Interleukin-2 active substances and their use

Abstract

To provide an interleukin-2 active substance and its use. [Solution] Disclosed are IL-2 activators containing IL-2 variants, as well as methods, compositions and uses thereof. The IL-2 activators described herein can be used to treat and / or prevent a variety of disorders and symptoms. Disclosed herein are IL-2 activators (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, some of the IL-2 activators described herein have one or more improved or desired properties compared to IL-2 activators containing wild-type IL-2.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits of U.S. Provisional Application No. 62 / 879,137, filed on 26 July 2019, and U.S. Provisional Application No. 62 / 983,061, filed on 28 February 2020. The contents of the aforementioned applications are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on July 20, 2020, is named P2029-7028WO_SL.txt and is 1,728,528 bytes in size. [Background technology]

[0003] Interleukin-2 (IL-2) is a cytokine that regulates the activity of the immune system. Cytokines are produced by leukocytes such as T cells, natural killer (NK) cells, dendritic cells, and macrophages in response to antigenic or pro-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, which distinguishes foreign antigens from host antigens. IL-2 mediates its effects by binding to the IL-2 receptor, which also activates downstream signaling events.

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

[0005] There remains an unmet need for novel IL-2 activators that demonstrate sufficient characteristics to develop safe and effective treatments for patients with a variety of diseases and conditions that are treatable with IL-2. [Overview of the project] [Means for solving the problem]

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

[0007] This disclosure is based at least in part on the discovery that by using combinations of IL-2 mutations that stabilize the protein, reduce its affinity for CD122 (e.g., CD122 / CD132 heterodimer) and / or reduce its affinity for CD25, or have only a minimal effect, regulatory T cell (Treg) activity can be selectively enhanced via the IL-2 pathway, thereby achieving favorable therapeutic effects for treating disorders and symptoms such as autoimmune diseases. IL-2 activators containing such mutations are suitable for treating symptoms arising from abnormal immune responses, such as autoimmune diseases.

