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-reference to related applications This application is a divisional application of Chinese Patent Application No. 202080067804.7, filed on July 24, 2020, entitled "Interleukin-2 Preparations and Their Uses," the original application being a national phase application with international application No. PCT / US2020 / 043416, filed on July 24, 2020. The contents of that international application are incorporated herein by reference in their entirety. This application claims the benefit of U.S. Provisional Application No. 62 / 879,137, filed on July 26, 2019, and U.S. Provisional Application No. 62 / 983,061, filed on February 28, 2020. The contents of the aforementioned applications are incorporated herein by reference in their entirety.

[0002] sequence list 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

[0003] 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.

[0004] 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.

[0005] 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

[0006] This disclosure provides at least in part an IL-2 formulation ( For exampleIL-2 variants, IL-2 fusion proteins, IL-2 complexes, and IL-2 conjugates, which contain one or more amino acid variations in IL-2. For example (replace), and includes one or more structural or functional properties disclosed herein. In one embodiment, a nucleic acid molecule encoding the IL-2 formulation, an expression vector, a host cell, and a composition are also provided. For example Pharmaceutical compositions, kits, containers, and methods for preparing the IL-2 formulations disclosed herein. The IL-2 formulations disclosed herein may be used (alone or in combination with other formulations or therapies) to treat, prevent, and / or diagnose diseases, such as those disclosed herein.

[0007] This disclosure is based, in at least part, on the discovery that a combination of mutations in IL-2 that stabilize proteins, reduce their affinity for CD122 (e.g., the CD122 / CD132 heterodimer), and / or reduce their affinity for CD25 or have no more than a minimal effect, can be used to selectively enhance regulatory T cell (Treg) activity through the IL-2 pathway, thereby achieving a favorable therapeutic effect in treating diseases and conditions, such as autoimmune diseases. IL-2 formulations containing such mutations are suitable for treating conditions caused by abnormal immune responses, such as autoimmune diseases.

[0008] Therefore, in some aspects, this disclosure provides an IL-2 formulation, for example, an IL-2 formulation having one or more of the following properties a)-x) (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 them): Expression in vitro and / or in vivo at higher or increased levels, such as an increase of approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 100%, or more, or an increase of approximately 0.5-fold. Approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or more, 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 example, by protein concentration determination; Accumulates in vitro and / or in vivo at lower or reduced levels, for example, 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 times, about 1 time, about 1.5 times, or about 2 times. Approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 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, or more, for example, relative to an IL-2 formulation containing wild-type IL-2 or an IL-2 formulation containing a reference IL-2 variant, determined, for example, by melting temperature analysis (e.g., using fluorescence assay), dynamic light scattering, and / or size exclusion chromatography; It exhibits enhanced or increased stability in vitro and / or in vivo, for example, an increase of approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 100%, or more, or an increase of approximately 0.5 times, approximately 1 time, approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, or approximately 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times, or more, 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 example by expression in yeast surface display, expression in mammalian cells, chromatography, circular dichroism determination or related spectroscopic techniques and / or melting temperature analysis (e.g., using fluorescence); It has an enhanced or increased half-life in vitro and / or in vivo, for example, an increase of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more, or greater than about 0.5 times, about 1 time, or about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times, or more, for example, relative to IL-2 formulations containing wild-type IL-2 or IL-2 formulations containing a reference IL-2 variant, for example, as determined by ELISA, flow cytometry, and / or mass spectrometry; Having a lower, reduced, or diminished turnover and / or clearance rate or level in the body, 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 times, about 1 Approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 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, or more, for example, relative to IL-2 formulations containing wild-type IL-2 or IL-2 formulations containing a reference IL-2 variant, as determined by ELISA, flow cytometry, and / or mass spectrometry; The binding affinity to CD25 (e.g., human CD25) is reduced or substantially unchanged, for example, by 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 (e.g., about 1% to about 20%, about 2% to about 15%, or about 5% to about 10%), or by a reduction or increase of 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 The levels may be reduced by approximately 0.5 times, approximately 1 time, approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 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, or more, or reduced or increased by no more than approximately 0.5 times, approximately 1 time, approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 times, or approximately 5 times, 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 example, by yeast surface display, biolayer interferometry (e.g., Octet binding), and / or surface plasmon resonance (e.g., Biacore); It binds to CD25 (e.g., human CD25) with low affinity, for example with a dissociation constant (K0) of about 5-500 pM. DFor example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 pM, or for example, about 10 pM to about 490 pM, about 20 pM to about 480 pM, about 30 pM to about 470 pM, about 40 pM to about 460 pM, about 50 pM to about 450 pM, about 60 pM to about 440 pM. pM, approximately 70 pM to approximately 430 pM, approximately 80 pM to approximately 420 pM, approximately 90 pM to approximately 410 pM, approximately 100 pM to approximately 400 pM, approximately 110 pM to approximately 390 pM, approximately 120 pM to approximately 380 pM, approximately 130 pM to approximately 370 pM, approximately 140 pM to approximately 360 pM, approximately 150 pM to approximately 350 pM, approximately 160 pM to approximately 340 pM, approximately 170 pM to approximately 330 pM, approximately 180 pM to approximately 320 pM, approximately 190 pM to approximately 310 pM, approximately 200 pM to approximately 300 pM, approximately 210 pM to approximately 290 pM, approximately 220 pM to approximately 280 pM, approximately 230 pM to approximately 270 pM, approximately 240 pM to approximately 260 pM, or for example, approximately 5 pM to approximately 450 pM. pM, approximately 5 pM to approximately 400 pM, approximately 5 pM to approximately 350 pM, approximately 5 pM to approximately 300 pM, approximately 5 pM to approximately 250 pM, approximately 5 pM to approximately 200 pM, approximately 5 pM to approximately 150 pM, approximately 5 pM to approximately 100 pM, approximately 5 pM to approximately 50 pM, or for example, approximately 10 pM to approximately 500 pM, approximately 20 pM to approximately 500 pM, approximately 50 pM to approximately 500 pM, approximately 100 pM to approximately 500 pM, approximately 150 pM to approximately 500 pM, approximately 200 pM to approximately 500 pM, approximately 250 pM to approximately 500 pM, approximately 300 pM to approximately 500 pM, approximately 350 pM to approximately 500 pM, approximately 400 pM to approximately 500 pM, approximately 450 pM to approximately 500 pM PM, or for example, greater than about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250.Approximately 300, 350, 400, 450, or 500 pM, as determined, for example, by yeast surface appearance; It 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. nM, about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM, about 5 to about 10 nM, about 5.5 to about 10 nM, about 6 to about 10 nM, about 6.5 to about 10 nM, about 7 to about 10 nM, about 7.5 to about 10 nM, about 8 to about 10 nM, about 8.5 to about 10 nM, about 9 to about 10 nM, or about 9.5 to about 10 nM, or for example about 0.1 to about 9.5 nM, about 0.5 to about 9 nM, about 1 to about 8.5 nM, about 1.5 to about 8 nM, about 2 to about 7.5 nM, about 2.5 to about 7 nM, about 3 to about 6.5 nM, about 3.5 to about 6 nM, about 4 to about 5.5 nM, or about 4.5 to about 5 nM, or for example greater than about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nM, for example determined by biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore); It has a reduced or decreased binding affinity to CD122 / CD132 heterodimers (e.g., human CD122 / CD132 heterodimers), 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 (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 Approximately 50%, approximately 10% to approximately 20%, approximately 20% to approximately 30%, approximately 30% to approximately 40%, approximately 10% to approximately 30%, or approximately 20% to approximately 40%), or reduced by approximately 0.5 times, approximately 1 time, approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 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, or more (e.g., approximately 0.5 times to approximately 5 times, approximately 1 time to approximately 4 times, or approximately 2 times to approximately 3 times), 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 example, as determined by yeast surface display, biolayer interferometry (e.g., Octet binding), and / or surface plasmon resonance (e.g., Biacore); It binds to CD122 / CD132 heterodimers with low affinity (e.g., human CD122 / CD132 heterodimer), for example, the 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 0.2 to approximately 19 nM, approximately 0.2 to approximately 18 nM, approximately 0.2 to approximately 17 nM, or approximately 0.2 to approximately 16 nM, for example, approximately 0.2 to approximately 15 nM, approximately 0.1 to approximately 4 nM, approximately 0.1 to approximately 3 nM, approximately 0.1 to approximately 2 nM, approximately 0.1 to approximately 1 nM, or about 0.1 to about 0.5 nM, or for example, about 0.1 to about 10 nM, about 0.5 to about 10 nM, about 1 to about 10 nM, about 1.5 to about 10 nM, about 2 to about 10 nM, about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM, about 5 to about 10 nM, about 5.5 to about 10 nM, about 6 to about 10 nM, about 6.5 to about 10 nM, about 7 to about 10 nM, about 7.5 to about 10 nM, about 8 to about 10 nM, about 8.5 to about 10 nM, about 9 to about 10 nM, or about 9.5 to about 10 nM, or for example, about 0.1 to about 9.5 nM, about 0.5 to about 9 nM, about 1 to about 8.5 nM, about 1.5 to about 8 nM, about 2 to about 7.5 nM, about 2.5 to about 7 nM, about 3 to about 6.5 nM, about 3.5 to about 6 nM, about 4 to about 5.5 nM, or about 4.5 to about 5 nM, or 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, as determined by yeast surface display; Binding CD122 / CD132 heterodimers with low affinity (e.g., human CD122 / CD132 heterodimer), for example, the dissociation constant (K... DThe range is approximately 0.2-300 nM, for example, approximately 0.2 nM, approximately 0.5 nM, approximately 1 nM, approximately 2 nM, approximately 5 nM, approximately 10 nM, approximately 15 nM, approximately 20 nM, approximately 25 nM, approximately 30 nM, approximately 40 nM, approximately 50 nM, approximately 60 nM, approximately 70 nM, approximately 80 nM, approximately 90 nM, approximately 100 nM, approximately 110 nM, approximately 120 nM, approximately 130 nM, approximately 140 nM, approximately 150 nM, approximately 160 nM, approximately 170 nM, approximately 180 nM, approximately 190 nM, approximately 200 nM, approximately 210 nM, approximately 220 nM, approximately 230 nM, approximately 240 nM, approximately 250 nM, approximately 260 nM, approximately 270 nM, approximately 280 nM. 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 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, approximately 1, approximately 2, approximately 5, approximately 10, approximately 15, approximately 20 nM, approximately 25 nM, approximately 30 nM, approximately 40 nM, approximately 50 nM, approximately 60 nM, approximately 70 nM, approximately 80 nM, approximately 90 nM, approximately 100 nM, approximately 110 nM, approximately 120 nM, approximately 130 nM, approximately 140 nM, approximately 150 nM, approximately 160 nM, approximately 170 nM, approximately 180 nM, approximately 190 nM, approximately 200 nM, approximately 210 nM, approximately 220 nM, approximately 230 nM, approximately 240 nM, approximately 250 nM, approximately 260 nM, approximately 270 nM, approximately 280 nM, approximately 290 nM, or greater than approximately 300 nM, for example, is determined by biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore). Selectively activate IL-2 signaling in T regulatory cells, for example, T helper ECs, in vitro and / or in vivo. 50 / Treg EC 50 Ratio: Greater than approximately 1, 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, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25, approximately 26, approximately 27, approximately 28, approximately 29, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 150, approximately 200, approximately 250, approximately 300, approximately 350, approximately 400, approximately 450, approximately 500 Approximately 600, approximately 700, approximately 800, approximately 900, approximately 1000, approximately 1500, approximately 2000, approximately 2500, or approximately 3000, or more, or for example, greater than 1 and approximately 1 to 2, approximately 2 to 3, approximately 3 to 4, approximately 4 to 5, greater than 1 and approximately 1 to 10, greater than 1 and approximately 1 to 20, greater than 1 and approximately 1 to 30, greater than 1 and approximately 1 to 40, greater than 1 and approximately 1 to 50, approximately 2 to 10, approximately 2 to 20, approximately 2 to 30, approximately 2 to 40, 2 to 50, approximately 5 to 10, approximately 5 to 20, approximately 5 to 30, approximately 5 to 40, approximately 5 to 50, approximately 10 to 20, approximately 10 to 30, approximately 10 to 40 Approximately 10 to 50, approximately 20 to 40, approximately 20 to 50, approximately 50 to 100, approximately 100 to 200, approximately 200 to 500, approximately 500 to 1000, approximately 1000 to 2000, or approximately 1000 to 3000, relative to an IL-2 formulation containing wild-type IL-2 or an IL-2 formulation containing a reference IL-2 variant, as determined by flow cytometry, for example. Selectively activate IL-2 signal transduction in T regulatory cells in vitro and / or in vivo, for example, NK cells with the following ECMO...50 / Treg EC 50 Ratio: Greater than approximately 1, 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, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25, approximately 26, approximately 27, approximately 28, approximately 29, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 150, approximately 200, approximately 250, approximately 300, approximately 350, approximately 400, approximately 450, approximately 500 Approximately 600, approximately 700, approximately 800, approximately 900, approximately 1000, approximately 1500, approximately 2000, approximately 2500, or approximately 3000, or more, or for example, greater than 1 and approximately 1 to 2, approximately 2 to 3, approximately 3 to 4, approximately 4 to 5, greater than 1 and approximately 1 to 10, greater than 1 and approximately 1 to 20, greater than 1 and approximately 1 to 30, greater than 1 and approximately 1 to 40, greater than 1 and approximately 1 to 50, approximately 2 to 10, approximately 2 to 20, approximately 2 to 30, approximately 2 to 40, 2 to 50, approximately 5 to 10, approximately 5 to 20, approximately 5 to 30, approximately 5 to 40, approximately 5 to 50, approximately 10 to 20, approximately 10 to 30, approximately 10 to 40 Approximately 10 to 50, approximately 20 to 40, approximately 20 to 50, approximately 50 to 100, approximately 100 to 200, approximately 200 to 500, approximately 500 to 1000, approximately 1000 to 2000, or approximately 1000 to 3000, relative to an IL-2 formulation containing wild-type IL-2 or an IL-2 formulation containing a reference IL-2 variant, as determined by flow cytometry, for example. (i) Enhanced or increased efficacy and / or capacity to induce or promote T regulatory cell activity, for example, relative to IL-2 formulations containing wild-type IL-2 or IL-2 formulations containing a reference IL-2 variant, for Treg EC 50 The reduction 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 inhibition assays; (ii) Reduced or diminished efficacy and / or ability to induce or promote T regulatory cell activity, for example, for Treg ECMO compared to IL-2 formulations containing wild-type IL-2 or IL-2 formulations 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, for example, as determined by flow cytometry, in vitro or in vivo T regulatory cell proliferation or expansion assays and / or T cell inhibition assays; Regulate (e.g., reduce (e.g., inhibit, block or neutralize) or increase (e.g., activate, initiate or enhance) one or more biological activities of T cells (e.g., Treg) in vitro, in vitro or in vivo; It exhibits the same or similar binding affinity or specificity as the IL-2 formulation described herein, or both; It exhibits the same or similar binding affinity or specificity, or both, with IL-2 formulations that contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) variations (e.g., substitutions) described herein; It exhibits the same or similar binding affinity or specificity as, or both, that of IL-2 formulations containing the amino acid sequences described herein; It exhibits the same or similar binding affinity or specificity as, or both, that of IL-2 formulations containing an amino acid sequence encoded by the nucleotide sequence described herein; Inhibition, for example, competitive inhibition of the binding of a second IL-2 agent to the IL-2 receptor, wherein the second IL-2 agent is the IL-2 agent described herein. It competes with a second IL-2 agent for binding to the IL-2 receptor, wherein the second IL-2 agent is the IL-2 agent described herein; It possesses one or more biological properties of the IL-2 formulation described herein; Having one or more structural properties of the IL-2 formulation described herein; or It has one or more pharmacokinetic properties of the IL-2 formulations described herein.

[0009] In one embodiment, the IL-2 formulation is expressed in vitro and / or in vivo at higher or increased levels, for example, 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 increase by approximately 0.5 times, approximately 1 time, approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 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 or more, 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 example, by a protein concentration assay. In one embodiment, the IL-2 formulation accumulates in vitro and / or in vivo at a lower or reduced level, 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 times, about 1 time, or about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times, or more, 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 example, by melting temperature analysis (e.g., using fluorescence assay), dynamic light scattering, and / or size exclusion chromatography.

[0010] In one embodiment, the IL-2 formulation has enhanced or increased stability in vitro and / or in vivo, for example, 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 times, about 1 time, about 1.5 times, about 2 times, or about 2. 5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times, or more, 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 example by expression in yeast surface display, expression in mammalian cells, chromatography, circular dichroism determination or related spectroscopic techniques and / or melting temperature analysis (e.g., using fluorescence).

[0011] In one embodiment, the IL-2 formulation has an enhanced or increased half-life in vitro and / or in vivo, for example, an increase of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more, or greater than about 0%. 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times, or more, for example, relative to an IL-2 formulation containing wild-type IL-2 or an IL-2 formulation containing a reference IL-2 variant, as determined by, for example, ELISA, flow cytometry, and / or mass spectrometry.

[0012] In one embodiment, the IL-2 formulation has a lower, reduced, or diminished turnover and / or clearance rate or level in vivo, 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. Approximately 0.5 times, approximately 1 time, approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 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, or more, for example, relative to IL-2 formulations containing wild-type IL-2 or IL-2 formulations containing a reference IL-2 variant, as determined by, for example, ELISA, flow cytometry, and / or mass spectrometry.

[0013] In one embodiment, the binding affinity of the IL-2 formulation to CD25 (e.g., human CD25) is reduced or substantially unchanged, for example, 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 (e.g., about 1% to about 20%, about 2% to about 15%, or about 5% to about 10%), or reduced 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%, or about 45%. %, or about 50%, or reduced by about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times, or more, or reduced or increased by no more than about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, or about 5 times, 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 example, by yeast surface display, biolayer interferometry (e.g., Octet binding), and / or surface plasmon resonance (e.g., Biacore). In one embodiment, the reduction or decrease in binding affinity for CD25 is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% less than the reduction or decrease in binding affinity for CD25. In one embodiment, there is no significant reduction or decrease in binding affinity for CD25.

[0014] In one embodiment, the IL-2 formulation binds to CD25 (e.g., human CD25) with low affinity, for example, with a dissociation constant (KD) of about 5-500 pM, such as about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 pM, or for example, about 10 pM to about 490 pM, about 20 pM to about 480 pM, about 30 pM, or about 500 pM, or for example, about 10 pM to about 490 pM, about 20 pM to about 480 pM, about 30 pM, or about 500 pM. pM to approximately 470 pM, approximately 40 pM to approximately 460 pM, approximately 50 pM to approximately 450 pM, approximately 60 pM to approximately 440 pM, approximately 70 pM to approximately 430 pM, approximately 80 pM to approximately 420 pM, approximately 90 pM to approximately 410 pM, approximately 100 pM to approximately 400 pM, approximately 110 pM to approximately 390 pM, approximately 120 pM to approximately 380 pM, approximately 130 pM to approximately 370 pM, approximately 140 pM to approximately 360 pM, approximately 150 pM to approximately 350 pM, approximately 160 pM to approximately 340 pM, approximately 170 pM to approximately 330 pM, approximately 180 pM to approximately 320 pM, approximately 190 pM to approximately 310 pM, approximately 200 pM to approximately 300 pM, approximately 210 pM to approximately 290 pM, approximately 220 pM to approximately 370 pM, approximately 40 pM to approximately 460 pM, approximately 50 pM to approximately 450 pM, approximately 60 pM to approximately 440 pM, approximately 70 pM to approximately 430 pM, approximately 80 pM to approximately 420 pM, approximately 90 pM to approximately 410 pM, approximately 100 pM to approximately 400 pM, approximately 110 pM to approximately 390 pM, approximately 200 pM to approximately 300 pM, approximately 210 pM to approximately 290 pM, approximately 220 pM to approximately 370 pM, approximately 400 pM to approximately 460 pM, approximately 50 pM to approximately 450 pM, approximately pM to about 280 pM, about 230 pM to about 270 pM, about 240 pM to about 260 pM, or for example, about 5 pM to about 450 pM, about 5 pM to about 400 pM, about 5 pM to about 350 pM, about 5 pM to about 300 pM, about 5 pM to about 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM, about 5 pM to about 100 pM, about 5 pM to about 50 pM, or for example, about 10 pM to about 500 pM, about 20 pM to about 500 pM, about 50 pM to about 500 pM, about 100 pM to about 500 pM, about 150 pM to about 500 pM, about 200 pM to about 500 pM, about 250 pM to about 500 pM, about 300 pM to about 500 pM, about 350 pM to about 500 pM, about 350 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 230 pM to about 270 pM, about 240 pM to about 260 pM, or for example, about 5 pM to about 450 pM, about 5 pM to about 400 pM, about 5 pM to about 450 pM, about 5 pM to about 450 pM, about 5 pM to about 350 pM, about 230 pM to pM to approximately 500 pM, approximately 400 pM to approximately 500 pM, approximately 450 pM to approximately 500 pM, or for example, greater than approximately 5, approximately 10, approximately 15, approximately 20, approximately 25, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 105.Approximately 110, 115, 120, 125, 130, 135, 140, 145, 150, 200, 250, 300, 350, 400, 450, or 500 pM, as determined, for example, by yeast surface display.

