 5, greater than 1 and about 1 to 10, greater than 1 and about 1 to 20, greater than 1 and about 1 to 30, greater than 1 and about 1 to 40, greater than 1 and about 1 to 50, about 2 to 10, about 2 to 20, about 2 to 30, about 2 to 40, 2 to 50, about 5 to 10, about 5 to 20, about 5 to 30, about 5 to 40, about 5 to 50, about 10 to 20, about 10 to 30, about 10 to 40 about 10 to 50, about 20 to 40, about 20 to 50, about 50 to 100, about 100 to 200, about 200 to 500, about 500 to 1000, about 1000 to 2000, or about 1000 to 3000, relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by flow cytometry. In an embodiment, the NK cells are CD45+CD3- cells that are CD56+and / or CD16+, e.g., as determined by flow cytometry. In an embodiment, the NK cells are CD45+CD3-CD56+cells, e.g., as determined by flow cytometry. In an embodiment, the Tregs are CD45+CD3+CD4+Foxp3+cells, e.g., as determined by flow cytometry.

[0048] In an embodiment, the IL-2 formulation has an enhanced or increased potency and / or ability to induce or promote T regulatory cell activity, e.g., EC 50The levels should be approximately 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or for example, reduced by approximately 0.5 times, 1 time, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, or more, as determined by flow cytometry, in vitro or in vivo T-regulatory cell proliferation or expansion assays, and / or T-cell suppression assays.

[0049] In one embodiment, the efficacy and / or ability of the IL-2 formulation to induce or promote T regulatory cell activity is reduced or diminished, for example, for Treg EC50, relative to an IL-2 formulation containing wild-type IL-2 or an IL-2 formulation containing a reference IL-2 variant. 50 To be approximately 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more higher, or for example, to be approximately 0.5 times, 1 time, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, or 4 times higher. 5 times, approximately 5 times, approximately 5.5 times, approximately 6 times, approximately 6.5 times, approximately 7 times, approximately 7.5 times, approximately 8 times, approximately 8.5 times, approximately 9 times, approximately 9.5 times, approximately 10 times, approximately 50 times, approximately 100 times, approximately 200 times, approximately 500 times, approximately 1000 times, approximately 2000 times, approximately 5000 times, approximately 10,000 times, approximately 15,000 times, approximately 20,000 times or more, as determined, for example, by flow cytometry, in vitro or in vivo T regulatory cell proliferation or expansion assays and / or T cell suppression assays. In one embodiment, the efficacy and / or ability of the IL-2 formulation to induce or promote T regulatory cell activity is reduced or diminished, for example, relative to an IL-2 formulation containing wild-type IL-2 or an IL-2 formulation containing a reference IL-2 variant, for ECGs of Tregs. 50 It must be approximately 100 times higher or more (e.g., as determined by flow cytometry, in vitro or in vivo T regulatory cell proliferation or expansion assays and / or T cell suppression assays), and must not activate or significantly activate NK cells.

[0050] In an embodiment, the IL-2 agent modulates (e.g., decreases (e.g., inhibits, blocks, or neutralizes) or increases (e.g., activates, initiates, or enhances) one or more biological activities of a T cell (e.g., a Treg) in vitro, ex vivo, or in vivo.

[0051] In an embodiment, the IL-2 agent displays the same or similar binding affinity or specificity, or both, as an IL-2 agent described herein.

[0052] In an embodiment, the IL-2 agent displays the same or similar binding affinity or specificity, or both, as an IL-2 agent comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) alterations (e.g., substitutions) described herein.

[0053] In an embodiment, the IL-2 agent displays the same or similar binding affinity or specificity, or both, as an IL-2 agent comprising an amino acid sequence described herein.

[0054] In an embodiment, the IL-2 agent displays the same or similar binding affinity or specificity, or both, as an IL-2 agent comprising an amino acid sequence encoded by a nucleotide sequence described herein.

[0055] In an embodiment, the IL-2 agent inhibits, e.g., competitively inhibits, 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.

[0056] In an embodiment, the IL-2 agent 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.

[0057] In an embodiment, the IL-2 agent has one or more biological properties of an IL-2 agent described herein.

[0058] In an embodiment, the IL-2 agent has one or more structural properties of an IL-2 agent described herein.

[0059] In an embodiment, the IL-2 agent has one or more pharmacokinetic properties of an IL-2 agent described herein.

[0060] In an embodiment, the interleukin-2 (IL-2) formulation comprises a human IL-2 variant comprising an amino acid change (e.g., 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, for example, T3, H16, I28, K35, R38, F42, E68, V69, Q74, D84, S87, N88, I92, C125, Q126, or combinations thereof, corresponding to wild-type human IL-2. In another embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position V69, Q74, or combinations thereof. In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at positions V69 and Q74. In an embodiment, the IL-2 formulation comprises the amino acid substitution V69A. In an embodiment, the IL-2 formulation comprises the amino acid substitution Q74P. In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position H16, I92, D84, or combinations thereof. In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position H16, optionally wherein the amino acid substitution is H16N, H16L, or H16D. In an embodiment, the IL-2 formulation comprises the amino acid substitution H16N. In an embodiment, the IL-2 formulation comprises the amino acid substitution H16L. In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position I92, optionally wherein the amino acid substitution is I92S. In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position D84, optionally wherein the amino acid substitution is D84V. In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position K35, R38, F42, E68, or combinations thereof. In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position K35, optionally wherein the amino acid substitution is K35E. In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position R38, optionally wherein the amino acid substitution is R38E, R38N, or R38Q. In an embodiment, the IL-2 formulation comprises the amino acid substitution R38N. In an embodiment, the IL-2 formulation comprises the amino acid substitution R38Q. In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position F42, optionally wherein the amino acid substitution is F42K or F42Q. In an embodiment, the IL-2 formulation comprises the amino acid substitution F42Q.

[0061] In an embodiment, the IL-2 formulation comprises one or more (e.g., two, three, four, or all) of (i)-(v):

[0062] (i) one or more (e.g., two, three, four, five, six, or seven) amino acid changes (e.g., substitutions) that decrease or are identified to decrease its affinity for CD122 (e.g., CD122 / CD132 heterodimer), e.g., a change (e.g., substitution) at position H16 (e.g., H16L, H16N, or H16D), a change (e.g., substitution) at position 128 (e.g., 128T or 128F), a change (e.g., substitution) at position D84 (e.g., D84V), a change (e.g., substitution) at position S87 (e.g., S87R), a change (e.g., substitution) at position N88 (e.g., N88S, N88L, or N88D), a change (e.g., substitution) at position 192 (e.g., 192S), and / or a change (e.g., substitution) at position Q126 (e.g., Q126T, Q126K, or Q126R);

[0063] (ii) one or more (e.g., two) amino acid changes (e.g., substitutions) that increase or are identified to increase the stability of the IL-2 preparation, e.g., a change (e.g., substitution) at position V69 (e.g., V69A) and / or a change (e.g., substitution) at Q74 (e.g., Q74P);

[0064] (iii) one or more (e.g., two, three, or four) amino acid changes (e.g., substitutions) that decrease or are identified to decrease its affinity for CD25, e.g., a change (e.g., substitution) at position K35 (e.g., K35E), a change (e.g., substitution) at position R38 (e.g., R38E, R38N, or R38Q), a change (e.g., substitution) at position F42 (e.g., F42K or F42Q), and / or a change (e.g., substitution) at position E68 (e.g., E68Q or E68N); or

[0065] (iv) one or more amino acid changes (e.g., substitutions) that decrease or are identified to decrease O-glycosylation of the IL-2 preparation, e.g., a change (e.g., substitution) at position T3 (e.g., T3A); or

[0066] (v) one or more amino acid changes (e.g., substitutions) that decrease or are identified to decrease improper disulfide pairing and / or aggregation of the IL-2 preparation (e.g., to increase stability), e.g., a change (e.g., substitution) at position C125 (e.g., C125S).

[0067] In one embodiment, the IL-2 formulation comprises (i). In one embodiment, the IL-2 formulation comprises (ii). In one embodiment, the IL-2 formulation comprises (iii). In one embodiment, the IL-2 formulation comprises (iv). In one embodiment, the IL-2 formulation comprises (v).

[0068] In one embodiment, the IL-2 formulation comprises (i) and (ii). In one embodiment, the IL-2 formulation comprises (i) and (iii). In one embodiment, the IL-2 formulation comprises (i) and (iv). In one embodiment, the IL-2 formulation comprises (i) and (v). In one embodiment, the IL-2 formulation comprises (ii) and (iii). In one embodiment, the IL-2 formulation comprises (ii) and (iv). In one embodiment, the IL-2 formulation comprises (ii) and (v). In one embodiment, the IL-2 formulation comprises (iii) and (iv). In one embodiment, the IL-2 formulation comprises (iii) and (v). In one embodiment, the IL-2 formulation comprises (iv) and (v).

[0069] In one embodiment, the IL-2 formulation comprises (i), (ii), and (iii). In one embodiment, the IL-2 formulation comprises (i), (ii), and (iv). In one embodiment, the IL-2 formulation comprises (i), (ii), and (v). In one embodiment, the IL-2 formulation comprises (i), (iii), and (iv). In one embodiment, the IL-2 formulation comprises (i), (iii), and (v). In one embodiment, the IL-2 formulation comprises (i), (iv), and (iv). In one embodiment, the IL-2 formulation comprises (ii), (iii), and (iv). In one embodiment, the IL-2 formulation comprises (ii), (iii), and (v). In one embodiment, the IL-2 formulation comprises (ii), (iv), and (iv). In one embodiment, the IL-2 formulation comprises (iii), (iv), and (v).

[0070] In one embodiment, the IL-2 formulation comprises (i), (ii), (iii), and (iv). In one embodiment, the IL-2 formulation comprises (i), (ii), (iii), and (v). In one embodiment, the IL-2 formulation comprises (i), (ii), (iv), and (v). In one embodiment, the IL-2 formulation comprises (i), (iii), (iv), and (v). In one embodiment, the IL-2 formulation comprises (ii), (iii), (iv), and (v).

[0071] In one embodiment, the IL-2 formulation comprises (i), (ii), (iii), (iv), and (v).

[0072] In one embodiment, the IL-2 formulation does not comprise (i). In one embodiment, the IL-2 formulation does not comprise (ii). In one embodiment, the IL-2 formulation does not comprise (iii). In one embodiment, the IL-2 formulation does not comprise (iv). In one embodiment, the IL-2 formulation does not comprise (v).

[0073] In one embodiment, the IL-2 formulation does not comprise (i) and (ii). In one embodiment, the IL-2 formulation does not comprise (i) and (iii). In one embodiment, the IL-2 formulation does not comprise (i) and (iv). In one embodiment, the IL-2 formulation does not comprise (i) and (v). In one embodiment, the IL-2 formulation does not comprise (ii) and (iii). In one embodiment, the IL-2 formulation does not comprise (ii) and (iv). In one embodiment, the IL-2 formulation does not comprise (ii) and (v). In one embodiment, the IL-2 formulation does not comprise (iii) and (iv). In one embodiment, the IL-2 formulation does not comprise (iii) and (v). In one embodiment, the IL-2 formulation does not comprise (iv) and (v).

[0074] In one embodiment, the IL-2 formulation does not comprise (i), (ii), and (iii). In one embodiment, the IL-2 formulation does not comprise (i), (ii), and (iv). In one embodiment, the IL-2 formulation does not comprise (i), (ii), and (v). In one embodiment, the IL-2 formulation does not comprise (i), (iii), and (iv). In one embodiment, the IL-2 formulation does not comprise (i), (iii), and (v). In one embodiment, the IL-2 formulation does not comprise (i), (iv), and (v). In one embodiment, the IL-2 formulation does not comprise (ii), (iii), and (iv). In one embodiment, the IL-2 formulation does not comprise (ii), (iii), and (v). In one embodiment, the IL-2 formulation does not comprise (ii), (iv), and (iv). In one embodiment, the IL-2 formulation does not comprise (iii), (iv), and (v).

[0075] In an embodiment, the IL-2 formulation does not comprise (i), (ii), (iii), and (iv). In an embodiment, the IL-2 formulation does not comprise (i), (ii), (iii), and (v). In an embodiment, the IL-2 formulation does not comprise (i), (ii), (iv), and (v). In an embodiment, the IL-2 formulation does not comprise (i), (iii), (iv), and (v). In an embodiment, the IL-2 formulation does not comprise (ii), (iii), (iv), and (v).

[0076] In an embodiment, the IL-2 formulation does not comprise (i), (ii), (iii), (iv), and (v).

[0077] In an embodiment, the IL-2 formulation comprises amino acid changes (e.g., substitutions) at:

[0078] (i) positions V69 and Q74, and / or at position K35; and

[0079] (ii) at position H16, I92, or D84; and optionally

[0080] (iii) at position R38, F42, E68, or a combination thereof.

[0081] In an embodiment, the IL-2 formulation comprises amino acid changes (e.g., substitutions) at:

[0082] (i) positions V69 and Q74, and / or at position K35; and

[0083] (ii) at position H16, I92, or D84; and

[0084] (iii) at position R38, F42, E68, or a combination thereof.

[0085] In an embodiment, the IL-2 formulation comprises amino acid changes (e.g., substitutions) at:

[0086] (i) positions V69 and Q74, and / or at position K35; and

[0087] (ii) at position H16, I92, or D84; or

[0088] (iii) at position R38, F42, E68, or a combination thereof.

[0089] In an embodiment, the IL-2 formulation comprises amino acid changes (e.g., substitutions) at:

[0090] (i) positions V69 and Q74, and / or at position K35; and

[0091] (ii) at position H16, I92, D84, or a combination thereof, and

[0092] (iii) at position R38, F42, E68, or a combination thereof.

[0093] In one embodiment, the IL-2 agent comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and H16, optionally wherein the amino acid substitutions are V69A, Q74P, and H16N or H16L, respectively, optionally wherein the amino acid substitutions are V69A, Q74P, and H16L. In one embodiment, the IL-2 agent comprises the amino acid substitutions V69A, Q74P, and H16L.

[0094] In one embodiment, the IL-2 agent comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and I92, optionally wherein the amino acid substitutions are V69A, Q74P, and I92S, respectively. In one embodiment, the IL-2 agent comprises the amino acid substitutions V69A, Q74P, and I92S.

[0095] In one embodiment, the IL-2 agent comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and D84, optionally wherein the amino acid substitutions are V69A, Q74P, and D84V, respectively. In one embodiment, the IL-2 agent comprises the amino acid substitutions V69A, Q74P, and D84V.

[0096] In one embodiment, the IL-2 agent comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, and R38Q, respectively.

[0097] In one embodiment, the IL-2 agent comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and F42, optionally wherein the amino acid substitutions are V69A, Q74P, and F42Q, respectively. In one embodiment, the IL-2 agent comprises the amino acid substitutions V69A, Q74P, and F42Q.

[0098] In one embodiment, the IL-2 agent comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, and R38N, respectively. In one embodiment, the IL-2 agent comprises the amino acid substitutions V69A, Q74P, and R38N.

[0099] In an embodiment, the IL-2 agent comprises an amino acid change (e.g., substitution) at position V69, Q74, K35, H16, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, K35E, H16N, and R38N, respectively. In an embodiment, the IL-2 agent comprises the amino acid substitutions V69A, Q74P, K35E, H16N, and R38N.

[0100] In an embodiment, the IL-2 agent comprises an amino acid change (e.g., substitution) at position V69, Q74, K35, H16, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, K35E, H16N, and R38N, respectively. In an embodiment, the IL-2 agent comprises the amino acid substitutions V69A, Q74P, K35E, H16N, and R38N.

[0101] In an embodiment, the IL-2 agent comprises an amino acid change (e.g., substitution) at position V69, Q74, K35, H16, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, K35E, H16N, and R38N, respectively. In an embodiment, the IL-2 agent comprises the amino acid substitutions V69A, Q74P, K35E, H16N, and R38N.

[0102] In an embodiment, the IL-2 agent comprises an amino acid change (e.g., substitution) at position V69, Q74, K35, H16, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, K35E, H16N, and R38N, respectively. In an embodiment, the IL-2 agent comprises the amino acid substitutions V69A, Q74P, K35E, H16N, and R38N.

[0103] In an embodiment, the IL-2 agent comprises an amino acid change (e.g., substitution) at position I28, E68, S87, N88, Q126, or a combination thereof. In an embodiment, the IL-2 agent comprises an amino acid change (e.g., substitution) at position I28, optionally wherein the amino acid substitution is I28T or I28F. In an embodiment, the IL-2 agent comprises the amino acid substitution I28T. In an embodiment, the IL-2 agent comprises the amino acid substitution I28F.

[0104] In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position E68, optionally wherein the amino acid substitution is E68Q or E68N. In an embodiment, the IL-2 formulation comprises the amino acid substitution E68Q. In an embodiment, the IL-2 formulation comprises the amino acid substitution E68N.

[0105] In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position S87, optionally wherein the amino acid substitution is S87R. In an embodiment, the IL-2 formulation comprises the amino acid substitution S87R.

[0106] In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position N88, optionally wherein the amino acid substitution is N88R, N88S, N88L, or N88D. In an embodiment, the IL-2 formulation comprises the amino acid substitution N88R. In an embodiment, the IL-2 formulation comprises the amino acid substitution N88S. In an embodiment, the IL-2 formulation comprises the amino acid substitution N88L. In an embodiment, the IL-2 formulation comprises the amino acid substitution N88D.

[0107] In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position Q126, optionally wherein the amino acid substitution is Q126T, Q126K, or Q126R. In an embodiment, the IL-2 formulation comprises the amino acid substitution Q126T. In an embodiment, the IL-2 formulation comprises the amino acid substitution Q126K. In an embodiment, the IL-2 formulation comprises the amino acid substitution Q126R.

[0108] In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position C125, optionally wherein the amino acid substitution is C125S. In an embodiment, the IL-2 formulation comprises the amino acid substitution C125S.

[0109] In an embodiment, the IL-2 formulation comprises an amino acid change (e.g., substitution) at position T3, optionally wherein the amino acid substitution is T3A. In an embodiment, the IL-2 formulation comprises the amino acid substitution T3A.

[0110] In an embodiment, the IL-2 formulation comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and C125, optionally wherein the amino acid substitutions are V69A, Q74P, and C125S, respectively. In an embodiment, the IL-2 formulation comprises the amino acid substitutions V69A, Q74P, and C125S.