[0008] Accordingly, in certain embodiments, the present disclosure provides IL-2 active materials having, for example, one or more of the following properties a) to 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). a) For example, by a protein concentration assay, it is expressed at higher or increased levels in vitro and / or in vivo compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, e.g., 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% It increases by approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more; or it is expressed by increasing 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; b) For example, when determined by melting temperature analysis (e.g., using fluorescence measurement), dynamic light scattering and / or size exclusion chromatography, aggregates at lower or reduced levels in vitro and / or in vivo compared to IL-2 active agents containing wild-type IL-2 or IL-2 active agents containing a reference IL-2 variant, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35% It decreases by approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or it aggregates by decreasing 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; c) For example, when determined by expression in yeast surface display, expression in mammalian cells, chromatography, circular dichroism or related spectroscopic techniques, and / or melting temperature analysis (e.g., using fluorescence measurement), enhancement or increase in in vitro and / or in vivo compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, It increases by approximately 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more; or it has stability that increases 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; d) For example, when determined by ELISA, flow cytometry and / or mass spectrometry, the enhancement or increase in in vitro and / or in vivo compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, Having a half-life that increases by approximately 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more; or having a half-life that increases 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; e) For example, when determined by ELISA, flow cytometry and / or mass spectrometry, compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 9 Having a lower, decreased, or reduced rate or level of in vivo turnover and / or clearance, reduced by 0%, approximately 95%, approximately 100%, or more, 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; f) For example, when determined by yeast surface display, biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore), compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, it is reduced by, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more (e.g., about 1% to about 20%, about 2% to about 15%, or about 5% to about 10%), or about 1%, about 2%, about 3%, A decrease or increase of approximately 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or less than 50%, or a decrease or increase of 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, Having a reduced, decreased, or substantially unchanged binding affinity to CD25 (e.g., human CD25) that has decreased by approximately 9 times, 9.5 times, 10 times or more, or decreased or increased 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 or less; g) For example, when determined by yeast surface display, low affinity for CD25 (e.g., human CD25), for example, about 5-500 pM, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450 or about 500 pM, or for example, about 10 pM ~about 490pM, about 20pM to about 480pM, about 30pM to about 470pM, about 40pM to about 460pM, about 50pM to about 450pM, about 6 0pM to about 440pM, about 70pM to about 430pM, about 80pM to about 420pM, about 90pM to about 410pM, about 100pM to about 400pM , about 110pM to about 390pM, about 120pM to about 380pM, about 130pM to about 370pM, about 140pM to about 360pM, about 150p M ~ about 350pM, about 160pM - about 340pM, about 170pM - about 330pM, about 180pM - about 320pM, about 190pM - about 310p M, approximately 200 pM to 300 pM, approximately 210 pM to 290 pM, approximately 220 pM to 280 pM, approximately 230 pM to 270 pM, approximately 240 pM to 260 pM, or for example, approximately 5 pM to 450 pM, approximately 5 pM to 400 pM, approximately 5 pM to 350 pM, approximately 5 pM to 300 pM, approximately 5 pM to 250 pM, approximately 5 pM to 200 pM, approximately 5 pM to 150 pM, approximately 5 pM to 100 pM, approximately 5 pM to 50 pM, or for example, approximately 10 pM to 500 pM, approximately 20 pM to 500 pM, approximately 50 pM to 500 pM, approximately 100 pM to 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, or 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,Dissociation constants (K, ) exceeding approximately 400, 450, or 500 pM D ) is used to join them; h) For example, when determined by biolayer interferometry (e.g., Octet coupling) and / or surface plasmon resonance (e.g., Biacore), low affinity for CD25 (e.g., human CD25), for example, about 0.1 to 10 nM, 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, Approximately 5, 6, 7, 8, 9, or 10 nM, or for example, approximately 0.1 to 9 nM, approximately 0.1 to 8 nM, approximately 0.1 to 7 nM, or approximately 0.1 to 6 nM, for example, approximately 0.1 to 5 nM, approximately 0.1 to 4 nM, approximately 0.1 to 3 nM, approximately 0.1 to 2 nM, approximately 0.1 to 1 nM, or approximately 0.1 to 0.5 nM, or for example, approximately 0.1 to 10 nM, approximately 0.5 to 10 nM, approximately 1 to 10 nM, approximately 1.5 to 1 0nM, approximately 2-10nM, approximately 2.5-10nM, approximately 3-10nM, approximately 3.5-10nM, approximately 4-10nM, approximately 4.5-10nM, approximately 5-10nM, approximately 5.5-10nM, approximately 6-10nM, approximately 6.5-10nM, approximately 7-10nM, approximately 7.5-10nM, approximately 8-10nM, approximately 8.5-10nM, approximately 9-10nM or approximately 9.5-10nM, or for example, approximately 0.1-9.5nM, approximately 0 Dissociation constants (K) greater than approximately 0.5 to 9 nM, approximately 1 to 8.5 nM, approximately 1.5 to 8 nM, approximately 2 to 7.5 nM, approximately 2.5 to 7 nM, approximately 3 to 6.5 nM, approximately 3.5 to 6 nM, approximately 4 to 5.5 nM, or approximately 4.5 to 5 nM, or 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 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, or approximately 10 nM. D ) is used to join them; i) For example, when determined by yeast surface display, biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore), compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 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 (for example, approximately 1% to approximately 50%, approximately 2% to approximately 40%, approximately 3% to approximately 30%, approximately 4% to approximately 20%, approximately 5% to approximately 10%, approximately 1% to approximately 40%, approximately 1% to approximately 30%, approximately 1 (Decreased by approximately 20%, 1% to 10%, 40% to 50%, 30% to 50%, 20% to 50%, 10% to 50%, 10% to 20%, 20% to 30%, 30% to 40%, 10% to 30%, or 20% to 40%), or 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, or 5 times It has a binding affinity for CD122 / CD132 heterodimers (e.g., human CD122 / CD132 heterodimers) that is reduced, decreased, or decreased by 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); j) For example, when determined by yeast surface display, low affinity for CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), for example, about 0.2~20 nM, for example, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, 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 approximately 0.1 to approximately 0.5 nM, or for example, approximately 0.1 to approximately 10 nM, approximately 0.5 to approximately 10 nM, approximately 1 to approximately 10 nM, approximately 1.5 to approximately 10 nM, approximately 2 to approximately 10 nM, approximately 2 0.5 to approximately 10 nM, approximately 3 to approximately 10 nM, approximately 3.5 to approximately 10 nM, approximately 4 to approximately 10 nM, approximately 4.5 to approximately 10 nM, approximately 5 to approximately 10 nM, approximately 5.5 to approximately 10 nM, approximately 6 to approximately 10 nM, approximately 6.5 to approximately 10 nM, approximately 7 to approximately 10 nM, approximately 7.5 to approximately 10 nM, approximately 8 to approximately 10 nM, approximately 8.5 to approximately 10 nM, approximately 9 to approximately 10 nM or approximately 9.5 to approximately 10 nM, or for example, approximately 0.1 to approximately 9.5 nM, approximately 0.5 to approximately 9 nM, approximately 1 to approximately 8.5 nM, approximately 1.5 to approximately 8 nM, approximately 2 to approximately 7.5 Dissociation constants (K) exceeding nM, approximately 2.5 to 7 nM, approximately 3 to 6.5 nM, approximately 3.5 to 6 nM, approximately 4 to 5.5 nM, or approximately 4.5 to 5 nM, or 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 D ) is used to join them; k) For example, when determined by biolayer interferometry (e.g., Octet coupling) and / or surface plasmon resonance (e.g., Biacore), low affinity for CD122 / CD132 heterodimers (e.g., human CD122 / CD132 heterodimers), for example, about 0.2 to 300 nM, for example, about 0.2 nM, about 0.5 nM, about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80nM, approximately 90nM, approximately 100nM, approximately 110nM, approximately 120nM, approximately 130nM, approximately 140nM, approximately 150nM, approximately 160nM, approximately 170nM, approximately 180nM, approximately 190nM, approximately 200nM, approximately 210nM, approximately 220nM, approximately 230nM, approximately 240nM, approximately 250nM, approximately 260nM, approximately 270nM, approximately 280nM, approximately 290nM, or approximately 300nM, or for example, approximately 0.2 to approximately 280nM, approximately 0.2 to approximately 260nM, approximately 0.2 to approximately 240nM, approximately 0.2 to approximately 220nM, approximately 0.2 to approximately 200nM, approximately 0.2 to approximately 180nM, approximately 0.2 to approximately 160nM, approximately 0.2 to approximately 140nM, approximately 0.2 to approximately 120nM, approximately 0.2 to approximately 100nM, approximately 0.2 to approximately 80nM, approximately 0.2 to approximately 60nM, approximately 0.2 to approximately 40nM, approximately 0.2 to approximately 20nM, or for example, approximately 0.5 to approximately 300nM, approximately 1 to approximately 300nM, approximately 5 to approximately 300nM, approximately 10 to approximately 300nM, approximately 20 to approximately 300nM, approximately 40 to approximately 300nM, approximately 60 to approximately 300nM, approximately 80 to approximately 300nM, approximately 100 to approximately 300nM, approximately 120 to approximately 300nM, approximately 140-300nM, approximately 160-300nM, approximately 180-300nM, approximately 200-300nM, approximately 220-300nM, approximately 240-300nM, approximately 260-300nM, approximately 280-300nM, or for example, approximately 0.5-280nM, approximately 1-260nM, approximately 5-240nM, approximately 10-220nM, approximately 20-200nM, approximately 40-180nM, approximately 60-160nM, approximately 80-140mM, approximately 100-120nM, or for example, approximately 0.2, approximately 0.Dissociation constants (K) exceeding 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 approximately 300 nM. D ) is used to join them; l) For example, when determined by flow cytometry, IL-2 activators containing wild-type IL-2 or IL-2 activators containing a reference IL-2 variant selectively activate IL-2 signaling in vitro and / or in vivo, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, 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 greater than approximately 3000, or for example, greater than 1 and approximately 1-2, approximately 2-3, approximately 3-4, approximately 4-5, greater than 1 and approximately 1-10, greater than 1 and approximately 1-20, greater than 1 and approximately 1-30, greater than 1 and approximately 1-40, greater than 1 and approximately 1-50, approximately 2-10, approximately 2 ~20, approximately 2~30, approximately 2~40, approximately 2~50, approximately 5~10, approximately 5~20, approximately 5~30, approximately 5~40, approximately 5~50, approximately 10~20, approximately 10~30, approximately 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 T Helper EC 50 / Treg EC 50 Having a ratio; m) For example, as determined by flow cytometry, selective activation of IL-2 signaling in vitro and / or in vivo for IL-2 activators containing wild-type IL-2 or IL-2 activators containing a reference IL-2 variant, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, 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 greater than approximately 3000, or for example, greater than 1 and approximately 1-2, approximately 2-3, approximately 3-4, approximately 4-5, greater than 1 and approximately 1-10, greater than 1 and approximately 1-20, greater than 1 and approximately 1-30, greater than 1 and approximately 1-40, greater than 1 and approximately 1-50, approximately 2-10, approximately 2 ~20, approximately 2~30, approximately 2~40, approximately 2~50, approximately 5~10, approximately 5~20, approximately 5~30, approximately 5~40, approximately 5~50, approximately 10~20, approximately 10~30, approximately 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 NK cells EC 50 / Treg EC 50 Having a ratio; (n)(i) For example, when determined by flow cytometry, in vitro or in vivo regulatory T cell proliferation or expansion assays, and / or T cell suppression assays, compared to an IL-2 agent containing wild-type IL-2 or an IL-2 agent containing a reference IL-2 variant, having the efficacy and / or ability to induce or promote enhanced or increased regulatory T cell activity, for example, decreased by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or, for example, decreased by about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold or more; EC for Treg 50 having; (ii) For example, when determined by flow cytometry, in vitro or in vivo regulatory T cell proliferation or expansion assays, and / or T cell suppression assays, compared to an IL-2 agent containing wild-type IL-2 or an IL-2 agent containing a reference IL-2 variant, having the efficacy and / or ability to induce or promote decreased or reduced regulatory T cell activity, for example, increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more, or, for example, decreased by about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 50-fold, about 100-fold, about 200-fold, about 500-fold, about 1000-fold, about 2000-fold, about 5000-fold, about 10,000-fold, about 15,000-fold, about 20,000-fold or more; EC for Treg 50Having; o) To modulate (e.g., reduce, inhibit, block, or neutralize) or increase (e.g., activate, initiate, or enhance) one or more biological activities of T cells (e.g., Tregs) in vitro, ex vivo, or in vivo; p) Exhibiting the same or similar binding affinity or binding specificity, or both, as the IL-2 active substances described herein; q) Exhibiting the same or similar binding affinity or binding specificity, or both, as an IL-2 active agent containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) changes (e.g., substitutions) described herein; r) Exhibiting the same or similar binding affinity or binding specificity, or both, of an IL-2 active substance containing the amino acid sequence described herein; s) Exhibiting the same or similar binding affinity or binding specificity, or both, of an IL-2 activator containing an amino acid sequence encoded by a nucleotide sequence described herein; t) Inhibiting the binding of a second IL-2 activator to the IL-2 receptor, for example, competitively (where the second IL-2 activator is an IL-2 activator as described herein); u) an activator that competes with a second IL-2 activator for binding to the IL-2 receptor, wherein the second IL-2 activator is an IL-2 activator described herein; v) Having one or more biological properties of IL-2 active substances described herein; w) Having one or more structural properties of IL-2 active substances described herein; or x) Having one or more pharmacokinetic properties of the IL-2 active substances described herein.