[0015] 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. nM, about 2 to about 10 nM, about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM, about 5 to about 10 nM, about 5.5 to about 10 nM, about 6 to about 10 nM, about 6.5 to about 10 nM, about 7 to about 10 nM, about 7.5 to about 10 nM, about 8 to about 10 nM, about 8.5 to about 10 nM, about 9 to about 10 nM, or about 9.5 to about 10 nM, or for example about 0.1 to about 9.5 nM, about 0.5 to about 9 nM, about 1 to about 8.5 nM, about 1.5 to about 8 nM, about 2 to about 7.5 nM, about 2.5 to about 7 nM, about 3 to about 6.5 nM, about 3.5 to about 6 nM, about 4 to about 5.5 nM, or about 4.5 to about 5 nM, or for example greater than about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nM, for example determined by biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore).

[0016] In one embodiment, the IL-2 formulation has a reduced or decreased binding affinity to CD122 / CD132 heterodimers (e.g., human CD122 / CD132 heterodimers), 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 (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 10%). 50%, approximately 10% to approximately 50%, approximately 10% to approximately 20%, approximately 20% to approximately 30%, approximately 30% to approximately 40%, approximately 10% to approximately 30%, or 20% to approximately 40%), or reduced by approximately 0.5 times, approximately 1 time, approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 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, about 9 times, about 9.5 times, about 10 times, or more (e.g., about 0.5 times to about 5 times, about 1 times to about 4 times, or about 2 times to about 3 times), 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 example, as determined by yeast surface display, biolayer interferometry (e.g., Octet binding), and / or surface plasmon resonance (e.g., Biacore). In one embodiment, the reduction or decrease in binding affinity to the CD122 / CD132 heterodimer is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 times greater than the reduction or decrease in binding affinity to CD25. In one embodiment, there is no significant reduction or decrease in binding affinity to CD25.

[0017] In one embodiment, the IL-2 formulation binds with low affinity to the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), for example, the 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 0.2 to approximately 19 nM, approximately 0.2 to approximately 18 nM, approximately 0.2 to approximately 17 nM, or approximately 0.2 to approximately 16 nM, for example, approximately 0.2 to approximately 15 nM, approximately 0.1 to approximately 4 nM, approximately 0.1 to approximately 3 nM, approximately 0.1 to approximately 2 nM, approximately 0.1 to approximately 1 nM, or about 0.1 to about 0.5 nM, or for example, about 0.1 to about 10 nM, about 0.5 to about 10 nM, about 1 to about 10 nM, about 1.5 to about 10 nM, about 2 to about 10 nM, about 2.5 to about 10 nM, about 3 to about 10 nM, about 3.5 to about 10 nM, about 4 to about 10 nM, about 4.5 to about 10 nM, about 5 to about 10 nM, about 5.5 to about 10 nM, about 6 to about 10 nM, about 6.5 to about 10 nM, about 7 to about 10 nM, about 7.5 to about 10 nM, about 8 to about 10 nM, about 8.5 to about 10 nM, about 9 to about 10 nM, or about 9.5 to about 10 nM, or for example, about 0.1 to about 9.5 nM, about 0.5 to about 9 nM, about 1 to about 8.5 nM, about 1.5 to about 8 nM, about 2 to about 7.5 nM, about 2.5 to about 7 nM, about 3 to about 6.5 nM, about 3.5 to about 6 nM, about 4 to about 5.5 nM, or about 4.5 to about 5 nM, or 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, as determined by yeast surface display.

[0018] In one embodiment, the IL-2 formulation binds to CD122 / CD132 heterodimers (e.g., human CD122 / CD132 heterodimers) with low affinity, for example, the dissociation constant (K). DThe range is approximately 0.2-300 nM, for example, approximately 0.2 nM, approximately 0.5 nM, approximately 1 nM, approximately 2 nM, approximately 5 nM, approximately 10 nM, approximately 15 nM, approximately 20 nM, approximately 25 nM, approximately 30 nM, approximately 40 nM, approximately 50 nM, approximately 60 nM, approximately 70 nM, approximately 80 nM, approximately 90 nM, approximately 100 nM, approximately 110 nM, approximately 120 nM, approximately 130 nM, approximately 140 nM, approximately 150 nM, approximately 160 nM, approximately 170 nM, approximately 180 nM, approximately 190 nM, approximately 200 nM, approximately 210 nM, approximately 220 nM, approximately 230 nM, approximately 240 nM, approximately 250 nM, approximately 260 nM, approximately 270 nM, approximately 280 nM. 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 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, approximately 1, approximately 2, approximately 5, approximately 10, approximately 15, approximately 20 nM, approximately 25 nM, approximately 30 nM, approximately 40 nM, approximately 50 nM, approximately 60 nM, approximately 70 nM, approximately 80 nM, approximately 90 nM, approximately 100 nM, approximately 110 nM, approximately 120 nM, approximately 130 nM, approximately 140 nM, approximately 150 nM, approximately 160 nM, approximately 170 nM, approximately 180 nM, approximately 190 nM, approximately 200 nM, approximately 210 nM, approximately 220 nM, approximately 230 nM, approximately 240 nM, approximately 250 nM, approximately 260 nM, approximately 270 nM, approximately 280 nM, approximately 290 nM, or greater than approximately 300 nM, for example, is determined by biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore).

[0019] In one embodiment, the IL-2 agent selectively activates IL-2 signal transduction in T regulatory cells in vitro and / or in vivo, for example, in T helper ECs with the following... 50 / Treg EC 50Ratio: Greater than approximately 1, 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, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25, approximately 26, approximately 27, approximately 28, approximately 29, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 150, approximately 200, approximately 250, approximately 300, approximately 350, approximately 400, approximately 450, approximately 500 Approximately 600, approximately 700, approximately 800, approximately 900, approximately 1000, approximately 1500, approximately 2000, approximately 2500, or approximately 3000, or more, or for example, greater than 1 and approximately 1 to 2, approximately 2 to 3, approximately 3 to 4, approximately 4 to 5, greater than 1 and approximately 1 to 10, greater than 1 and approximately 1 to 20, greater than 1 and approximately 1 to 30, greater than 1 and approximately 1 to 40, greater than 1 and approximately 1 to 50, approximately 2 to 10, approximately 2 to 20, approximately 2 to 30, approximately 2 to 40, 2 to 50, approximately 5 to 10, approximately 5 to 20, approximately 5 to 30, approximately 5 to 40, approximately 5 to 50, approximately 10 to 20, approximately 10 to 30, approximately 10 to 40 Approximately 10 to 50, approximately 20 to 40, approximately 20 to 50, approximately 50 to 100, approximately 100 to 200, approximately 200 to 500, approximately 500 to 1000, approximately 1000 to 2000, or approximately 1000 to 3000, relative to an IL-2 formulation containing wild-type IL-2 or an IL-2 formulation containing a reference IL-2 variant, as determined by flow cytometry, for example. In one embodiment, T helper cells are CD45+CD3+CD4+Foxp3- cells, as determined by flow cytometry, for example. In one embodiment, Tregs are CD45+CD3+CD4+Foxp3+ cells, as determined by flow cytometry, for example.

[0020] In one embodiment, the IL-2 agent selectively activates IL-2 signal transduction in T regulatory cells in vitro and / or in vivo, for example, NK cell ECMO cells with the following characteristics. 50 / Treg EC 50Ratio: Greater than approximately 1, 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, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25, approximately 26, approximately 27, approximately 28, approximately 29, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 150, approximately 200, approximately 250, approximately 300, approximately 350, approximately 400, approximately 450, approximately 500 Approximately 600, approximately 700, approximately 800, approximately 900, approximately 1000, approximately 1500, approximately 2000, approximately 2500, or approximately 3000, or more, or for example, greater than 1 and approximately 1 to 2, approximately 2 to 3, approximately 3 to 4, approximately 4 to 5, greater than 1 and approximately 1 to 10, greater than 1 and approximately 1 to 20, greater than 1 and approximately 1 to 30, greater than 1 and approximately 1 to 40, greater than 1 and approximately 1 to 50, approximately 2 to 10, approximately 2 to 20, approximately 2 to 30, approximately 2 to 40, 2 to 50, approximately 5 to 10, approximately 5 to 20, approximately 5 to 30, approximately 5 to 40, approximately 5 to 50, approximately 10 to 20, approximately 10 to 30, approximately 10 to 40 Approximately 10 to 50, approximately 20 to 40, approximately 20 to 50, approximately 50 to 100, approximately 100 to 200, approximately 200 to 500, approximately 500 to 1000, approximately 1000 to 2000, or approximately 1000 to 3000, relative to an IL-2 formulation containing wild-type IL-2 or an IL-2 formulation containing a reference IL-2 variant, as determined by flow cytometry, for example. In one embodiment, NK cells are CD45+CD3- cells that are CD56+ and / or CD16+, as determined by flow cytometry, for example. In one embodiment, NK cells are CD45+CD3-CD56+ cells, as determined by flow cytometry, for example. In one embodiment, Tregs are CD45+CD3+CD4+Foxp3+ cells, as determined by flow cytometry, for example.

[0021] In one embodiment, the efficacy and / or ability of the IL-2 formulation to induce or promote T regulatory cell activity is enhanced or increased, 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 Treg 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 lower, 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.

[0022] 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.

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

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

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

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

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

[0028] In one embodiment, the IL-2 formulation inhibits, for example, competitively inhibits the binding of a second IL-2 formulation to the IL-2 receptor, wherein the second IL-2 formulation is the IL-2 formulation described herein.

[0029] In one embodiment, the IL-2 formulation competes with a second IL-2 formulation for binding to the IL-2 receptor, wherein the second IL-2 formulation is the IL-2 formulation described herein.

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

[0031] In one embodiment, the IL-2 formulation has one or more structural properties of the IL-2 formulation described herein.

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

[0033] In one embodiment, the interleukin-2 (IL-2) formulation comprises a human IL-2 variant containing amino acid variations (e.g., substitutions) at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all) corresponding to, for example, wild-type human IL-2: T3, H16, I28, K35, R38, F42, E68, V69, Q74, D84, S87, N88, I92, C125, Q126, or combinations thereof. In another embodiment, the IL-2 formulation contains amino acid variations (e.g., substitutions) at positions V69, Q74, or combinations thereof. In one embodiment, the IL-2 formulation contains amino acid variations (e.g., substitutions) at positions V69 and Q74. In one embodiment, the IL-2 formulation contains the amino acid substitution V69A. In one embodiment, the IL-2 formulation contains the amino acid substitution Q74P. In one embodiment, the IL-2 formulation contains an amino acid change (e.g., substitution) at positions H16, I92, D84, or combinations thereof. In one embodiment, the IL-2 formulation contains an amino acid change (e.g., substitution) at position H16, optionally wherein the amino acid substitution is H16N, H16L, or H16D. In one embodiment, the IL-2 formulation contains the amino acid substitution H16N. In one embodiment, the IL-2 formulation contains the amino acid substitution H16L. In one embodiment, the IL-2 formulation contains an amino acid change (e.g., substitution) at position I92, optionally wherein the amino acid substitution is I92S. In one embodiment, the IL-2 formulation contains an amino acid change (e.g., substitution) at position D84, optionally wherein the amino acid substitution is D84V. In one embodiment, the IL-2 formulation contains an amino acid change (e.g., substitution) at positions K35, R38, F42, E68, or combinations thereof. In one embodiment, the IL-2 formulation contains an amino acid change (e.g., substitution) at position K35, optionally wherein the amino acid substitution is K35E. In one embodiment, the IL-2 formulation contains an amino acid change (e.g., substitution) at position R38, optionally wherein the amino acid substitution is R38E, R38N, or R38Q. In one embodiment, the IL-2 formulation contains the amino acid substitution R38N. In one embodiment, the IL-2 formulation contains the amino acid substitution R38Q. In one embodiment, the IL-2 formulation contains an amino acid change (e.g., substitution) at position F42, optionally wherein the amino acid substitution is F42K or F42Q. In one embodiment, the IL-2 formulation contains the amino acid substitution F42Q.

[0034] In one implementation, the IL-2 formulation comprises one or more of (i)-(v) (e.g., two, three, four, or all): (i) One or more (e.g., two, three, four, five, six or seven) amino acid changes (e.g., substitutions) that reduce or are identified as reducing their affinity for CD122 (e.g., CD122 / CD132 heterodimer), such as changes at position H16 (e.g., substitutions) (e.g., H16L, H16N or H16D), changes at position I28 (e.g., substitutions) (e.g., I28T or I28F), changes at position D84 (e.g., substitutions) (e.g., D84V), changes at position S87 (e.g., substitutions) (e.g., S87R), changes at position N88 (e.g., substitutions) (e.g., N88S, N88L or N88D), changes at position I92 (e.g., substitutions) (e.g., I92S) and / or changes at position Q126 (e.g., substitutions) (e.g., Q126T, Q126K or Q126R); (ii) One or more (e.g., two) amino acid changes (e.g., substitutions) that increase or are identified as increasing the stability of the IL-2 formulation, such as changes at position V69 (e.g., substitutions) (e.g., V69A) and / or changes at position Q74 (e.g., substitutions) (e.g., Q74P); (iii) One or more (e.g., two, three, or four) amino acid changes (e.g., substitutions) that reduce or are identified as reducing their affinity for CD25, such as changes at position K35 (e.g., substitutions) (e.g., K35E), changes at position R38 (e.g., substitutions) (e.g., R38E, R38N, or R38Q), changes at position F42 (e.g., substitutions) (e.g., F42K or F42Q), and / or changes at position E68 (e.g., substitutions) (e.g., E68Q or E68N); or (iv) One or more amino acid changes (e.g., substitutions) that reduce or are identified as reducing O-glycosylation of IL-2 formulations, such as changes at position T3 (e.g., substitutions) (e.g., T3A); or (v) One or more amino acid changes (e.g., substitutions) that reduce or are identified as reducing incorrect disulfide bond pairing and / or aggregation in IL-2 formulations (e.g., to improve stability), such as changes at position C125 (e.g., substitutions) (e.g., C125S).

[0035] 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).

[0036] 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).

[0037] 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 (v). In one embodiment, the IL-2 formulation comprises (ii), (iii), and (iv). In one embodiment, the IL-2 formulation comprises (ii), (iv), and (iv). In one embodiment, the IL-2 formulation comprises (iii), (iv), and (v).

[0038] 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).

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

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

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

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

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

[0044] In one implementation, the IL-2 formulation does not contain (i), (ii), (iii), (iv), and (v).

[0045] In one embodiment, the IL-2 formulation comprises amino acid changes (e.g., substitutions): (i) at positions V69 and Q74, and / or at position K35; and (ii) at position H16, I92, or D84; and optionally (iii) at positions R38, F42, E68 or a combination thereof.

[0046] In one embodiment, the IL-2 formulation comprises amino acid changes (e.g., substitutions): (i) at positions V69 and Q74, and / or at position K35; and (ii) at positions H16, I92, or D84; and (iii) at positions R38, F42, E68 or a combination thereof.

[0047] In one embodiment, the IL-2 formulation comprises amino acid changes (e.g., substitutions): (i) at positions V69 and Q74, and / or at position K35; and (ii) at position H16, I92, or D84; or (iii) at positions R38, F42, E68 or a combination thereof.

[0048] In one embodiment, the IL-2 formulation comprises amino acid changes (e.g., substitutions): (i) at positions V69 and Q74; and / or at position K35; and (ii) at positions H16, I92, D84 or a combination thereof, and (iii) at positions R38, F42, E68 or a combination thereof.

[0049] In one embodiment, the IL-2 formulation 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, optionally wherein the amino acid substitutions are V69A, Q74P, and H16L. In one embodiment, the IL-2 formulation comprises amino acid substitutions V69A, Q74P, and H16L.

[0050] In one embodiment, the IL-2 formulation 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 formulation comprises amino acid substitutions V69A, Q74P, and I92S.

[0051] In one embodiment, the IL-2 formulation 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 formulation comprises amino acid substitutions V69A, Q74P, and D84V.

[0052] In one embodiment, the IL-2 formulation contains amino acid changes (e.g., substitutions) at positions V69, Q74, and R38, optionally wherein the amino acid substitutions are V69A, Q74P, and R38Q, respectively.

[0053] In one embodiment, the IL-2 formulation 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 formulation comprises amino acid substitutions V69A, Q74P, and F42Q.

[0054] In one embodiment, the IL-2 formulation 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 formulation comprises amino acid substitutions V69A, Q74P, and R38N.

[0055] In one embodiment, the IL-2 formulation 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 one embodiment, the IL-2 formulation comprises amino acid substitutions V69A, Q74P, and R38E.

[0056] In one embodiment, the IL-2 formulation comprises amino acid variations (e.g., substitutions) at positions V69, Q74, K35, and H16, optionally wherein the amino acid substitutions are V69A, Q74P, K35E, and H16N or H16L, respectively. In one embodiment, the IL-2 formulation comprises amino acid substitutions of V69A, Q74P, K35E, and H16N or H16L. In one embodiment, the IL-2 formulation comprises amino acid substitutions of V69A, Q74P, K35E, and H16N. In one embodiment, the IL-2 formulation comprises amino acid substitutions of V69A, Q74P, K35E, and H16L.

[0057] In one embodiment, the IL-2 formulation 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 one embodiment, the IL-2 formulation comprises amino acid substitutions V69A, Q74P, K35E, H16N, and R38N.

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

[0059] In one embodiment, the IL-2 formulation contains an amino acid change (e.g., substitution) at positions I28, E68, S87, N88, Q126, or combinations thereof. In one embodiment, the IL-2 formulation contains an amino acid change (e.g., substitution) at position I28, optionally wherein the amino acid substitution is I28T or I28F. In one embodiment, the IL-2 formulation contains the amino acid substitution I28T. In one embodiment, the IL-2 formulation contains the amino acid substitution I28F.

[0060] In one embodiment, the IL-2 formulation contains an amino acid change (e.g., substitution) at position E68, optionally wherein the amino acid substitution is E68Q or E68N. In one embodiment, the IL-2 formulation contains the amino acid substitution E68Q. In one embodiment, the IL-2 formulation contains the amino acid substitution E68N.

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

[0062] In one 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 one embodiment, the IL-2 formulation comprises an amino acid-substituted N88R. In one embodiment, the IL-2 formulation comprises an amino acid-substituted N88S. In one embodiment, the IL-2 formulation comprises an amino acid-substituted N88L. In one embodiment, the IL-2 formulation comprises an amino acid-substituted N88D.

[0063] In one 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 one embodiment, the IL-2 formulation comprises the amino acid substitution Q126T. In one embodiment, the IL-2 formulation comprises the amino acid substitution Q126K. In one embodiment, the IL-2 formulation comprises the amino acid substitution Q126R.

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

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

[0066] In one 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 one embodiment, the IL-2 formulation comprises amino acid substitutions V69A, Q74P, and C125S.

[0067] In one embodiment, the IL-2 formulation contains an amino acid variation (e.g., substitution) at positions T3, H16, I92, or combinations thereof, optionally wherein the amino acid substitutions are T3A, H16N, and I92S, respectively.