[0111] In an embodiment, the IL-2 agent comprises an amino acid change (e.g., substitution) at position T3, H16, I92, or a combination thereof, optionally wherein the amino acid substitutions are T3A, H16N, and I92S, respectively.

[0112] In an embodiment, the IL-2 agent comprises an amino acid change (e.g., substitution) at position H16, V69, Q74, and C125, optionally wherein the amino acid substitutions are H16N, V69A, Q74P, and C125S, respectively. In an embodiment, the IL-2 agent comprises the amino acid substitutions H16N, V69A, Q74P, and C125S.

[0113] In an embodiment, the IL-2 agent comprises an amino acid change (e.g., substitution) at position H16, V69, Q74, and C125, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, and C125S, respectively. In an embodiment, the IL-2 agent comprises the amino acid substitutions H16L, V69A, Q74P, and C125S. Various technical effects are associated with IL-2 agents comprising the combination of the above-described amino acid changes. Without wishing to be bound by theory, it is believed that in an embodiment, an IL-2 agent comprising the amino acid substitutions H16L, V69A, Q74P, and C125S can have at least one or more of the following advantageous properties: (i) reduced binding affinity for CD122 and / or CD132 compared to other T cell types, which increases the efficacy and selectivity of the IL-2 agent for regulatory T cells (Tregs); (ii) significant stabilization, e.g., due to the presence of the stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity for CD25 (or no more than a minimal impact on it), which can extend the life of the IL-2 agent; (iv) substantially no promotion of 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 incorrect disulfide pairing and improved stability, e.g., due to the presence of the C125S mutation. In an embodiment, the IL-2 agent comprising the H16L mutation has reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types compared to IL-2 agents comprising other H16 mutations. These properties make the IL-2 agent comprising the amino acid substitutions H16L, V69A, Q74P, and C125S particularly suitable for use in treating diseases and disorders caused by aberrant immune responses, such as autoimmune diseases.

[0114] Accordingly, in one embodiment, an IL-2 preparation comprising the amino acid substitutions H16L, V69A, Q74P, and C125S has one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties relative to wild-type IL-2 or a reference IL-2 variant that does not comprise the following amino acid substitutions: (i) enhanced or increased in vitro or in vivo stability; (ii) reduced or decreased in vitro and / or in vivo binding capacity and / or binding affinity for human CD122; (iii) reduced or decreased in vitro and / or in vivo binding capacity and / or binding affinity for human CD132; (iv) reduced or decreased in vitro and / or in vivo affinity for the heterodimeric (i.e., human CD122 / CD132 heterodimer) IL-2 receptor composed of human CD122 and human CD132; (v) reduced or decreased (e.g., moderately reduced or decreased) in vitro and / or in vivo binding capacity and / or binding affinity for human CD25; (vi) selectively binds to regulatory T cells (e.g., Foxp3 + T cells); (vii) selectively activates IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased capacity to induce or promote Treg expansion, activity, survival, and / or proliferation.

[0115] In one embodiment, the IL-2 preparation comprises amino acid changes (e.g., substitutions) at positions H16, V69, Q74, I92, and C125, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, I92S, and C125S, respectively. In one embodiment, the IL-2 preparation comprises the amino acid substitutions H16L, V69A, Q74P, I92S, and C125S.

[0116] In one embodiment, the IL-2 preparation comprises amino acid changes (e.g., substitutions) at positions T3, V69, Q74, and C125, optionally wherein the amino acid substitutions are T3A, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 preparation comprises the amino acid substitutions T3A, V69A, Q74P, and C125S.

[0117] In one embodiment, the IL-2 preparation comprises amino acid changes (e.g., substitutions) at positions T3, H16, V69, Q74, and C125, optionally wherein the amino acid substitutions are T3A, H16N or H16L, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 preparation comprises the amino acid substitutions T3A, H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 preparation comprises the amino acid substitutions T3A, H16L, V69A, Q74P, and C125S.

[0118] In one embodiment, the IL-2 agent comprises an amino acid change (e.g., substitution) at positions T3, V69, Q74, I92, and C125, optionally wherein the amino acid substitutions are T3A, V69A, Q74P, I92S, and C125S, respectively. In one embodiment, the IL-2 agent comprises the amino acid substitutions T3A, V69A, Q74P, I92S, and C125S. In one embodiment, the IL-2 agent comprises the amino acid substitutions T3A, V69A, Q74P, I92S, and C125S.

[0119] In one embodiment, the IL-2 agent comprises a human IL-2 variant comprising an amino acid sequence 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, 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 an amino acid sequence differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0120] In one embodiment, the amino acid change (e.g., substitution) provides the IL-2 formulation 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 formulation that does not comprise the amino acid change (e.g., substitution):

[0121] (i) enhanced or increased expression of the IL-2 formulation;

[0122] (ii) inhibited or decreased aggregation of the IL-2 formulation;

[0123] (iii) enhanced or increased stability of the IL-2 formulation;

[0124] (iv) prolonged or increased half-life of the IL-2 formulation;

[0125] (v) inhibited or decreased turnover and / or clearance of the IL-2 formulation;

[0126] (vi) inhibited or decreased (e.g., moderately inhibited or decreased) or substantially unchanged binding of the IL-2 formulation to human CD25;

[0127] (vii) inhibited or decreased affinity of the IL-2 formulation for human CD122;

[0128] (viii) inhibited or decreased affinity of the IL-2 formulation for human CD132; or

[0129] (ix) inhibited or decreased affinity of the IL-2 formulation for the dimeric IL-2 receptor composed of human CD122 and human CD132;

[0130] (x) selective binding to regulatory T cells (e.g., Foxp3+ T cells);

[0131] (xi) selective activation of the IL-2 signaling pathway in Tregs; or

[0132] (xii) enhanced or increased, or decreased or decreased, ability to induce or promote Treg expansion, activity, survival, and / or proliferation.

[0133] In one embodiment, the IL-2 formulation 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) render the IL-2 formulation having at least one or more of the following properties relative to a reference IL-2 formulation that does not comprise the amino acid changes (e.g., substitutions):

[0134] (i) enhanced or increased expression of the IL-2 formulation;

[0135] (ii) inhibited or reduced aggregation of the IL-2 formulation;

[0136] (iii) enhanced or increased stability of the IL-2 formulation;

[0137] (iv) prolonged or increased half-life of the IL-2 formulation;

[0138] (v) inhibited or reduced turnover and / or clearance of the IL-2 formulation;

[0139] (vi) inhibited or reduced (e.g., moderately inhibited or reduced) or substantially unchanged binding of the IL-2 formulation to human CD25;

[0140] (vii) inhibited or reduced affinity of the IL-2 formulation for human CD122;

[0141] (viii) inhibited or reduced affinity of the IL-2 formulation for human CD132;

[0142] (ix) inhibited or reduced affinity of the IL-2 formulation for the dimeric IL-2 receptor composed of human CD122 and human CD132;

[0143] (x) selective binding to regulatory T cells (e.g., Foxp3+ T cells);

[0144] (xi) selective activation of the IL-2 signaling pathway in Tregs; and / or

[0145] (xii) enhanced or increased, or reduced or decreased, ability to induce or promote Treg expansion, activity, survival, and / or proliferation.

[0146] In one embodiment, the human IL-2 variant comprises the amino acid change (e.g., substitution):

[0147] (i) C125S;

[0148] (ii) V69A, Q74P and C125S;

[0149] (iii) H16D, V69A, Q74P and C125S;

[0150] (iv) H16N, V69A, Q74P and C125S;

[0151] (v) H16L, V69A, Q74P and C125S;

[0152] (vi) I28T, V69A, Q74P and C125S;

[0153] (vii) V69A, Q74P, D84V and C125S;

[0154] (viii) V69A, Q74P, S87R and C125S;

[0155] (ix) V69A, Q74P, N88L and C125S;

[0156] (x) V69A, Q74P, N88S and C125S;

[0157] (xi) V69A, Q74P, I92S and C125S;

[0158] (xii) K35E, V69A, Q74P and C125S;

[0159] (xiii) K35E, H16N, V69A, Q74P and C125S;

[0160] (xiv) K35E, H16L, V69A, Q74P and C125S;

[0161] (xv) K35E, D84V, V69A, Q74P and C125S;

[0162] (xvi) K35E, I92S, V69A, Q74P and C125S;

[0163] (xvii) R38Q, V69A, Q74P and C125S;

[0164] (xviii) R38Q, H16N, V69A, Q74P and C125S;

[0165] (xix) R38Q, H16L, V69A, Q74P and C125S;

[0166] (xx) R38Q, D84V, V69A, Q74P and C125S;

[0167] (xxi) R38Q, I92S, Q74P and C125S;

[0168] (xxii) R38N, V69A, Q74P and C125S;

[0169] (xxiii) R38N, H16N, V69A, Q74P and C125S;

[0170] (xxiv) R38N, H16L, V69A, Q74P and C125S;

[0171] (xxv) R38N, D84V, V69A, Q74P and C125S;

[0172] (xxvi) R38N, I92S, Q74P and C125S;

[0173] (xxvii) R38E, V69A, Q74P and C125S;

[0174] (xxviii) F42K, V69A, Q74P and C125S;

[0175] (xxix) F42Q, V69A, Q74P and C125S;

[0176] (xxx) F42A, Y45A, L72G, N88D, V69A, Q74P and C125S;

[0177] (xxxi) R38N, S87R, V69A, Q74P and C125S;

[0178] (xxxii) R38E, H16N, V69A, Q74P and C125S;

[0179] (xxxiii) R38E, D84V, V69A, Q74P and C125S;

[0180] (xxxiv) R38E, S87R, V69A, Q74P and C125S;

[0181] (xxxv) R38E, I92S, V69A, Q74P and C125S;

[0182] (xxxvi) F42Q, H16N, V69A, Q74P and C125S;

[0183] (xxxvii) F42Q, I92S, V69A, Q74P and C125S; or

[0184] (xxxviii) K35E, R38N, H16N, V69A, Q74P and C125S.

[0185] (xxxix) T3A, H16N, V69A, Q74P and C125S;

[0186] (xl) T3A, H16L, V69A, Q74P and C125S; or

[0187] (xli) T3A, V69A, Q74P, I92S and C125S.

[0188] In one embodiment, the IL-2 formulation comprises a human IL-2 variant comprising an amino acid sequence 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, SEQ ID NO: 38, SEQ ID NO: 1000, SEQ ID NO: 1001, or 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 an amino acid sequence differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom; a polypeptide linker described herein; and a non-IL-2 moiety described herein; wherein the IL-2 formulation exhibits at least one or more of the following properties relative to a reference IL-2 formulation that does not comprise a human IL-2 polypeptide variant:

[0189] (i) enhanced or increased expression of the IL-2 formulation;

[0190] (ii) inhibited or reduced aggregation of the IL-2 formulation;

[0191] (iii) enhanced or increased stability of the IL-2 formulation;

[0192] (iv) prolonged or increased half-life of the IL-2 formulation;

[0193] (v) inhibited or reduced turnover and / or clearance of the IL-2 agent;

[0194] (vi) inhibited or reduced (e.g., moderately inhibited or reduced) or substantially unchanged binding of the IL-2 agent to human CD25;

[0195] (vii) inhibited or reduced affinity of the IL-2 agent for human CD122;

[0196] (viii) inhibited or reduced affinity of the IL-2 agent for human CD132;

[0197] (ix) inhibited or reduced affinity of the IL-2 agent for the dimeric IL-2 receptor composed of human CD122 and human CD132;

[0198] (x) selectively binds to regulatory T cells (e.g., Foxp3+ T cells);

[0199] (xi) selective activation of the IL-2 signaling pathway in Tregs; and / or

[0200] (xii) enhanced or increased, or reduced or decreased, ability to induce or promote Treg expansion, activity, and / or proliferation.

[0201] A variety of technical effects are associated with IL-2 agents comprising the amino acid sequence of SEQ ID NO: 5. Without wishing to be bound by theory, it is believed that in one embodiment, IL-2 agents comprising the amino acid sequence of SEQ ID NO: 5 can have at least one or more of the following advantageous properties: (i) reduced binding affinity for CD122 and / or CD132 compared to other T cell types, which increases the efficacy and selectivity of the IL-2 agent for regulatory T cells (Tregs); (ii) significantly stabilized, e.g., due to the presence of stabilizing V69A and Q74P mutations; (iii) have reduced or decreased binding ability and / or binding affinity for CD25 (or no more than a minimal impact on it), which can extend the life of the IL-2 agent; (iv) substantially do not promote 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) have reduced incorrect disulfide pairing and increased stability, e.g., due to the presence of the C125S mutation. In one embodiment, IL-2 agents comprising the H16L mutation have reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types compared to IL-2 agents comprising other H16 mutations. These properties make IL-2 agents comprising the amino acid sequence of SEQ ID NO: 5 particularly suitable for treating diseases and disorders caused by aberrant immune responses, e.g., autoimmune diseases.

[0202] Thus, in one embodiment, the IL-2 preparation comprising the amino acid sequence of SEQ ID NO: 5 has one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties relative to wild-type IL-2 or a reference IL-2 variant that does not comprise the amino acid substitution: (i) enhanced or increased in vitro or in vivo stability; (ii) reduced or decreased in vitro and / or in vivo binding capacity and / or binding affinity for human CD122; (iii) reduced or decreased in vitro and / or in vivo binding capacity and / or binding affinity for human CD132; (iv) reduced or decreased in vitro and / or in vivo affinity for a heterodimeric (i.e., human CD122 / CD132 heterodimer) IL-2 receptor composed of human CD122 and human CD132; (v) reduced or decreased (e.g., moderately reduced or decreased) in vitro and / or in vivo binding capacity and / or binding affinity for human CD25; (vi) selectively binds to regulatory T cells (e.g., Foxp3 + T cells); (vii) selectively activates IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased capacity to induce or promote Treg expansion, activity, survival, and / or proliferation.

[0203] In one embodiment, the reference IL-2 preparation 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 preparation comprises the amino acid sequence of SEQ ID NO: 1031. In one embodiment, the reference IL-2 preparation comprises the amino acid sequence of SEQ ID NO: 1. In one embodiment, the reference IL-2 preparation comprises the amino acid sequence of SEQ ID NO: 2.

[0204] In an embodiment, the IL-2 formulation comprises a human IL-2 variant described herein fused to a non-IL-2 moiety described herein via a linker, wherein the linker is a polypeptide linker, optionally wherein the polypeptide linker is a flexible linker, a rigid linker, or a cleavable linker. In an embodiment, the polypeptide linker is a Gly-Ser linker (e.g., a (G4S)nlinker, wherein n = 1, 2, 3, 4, 5, 6, or greater (SEQ ID NO: 1020)), a proline-rich extension linker (e.g., V1 GPc, V2, GPGc, V3 GcGcP, Cellulase linker 4, Cellulase linker 4), a rigid linker (e.g., A(EAAAK)nA, wherein n = 2, 3, 4, 5, or greater (SEQ ID NO: 1021); REPR_12), a non-GS linker (e.g., (GGGSA)n, wherein n = 1, 2, 3, 4, 5, or greater (SEQ ID NO: 1022)), or an immunoglobulin hinge region or portion thereof. In an 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 an embodiment, the polypeptide linker is a Gly-Ser linker comprising (G4S)4(SEQ ID NO: 48). In an 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 an embodiment, the polypeptide linker comprises the amino acid sequence of SEQ ID NO: 48.

[0205] In an embodiment, the non-IL-2 moiety is an immunoglobulin Fc region, or a fragment or portion (e.g., a functional fragment) thereof. In an 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 an embodiment, the IgG Fc region comprises a wild-type human IgG1 Fc region (e.g., IgG1 m3 allotype), a wild-type IgG2 Fc region, or a wild-type human IgG4 Fc region, or a fragment or portion thereof.

[0206] In an embodiment, the IgG Fc region comprises a mutated IgGl or a mutated IgG4 Fc region, or a fragment or portion thereof. In an embodiment, the IgG Fc region comprises one or more (e.g., two, three, four, or five) mutations, for example, one or more (e.g., two, three, four, or five) mutations described herein.

[0207] In an embodiment, the IgG Fc region comprises a mutated IgG4 Fc region, or a fragment or portion thereof, wherein the mutated IgG4 Fc region is human.

[0208] In an embodiment, the mutated IgG4 Fc region, or a fragment or portion thereof, comprises an amino acid change (e.g., substitution) at Ser228 numbered according to the EU numbering, optionally wherein the amino acid change (e.g., substitution) at Ser228 is S228P. In an embodiment, the mutated IgG4 Fc region comprises the amino acid substitution S228P.

[0209] In an embodiment, the mutated IgG4 Fc region, or a fragment or portion thereof, comprises an amino acid change (e.g., substitution) at Arg409 numbered according to the EU numbering, optionally wherein the amino acid change (e.g., substitution) at Arg409 is R409K. In an embodiment, the mutated IgG4 Fc region comprises the amino acid substitution R409K.

[0210] In an embodiment, the mutated IgG4 Fc region, or a fragment or portion thereof, comprises amino acid changes (e.g., substitutions) at Thr307, Gln311, and Ala378 numbered according to the EU numbering, optionally wherein the amino acid changes (e.g., substitutions) are T307Q, Q311V, and A378V, respectively. In an embodiment, the mutated IgG4 Fc region comprises the amino acid substitutions T307Q, Q311V, and A378V.

[0211] In an embodiment, the mutated 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 from the same 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.

[0212] In an embodiment, the IgG Fc region comprises a mutated IgG1 Fc region, or fragment or portion thereof, wherein the mutated IgG1 Fc region is human. In an embodiment, the mutated IgG1 Fc region (e.g., comprising a N297G substitution) has an IgG1 m3 allotype.

[0213] In an embodiment, the mutated IgG1 Fc region, or fragment or portion thereof, comprises an amino acid change (e.g., substitution) at Asn297, numbered according to the EU numbering scheme, optionally wherein the amino acid change (e.g., substitution) at Asn297 is N297G. In an embodiment, the mutated IgG1 Fc region comprises the amino acid substitution N297G.

[0214] In an embodiment, the mutated IgG1 Fc region, or fragment or portion thereof, comprises an amino acid change (e.g., substitution) at Leu234, Leu235, and Pro329, numbered according to the EU numbering scheme, optionally wherein the amino acid change (e.g., substitution) is L234A, L235A, and P329G, respectively. In an embodiment, the mutated IgG1 Fc region comprises the amino acid substitutions L234A, L235A, and P329G.