[0009] In one embodiment, the IL-2 activator is expressed at higher or increased levels in vitro and / or in vivo compared to, for example, an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, as determined by a protein concentration assay, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%. It is expressed by increasing by approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or by increasing 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. In one embodiment, when the IL-2 active substance is determined, for example, by melting temperature analysis (e.g., using fluorescence measurement), dynamic light scattering and / or size exclusion chromatography, it aggregates at lower or reduced levels in vitro and / or in vivo compared to an IL-2 active substance containing wild-type IL-2 or an IL-2 active substance containing a reference IL-2 variant, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%. It decreases by approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or it aggregates by decreasing 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.

[0010] In one embodiment, when the IL-2 activator is determined, for example, by expression in yeast surface display, expression in mammalian cells, chromatography, circular dichroism or related spectroscopic techniques, and / or melting temperature analysis (e.g., using fluorescence measurement), it is enhanced or increased in vitro and / or in vivo compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about It increases by 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 has stability that increases 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.

[0011] In one embodiment, when the IL-2 active agent is determined, for example, by ELISA, flow cytometry and / or mass spectrometry, it is enhanced or increased in vitro and / or in vivo compared to an IL-2 active agent containing wild-type IL-2 or an IL-2 active agent containing a reference IL-2 variant, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about It has a half-life that increases by 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 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.

[0012] In one embodiment, when the IL-2 active agent is determined, for example, by ELISA, flow cytometry and / or mass spectrometry, the concentrations compared to an IL-2 active agent containing wild-type IL-2 or an IL-2 active agent containing a reference IL-2 variant are, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, and about 80%. Having a lower, decreased, or reduced rate or level of in vivo turnover and / or clearance, 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.

[0013] In one embodiment, when the IL-2 active agent is determined, for example, by yeast surface display, biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore), it is reduced by, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more (e.g., about 1% to about 20%, about 2% to about 15%, or about 5% to about 10%) compared to an IL-2 active agent containing wild-type IL-2 or an IL-2 active agent containing a reference IL-2 variant. , decreased or increased by approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, or approximately 50% or less, or 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, 5.5 times, approximately 6 times, approximately 6.5 times, approximately 7 times, approximately 7.5 times, approximately 8 times, approximately The binding affinity to CD25 (e.g., human CD25) is reduced, decreased, or substantially unchanged by 8.5 times, approximately 9 times, approximately 9.5 times, approximately 10 times or more, or 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 or less. In one embodiment, the reduction or decrease in binding affinity to CD25 is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% lower than the reduction or decrease in binding affinity to CD25. In one embodiment, the binding affinity to CD25 is substantially not reduced or decreased.

[0014] In one embodiment, the IL-2 active substance, when determined by, for example, yeast surface display, has low affinity for CD25 (e.g., human CD25), and is, for example, about 5 to 500 pM, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 200, about 250, about 300, about 350, about 400, about 450 or Approximately 500 pM, or for example, approximately 10 pM to 490 pM, approximately 20 pM to 480 pM, approximately 30 pM to 470 pM, approximately 40 pM to 460 pM, approximately 50 pM to 450 pM, approximately 60 pM to 440 pM, approximately 70 pM to 430 pM, approximately 80 pM to 420 pM, approximately 90 pM to 410pM, about 100pM to about 400pM, about 110pM to about 390pM, about 120pM to about 380pM, about 130pM to about 370pM, Approximately 140pM to approximately 360pM, approximately 150pM to approximately 350pM, approximately 160pM to approximately 340pM, approximately 170pM to approximately 330pM, approximately 180pM to 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, 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 500 pM, approximately 150 pM to 500 pM, approximately 200 pM to 500 pM, approximately 250 pM to 500 pM, approximately 300 pM to 500 pM, approximately 350 pM to 500 pM, approximately 400 pM to 500 pM, approximately 450 pM to 500 pM, or for example, approximately 5, approximately 1 0, 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,Dissociation constants (K, ) exceeding approximately 200, 250, 300, 350, 400, 450, or 500 pM D They are joined together using ).

[0015] In one embodiment, the IL-2 active substance is determined, for example, by biolayer interferometry (e.g., Octet bonding) and / or surface plasmon resonance (e.g., Biacore), with low affinity for CD25 (e.g., human CD25), for example, about 0.1 to 10 nM, 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 , 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 0.1 to approximately 9 nM, approximately 0.1 to approximately 8 nM, approximately 0.1 to approximately 7 nM or approximately 0.1 to approximately 6 nM, for example, approximately 0.1 to approximately 5 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 approximately 0.1 to approximately 0.5 nM, or for example, approximately 0.1 to approximately 10 nM, approximately 0.5 to approximately 10 nM, approximately 1 to approximately 10 n M, approximately 1.5 to 10 nM, approximately 2 to 10 nM, approximately 2.5 to 10 nM, approximately 3 to 10 nM, approximately 3.5 to 10 nM, approximately 4 to 10 nM, approximately 4.5 to 10 nM, approximately 5 to 10 nM, approximately 5.5 to 10 nM, approximately 6 to 10 nM, approximately 6.5 to 10 nM, approximately 7 to 10 nM, approximately 7.5 to 10 nM, approximately 8 to 10 nM, approximately 8.5 to 10 nM, approximately 9 to 10 nM or approximately 9.5 to 10 nM, or for example, approximately 0.1 to 9.5 Dissociation constants (K) greater than approximately nM, 0.5 to 9 nM, 1 to 8.5 nM, 1.5 to 8 nM, 2 to 7.5 nM, 2.5 to 7 nM, 3 to 6.5 nM, 3.5 to 6 nM, 4 to 5.5 nM, or 4.5 to 5 nM, or for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nM D They are joined together using ).