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

[0069] In one embodiment, the IL-2 formulation contains amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, and C125S, respectively. In another embodiment, the IL-2 formulation contains amino acid substitutions H16L, V69A, Q74P, and C125S. Various technical effects are associated with IL-2 formulations containing the above-described combinations of amino acid changes. Without being bound by theory, it is believed that in one embodiment, an IL-2 formulation comprising amino acid substitutions for H16L, V69A, Q74P, and C125S may have at least one 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 stability, for example, attributable to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or diminished binding capacity and / or binding affinity for CD25 (or with an effect not exceeding a minimum), which prolongs the lifespan of the IL-2 formulation; (iv) substantially does not promote the expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) reduced incorrect disulfide bond pairing and improved stability, for example, attributable to the presence of C125S mutations. In one implementation, IL-2 formulations containing H16L mutations exhibit reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs compared to IL-2 formulations containing other H16 mutations. These properties make IL-2 formulations containing amino acid substitutions for H16L, V69A, Q74P, and C125S particularly suitable for treating diseases and conditions caused by abnormal immune responses, such as autoimmune diseases.

[0070] Therefore, in one embodiment, IL-2 formulations containing amino acid substitutions H16L, V69A, Q74P, and C125S, relative to wild-type IL-2 or a reference IL-2 variant that does not contain the following amino acid substitutions, particularly possess one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, or all of them): (i) enhanced or increased in vitro or in vivo stability; (ii) decreased or reduced in vitro and / or in vivo binding capacity and / or binding affinity to human CD122; (iii) in vitro and / or in vivo... (iv) Decreased or reduced binding capacity and / or binding affinity of the IL-2 variant to the IL-2 receptor composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) Decreased or reduced binding capacity and / or binding affinity of the IL-2 variant to human CD25 in vitro and / or in vivo (e.g., moderately decreased or reduced); (vi) Selective binding to regulatory T cells (e.g., Foxp3). + (vii) selectively activate the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhance or increase the ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0071] In one embodiment, the IL-2 formulation 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 formulation comprises amino acid substitutions H16L, V69A, Q74P, I92S, and C125S.

[0072] In one embodiment, the IL-2 formulation 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 formulation comprises amino acid substitutions T3A, V69A, Q74P, and C125S.

[0073] In one embodiment, the IL-2 formulation 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 formulation comprises amino acid substitutions of T3A, H16N, V69A, Q74P, and C125S. In another embodiment, the IL-2 formulation comprises amino acid substitutions of T3A, H16L, V69A, Q74P, and C125S.

[0074] In one embodiment, the IL-2 formulation 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 one embodiment, the IL-2 formulation comprises amino acid substitutions of T3A, V69A, Q74P, I92S, and C125S. In another embodiment, the IL-2 formulation comprises amino acid substitutions of T3A, V69A, Q74P, I92S, and C125S.

[0075] In one embodiment, the IL-2 formulation comprises a human IL-2 variant comprising an amino acid sequence selected from the following: 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 their functional fragments, or amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence similarity, or amino acid sequences having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids similarity.

[0076] In one embodiment, relative to a reference IL-2 formulation that does not contain amino acid changes (e.g., substitutions), the amino acid changes (e.g., substitutions) provide the IL-2 formulation with at least one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or all of them): (i) Enhanced or increased expression of IL-2 formulations; (ii) Inhibits or reduces the aggregation of IL-2 agents; (iii) Enhanced or increased stability of IL-2 formulations; (iv) Prolonging or increasing the half-life of IL-2 formulations; (v) The turnover and / or clearance of IL-2 inhibitory or reduced agents; (vi) IL-2 formulations that inhibit or reduce (e.g., moderately inhibit or reduce) or remain substantially unchanged in binding to human CD25; (vii) The affinity of IL-2 inhibitors or reduced agents for human CD122; (viii) The affinity of IL-2 inhibitors or reduced agents for human CD132; or (ix) The affinity of IL-2 inhibitors or reducers for the dimerized IL-2 receptor composed of human CD122 and human CD132; (x) Selectively binds to regulatory T cells (e.g., Foxp3+ T cells); (xi)Selective activation of the IL-2 signaling pathway in Treg; or (xii) The ability to enhance or increase, or decrease or reduce, the ability to induce or promote the amplification, activity, survival and / or proliferation of Tregs.

[0077] In one embodiment, the IL-2 formulation comprises a human IL-2 variant comprising one or more amino acid variations (e.g., substitutions) selected from H16D, H16N, H16L, I28T, K35E, R38Q, R38N, R38E, F42K, F42Q, V69A, Q74P, D84V, S87R, N88L, N88S, I92S, and C125S; a polypeptide linker as described herein; and a non-IL-2 portion as described herein; wherein the amino acid variations (e.g., substitutions) cause the IL-2 formulation to have at least one or more of the following properties relative to a reference IL-2 formulation that does not contain said amino acid variations (e.g., substitutions): (i) Enhanced or increased expression of IL-2 formulations; (ii) Inhibits or reduces the aggregation of IL-2 agents; (iii) Enhanced or increased stability of IL-2 formulations; (iv) Prolonging or increasing the half-life of IL-2 formulations; (v) The turnover and / or clearance of IL-2 inhibitory or reduced agents; (vi) IL-2 formulations that inhibit or reduce (e.g., moderately inhibit or reduce) or remain substantially unchanged in binding to human CD25; (vii) The affinity of IL-2 inhibitors or reduced agents for human CD122; (viii) The affinity of IL-2 inhibitors or reduced agents for human CD132; (ix) The affinity of IL-2 inhibitors or reducers for the dimerized IL-2 receptor composed of human CD122 and human CD132; (x) Selectively binds to regulatory T cells (e.g., Foxp3+ T cells); (xi) Selective activation of the IL-2 signaling pathway in Treg; and / or (xii) The ability to enhance, increase, decrease, or reduce the induction or promotion of Treg amplification, activity, survival, and / or proliferation.

[0078] In one implementation, the human IL-2 variant includes amino acid changes (e.g., substitutions): (i)C125S; (ii) V69A, Q74P and C125S; (iii) H16D, V69A, Q74P and C125S; (iv) H16N, V69A, Q74P and C125S; (v)H16L, V69A, Q74P and C125S; (vi) I28T, V69A, Q74P and C125S; (vii) V69A, Q74P, D84V and C125S; (viii) V69A, Q74P, S87R and C125S; (ix) V69A, Q74P, N88L and C125S; (x)V69A, Q74P, N88S and C125S; (xi) V69A, Q74P, I92S and C125S; (xii) K35E, V69A, Q74P and C125S; (xiii) K35E, H16N, V69A, Q74P and C125S; (xiv) K35E, H16L, V69A, Q74P and C125S; (xv) K35E, D84V, V69A, Q74P and C125S; (xvi) K35E, I92S, V69A, Q74P and C125S; (xvii) R38Q, V69A, Q74P and C125S; (xviii) R38Q, H16N, V69A, Q74P and C125S; (xix) R38Q, H16L, V69A, Q74P and C125S; (xx) R38Q, D84V, V69A, Q74P and C125S; (xxi) R38Q, I92S, Q74P and C125S; (xxii) R38N, V69A, Q74P and C125S; (xxiii) R38N, H16N, V69A, Q74P and C125S; (xxiv) R38N, H16L, V69A, Q74P and C125S; (xxv) R38N, D84V, V69A, Q74P and C125S; (xxvi) R38N, I92S, Q74P and C125S; (xxvii) R38E, V69A, Q74P and C125S; (xxviii) F42K, V69A, Q74P and C125S; (xxix) F42Q, V69A, Q74P and C125S; (xxx) F42A, Y45A, L72G, N88D, V69A, Q74P and C125S; (xxxi) R38N, S87R, V69A, Q74P and C125S; (xxxii) R38E, H16N, V69A, Q74P and C125S; (xxxiii) R38E, D84V, V69A, Q74P and C125S; (xxxiv) R38E, S87R, V69A, Q74P and C125S; (xxxv) R38E, I92S, V69A, Q74P and C125S; (xxxvi) F42Q, H16N, V69A, Q74P and C125S; (xxxvii) F42Q, I92S, V69A, Q74P, and C125S; or (xxxviii) K35E, R38N, H16N, V69A, Q74P and C125S.

[0079] (xxxix) T3A, H16N, V69A, Q74P and C125S; (XL) T3A, H16L, V69A, Q74P, and C125S; or (xli) T3A, V69A, Q74P, I92S and C125S.

[0080] In one embodiment, the IL-2 formulation comprises a human IL-2 variant comprising an amino acid sequence selected from the following: 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 with it, or an amino acid sequence having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids similar to it; the polypeptide linker described herein; and the non-IL-2 portion described herein; wherein the IL-2 formulation exhibits at least one or more of the following properties relative to a reference IL-2 formulation not containing a human IL-2 polypeptide variant: (i) Enhanced or increased expression of IL-2 formulations; (ii) Inhibits or reduces the aggregation of IL-2 agents; (iii) Enhanced or increased stability of IL-2 formulations; (iv) Prolonging or increasing the half-life of IL-2 formulations; (v) The turnover and / or clearance of IL-2 inhibitory or reduced agents; (vi) IL-2 formulations that inhibit or reduce (e.g., moderately inhibit or reduce) or remain substantially unchanged in binding to human CD25; (vii) The affinity of IL-2 inhibitors or reduced agents for human CD122; (viii) The affinity of IL-2 inhibitors or reduced agents for human CD132; (ix) The affinity of IL-2 inhibitors or reducers for the dimerized IL-2 receptor composed of human CD122 and human CD132; (x) Selectively binds to regulatory T cells (e.g., Foxp3+ T cells); (xi) Selective activation of the IL-2 signaling pathway in Treg; and / or (xii) The ability to enhance or increase, or decrease or reduce, the ability to induce or promote Treg amplification, activity and / or proliferation.

[0081] Multiple technical effects are associated with IL-2 formulations containing the amino acid sequence of SEQ ID NO:5. Not wishing to be bound by theory, it is believed that in one embodiment, an IL-2 formulation containing the amino acid sequence of SEQ ID NO:5 may have at least one 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 stability, for example, attributable to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity for CD25 (or an effect on it not exceeding a minimum), which prolongs the lifespan of the IL-2 formulation; (iv) substantially does not promote the expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) reduced incorrect disulfide pairing and increased stability, for example, attributable to the presence of C125S mutations. In one embodiment, compared to IL-2 agents containing other H16 mutations, IL-2 agents containing the H16L mutation exhibit reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than for other T cell types. These properties make IL-2 agents containing the amino acid sequence of SEQ ID NO:5 particularly suitable for treating diseases and conditions caused by abnormal immune responses, such as autoimmune diseases.

[0082] Therefore, in one embodiment, relative to wild-type IL-2 or a reference IL-2 variant that does not contain the said amino acid substitution, SEQ ID is included. IL-2 formulations with the amino acid sequence NO:5 particularly possess one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, or all): (i) enhanced or increased in vitro or in vivo stability; (ii) decreased or reduced binding capacity and / or binding affinity to human CD122 in vitro and / or in vivo; (iii) decreased or reduced binding capacity and / or binding affinity to human CD132 in vitro and / or in vivo; (iv) decreased or reduced affinity of the IL-2 variant to the heterodimer of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) IL-2 receptor in vitro and / or in vivo; (v) decreased or reduced binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo (e.g., moderately decreased or reduced); (vi) selective binding to regulatory T cells (e.g., Foxp3). + (vii) selectively activate the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhance or increase the ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0083] In one embodiment, the reference IL-2 formulation 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 formulation comprises the amino acid sequence of SEQ ID NO:1031. In one embodiment, the reference IL-2 formulation comprises the amino acid sequence of SEQ ID NO:1. In one embodiment, the reference IL-2 formulation comprises the amino acid sequence of SEQ ID NO:2.

[0084] In one embodiment, the IL-2 formulation comprises a human IL-2 variant described herein fused to a non-IL-2 portion thereof via a linker, wherein the linker is a peptide linker, optionally wherein the peptide linker is a flexible linker, a rigid linker, or a cleavable linker. In one embodiment, the peptide linker is a Gly-Ser linker (e.g., (G4S)n linker, where n = 1, 2, 3, 4, 5, 6, or greater (SEQ ID NO: 1020)), a proline-rich extended linker (e.g., V1 GPc, V2, GPGc, V3 GcGcP, cellulase linker 4, cellulase linker 4), a rigid linker (e.g., A(EAAAK)nA, where n = 2, 3, 4, 5, or greater (SEQ ID NO: 1021); REPR_12), a non-GS linker (e.g., (GGGSA)n, where n = 1, 2, 3, 4, 5, or greater (SEQ ID NO: 1022)), or an immunoglobulin hinge region or a portion thereof. In one embodiment, the peptide linker is a Gly-Ser linker comprising (G4S)1 (SEQ ID NO:1023), (G4S)2 (SEQ ID NO:1024), (G4S)3 (SEQ ID NO:1025), (G4S)4 (SEQ ID NO:48), (G4S)5 (SEQ ID NO:1026), or (G4S)6 (SEQ ID NO:1027). In one embodiment, the peptide linker is a Gly-Ser linker comprising (G4S)4 (SEQ ID NO:48). In one embodiment, the peptide linker comprises an amino acid sequence selected from SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, or SEQ ID NO:55. In one embodiment, the peptide linker comprises the amino acid sequence of SEQ ID NO:48.

[0085] In one embodiment, the non-IL-2 portion is an immunoglobulin Fc region, or a fragment or portion thereof (e.g., a functional fragment). In one embodiment, the immunoglobulin Fc region comprises an IgG Fc region, an IgD Fc region, an IgA Fc region, an IgM Fc region, or an IgE Fc region, or a fragment or portion thereof. In one embodiment, the IgG Fc region comprises a wild-type human IgG1 Fc region (e.g., an IgG1 m3 allotype), a wild-type IgG2 Fc region, or a wild-type human IgG4 Fc region, or a fragment or portion thereof.

[0086] In one embodiment, the IgG Fc region comprises a mutant IgG1 or mutant IgG4 Fc region, or a fragment or portion thereof. In one embodiment, the IgG Fc region comprises one or more (e.g., two, three, four, or five) mutations, such as one or more (e.g., two, three, four, or five) mutations described herein.

[0087] In one implementation, the IgG Fc region includes a mutated IgG4 Fc region, or a fragment or portion thereof, wherein the mutated IgG4 Fc region is human.

[0088] In one embodiment, the mutant IgG4 Fc region, or a fragment or portion thereof, comprises an amino acid change (e.g., substitution) at Ser228, numbered according to EU numbering, optionally wherein the amino acid change (e.g., substitution) at Ser228 is S228P. In one embodiment, the mutant IgG4 Fc region comprises the amino acid substitution S228P.

[0089] In one embodiment, the mutant IgG4 Fc region or a fragment or portion thereof comprises an amino acid change (e.g., substitution) at Arg409, numbered according to EU numbering, optionally wherein the amino acid change (e.g., substitution) at Arg409 is R409K. In one embodiment, the mutant IgG4 Fc region comprises the amino acid substitution R409K.

[0090] In one embodiment, the mutant IgG4 Fc region, or a fragment or portion thereof, comprises amino acid changes (e.g., substitutions) at Thr307, Gln311, and Ala378, numbered according to EU numbering, optionally wherein the amino acid changes (e.g., substitutions) are T307Q, Q311V, and A378V, respectively. In one embodiment, the mutant IgG4 Fc region comprises amino acid substitutions T307Q, Q311V, and A378V.

[0091] In one embodiment, the mutant IgG4 Fc region comprises an amino acid sequence selected from SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it, or an amino acid sequence having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids similar to it.

[0092] In one embodiment, the IgG Fc region comprises a mutated IgG1 Fc region, or a fragment or portion thereof, wherein the mutated IgG1 Fc region is human. In one embodiment, the mutated IgG1 Fc region (e.g., comprising an N297G substitution) has an IgG1m3 allotype.

[0093] In one embodiment, the mutant IgG1 Fc region, or a fragment or portion thereof, comprises an amino acid change (e.g., substitution) at Asn297, numbered according to EU numbering, optionally wherein the amino acid change (e.g., substitution) at Asn297 is N297G. In one embodiment, the mutant IgG1 Fc region comprises the amino acid substitution N297G.

[0094] In one embodiment, the mutant IgG1 Fc region, or a fragment or portion thereof, comprises amino acid changes (e.g., substitutions) at Leu234, Leu235, and Pro329, numbered according to EU numbering, optionally wherein the amino acid changes (e.g., substitutions) are L234A, L235A, and P329G, respectively. In one embodiment, the mutant IgG1 Fc region comprises amino acid substitutions L234A, L235A, and P329G.

[0095] In one embodiment, the mutant IgG1 Fc region, or a fragment or portion thereof, comprises amino acid changes (e.g., substitutions) at Thr307, Gln311, and Ala378, numbered according to EU numbering, optionally wherein the amino acid changes (e.g., substitutions) are T307Q, Q311V, and A378V, respectively. In one embodiment, the mutant IgG1 Fc region comprises amino acid substitutions T307Q, Q311V, and A378V.

[0096] In one embodiment, the mutant IgG1 Fc region comprises an amino acid sequence selected from SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:1003, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it, or differing from it by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids. In one embodiment, the mutated IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:1003, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it, or differs from it by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids. In one embodiment, the mutated IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:1003.

[0097] In one implementation, the non-IL-2 portion inhibits or reduces the ability of the IL-2 formulation to induce Fc receptor-mediated immune effector function.

[0098] In one embodiment, the IL-2 formulation comprises an IL-2 variant comprising an amino acid sequence selected from the following: 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:25, SEQ ID NO:3 ... 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 via the Gly-Ser linker to an IgG Fc region comprising an amino acid sequence selected from: SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:1003.

[0099] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the following: 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:8 ... 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.

[0100] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the following: 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: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: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.

[0101] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the following: 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 ...0, 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:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO: 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.

[0102] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the following: 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:19 ... 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.

[0103] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the following: 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:23 ... 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.

[0104] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the following: 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: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:273, 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:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO: 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.

[0105] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the following: 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:31 ... 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.

[0106] In one embodiment, the IL-2 formulation comprises an amino acid sequence selected from the following: 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 ...0, 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:340, SEQ ID NO:341, SEQ ID NO:342, SEQ ID NO:343, SEQ ID NO:344, SEQ ID NO: 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.

[0107] In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:59 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:97 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:135 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:173 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:211 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:249 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:287 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:325 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:66 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:104 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:142 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:180 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:218 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:256 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:294 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:332 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:60 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:98 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:136 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:174 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:212 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:250 or a functional fragment thereof.In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:288 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:326 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:69 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:107 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:145 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:183 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:221 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:259 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:297 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:335 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:1004 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:1005 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:1006 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:1007 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:1008 or a functional fragment thereof. In one embodiment, the IL-2 formulation comprises the amino acid sequence of SEQ ID NO:1009 or a functional fragment thereof.

[0108] Several technical effects are associated with IL-2 formulations containing the amino acid sequence SEQ ID NO:1008. Not wishing to be bound by theory, it is believed that in one embodiment, an IL-2 formulation containing the amino acid sequence SEQ ID NO:1008 may have at least one 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 stability, for example, attributable to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity for CD25 (or an effect on it not exceeding a minimum), which prolongs the lifespan of the IL-2 formulation; (iv) substantially does not promote the expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) reduced incorrect disulfide pairing and increased stability, for example, attributable to the presence of C125S mutations. In one implementation, compared to IL-2 formulations containing other H16 mutations, IL-2 formulations containing the H16L mutation exhibit reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types. These properties make IL-2 variants containing the amino acid sequence SEQ ID NO:1008 particularly suitable for treating diseases and conditions caused by abnormal immune responses, such as autoimmune diseases.

[0109] Therefore, in one embodiment, relative to wild-type IL-2 or a reference IL-2 variant that does not contain the said amino acid substitution, SEQ ID is included. IL-2 formulations with the amino acid sequence NO:5 particularly possess one or more of the following properties (e.g., 2, 3, 4, 1008, 6, 7, or all): (i) enhanced or increased in vitro or in vivo stability; (ii) decreased or reduced binding capacity and / or binding affinity to human CD122 in vitro and / or in vivo; (iii) decreased or reduced binding capacity and / or binding affinity to human CD132 in vitro and / or in vivo; (iv) decreased or reduced affinity of the IL-2 variant to the heterodimer of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) IL-2 receptor in vitro and / or in vivo; (v) decreased or reduced binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo (e.g., moderately decreased or reduced); (vi) selective binding to regulatory T cells (e.g., Foxp3). + (vii) To selectively activate the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; (viii) To enhance or increase the ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0110] In one embodiment, the IL-2 formulation forms a dimer (e.g., a homodimer or a heterodimer).