[0215] In an embodiment, the mutated IgG1 Fc region, or fragment or portion thereof, comprises an amino acid change (e.g., substitution) at Thr307, Gln311, and Ala378, numbered according to the EU numbering scheme, optionally wherein the amino acid change (e.g., substitution) is T307Q, Q311V, and A378V, respectively. In an embodiment, the mutated IgG1 Fc region comprises the amino acid substitutions T307Q, Q311V, and A378V.

[0216] In one embodiment, the mutated IgGl Fc region comprises the amino acid sequence of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 1003, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom. In one embodiment, the mutated IgGl 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 therefrom. In one embodiment, the mutated IgGl Fc region comprises the amino acid sequence of SEQ ID NO: 1003.

[0217] In one embodiment, the non-IL-2 moiety inhibits or reduces the ability of the IL-2 preparation to elicit Fc receptor-mediated immune effector functions.

[0218] In one embodiment, the IL-2 formulation comprises an IL-2 variant comprising an amino acid sequence 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 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 formulation 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 to an IgG Fc region comprising an amino acid sequence selected from the group consisting of: 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.

[0219] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, 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.

[0220] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the group consisting of 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.

[0221] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, 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.

[0222] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, 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.

[0223] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, 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.

[0224] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the group consisting of SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273, 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.

[0225] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, 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.

[0226] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, SEQ ID NO: 349, 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.

[0227] 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.

[0228] A variety of technical effects are associated with IL-2 preparations comprising the amino acid sequence of SEQ ID NO: 1008. Without wishing to be bound by theory, it is believed that in one embodiment, IL-2 preparations comprising the amino acid sequence of SEQ ID NO: 1008 can have at least one or more of the following advantageous properties: (i) reduced binding affinity for CD122 and / or CD132 compared to other T cell types, which increases the efficacy and selectivity of the IL-2 preparation for regulatory T cells (Tregs); (ii) significant stabilization, e.g., due to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity for CD25 (or no more than a minimal impact thereon), which can extend the life of the IL-2 preparation; (iv) substantially no promotion of T effector cell and / or natural killer (NK) cell expansion, activation, survival, and / or proliferation in vitro and / or in vivo; and / or (v) reduced incorrect disulfide pairing and increased stability, e.g., due to the presence of the C125S mutation. In one embodiment, the IL-2 preparation comprising the H16L mutation has reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types compared to IL-2 preparations comprising other H16 mutations. These properties make IL-2 variants comprising the amino acid sequence of SEQ ID NO: 1008 particularly suitable for use in treating diseases and disorders caused by aberrant immune responses, e.g., autoimmune diseases.

[0229] Thus, in one embodiment, the IL-2 preparation comprising the amino acid sequence of SEQ ID NO: 5 has, inter alia, one or more (e.g., 2, 3, 4, 1008, 6, 7, or all) of the following properties relative 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 for human CD122 in vitro and / or in vivo; (iii) reduced or decreased binding capacity and / or binding affinity for human CD132 in vitro and / or in vivo; (iv) reduced or decreased affinity of the IL-2 variant for a heterodimeric (i.e., human CD122 / CD132 heterodimer) IL-2 receptor composed of human CD122 and human CD132 in vitro and / or in vivo; (v) reduced or decreased (e.g., moderately reduced or decreased) binding capacity and / or binding affinity for human CD25 in vitro and / or in vivo; (vi) selective binding to regulatory T cells (e.g., Foxp3 + T cells); (vii) selective activation of IL-2 signaling pathways in T regulatory cells (Tregs) in vitro or in vivo; (viii) enhanced or increased ability to induce or promote Treg expansion, activity, survival, and / or proliferation in vitro or in vivo.

[0230] In one embodiment, the IL-2 agent forms a dimer (e.g., a homodimer or a heterodimer).

[0231] 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.

[0232] In some aspects, the disclosure provides a pharmaceutical composition comprising the IL-2 agent described and a pharmaceutically acceptable carrier. In some aspects, the disclosure provides a nucleic acid encoding the IL-2 agent described herein. In some aspects, the disclosure provides a vector (e.g., an expression vector) comprising a nucleic acid encoding the IL-2 agent described herein. In some aspects, the disclosure provides a cell (e.g., an isolated cell) comprising a nucleic acid encoding the IL-2 agent described herein or a vector (e.g., an expression vector) comprising a nucleic acid encoding the IL-2 agent described herein.

[0233] In some aspects, the disclosure provides a method of producing an IL-2 agent, comprising culturing (e.g., maintaining) a cell comprising a nucleic acid encoding the IL-2 agent described herein or a vector (e.g., an expression vector) comprising a nucleic acid encoding the 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.

[0234] In some aspects, the disclosure provides a method of enhancing Treg expansion, activity, survival, and / or proliferation, comprising contacting a Treg cell or a population of Treg cells (e.g., in vitro, ex vivo, or in vivo) with an effective amount of an IL-2 agent described herein or a pharmaceutical composition comprising the IL-2 agent, or administering to a subject in need thereof an effective amount of an IL-2 agent described herein or a pharmaceutical composition comprising the IL-2 agent. The IL-2 agent may, for example, comprise 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.

[0235] In some aspects, the present disclosure provides a method of selectively activating IL-2 signaling pathway in T regulatory cells (Tregs), comprising contacting a Treg cell or a population of Treg cells (e.g., in vitro, ex vivo, or in vivo) with an effective amount of an IL-2 agent described herein or a pharmaceutical composition comprising the IL-2 agent, or administering to a subject in need thereof an effective amount of an IL-2 agent described herein or a pharmaceutical composition comprising the IL-2 agent. The IL-2 agent may, for example, comprise 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.

[0236] In some aspects, the present disclosure provides a method of inducing immune tolerance in a subject in need thereof, comprising administering an effective amount of an IL-2 agent described herein or a pharmaceutical composition comprising the IL-2 agent. The IL-2 agent may, for example, comprise 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.

[0237] In some aspects, the present disclosure provides a method of treating a subject having a disease (e.g., a disease described herein, e.g., an autoimmune disease, lupus nephritis, autoimmune hepatitis, nephrotic syndrome, or cancer), comprising administering to the subject an effective amount of an IL-2 agent described herein or a pharmaceutical composition comprising the IL-2 agent. The IL-2 agent may, for example, comprise 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.

[0238] In some aspects, the present disclosure provides an IL-2 agent or composition for use in a method of treating a subject having a disease (e.g., a disease 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 described herein or a pharmaceutical composition comprising the IL-2 agent. The IL-2 agent may, for example, comprise 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.

[0239] In some aspects, the present disclosure provides a use of an IL-2 preparation or composition in the manufacture of a medicament for treating a subject having a disease (e.g., a disease described herein, e.g., an autoimmune disease, lupus nephritis, autoimmune hepatitis, nephrotic syndrome, or a cancer) comprising administering to the subject an IL-2 agent described herein or a pharmaceutical composition comprising the IL-2 agent. The IL-2 preparation may, for example, comprise 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 preparation comprises the amino acid substitutions H16L, V69A, Q74P, and C125S.

[0240] In some aspects, the present disclosure provides a kit comprising an IL-2 preparation described herein or a pharmaceutical composition containing the IL-2 preparation, and instructions for use. The IL-2 preparation may, for example, comprise 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 preparation comprises the amino acid substitutions H16L, V69A, Q74P, and C125S.

[0241] In some aspects, the present disclosure provides a container comprising an IL-2 preparation described herein or a pharmaceutical composition containing the IL-2 preparation. The IL-2 preparation may, for example, comprise 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 preparation comprises the amino acid substitutions H16L, V69A, Q74P, and C125S. BRIEF DESCRIPTION OF DRAWINGS

[0242] Figure 1A The provided schematic illustrates the domain structure of exemplary, non-limiting embodiments of the IL-2 preparations provided herein. As shown, the IL-2 preparations comprise an IL-2 moiety or variant (also referred to herein as a “mutein”), a peptide linker, a hinge sequence-containing Fc, and CH2 and CH3 domains of an antibody. Figure 1B A depiction of the amino acid sequence of human IL-2 (SEQ ID NO: 1030) is provided, showing exemplary, non-limiting positions that, when mutated, result in effects on IL-2 receptor binding and IL-2-mediated signaling activity in vitro and in vivo.

[0243] Figure 2A schematic illustration of a cell-based method to generate IL-2 variant libraries and select stable and active clones from these libraries using yeast surface display. IL-2 mutations or variants of IL-2 expressed from initial clones are generated by DNA synthesis or error-prone PCR and transformed into yeast cells. Yeast cells are stained with anti-Myc antibody and fluorescent secondary antibody to determine IL-2 expression (x-axis) and with recombinant CD25, anti-6xHis antibody ("6xHis" disclosed as SEQ ID NO: 1028) and fluorescent secondary antibody to measure bound CD25 (y-axis). In some forms 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 showing high expression and high binding activity.

[0244] Figure 3A The provided graphs illustrate the results of a method to determine 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 a range of concentrations of CD25 extracellular domain tagged with 6xHis ("6xHis" disclosed as SEQ ID NO: 1028). Bound CD25 was measured by staining with anti-6xHis antibody ("6xHis" disclosed as SEQ ID NO: 1028) and fluorescent secondary antibody. Several exemplary IL-2 muteins are shown. Curve fitting was used to determine the binding affinity (K D ) and maximum binding signal (data not shown). Figure 3B The provided graphs illustrate the relative binding capacity of selected IL-2 muteins (maximum binding signal normalized to IL-2 expression level).

[0245] Figure 4A The provided graphs illustrate the thermal denaturation (melting curve) of selected IL-2 preparations (IL-2-Fc fusion proteins) as determined by SYPRO Orange fluorescence. The native IL-2-Fc fusion shows a maximum signal at low temperature, indicating the presence of unfolded protein, while the V69A / Q74P mutein shows a unfolding event as the temperature is raised. Figure 4B HPLC size exclusion chromatograms are provided showing that the majority of the native IL-2-Fc fusion elutes from the column very early (>670 kDa), indicating aggregated unfolded protein. In contrast, the V69A / Q74P IL-2-Fc elutes as a single peak at the expected time for an 84 kDa protein.

[0246] Figures 5A-5BProvided is a scatter plot showing the results of a yeast cell sorting operation to identify mutations that affect interaction with the CD122 and / or CD132 IL-2 receptor. Yeast expressing a library of IL-2 variants on their surface were stained with the indicated concentration of CD122 / CD132 Fc heterodimer, 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 recovered, and the IL-2 mutant proteins enriched in these populations were determined by a combination of Sanger sequencing and next-generation sequencing.

[0247] Figure 6A Provided is a graph showing the results of a method to determine the fractional saturation of yeast expressing the indicated IL-2 mutant protein on their surface after titration with the indicated concentration of CD122 / CD132 Fc heterodimer. All mutant proteins described contain V69A / Q74P in addition to the mutations shown. Bound CD122 / CD132 was labeled using an anti-human Fc fluorescent secondary antibody, and measurements were made using an Accuri C6 flow cytometer. Fractional saturation was calculated by fitting each curve to a 4-parameter dose response to estimate the maximum binding signal for each curve, and then normalized so that the estimated maximum value is defined as 1. Figure 6B Provided is a graph showing the results of the same method except that the selected mutant protein was incubated with recombinant CD25 extracellular domain tagged with a 6xHis tag ("6xHis" disclosed as SEQ ID NO: 1028), and bound CD25 was detected with an anti-6xHis antibody ("6xHis" disclosed as SEQ ID NO: 1028). Figure 6A

[0248] Figure 7 Provided is a series of graphs illustrating the affinity of IL-2-Fc fusion proteins containing different IL-2 variants (as indicated in the figure) for CD122 / CD132 Fc heterodimer and CD25 extracellular domain, measured on an Octet BioLayer Interferometer. IL-2 variants contain V69A / Q74P as well as the indicated mutations. IL-2-Fc fusion proteins were immobilized on anti-human Fc capture tips, and then incubated with a range of concentrations of the indicated IL-2 receptor. Association and dissociation phase kinetics were used to estimate binding affinity. Excess irrelevant antibody was used to prevent non-specific binding or capture of CD122 / CD132 Fc protein by the tips.

[0249] Figure 8 Provided is a schematic illustrating the gating strategy used to identify IL-2 sensitive cell populations from human PBMCs and the corresponding flow cytometry data. Singlet lymphocytes were identified based on forward and side scatter. Populations were defined as: T regulatory cells (CD4+CD25 高 ​Foxp3+), CD25 高 T helper cells (CD4+CD25 高 Foxp3-) and natural killer cells (CD3-CD56+).

[0250] Figures 9A-9D The provided graph illustrates the IL-2 signaling response in IL-2 sensitive cell populations in human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce the affinity of the CD122 / CD132 dimer, as determined by the degree of STAT5 phosphorylation. Figure 9A Tregs; Figure 9B CD25+(high) T helper cells; Figure 9C NK cells; Figure 9D CD8+cytotoxic T cells), as determined by the degree of STAT5 phosphorylation. Cells at the indicated concentrations were treated for 30 minutes with IL-2-Fc fusion proteins containing the V69A / Q74 mutations and the indicated mutations or with the IL-2 N88D mutein fused to the C-terminus of a non-binding antibody (C-terminal N88D). The inactivated IL-2-Fc fusion proteins contain several mutations to reduce their IL-2 signaling activity (F42A, Y45A, L72G, N88D, V69A, Q74P). Following treatment, cells were fixed with formaldehyde, permeabilized with cold methanol and stained for surface markers and STAT5 transcription factor phosphorylated at Tyr694 (pSTAT5). Each population was identified based on the gating described. Figure 8 The signaling activity of selected muteins on CD8+cytotoxic T cells was also assessed. These cells were gated as in Figure 8 , with the exception that the CD8 surface marker was used instead of CD4. The median pSTAT5 levels (median fluorescence intensity, MFI) of each concentration of IL-2-Fc fusion proteins tested in each cell population are shown. Curve fitting was performed using GraphPad Prism v5.03, 4-parameter fit log (agonist) vs. response.

[0251] Figures 10A-10C The provided graph depicts the IL-2 signaling response in IL-2 sensitive cell populations in human PBMCs after treatment with IL-2-Fc fusions containing mutations that reduce the affinity for CD25. Figure 10A Tregs; Figure 10B CD25+(high) T helper cells; Figure 10C NK cells. Treatment and analysis of human PBMCs was as shown in Figure 9. The median pSTAT5 levels (MFI) of each treatment in each human population are shown. To highlight the effect on EC 50 s, the signal within each mutein was normalized from 0 to 1 over the concentration range of IL-2-Fc treatment.

[0252] Figures 11A-11C The provided graphs depict IL-2 signaling responses in IL-2 sensitive cell populations in human PBMCs following treatment with IL-2-Fc fusions containing pairs of mutations that reduce affinity for both CD25 and CD122 / CD132 dimers Figure 11A , Tregs; Figure 11B , CD25+(high) T helper cells; Figure 11C , NK cells. Treatment and analysis of human PBMCs are as described in Figure 9. As shown, IL-2-Fc fusion proteins containing various IL-2 muteins are separated into upper and lower panels for clarity. Median pSTAT5 levels (MFI) are shown for each treatment in each population.

[0253] Figures 12A-12C The provided graphs illustrate in vivo expansion of Tregs, measured as percentage of total CD3+T cells, in Tg32 mice treated with IL-2-Fc H16N Figure 12A ) or C-terminal N88D Figure 12B ). CohohTg32 mice were dosed with the indicated amount of each IL-2 Fc fusion protein by tail vein injection (dose levels are approximately equimolar). At the indicated time points, lymphocyte populations were analyzed, and Tregs were defined as CD45+CD3+CD4+CD25 高 CD127- cells. Figure 12A and Figure 12B Data in and are the average of three mice per treatment group. Figure 12C Data from individual mice at the highest dose of each IL-2-Fc fusion protein tested are shown.

[0254] Figures 13A-13C The provided graphs illustrate changes in CD4+T helper cell levels, measured as percentage of total CD3+T cells, in Tg32 mice treated with IL-2-Fc H16N Figure 13A ) or C-terminal N88D Figure 13B ). Mice were dosed as in Figure 12. CD4+T helper cells were defined as CD45+CD3+CD4+cells that were not CD25 高 CD127-. Figure 13A and Figure 13B Data in and are the average of three mice per treatment group. Figure 13C Data from individual mice at the highest dose of each IL-2-Fc fusion protein tested are shown.

[0255] Figures 14A-14C The provided graphs illustrate changes in CD4+T helper cell levels, measured as percentage of total CD3+T cells, in Tg32 mice treated with IL-2-Fc H16N Figure 14A ) or C-terminal N88D Figure 14BThe change in CD8+ cytotoxic T cell levels in Tg32 mice treated with [the drug] was measured as the percentage of total CD3+ T cells. Mice were administered the drug as shown in Figure 12. Cytotoxic T cells were defined as CD45+CD3+CD8+ cells. Figure 14A and Figure 14B The data in the figure are the average values ​​of three mice in each treatment group. Figure 14C This shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested.

[0256] Figures 15A-15C The provided chart illustrates the use of IL-2-Fc H16N ( Figure 15A ) or C-terminal N88D ( Figure 15B The change in NK cell levels in Tg32 mice treated with [the drug] was measured as the percentage of total CD45+ lymphocytes. Mice were administered the drug as shown in Figure 12. NK cells were defined as CD45+CD3-CD56+ cells. Figure 15A and Figure 15B The data in the table represent the average values ​​of three mice in each treatment group. Under each condition, the percentage of NK cells in each mouse was standardized so that the pretreatment value was 1. Figure 15C This shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested.

[0257] Figures 16A-16B The provided charts illustrate the differences between CD122 / CD132Fc heterodimers or CD25 extracellular domains within a certain concentration range and those containing only the V69A / Q74P mutation (wild type). Figure 16A ) or contains inactivating mutations (42A, Y45A, L72G, N88D, V69A, Q74P; inactivation; Figure 16B The binding kinetics of the IL-2-Fc fusion protein (anchored to the tip of the anti-human Fc Octet) were studied. The binding kinetics were used to estimate the KB of each interaction. D .