[0016] In one embodiment, when the IL-2 active agent is determined, for example, by yeast surface display, biolayer interferometry (e.g., Octet binding) and / or surface plasmon resonance (e.g., Biacore), it is present in concentrations of, for example, about 1%, about 2%, about 3%, about 4%, compared to an IL-2 active agent containing wild-type IL-2 or an IL-2 active agent containing a reference IL-2 variant. 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 (for example, approximately 1% to approximately 50%, approximately 2% to approximately 40%, approximately 3% to approximately 30%, approximately 4% to approximately 20%, approximately 5% to approximately 10%, approximately 1% to approximately 40%, approximately 1 (Decreased by approximately 30%, 1% to 20%, 1% to 10%, 40% to 50%, 30% to 50%, 20% to 50%, 10% to 50%, 10% to 20%, 20% to 30%, 30% to 40%, 10% to 30%, or 20% to 40%) or approximately 0.5 times, 1 time, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times , has a binding affinity to the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer) that is reduced, decreased, or decreased by approximately 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 (e.g., approximately 0.5 times to 5 times, approximately 1 time to 4 times, or approximately 2 times to 3 times). 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 higher than the reduction or decrease in binding affinity to CD25. In one embodiment, the binding affinity to CD25 is substantially not reduced or decreased.

[0017] In one embodiment, the IL-2 active substance, as determined by, for example, yeast surface display, has low affinity for the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), for example, about 0.2 to 20 nM, for example, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , 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 approximately 0.1 to approximately 0.5 nM, or for example, approximately 0.1 to approximately 10 nM, approximately 0.5 to approximately 10 nM, approximately 1 to approximately 10 nM, approximately 1.5 to approximately 10 nM, approximately 2 ~approximately 10nM, approximately 2.5~approximately 10nM, approximately 3~approximately 10nM, approximately 3.5~approximately 10nM, approximately 4~approximately 10nM, approximately 4.5~approximately 10nM, approximately 5~approximately 10nM, approximately 5.5~approximately 10nM, approximately 6~approximately 10nM, approximately 6.5~approximately 10nM, approximately 7~approximately 10nM, approximately 7.5~approximately 10nM, approximately 8~approximately 10nM, approximately 8.5~approximately 10nM, approximately 9~approximately 10nM or approximately 9.5~approximately 10nM, or for example, approximately 0.1~approximately 9.5nM, approximately 0.5~approximately 9nM, approximately 1~approximately 8.5nM, approximately 1.5~approximately 8nM, approximately 2~ Dissociation constants (K) exceeding approximately 7.5 nM, approximately 2.5 to 7 nM, approximately 3 to 6.5 nM, approximately 3.5 to 6 nM, approximately 4 to 5.5 nM, or approximately 4.5 to 5 nM, or 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. D They are joined together using ).

[0018] In one embodiment, the IL-2 active substance is determined, for example, by biolayer interferometry (e.g., Octet bonding) and / or surface plasmon resonance (e.g., Biacore), to have low affinity for the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), with concentrations of approximately 0.2 to 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, and approximately 50 nM. , approximately 60nM, approximately 70nM, approximately 80nM, approximately 90nM, approximately 100nM, approximately 110nM, approximately 120nM, approximately 130nM, approximately 140nM, approximately 150nM, approximately 160nM, approximately 170nM, approximately 180nM, approximately 190nM, approximately 200nM, approximately 210nM, approximately 220nM, approximately 230nM, approximately 240nM, approximately 250nM, approximately 260nM, approximately 270nM, approximately 280nM, approximately 290nM or approximately 300nM, or for example, approximately 0.2 to approximately 280nM, approximately 0.2 to approximately 260nM, approximately 0.2 to approximately 240nM, approximately 0.2 to approximately 220 nM, approximately 0.2 to approximately 200 nM, approximately 0.2 to approximately 180 nM, approximately 0.2 to approximately 160 nM, approximately 0.2 to approximately 140 nM, approximately 0.2 to approximately 120 nM, approximately 0.2 to approximately 100 nM, approximately 0.2 to approximately 80 nM, approximately 0.2 to approximately 60 nM, approximately 0.2 to approximately 40 nM, approximately 0.2 to approximately 20 nM, or for example, approximately 0.5 to approximately 300 nM, approximately 1 to approximately 300 nM, approximately 5 to approximately 300 nM, approximately 10 to approximately 300 nM, approximately 20 to approximately 300 nM, approximately 40 to approximately 300 nM, approximately 60 to approximately 300 nM, approximately 80 to approximately 300 nM, approximately 100 to approximately 300 nM, approximately 1 20 to approximately 300 nM, approximately 140 to approximately 300 nM, approximately 160 to approximately 300 nM, approximately 180 to approximately 300 nM, approximately 200 to approximately 300 nM, approximately 220 to approximately 300 nM, approximately 240 to approximately 300 nM, approximately 260 to approximately 300 nM, approximately 280 to approximately 300 nM, or for example, approximately 0.5 to approximately 280 nM, approximately 1 to approximately 260 nM, approximately 5 to approximately 240 nM, approximately 10 to approximately 220 nM, approximately 20 to approximately 200 nM, approximately 40 to approximately 180 nM, approximately 60 to approximately 160 nM, approximately 80 to approximately 140 nmM, approximately 100 to approximately 120 nM, or for example, approximately 0.2, approximately 0.Dissociation constants (K) exceeding 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 approximately 300 nM. D They are joined together using ).

[0019] In one embodiment, the IL-2 activator, when determined, for example by flow cytometry, is an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, which selectively activates IL-2 signaling in vitro and / or in vivo, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 1 0, 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 4 00, approximately 450, approximately 500, approximately 600, approximately 700, approximately 800, approximately 900, approximately 1000, approximately 1500, approximately 2000, approximately 2500 or greater than approximately 3000, or for example, greater than 1 and approximately 1-2, approximately 2-3, approximately 3-4, approximately 4-5, greater than 1 and approximately 1-10, greater than 1 and approximately 1-20, greater than 1 and approximately 1-30, greater than 1 and approximately 1-40, greater than 1 and approximately 1-50, approximately 2-1 0, approximately 2-20, approximately 2-30, approximately 2-40, approximately 2-50, approximately 5-10, approximately 5-20, approximately 5-30, approximately 5-40, approximately 5-50, approximately 10-20, approximately 10-30, approximately 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 T Helper EC 50 / Treg EC 50The ratio is as follows: In one embodiment, the T helper cells are CD45+CD3+CD4+Foxp3- cells, determined, for example, by flow cytometry. In one embodiment, the Treg cells are CD45+CD3+CD4+Foxp3+ cells, determined, for example, by flow cytometry.

[0020] In one embodiment, the IL-2 activator, when determined, for example by flow cytometry, is an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, which selectively activates IL-2 signaling in vitro and / or in vivo, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 1 0, 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 4 00, approximately 450, approximately 500, approximately 600, approximately 700, approximately 800, approximately 900, approximately 1000, approximately 1500, approximately 2000, approximately 2500 or greater than approximately 3000, or for example, greater than 1 and approximately 1-2, approximately 2-3, approximately 3-4, approximately 4-5, greater than 1 and approximately 1-10, greater than 1 and approximately 1-20, greater than 1 and approximately 1-30, greater than 1 and approximately 1-40, greater than 1 and approximately 1-50, approximately 2- 10, approximately 2-20, approximately 2-30, approximately 2-40, approximately 2-50, approximately 5-10, approximately 5-20, approximately 5-30, approximately 5-40, approximately 5-50, approximately 10-20, approximately 10-30, approximately 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 NK cells EC 50 / Treg EC 50The ratio is as follows: In one embodiment, NK cells are CD45+CD3- cells which are CD56+ and / or CD16+ as determined, for example, by flow cytometry. In one embodiment, NK cells are CD45+CD3-CD56+ cells as determined, for example, by flow cytometry. In one embodiment, Treg cells are CD45+CD3+CD4+Foxp3+ cells as determined, for example, by flow cytometry.