[0111] In one embodiment, the IL-2 formulation comprises an IL-2 fusion protein. In one embodiment, the IL-2 formulation comprises an IL-2 formulation / anti-IL-2 antibody complex. In one embodiment, the IL-2 formulation comprises a conjugate.

[0112] In some aspects, this disclosure provides a pharmaceutical composition comprising the described IL-2 formulation and a pharmaceutically acceptable carrier. In some aspects, this disclosure provides a nucleic acid encoding the described IL-2 formulation. In some aspects, this disclosure provides a carrier (e.g., an expression vector) comprising a nucleic acid encoding the described IL-2 formulation. In some aspects, this disclosure provides a cell (e.g., isolated cells) comprising a nucleic acid encoding the described IL-2 formulation or a carrier (e.g., an expression vector) comprising a nucleic acid encoding the described IL-2 formulation.

[0113] In some aspects, this disclosure provides a method for producing an IL-2 formulation, comprising culturing (e.g., maintaining) cells containing nucleic acids encoding the IL-2 formulation described herein, or cells containing a vector (e.g., an expression vector) encoding nucleic acids of the IL-2 formulation described herein, under conditions that allow expression of the IL-2 formulation. In one embodiment, the method further includes obtaining the IL-2 formulation. In one embodiment, the method further includes purifying the IL-2 formulation.

[0114] In some aspects, this disclosure provides methods for enhancing the expansion, activity, survival, and / or proliferation of regulatory T cells (Tregs), comprising contacting Treg cells or populations of Treg cells (e.g., in vitro, ex vivo, or in vivo) with an effective amount of the IL-2 formulation described herein or a pharmaceutical composition comprising said IL-2 formulation, or administering to a subject in need of such treatment an effective amount of the IL-2 formulation described herein or a pharmaceutical composition comprising said IL-2 formulation. The IL-2 formulation may, for example, comprise amino acid substitutions for H16L, V69A, Q74P, and C125S, or amino acid substitutions for H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 formulation comprises amino acid substitutions for H16L, V69A, Q74P, and C125S.

[0115] In some aspects, this disclosure provides a method for selectively activating the IL-2 signaling pathway in regulatory T cells (Tregs), comprising contacting Treg cells or a population of Treg cells (e.g., in vitro, ex vivo, or in vivo) with an effective amount of the IL-2 formulation described herein or a pharmaceutical composition comprising said IL-2 formulation, or administering to a subject in need of such an effective amount of the IL-2 formulation described herein or a pharmaceutical composition comprising said IL-2 formulation. The IL-2 formulation may, for example, comprise amino acid substitutions for H16L, V69A, Q74P, and C125S, or amino acid substitutions for H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 formulation comprises amino acid substitutions for H16L, V69A, Q74P, and C125S.

[0116] In some aspects, this disclosure provides a method for inducing immune tolerance in a desired subject, comprising administering an effective amount of the IL-2 formulation described herein or a pharmaceutical composition comprising said IL-2 formulation. The IL-2 formulation may, for example, comprise amino acid substitutions for H16L, V69A, Q74P, and C125S, or amino acid substitutions for H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 formulation comprises amino acid substitutions for H16L, V69A, Q74P, and C125S.

[0117] In some aspects, this disclosure provides a method of treating a subject suffering from a disease (such as those described herein, such as an autoimmune disease, lupus nephritis, autoimmune hepatitis, nephrotic syndrome, or cancer), comprising administering to the subject an effective amount of an IL-2 formulation described herein or a pharmaceutical composition comprising said IL-2 formulation. The IL-2 formulation may, for example, comprise amino acid substitutions for H16L, V69A, Q74P, and C125S, or amino acid substitutions for H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 formulation comprises amino acid substitutions for H16L, V69A, Q74P, and C125S.

[0118] In some aspects, this disclosure provides an IL-2 formulation or composition for use in treating a subject suffering from a disease (such as those described herein, such as autoimmune diseases, 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 formulation may, for example, comprise amino acid substitutions for H16L, V69A, Q74P, and C125S, or amino acid substitutions for H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 formulation comprises amino acid substitutions for H16L, V69A, Q74P, and C125S.

[0119] In some aspects, this disclosure provides the use of an IL-2 formulation or composition in a medicament for preparing a medicine for treating a subject suffering from a disease (such as those described herein, such as an autoimmune disease, lupus nephritis, autoimmune hepatitis, nephrotic syndrome, or cancer), said method comprising administering to said subject an IL-2 formulation or a pharmaceutical composition comprising said IL-2 formulation. The IL-2 formulation may, for example, comprise amino acid substitutions for H16L, V69A, Q74P, and C125S, or amino acid substitutions for H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 formulation comprises amino acid substitutions for H16L, V69A, Q74P, and C125S.

[0120] In some aspects, this disclosure provides a kit comprising the IL-2 formulation described herein or a pharmaceutical composition containing the IL-2 formulation, along with instructions for use. The IL-2 formulation may, for example, comprise amino acid substitutions for H16L, V69A, Q74P, and C125S, or amino acid substitutions for H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 formulation comprises amino acid substitutions for H16L, V69A, Q74P, and C125S.

[0121] In some aspects, this disclosure provides containers that contain the IL-2 formulation described herein or pharmaceutical compositions containing the IL-2 formulation. The IL-2 formulation may, for example, contain amino acid substitutions of H16L, V69A, Q74P, and C125S, or amino acid substitutions of H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 formulation contains amino acid substitutions of H16L, V69A, Q74P, and C125S. Attached Figure Description

[0122] Figure 1A The provided schematic diagram illustrates the domain structure of exemplary, non-limiting embodiments of the IL-2 formulations provided herein. As shown, the IL-2 formulation comprises an IL-2 moiety or variant (also referred to herein as a "mutein"), a peptide linker, an Fc domain containing a hinge sequence, and CH2 and CH3 domains of an antibody. Figure 1B A description of the amino acid sequence of human IL-2 (SEQ ID NO: 1030) is provided, showing exemplary non-restrictive positions that, when mutated, result in effects on IL-2 receptor binding and IL-2-mediated signal transduction activity in vitro and in vivo.

[0123] Figure 2The schematic diagram illustrates a cell-based method for generating IL-2 variant libraries and selecting stable and active clones from these libraries using yeast surface visualization. IL-2 mutants or IL-2 variants expressed by the initial clone are generated via 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 bound CD25 is measured using recombinant CD25, anti-6xHis antibody (“6xHis” disclosed as SEQ ID NO:1028), and fluorescent secondary antibody staining (y-axis). In some forms of experiments, HA-tags are used in addition to or as an alternative to the Myc-tag. Fluorescently activated cell sorting is used to enrich IL-2 variants exhibiting high expression and high binding activity.

[0124] Figure 3A The provided graphs illustrate the results of a method using IL-2 receptor titration to determine the affinity and binding capacity of IL-2 mutant proteins displayed on yeast surfaces. Yeast clones expressing the IL-2 mutant proteins shown were incubated with a series of concentrations of the extracellular domain of CD25 tagged with 6xHis (“6xHis” disclosed as SEQ ID NO:1028). Binding of CD25 was measured by staining with an anti-6xHis antibody (“6xHis” disclosed as SEQ ID NO:1028) and a fluorescent secondary antibody. Several exemplary IL-2 mutant proteins are shown. Curve fitting was used to determine binding affinity (K0). D ) and the maximum binding signal (data not shown). Figure 3B The provided charts illustrate the relative binding capacity of the selected IL-2 mutant proteins (maximum binding signal normalized to IL-2 expression levels).

[0125] Figure 4A The provided graphs illustrate the thermal denaturation (melting curves) of the selected IL-2 formulation (IL-2-Fc fusion protein) as measured by SYPRO orange fluorescence. The native IL-2-Fc fusion protein shows a maximum signal at low temperatures, indicating the presence of an unfolded protein, while the V69A / Q74P mutant protein shows unfolding events as the temperature increases. Figure 4B HPLC size exclusion chromatograms were provided, showing that most of the native IL-2-Fc fusion complex eluted from the column very early (>670 kDa), indicating unfolded protein aggregation. In contrast, V69A / Q74P IL-2-Fc eluted as a single peak at the expected time of 84 kDa for the protein.

[0126] Figures 5A-5BThe provided scatter plot shows the results of a yeast cell sorting operation used to identify mutations affecting interaction with the CD122 and / or CD132 IL-2 receptors. Yeast cells containing IL-2 variant libraries expressing their surface were stained with a specified concentration of CD122 / CD132 Fc heterodimers, and receptor binding was detected using a fluorescent anti-human Fc secondary antibody. Surface IL-2 expression was detected using an anti-Myc antibody and a fluorescent secondary antibody. Cells within indicated gates were sorted and recovered, and enriched IL-2 mutant proteins in these populations were identified using a combination of Sanger sequencing and next-generation sequencing.

[0127] Figure 6A The provided graphs show the results of a method for determining the fractional saturation of yeast expressing a specified IL-2 mutant protein on its surface after titration with a specified concentration of CD122 / CD132 Fc heterodimer. All mutant proteins described, except for the shown mutant, contain V69A / Q74P. CD122 / CD132 bound with anti-human Fc fluorescent secondary antibody were labeled, and measurements were performed 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 was defined as 1. Figure 6B The provided charts show that, in addition to incubating the selected mutant protein with the recombinant CD25 extracellular domain tagged with 6xHis (“6xHis” is disclosed as SEQ ID NO:1028), the bound CD25 was detected using an anti-6xHis antibody (“6xHis” is disclosed as SEQ ID NO:1028) and other antibodies. Figure 6A The same method yields the same result.

[0128] Figure 7 A series of graphs are provided illustrating the affinity of IL-2-Fc fusion proteins containing different IL-2 variants (as shown in the figures) for the CD122 / CD132 Fc heterodimer and the CD25 extracellular domain, measured on an Octet biolayer interferometer. The IL-2 variants include V69A / Q74P and specified mutations. The IL-2-Fc fusion proteins were immobilized on anti-human Fc capture tips and then incubated with a range of indicator IL-2 receptors. Binding and dissociation phase kinetics were used to estimate binding affinity. An excess of irrelevant antibody was used to prevent nonspecific binding or capture of the CD122 / CD132 Fc protein by the tip.

[0129] Figure 8 The provided diagram illustrates the gating strategy and corresponding flow cytometry data used to identify IL-2-sensitive cell populations from human PBMCs. Singlet lymphocytes were identified based on forward and side scattering. The population was defined as: T regulatory cells (CD4+CD25+). 高Foxp3+), CD25 高 T helper cells (CD4+CD25) 高 Foxp3- and natural killer cells (CD3-CD56+).

[0130] Figures 9A-9D The provided charts illustrate the IL-2 signaling response in an IL-2-sensitive cell population in human PBMCs after treatment with an IL-2-Fc fusion variant containing a mutation that reduces CD122 / CD132 dimer affinity. Figure 9A ,Treg; Figure 9B CD25+ (high) T helper cells; Figure 9C NK cells; Figure 9D CD8+ cytotoxic T cells, determined by the degree of STAT5 phosphorylation. Cells of indicated concentrations were treated for 30 min with IL-2-Fc fusion proteins containing V69A / Q74 mutations and specified mutations, or with IL-2 N88D mutant proteins fused to the C-terminus of unbound antibodies (C-terminal N88D). Inactivated IL-2-Fc fusion proteins contained several mutations to reduce their IL-2 signaling activity (F42A, Y45A, L72G, N88D, V69A, Q74P). After treatment, cells were fixed with formaldehyde, permeabilized with cold methanol, and stained for surface markers and STAT5 transcription factors phosphorylated at Tyr694 (pSTAT5). Populations were based on... Figure 8 The gating was used for identification. The signal transduction activity of selected mutant proteins on CD8+ cytotoxic T cells was also evaluated. These cells, such as... Figure 8 Gating was performed using CD8 surface markers instead of CD4. Median pSTAT5 levels (median fluorescence intensity, MFI) of various concentrations of IL-2-Fc fusion protein tested in different cell populations are shown. Curve fitting was performed using GraphPad Prism v5.03, with 4 parameters fitted to log(agonist) versus response.

[0131] Figures 10A-10C The provided chart depicts the IL-2-sensitive cell population in human PBMCs after treatment with an IL-2-Fc fusion containing a mutation that reduces affinity for CD25. Figure 10A ,Treg; Figure 10B CD25+ (high) T helper cells; Figure 10C IL-2 signaling transduction in NK cells. The processing and analysis of human PBMCs are shown in Figure 9. The median pSTAT5 level (MFI) for each treatment is shown in each population. To highlight the effects on EC... 50 The function is to standardize the signals in each mutant protein from 0 to 1 within the concentration range of IL-2-Fc treatment.

[0132] Figure 11A-11C The provided chart depicts the IL-2-sensitive cell population in human PBMCs after treatment with an IL-2-Fc fusion containing paired mutations that reduce affinity for the CD25 and CD122 / CD132 dimers. Figure 11A ,Treg; Figure 11B CD25+ (high) T helper cells; Figure 11C The IL-2 signaling transduction response in NK cells was analyzed. The processing and analysis of human PBMCs are shown in Figure 9. For clarity, the IL-2-Fc fusion protein containing various IL-2 mutant proteins is divided into upper and lower subplots. The median pSTAT5 level (MFI) for each treatment in each population is shown.

[0133] Figures 12A-12C The provided chart illustrates the use of IL-2-Fc H16N ( Figure 12A ) or C-terminal N88D ( Figure 12B In Tg32 mice treated with IL-2Fc fusion protein, Tregs were amplified in vivo and measured as a percentage of total CD3+ T cells. Specific amounts of each IL-2Fc fusion protein (approximately equimolar dose levels) were administered to homozygous Tg32 mice via tail vein injection. Lymphocyte populations were analyzed at specified time points, defining Tregs as CD45+CD3+CD4+CD25+. 高 CD127 cells. Figure 12A and Figure 12B The data in the figure are the average values ​​of three mice in each treatment group. Figure 12C Data from individual mice are shown at the highest dose of each IL-2-Fc fusion protein tested.

[0134] Figures 13A-13C The provided chart illustrates the use of IL-2-Fc H16N ( Figure 13A ) or C-terminal N88D ( Figure 13B Changes in CD4+ T helper cell levels in Tg32 mice treated with [the drug] were measured as a percentage of total CD3+ T cells. Mice were administered the drug as shown in Figure 12. CD4+ T helper cells were defined as CD45+CD3+CD4+ cells instead of CD25+ cells. 高 CD127-. Figure 13A and Figure 13B The data in the figure are the average values ​​of three mice in each treatment group. Figure 13C This shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested.

[0135] Figures 14A-14C The provided chart illustrates the use of 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.

[0136] 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.

[0137] 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 .

[0138] 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; Figure 17C-17DSerum was collected from mice treated with the IL-2-Fc fusion protein. 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% starting material was defined as the amount detectable in plasma 1 hour after injection. Note that the x-axis is categorical, not scaled over time.

[0139] Figures 18A-18D The expansion of immune cells in vivo after administration of an exemplary IL-2 Fc fusion protein in humanized mice was described. Figure 18A The schematic diagram of the experimental design shown illustrates blood samples drawn from humanized mice administered the IL-2 Fc fusion peptide and the control peptide at different time points. Flow cytometry was used to measure various lymphocyte populations at each specified time point. Figure 18B The Y-axis shows the fold expansion of T-regulated cells for each IL-2 Fc fusion peptide, and the X-axis indicates the corresponding dose (low or high). Figure 18C The Y-axis shows the fold expansion of T helper cells for each IL-2Fc fusion peptide, and the X-axis indicates the corresponding dose (low or high). Figure 18D The Y-axis shows the fold increase in NK cells for each IL-2 Fc fusion peptide, and the X-axis indicates the corresponding dose (low or high). Investigational IL-2 Fc fusion peptides, such as... Figure 18B-18D The X-axis, from left to right, is shown below: control monoclonal antibody (Motavizumab), inactivated IL-2, IL-2 mutant protein containing the N88D mutation, wild-type IL-2, IL-2 mutant protein containing the mutation H16N / V69A / Q74P / C125S (SEQ ID NO:1007), and IL-2 mutant protein containing the mutation H16L / V69A / Q74P / C125S (SEQ ID NO:1008).

[0140] Figures 19A-19B The persistence and effective half-life of the exemplary IL-2 fusion protein in Tg32 mice were depicted. Figure 19A The concentration of the indicated mutant combination of IL-2 fusion protein in mouse blood is shown on the Y-axis, and the number of days after administration is shown on the X-axis. Figure 19B The study presents a comparison of the half-lives of IL-2 fusion proteins with and without other mutations in the indicated IL-2 moiety in the Fc region. The concentration of the indicated IL-2 fusion protein in the blood is shown on the Y-axis, and the number of days after administration is shown on the X-axis.

[0141] Figure 20 The pharmacokinetic profile of an exemplary IL-2-Fc fusion protein (containing the mutant H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused with the IgG1 Fc N297G allotype m3)) in cynomolgus monkeys is described. Serum IL-2-Fc fusion protein levels were measured over time after weekly injections of 100 µg / kg IL-2-Fc fusion protein in four monkeys (numbered 3501, 3502, 3503, and 3504).

[0142] Figures 21A-21B The effects of an exemplary IL-2-Fc fusion protein (containing mutant H16L / V69A / Q74P / C125S (SEQ ID NO:1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on the expansion and proliferation of regulatory T cells in cynomolgus monkeys are described. Figure 21A The changes in T regulatory cell expansion over time following four weekly injections of 100 μg / kg of IL-2-Fc fusion protein are presented as fold changes relative to baseline (baseline = before administration). Figure 21B The study showed that Ki67 levels decreased over time after four weekly injections of 100 μg / kg IL-2-Fc fusion protein. + The percentage of T regulatory cells (a measure of proliferating T regulatory cells) is standardized against the total number of T regulatory cells.

[0143] Figures 22A-22D This study describes the effect of an exemplary IL-2-Fc fusion protein (containing mutant H16L / V69A / Q74P / C125S (SEQ ID NO:1008) (IL2-118 fused with IgG1 Fc N297G isoform m3)) on circulating immune cells in cynomolgus monkeys after four weekly injections of 100 µg / kg IL-2-Fc fusion protein. Figure 22A The effect of the IL-2-Fc fusion protein on the number of NK cells over time is presented. Figure 22B The effects on cytotoxic T cells over time are shown. Figure 22C The effects on T helper cells over time are shown, while Figure 22D The effects on total T cells over time are presented. For each cell type, the data are shown as fold changes from baseline (baseline = before administration).

[0144] Figures 23A-23C This paper illustrates the role of the exemplary IL-2-Fc fusion protein described herein in the progression of disease in a mouse model of systemic lupus erythematosus with kidney involvement resembling lupus nephritis. Figure 23A Proteinuria scores, measured weekly in mice after treatment with either 40 μg / kg of the exemplary IL-2-Fc fusion protein or the PBS carrier control, were presented, administered every 3 days from 3 weeks of age until 18 weeks of age. Proteinuria scores are shown on the Y-axis, and mouse age in weeks is shown on the X-axis. Figure 23B The presented series of charts illustrates proteinuria scores in individual mice treated with either the load control or the exemplary IL-2-Fc fusion protein (as shown on the X-axis) on the Y-axis. From left to right, the first subplot depicts the proteinuria score at 11 weeks of age, the middle subplot depicts the score at 12 weeks of age, and the last subplot depicts the score at 13 weeks of age. Figure 23C The Y-axis shows quantified glomerular damage in individual mice treated with either the load control or the exemplary IL-2-Fc fusion protein (as shown on the X-axis) at the end of the study (when the mice reached 18 weeks of age). Invention Details This document discloses IL-2 formulations (e.g., IL-2 variants, IL-2 fusion proteins, IL-2 complexes, or IL-2 conjugates) having one or more of the structural and / or functional properties described herein. Advantageously, several IL-2 formulations described herein possess one or more improved or desired properties compared to IL-2 formulations containing wild-type IL-2. Without wishing to be bound by theory, it is believed that in one embodiment, the IL-2 formulations described herein selectively enhance regulatory T cell (Treg) activity via the IL-2 pathway. Nucleic acid molecules encoding the IL-2 formulations, expression vectors, host cells, compositions (e.g., pharmaceutical compositions), kits, containers, and methods for preparing the IL-2 formulations are also provided. The IL-2 formulations and pharmaceutical compositions disclosed herein can be used (alone or in combination with other formulations or modalities) to treat, prevent, and / or diagnose diseases and conditions, such as those related to T cell activity, for example, the diseases or conditions described herein (e.g., autoimmune diseases described herein).