[0258] Figures 17A-17D The provided charts illustrate the clearance kinetics of the IL-2-Fc fusion protein in mice. Figure 12 shows the clearance kinetics of the IL-2-Fc fusion protein at different doses containing the V69A / Q74P / H16N mutation or the C-terminal N88D. Figures 17A-17B ) or contains inactivating mutations (42A, Y45A, L72G, N88D, V69A, Q74P; inactivation; Figures 17C-17DSerum was collected from mice treated with IL-2-Fc fusion proteins of the disclosure. The amount of IL-2-Fc or C-terminal N88D presented 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 Immuno Research 109-035-008). 100% of starting material was defined as the amount detectable in plasma 1 hour post-injection. Note that the x-axis is categorical, not scaled by time.

[0259] Figures 18A-18D Expansion of immune cells in vivo after administration of exemplary IL-2 Fc fusion proteins in humanized mice is depicted. Figure 18A A schematic of the experimental design shown displays different time points at which blood was drawn from humanized mice administered IL-2 Fc fusion polypeptides and control polypeptides. Flow cytometry was used to measure various lymphocyte populations at each indicated time point. Figure 18B The fold expansion of T regulatory cells for each IL-2 Fc fusion polypeptide is presented on the Y-axis, and its corresponding dose (low or high) is indicated on the X-axis. Figure 18C The fold expansion of T helper cells for each IL-2 Fc fusion polypeptide is presented on the Y-axis, and its corresponding dose (low or high) is indicated on the X-axis. Figure 18D The fold expansion of NK cells for each IL-2 Fc fusion polypeptide is presented on the Y-axis, and its corresponding dose (low or high) is indicated on the X-axis. The IL-2 Fc fusion polypeptides under investigation are as follows: Figures 18B-18D The X-axis from left to right as follows: control monoclonal antibody (Motavizumab), inactivated IL-2, IL-2 mutein comprising N88D mutation, wild-type IL-2, IL-2 mutein comprising mutations H16N / V69A / Q74P / C125S (SEQ ID NO: 1007), and IL-2 mutein comprising mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008).

[0260] Figures 19A-19B The persistence and effective half-life of exemplary IL-2 fusion proteins in Tg32 mice is depicted. Figure 19A The concentration of IL-2 fusion proteins with the indicated combination of mutations in the mouse blood is presented on the Y-axis, and the number of days post-dosing at which the sample was taken is presented on the X-axis. Figure 19B A comparison of the half-life of IL-2 fusion proteins with the indicated combination of mutations in the IL-2 portion with or without additional mutations in the Fc region is presented. The concentration of the indicated IL-2 fusion proteins in the blood is presented on the Y-axis, and the number of days post-dosing is presented on the X-axis.

[0261] Figure 20 Pharmacokinetic profiles of exemplary IL-2-Fc fusion proteins (comprising mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgGl Fc N297G allotype m3)) in cynomolgus monkeys are described. Serum IL-2-Fc fusion protein levels were measured over time following weekly injections of 100 pg / kg IL-2-Fc fusion protein in four monkeys (numbered 3501, 3502, 3503, and 3504).

[0262] Figures 21A-21B Effects of exemplary IL-2-Fc fusion proteins (comprising mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgGl Fc N297G allotype m3)) on the expansion and proliferation of regulatory T cells in cynomolgus monkeys are described. Figure 21A Changes in the expansion of T regulatory cells over time following weekly injections of 100 pg / kg of IL-2-Fc fusion protein are presented as fold change relative to baseline (baseline = pre-dose). Figure 21B Ki67 expression in T regulatory cells over time following weekly injections of 100 pg / kg IL-2-Fc fusion protein is shown. + Percentages of T regulatory cells (a measure of the amount of proliferating T regulatory cells) were normalized to total T regulatory cells.

[0263] Figures 22A-22D Effects of exemplary IL-2-Fc fusion proteins (comprising mutations H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgGl Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys following weekly injections of 100 pg / kg IL-2-Fc fusion protein are illustrated. Figure 22A Effects of IL-2-Fc fusion proteins on the number of NK cells over time are presented, Figure 22B Effects on cytotoxic T cells over time are presented, Figure 22C Effects on T helper cells over time are presented, and Figure 22D Effects on total T cells over time are presented. For each cell type, data is shown as fold change from baseline (baseline = pre-dose).

[0264] Figures 23A-23C Effects of exemplary IL-2-Fc fusion proteins described herein on disease progression in a murine model of systemic lupus erythematosus with kidney involvement similar to lupus nephritis are illustrated. Figure 23AA series of graphs is presented illustrating proteinuria scores in individual mice treated with either a control for vehicle or an exemplary IL-2-Fc fusion protein (as indicated on the X-axis) on a Y-axis. From left to right, the first panel depicts proteinuria scores at 11 weeks of age, the middle panel depicts scores at 12 weeks of age, and the last panel depicts scores at 13 weeks of age. Figure 23B A series of graphs is presented illustrating proteinuria scores in individual mice treated with either a control for vehicle or an exemplary IL-2-Fc fusion protein (as indicated on the X-axis) on a Y-axis. From left to right, the first panel depicts proteinuria scores at 11 weeks of age, the middle panel depicts scores at 12 weeks of age, and the last panel depicts scores at 13 weeks of age. Figure 23C Quantified glomerular injury in individual mice treated with either a control for vehicle or an exemplary IL-2-Fc fusion protein (as indicated on the X-axis) at the end of the study (when mice reached 18 weeks of age) is presented on a Y-axis. DETAILED DESCRIPTION

[0266] 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, several of the IL-2 agents described herein have one or more improved or desirable properties compared to IL-2 agents comprising wild-type IL-2. Without wishing to be bound by theory, it is believed that in one embodiment, the IL-2 agents described herein 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 of making the IL-2 agents. The IL-2 agents and pharmaceutical compositions disclosed herein can be used (alone or in combination with other agents or therapeutic modalities) to treat, prevent, and / or diagnose diseases and conditions, e.g., diseases and conditions associated with T cell activity, e.g., the diseases or conditions described herein (e.g., the autoimmune conditions described herein).

[0267] Immune responses are generally controlled by the recognition of specific foreign or self antigens, communication between innate and adaptive immune pathways, cross-talk between B and T cells, and other factors. Some autoimmune diseases are characterized by the widespread recognition of self antigens. These diseases can be treated by therapies that broadly enhance the processes that protect self antigens from the immune system. Tregs are a type of T cell that recognizes self antigens. In response to antigenic stimulation, they release immunosuppressive cytokines and directly inhibit other T cells through cell-cell contact. Impaired Treg activity results in a variety of autoimmune diseases (e.g., cells that are too few or less active). IL-2 is a cytokine that leads to the expansion and activation of many cell types, but Tregs are generally more sensitive to IL-2 than other cell types. The administration of low-dose IL-2 has been shown to be associated with preferential, sustained in vivo expansion of Tregs and improvement in the presentation of chronic graft-versus-host disease (GVHD) in a significant proportion of patients (Koreth et al., N Engl J Med. 2011; 365(22):2055-2066). In one embodiment, the IL-2 formulations described herein provide a long-acting immunomodulator (e.g., immunosuppressant) for a number of disorders (e.g., autoimmune indications).

[0268] The present disclosure is based, at least in part, on the discovery that IL-2 formulations comprising human IL-2 polypeptides having specific combinations of amino acid substitutions described herein can have advantageous technical effects, e.g., increasing the stability of the IL-2 formulation and / or providing selective activation of regulatory T cells. The IL-2 formulations described herein generally require CD25 for efficient signaling through the IL-2 receptor, making them highly selective for Tregs. IL-2 signaling promotes Treg suppressive function and drives proliferation. Without wishing to be bound by theory, it is believed that Tregs activated by the IL-2 formulations described herein can suppress autoimmune activity through a variety of mechanisms.

[0269] In one embodiment, the IL-2 formulations described herein selectively bind to and activate regulatory T cells while sparing other immune cell types (e.g., CD25 高T cells and NK cells. Without wishing to be bound by theory, it is believed that in an embodiment, the amino acid substitutions described herein can facilitate the ability of the IL-2 preparation to maintain an active conformation and modulate the binding affinity of the IL-2 preparation to the dimeric receptor comprising IL-2Rβ (CD122) and IL-2Rγ (CD132) and the trimeric receptor comprising IL-2Rα (CD25) and CD122 and CD132. In an embodiment, the IL-2 preparations described herein have optimal affinity for selective binding and activation of IL-2 signaling in T regulatory cells, resulting in selective T regulatory cell activation and expansion in vitro and in vivo. Without wishing to be bound by theory, it is believed that in an embodiment, binding of IL-2 to the IL-2 receptor is the primary pathway for clearance of IL-2 in vivo. For example, the IL-2 preparations described herein that have reduced affinity for the dimeric and trimeric IL-2 receptors show extended half-lives, which suggests that reducing the affinity for the IL-2 receptor reduces the clearance rate of the IL-2 preparation in vivo. The IL-2 preparations described herein, for example those with amino acid substitutions that increase stability and reduce affinity for the IL-2 receptor, can selectively activate T regulatory cells and exhibit increased half-life in vivo. The IL-2 preparations described herein, for example those with mutations that prevent CD25 binding, can have improved half-life in vivo. In an embodiment, the IL-2 preparation does not promote or substantially does not promote 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, it is believed that in an embodiment, the IL-2 preparations described herein can have a greater therapeutic window than low-dose IL-2.

[0270] There are various technical effects associated with the presence of certain sets 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, it is believed that 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 significantly stabilized, e.g., due to the presence of the stabilizing V69A and Q74P mutations. For example, it was unexpectedly discovered that the V69A and Q74P substitutions do not significantly increase (or significantly decrease) the binding affinity of the IL-2 agent to CD25, but rather stabilize the IL-2 agent in an active conformation sufficient to bind to CD25. Without wishing to be bound by theory, it is also believed that in one embodiment, an IL-2 agent comprising the above-mentioned mutations has reduced binding affinity to CD122 and / or CD132, which increases the efficacy and selectivity (compared to other T cell types) of the IL-2 agent for regulatory T cells (Tregs). Thus, an IL-2 agent comprising these mutations is generally stabilized and selectively activates regulatory T cells (Tregs). Without wishing to be bound by theory, it is further believed that in one embodiment, an IL-2 agent comprising the above-mentioned mutations has reduced or decreased binding capacity and / or binding affinity to CD25, which improves the longevity of the IL-2 agent. Without wishing to be bound by theory, it is also believed that in one embodiment, an IL-2 agent comprising these mutations does not substantially promote in vitro and / or in vivo expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer cells (NK). In one embodiment, an IL-2 agent comprising the H16L mutation has reduced binding affinity to CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types compared to an IL-2 agent comprising other H16 mutations. These properties make an IL-2 agent comprising the above-mentioned mutations particularly suitable for treating a disease or disorder caused by an aberrant immune response, e.g., an autoimmune disease.

[0271] Thus, in one embodiment, an IL-2 agent (e.g., an IL-2 variant or IL-2 fusion protein) comprising a combination of 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 one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties (among others) relative to wild-type IL-2 or a reference IL-2 agent that does not comprise the amino acid substitutions:

[0272] (i) enhanced or increased stability in vitro or in vivo;

[0273] (ii) reduced or decreased binding capacity and / or binding affinity for human CD122 in vitro and / or in vivo;

[0274] (iii) reduced or decreased binding capacity and / or binding affinity for human CD132 in vitro and / or in vivo;

[0275] (iv) reduced or decreased affinity for a heterodimeric (i.e., human CD122 / CD132 heterodimeric) IL-2 receptor composed of human CD122 and human CD132 in vitro and / or in vivo by the IL-2 preparation;

[0276] (v) reduced or decreased (e.g., moderately reduced or decreased) binding capacity and / or binding affinity for human CD25 in vitro and / or in vivo;

[0277] (vi) selectively binds to regulatory T cells (e.g., Foxp3 + T cells);

[0278] (vii) selectively activates IL-2 signaling pathways in T regulatory cells (Tregs) in vitro or in vivo; or

[0279] (viii) enhanced or increased ability to induce or promote Treg expansion, activity, survival, and / or proliferation.

[0280] Definitions

[0281] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the articles. By the use of the term "or" as used herein is meant "and / or", unless otherwise indicated by context.

[0282] The term "or / otherwise" as used herein is meant "and / or", unless otherwise indicated by context, and is used interchangeably therewith.

[0283] "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured considering the nature of the quantity and the precision of the measuring instrument used to measure the quantity. An exemplary degree of error is within 20 percent (%), typically within 10 percent, and more typically within 5 percent of a given value or range of values. When "about" or "approximately" precedes a series of numbers or a range of numbers, it is understood that "about" or "approximately" can modify each number in the series or range. Similarly, when "at least," "greater than," "less than," "not greater than," "not less than," or "within" precedes a series of numbers or a range of numbers, it is understood that "at least," "greater than," "less than," "not greater than," "not less than," or "within" can modify each number in the series or range. As used herein, ranges include the upper and lower limits.

[0284] The compositions and methods described herein include polypeptides and nucleic acids having the specified sequences, or polypeptides and nucleic acids having sequences that are substantially identical to or similar to the specified sequences (e.g., at least 85%, 90%, 95%, or more identical to the specified sequences).

[0285] In the context of describing an amino acid sequence, the term "substantially identical" as used herein means that a first amino acid contains a sufficient or minimum number of amino acid residues that are either i) identical to, or ii) conservative substitutions of, aligned amino acid residues in a second amino acid sequence, such that the first and second amino acid sequences can have shared domains and / or shared functional activities. For example, an amino acid sequence that comprises a shared domain that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence (e.g., a sequence provided herein).

[0286] In the context of describing a nucleotide sequence, the term "substantially identical" as used herein means that a first nucleic acid sequence comprises a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence, such that the first and second nucleotide sequences encode polypeptides with identical functional activities, or encode identical structural polypeptide domains or identical functional polypeptide activities. For example, a nucleotide sequence that comprises at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence (e.g., a sequence provided herein).

[0287] The term "functional variant" refers to a polypeptide that has a substantially identical amino acid sequence to or is encoded by a substantially identical nucleotide sequence to a native sequence, and is capable of one or more activities of the native sequence.

[0288] Calculations of homology or sequence identity between sequences (these terms are used interchangeably herein) are performed as follows.

[0289] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the reference sequence is at least 30%, e.g., at least 40%, 50%, 60%, e.g., at least 70%, 80%, 90%, 100% the length of the reference sequence for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence then the molecules are identical at that position.

[0290] The percent identity between two sequences is related to the number of positions shared by the two sequences, taking into account the number and length of gaps required to achieve an optimal alignment of the two sequences.

[0291] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)), as implemented in the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, with either weight of gaps as 16, 14, 12, 10, 8, 6, or 4 and the length weight as 1, 2, 3, 4, 5, or 6. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and the length weight of 1, 2, 3, 4, 5, or 6. One suitable set of parameters (and the set that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, and a gap extend penalty of 4, and a translation gap penalty of 5.

[0292] The percent identity between two sequences is related to the number of positions shared by the two sequences, taking into account the number and length of gaps required to achieve an optimal alignment of the two sequences.

[0293] The nucleic acid and protein sequences disclosed herein can also be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993) available through www.ncbi.nlm.nih.gov. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to nucleic acids described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0294] As used herein, the term "hybridize" or "hybridization" under low, medium, high, or very high stringency conditions refers to the conditions under which the hybridization and washing occur. Guidance in regard to conditions suitable for hybridization reactions can be found in Current Protocols in Molecular Biology (John Wiley & Sons, New York, 1989, 6.3.1-6.3.6), which is incorporated herein by reference. Both aqueous and nonaqueous methods are described in this reference, either of which can be used. The particular hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at 50°C (the wash temperature can be increased to 55°C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C, and preferably 4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS, 65°C, followed by one or more washes in 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions 4) are suitable conditions and are to be used unless otherwise specified.

[0295] It is understood that the molecules described herein can have other conservative or non-essential amino acid substitutions that do not have a substantial effect on the function of the molecule.

[0296] The term "amino acid" encompasses all molecules comprising an amino functional group and an acid functional group and capable of being included in a polymer formed from naturally occurring amino acids, whether natural or synthetic molecules. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D- and L- mirror image isomers and peptidomimetics.

[0297] A "conservative amino acid substitution" is where one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0298] The terms "polypeptide," "peptide," and "protein" (if single chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The terms also encompass modified amino acid polymers; for example, glycosylated, lipidated, acetylated, phosphorylated, or otherwise modified polymers, as well as conjugates with labeling components. The polypeptides can be isolated from natural sources, produced by recombinant techniques from prokaryotic or eukaryotic hosts, or be products of synthetic procedures.

[0299] As will be recognized by those of skill in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of the present application. 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) of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or greater than 100 amino acids in length is provided herein. In another example, any protein that comprises a sequence stretch of about 20, about 30, about 40, about 50, or about 100 amino acids that is about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100% identical to any sequence described herein can be used in the present application. In one embodiment, a protein sequence used in accordance with the present disclosure comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any sequence provided or referenced herein.

[0300] The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence," and "polynucleotide" are used interchangeably herein. They refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs. A polynucleotide can be single-stranded or double-stranded, and if single-stranded, can be the coding strand or the non-coding (anti- sense) strand. A polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Nucleotide sequences can be interspersed with non-nucleotide components. A polynucleotide can be further modified, such as by conjugation with a labeling component. The nucleic acid can be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin, i.e., not naturally occurring or associated with another polynucleotide in a natural arrangement.

[0301] The term "isolated" as used herein refers to a substance that is removed from its original or natural environment (e.g., from a natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide that is present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials of the natural system, is isolated. Such polynucleotides can be part of a vector and / or such polynucleotides or polypeptides can be part of a composition, but are still isolated in that the vector or composition is not part of the natural environment of the polynucleotide or polypeptide.

[0302] As used herein, the term "treatment" of a disorder (e.g., myeloma) refers, in one embodiment, to a subject (e.g., a human) having a disorder (e.g., myeloma) and / or experiencing symptoms of a disorder (e.g., myeloma) will, upon receiving an antibody molecule, undergo a less severe level of symptoms and / or recover more quickly than if the antibody molecule had not been received. In one embodiment, when treating myeloma, a bone marrow biopsy will show fewer clonal plasma cells after effective treatment of myeloma. For example, after administration of an antibody molecule described herein to effectively treat myeloma, a diagnostic assay will detect fewer clonal plasma cells in a biological sample from the subject. Other assays, a urine test or a blood test, can also be used to monitor treatment of a patient, or to detect the presence of myeloma symptoms in a subject after treatment of myeloma, e.g., a decrease in (or absence of) the presence. In one embodiment, when treating myeloma, the level of beta 2 microglobulin (β2Μ) in serum or urine will decrease after effective treatment of myeloma. Treatment can be, for example, partial or complete alleviation, amelioration, relief, inhibition, or reduction in severity and / or frequency of a disorder (e.g., myeloma), and can optionally delay onset or progression of one or more consequences or symptoms, features, and / or causes of a disorder (e.g., myeloma). In one embodiment, treatment is of a subject who does not show signs of a disorder (e.g., myeloma) and / or shows only early signs of a disorder (e.g., kidney disease). In one embodiment, treatment is of a subject who has one or more established signs of a disorder (e.g., myeloma). In one embodiment, treatment is of a subject diagnosed with a disease (e.g., myeloma).