[0021] In one embodiment, the IL-2 activator (i) has the potency and / or ability to induce or promote enhanced or increased regulatory T cell activity compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, as determined by, for example, flow cytometry, in vitro or in vivo regulatory T cell proliferation or expansion assays and / or T cell suppression assays, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20% EC to Treg has decreased by approximately 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or for example, 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. 50 It holds.

[0022] In one embodiment, the IL-2 activator, as determined by, for example, flow cytometry, in vitro or in vivo regulatory T cell proliferation or expansion assays, and / or T cell suppression assays, has the potency and / or ability to induce or promote reduced or diminished regulatory T cell activity compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, for example, at concentrations 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%, EC relative to Treg has increased by approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or has decreased by, for example, 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, 50 times, 100 times, 200 times, 500 times, 1000 times, 2000 times, 5000 times, 10,000 times, 15,000 times, 20,000 times, or more. 50 In one embodiment, the IL-2 activator has reduced or diminished potency and / or ability to induce or promote regulatory T cell activity (as determined by, for example, flow cytometry, in vitro or in vivo regulatory T cell proliferation or expansion assays, and / or T cell suppression assays) compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, for example, having an EC50 of about 100 times or more against Tregs, and not activating or significantly activating NK cells.

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

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

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

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

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

[0028] In one embodiment, an IL-2 activator inhibits, for example, competitively inhibiting the binding of a second IL-2 activator to an IL-2 receptor (where the second IL-2 activator is an IL-2 activator as described herein).

[0029] In one embodiment, the IL-2 activator is an activator that competes with a second IL-2 activator for binding to the IL-2 receptor, the second IL-2 activator being an IL-2 activator as described herein.

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

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

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

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

[0034] In one embodiment, the IL-2 active substance comprises one or more (e.g., 2, 3, 4, or all) amino acid changes from (i) to (v): (i) One or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid changes (e.g., substitutions) that reduce or are identified as reducing the affinity for CD122 (e.g., CD122 / CD132 heterodimer), e.g., changes (e.g., substitutions) at the positions of H16 (e.g., H16L, H16N, or H16D), I28 (e.g., I28T, or I28F), D84 (e.g., D84V), S87 (e.g., S87R), N88 (e.g., N88S, N88L, or N88D), I92 (e.g., I92S), and / or Q126 (e.g., Q126T, Q126K, or Q126R); (ii) One or more (e.g., two) amino acid changes (e.g., substitutions) that are identified as increasing or increasing the stability of the IL-2 active substance, e.g., changes (e.g., substitutions) at the positions of V69 (e.g., V69A) and / or Q74 (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 affinity for CD25, e.g., changes (e.g., substitutions) at the positions of K35 (e.g., K35E), R38 (e.g., R38E, R38N, or R38Q), F42 (e.g., F42K, or F42Q), and / or E68 (e.g., E68Q, or E68N); or (iv) One or more amino acid changes (e.g., substitutions) that reduce or are identified as reducing the O-glycosylation of IL-2 activators, e.g., changes at the position of T3 (e.g., T3A) (e.g., substitutions); or (v) One or more amino acid changes (e.g., substitutions) identified as reducing or decreasing the erroneous disulfide pair formation and / or aggregation of the IL-2 active substance (e.g., to improve stability), e.g., changes at the position of C125 (e.g., C125S) (e.g., substitutions).

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

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

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

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

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

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

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

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

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

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

[0045] In one embodiment, the IL-2 active substance undergoes amino acid changes (e.g., substitution), (i) Positions V69 and Q74, and / or position K35; and (ii) Position H16, I92, or D84; if necessary, (iii) Positions R38, F42, E68, or combinations thereof It is included in.

[0046] In one embodiment, the IL-2 active substance undergoes amino acid changes (e.g., substitution), (i) Positions V69 and Q74, and / or position K35; and (ii) Positions H16, I92, or D84; (iii) Positions R38, F42, E68, or combinations thereof It is included in.

[0047] In one embodiment, the IL-2 active substance undergoes amino acid changes (e.g., substitution), (i) Positions V69 and Q74, and / or position K35; and (ii) Position H16, I92, or D84; or (iii) Positions R38, F42, E68, or combinations thereof It is included in.

[0048] In one embodiment, the IL-2 active substance undergoes amino acid changes (e.g., substitution), (i) Positions V69 and Q74, and / or position K35; and (ii) Positions H16, I92, D84 or any combination thereof; and (iii) Positions R38, F42, E68, or combinations thereof It is included in.

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

[0050] In one embodiment, the IL-2 active substance includes amino acid changes (e.g., substitutions) at positions V69, Q74, and I92, and optionally the amino acid substitutions are V69A, Q74P, and I92S, respectively. In one embodiment, the IL-2 active substance includes amino acid substitutions V69A, Q74P, and I92S.

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

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

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

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

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

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

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

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

[0059] In one embodiment, the IL-2 active substance includes an amino acid change (e.g., substitution) at position I28, E68, S87, N88, Q126, or a combination thereof. In one embodiment, the IL-2 active substance includes an amino acid change (e.g., substitution) at position I28, and the amino acid substitution is I28T or I28F as needed. In one embodiment, the IL-2 active substance includes the amino acid substitution I28T. In one embodiment, the IL-2 active substance includes the amino acid substitution I28F.

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

[0061] In one embodiment, the IL-2 active substance includes an amino acid change (e.g., substitution) at position S87, and optionally the amino acid substitution is S87R. In one embodiment, the IL-2 active substance includes the amino acid substitution S87R.

[0062] In one embodiment, the IL-2 active substance includes an amino acid change (e.g., substitution) at position N88, and the amino acid substitution is optionally N88R, N88S, N88L, or N88D. In one embodiment, the IL-2 active substance includes the amino acid substitution N88R. In one embodiment, the IL-2 active substance includes the amino acid substitution N88S. In one embodiment, the IL-2 active substance includes the amino acid substitution N88L. In one embodiment, the IL-2 active substance includes the amino acid substitution N88D.

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

[0064] In one embodiment, the IL-2 active substance includes an amino acid change (e.g., substitution) at position C125, and optionally the amino acid substitution is C125S. In one embodiment, the IL-2 active substance includes the amino acid substitution C125S.

[0065] In one embodiment, the IL-2 active substance includes an amino acid change (e.g., substitution) at position T3, and optionally the amino acid substitution is T3A. In one embodiment, the IL-2 active substance includes the amino acid substitution T3A.