[0145] Immune responses are typically controlled by the recognition of specific exogenous or self-antigens, communication between innate and adaptive immune pathways, crosstalk between B cells and T cells, and other factors. Some autoimmune diseases are characterized by the widespread recognition of self-antigens. These diseases can be treated with therapies that broadly enhance the processes that protect self-antigens from attack by the immune system. Tregs are T cells that recognize self-antigens. In response to antigenic stimulation, they release immunosuppressive cytokines and directly suppress other T cells through cell-to-cell contact. Impaired Treg activity can lead to a variety of autoimmune diseases (e.g., insufficient or low-activity cells). IL-2 is a cytokine that can lead to the expansion and activation of many cell types, but Tregs are generally more sensitive to IL-2 than other cell types. Administration of low-dose IL-2 has been shown to be associated with preferential, sustained in vivo expansion of Treg cells and improvement in 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 long-acting immunomodulators (e.g., immunosuppressants) for a number of conditions (e.g., autoimmune indications).

[0146] This disclosure is based, in at least part, on the finding that IL-2 formulations containing a specific combination of human IL-2 peptides having the amino acid substitutions described herein can have advantageous technical effects, such as increased stability of the IL-2 formulation and / or providing selective activation of regulatory T cells. The IL-2 formulations described herein typically require CD25 for efficient signal transduction via the IL-2 receptor, making them highly selective for Tregs. IL-2 signal transduction 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.

[0147] In one embodiment, it was found that the IL-2 preparations described herein selectively bind to and activate regulatory T cells, while also inhibiting other immune cell types (e.g., CD25). 高(T cells and NK cells) lack function. Not wishing to be bound by theory, it is believed that in one embodiment, the amino acid substitutions described herein can promote the maintenance of the active conformation of the IL-2 formulation and modulate the binding affinity of the IL-2 formulation to dimeric receptors containing IL-2Rβ (CD122) and IL-2Rγ (CD132) and trimeric receptors containing IL-2Rα (CD25) and CD122 and CD132. In one embodiment, the IL-2 formulation described herein has optimal affinity for selectively binding to and activating IL-2 signaling in regulatory T cells, resulting in selective activation and expansion of regulatory T cells in vitro and in vivo. Not wishing to be bound by theory, it is believed that in one embodiment, IL-2 binding to the IL-2 receptor is the primary pathway for IL-2 clearance in vivo. For example, the IL-2 formulations described herein with reduced affinity for both dimeric and trimeric IL-2 receptors exhibit prolonged half-lives, suggesting that reduced affinity for IL-2 receptors decreases the in vivo clearance of the IL-2 formulation. The IL-2 formulations described herein, such as those with amino acid substitutions that increase stability and decrease affinity for the IL-2 receptor, can selectively activate regulatory T cells and exhibit an increased in vivo half-life. The IL-2 formulations described herein, such as those with mutations that block CD25 binding, can have an improved in vivo half-life. In one embodiment, the IL-2 formulation does not promote, or substantially does not promote, the expansion, activation, survival, and / or proliferation of T effector cells and / or NK cells in vitro and / or in vivo. Without wishing to be bound by theory, it is believed that in one embodiment, the IL-2 formulations described herein may have a larger therapeutic window than low-dose IL-2.

[0148] Various technical effects exist associated with the presence of the specific mutant sets described herein, for example, a set of mutations comprising an amino acid substitution at position H16, combined with amino acid substitutions at positions V69, Q74, and C125 (e.g., H16L, V69A, Q74P, and C125S). Without wishing to be bound by theory, it is believed that in one embodiment, IL-2 formulations comprising H16L, V69A, Q74P, and C125S (e.g., IL-2 variants or IL-2 fusion proteins) are significantly stable, for example, due to the presence of stabilizing V69A and Q74P mutations. For example, it has been unexpectedly found that V69A and Q74P substitutions do not significantly increase (or significantly decrease) the binding affinity of the IL-2 formulation to CD25, but rather stabilize the IL-2 formulation in an active conformation sufficient to bind to CD25. Not wanting to be bound by theory, it is also believed that in one embodiment, the IL-2 formulation containing the above-mentioned mutations has reduced binding affinity for CD122 and / or CD132, which increases the efficacy and selectivity of the IL-2 formulation for regulatory T cells (Tregs) (compared to other T cell types). Therefore, IL-2 formulations containing these mutations are generally stable and selectively activate regulatory T cells (Tregs). Not wanting to be bound by theory, it is further believed that in one embodiment, the IL-2 formulation containing the above-mentioned mutations has reduced or decreased binding capacity and / or binding affinity for CD25, which improves the lifespan of the IL-2 formulation. Not wanting to be bound by theory, it is also believed that in one embodiment, the IL-2 formulation containing these mutations substantially does not promote the in vitro and / or in vivo expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells. In one implementation, compared to IL-2 agents containing other H16 mutations, IL-2 agents containing the H16L mutation exhibit reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types. These properties make IL-2 agents containing the aforementioned mutations particularly suitable for treating diseases or conditions caused by abnormal immune responses, such as autoimmune diseases.

[0149] Therefore, in one embodiment, an IL-2 formulation (e.g., an IL-2 variant or IL-2 fusion protein) comprising a combination of amino acid substitutions at position H16 and amino acid substitutions at positions V69, Q74, and C125 (e.g., H16L, V69A, Q74P, and C125S) has, in particular, one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, or all of them) relative to wild-type IL-2 or a reference IL-2 formulation that does not contain the aforementioned amino acid substitutions. (i) Enhanced or increased stability in vitro or in vivo; (ii) Reduced or decreased binding capacity and / or binding affinity to human CD122 in vitro and / or in vivo; (iii) Reduced or decreased binding capacity and / or binding affinity to human CD132 in vitro and / or in vivo; (iv) The affinity of IL-2 formulations for the IL-2 receptor, which is reduced or decreased in vitro and / or in vivo, for the heterodimer composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer). (v) Reduced or diminished (e.g., moderately reduced or diminished) binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo. (vi) Selectively binds 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) Enhances or increases the ability to induce or promote Treg amplification, activity, survival and / or proliferation.

[0150] definition This article uses the articles “a” and “a kind” to indicate one or more kinds (i.e., at least one kind) as the grammatical object of the article.

[0151] Unless the context clearly indicates otherwise, the term “or” as used herein means “and / or” and may be used interchangeably with it.

[0152] "Approximately" and "about" should generally indicate an acceptable degree of error in the measured quantity, taking into account the nature or precision of the measurement. Exemplary error levels are within 20% of a given range or value, typically within 10%, and more often within 5%. When "approximately" or "about" precedes a series of numbers or a range, it should be understood that "approximately" or "about" modifies each number in that series or range. Similarly, when "at least," "greater than," "not greater than," "less than," "not less than," or "within" precedes a series of numbers or a range, it should be understood that "at least," "greater than," "not greater than," "less than," "not less than," or "within" modifies each number in that series or range. As used herein, ranges include upper and lower limits.

[0153] The compositions and methods described herein include polypeptides and nucleic acids having a specified sequence, or polypeptides and nucleic acids having a sequence substantially identical or similar to the specified sequence (e.g., at least 85%, 90%, 95% or higher identical to the specified sequence).

[0154] In describing amino acid sequences, the term "substantially identical" as used herein means that the first amino acid contains a sufficient or minimum number of amino acid residues that: i) are identical to the aligned amino acid residues in the second amino acid sequence, or ii) have conserved substitutions of the aligned amino acid residues in the second amino acid sequence, such that the first and second amino acid sequences may have a common domain and / or common functional activity. For example, an amino acid sequence containing a common 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., the sequence provided herein).

[0155] In describing nucleotide sequences, the term "substantially identical" as used herein means that the first nucleic acid sequence contains a sufficient or minimum number of nucleotides identical to the aligned nucleotides in the second nucleic acid sequence, such that the first and second nucleotide sequences encode polypeptides with the same functional activity, or encode the same structural polypeptide domains or the same functional polypeptide activity. For example, a nucleotide sequence containing at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference sequence (e.g., the sequence provided herein).

[0156] The term "functional variant" refers to a polypeptide that has a substantially identical amino acid sequence to the natural sequence or is encoded by a substantially identical nucleotide sequence and is capable of having one or more activities of the natural sequence.

[0157] The calculation of sequence homology or sequence identity (these terms are used interchangeably in this paper) is performed as follows.

[0158] To determine the percentage similarity between two amino acid sequences or two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). In a typical embodiment, the reference sequence used for comparison is at least 30% of its length, for example, at least 40%, 50%, 60%, or at least 70%, 80%, 90%, or 100%. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When the amino acid residues or nucleotides occupying a position in the first sequence are the same as those at the corresponding position in the second sequence, the molecules are identical at that position.

[0159] Considering the number of gaps required for optimal alignment of two sequences and the length of each gap, the percentage similarity between two sequences is related to the number of common positions in the sequences.

[0160] Sequence comparison and determination of percentage identity between two sequences can be accomplished using mathematical algorithms. In one implementation, the percentage identity between two amino acid sequences can be determined using Needleman and Wunsch (…). J. Mol. Biol. The algorithm (48):444-453 (1970) is determined and incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com). It uses a Blossum 62 matrix or a PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In some implementations, the percentage of similarity between two nucleotide sequences is calculated using the GAP program in the GCG software package (available at http: / / www.gcg.com) with an NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. A suitable set of parameters (and, unless otherwise specified, the parameter set to be used) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a decoding gap penalty of 5.

[0161] The percentage of similarity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), which has been incorporated into the ALIGN program (version 2.0) using the PAM120 weighted residue table with a nick length penalty of 12 and a nick penalty of 4.

[0162] The nucleic acid and protein sequences disclosed in this paper can also be used as "query sequences" to search public databases for purposes such as identifying other family components or related sequences. Such searches can be performed using Altschul et al., 1990. J . Mol . Biol. The NBLAST and XBLAST programs (version 2.0) of 215:403-10 can be used to run. The NBLAST program can be used for BLAST nucleotide searches with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acids described herein. The XBLAST program can be used for BLAST protein searches with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules described herein. For gap alignment results for comparative purposes, refer to Altschul et al., 1997. Nucleic Acids Res.The use of gap BLAST is described in 25:3389-3402. When using BLAST and gap BLAST procedures, the default parameters of each procedure (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0163] As used herein, the term "hybridization under low, medium, high, or very high stringency conditions" refers to the conditions for hybridization and washing. Instructions for conducting hybridization reactions can be found in [link to relevant documentation]. Current Protocols in Molecular Biology (The New Laboratory Manual of Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6), is included in this paper as a reference. This reference describes both aqueous and non-aqueous methods, which can be used. The specific hybridization conditions discussed in this paper are as follows: 1) Low-strict hybridization conditions, at approximately 45°C. In 6X sodium chloride / sodium citrate (SSC), then in 0.2X SSC, 0.1% SDS for at least 50 Perform two washes at temperature C (washing temperature can be increased to 55°C). C is suitable for low-strict hybridization conditions); 2) medium-strict hybridization conditions, at approximately 45 In approximately 6X-SSC at C, then in 0.2X SSC, 0.1% SDS (60 C) Perform one or more washes; 3) Under highly stringent hybridization conditions, at approximately 45 In approximately 6X-SSC at C, then in 0.2X SSC, 0.1% SDS (65 C) Perform one or more washes, and preferably 4) the extremely stringent hybridization conditions are 0.5M sodium phosphate, 7% SDS, 65 C, then 0.2X SSC, 1% SDS (65 C) Perform one or more washes. Extremely stringent condition 4) is the appropriate condition and should be used (unless otherwise stated).

[0164] It should be understood that the molecules described herein may have other conserved or non-essential amino acid substitutions, which do not substantially affect the molecular function.

[0165] The term "amino acid" encompasses all molecules containing both amino and acid functional groups that can be incorporated into polymers formed from naturally occurring amino acids, whether natural or synthetic. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives, and congeners; amino acid analogs having variant side chains; and any of the stereoisomers listed above. As used herein, the term "amino acid" includes D- and L-mirror isomers and peptide mimics.

[0166] "Conservative amino acid substitution" refers to the substitution of one amino acid residue by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with basic side chains (such as lysine, arginine, and histidine); amino acids with acidic side chains (such as aspartic acid and glutamic acid); amino acids with nonpolar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine); amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan); amino acids with β-branched side chains (such as threonine, valine, and isoleucine); and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0167] The terms “polypeptide,” “peptide,” and “protein” (if single-chain) are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may intercalate non-amino acids. The term also includes polymers of modified amino acids; for example, those subjected to disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation, such as coupling with a labeled component. Polypeptides may be isolated from natural sources, may be produced from a prokaryotic or eukaryotic host using recombinant techniques, or may be products of synthetic methods.

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

[0169] The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” and “polynucleotide” are used interchangeably herein. They refer to any length of nucleotide polymeric form, whether deoxyribonucleotides or ribonucleotides or their analogues. Polynucleotides can be single-stranded or double-stranded, and if single-stranded, can be coding or non-coding (antisense) strands. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogues. Non-nucleotide components may be intercalated in the nucleotide sequence. Polynucleotides may be further modified after polymerization, such as by coupling with labeled components. Nucleic acids can be recombinant polynucleotides, or genomic, cDNA, semi-synthetic, or synthetically derived polynucleotides, i.e., not naturally occurring or linked to another polynucleotide in a non-natural arrangement.

[0170] As used herein, the term "isolated" means that a substance is separated from its native or natural environment (e.g., from the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but a polynucleotide or polypeptide isolated from some or all of the coexisting substances in the natural system is isolated. Such a polynucleotide may be a part of a carrier and / or such a polynucleotide or polypeptide may be a part of a composition, but it is still isolated because the carrier or composition is not part of the natural environment in which the polynucleotide or polypeptide exists.

[0171] As used herein, the term "treating" a condition (e.g., myeloma) means, in one embodiment, that a subject (e.g., a human) with a condition (e.g., myeloma) and / or experiencing symptoms of a condition (e.g., myeloma) will experience less severe symptoms and / or recover more quickly after receiving an antibody molecule than if they had not received the antibody molecule. In one embodiment, when treating myeloma, a bone marrow biopsy will show fewer clonal plasma cells after effective treatment of the myeloma. For example, after administering the antibody molecule described herein to effectively treat myeloma, diagnostic assays will detect fewer clonal plasma cells in the subject's biological sample. Other assays, urine tests, or blood tests may also be used to monitor a patient's treatment or to detect the presence of myeloma symptoms in a subject after treatment, such as a reduction (or absence). In one embodiment, when treating myeloma, the level of β2-microglobulin (β2M) in serum or urine will decrease after effective treatment of the myeloma. Treatment may, for example, partially or completely alleviate, improve, relieve, suppress, or reduce the severity and / or incidence of a condition, and may optionally delay the onset or development of one or more consequences, symptoms, features, and / or causes of a condition (e.g., myeloma). In one embodiment, treatment is given to subjects who do not exhibit certain signs of a condition (e.g., myeloma) and / or who exhibit only early signs of a condition (e.g., kidney disease). In one embodiment, treatment is given to subjects who have one or more identified signs of a condition (e.g., myeloma). In one embodiment, treatment is given to subjects diagnosed with a disease (e.g., myeloma).

[0172] As used in this article, the term preventive disease (e.g., myeloma) means that if a subject (e.g., a person) receives antibody molecules, that subject (e.g., a person) is less likely to develop the disease (e.g., myeloma).

[0173] The various aspects of the compositions and methods described herein are further described in detail below. Other definitions are provided throughout the specification.

[0174] IL-2 preparations This 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 may have one or more of the structural and / or functional properties described herein. In one embodiment, the IL-2 formulation comprises an IL-2 variant that includes one or more amino acid changes (e.g., substitutions) described herein. In one embodiment, the IL-2 formulation comprises an IL-2 variant that includes one or more amino acid changes (e.g., substitutions) described in Table 9. In one embodiment, the IL-2 formulation comprises an IL-2 variant that includes the amino acid sequence described in Table 9, or a portion thereof. In one 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, may provide one or more desired biological properties described herein. In one embodiment, the IL-2 formulation may modulate (e.g., increase) Treg proliferation, survival, activation, and / or function. In one embodiment, the modulation is selective or specific to Tregs. For example, the IL-2 formulation may modulate activity in Tregs but has limited or no ability to promote activity in non-regulatory T cells. In one embodiment, the IL-2 formulation comprises a polypeptide (sometimes referred to herein as an "IL-2 polypeptide reagent").

[0175] IL-2 variants In one embodiment, the IL-2 formulation comprises an IL-2 variant, such as the IL-2 variant described herein.

[0176] In one embodiment, the IL-2 variant comprises the IL-2 polypeptide (e.g., human IL-2 polypeptide) described herein or a functional fragment thereof. In one embodiment, the IL-2 variant comprises one or more amino acid variations (e.g., substitutions) described in Table 9. In one embodiment, the IL-2 variant comprises or consists of the 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, for example, in Table 10.

[0177] Without being bound by theory, it is believed that in one implementation, the IL-2 variants described herein have reduced binding affinity for human CD25 and / or reduced binding affinity for human CD122 / CD132 relative to wild-type human IL-2 or a reference IL-2 variant, and may have improved binding and / or selectivity for regulatory T cell (Treg) activation 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 peptides to identify IL-2 variants with binding affinity for human CD25 and / or human CD122 / CD132 within the desired range.

[0178] In one embodiment, the IL-2 variant has one or more of the properties described herein (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more), such as properties that are different and / or improved 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 variations (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 the assays described herein): i) Altered in vitro and / or in vivo expression (e.g., enhancement or increase); ii) altered (e.g., reduced or decreased) in vitro and / or in vivo accumulations; iii) Changes (e.g., enhanced or increased) in vitro and / or in vivo stability; iv) altered (e.g., enhanced or increased) in vitro and / or in vivo half-life; v) Altered (e.g., reduced or decreased) in vivo turnover and / or clearance rates; vi) Altered (e.g., reduced or diminished) sensitivity to protein hydrolysis in vitro and / or in vivo. vii) Changes (e.g., enhanced or increased) in vitro and / or in vivo resistance to proteolysis; viii) Changes (e.g., reduced or decreased) in vitro and / or in vivo binding capacity and / or binding affinity to human CD25; (ix) altered (e.g., reduced or decreased) in vitro and / or in vivo binding capacity and / or binding affinity to human CD132; x) altered (e.g., reduced or decreased) in vitro and / or in vivo binding capacity and / or binding affinity to the dimer IL-2 receptor containing human CD122 and human CD132. xi) changes (e.g., enhanced, increased, reduced, decreased, and / or selective) in vitro and / or in vivo binding to Treg; xii) altered (e.g., enhanced, increased, reduced, decreased, and / or selective) activation of the IL-2 signaling pathway in in vitro and / or in vivo Tregs; xiii) The ability to induce or promote Treg amplification, activity, survival and / or proliferation in vitro and / or in vivo by alterations (e.g., enhancement, increase, decrease, reduction and / or selectivity).

[0179] In one embodiment, the IL-2 variant has varied (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 relative to wild-type IL-2 (e.g., in bacterial or mammalian cells). In one embodiment, the IL-2 variant has enhanced or increased expression relative to a reference IL-2 variant (e.g., in bacterial or mammalian cells). In one embodiment, the expression of the IL-2 variant is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the expression of the IL-2 variant is increased by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more. In one embodiment, the IL-2 variant is expressed in vitro and / or in vivo at higher or increased levels, for example, 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, 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 example, as determined by a protein concentration assay. In one implementation, the IL-2 variant is expressed at a higher or increased level, for example, by about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times or more, 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 example, as determined by protein concentration assay.