[0303] As used herein, the term "prevention" of a disease (e.g., myeloma) refers to a subject (e.g., a human) who is less likely to develop the disease (e.g., myeloma) if the subject (e.g., human) receives an antibody molecule.

[0304] Various aspects of the compositions and methods described herein are described in further detail below. Additional definitions are provided throughout the specification.

[0305] IL-2 formulations

[0306] The present disclosure provides IL-2 formulations, including but not limited to IL-2 variants, IL-2 fusion proteins, IL-2 complexes, and IL-2 conjugates. For example, the IL-2 formulations described herein can have one or more structural and / or functional properties described herein. In an embodiment, the IL-2 formulation comprises an IL-2 variant comprising one or more amino acid changes (e.g., substitutions) described herein. In an embodiment, the IL-2 formulation comprises an IL-2 variant comprising one or more amino acid changes (e.g., substitutions) described in Table 9. In an embodiment, the IL-2 formulation comprises an IL-2 variant comprising an amino acid sequence described in Table 9, or a portion thereof. In an embodiment, the IL-2 formulation, or a portion thereof, is encoded by a nucleic acid comprising a nucleotide sequence described herein (e.g., Table 10). One or more amino acid changes (e.g., substitutions) alone or in combination can provide one or more desirable biological properties described herein. In an embodiment, the IL-2 formulation can modulate (e.g., increase) Treg proliferation, survival, activation, and / or function. In an embodiment, the modulation is selective or specific for Tregs. For example, the IL-2 formulation can be capable of modulating activity in Tregs, but have limited or lack the ability to promote activity in non-regulatory T cells. In an embodiment, the IL-2 formulation comprises a polypeptide (sometimes referred to herein as an “IL-2 polypeptide agent”).

[0307] IL-2 variants

[0308] In an embodiment, the IL-2 formulation comprises an IL-2 variant, e.g., an IL-2 variant described herein.

[0309] In an 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 an embodiment, the IL-2 variant comprises one or more amino acid changes (e.g., substitutions) described in Table 9. In an embodiment, the IL-2 variant comprises or consists of an amino acid sequence described in Table 9, or a functional fragment thereof. In an embodiment, the IL-2 variant is encoded by a nucleic acid comprising a nucleotide sequence described herein, e.g., in Table 10.

[0310] Without wishing to be bound by theory, it is believed that in one embodiment, the IL-2 variants described herein have reduced human CD25 and / or reduced human CD122 / CD132 binding affinity relative to wild-type human IL-2 or a reference IL-2 variant, can have improved binding and efficacy and / or selectivity to activate regulatory T cells (Tregs) compared 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 affinity to human CD25 and / or human CD122 / CD132 within a desired range.

[0311] 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 described herein, e.g., different and / or improved properties relative to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant comprises 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 relative 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 relative to wild-type IL-2 or a reference IL-2 variant (e.g., determined by an assay described herein):

[0312] i) altered (e.g., enhanced or increased) expression in vitro and / or in vivo;

[0313] ii) altered (e.g., reduced or decreased) aggregation in vitro and / or in vivo;

[0314] iii) altered (e.g., enhanced or increased) stability in vitro and / or in vivo;

[0315] iv) altered (e.g., enhanced or increased) half-life in vitro and / or in vivo;

[0316] v) altered (e.g., reduced or decreased) turnover and / or clearance in vivo;

[0317] vi) altered (e.g., reduced or decreased) susceptibility to proteolysis in vitro and / or in vivo;

[0318] vii) altered (e.g., enhanced or increased) resistance to proteolysis in vitro and / or in vivo;

[0319] viii) altered (e.g., reduced or decreased) binding capacity and / or binding affinity for human CD25 in vitro and / or in vivo;

[0320] ix) altered (e.g., reduced or decreased) binding capacity and / or binding affinity for human CD132 in vitro and / or in vivo;

[0321] x) altered (e.g., reduced or decreased) binding capacity and / or binding affinity for a dimeric IL-2 receptor comprising human CD122 and human CD132 in vitro and / or in vivo;

[0322] xi) altered (e.g., enhanced, increased, decreased, reduced, and / or selective) binding to Tregs in vitro and / or in vivo;

[0323] xii) altered (e.g., enhanced, increased, decreased, reduced, and / or selective) activation of IL-2 signaling pathway in Tregs in vitro and / or in vivo;

[0324] xiii) altered (e.g., enhanced, increased, decreased, reduced, and / or selective) ability to induce or promote Treg expansion, activity, survival, and / or proliferation in vitro and / or in vivo.

[0325] In one embodiment, the IL-2 variant has altered (e.g., enhanced or increased) expression in vitro and / or in vivo relative to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has enhanced or increased expression (e.g., in bacterial or mammalian cells) relative to wild-type IL-2. In one embodiment, the IL-2 variant has enhanced or increased expression (e.g., in bacterial or mammalian cells) relative to a reference IL-2 variant. In one embodiment, the IL-2 variant has about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more, increased expression. In one embodiment, the IL-2 variant has 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, increased expression. 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, increased, e.g., relative to an IL-2 preparation comprising wild-type IL-2 or an IL-2 preparation comprising a reference IL-2 variant, e.g., as determined by a protein concentration assay. 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, increased, e.g., relative to an IL-2 preparation comprising wild-type IL-2 or an IL-2 preparation comprising a reference IL-2 variant, e.g., as determined by a protein concentration assay.

[0326] In one embodiment, the IL-2 variant has altered (e.g., reduced or decreased) aggregation in vitro and / or in vivo relative to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has reduced or decreased aggregation relative to wild-type IL-2. In one embodiment, the IL-2 variant has reduced or decreased aggregation relative to a reference IL-2 variant. In one embodiment, the IL-2 variant has about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more reduction in aggregation. In one embodiment, the IL-2 variant has 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 reduction in aggregation. In one embodiment, an IL-2 formulation comprising an IL-2 variant described herein aggregates at a lower or decreased 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 reduction, e.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by melting temperature analysis (e.g., using fluorescence), dynamic light scattering, and / or size exclusion chromatography. In one embodiment, an IL-2 formulation comprising an IL-2 variant described herein aggregates at a lower or decreased level, e.g., about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more reduction, e.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by melting temperature analysis (e.g., using fluorescence), dynamic light scattering, and / or size exclusion chromatography.

[0327] In one embodiment, the IL-2 variant has altered (e.g., enhanced or increased) stability in vitro and / or in vivo relative to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has enhanced or increased stability relative to wild-type IL-2. In one embodiment, the IL-2 variant has enhanced or increased stability relative to a reference IL-2 variant. In one embodiment, the IL-2 variant has about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more, increased stability. In one embodiment, the IL-2 variant has 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, increased stability. In one embodiment, an IL-2 formulation comprising an IL-2 variant described herein 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 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, 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, increased stability, e.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by yeast surface display, circular dichroism or related spectroscopic techniques, and / or melting temperature analysis (e.g., using a fluorimetric method).

[0328] In one embodiment, the IL-2 variant has an altered (e.g., enhanced or increased) half-life in vitro and / or in vivo relative 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 relative to wild-type IL-2. In one embodiment, the IL-2 variant has an enhanced or increased half-life relative 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 formulation comprising an IL-2 variant described herein has an enhanced or increased half-life in vitro and / or in vivo, e.g., increased by 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, e.g., greater than 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.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by ELISA, flow cytometry, and / or mass spectrometry.

[0329] In one embodiment, the IL-2 variant has altered (e.g., reduced or decreased) turnover in vitro and / or in vivo relative to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has reduced or decreased turnover relative to wild-type IL-2. In one embodiment, the IL-2 variant has reduced or decreased turnover relative to a reference IL-2 variant. In one embodiment, the IL-2 variant has about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more, decreased turnover. In one embodiment, the IL-2 variant has 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, decreased turnover. In one embodiment, an IL-2 formulation comprising an IL-2 variant described herein has a lower, reduced, or decreased turnover rate or level and / or clearance rate 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, or, 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, decreased, e.g., relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by ELISA, flow cytometry, and / or mass spectrometry.

[0330] In one embodiment, the IL-2 has altered (e.g., reduced or decreased) susceptibility to proteolysis in vitro and / or in vivo relative 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 relative to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 variant has reduced or decreased susceptibility to proteolysis relative to a reference IL-2 variant. In one embodiment, the IL-2 variant has about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more, decreased susceptibility to proteolysis. In one embodiment, the IL-2 variant has 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, decreased susceptibility to proteolysis.

[0331] In one embodiment, the IL-2 variant has altered (e.g., enhanced or increased) resistance to proteolysis in vitro and / or in vivo relative 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 relative to wild-type IL-2. In one embodiment, the IL-2 variant has enhanced or increased resistance to proteolysis relative to a reference IL-2 variant. In one embodiment, the IL-2 variant has about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more, increased resistance to proteolysis. In one embodiment, the IL-2 variant has 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, increased resistance to proteolysis.

[0332] In one embodiment, the IL-2 variant has an altered (e.g., reduced or decreased) binding capacity and / or binding affinity for human CD25 in vitro and / or in vivo relative to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has a reduced or decreased binding capacity and / or binding affinity for human CD25 relative to wild-type human IL-2. In one embodiment, the IL-2 variant has a reduced or decreased binding capacity and / or binding affinity for human CD25 relative to a reference human IL-2 variant. In one embodiment, the IL-2 variant has a reduced binding capacity and / or binding affinity for human CD25 by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the IL-2 variant has a reduced binding capacity and / or binding affinity for human CD25 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 preparation comprising an IL-2 variant described herein has a reduced or decreased binding affinity for CD25 (e.g., human CD25), e.g., by 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 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.g., relative to an IL-2 preparation comprising wild-type IL-2 or an IL-2 preparation comprising a reference IL-2 variant, e.g., as determined by yeast surface display, surface plasmon resonance (e.g., Biacore), and / or bio-layer interferometry (e.g., Octet binding).

[0333] In one embodiment, the IL-2 variant binds CD25 (e.g., human CD25) with low affinity, e.g., a dissociation constant (K D) is about 5-500 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 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, e.g., about 10 to about 20 pM, about 20 to about 30 pM, or about 30 to about 40 pM, e.g., 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, e.g., 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, e.g., as determined by yeast surface display, surface plasmon resonance (e.g., Biacore), and / or bio-layer interferometry (e.g., Octet binding).

[0334] In one embodiment, the IL-2 variant binds CD25 (e.g., human CD25) with low affinity, e.g., a dissociation constant (K D) is about 0.1-10 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-about 5 nM, about 0.5-about 2 nM, about 1-1.5 nM, about 0.1-about 0.2 nM, e.g., about 0.2-about 0.3 nM, about 0.3-about 0.4 nM, or about 0.4-about 0.5 nM, e.g., about 0.5-about 0.6 nM, about 0.6-about 0.7 nM, about 0.7-about 0.8 nM, about 0.8-about 0.9 nM, about 0.9-about 1 nM, about 1-about 1.5 nM, about 1.5-about 2 nM, about 2.5-about 3 nM, about 3.5-about 4 nM, about 4-about 4.5 nM, about 4.5-about 5 nM, about 5-about 6 nM, about 6-about 7 nM, about 7-about 8 nM, about 8-about 9 nM, or about 9-about 10 nM, or e.g., 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, e.g., as determined by surface plasmon resonance (e.g., Biacore) and / or bio-layer interferometry (e.g., Octet binding).

[0335] In one embodiment, the IL-2 variant has an altered (e.g., reduced or decreased) binding capacity and / or binding affinity for human CD132 in vitro and / or in vivo relative to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has a reduced or decreased binding capacity and / or binding affinity for human CD132 relative to wild-type IL-2. In one embodiment, the IL-2 variant has a reduced or decreased binding capacity and / or binding affinity for human CD132 relative to a reference human IL-2 variant. In one embodiment, the IL-2 variant has a reduction of about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more in binding capacity and / or binding affinity for human CD132. In one embodiment, the IL-2 variant has a reduction of 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 binding capacity and / or binding affinity for human CD132.

[0336] In one embodiment, the IL-2 variant has an altered (e.g., reduced or decreased) binding capacity and / or binding affinity for a human dimeric IL-2 receptor comprising human CD122 and human CD132, in vitro and / or in vivo, relative to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has a reduced or decreased binding capacity and / or binding affinity for a human dimeric IL-2 receptor comprising human CD122 and human CD132, relative to wild-type IL-2. In one embodiment, the IL-2 variant has a reduced or decreased binding capacity and / or binding affinity for a human dimeric IL-2 receptor comprising human CD122 and human CD132, relative to a reference IL-2 variant. In one embodiment, the IL-2 variant has a reduction of about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more in binding capacity and / or binding affinity for a human dimeric IL-2 receptor comprising human CD122 and human CD132. In one embodiment, the IL-2 variant has a reduction of 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 binding capacity and / or binding affinity for a human dimeric IL-2 receptor comprising human CD122 and human CD132.

[0337] In one embodiment, the IL-2 variant has a reduced or decreased binding affinity for a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer), for example, a reduction of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more, or a reduction of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, or more, for example, relative to an IL-2 preparation comprising wild-type IL-2 or an IL-2 preparation comprising a reference IL-2 variant, as determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore), and / or bio-layer interferometry (e.g., Octet binding).

[0338] In one embodiment, the IL-2 variant binds a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with a low affinity, for example, a dissociation constant (K D) is about 0.2-20 nM, e.g., about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4. about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 20 nM, or e.g., about 0.5 to about 15 nM, about 1 to about 10 nM, about 2 to about 5 nM, about 0.2 to about 0.3 nM, about 0.3 to about 0.4 nM, about 0.4 to about 0.5 nM, about 0.5 to about 0.6 nM, about 0.6 to about 0.7 nM, about 0.7 to about 0.8 nM, about 0.8 to about 0.9 nM, about 0.9 to about 1 nM, about 1 to about 1.1 nM, about 1.1 to about 1.2 nM, about 1.2 to about 1.3 nM, about 1.3 to about 1.4 nM, about 1.4 to about 1.5 nM, about 1.5 to about 2 nM, about 2 to about 3 nM, about 3 to about 4 nM, about 4 to about 5 nM, about 5 to about 6 nM, about 6 to about 7 nM, about 7 to about 8 nM, about 8 to about 9 nM, about 9 to about 10 nM, about 10 to about 11 nM, about 11 to about 12 nM, about 12 to about 13 nM, about 13 to about 14 nM, about 14 to about 15 nM, about 15 to about 16 nM, about 16 to about 17 nM, about 17 to about 18 nM, about 18 to about 19 nM, or about 19 to about 20 nM, or e.g., 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, e.g., as determined by yeast surface display.

[0339] In one embodiment, the IL-2 variant binds to a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with a low affinity, e.g., a dissociation constant (K D) is about 0.2-300 nM, e.g., about 0.2 nM, about 0.5 nM, about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 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 e.g., about 0.5 to about 15 nM, about 1 to about 10 nM, about 2 to about 5 nM, about 0.2 nM to about 0.5 nM, about 0.5 nM to about 1 nM, about 1 to about 2 nM, about 2 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 15 nM, about 15 nM to about 20 nM, about 20 nM to about 25 nM, about 25 to about 30 nM, about 30 nM to about 40 nM, about 40 nM to about 50 nM, about 50 to about 60 nM, about 60 to about 70 nM, about 70 nM to about 80 nM, about 80 nM to about 90 nM, about 90 nM to about 100 nM, about 100 nM to about 110 nM, about 110 nM to about 120 nM, about 120 nM to about 130 nM, about 130 nM to about 140 nM, about 140 nM to about 150 nM, about 150 nM to about 160 nM, about 160 nM to about 170 nM, about 170 nM to about 180 nM, about 180 nM to about 190 nM, about 190 nM to about 200 nM, about 200 nM to about 210 nM, about 210 nM to about 220 nM, about 220 nM to about 230 nM, about 230 nM to about 240 nM, about 240 nM to about 250 nM, about 250 nM to about 260 nM, about 260 nM to about 270 nM, about 270 nM to about 280 nM, about 280 nM to about 290 nM, or about 290 nM to about 300 nM, or e.g., greater than about 0.2, about 0.5, 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, e.g., as determined by surface plasmon resonance (e.g., Biacore) and / or bio-layer interferometry (e.g., Octet binding).

[0340] 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 relative 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 relative to wild-type IL-2. In one embodiment, the IL-2 variant has selective binding to Tregs relative to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 variant has enhanced or increased binding to Tregs relative to a reference IL-2 variant. In one embodiment, the IL-2 variant has selective binding to Tregs relative to a reference IL-2 variant. In one embodiment, the 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, the 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.

[0341] 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 relative 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 relative to wild-type IL-2. In one embodiment, the IL-2 variant has selective activation of the IL-2 signaling pathway in Tregs relative 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 relative to a reference IL-2 variant. In one embodiment, the IL-2 variant has selective activation of the IL-2 signaling pathway in Tregs relative to a reference IL-2 variant. In one embodiment, the activation of the IL-2 signaling pathway in Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the 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.

[0342] In one embodiment, the IL-2 variant selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, e.g., has a T helper EC50 / Treg EC50 ratio of greater than 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, relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by flow cytometry.

[0343] In one embodiment, the IL-2 variant selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, e.g., has an NK cell EC50 / Treg EC50 ratio of greater than, 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 greater, or, e.g., greater than 1 and about 1-2, about 2-3, about 3-4, about 4-5, greater than 1 and about 1-10, greater than 1 and about 1-20, greater than 1 and about 1-30, greater than 1 and about 1-40, greater than 1 and about 1-50, about 2-10, about 2-20, about 2-30, about 2-40, 2-50, about 5-10, about 5-20, about 5-30, about 5-40, about 5-50, about 10-20, about 10-30, about 10-40 about 10-50, about 20-40, about 20-50, about 50-100, about 100-200, about 200-500, about 500-1000, about 1000-2000, or about 1000-3000, relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant, e.g., as determined by flow cytometry.