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

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

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

[0069] In one embodiment, the IL-2 active substance includes amino acid changes (e.g., substitutions) at positions H16, V69, Q74, and C125, and optionally the amino acid substitutions are H16L, V69A, Q74P, and C125S, respectively. In one embodiment, the IL-2 active substance includes the amino acid substitutions H16L, V69A, Q74P, and C125S. Various technical effects are associated with IL-2 active substances including the aforementioned combinations of amino acid changes. While we do not wish to be bound by theory, in one embodiment, IL-2 activators containing amino acid substitutions H16L, V69A, Q74P, and C125S may possess at least one or more of the following advantageous properties: (i) reduced binding affinity to CD122 and / or CD132, increasing the potency and selectivity of the IL-2 activator on regulatory T cells (Tregs) compared to other T cell types; (ii) significantly more stable, for example, due to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity to CD25 (or having a minimal or no effect thereon), improving the lifespan of the IL-2 activator; (iv) not substantially promoting the expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) reduced erroneous disulfide pair formation and improved stability, for example, due to the presence of the C125S mutation. In one embodiment, IL-2 activators containing the H16L mutation exhibit reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity towards Treg cells compared to other T cell types, compared to IL-2 activators containing other H16 mutations. These properties make IL-2 activators containing amino acid substitutions H16L, V69A, Q74P, and C125S particularly suitable for treating disorders and symptoms resulting from abnormal immune responses, such as autoimmune diseases.

[0070] Therefore, in one embodiment, an IL-2 active agent containing amino acid substitutions H16L, V69A, Q74P, and C125S has, among other things, one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties compared to wild-type IL-2 or a reference IL-2 variant that does not contain amino acid substitutions: (i) enhanced or increased stability in vitro or in vivo; (ii) decreased or reduced binding ability and / or binding affinity to human CD122 in vitro and / or in vivo; (iii) in vitro and (iv) Reduced or decreased binding capacity and / or binding affinity to human CD132 in vivo; (v) Reduced or decreased affinity of IL-2 variants to heterodimeric IL-2 receptors composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) Reduced or decreased (e.g., moderately reduced or decreased) binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo; (vi) Regulatory T cells (e.g., Foxp3 + (vii) selective binding to T cells; (vii) selective activation of the IL-2 signaling pathway in regulatory T cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote the enlargement, activity, survival and / or proliferation of Tregs.

[0071] In one embodiment, the IL-2 active substance includes amino acid changes (e.g., substitutions) at positions H16, V69, Q74, I92, and C125, and optionally the amino acid substitutions are H16L, V69A, Q74P, I92S, and C125S, respectively. In one embodiment, the IL-2 active substance includes amino acid substitutions H16L, V69A, Q74P, I92S, and C125S.

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

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

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

[0075] In one embodiment, the IL-2 active substance is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 3 5. A human IL-2 variant comprising an amino acid sequence selected from SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 1000, SEQ ID NO: 1001, SEQ ID NO: 1002, or a functional fragment thereof, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity therewith, or comprising an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 or fewer amino acids different therefrom.

[0076] In one embodiment, the amino acid change (e.g., substitution) provides the IL-2 activator with at least one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or all) of the following properties compared to a reference IL-2 activator that does not have the amino acid change (e.g., substitution), as described in any one of the preceding items: (i) Enhancement or increase in the expression of IL-2 activators; (ii) Inhibition or reduction of aggregation of IL-2 active substances; (iii) Enhancement or increase in the stability of IL-2 active substances; (iv) Enhancement or increase in the half-life of IL-2 activators; (v) Inhibition or reduction of the turnover and / or clearance of IL-2 activators; (vi) Inhibition or reduction (e.g., moderate inhibition or reduction) of the binding of IL-2 activators to human CD25, or substantially no change; (vii) Inhibition or reduction of the affinity of IL-2 activators for human CD122; (viii) Inhibition or reduction of the affinity of IL-2 activators for human CD132; or (ix) Inhibition or reduction of the affinity of IL-2 activators for the dimeric IL-2 receptor composed of human CD122 and human CD132; (x) Selective binding to regulatory T cells (e.g., Foxp3+ T cells); (xi) Selective activation of the IL-2 signaling pathway in Treg; and / or (xii) Enhancement or increase, or decrease or reduction, of the ability to induce or promote the enlargement, activity, survival and / or proliferation of Tregs.

[0077] In one embodiment, the IL-2 active substance comprises one or more amino acid changes (e.g., substitutions) selected from H16D, H16N, H16L, I28T, K35E, R38Q, R38N, R38E, F42K, F42Q, V69A, Q74P, D84V, S87R, N88L, N88S, I92S, C125S; a polypeptide linker as described herein; and a human IL-2 variant comprising a non-IL-2 moiety as described herein, wherein the amino acid changes (e.g., substitutions) provide the IL-2 active substance with at least one or more of the following properties compared to a reference IL-2 active substance without amino acid changes (e.g., substitutions): (i) Enhancement or increase in the expression of IL-2 activators; (ii) Inhibition or reduction of aggregation of IL-2 active substances; (iii) Enhancement or increase in the stability of IL-2 active substances; (iv) Enhancement or increase in the half-life of IL-2 activators; (v) Inhibition or reduction of the turnover and / or clearance of IL-2 activators; (vi) Inhibition or reduction (e.g., moderate inhibition or reduction) of the binding of IL-2 activators to human CD25, or substantially no change; (vii) Inhibition or reduction of the affinity of IL-2 activators for human CD122; (viii) Inhibition or reduction of the affinity of IL-2 activators for human CD132; (ix) Inhibition or reduction of the affinity of IL-2 activators for the dimeric IL-2 receptor composed of human CD122 and human CD132; (x) Selective binding to regulatory T cells (e.g., Foxp3+ T cells); (xi) Selective activation of the IL-2 signaling pathway in Treg; and / or (xii) Enhancement or increase, or decrease or reduction, of the ability to induce or promote the enlargement, activity, survival and / or proliferation of Tregs.

[0078] In one embodiment, the human IL-2 variant is Amino acid changes (e.g., substitutions): (i)C125S; (ii) V69A, Q74P and C125S; (iii) H16D, V69A, Q74P and C125S; (iv) H16N, V69A, Q74P and C125S; (v) H16L, V69A, Q74P and C125S; (vi) I28T, V69A, Q74P and C125S; (vii)V69A, Q74P, D84V and C125S; (viii) V69A, Q74P, S87R and C125S; (ix) V69A, Q74P, N88L and C125S; (x)V69A, Q74P, N88S and C125S; (xi)V69A, Q74P, I92S and C125S; (xii) K35E, V69A, Q74P and C125S; (xiii) K35E, H16N, V69A, Q74P and C125S; (xiv) K35E, H16L, V69A, Q74P and C125S; (xv)K35E, D84V, V69A, Q74P and C125S; (xvi)K35E, I92S, V69A, Q74P and C125S; (xvii) R38Q, V69A, Q74P and C125S; (xviii) R38Q, H16N, V69A, Q74P and C125S; (xix) R38Q, H16L, V69A, Q74P and C125S; (xx)R38Q, D84V, V69A, Q74P and C125S; (xxi)R38Q, I92S, Q74P and C125S; (xxii) R38N, V69A, Q74P and C125S; (xxiii) R38N, H16N, V69A, Q74P and C125S; (xxiv) R38N, H16L, V69A, Q74P and C125S; (xxv)R38N, D84V, V69A, Q74P and C125S; (xxvi)R38N, I92S, Q74P and C125S; (xxvii) R38E, V69A, Q74P and C125S; (xxviii)F42K, V69A, Q74P and C125S; (xxix)F42Q, V69A, Q74P and C125S; (xxx)F42A, Y45A, L72G, N88D, V69A, Q74P and C125S; (xxxi)R38N, S87R, V69A, Q74P and C125S; (xxxii) R38E, H16N, V69A, Q74P and C125S; (xxxiii) R38E, D84V, V69A, Q74P and C125S; (xxxiv) R38E, S87R, V69A, Q74P and C125S; (xxxv) R38E, I92S, V69A, Q74P and C125S; (xxxvi)F42Q, H16N, V69A, Q74P and C125S; (xxxvii)F42Q, I92S, V69A, Q74P and C125S; (xxxviii) K35E, R38N, H16N, V69A, Q74P and C125S; (xxxix) T3A, H16N, V69A, Q74P and C125S; (xl)T3A, H16L, V69A, Q74P and C125S; or (xli)T3A, V69A, Q74P, I92S and C125S Includes.