[0180] In one embodiment, the IL-2 variant has varying (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 aggregation of the IL-2 variant is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the aggregation of the IL-2 variant is reduced by about 0.5 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more. In one embodiment, an IL-2 formulation comprising the IL-2 variant described herein accumulates in vitro and / or in vivo at a lower or reduced level, 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, for example, 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 melting temperature analysis (e.g., using fluorescence), dynamic light scattering, and / or size exclusion chromatography. In one embodiment, IL-2 formulations containing the IL-2 variant described herein aggregate at lower or reduced levels, for example, by about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times or more, for example, relative to IL-2 formulations containing wild-type IL-2 or IL-2 formulations containing a reference IL-2 variant, for example, as determined by melting temperature analysis (e.g., using fluorescence), dynamic light scattering and / or size exclusion chromatography.

[0181] In one embodiment, the IL-2 variant has varying (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 stability of the IL-2 variant is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the stability of the IL-2 variant is increased by about 0.5 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more. In one embodiment, the IL-2 formulation comprising the IL-2 variant described herein has enhanced or increased stability in vitro and / or in vivo, for example, by an increase of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or for example, by an increase of about 0.5 times. Approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or more, 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 example, as determined by yeast surface display, circular dichroism or related spectroscopic techniques and / or melting temperature analysis (e.g., using fluorescence).

[0182] In one embodiment, the IL-2 variant has a variable (e.g., enhanced or increased) half-life relative to wild-type IL-2 or a reference IL-2 variant in vitro and / or in vivo. 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 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more. In one embodiment, an IL-2 formulation comprising the IL-2 variant described herein has an enhanced or increased half-life in vitro and / or in vivo, for example, an increase of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or For example, greater than about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times or more, 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 example, as determined by ELISA, flow cytometry and / or mass spectrometry.

[0183] In one embodiment, the IL-2 variant has a varied (e.g., reduced or decreased) turnover relative to wild-type IL-2 or a reference IL-2 variant in vitro and / or in vivo. In one embodiment, the IL-2 variant has a reduced or decreased turnover relative to wild-type IL-2. In one embodiment, the IL-2 variant has a reduced or decreased turnover relative to a reference IL-2 variant. In one embodiment, the turnover of the IL-2 variant is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the turnover of the IL-2 variant is reduced by about 0.5 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more. In one embodiment, an IL-2 formulation comprising the IL-2 variant described herein has a lower, reduced, or diminished turnover rate or level and / or clearance in vivo, 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 for example, a reduction of about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times or more, 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 example, by ELISA, flow cytometry and / or mass spectrometry.

[0184] In one embodiment, the IL-2 exhibits varying (e.g., reduced or diminished) sensitivity to proteolysis relative to wild-type IL-2 or a reference IL-2 variant in vitro and / or in vivo. In one embodiment, the IL-2 variant exhibits reduced or diminished sensitivity to proteolysis relative to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 variant exhibits reduced or diminished sensitivity to proteolysis relative to a reference IL-2 variant. In one embodiment, the IL-2 variant exhibits a reduction in sensitivity to proteolysis of approximately 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or approximately 100%, or more. In one embodiment, the IL-2 variant exhibits a reduction in sensitivity to proteolysis of approximately 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or approximately 10-fold, or more.

[0185] In one embodiment, the IL-2 variant has varying (e.g., enhanced or increased) resistance to proteolysis relative to wild-type IL-2 or a reference IL-2 variant, both in vitro and / or in vivo. 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 exhibits increased resistance to proteolysis 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 exhibits increased resistance to proteolysis by about 0.5 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more.

[0186] In one embodiment, the IL-2 variant has a varied (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 binding capacity and / or binding affinity of the IL-2 variant for human CD25 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the binding ability and / or binding affinity of the IL-2 variant to human CD25 is reduced by about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 times or more. In one embodiment, the binding affinity of an IL-2 formulation comprising the IL-2 variant described herein to CD25 (e.g., human CD25) is reduced or decreased, for example, 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, about 1 time. Approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 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, or more, for example, relative to IL-2 formulations containing wild-type IL-2 or IL-2 formulations containing a reference IL-2 variant, as determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore), and / or biolayer interferometry (e.g., Octet binding).

[0187] In one implementation, the IL-2 variant binds to CD25 (e.g., human CD25) with low affinity, for example, the dissociation constant (K). DThe range is approximately 5-500 pM, for example, approximately 5, approximately 10, approximately 15, approximately 20, approximately 25, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 105, approximately 110, approximately 115, approximately 120, approximately 125, approximately 130, approximately 135, approximately 140, approximately 145, approximately 150, approximately 200, approximately 250, approximately 300, approximately 350, approximately 400, approximately 450, or approximately 500 pM, or for example, approximately 10-approximately 400 pM, approximately 20-approximately 300 pM, approximately 50-approximately 200 pM, approximately 100-approximately 150 pM, approximately 5-approximately 10 pM, for example, about 10-20 pM, about 20-30 pM, or about 30-40 pM, for example, about 40-50 pM, about 50-60 pM, about 60-70 pM, about 70-80 pM, about 80-90 pM, about 90-100 pM, about 100-110 pM, about 110-120 pM, about 120-130 pM, about 130-140 pM, about 140-150 pM, about 150-200 pM, about 200-250 pM, about 250-300 pM, about 300-350 pM, about 350-400 pM, about 400-500 pM. pM, or for example, greater than about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 pM, determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding).

[0188] In one implementation, the IL-2 variant binds to CD25 (e.g., human CD25) with low affinity, for example, the dissociation constant (K). DThe value is approximately 0.1–10 nM, for example, 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 1.5, approximately 2, approximately 2.5, approximately 3, approximately 3.5, approximately 4, approximately 4.5, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, or approximately 10 nM, or for example, approximately 0.2–approximately 5 nM, approximately 0.5–approximately 2 nM, approximately 1–1.5 nM, approximately 0.1–approximately 0.2 nM, for example, approximately 0.2–approximately 0.3 nM, approximately 0.3–approximately 0.4 nM, or approximately 0.4–approximately 0.5 nM, for example, approximately 0.5–approximately 0.6 nM, approximately 0.6–approximately 0.7 nM, approximately 0.7–approximately 0.8 nM, approximately 0.8–approximately 0.9 nM. nM, about 0.9 to about 1 nM, about 1 to about 1.5 nM, about 1.5 to about 2 nM, about 2.5 to about 3 nM, about 3.5 to about 4 nM, about 4 to about 4.5 nM, about 4.5 to about 5 nM, about 5 to about 6 nM, about 6 to about 7 nM, about 7 to about 8 nM, about 8 to about 9 nM, or about 9 to about 10 nM, or for example, greater than about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nM, for example, determined by surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding).

[0189] In one embodiment, the IL-2 variant has a varied (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 reduced binding capacity and / or binding affinity for human CD132 by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one implementation, the IL-2 variant has a binding capacity and / or binding affinity to human CD132 that is reduced by about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 or more.

[0190] In one embodiment, the IL-2 variant has, in vitro and / or in vivo, a varied (e.g., reduced or decreased) binding capacity and / or binding affinity for human dimeric IL-2 receptors comprising human CD122 and human CD132, 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 dimeric IL-2 receptors 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 human dimeric IL-2 receptors comprising human CD122 and human CD132, relative to a reference IL-2 variant. In one embodiment, the IL-2 variant exhibits a reduced binding affinity and / or binding affinity to human dimerized IL-2 receptors comprising human CD122 and human CD132 by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In another embodiment, the IL-2 variant exhibits a reduced binding affinity and / or binding affinity to human dimerized IL-2 receptors comprising human CD122 and human CD132 by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0191] In one embodiment, the binding affinity of the IL-2 variant to CD122 / CD132 heterodimers (e.g., human CD122 / CD132 heterodimers) is reduced or decreased, for example, 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%. Approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or more, for example, relative to an IL-2 formulation containing wild-type IL-2 or an IL-2 formulation containing a reference IL-2 variant, as determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore), and / or biolayer interferometry (e.g., Octet binding).

[0192] In one implementation, the IL-2 variant binds to the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer) with low affinity, for example, the dissociation constant (K). DThe value 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, approximately 1–approximately 10 nM, approximately 2–approximately 5 nM, approximately 0.2–approximately 0.3 nM, approximately 0.3–approximately 0.4 nM, approximately 0.4–approximately 0.5 nM, approximately 0.5–approximately 0.6 nM, approximately 0.6–approximately 0.7 nM. nM, about 0.7-0.8 nM, about 0.8-0.9 nM, about 0.9-1 nM, about 1-1.1 nM, about 1.1-1.2 nM, about 1.2-1.3 nM, about 1.3-1.4 nM, about 1.4-1.5 nM, about 1.5-2 nM, about 2-3 nM, about 3-4 nM, about 4-5 nM, about 5-6 nM, about 6-7 nM, about 7-8 nM, about 8-9 nM, about 9-10 nM, about 10-11 nM, about 11-12 nM, about 12-13 nM, about 13-14 nM, about 14-15 nM, about 15-16 nM, about 16-17 nM, about 17-about 18 nM, about 18-about 19 nM, or about 19-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.

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

[0194] In one embodiment, the IL-2 variant has varying (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 implementation, the binding to Treg is increased by about 0.5 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more.

[0195] In one embodiment, the IL-2 variant has, in vitro and / or in vivo, varying (e.g., enhanced, increased, and / or selective) activation of the IL-2 signaling pathway in Tregs 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 Treg is increased by about 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more.

[0196] In one implementation, the IL-2 variant selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, for example, those with the following T helper EC50 / Treg... EC50 ratio: 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 formulation containing wild-type IL-2 or an IL-2 formulation containing a reference IL-2 variant, as determined, for example, by flow cytometry.

[0197] In one implementation, the IL-2 variant selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, for example, having an NK cell EC50 / Treg EC50 ratio greater than, for example, about 1, about 2, about 3, about 4, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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 for example, greater than 1 and about 1-2, about 2-3, about 3-4, about 4-5, greater than 1 and about 1-10, greater than 1 and about 1-20, greater than 1 and about 1-30, greater than 1 and about 1-40, greater than 1 and about 1-50, about 2-10, about 2-20, about 2-30, about 2-40, 2-50, about 5-10, about 5-20, about 5-30, about 5-40, about 5-50, about 10-20, about 10-30, about 10-40 Approximately 10-50, approximately 20-40, approximately 20-50, approximately 50-100, approximately 100-200, approximately 200-500, approximately 500-1000, approximately 1000-2000, or approximately 1000-3000, relative to an IL-2 formulation containing wild-type IL-2 or an IL-2 formulation containing a reference IL-2 variant, as determined by flow cytometry, for example.

[0198] In one embodiment, the IL-2 variant has, relative to wild-type IL-2 or a reference IL-2 variant, a variable (e.g., enhanced, increased, and / or selective) ability to induce or promote Treg amplification, activity, survival, and / or proliferation in vitro and / or in vivo. In one embodiment, the IL-2 variant has an enhanced or increased ability to induce or promote Treg amplification, 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 amplification, 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 amplification, 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 amplification, activity, survival, and / or proliferation relative to a reference IL-2 variant. In one embodiment, the ability to induce or promote Treg amplification, 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 another embodiment, the ability to induce or promote Treg amplification, 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.

[0199] In one implementation, the efficacy and / or ability of the IL-2 variant to induce or promote T regulatory cell activity is enhanced or increased, 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 Treg EC 50 It should be 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, reduced by about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times or more, for example, as determined by flow cytometry.

[0200] In one implementation, the efficacy and / or ability of the IL-2 variant to induce or promote T regulatory cell activity is reduced or diminished, for example, for Treg ECGs, 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, or 3 times lower. Approximately 3.5 times, approximately 4 times, approximately 4.5 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, or approximately 20,000 times or more, for example, as determined by flow cytometry.

[0201] In one embodiment, the T helper cells described herein are CD45+CD3+CD4+Foxp3- cells, for example, as determined by flow cytometry. In one embodiment, the Treg cells described herein are CD45+CD3+CD4+Foxp3+ cells, for example, as determined by flow cytometry. In one embodiment, the NK cells described herein are CD45+CD3- cells, which are CD56+ and / or CD16+, for example, as determined by flow cytometry. In one embodiment, the NK cells described herein are CD45+CD3-CD56+ cells, for example, as determined by flow cytometry.

[0202] In one implementation, the 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.

[0203] In one embodiment, the reference IL-2 variant comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the IL-2 variant described herein. In one embodiment, the reference IL-2 variant comprises the amino acid sequence of SEQ ID NO:1 (IL-2C125S). In one embodiment, the IL-2 variant comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 98% identity with 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 variations (e.g., substitutions) described herein.

[0204] For the purposes of this 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: MYRMQLLSCIALSLALVTNS / A1 / P2 / T3 / S4 / S5 / S6 / T7 / K8 / K9 / T10 / Q11 / L12 / Q13 / L14 / E15 / H16 / L17 / L18 / L19 / D 20 / 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 / L7 2 / 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; UniprotP60568; signal peptide) underlined The corresponding amino acid sequence without the signal peptide is shown in SEQ ID NO:1031.

[0205] In one embodiment, the IL-2 formulation contains one or more amino acid variations (e.g., substitutions) at one or more positions corresponding to human IL-2 (e.g., containing the amino acid sequence of SEQ ID NO: 1031).

[0206] In one 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 / N 33 / P34 / X35 / L36 / T37 / X38 / M39 / L40 / T41 / X42 / K43 / F44 / Y45 / M46 / P47 / K48 / K49 / A50 / T 51 / E52 / L53 / K54 / H55 / L56 / Q57 / C58 / L59 / E60 / E61 / E62 / L63 / K64 / P65 / L66 / E67 / X68 / X6 9 / L70 / N71 / L72 / A73 / X74 / S75 / K76 / N77 / F78 / H79 / L80 / R81 / P82 / R83 / X84 / L85 / I86 / X8 7 / X88 / I89 / N90 / V91 / X92 / V93 / L94 / E95 / L96 / K97 / G98 / S99 / E100 / T101 / T102 / F103 / M10 4 / C105 / E106 / Y107 / A108 / D109 / E110 / T111 / A112 / T113 / I114 / V115 / E116 / F117 / L118 / N 119 / R120 / W121 / I122 / T123 / F124 / X125 / X126 / S127 / I128 / I129 / S130 / T131 / L132 / T133 (SEQ ID NO:1032), 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 contain the amino acid sequence of SEQ ID NO: 1 or 1031. In one embodiment, the IL-2 variant comprises or consists of the following: the IL-2 variant amino acid sequence described herein.

[0207] In one embodiment, the IL-2 variant contains amino acid changes (e.g., substitutions) at one or more of the positions described herein (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all). In another embodiment, the IL-2 variant contains amino acid changes (e.g., substitutions) at one or more of the positions selected from T3, H16, I28, K35, R38, F42, E68, V69, Q74, D84, S87, N88, I92, C125, or Q126 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all).

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

[0209] In one embodiment, the IL-2 variant contains an amino acid change (e.g., substitution) at positions V69, Q74, or a combination thereof. In one embodiment, the IL-2 variant contains an amino acid change (e.g., substitution) at positions V69 and Q74. In one embodiment, the IL-2 variant contains the amino acid substitution V69A. In one embodiment, the IL-2 variant contains the amino acid substitution Q74P.

[0210] In one embodiment, the IL-2 variant contains an amino acid change (e.g., substitution) at position H16, I92, D84, or a combination thereof. In one embodiment, the IL-2 variant contains an amino acid change (e.g., substitution) at position H16, optionally wherein the amino acid substitution is H16N, H16L, or H16D. In one embodiment, the IL-2 variant contains the amino acid substitution H16N. In one embodiment, the IL-2 variant contains the amino acid substitution H16L. In one embodiment, the IL-2 variant contains the amino acid substitution H16D.

[0211] In one embodiment, the IL-2 variant contains an amino acid change (e.g., substitution) at position I92, optionally wherein the amino acid substitution is I92S. In one embodiment, the IL-2 variant contains the amino acid substitution I92S.

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

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

[0214] In one embodiment, the IL-2 variant includes an amino acid change (e.g., substitution) at position R38, optionally wherein the amino acid substitution is R38E, R38N, or R38Q. In one embodiment, the IL-2 variant includes the amino acid substitution R38N. In one embodiment, the IL-2 variant includes the amino acid substitution R38Q.

[0215] In one embodiment, the IL-2 variant includes an amino acid change (e.g., substitution) at position F42, optionally wherein the amino acid substitution is F42K or F42Q. In one embodiment, the IL-2 variant includes the amino acid substitution F42K. In one embodiment, the IL-2 variant includes the amino acid substitution F42Q.

[0216] In one 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) one, two, or all of positions H16, I92, or D84. In one embodiment, the IL-2 variant also comprises amino acid changes (e.g., substitutions) at one, two, or all of positions R38, F42, or E68.

[0217] In one embodiment, the IL-2 variant comprises 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.

[0218] In one 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) at (b) one, two, or all of positions R38, F42, or E68.

[0219] In one embodiment, the IL-2 variant includes 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 one embodiment, the IL-2 variant includes amino acid substitutions of V69A, Q74P, and H16N or H16L. In one embodiment, the IL-2 variant includes amino acid substitutions of V69A, Q74P, and H16N. In one embodiment, the IL-2 variant includes amino acid substitutions of V69A, Q74P, and H16L.

[0220] In one embodiment, the IL-2 variant includes 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 variant includes amino acid substitutions V69A, Q74P, and I92S.

[0221] In one embodiment, the IL-2 variant includes 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 variant includes amino acid substitutions V69A, Q74P, and D84V.

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

[0223] In one embodiment, the IL-2 variant includes 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 variant includes amino acid substitutions V69A, Q74P, and F42Q.

[0224] In one embodiment, the IL-2 variant includes 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 variant includes amino acid substitutions V69A, Q74P, and R38N.

[0225] In one embodiment, the IL-2 variant includes 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 one embodiment, the IL-2 variant includes amino acid substitutions V69A, Q74P, and R38E.

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

[0227] In one embodiment, the IL-2 variant includes 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 another embodiment, the IL-2 variant includes amino acid substitutions V69A, Q74P, K35E, H16N, and R38N.

[0228] In one embodiment, the IL-2 variant includes 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 one embodiment, the IL-2 variant includes amino acid substitutions of V69A, Q74P, H16N, and R38N or R38Q. In one embodiment, the IL-2 variant includes amino acid substitutions of V69A, Q74P, H16N, and R38N. In one embodiment, the IL-2 variant includes amino acid substitutions of V69A, Q74P, H16N, and R38Q.

[0229] In one embodiment, the IL-2 variant contains amino acid changes (e.g., substitutions) at positions I28, E68, S87, N88, Q126, or combinations thereof.

[0230] In one embodiment, the IL-2 variant includes an amino acid change (e.g., substitution) at position I28, optionally wherein the amino acid substitution is I28T or I28F. In one embodiment, the IL-2 variant includes the amino acid substitution I28T. In one embodiment, the IL-2 variant includes the amino acid substitution I28F.

[0231] In one embodiment, the IL-2 variant includes an amino acid change (e.g., substitution) at position E68, optionally wherein the amino acid substitution is E68Q or E68N. In one embodiment, the IL-2 variant includes the amino acid substitution E68Q. In one embodiment, the IL-2 variant includes the amino acid substitution E68N.

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

[0233] In one embodiment, the IL-2 variant includes an amino acid change (e.g., substitution) at position N88, optionally wherein the amino acid substitution is N88S, N88L, or N88D. In one embodiment, the IL-2 variant includes amino acid substitution N88S. In one embodiment, the IL-2 variant includes amino acid substitution N88L. In one embodiment, the IL-2 variant includes amino acid substitution N88D.

[0234] In one embodiment, the IL-2 variant includes an amino acid change (e.g., substitution) at position Q126, optionally wherein the amino acid substitution is Q126T, Q126K, or Q126R. In one embodiment, the IL-2 variant includes an amino acid substitution of Q126T, Q126K, or Q126R. In one embodiment, the IL-2 variant includes an amino acid substitution of Q126T. In one embodiment, the IL-2 variant includes an amino acid substitution of Q126K. In one embodiment, the IL-2 variant includes an amino acid substitution of Q126R.