[0344] In one embodiment, the IL-2 variant has an altered (e.g., enhanced, increased, and / or selective) ability to induce or promote Treg expansion, activity, survival, and / or proliferation in vitro and / or in vivo relative 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 Treg expansion, activity, survival, and / or proliferation relative to wild-type IL-2. In one embodiment, the IL-2 variant has a selective ability to induce or promote Treg expansion, activity, survival, and / or proliferation relative to wild-type IL-2. In one embodiment, the IL-2 variant has an enhanced or increased ability to induce or promote Treg expansion, activity, survival, and / or proliferation relative to a reference IL-2 variant. In one embodiment, the IL-2 variant has a selective ability to induce or promote Treg expansion, activity, survival, and / or proliferation relative to a reference IL-2 variant. In one embodiment, the ability to induce or promote Treg expansion, activity, survival, and / or proliferation is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the ability to induce or promote Treg expansion, activity, survival, and / or proliferation 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.

[0345] In one embodiment, the IL-2 variant has an enhanced or increased efficacy and / or ability to induce or promote Treg activity, e.g., EC50, relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant. 50 In one embodiment, the IL-2 variant has a decreased or reduced ability to induce or promote Treg activity, e.g., EC50, relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant. In one embodiment, the ability to induce or promote Treg activity is decreased by 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, e.g., 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.g., as determined by flow cytometry.

[0346] In one embodiment, the IL-2 variant has a decreased or reduced efficacy and / or ability to induce or promote Treg activity, e.g., EC50, relative to an IL-2 formulation comprising wild-type IL-2 or an IL-2 formulation comprising a reference IL-2 variant.50 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, for example, 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, about 50-fold, about 100-fold, about 200-fold, about 500-fold, about 1000-fold, about 2000-fold, about 5000-fold, about 10,000-fold, about 15,000-fold, or about 20,000-fold or more, e.g., as determined by flow cytometry.

[0347] In one embodiment, a T helper cell described herein is a CD45+CD3+CD4+Foxp3- cell, e.g., as determined by flow cytometry. In one embodiment, a Treg described herein is a CD45+CD3+CD4+Foxp3+ cell, e.g., as determined by flow cytometry. In one embodiment, an NK cell described herein is a CD45+CD3- cell that is CD56+and / or CD16+, e.g., as determined by flow cytometry. In one embodiment, an NK cell described herein is a CD45+CD3-CD56+ cell, e.g., as determined by flow cytometry.

[0348] In one embodiment, an IL-2 variant has one or more of the same or substantially the same structural and / or functional properties as wild-type IL-2 or a reference IL-2 variant.

[0349] In one embodiment, a 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, a reference IL-2 variant comprises the amino acid sequence of SEQ ID NO: 1 (IL-2 C125S). In one embodiment, an 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 comprises one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid changes (e.g., substitutions) described herein.

[0350] For purposes of the present disclosure, IL-2 variant position numbering begins with the first amino acid following the signal peptide of the exemplary wild-type (WT) human IL-2 polypeptide:

[0351] MYRMQLLSCIALSLALVTNS / A1 / P2 / T3 / S4 / S5 / S6 / T7 / K8 / K9 / T10 / Q11 / L12 / Q13 / L14 / E15 / H16 / L17 / L18 / L19 / D20 / L21 / Q22 / M23 / I24 / L25 / N26 / G27 / I28 / N29 / N30 / Y31 / K32 / N33 / P34 / K35 / L36 / T37 / R38 / M39 / L40 / T41 / F42 / K43 / F44 / Y45 / M46 / P47 / K48 / K49 / A50 / T51 / E52 / L53 / K54 / H55 / L56 / Q57 / C58 / L59 / E60 / E61 / E62 / L63 / K64 / P65 / L66 / E67 / E68 / V69 / L70 / N71 / L72 / A73 / Q74 / S75 / K76 / N77 / F78 / H79 / L80 / R81 / P82 / R83 / D84 / L85 / I86 / S87 / N88 / I89 / N90 / V91 / I92 / V93 / L94 / E95 / L96 / K97 / G98 / S99 / E100 / T101 / T102 / F103 / M104 / C105 / E106 / Y107 / A108 / D109 / E110 / T111 / A112 / T113 / I114 / V115 / E116 / F117 / L118 / N119 / R120 / W121 / I122 / T123 / F124 / C125 / Q126 / S127 / I128 / I129 / S130 / T131 / L132 / T133(SEQ ID NO:360; Uniprot P60568; signal peptide underlined ). The corresponding amino acid sequence without the signal peptide is set forth in SEQ ID NO: 1031.

[0352] In one embodiment, the IL-2 formulation comprises one or more amino acid changes (e.g., substitutions) at one or more positions corresponding to human IL-2 (e.g., comprising the amino acid sequence of SEQ ID NO: 1031).

[0353] In an embodiment, the IL-2 variant comprises the amino acid sequence: A1 / P2 / X3 / S4 / S5 / S6 / T7 / K8 / K9 / T10 / Q11 / L12 / Q13 / L14 / E15 / X16 / L17 / L18 / L19 / D20 / L21 / Q22 / M23 / I24 / L25 / N26 / G27 / X28 / N29 / N30 / Y31 / K32 / N33 / P34 / X35 / L36 / T37 / X38 / M39 / L40 / T41 / X42 / K43 / F44 / Y45 / M46 / P47 / K48 / K49 / A50 / T51 / E52 / L53 / K54 / H55 / L56 / Q57 / C58 / L59 / E60 / E61 / E62 / L63 / K64 / P65 / L66 / E67 / X68 / X69 / L70 / N71 / L72 / A73 / X74 / S75 / K76 / N77 / F78 / H79 / L80 / R81 / P82 / R83 / X84 / L85 / I86 / X87 / X88 / I89 / N90 / V91 / X92 / V93 / L94 / E95 / L96 / K97 / G98 / S99 / E100 / T101 / T102 / F103 / M104 / C105 / E106 / Y107 / A108 / D109 / E110 / T111 / A112 / T113 / I114 / V115 / E116 / F117 / L118 / N119 / R120 / W121 / I122 / T123 / F124 / X125 / X126 / S127 / I128 / I129 / S130 / T131 / L132 / T133 (SEQ ID NO: 1032),

[0354] 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, provided that the IL-2 variant does not comprise the amino acid sequence of SEQ ID NO: 1 or 1031. In an embodiment, the IL-2 variant comprises or consists of an IL-2 variant amino acid sequence described herein.

[0355] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) positions described herein. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) positions selected from T3, H16, I28, K35, R38, F42, E68, V69, Q74, D84, S87, N88, I92, C125, or Q126.

[0356] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position T3. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position H16. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position I28. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position K35. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position R38. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position F42. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position E68. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position V69. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position Q74. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position D84. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position S87. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position N88. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position I92. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position C125. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position Q126.

[0357] In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position V69, Q74, or a combination thereof. In one embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at positions V69 and Q74. In one embodiment, the IL-2 variant comprises the amino acid substitution V69A. In one embodiment, the IL-2 variant comprises the amino acid substitution Q74P.

[0358] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position H16, I92, D84, or a combination thereof. In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position H16, optionally wherein the amino acid substitution is H16N, H16L, or H16D. In an embodiment, the IL-2 variant comprises the amino acid substitution H16N. In an embodiment, the IL-2 variant comprises the amino acid substitution H16L. In an embodiment, the IL-2 variant comprises the amino acid substitution H16D.

[0359] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position I92, optionally wherein the amino acid substitution is I92S. In an embodiment, the IL-2 variant comprises the amino acid substitution I92S.

[0360] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position D84, optionally wherein the amino acid substitution is D84V. In an embodiment, the IL-2 variant comprises the amino acid substitution D84V.

[0361] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position K35, R38, F42, E68, or a combination thereof. In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position K35, optionally wherein the amino acid substitution is K35E. In an embodiment, the IL-2 variant comprises the amino acid substitution K35E.

[0362] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position R38, optionally wherein the amino acid substitution is R38E, R38N, or R38Q. In an embodiment, the IL-2 variant comprises the amino acid substitution R38N. In an embodiment, the IL-2 variant comprises the amino acid substitution R38Q.

[0363] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position F42, optionally wherein the amino acid substitution is F42K or F42Q. In an embodiment, the IL-2 variant comprises the amino acid substitution F42K. In an embodiment, the IL-2 variant comprises the amino acid substitution F42Q.

[0364] In an embodiment, the IL-2 variant comprises the following amino acid changes (e.g., substitutions): (i) at (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35; (ii) at one, two, or all of positions H16, I92, or D84. In an embodiment, the IL-2 variant further comprises amino acid changes (e.g., substitutions) at one, two, or all of positions R38, F42, or E68.

[0365] In an embodiment, the IL-2 variant comprises the following amino acid changes (e.g., substitutions): (i) at (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35; (ii) at (a) one, two, or all of positions H16, I92, or D84; or (b) one, two, or all of positions R38, F42, or E68.

[0366] In an embodiment, the IL-2 variant comprises the following amino acid changes (e.g., substitutions): (i) at (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35; (ii) at (a) one, two, or all of positions H16, I92, or D84; and (b) one, two, or all of positions R38, F42, or E68.

[0367] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and H16, optionally wherein the amino acid substitutions are V69A, Q74P, and H16N or H16L, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and H16N or H16L. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and H16N. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and H16L.

[0368] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and I92, optionally wherein the amino acid substitutions are V69A, Q74P, and I92S, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and I92S.

[0369] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and D84, optionally wherein the amino acid substitutions are V69A, Q74P, and D84V, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and D84V.

[0370] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, and R38Q, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and R38Q.

[0371] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and F42, optionally wherein the amino acid substitutions are V69A, Q74P, and F42Q, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and F42Q.

[0372] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, and R38N, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and R38N.

[0373] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, and R38E, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and R38E.

[0374] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, K35, and H16, optionally wherein the amino acid substitutions are V69A, Q74P, K35E, and H16N, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, K35E, and H16N.

[0375] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, K35, H16, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, K35E, H16N, and R38N, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, K35E, H16N, and R38N.

[0376] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions V69, Q74, H16, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, H16N, and R38N or R38Q, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, H16N, and R38N or R38Q. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, H16N, and R38N. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, H16N, and R38Q.

[0377] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions I28, E68, S87, N88, Q126, or combinations thereof.

[0378] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions I28, E68, S87, N88, Q126, or combinations thereof.

[0379] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions I28, E68, S87, N88, Q126, or combinations thereof.

[0380] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions I28, E68, S87, N88, Q126, or combinations thereof.

[0381] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions I28, E68, S87, N88, Q126, or combinations thereof.

[0382] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position Q126, optionally wherein the amino acid substitution is Q126T, Q126K, or Q126R. In an embodiment, the IL-2 variant comprises the amino acid substitution Q126T, Q126K, or Q126R. In an embodiment, the IL-2 variant comprises the amino acid substitution Q126T, Q126K, or Q126R. In an embodiment, the IL-2 variant comprises the amino acid substitution Q126T. In an embodiment, the IL-2 variant comprises the amino acid substitution Q126K. In an embodiment, the IL-2 variant comprises the amino acid substitution Q126R.

[0383] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position C125, optionally wherein the amino acid substitution is C125S. In an embodiment, the IL-2 variant comprises the amino acid substitution C125S.

[0384] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at position T3, optionally wherein the amino acid substitution is T3A. In an embodiment, the IL-2 variant comprises the amino acid substitution T3A.

[0385] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at positions V69, Q74, and C125, optionally wherein the amino acid substitutions are V69A, Q74P, and C125S, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions V69A, Q74P, and C125S.

[0386] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at positions T3, H16, I92, or a combination thereof, optionally wherein the amino acid substitutions are T3A, H16N, and I92S, respectively.

[0387] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at positions H16, V69, Q74, and C125, optionally wherein the amino acid substitutions are H16N, V69A, Q74P, and C125S, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions H16N, V69A, Q74P, and C125S.

[0388] In an embodiment, the IL-2 variant comprises an amino acid change (e.g., substitution) at positions H16, V69, Q74, and C125, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, and C125S, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions H16L, V69A, Q74P, and C125S.

[0389] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions H16, V69, Q74, I92, and C125, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, I92S, and C125S, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions H16L, V69A, Q74P, I92S, and C125S.

[0390] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions T3, V69, Q74, and C125, optionally wherein the amino acid substitutions are T3A, V69A, Q74P, and C125S, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions T3A, V69A, Q74P, and C125S.

[0391] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions T3, H16, V69, Q74, and C125, optionally wherein the amino acid substitutions are T3A, H16N or H16L, V69A, Q74P, and C125S, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions T3A, H16N, V69A, Q74P, and C125S. In an embodiment, the IL-2 variant comprises the amino acid substitutions T3A, H16L, V69A, Q74P, and C125S.

[0392] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions T3, V69, Q74, I92, and C125, optionally wherein the amino acid substitutions are T3A, V69A, Q74P, I92S, and C125S, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions T3A, V69A, Q74P, I92S, and C125S. In an embodiment, the IL-2 variant comprises the amino acid substitutions T3A, V69A, Q74P, I92S, and C125S.

[0393] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions H16, K35, V69, and Q74, optionally wherein the amino acid substitutions are H16L, K35E, V69A, and Q74P, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions H16L, K35E, V69A, and Q74P.

[0394] In an embodiment, the IL-2 variant comprises amino acid changes (e.g., substitutions) at positions H16, R38, V69A, and Q74P, optionally wherein the amino acid substitutions are H16L, R38Q, V69A, and Q74P, respectively. In an embodiment, the IL-2 variant comprises the amino acid substitutions H16L, R38Q, V69A, and Q74P.

[0395] In an embodiment, the IL-2 variant comprises the amino acid substitutions H16L, V69A, Q74P, and C125S. In an embodiment, the IL-2 variant comprises the amino acid substitutions H16N, V69A, Q74P, and C125S.

[0396] There are various technical effects associated with the presence of certain sets 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, it is believed that in one embodiment, IL-2 variants comprising the above-mentioned mutations have reduced binding affinity for CD122 and / or CD132, which increases the efficacy and selectivity (compared to other T cell types) of the IL-2 variant for regulatory T cells (Tregs). Without wishing to be bound by theory, it is further believed that in one embodiment, IL-2 variants comprising the above-mentioned mutations are significantly stabilized, e.g., due to the presence of the stabilizing V69A and Q74P mutations. For example, it was unexpectedly discovered that the V69A and Q74P substitutions do not significantly increase the binding affinity of the IL-2 variant for CD25, but rather stabilize the IL-2 variant in an active conformation sufficient to bind to CD25. Thus, IL-2 variants comprising these mutations are selectively able to activate regulatory T cells (Tregs) and are significantly stabilized. Without wishing to be bound by theory, it is further believed that in one embodiment, IL-2 variants comprising the above-mentioned mutations have reduced or decreased binding capacity and / or binding affinity for CD25, which improves the longevity of the IL-2 variant. Without wishing to be bound by theory, it is further believed that in one embodiment, IL-2 variants comprising these mutations do not substantially promote in vitro and / or in vivo expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer cells (NK). Without wishing to be bound by theory, it is further believed that in one embodiment, IL-2 variants comprising the above-mentioned mutations have reduced incorrect disulfide bond pairing and improved stability, e.g., due to the presence of the C125S mutation. In one embodiment, IL-2 preparations comprising the H16L mutation have reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types compared to IL-2 preparations comprising other H16 mutations. These properties make IL-2 variants comprising these mutations particularly suitable for treating diseases or disorders caused by aberrant immune responses, e.g., autoimmune diseases.

[0397] Accordingly, in one embodiment, an IL-2 variant (e.g., IL-2 variant or IL-2 fusion protein) comprising a combination of amino acid substitutions at position H16 and positions V69, Q74, and C125 (e.g., H16L, V69A, Q74P, and C125S) has (among others) one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties relative to wild-type IL-2 or a reference IL-2 variant that does not comprise the amino acid substitutions: (i) enhanced or increased in vitro or in vivo stability; (ii) reduced or decreased in vitro and / or in vivo binding capacity and / or binding affinity to human CD122; (iii) reduced or decreased in vitro and / or in vivo binding capacity and / or binding affinity to human CD132; (iv) reduced or decreased in vitro and / or in vivo 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); (v) reduced or decreased in vitro and / or in vivo binding capacity and / or binding affinity to human CD25; (vi) selective binding to regulatory T cells (e.g., Foxp3 + T cells); (vii) selective activation of IL-2 signaling pathway in T regulatory cells (Treg) in vitro or in vivo; or (viii) enhanced or increased capacity to induce or promote Treg expansion, activity, survival, and / or proliferation.

[0398] In one embodiment, the IL-2 variant comprises or consists of an amino acid sequence 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, SEQ ID NO: 38, SEQ ID NO: 1000, SEQ ID NO: 1001, SEQ ID NO: 1002, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.

[0399] 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 an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids therefrom. In one embodiment, the IL-2 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 an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids therefrom. In one embodiment, the IL-2 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 an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids therefrom. In one embodiment, the IL-2 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 an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids therefrom. In one embodiment, the IL-2 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 an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids therefrom.In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, or a functional fragment thereof. In an 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 an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof. In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 5, or a functional fragment thereof. In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 11, or a functional fragment thereof. In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 1000, or a functional fragment thereof. In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 1001, or a functional fragment thereof. In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 1002, or a functional fragment thereof.

[0400] In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, or a functional fragment thereof. In an 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 an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof. In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 5, or a functional fragment thereof. In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 11, or a functional fragment thereof. In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 1000, or a functional fragment thereof. In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 1001, or a functional fragment thereof. In an embodiment, the IL-2 variant comprises or consists of the amino acid sequence of SEQ ID NO: 1002, or a functional fragment thereof.

[0401] Without wishing to be bound by theory, it is believed that in one embodiment, the IL-2 variant comprising or consisting of the amino acid sequence of SEQ ID NO: 5, or a functional fragment thereof, can have at least one or more of the following advantageous properties: (i) reduced binding affinity for CD122 and / or CD132 compared to other T cell types, which increases the efficacy and selectivity of the IL-2 formulation for regulatory T cells (Tregs); (ii) significantly stabilized, e.g., due to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity for CD25, which can prolong the life of the IL-2 formulation; (iv) substantially no promotion of T effector cell and / or natural killer (NK) cell expansion, activation, survival, and / or proliferation in vitro and / or in vivo; and / or (v) reduced incorrect disulfide pairing and increased stability, e.g., due to the presence of the C125S mutation. In one embodiment, the IL-2 formulation comprising the H16L mutation has reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types compared to the IL-2 formulation comprising other H16 mutations. These properties make the IL-2 variant comprising or consisting of the amino acid sequence of SEQ ID NO: 5 particularly suitable for treating diseases and disorders caused by aberrant immune responses, e.g., autoimmune diseases.