[0079] In one embodiment, the IL-2 active substance is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 1000, SEQ ID NO: 1001, or SEQ ID NO: 1002, or a functional fragment thereof. An interleukin-2 (IL-2) activator comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids different thereto from an amino acid sequence; a polypeptide linker as described herein; and an interleukin-2 (IL-2) activator comprising a non-IL-2 moiety as described herein, wherein the IL-2 activator exhibits at least one or more of the following properties compared to a reference IL-2 activator that does not contain a human IL-2 polypeptide variant: (i) Enhancement or increase in the expression of IL-2 activators; (ii) Inhibition or reduction of aggregation of IL-2 active substances; (iii) Enhancement or increase in the stability of IL-2 active substances; (iv) Enhancement or increase in the half-life of IL-2 activators; (v) Inhibition or reduction of the turnover and / or clearance of IL-2 activators; (vi) Inhibition or reduction (e.g., moderate inhibition or reduction) of the binding of IL-2 activators to human CD25, or substantially no change; (vii) Inhibition or reduction of the affinity of IL-2 activators for human CD122; (viii) Inhibition or reduction of the affinity of IL-2 activators for human CD132; (ix) Inhibition or reduction of the affinity of IL-2 activators for the dimeric IL-2 receptor composed of human CD122 and human CD132; (x) Selective binding to regulatory T cells (e.g., Foxp3+ T cells); (xi) Selective activation of the IL-2 signaling pathway in Treg; and / or (xii) Enhancement or increase, or decrease or reduction, of the ability to induce or promote the enlargement, activity, and / or proliferation of Tregs.

[0080] Various technical effects are associated with IL-2 activators containing the amino acid sequence of SEQ ID NO: 5. While we do not wish to be bound by theory, in one embodiment, an IL-2 activator comprising the amino acid sequence of Sequence ID No. 5 may possess at least one or more of the following advantageous properties: (i) reduced binding affinity to CD122 and / or CD132, increasing the potency and selectivity of the IL-2 activator on regulatory T cells (Tregs) compared to other T cell types; (ii) significantly more stable, for example, due to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity to CD25 (or having a minimal or less effect thereon), improving the lifespan of the IL-2 activator; (iv) substantially not promoting the expansion, activation, survival and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) reduced false disulfide pair formation and improved stability, for example, due to the presence of the C125S mutation. In one embodiment, an IL-2 activator containing the H16L mutation exhibits reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity towards Treg cells compared to other T cell types, compared to other IL-2 activators containing the H16 mutation. These properties make the IL-2 activator containing the amino acid sequence of SEQ ID NO: 5 particularly suitable for treating disorders and symptoms resulting from abnormal immune responses, such as autoimmune diseases.

[0081] Therefore, in one embodiment, an IL-2 activator containing the amino acid sequence of SEQ ID NO: 5 has, among other things, one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties compared to wild-type IL-2 or a reference IL-2 variant that does not contain amino acid substitutions: (i) enhanced or increased stability in vitro or in vivo; (ii) decreased or reduced binding ability and / or binding affinity to human CD122 in vitro and / or in vivo; (iii) (iv) Reduced or decreased binding capacity and / or binding affinity of human CD132; (v) Reduced or decreased affinity of IL-2 variants to heterodimeric IL-2 receptors composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) Reduced or decreased (e.g., moderately reduced or decreased) binding capacity and / or binding affinity of human CD25 in vitro and / or in vivo; (vi) Regulatory T cells (e.g., Foxp3 + (vii) selective binding to T cells; (vii) selective activation of the IL-2 signaling pathway in regulatory T cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote the enlargement, activity, survival and / or proliferation of Tregs.

[0082] In one embodiment, the reference IL-2 activator includes 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 activator includes the amino acid sequence of SEQ ID NO: 1031. In one embodiment, the reference IL-2 activator includes the amino acid sequence of SEQ ID NO: 1. In one embodiment, the reference IL-2 activator includes the amino acid sequence of SEQ ID NO: 2.

[0083] In one embodiment, the IL-2 active substance comprises a human IL-2 variant described herein fused to a non-IL-2 portion described herein by a linker, the linker being a polypeptide linker, and optionally the polypeptide linker being a flexible linker, a rigid linker, or a cleavable linker. In one embodiment, the polypeptide linker is a Gly-Ser linker (e.g., (G4S)) n Linkers, where n=1, 2, 3, 4, 5, 6 or more (SEQ ID NO: 1020)), proline-rich extensional linkers (e.g., V1 GPc, V2, GPGc, V3 GcGcP, cellulase linker 4, cellulase linker 4), rigid linkers (e.g., A(EAAAK) n A is where n=2, 3, 4, 5 or more (SEQ ID NO: 1021), REPR_12, non-GS linker (e.g., (GGGSA)) n Here, n = 1, 2, 3, 4, 5 or more (SEQ ID NO: 1022), or an immunoglobulin hinge region or part thereof. In one embodiment, the polypeptide linker is a Gly-Ser linker comprising (G4S)1 (SEQ ID NO: 1023), (G4S)2 (SEQ ID NO: 1024), (G4S)3 (SEQ ID NO: 1025), (G4S)4 (SEQ ID NO: 48), (G4S)5 (SEQ ID NO: 1026), or (G4S)6 (SEQ ID NO: 1027). In one embodiment, the polypeptide linker is a Gly-Ser linker comprising (G4S)4 (SEQ ID NO: 48). In one embodiment, the polypeptide linker comprises an amino acid sequence selected from SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 55. In one embodiment, the polypeptide linker comprises the amino acid sequence of SEQ ID NO: 48.

[0084] In one embodiment, the non-IL-2 portion is an immunoglobulin Fc region, or a fragment or part thereof (e.g., a functional fragment). In one embodiment, the immunoglobulin Fc region includes 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 part thereof. In one embodiment, the IgG Fc region includes a wild-type human IgG1 Fc region (e.g., IgG1 m3 allotype), a wild-type IgG2 Fc region, or a wild-type human IgG4 Fc region, or a fragment or part thereof.

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

[0086] In one embodiment, the IgG Fc region includes a mutant IgG4 Fc region or a fragment or part thereof, wherein the mutant IgG4 Fc region is human.

[0087] In one embodiment, the mutant IgG4 Fc region or a fragment or part thereof includes an amino acid change (e.g., substitution) at Ser228 according to EU numbering, and optionally the amino acid change (e.g., substitution) at Ser228 is S228P. In one embodiment, the mutant IgG4 Fc region includes the amino acid substitution S228P.