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

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

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

[0238] In one embodiment, the IL-2 variant contains 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.

[0239] In one embodiment, the IL-2 variant includes amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125, optionally wherein the amino acid substitutions are H16N, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 variant includes amino acid substitutions H16N, V69A, Q74P, and C125S.

[0240] In one embodiment, the IL-2 variant includes amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 variant includes amino acid substitutions H16L, V69A, Q74P, and C125S.

[0241] In one embodiment, the IL-2 variant includes 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 another embodiment, the IL-2 variant includes amino acid substitutions H16L, V69A, Q74P, I92S, and C125S.

[0242] In one embodiment, the IL-2 variant includes 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 variant includes amino acid substitutions T3A, V69A, Q74P, and C125S.

[0243] In one embodiment, the IL-2 variant includes 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 variant includes amino acid substitutions of T3A, H16N, V69A, Q74P, and C125S. In another embodiment, the IL-2 variant includes amino acid substitutions of T3A, H16L, V69A, Q74P, and C125S.

[0244] In one embodiment, the IL-2 variant includes 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 another embodiment, the IL-2 variant includes amino acid substitutions of T3A, V69A, Q74P, I92S, and C125S. In yet another embodiment, the IL-2 variant includes amino acid substitutions of T3A, V69A, Q74P, I92S, and C125S.

[0245] In one embodiment, the IL-2 variant includes 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 one embodiment, the IL-2 variant includes amino acid substitutions H16L, K35E, V69A, and Q74P.

[0246] In one embodiment, the IL-2 variant includes 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 one embodiment, the IL-2 variant includes amino acid substitutions H16L, R38Q, V69A, and Q74P.

[0247] In one embodiment, the IL-2 variant comprises amino acid substitutions for H16L, V69A, Q74P, and C125S. In another embodiment, the IL-2 variant comprises amino acid substitutions for H16N, V69A, Q74P, and C125S.

[0248] Various technical effects exist associated with the presence of the specific mutant sets described herein, for example, a set of mutations comprising an amino acid substitution at position H16, combined with amino acid substitutions at positions V69, Q74, and C125 (e.g., H16L, V69A, Q74P, and C125S). Without wishing to be bound by theory, it is believed that in one embodiment, the IL-2 variant containing the above-described mutations has a reduced binding affinity for CD122 and / or CD132, which increases the efficacy and selectivity of the IL-2 variant for regulatory T cells (Tregs) (compared to other T cell types). Without wishing to be bound by theory, it is also believed that in one embodiment, the IL-2 variant containing the above-described mutations is significantly stable, for example, attributable to the presence of stabilizing V69A and Q74P mutations. For example, it was unexpectedly found that 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. Therefore, IL-2 variants containing these mutations can selectively activate and significantly stabilize regulatory T cells (Tregs). Not wishing to be bound by theory, it is further believed that in one embodiment, IL-2 variants containing the aforementioned mutations have reduced or decreased binding affinity to CD25, which improves the lifespan of the IL-2 variant. Not wishing to be bound by theory, it is also believed that in one embodiment, IL-2 variants containing these mutations substantially do not promote the in vitro and / or in vivo expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells. Not wishing to be bound by theory, it is also believed that in one embodiment, IL-2 variants containing the aforementioned mutations have reduced incorrect disulfide pairing and improved stability, for example, attributable to the presence of the C125S mutation. In one embodiment, compared to IL-2 formulations containing other H16 mutations, IL-2 formulations containing the H16L mutation have reduced binding affinity to CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types. These properties make IL-2 variants containing these mutations particularly suitable for treating diseases or conditions caused by abnormal immune responses, such as autoimmune diseases.

[0249] Therefore, in one embodiment, an IL-2 variant (e.g., an IL-2 variant or IL-2 fusion protein) comprising a combination of amino acid substitutions at position H16 and at positions V69, Q74, and C125 (e.g., H16L, V69A, Q74P, and C125S) has, in particular, one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, or all of them) relative to wild-type IL-2 or a reference IL-2 variant that does not contain the aforementioned amino acid substitutions. (iii) Reduced or decreased binding capacity and / or binding affinity to human CD122 in vitro and / or in vivo; (iv) Reduced or decreased affinity of IL-2 variants in vitro and / or in vivo for heterodimeric IL-2 receptors composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer); (v) Reduced or decreased binding capacity and / or binding affinity of IL-2 variants in vitro and / or in vivo for human CD25; (vi) Selective binding to regulatory T cells (e.g., Foxp3). + (vii) selectively activate the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhance or increase the ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0250] In one embodiment, the IL-2 variant comprises or consists of an amino acid sequence selected from the following: 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: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:3 ... 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 amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence similarity, or amino acid sequences having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids similarity.

[0251] In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequences: 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 with it, or an amino acid sequence having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids similar to it. In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequences: 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 with it, or an amino acid sequence having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids similar to it. In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequences: 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 with it, or an amino acid sequence having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids similar to it. In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequences: 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 with it, or an amino acid sequence having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids similar to it. In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequences: 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 with it, or an amino acid sequence having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids similar to it.In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequences: the amino acid sequence of SEQ ID NO:1002, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it, or an amino acid sequence having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids similar to it.

[0252] In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequences: the amino acid sequence of any one of SEQ ID NO:4, 5, 11, 1000, 1001, or 1002, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequences: the amino acid sequence of SEQ ID NO:4 or 5, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:4, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:5, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:11, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:1000, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:1001, or a functional fragment thereof. In one embodiment, the IL-2 variant comprises or consists of the following amino acid sequence: the amino acid sequence of SEQ ID NO:1002 or a functional fragment thereof.

[0253] Without being bound by theory, it is believed that in one embodiment, an IL-2 variant comprising the amino acid sequence of SEQ ID NO:5 or a functional fragment thereof, or composed thereof, may have at least one 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 stability, for example, attributable to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity for CD25, which prolongs the lifespan of the IL-2 formulation; (iv) substantially does not promote the expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) reduced incorrect disulfide pairing and increased stability, for example, attributable to the presence of C125S mutations. In one implementation, compared to IL-2 agents containing other H16 mutations, IL-2 agents containing the H16L mutation exhibit reduced binding affinity for CD122 and / or CD132 and / or higher efficacy and selectivity for Tregs than other T cell types. These properties make IL-2 variants containing the amino acid sequence of SEQ ID NO:5 or composed thereof particularly suitable for treating diseases and conditions caused by abnormal immune responses, such as autoimmune diseases.

[0254] Therefore, in one embodiment, an IL-2 variant comprising or consisting of the following amino acid sequences: amino acid sequence SEQ ID NO:5 or a functional fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it, or an amino acid sequence having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids similar to it, relative to wild-type IL-2 or a reference IL-2 variant not containing the said amino acid substitutions, particularly having one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7 or all of them): (i) enhanced or increased in vitro (ii) Decreased or reduced binding capacity and / or binding affinity to human CD122 in vitro and / or in vivo; (iii) Decreased or reduced binding capacity and / or binding affinity to human CD132 in vitro and / or in vivo; (iv) Decreased or reduced affinity of IL-2 variants for heterodimeric IL-2 receptors composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) Decreased or reduced or substantially unchanged binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo; (vi) Selective binding to regulatory T cells (e.g., Foxp3).+ (vii) selectively activate the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhance or increase the ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0255] As further described herein, this disclosure provides IL-2 fusion proteins, IL-2 complexes, and IL-2 conjugates comprising IL-2 variants described herein. In one embodiment, one or more different and / or improved properties attributable to the IL-2 variants described herein are maintained, transferred, or conferred upon the IL-2 fusion protein, IL-2 complex, or IL-2. For the purposes of this disclosure, the terms “IL-2 variant” and “IL-2 mutant protein” are used interchangeably herein.

[0256] In one embodiment, the IL-2 variant comprises a polypeptide (sometimes referred to herein as an "IL-2 variant polypeptide"). This disclosure provides isolated nucleic acid molecules encoding the IL-2 variants described herein, as well as their vectors and host cells. The nucleic acid molecules include, but are not limited to, RNA, genomic DNA, and cDNA.

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

[0258] In one embodiment, the IL-2 fusion protein comprises an IL-2 variant, such as the IL-2 variants described herein. In one embodiment, the IL-2 fusion protein comprises one or more amino acid variations (e.g., substitutions) described in Table 9. In one embodiment, the IL-2 fusion protein comprises the 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, for example, in Table 10.

[0259] 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 IL-2 fusion proteins containing wild-type human IL-2 or reference IL-2 fusion proteins, may have improved efficacy and / or selectivity for binding to and activating regulatory T cells (Tregs) compared to IL-2 fusion proteins containing wild-type human IL-2 or other IL-2 fusion proteins.

[0260] 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, such as different and / or improved properties relative to an IL-2 fusion protein containing 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 alterations (e.g., substitutions) capable of providing different and / or improved properties relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all) of the following different and / or improved properties (e.g., as determined by the tests described herein) relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein: i) Altered in vitro and / or in vivo expression (e.g., enhancement or increase); ii) altered (e.g., reduced or decreased) in vitro and / or in vivo accumulations; iii) Changes (e.g., enhanced or increased) in vitro and / or in vivo stability; iv) altered (e.g., enhanced or increased) in vitro and / or in vivo half-life; v) Altered (e.g., reduced or decreased) in vivo turnover and / or clearance rates; vi) Altered (e.g., reduced or diminished) sensitivity to protein hydrolysis in vitro and / or in vivo. vii) Changes (e.g., enhanced or increased) in vitro and / or in vivo resistance to proteolysis; viii) Changes (e.g., reduced or decreased) in vitro and / or in vivo binding capacity and / or binding affinity to human CD25; (ix) altered (e.g., reduced or decreased) in vitro and / or in vivo binding capacity and / or binding affinity to human CD132; x) altered (e.g., reduced or decreased) in vitro and / or in vivo binding capacity and / or binding affinity to the dimer IL-2 receptor containing human CD122 and human CD132. xi) changes (e.g., enhanced, increased, reduced, decreased, and / or selective) in vitro and / or in vivo binding to Treg; xii) altered (e.g., enhanced, increased, reduced, and / or selective) activation of the IL-2 signaling pathway in in vitro and / or in vivo Tregs; or xiii) The ability to induce or promote Treg amplification, activity, survival and / or proliferation in vitro and / or in vivo by alterations (e.g., enhancement, increase, decrease, reduction and / or selectivity).

[0261] In one embodiment, the IL-2 fusion protein has varied (e.g., enhanced or increased) expression in vitro and / or in vivo relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has enhanced or increased expression (e.g., in bacterial or mammalian cells) relative to an IL-2 fusion protein containing 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 in vitro and / or in vivo at higher or increased levels, for example, 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, for example, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, as determined by protein concentration assay. In one embodiment, the IL-2 fusion protein is expressed at a higher or increased level, for example, by about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times or more, for example, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, as determined, for example, by protein concentration assay.

[0262] In one embodiment, the IL-2 fusion protein exhibits varied (e.g., reduced or decreased) aggregation in vitro and / or in vivo relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein exhibits reduced or decreased aggregation relative to wild-type IL-2. In one embodiment, the IL-2 fusion protein exhibits 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 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more. In one embodiment, the IL-2 fusion protein aggregates in vitro and / or in vivo at a lower or reduced level, 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, for example, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, 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 reduced level, for example, by about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times or more, for example, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, as determined by, for example, melting temperature analysis (e.g., using fluorescence), dynamic light scattering and / or size exclusion chromatography.

[0263] In one embodiment, the IL-2 fusion protein exhibits varying (e.g., enhanced or increased) stability in vitro and / or in vivo relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein exhibits enhanced or increased stability relative to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein exhibits enhanced or increased stability relative to a reference IL-2 fusion protein. In one embodiment, the stability of the IL-2 fusion protein is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the stability of the IL-2 fusion protein is increased by about 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more. In one embodiment, the IL-2 fusion protein exhibits enhanced or increased stability in vitro and / or in vivo, for example, by an increase of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more, or for example, by an increase of about 0.5 times, about 1 time. Approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 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 or more, for example, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, as determined by yeast surface display, circular dichroism or related spectroscopy and / or melting temperature analysis (e.g., using fluorescence).

[0264] In one embodiment, the IL-2 fusion protein has a variable (e.g., enhanced or increased) half-life in vitro and / or in vivo relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has an enhanced or increased half-life relative to an IL-2 fusion protein containing 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 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more. In one embodiment, the IL-2 fusion protein has an enhanced or increased half-life in vitro and / or in vivo, for example, an increase of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or for example, Greater than approximately 0.5 times, approximately 1 time, approximately 1.5 times, approximately 2 times, approximately 2.5 times, approximately 3 times, approximately 3.5 times, approximately 4 times, approximately 4.5 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 or more, for example, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, as determined by ELISA, flow cytometry and / or mass spectrometry.

[0265] In one embodiment, the IL-2 fusion protein has a varied (e.g., reduced or decreased) turnover in vitro and / or in vivo relative to an IL-2 fusion protein containing 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 a reference IL-2 fusion protein. In one embodiment, the turnover of the IL-2 fusion protein is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the turnover of the IL-2 fusion protein is reduced by about 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more. In one embodiment, the IL-2 fusion protein has a lower, reduced, or diminished turnover rate or level and / or in vivo clearance rate, for example, 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. More, or for example, reduced by about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times or more, for example, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, by ELISA, flow cytometry and / or mass spectrometry.

[0266] In one embodiment, the IL-2 fusion protein provided in this disclosure comprises, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, a property that exhibits varying (e.g., reduced or decreased) proteolytic sensitivity in vitro and / or in vivo. In one embodiment, the IL-2 fusion protein exhibits reduced or decreased proteolytic sensitivity relative to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 fusion protein has reduced or decreased proteolytic sensitivity relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein exhibits a reduction in proteolytic sensitivity of 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 exhibits a reduction in proteolytic sensitivity of about 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times, or more.

[0267] In one embodiment, the IL-2 fusion protein has varying (e.g., enhanced or increased) proteolytic resistance in vitro and / or in vivo, relative to an IL-2 fusion protein containing 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 containing 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 exhibits increased resistance to proteolysis 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 exhibits increased resistance to proteolysis by about 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more.

[0268] In one embodiment, the IL-2 fusion protein, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, has varied (e.g., reduced or decreased) binding capacity and / or binding affinity for human CD25 in vitro and / or in vivo. In one embodiment, the IL-2 fusion protein has 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 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's binding capacity and / or binding affinity for human CD25 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the binding ability and / or binding affinity of the IL-2 fusion protein to human CD25 is reduced by about 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more. In one embodiment, the binding affinity of the IL-2 fusion protein to CD25 (e.g., human CD25) is reduced or decreased, for example, 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 times, about 1 time, or about 10 times. 0.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times, or more, for example, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore), and / or biolayer interferometry (e.g., Octet binding).

[0269] In one implementation, the IL-2 fusion protein binds to CD25 (e.g., human CD25) with low affinity, for example, at a dissociation constant (K). DThe pM range is approximately 5-500 pM, for example, approximately 5, approximately 10, approximately 15, approximately 20, approximately 25, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 105, approximately 110, approximately 115, approximately 120, approximately 125, approximately 130, approximately 135, approximately 140, approximately 145, approximately 150, approximately 200, approximately 250, approximately 300, approximately 350, approximately 400, approximately 450, or approximately 500 pM, or for example, approximately 10-approximately 400 pM, approximately 20-approximately 300 pM, approximately 50-approximately 200 pM, approximately 100-approximately 150 pM. pM, approximately 5-10 pM, for example, approximately 10-20 pM, approximately 20-30 pM, or approximately 30-40 pM, for example, approximately 40-50 pM, approximately 50-60 pM, approximately 60-70 pM, approximately 70-80 pM, approximately 80-90 pM, approximately 90-100 pM, approximately 100-110 pM, approximately 110-120 pM, approximately 120-130 pM, approximately 130-140 pM, approximately 140-150 pM, approximately 150-200 pM, approximately 200-250 pM, approximately 250-300 pM, approximately 300-350 pM, approximately 350-400 pM, approximately 400-500 pM pM, or for example, greater than about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 pM, determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding).

[0270] In one implementation, the IL-2 fusion protein binds to CD25 (e.g., human CD25) with low affinity, for example, at a dissociation constant (K). DThe value is approximately 0.1–10 nM, for example, 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 1.5, approximately 2, approximately 2.5, approximately 3, approximately 3.5, approximately 4, approximately 4.5, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, or approximately 10 nM, or for example, approximately 0.2–approximately 5 nM, approximately 0.5–approximately 2 nM, approximately 1–1.5 nM, approximately 0.1–approximately 0.2 nM, for example, approximately 0.2–approximately 0.3 nM, approximately 0.3–approximately 0.4 nM, or approximately 0.4–approximately 0.5 nM, for example, approximately 0.5–approximately 0.6 nM, approximately 0.6–approximately 0.7 nM, approximately 0.7–approximately 0.8 nM, approximately 0.8–approximately 0.9 nM. nM, about 0.9 to about 1 nM, about 1 to about 1.5 nM, about 1.5 to about 2 nM, about 2.5 to about 3 nM, about 3.5 to about 4 nM, about 4 to about 4.5 nM, about 4.5 to about 5 nM, about 5 to about 6 nM, about 6 to about 7 nM, about 7 to about 8 nM, about 8 to about 9 nM, or about 9 to about 10 nM, or for example, greater than about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nM, for example, determined by surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding).

[0271] In one embodiment, the IL-2 fusion protein has varying (e.g., reduced or decreased) binding capacity and / or affinity for human CD132 in vitro and / or in vivo, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has reduced or decreased binding capacity and / or affinity for human CD132, relative to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has reduced or decreased binding capacity and / or affinity for human CD132, relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein's binding capacity and / or affinity for human CD132 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In one embodiment, the binding ability and / or binding affinity of the IL-2 fusion protein to human CD132 is reduced by about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 or more.

[0272] In one embodiment, the IL-2 fusion protein, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, has varied (e.g., reduced or decreased) binding capacity and / or binding affinity for human dimeric IL-2 receptors containing human CD122 and human CD132 in vitro and / or in vivo. In one embodiment, the IL-2 fusion protein has reduced or decreased binding capacity and / or binding affinity for human dimeric IL-2 receptors containing human CD122 and human CD132, relative to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has reduced or decreased binding capacity and / or binding affinity for human dimeric IL-2 receptors containing human CD122 and human CD132, relative to a reference IL-2 fusion protein. In one embodiment, the binding affinity and / or binding affinity of the IL-2 fusion protein to the human dimeric IL-2 receptor comprising human CD122 and human CD132 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% or more. In another embodiment, the binding affinity and / or binding affinity of the IL-2 fusion protein to the human dimeric IL-2 receptor comprising human CD122 and human CD132 is reduced by about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or about 10-fold or more.

[0273] In one embodiment, the IL-2 fusion protein has varying (e.g., enhanced, increased, and / or selective) binding to Tregs in vitro and / or in vivo, relative to an IL-2 fusion protein containing 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 containing 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 implementation, the binding to Treg is increased by about 0.5 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more.

[0274] In one embodiment, the binding affinity of the IL-2 fusion protein to the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer) is reduced or decreased, for example, 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 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times, or more, for example, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore), and / or biolayer interferometry (e.g., Octet binding).

[0275] 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). DThe value 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, approximately 1–approximately 10 nM, approximately 2–approximately 5 nM, approximately 0.2–approximately 0.3 nM, approximately 0.3–approximately 0.4 nM, approximately 0.4–approximately 0.5 nM, approximately 0.5–approximately 0.6 nM, approximately 0.6–approximately 0.7 nM. nM, about 0.7-0.8 nM, about 0.8-0.9 nM, about 0.9-1 nM, about 1-1.1 nM, about 1.1-1.2 nM, about 1.2-1.3 nM, about 1.3-1.4 nM, about 1.4-1.5 nM, about 1.5-2 nM, about 2-3 nM, about 3-4 nM, about 4-5 nM, about 5-6 nM, about 6-7 nM, about 7-8 nM, about 8-9 nM, about 9-10 nM, about 10-11 nM, about 11-12 nM, about 12-13 nM, about 13-14 nM, about 14-15 nM, about 15-16 nM, about 16-17 nM, about 17-about 18 nM, about 18-about 19 nM, or about 19-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.