[0402] Thus, in one embodiment, the IL-2 variant comprising or consisting of the amino acid sequence of SEQ ID NO: 5, or a functional fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity thereto, or an amino acid sequence differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom, has particularly one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties relative to wild-type IL-2 or a reference IL-2 variant that does not comprise the amino acid substitutions: (i) enhanced or increased in vitro or in vivo stability; (ii) reduced or decreased in vitro and / or in vivo binding capacity and / or binding affinity for human CD122; (iii) reduced or decreased in vitro and / or in vivo binding capacity and / or binding affinity for human CD132; (iv) reduced or decreased affinity of the IL-2 variant for the heterodimeric IL-2 receptor composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) reduced or decreased or substantially unchanged in vitro and / or in vivo binding capacity and / or binding affinity for human CD25; (vi) selective binding to regulatory T cells (e.g., Foxp3+ (vii) selectively activate IL-2 signaling pathways in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote Treg expansion, activity, survival, and / or proliferation.

[0403] As further described herein, the present disclosure provides IL-2 fusion proteins, IL-2 complexes, and IL-2 conjugates comprising the IL-2 variants described herein. In one embodiment, one or more of the different and / or improved properties attributed 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 the present disclosure, the terms “IL-2 variant” and “IL-2 mutein” are used interchangeably herein.

[0404] 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. The nucleic acid molecules include, but are not limited to, RNA, genomic DNA, and cDNA.

[0405] IL-2 fusion proteins

[0406] In one embodiment, the IL-2 formulation comprises an IL-2 fusion protein, e.g., an IL-2 fusion protein described herein.

[0407] 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) described in Table 9. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence described in Table 9, or a functional fragment thereof. In one embodiment, the IL-2 variant is encoded by a nucleic acid comprising a nucleotide sequence described herein, e.g., in Table 10.

[0408] Without wishing to be bound by theory, it is believed that in one embodiment, the IL-2 fusion proteins described herein having reduced human CD25 and / or reduced human CD122 / CD132 binding affinity relative to an IL-2 fusion protein comprising wild-type human IL-2 or a reference IL-2 fusion protein can have improved efficacy and / or selectivity for binding to and activating regulatory T cells (Tregs) compared to an IL-2 fusion protein comprising wild-type human IL-2 or other IL-2 fusion proteins.

[0409] In one embodiment, the IL-2 fusion protein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) properties described herein, e.g., a different and / or improved property relative 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 comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid changes (e.g., substitutions) that are capable of providing a different and / or improved property relative 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 in an assay described herein) relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein:

[0410] i) altered (e.g., enhanced or increased) expression in vitro and / or in vivo;

[0411] ii) altered (e.g., reduced or decreased) aggregation in vitro and / or in vivo;

[0412] iii) altered (e.g., enhanced or increased) stability in vitro and / or in vivo;

[0413] iv) altered (e.g., enhanced or increased) half-life in vitro and / or in vivo;

[0414] v) altered (e.g., reduced or decreased) turnover and / or clearance in vivo;

[0415] vi) altered (e.g., reduced or decreased) susceptibility to proteolysis in vitro and / or in vivo;

[0416] vii) altered (e.g., enhanced or increased) resistance to proteolysis in vitro and / or in vivo;

[0417] viii) altered (e.g., reduced or decreased) binding capacity and / or binding affinity for human CD25 in vitro and / or in vivo;

[0418] ix) altered (e.g., reduced or decreased) binding capacity and / or binding affinity for human CD132 in vitro and / or in vivo;

[0419] x) altered (e.g., reduced or decreased) binding capacity and / or binding affinity for a dimeric IL-2 receptor comprising human CD122 and human CD132 in vitro and / or in vivo;

[0420] xi) altered (e.g., enhanced, increased, decreased, reduced, and / or selective) binding to Tregs in vitro and / or in vivo;

[0421] xii) altered (e.g., enhanced, increased, decreased, reduced, and / or selective) activation of IL-2 signaling pathway in Tregs in vitro and / or in vivo; or

[0422] xiii) altered (e.g., enhanced, increased, decreased, reduced, and / or selective) ability to induce or promote Treg expansion, activity, survival, and / or proliferation in vitro and / or in vivo.

[0423] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced or increased) expression in vitro and / or in vivo relative to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has enhanced or increased expression (e.g., in bacterial or mammalian cells) relative to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has enhanced or increased expression (e.g., in bacterial or mammalian cells) relative to a reference IL-2 fusion protein. In one embodiment, the 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, the 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., increased by 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., relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by a protein concentration assay. In one embodiment, the IL-2 fusion protein is expressed at a higher or increased level, e.g., 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.g., relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by a protein concentration assay.

[0424] In one embodiment, the IL-2 fusion protein has altered (e.g., reduced or decreased) aggregation 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. In one embodiment, the IL-2 fusion protein has reduced or decreased aggregation relative to wild-type IL-2. In one embodiment, the IL-2 fusion protein has reduced or decreased aggregation relative to a reference IL-2 fusion protein. In one embodiment, the 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, the 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 decreased level in vitro and / or in vivo, e.g., decreased by 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., relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by melting temperature analysis (e.g., using fluorescence), dynamic light scattering, and / or size exclusion chromatography. In one embodiment, the IL-2 fusion protein aggregates at a lower or decreased level, e.g., decreased 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.g., relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by melting temperature analysis (e.g., using fluorescence), dynamic light scattering, and / or size exclusion chromatography.

[0425] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced or increased) stability 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. In one embodiment, the IL-2 fusion protein has enhanced or increased stability relative to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has enhanced or increased stability relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more, increased stability. In one embodiment, the IL-2 fusion protein has 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, increased stability. 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 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, 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, increased, e.g., relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by yeast surface display, circular dichroism or related spectroscopic techniques, and / or melting temperature analysis (e.g., using a fluorometric method).

[0426] In one embodiment, the IL-2 fusion protein has an altered (e.g., enhanced or increased) half-life in vitro and / or in vivo relative 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 relative 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 relative 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., by 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, e.g., greater than 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.g., relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by ELISA, flow cytometry, and / or mass spectrometry.

[0427] In one embodiment, the IL-2 fusion protein has an altered (e.g., reduced or decreased) turnover 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. In one embodiment, the IL-2 fusion protein has a reduced or decreased turnover relative to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has a reduced or decreased turnover relative to a reference IL-2 fusion protein. In one embodiment, the turnover of the IL-2 fusion protein is decreased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the turnover of the IL-2 fusion protein is decreased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, the IL-2 fusion protein has a lower, reduced, or decreased turnover rate or level and / or in vivo clearance rate in vivo, e.g., decreased by 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, e.g., decreased 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.g., relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by ELISA, flow cytometry, and / or mass spectrometry.

[0428] In one embodiment, the IL-2 fusion protein provided by the present disclosure comprises a property of having altered (e.g., reduced or decreased) proteolytic susceptibility 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. In one embodiment, the IL-2 fusion protein has reduced or decreased susceptibility to proteolysis relative to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 fusion protein has reduced or decreased proteolytic susceptibility relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more, decreased susceptibility to proteolysis. In one embodiment, the IL-2 fusion protein has 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, decreased susceptibility to proteolysis.

[0429] In one embodiment, the IL-2 fusion protein provided by the present disclosure comprises a property of having altered (e.g., enhanced or increased) proteolytic resistance 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. In one embodiment, the IL-2 fusion protein has enhanced or increased proteolytic resistance relative to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has enhanced or increased proteolytic resistance relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more, increased resistance to proteolysis. In one embodiment, the IL-2 fusion protein has 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, increased resistance to proteolysis.

[0430] In one embodiment, the IL-2 fusion protein has an altered (e.g., reduced or decreased) binding capacity and / or binding affinity for human CD25 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. In one embodiment, the IL-2 fusion protein has a reduced or decreased binding capacity and / or binding affinity for human CD25 relative to wild-type human IL-2. In one embodiment, the IL-2 fusion protein has a reduced or decreased binding capacity and / or binding affinity for human CD25 relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has a binding capacity and / or binding affinity for human CD25 that 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 IL-2 fusion protein has a binding capacity and / or binding affinity for human CD25 that 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 affinity for CD25 (e.g., human CD25), e.g., reduced by 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 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, e.g., relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by yeast surface display, surface plasmon resonance (e.g., Biacore), and / or bio-layer interferometry (e.g., Octet binding).

[0431] In one embodiment, the IL-2 fusion protein binds CD25 (e.g., human CD25) with low affinity, e.g., a dissociation constant (K D) is about 5-500 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 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, e.g., about 10 to about 20 pM, about 20 to about 30 pM, or about 30 to about 40 pM, e.g., 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, e.g., 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, e.g., as determined by yeast surface display, surface plasmon resonance (e.g., Biacore), and / or bio-layer interferometry (e.g., Octet binding).

[0432] In one embodiment, the IL-2 fusion protein binds CD25 (e.g., human CD25) with low affinity, e.g., a dissociation constant (K D) is about 0.1-10 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-about 5 nM, about 0.5-about 2 nM, about 1-1.5 nM, about 0.1-about 0.2 nM, e.g., about 0.2-about 0.3 nM, about 0.3-about 0.4 nM, or about 0.4-about 0.5 nM, e.g., about 0.5-about 0.6 nM, about 0.6-about 0.7 nM, about 0.7-about 0.8 nM, about 0.8-about 0.9 nM, about 0.9-about 1 nM, about 1-about 1.5 nM, about 1.5-about 2 nM, about 2.5-about 3 nM, about 3.5-about 4 nM, about 4-about 4.5 nM, about 4.5-about 5 nM, about 5-about 6 nM, about 6-about 7 nM, about 7-about 8 nM, about 8-about 9 nM, or about 9-about 10 nM, or e.g., 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, e.g., as determined by surface plasmon resonance (e.g., Biacore) and / or bio-layer interferometry (e.g., Octet binding).

[0433] In one embodiment, the IL-2 fusion protein has an altered (e.g., reduced or decreased) binding capacity and / or binding affinity for human CD132 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. In one embodiment, the IL-2 fusion protein has a reduced or decreased binding capacity and / or binding affinity for human CD132 relative 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 capacity and / or binding affinity for human CD132 relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has a reduction of about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more in binding capacity and / or binding affinity for human CD132. In one embodiment, the IL-2 fusion protein has a reduction of 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 binding capacity and / or binding affinity for human CD132.

[0434] In one embodiment, the IL-2 fusion protein has an altered (e.g., reduced or decreased) binding capacity and / or binding affinity for the human dimeric IL-2 receptor comprising human CD122 and human CD132 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. In one embodiment, the IL-2 fusion protein has a reduced or decreased binding capacity and / or binding affinity for the human dimeric IL-2 receptor comprising human CD122 and human CD132 relative 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 capacity and / or binding affinity for the human dimeric IL-2 receptor comprising human CD122 and human CD132 relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has a binding capacity and / or binding affinity for the human dimeric IL-2 receptor comprising human CD122 and human CD132 that 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 IL-2 fusion protein has a binding capacity and / or binding affinity for the human dimeric IL-2 receptor comprising human CD122 and human CD132 that 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.

[0435] In one embodiment, the IL-2 fusion protein has an altered (e.g., enhanced, increased, and / or selective) binding to Tregs 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. In one embodiment, the IL-2 fusion protein has an enhanced or increased binding to Tregs relative to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has a selective binding to Tregs relative to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 fusion protein has an enhanced or increased binding to Tregs relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has a selective binding to Tregs relative to a reference IL-2 fusion protein. In one embodiment, the 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, the 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.

[0436] In one embodiment, the IL-2 fusion protein has reduced or decreased binding affinity for a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer), e.g., by 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 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.g., relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein, e.g., as determined by yeast surface display, surface plasmon resonance (e.g., Biacore), and / or bio-layer interferometry (e.g., Octet binding).

[0437] In one embodiment, the IL-2 fusion protein binds a CD122 / CD132 heterodimer (e.g., a human CD122 / CD132 heterodimer) with low affinity, e.g., a dissociation constant (K DThe molecular weight is approximately 0.2–20 nM, for example, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 1, approximately 1.1, approximately 1.2, approximately 1.3, approximately 1.4, approximately 1.5, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, or approximately 20 nM, or for example, approximately 0.5–approximately 15 nM. 1 - about 10 nM, about 2 - about 5 nM, about 0.2 - about 0.3 nM, about 0.3 - about 0.4 nM, about 0.4 - about 0.5 nM, about 0.5 - about 0.6 nM, about 0.6 - about 0.7 nM, about 0.7 - about 0.8 nM, about 0.8 - about 0.9 nM, about 0.9 - about 1 nM, about 1 - about 1.1 nM, about 1.1 - about 1.2 nM, about 1.2 - about 1.3 nM, about 1.3 - about 1.4 nM, about 1.4 - about 1.5 nM M, approximately 1.5-2 nM, approximately 2-3 nM, approximately 3-4 nM, approximately 4-5 nM, approximately 5-6 nM, approximately 6-7 nM, approximately 7-8 nM, approximately 8-9 nM, approximately 9-10 nM, approximately 10-11 nM, approximately 11-12 nM, approximately 12-13 nM, approximately 13-14 nM, approximately 14-15 nM, approximately 15-16 nM, approximately 16-17 nM, approximately 17-18 nM, approximately 18-19 nM Or about 19 to about 20 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, for example, determined by yeast surface display.

[0438] In one implementation, the IL-2 fusion protein binds to the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer) with low affinity, for example, the dissociation constant (K). D) is about 0.2-300 nM, e.g., about 0.2 nM, about 0.5 nM, about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 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 e.g., about 0.5 to about 15 nM, about 1 to about 10 nM, about 2 to about 5 nM, about 0.2 nM to about 0.5 nM, about 0.5 nM to about 1 nM, about 1 to about 2 nM, about 2 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 15 nM, about 15 nM to about 20 nM, about 20 nM to about 25 nM, about 25 to about 30 nM, about 30 nM to about 40 nM, about 40 nM to about 50 nM, about 50 to about 60 nM, about 60 to about 70 nM, about 70 nM to about 80 nM, about 80 nM to about 90 nM, about 90 nM to about 100 nM, about 100 nM to about 110 nM, about 110 nM to about 120 nM, about 120 nM to about 130 nM, about 130 nM to about 140 nM, about 140 nM to about 150 nM, about 150 nM to about 160 nM, about 160 nM to about 170 nM, about 170 nM to about 180 nM, about 180 nM to about 190 nM, about 190 nM to about 200 nM, about 200 nM to about 210 nM, about 210 nM to about 220 nM, about 220 nM to about 230 nM, about 230 nM to about 240 nM, about 240 nM to about 250 nM, about 250 nM to about 260 nM, about 260 nM to about 270 nM, about 270 nM to about 280 nM, about 280 nM to about 290 nM, or about 290 nM to about 300 nM, or e.g., greater than about 0.2, about 0.5, 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, e.g., as determined by surface plasmon resonance (e.g., Biacore) and / or bio-layer interferometry (e.g., Octet binding).

[0439] 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 relative 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 relative to an IL-2 fusion protein comprising wild-type IL-2. In one embodiment, the IL-2 fusion protein has selective binding to Tregs relative 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 relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has selective binding to Tregs relative to a reference IL-2 fusion protein. In one embodiment, the 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, the 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.

[0440] In one embodiment, the IL-2 fusion protein selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, e.g., has a T helper EC50 / Treg EC50 ratio of greater than 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 greater, 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.

[0441] In one embodiment, the IL-2 fusion protein selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, e.g., has a T helper EC50 / Treg EC50 ratio of greater than 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 greater, 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.

[0442] In one embodiment, the IL-2 fusion protein selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, e.g., has an NK cell EC50 / Treg EC50 ratio of greater than, 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 greater, or, e.g., greater than 1 and about 1-2, about 2-3, about 3-4, about 4-5, greater than 1 and about 1-10, greater than 1 and about 1-20, greater than 1 and about 1-30, greater than 1 and about 1-40, greater than 1 and about 1-50, about 2-10, about 2-20, about 2-30, about 2-40, 2-50, about 5-10, about 5-20, about 5-30, about 5-40, about 5-50, about 10-20, about 10-30, about 10-40 about 10-50, about 20-40, about 20-50, about 50-100, about 100-200, about 200-500, about 500-1000, about 1000-2000, or about 1000-3000, 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.

[0443] In one embodiment, the IL-2 fusion protein has an altered (e.g., enhanced, increased, and / or selective) ability to induce or promote Treg expansion, activity, survival, and / or proliferation 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. In one embodiment, the IL-2 fusion protein has an enhanced or increased ability to induce or promote Treg expansion, activity, survival, and / or proliferation relative 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 Treg expansion, activity, survival, and / or proliferation relative 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 Treg expansion, activity, survival, and / or proliferation relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has a selective ability to induce or promote Treg expansion, activity, survival, and / or proliferation relative to a reference IL-2 fusion protein. In one embodiment, the ability to induce or promote Treg expansion, activity, survival, and / or proliferation is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the ability to induce or promote Treg expansion, activity, survival, and / or proliferation 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.

[0444] In one embodiment, the IL-2 fusion protein has an enhanced or increased efficacy and / or ability to induce or promote T regulatory cell activity, e.g., EC 50 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, e.g., 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, e.g., as determined by flow cytometry.

[0445] In one embodiment, the IL-2 fusion protein has reduced or decreased efficacy and / or ability to induce or promote T regulatory cell activity, e.g., EC50for Tregs, relative to an IL-2 fusion protein comprising wild-type IL-2 or a reference IL-2 fusion protein. 50 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, 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, or about 20,000-fold or more, e.g., as determined by flow cytometry.

[0446] In one embodiment, the T helper cell described herein is a CD45+CD3+CD4+Foxp3- cell, e.g., as determined by flow cytometry. In one embodiment, the Treg described herein is a CD45+CD3+CD4+Foxp3+ cell, e.g., as determined by flow cytometry. In one embodiment, the NK cell described herein is a CD45+CD3- cell that is CD56+and / or CD16+, e.g., as determined by flow cytometry. In one embodiment, the NK cell described herein is a CD45+CD3-CD56+ cell, e.g., as determined by flow cytometry.