[0276] 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). DThe range is approximately 0.2-300 nM, for example, approximately 0.2 nM, approximately 0.5 nM, approximately 1 nM, approximately 2 nM, approximately 5 nM, approximately 10 nM, approximately 15 nM, approximately 20 nM, approximately 25 nM, approximately 30 nM, approximately 40 nM, approximately 50 nM, approximately 60 nM, approximately 70 nM, approximately 80 nM, approximately 90 nM, approximately 100 nM, approximately 110 nM, approximately 120 nM, approximately 130 nM, approximately 140 nM, approximately 150 nM, approximately 160 nM, approximately 170 nM, approximately 180 nM, approximately 190 nM, approximately 200 nM, approximately 210 nM, approximately 220 nM, approximately 230 nM, approximately 240 nM, approximately 250 nM, approximately 260 nM, approximately 270 nM, approximately 280 nM. nM, approximately 290 nM, or approximately 300 nM, or for example, approximately 0.5-approximately 15 nM, approximately 1-approximately 10 nM, approximately 2-approximately 5 nM, approximately 0.2 nM-approximately 0.5 nM, approximately 0.5 nM-approximately 1 nM, approximately 1-approximately 2 nM, approximately 2 nM-approximately 5 nM, approximately 5 nM-approximately 10 nM, approximately 10 nM-approximately 15 nM, approximately 15 nM-approximately 20 nM, approximately 20 nM-approximately 25 nM, approximately 25-approximately 30 nM, approximately 30 nM-approximately 40 nM, approximately 40 nM-approximately 50 nM, approximately 50-approximately 60 nM, approximately 60-approximately 70 nM, approximately 70 nM-approximately 80 nM, approximately 80 nM-approximately 90 nM, approximately 90 nM-approximately 100 nM, approximately 100 nM-approximately 110 nM nM, approximately 110 nM - approximately 120 nM, approximately 120 nM - approximately 130 nM, approximately 130 nM - approximately 140 nM, approximately 140 nM - approximately 150 nM, approximately 150 nM - approximately 160 nM, approximately 160 nM - approximately 170 nM, approximately 170 nM - approximately 180 nM, approximately 180 nM - approximately 190 nM, approximately 190 nM - approximately 200 nM, approximately 200 nM - approximately 210 nM, approximately 210 nM - approximately 220 nM, approximately 220 nM - approximately 230 nM, approximately 230 nM - approximately 240 nM, approximately 240 nM - approximately 250 nM, approximately 250 nM - approximately 260 nM, approximately 260 nM - approximately 270 nM, approximately 270 nM - approximately 280 nM, approximately 280 nM - approximately 290 nM, or approximately 290 nM - approximately 300 nM, or for example, greater than approximately 0.2, approximately 0.5, approximately 1, approximately 2, approximately 5, approximately 10, approximately 15, approximately 20 nM, approximately 25 nM, approximately 30 nM, approximately 40 nM, approximately 50 nM, approximately 60 nM, approximately 70 nM, approximately 80 nM, approximately 90 nM, approximately 100 nM, approximately 110 nM, approximately 120 nM, approximately 130 nM, approximately 140 nM, approximately 150 nM, approximately 160 nM, approximately 170 nM, approximately 180 nM, approximately 190 nM, approximately 200 nM, approximately 210 nM, approximately 220 nM, approximately 230 nM, approximately 240 nM, approximately 250 nM, approximately 260 nM, approximately 270 nM, approximately 280 nM, approximately 290 nM, or greater than approximately 300 nM, for example, is determined by surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding).

[0277] In one embodiment, the IL-2 fusion protein has varying (e.g., enhanced, increased, and / or selective) binding to Tregs in vitro and / or in vivo, relative to an IL-2 fusion protein containing 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 containing 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 implementation, the binding to Treg is increased by about 0.5 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or about 10 times or more.

[0278] In one embodiment, the IL-2 fusion protein, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, exhibits altered (e.g., enhanced, increased, and / or selective) activation of the IL-2 signaling pathway in Tregs in vitro and / or in vivo. In one embodiment, the IL-2 fusion protein exhibits enhanced or increased activation of the IL-2 signaling pathway in Tregs relative to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein exhibits selective activation of the IL-2 signaling pathway in Tregs relative to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein exhibits enhanced or increased activation of the IL-2 signaling pathway in Tregs relative to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein exhibits selective activation of the IL-2 signaling pathway in Tregs relative to a reference IL-2 fusion protein. In one embodiment, the activation of the IL-2 signaling pathway in Treg is increased by approximately 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or approximately 100%, or more. In another embodiment, the activation of the IL-2 signaling pathway in Treg is increased by approximately 0.5 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or approximately 10 times or more.

[0279] In one embodiment, the IL-2 fusion protein selectively activates IL-2 signal transduction in T regulatory cells in vitro and / or in vivo, for example, having the following T helper EC50 / Treg... EC50 ratio: 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 containing wild-type IL-2 or a reference IL-2 fusion protein, for example, determined by flow cytometry.

[0280] In one embodiment, the IL-2 fusion protein selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, for example, having an NK cell EC50 / Treg EC50 ratio greater than, for example, about 1, about 2, about 3, about 4, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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 for example, greater than 1 and about 1-2, about 2-3, about 3-4, about 4-5, greater than 1 and about 1-10, greater than 1 and about 1-20, greater than 1 and about 1-30, greater than 1 and about 1-40, greater than 1 and about 1-50, about 2-10, about 2-20, about 2-30, about 2-40, 2-50, about 5-10, about 5-20, about 5-30, about 5-40, about 5-50, about 10-20, about 10-30, about 10-40 Approximately 10-50, approximately 20-40, approximately 20-50, approximately 50-100, approximately 100-200, approximately 200-500, approximately 500-1000, approximately 1000-2000, or approximately 1000-3000, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, as determined by flow cytometry.

[0281] In one embodiment, the IL-2 fusion protein, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, has a variable (e.g., enhanced, increased, and / or selective) ability to induce or promote Treg amplification, activity, survival, and / or proliferation in vitro and / or in vivo. In one embodiment, the IL-2 fusion protein has an enhanced or increased ability to induce or promote Treg amplification, activity, survival, and / or proliferation relative to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has a selective ability to induce or promote Treg amplification, activity, survival, and / or proliferation relative to 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 amplification, activity, survival, and / or proliferation relative to a reference IL-2 fusion protein. In one embodiment, the ability to induce or promote Treg amplification, 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 another embodiment, the ability to induce or promote Treg amplification, 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.

[0282] In one embodiment, the efficacy and / or ability of the IL-2 fusion protein to induce or promote T-regulatory cell activity is enhanced or increased, for example, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for Treg EC 50 It should be 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, reduced by about 0.5 times, about 1 time, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about 10 times or more, for example, as determined by flow cytometry.

[0283] In one embodiment, the efficacy and / or ability of the IL-2 fusion protein to induce or promote T-regulatory cell activity is reduced or diminished, for example, for Treg ECMO cells, relative to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein. 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, or 3 times lower. Approximately 3.5 times, approximately 4 times, approximately 4.5 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, or approximately 20,000 times or more, for example, as determined by flow cytometry.

[0284] In one embodiment, the T helper cells described herein are CD45+CD3+CD4+Foxp3- cells, for example, as determined by flow cytometry. In one embodiment, the Treg cells described herein are CD45+CD3+CD4+Foxp3+ cells, for example, as determined by flow cytometry. In one embodiment, the NK cells described herein are CD45+CD3- cells, which are CD56+ and / or CD16+, for example, as determined by flow cytometry. In one embodiment, the NK cells described herein are CD45+CD3-CD56+ cells, for example, as determined by flow cytometry.

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

[0286] In one embodiment, the reference IL-2 fusion protein comprises an amino acid sequence having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the IL-2 fusion protein described herein. In one embodiment, the reference IL-2 fusion protein comprises an IL-2 variant comprising the amino acid sequence of SEQ ID NO:57. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 98% identity with 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 variations (e.g., substitutions) described herein.

[0287] In one embodiment, the IL-2 fusion protein comprises the IL-2 polypeptide described herein (e.g., human IL-2 polypeptide). In one embodiment, the IL-2 fusion protein is encoded by a nucleic acid comprising the nucleotide sequence described herein.

[0288] In one embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at one or more of the IL-2 molecules selected herein (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all of them). In another embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at one or more of the IL-2 molecules selected from T3, H16, I28, K35, R38, F42, E68, V69, Q74, D84, S87, N88, I92, C125, or Q126 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all of them).

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

[0290] In one embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at positions V69, Q74, or both in IL-2. In one embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at positions V69 and Q74 in IL-2. In one embodiment, the IL-2 fusion protein contains an amino acid substitution V69A in IL-2. In one embodiment, the IL-2 fusion protein contains an amino acid substitution Q74P in IL-2.

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

[0292] In one embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at position I92 in IL-2, optionally wherein the amino acid substitution is I92S. In one embodiment, the IL-2 fusion protein contains the amino acid substitution I92S in IL-2.

[0293] In one embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at position D84 in IL-2, optionally wherein the amino acid substitution is D84V. In one embodiment, the IL-2 fusion protein contains an amino acid substitution in IL-2 of D84V.

[0294] In one embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at positions K35, R38, F42, E68, or combinations thereof in IL-2. In one embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at position K35 in IL-2, optionally wherein the amino acid substitution is K35E. In one embodiment, the IL-2 fusion protein contains the amino acid substitution K35E in IL-2.

[0295] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein contains the amino acid substitution R38N in IL-2. In one embodiment, the IL-2 fusion protein contains the amino acid substitution R38Q in IL-2.

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

[0297] In one embodiment, the IL-2 fusion protein comprises the following amino acid changes (e.g., substitutions): (i) at (a) positions V69 and Q74, (b) at position K35, or (c) at positions V69, Q74, and K35; and (ii) at one, two, or all of positions H16, I92, or D84. In one embodiment, the IL-2 fusion protein also comprises amino acid changes (e.g., substitutions) at one, two, or all of positions R38, F42, or E68 in IL-2.

[0298] In one embodiment, the IL-2 fusion protein comprises the following amino acid variations (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.

[0299] In one embodiment, the IL-2 fusion protein comprises the following amino acid variations (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 (a) positions H16, I92, or D84; and (b) at one, two, or all of positions R38, F42, or E68.

[0300] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein comprises amino acid substitutions of V69A, Q74P, and H16N or H16L in IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions of V69A, Q74P, and H16N in IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions of V69A, Q74P, and H16L in IL-2.

[0301] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and I92S in IL-2.

[0302] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and D84V in IL-2.

[0303] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and R38Q in IL-2.

[0304] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and F42Q in IL-2.

[0305] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and R38N in IL-2.

[0306] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and R38E in IL-2.

[0307] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, K35E, and H16N in IL-2.

[0308] In one embodiment, the IL-2 fusion protein contains 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 another embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, K35E, H16N, and R38N in IL-2.

[0309] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein comprises amino acid substitutions of V69A, Q74P, H16N, and R38N or R38Q in IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions of V69A, Q74P, H16N, and R38N in IL-2. In one embodiment, the IL-2 fusion protein comprises amino acid substitutions of V69A, Q74P, H16N, and R38Q in IL-2.

[0310] In one embodiment, the IL-2 fusion protein contains amino acid changes (e.g., substitutions) at positions I28, E68, S87, N88, Q126, or combinations thereof in IL-2.

[0311] In one embodiment, the IL-2 fusion protein includes an amino acid change (e.g., substitution) at position I28 in IL-2, optionally wherein the amino acid substitution is I28T or I28F. In one embodiment, the IL-2 fusion protein includes the amino acid substitution I28T in IL-2. In one embodiment, the IL-2 fusion protein includes the amino acid substitution I28F in IL-2.

[0312] In one embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at position E68 in IL-2, optionally wherein the amino acid substitution is E68Q or E68N. In one embodiment, the IL-2 fusion protein contains the amino acid substitution E68Q in IL-2. In one embodiment, the IL-2 fusion protein contains the amino acid substitution E68N in IL-2.

[0313] In one embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at position S87 in IL-2, optionally wherein the amino acid substitution is S87R. In one embodiment, the IL-2 fusion protein contains the amino acid substitution S87R in IL-2.

[0314] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein contains the amino acid substitution N88S in IL-2. In one embodiment, the IL-2 fusion protein contains the amino acid substitution N88L in IL-2. In one embodiment, the IL-2 fusion protein contains the amino acid substitution N88D in IL-2.

[0315] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein contains the amino acid substitution Q126T, Q126K, or Q126R in IL-2. In one embodiment, the IL-2 fusion protein contains the amino acid substitution Q126T in IL-2. In one embodiment, the IL-2 fusion protein contains the amino acid substitution Q126K in IL-2. In one embodiment, the IL-2 fusion protein contains the amino acid substitution Q126R in IL-2.

[0316] In one embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at position C125 in IL-2, optionally wherein the amino acid substitution is C125S. In one embodiment, the IL-2 fusion protein contains the amino acid substitution C125S in IL-2.

[0317] In one embodiment, the IL-2 fusion protein contains an amino acid change (e.g., substitution) at position T3 in IL-2, optionally wherein the amino acid substitution is T3A. In one embodiment, the IL-2 fusion protein contains the amino acid substitution T3A in IL-2.

[0318] In one embodiment, the IL-2 fusion protein contains amino acid changes (e.g., substitutions) at positions V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 fusion protein comprises the amino acid substitutions V69A, Q74P, and C125S in IL-2.

[0319] In one embodiment, the IL-2 fusion protein contains amino acid changes (e.g., substitutions) at positions T3, H16, I92, or combinations thereof in IL-2, optionally wherein the amino acid substitutions are T3A, H16N, and I92S in IL-2, respectively.

[0320] In one embodiment, the IL-2 fusion protein contains amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are H16N, V69A, Q74P, and C125S in IL-2, respectively. In one embodiment, the IL-2 fusion protein comprises the amino acid substitutions H16N, V69A, Q74P, and C125S in IL-2.

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

[0322] In one embodiment, the IL-2 fusion protein contains amino acid changes (e.g., substitutions) at positions H16, V69, Q74, I92, and C125 in IL-2, optionally wherein the amino acid substitutions are H16L, V69A, Q74P, I92S, and C125S in IL-2, respectively. In another embodiment, the IL-2 fusion protein comprises the amino acid substitutions H16L, V69A, Q74P, I92S, and C125S in IL-2.

[0323] In one embodiment, the IL-2 fusion protein contains amino acid changes (e.g., substitutions) at positions T3, V69, Q74, and C125 in IL-2, optionally wherein the amino acid substitutions are T3A, V69A, Q74P, and C125S in IL-2, respectively. In one embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, V69A, Q74P, and C125S in IL-2.

[0324] In one embodiment, the IL-2 fusion protein contains 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 one embodiment, the IL-2 fusion protein contains amino acid substitutions of T3A, H16L, V69A, Q74P, and C125S in IL-2. In another embodiment, the IL-2 fusion protein contains amino acid substitutions of T3A, H16L, V69A, Q74P, and C125S in IL-2.

[0325] In one embodiment, the IL-2 fusion protein comprises amino acid changes (e.g., substitutions) at the T3, V69, Q74, I92, and C125 positions in IL-2, optionally wherein the amino acid substitutions are T3A, V69A, Q74P, I92S, and C125S in IL-2, respectively. In one embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, V69A, Q74P, I92S, and C125S in IL-2. In another embodiment, the IL-2 fusion protein comprises the amino acid substitutions T3A, V69A, Q74P, I92S, and C125S in IL-2.

[0326] In one embodiment, the IL-2 fusion protein contains amino acid changes (e.g., substitutions) at positions H16, K35, V69, and Q74 in IL-2, optionally wherein the amino acid substitutions are H16L, K35E, V69A, and Q74P in IL-2, respectively. In one embodiment, the IL-2 fusion protein comprises the amino acid substitutions H16L, K35E, V69A, and Q74P in IL-2.

[0327] In one embodiment, the IL-2 fusion protein contains amino acid changes (e.g., substitutions) at positions H16, R38, V69A, and Q74P in IL-2, optionally wherein the amino acid substitutions are H16L, R38Q, V69A, and Q74P, respectively. In one embodiment, the IL-2 fusion protein comprises the amino acid substitutions H16L, R38Q, V69A, and Q74P in IL-2.

[0328] In one embodiment, the IL-2 fusion protein comprises amino acid substitutions of H16L, V69A, Q74P, and C125S in IL-2.

[0329] Without being bound by theory, it is believed that in one embodiment, the IL-2 fusion protein comprising amino acid substitutions for H16L, V69A, Q74P, and C125S may have at least one 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 stability, for example, attributable to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity for CD25, which prolongs the lifespan of the IL-2 formulation; (iv) substantially does not promote the expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) reduced incorrect disulfide pairing and improved stability, for example, attributable to the presence of the C125S mutation. In one implementation, IL-2 agents containing 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 containing other H16 mutations. These properties make IL-2 variants containing amino acid substitutions for H16L, V69A, Q74P, and C125S particularly suitable for treating diseases and conditions caused by abnormal immune responses, such as autoimmune diseases.

[0330] Therefore, in one embodiment, IL-2 fusion proteins containing amino acid substitutions H16L, V69A, Q74P, and C125S, relative to wild-type IL-2 or a reference IL-2 variant that does not contain the following amino acid substitutions, particularly possess one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, or all of them): (i) enhanced or increased in vitro or in vivo stability; (ii) decreased or reduced in vitro and / or in vivo binding capacity and / or binding affinity to human CD122; (iii) (iv) Decreased or reduced binding capacity and / or binding affinity of IL-2 variants to human CD132 heterodimers (i.e., human CD122 / CD132 heterodimers) in vitro and / or in vivo; (v) Decreased or reduced binding capacity and / or binding affinity of IL-2 variants to human CD25 in vitro and / or in vivo, or substantially unchanged; (vi) Selective binding to regulatory T cells (e.g., Foxp3 cells). + (vii) selectively activate the IL-2 signaling pathway in T regulatory cells (Tregs) in vitro or in vivo; or (viii) enhance or increase the ability to induce or promote the expansion, activity, survival and / or proliferation of Tregs.

[0331] In one embodiment, the IL-2 fusion protein comprises an IL-2 variant comprising an amino acid sequence selected from the following: 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:3 ... 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 amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence similarity, or amino acid sequences having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids similarity.

[0332] In one embodiment, the IL-2 fusion protein comprises an IL-2 variant comprising or consisting of the following amino acid sequences: 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 with...

Claims

1. A variant of interleukin-2 (IL-2), comprising: (i) amino acid substitution H16L or H16N, and / or amino acid substitution I92S, and (ii) Amino acid substitutions for V69A, Q74P, and C125S. It corresponds to human IL-2 (SEQ ID NO:1031).

2. The IL-2 variant of claim 1, further comprising an amino acid substitution for T3A.

3. The IL-2 variant of claim 1 or 2, comprising an amino acid sequence of any one of SEQ ID NO: 4, 5, 11, 1000, 1001 or 1002, an amino acid sequence having at least 95% identity with it, or an amino acid sequence having approximately 1, 2, 3, 4 or 5 amino acids similar to it, or a functional fragment thereof.

4. An interleukin-2 (IL-2) variant comprising the amino acid sequence SEQ ID NO:

4.

5. The IL-2 variant according to any one of claims 1-4, which selectively stimulates regulatory T cells (Tregs).

6. An IL-2 fusion protein comprising an IL-2 variant as claimed in any one of claims 1-5.

7. The IL-2 fusion protein of claim 6, further comprising an Fc region.

8. The IL-2 fusion protein of claim 7, wherein the Fc region comprises the Fc region of IgG1 allotype m3, which comprises N297G substitution according to EU numbering.

9. The IL-2 fusion protein of claim 7 or 8, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:1003, or an amino acid sequence having at least 95% identity with it, or an amino acid sequence having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids similar to it, or a functional fragment thereof.

10. The IL-2 fusion protein of claim 7, wherein the Fc region is fused to the C-terminus of the IL-2 variant.

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