[0447] 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.

[0448] In an embodiment, the reference IL-2 fusion protein comprises an amino acid sequence that is about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identical to an IL-2 fusion protein described herein. In an embodiment, the reference IL-2 fusion protein comprises an IL-2 variant comprising the amino acid sequence of SEQ ID NO: 57. In an 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 comprises one or more (2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid changes (e.g., substitutions) described herein.

[0449] In an embodiment, the IL-2 fusion protein comprises an IL-2 polypeptide (e.g., a human IL-2 polypeptide) described herein. In an embodiment, the IL-2 fusion protein is encoded by a nucleic acid comprising a nucleotide sequence described herein.

[0450] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at a position in one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) of the IL-2s described herein. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) positions selected from T3, H16, I28, K35, R38, F42, E68, V69, Q74, D84, S87, N88, I92, C125, or Q126 in IL-2.

[0451] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position T3 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position H16 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position I28 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position K35 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position R38 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position F42 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position E68 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position V69 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position Q74 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position D84 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position S87 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position N88 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position I92 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position C125 in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position Q126 in IL-2.

[0452] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position V69, Q74, or both in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at positions V69 and Q74 in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution V69A in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution Q74P in IL-2.

[0453] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position H16, I92, D84, or a combination thereof in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position H16 in IL-2, optionally wherein the amino acid substitution is H16N, H16L, or H16D. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution H16N in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution H16L in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution H16D in IL-2.

[0454] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position I92 in IL-2, optionally wherein the amino acid substitution is I92S. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution I92S in IL-2.

[0455] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position D84 in IL-2, optionally wherein the amino acid substitution is D84V. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution D84V in IL-2.

[0456] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position K35, R38, F42, E68, or a combination thereof in IL-2. In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position K35 in IL-2, optionally wherein the amino acid substitution is K35E. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution K35E in IL-2.

[0457] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position R38 in IL-2, optionally wherein the amino acid substitution is R38E, R38N, or R38Q. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution R38N in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution R38Q in IL-2.

[0458] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position F42 in IL-2, optionally wherein the amino acid substitution is F42K or F42Q. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution F42K in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution F42Q in IL-2.

[0459] In an embodiment, the IL-2 fusion protein comprises the following amino acid changes (e.g., substitutions): (i) at (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35 in IL-2; (ii) at one, two, or all of positions H16, I92, or D84 in IL-2. In an embodiment, the IL-2 fusion protein further comprises amino acid changes (e.g., substitutions) at one, two, or all of positions R38, F42, or E68 in IL-2.

[0460] In an embodiment, the IL-2 fusion protein comprises the following amino acid changes (e.g., substitutions): (i) at (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35 in IL-2; (ii) at (a) one, two, or all of positions H16, I92, or D84; or (b) one, two, or all of positions R38, F42, or E68 in IL-2.

[0461] In an embodiment, the IL-2 fusion protein comprises the following amino acid changes (e.g., substitutions): (i) at (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35 in IL-2; (ii) at (a) one, two, or all of positions H16, I92, or D84; and (b) one, two, or all of positions R38, F42, or E68 in IL-2.

[0462] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and H16 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, and H16N or H16L, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and H16N or H16L in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and H16N in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and H16L in IL-2.

[0463] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and I92 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, and I92S, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and I92S in IL-2.

[0464] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and D84 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, and D84V, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and D84V in IL-2.

[0465] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and R38 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, and R38Q, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and R38Q in IL-2.

[0466] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and F42 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, and F42Q, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and F42Q in IL-2.

[0467] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and R38 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, and R38N, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and R38N in IL-2.

[0468] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions V69, Q74, and R38 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, and R38E, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and R38E in IL-2.

[0469] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions V69, Q74, K35, and H16 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, K35E, and H16N, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, K35E, and H16N in IL-2.

[0470] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions V69, Q74, K35, H16, and R38 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, K35E, H16N, and R38N, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, K35E, H16N, and R38N in IL-2.

[0471] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions V69, Q74, H16, and R38 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, H16N, and R38N or R38Q, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, H16N, and R38N or R38Q in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, H16N, and R38N in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, H16N, and R38Q in IL-2.

[0472] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions I28, E68, S87, N88, Q126, or a combination thereof in IL-2.

[0473] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at position I28 in IL-2, optionally wherein the amino acid substitution is I28T or I28F. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution I28T in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution I28F in IL-2.

[0474] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at position E68 in IL-2, optionally wherein the amino acid substitution is E68Q or E68N. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution E68Q in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution E68N in IL-2.

[0475] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at position S87 in IL-2, optionally wherein the amino acid substitution is S87R. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution S87R in IL-2.

[0476] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position N88 in IL-2, optionally wherein the amino acid substitution is N88S, N88L, or N88D. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution N88S, N88L, or N88D in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution N88S in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution N88L in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution N88D in IL-2.

[0477] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position Q126 in IL-2, optionally wherein the amino acid substitution is Q126T, Q126K, or Q126R. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution Q126T, Q126K, or Q126R in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution Q126T, Q126K, or Q126R in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution Q126T in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution Q126K in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution Q126R in IL-2.

[0478] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position C125 in IL-2, optionally wherein the amino acid substitution is C125S. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution C125S in IL-2.

[0479] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at position T3 in IL-2, optionally wherein the amino acid substitution is T3A. In an embodiment, the IL-2 fusion protein comprises the amino acid substitution T3A in IL-2.

[0480] In an embodiment, the IL-2 fusion protein comprises an amino acid change (e.g., substitution) at positions V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, and C125S, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and C125S in IL-2.

[0481] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, and C125S in IL-2, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions H16L, V69A, Q74P, and C125S in IL-2.

[0482] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, and C125S in IL-2, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions H16L, V69A, Q74P, and C125S in IL-2.

[0483] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, and C125S in IL-2, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions H16L, V69A, Q74P, and C125S in IL-2.

[0484] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, and C125S in IL-2, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions H16L, V69A, Q74P, and C125S in IL-2.

[0485] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, and C125S in IL-2, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions H16L, V69A, Q74P, and C125S in IL-2.

[0486] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions T3, H16, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are T3A, H16N or H16L, V69A, Q74P, and C125S in IL-2, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, H16N, V69A, Q74P, and C125S in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, H16L, V69A, Q74P, and C125S in IL-2.

[0487] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions T3, H16, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are T3A, H16N or H16L, V69A, Q74P, and C125S in IL-2, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, H16N, V69A, Q74P, and C125S in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, H16L, V69A, Q74P, and C125S in IL-2.

[0488] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions T3, H16, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are T3A, H16N or H16L, V69A, Q74P, and C125S in IL-2, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, H16N, V69A, Q74P, and C125S in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, H16L, V69A, Q74P, and C125S in IL-2.

[0489] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions T3, H16, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are T3A, H16N or H16L, V69A, Q74P, and C125S in IL-2, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, H16N, V69A, Q74P, and C125S in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, H16L, V69A, Q74P, and C125S in IL-2.

[0490] In an embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at positions T3, H16, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are T3A, H16N or H16L, V69A, Q74P, and C125S in IL-2, respectively. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, H16N, V69A, Q74P, and C125S in IL-2. In an embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, H16L, V69A, Q74P, and C125S in IL-2.

[0491] Without wishing to be bound by theory, it is believed that in one embodiment, IL-2 fusion proteins comprising the amino acid substitutions H16L, V69A, Q74P, and C125S can have at least one or more of the following advantageous properties: (i) reduced binding affinity for CD122 and / or CD132 compared to other T cell types, which increases the efficacy and selectivity of the IL-2 formulation for regulatory T cells (Tregs); (ii) significant stabilization, e.g., due to the presence of the stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity for CD25, which can prolong the life of the IL-2 formulation; (iv) substantially no promotion of T effector cell and / or natural killer (NK) cell expansion, activation, survival, and / or proliferation in vitro and / or in vivo; and / or (v) reduced incorrect disulfide pairing and improved stability, e.g., due to the presence of the C125S mutation. In one embodiment, the IL-2 formulation comprising the H16L mutation has reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types compared to IL-2 formulations comprising other H16 mutations. These properties make IL-2 variants comprising the amino acid substitutions H16L, V69A, Q74P, and C125S particularly suitable for use in treating diseases and disorders caused by aberrant immune responses, e.g., autoimmune diseases.

[0492] Thus, in one embodiment, IL-2 fusion proteins comprising the amino acid substitutions H16L, V69A, Q74P, and C125S have, inter alia, one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties relative to wild-type IL-2 or a reference IL-2 variant that does not comprise the following amino acid substitutions: (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 CD132 in vitro and / or in vivo; (iv) reduced or decreased affinity of the IL-2 variant for the heterodimeric (i.e., human CD122 / CD132 heterodimer) IL-2 receptor composed of human CD122 and human CD132 in vitro and / or in vivo; (v) reduced or decreased or substantially unchanged binding capacity and / or binding affinity for human CD25 in vitro and / or in vivo; (vi) selective binding to regulatory T cells (e.g., Foxp3 + T cells); (vii) selective activation of the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote Treg expansion, activity, survival, and / or proliferation.

[0493] In one embodiment, the IL-2 fusion protein comprises an IL-2 variant comprising an amino acid sequence 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, SEQ ID NO: 38, SEQ ID NO: 1000, SEQ ID NO: 1001, SEQ ID NO: 1002, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity thereto, or differing therefrom by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.

[0494] In one embodiment, the IL-2 fusion protein comprises an IL-2 variant comprising or consisting 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 an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids therefrom. In one embodiment, the IL-2 fusion protein comprises an IL-2 variant comprising or consisting 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 an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids therefrom. In one embodiment, the IL-2 fusion protein comprises 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 an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids therefrom. In one embodiment, the IL-2 fusion protein comprises 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 an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids therefrom. In one embodiment, the IL-2 fusion protein comprises 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 an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids therefrom.In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 4 or 5, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 5, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 11, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1000, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1001, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1002, or a functional fragment thereof.

[0495] In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 4, 5, 11, 1000, 1001, or 1002, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 4 or 5, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 5, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 11, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1000, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1001, or a functional fragment thereof. In an embodiment, the IL-2 fusion protein comprises an amino acid sequence of SEQ ID NO: 1002, or a functional fragment thereof.

[0496] Without wishing to be bound by theory, it is believed that in one embodiment, the IL-2 fusion protein comprising the amino acid sequence of SEQ ID NO: 5, or a functional fragment thereof, can have at least one or more of the following advantageous properties: (i) reduced binding affinity for CD122 and / or CD132 compared to other T cell types, which increases the efficacy and selectivity of the IL-2 formulation for regulatory T cells (Tregs); (ii) significantly stabilized, e.g., due to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity for CD25, which can prolong the life of the IL-2 formulation; (iv) substantially no promotion of T effector cell and / or natural killer (NK) cell expansion, activation, survival, and / or proliferation in vitro and / or in vivo; and / or (v) reduced incorrect disulfide pairing and increased stability, e.g., due to the presence of the C125S mutation. In one embodiment, the IL-2 formulation comprising the H16L mutation has reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types compared to IL-2 formulations comprising other H16 mutations. These properties make the IL-2 fusion protein comprising the amino acid sequence of SEQ ID NO: 5 particularly suitable for use in treating diseases and disorders caused by aberrant immune responses, e.g., autoimmune diseases.

[0497] Thus, in one embodiment, the IL-2 fusion protein comprising the amino acid sequence of SEQ ID NO: 5, or a functional fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity thereto, or an amino acid sequence differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom, has one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties relative to wild-type IL-2 or a reference IL-2 fusion protein not comprising the amino acid substitutions: (i) enhanced or increased in vitro or in vivo stability; (ii) reduced or decreased in vitro and / or in vivo binding capacity and / or binding affinity for human CD122; (iii) reduced or decreased in vitro and / or in vivo binding capacity and / or binding affinity for human CD132; (iv) reduced or decreased in vitro and / or in vivo affinity of the IL-2 fusion protein for the heterodimeric IL-2 receptor composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer); (v) reduced or decreased or substantially unchanged in vitro and / or in vivo binding capacity and / or binding affinity for human CD25; (vi) selective binding to regulatory T cells (e.g., Foxp3+ (vii) selectively activate IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote Treg expansion, activity, survival and / or proliferation.

[0498] In one embodiment, the IL-2 fusion proteins described herein comprise an Fc region, e.g., an Fc region having one or more mutations described herein and / or having one or more structural or functional properties described herein. Without wishing to be bound by theory, it is believed that in one embodiment, the Fc regions described herein can reduce (e.g., prevent) renal clearance of the IL-2 preparation and / or prolong the half-life of the IL-2 preparation (e.g., via FcRn).

[0499] As used herein, the term “fusion protein” refers to a protein comprising two or more protein or peptide components. The two or more protein or peptide components can be obtained from different sources or encoded by different genes. Fusion proteins are also sometimes referred to as chimeric proteins. Fc fusion proteins (also referred to as Fc-chimeric fusion proteins, Fc-Ig, Ig-based chimeric fusion proteins, or Fc-tag proteins) can include an Fc region of an immunoglobulin (e.g., an Fc region described herein) linked (e.g., fused) to a protein or peptide. The Fc region can be linked (e.g., genetically fused) directly or indirectly (e.g., via a linker) to the protein or peptide. In one embodiment, the Fc region is derived from an Fc region of an IgG, e.g., a human IgG, e.g., IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc region is derived from an Fc region of IgG1, e.g., a human IgG1.

[0500] IL-2 fusion proteins can include an IL-2 variant (e.g., an IL-2 variant described herein) or functional fragment thereof linked (e.g., fused) to a protein or peptide. In one embodiment, the IL-2 fusion protein is an IL-2-Fc fusion protein, e.g., further comprising an Fc region of an immunoglobulin (e.g., an Fc region described herein) linked (e.g., fused) to an IL-2 polypeptide (e.g., an IL-2 variant described herein) or functional fragment thereof. In one embodiment, the IL-2 fusion protein is not an IL-2-Fc fusion protein, e.g., an IL-2 fusion variant described herein or functional fragment thereof is linked (e.g., fused) to a protein or peptide other than an Fc region of an IgG (e.g., a human IgG, e.g., IgG1, IgG2, IgG3, or IgG4).

[0501] In one embodiment, the IL-2 fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0502] In one embodiment, the IL-2 fusion protein comprises an amino acid sequence selected from the group consisting of: 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 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 an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0503] In one embodiment, the IL-2 fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, or SEQ ID NO: 169, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0504] In one embodiment, the IL-2 fusion protein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, or SEQ ID NO: 207, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0505] In one embodiment, the IL-2 fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, or SEQ ID NO: 245, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0506] In one embodiment, the IL-2 fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, or SEQ ID NO: 283, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0507] In one embodiment, the IL-2 fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, or SEQ ID NO: 321, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or an amino acid sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids therefrom.

[0508] In one embodiment, the IL-2 fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 357, SEQ ID NO: 358, or SEQ ID NO: 359, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or an amino acid sequence that differs by no more than 1,...

Claims

1. An interleukin 2 (IL-2) variant, the amino acid substitutions relative to SEQ ID NO: 1 are H16L, V69A, and Q74P.

2. An interleukin 2 (IL-2) variant, the amino acid substitutions relative to SEQ ID NO: 1 are T3A, H16L, V69A, and Q74P.

3. The IL-2 variant of claim 1, comprising the amino acid sequence of any one of SEQ ID NO: 5 or 1001.

4. An IL-2 variant, comprising the amino acid sequence of SEQ ID NO:

5.

5. An IL-2 fusion protein, consisting of the IL-2 variant of any one of claims 1-4, an Fc region, and a linker, wherein the Fc region is fused to the C-terminus of the IL-2 variant through the linker.

6. The IL-2 fusion protein of claim 5, wherein the Fc region comprises the Fc region of IgG1 allotype m3, which comprises a N297G substitution according to the EU numbering.

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

8. The IL-2 fusion protein of claim 5, wherein the amino acid sequence of the linker is SEQ ID NO:

48.

9. The IL-2 fusion protein of claim 6, wherein the amino acid sequence of the IL-2 fusion protein is SEQ ID NO: 1005 or 1008.

10. An IL-2 fusion protein, wherein the amino acid sequence of the IL-2 fusion protein is SEQ ID NO: 1008.

11. The IL-2 fusion protein of any one of claims 5-10, which forms a dimer.

12. An IL-2 complex, comprising the IL-2 variant of any one of claims 1-4 and an anti-IL-2 antibody molecule.

13. A pharmaceutical composition, comprising the IL-2 variant of any one of claims 1-4 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition, comprising the IL-2 fusion protein of claim 5 and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition, comprising the IL-2 complex of claim 12 and a pharmaceutically acceptable carrier.

16. A nucleic acid, encoding the IL-2 variant of any one of claims 1-4.

17. A nucleic acid, encoding the IL-2 fusion protein of claim 5.

18. A nucleic acid, encoding the IL-2 complex of claim 12.

19. A vector, comprising the nucleic acid of claim 16.

20. A vector, comprising the nucleic acid of claim 17.

21. A vector, comprising the nucleic acid of claim 18.

22. A cell, comprising the nucleic acid of claim 16.

23. A cell, comprising the nucleic acid of claim 17.

24. A cell, comprising the nucleic acid of claim 18.

25. A method of producing an IL-2 variant, comprising culturing the cell of claim 22 under conditions that allow expression of the IL-2 variant.

26. A method of producing an IL-2 fusion protein, comprising culturing the cell of claim 23 under conditions that allow expression of the IL-2 fusion protein.

27. A method of producing an IL-2 complex, comprising culturing the cell of claim 24 under conditions that allow expression of the IL-2 complex.

28. Use of the IL-2 variant of any one of claims 1-4 in the manufacture of a medicament for treating lupus nephritis.

29. Use of the IL-2 fusion protein of claim 5 in the manufacture of a medicament for treating lupus nephritis.

30. Use of the IL-2 complex of claim 12 in the manufacture of a medicament for treating lupus nephritis.

31. A kit comprising the IL-2 variant of any one of claims 1-4 and instructions for use.

32. A kit comprising the IL-2 fusion protein of claim 5 and instructions for use.

33. A kit comprising the IL-2 complex of claim 12 and instructions for use.

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