[0088] In one embodiment, the mutant IgG4 Fc region or a fragment or part thereof contains an amino acid change (e.g., substitution) to Arg409 according to EU numbering, and optionally the amino acid change (e.g., substitution) of Arg409 is R409K. In one embodiment, the mutant IgG4 Fc region contains the amino acid substitution R409K.

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

[0090] In one embodiment, the mutant IgG4 Fc region contains 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 of sequence identity therewith, or an amino acid sequence that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 or fewer amino acids.

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

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

[0093] In one embodiment, the mutant IgG1 Fc region or a fragment or part thereof includes amino acid changes (e.g., substitutions) to Leu234, Leu235, and Pro329 according to EU numbering, and optionally the amino acid changes (e.g., substitutions are L234A, L235A, and P329G, respectively). In one embodiment, the mutant IgG1 Fc region includes amino acid substitutions L234A, L235A, and P329G.

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

[0095] In one embodiment, the mutant IgG1 Fc region contains 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 of sequence identity thereto, or an amino acid sequence that differs from it by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids or less. In one embodiment, the mutant IgG1 Fc region contains an amino acid sequence that has sequence identity with or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the amino acid sequence of sequence 1003, or less, or a sequence that differs from it by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids or less. In one embodiment, the mutant IgG1 Fc region contains the amino acid sequence of sequence 1003.

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

[0097] In one embodiment, the IL-2 active substance is SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and The IL-2 variant comprises an amino acid sequence selected from SEQ ID NO: 38, SEQ ID NO: 1000, SEQ ID NO: 1001, or SEQ ID NO: 1002, or a functional fragment thereof, the IL-2 activator comprises a Gly-Ser linker, which optionally comprises (G4S)4 (SEQ ID NO: 48), and the IL-2 variant is fused by 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.

[0098] In one embodiment, the IL-2 active substance comprises an amino acid sequence selected from SEQ ID NOs. 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 1004, 1005, 1006, 1007, 1008, or 1009, or a functional fragment thereof.

[0099] In one embodiment, the IL-2 active substance comprises an amino acid sequence selected from SEQ ID NOs: 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, or 131, or a functional fragment thereof.

[0100] In one embodiment, the IL-2 active substance comprises an amino acid sequence selected from SEQ ID NOs: 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, or 169, or a functional fragment thereof.

[0101] In one embodiment, the IL-2 active substance comprises an amino acid sequence selected from SEQ ID NOs: 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, or 207, or a functional fragment thereof.

[0102] In one embodiment, the IL-2 active substance comprises an amino acid sequence selected from SEQ ID NOs: 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, or 245, or a functional fragment thereof.

[0103] In one embodiment, the IL-2 active substance comprises an amino acid sequence selected from SEQ ID NOs: 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, or 283, or a functional fragment thereof.

[0104] In one embodiment, the IL-2 active substance includes an amino acid sequence selected from SEQ ID NOs: 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, or 321, or a functional fragment thereof.

[0105] In one embodiment, the IL-2 active substance comprises an amino acid sequence selected from SEQ ID NOs: 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, or 359, or a functional fragment thereof.

[0106] In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 59 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 97 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 135 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 173 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 211 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 249 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 287 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 325 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 66 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 104 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 142 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 180 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 218 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 256 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 294 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 332 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 60 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 98 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 136 or a functional fragment thereof.In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 174 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 212 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 250 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 288 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 326 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 69 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 107 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 145 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 183 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 221 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 259 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 297 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 335 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 1004 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 1005 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 1006 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 1007 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 1008 or a functional fragment thereof. In one embodiment, the IL-2 activator includes the amino acid sequence of SEQ ID NO: 1009 or a functional fragment thereof.

[0107] Various technical effects are associated with IL-2 activators containing the amino acid sequence of SEQ ID NO: 1008. While we do not wish to be bound by theory, in one embodiment, an IL-2 activator comprising the amino acid sequence of Sequence ID No. 1008 may possess at least one or more of the following advantageous properties: (i) reduced binding affinity to CD122 and / or CD132, increasing the potency and selectivity of the IL-2 activator on regulatory T cells (Tregs) compared to other T cell types; (ii) significantly more stable, for example, due to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity to CD25 (or having a minimal or less effect thereon), improving the lifespan of the IL-2 activator; (iv) substantially not promoting the expansion, activation, survival and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) reduced false disulfide pair formation and improved stability, for example, due to the presence of the C125S mutation. In one embodiment, an IL-2 activator containing the H16L mutation exhibits reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Treg cells compared to other T cell types, compared to other IL-2 activators containing the H16 mutation. These properties make the IL-2 activator containing the amino acid sequence of SEQ ID NO: 1008 particularly suitable for treating disorders and symptoms resulting from abnormal immune responses, such as autoimmune diseases.

[0108] Therefore, in one embodiment, an IL-2 active agent containing the amino acid sequence of SEQ ID NO: 1008 has, among other things, one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, or all of them) compared to wild-type IL-2 or a reference IL-2 variant that does not contain amino acid substitutions: (i) enhanced or increased stability in vitro or in vivo; (ii) reduced or decreased binding ability and / or binding affinity to human CD122 in vitro and / or in vivo; (iii) reduced binding ability and / or binding affinity in vitro and / or in vivo. (iv) Reduced or decreased binding capacity and / or binding affinity to human CD132 in vivo; (v) Reduced or decreased affinity of IL-2 variants to heterodimeric IL-2 receptors composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) Reduced or decreased (e.g., moderately reduced or decreased) binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo; (vi) Regulatory T cells (e.g., Foxp3 + (vii) selective binding to T cells; (vii) selective activation of the IL-2 signaling pathway in regulatory T cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote the enlargement, activity, survival and / or proliferation of Tregs.

[0109] In one embodiment, the IL-2 active substance forms a dimer (e.g., a homodimer or a heterodimer).

[0110] In one embodiment, the IL-2 activator comprises an IL-2 fusion protein. In one embodiment, the IL-2 activator comprises an IL-2 activator / anti-IL-2 antibody complex. In one embodiment, the IL-2 activator comprises a conjugate.

[0111] In some embodiments, the Disclosure provides a pharmaceutical composition comprising a described IL-2 activator and a pharmaceutically acceptable carrier. In some embodiments, the Disclosure provides a nucleic acid encoding an IL-2 activator as described herein. In some embodiments, the Disclosure provides a vector (e.g., an expression vector) comprising a nucleic acid encoding an IL-2 activator as described herein. In some embodiments, the Disclosure provides a cell (e.g., an isolated cell) or a vector (e.g., an expression vector) comprising a nucleic acid encoding an IL-2 activator as described herein.

[0112] In some embodiments, the Disclosure provides a method for producing an IL-2 activator, comprising the step of culturing (e.g., maintaining) cells containing nucleic acids encoding an IL-2 activator as described herein, or vectors (e.g., expression vectors) containing nucleic acids encoding an IL-2 activator as described herein, under conditions that enable the expression of the IL-2 activator. In one embodiment, the Method further comprises the step of obtaining an IL-2 activator. In one embodiment, the Method further comprises the step of purifying the IL-2 activator.

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

[0114] In some embodiments, the present disclosure provides a method for selectively activating the IL-2 signaling pathway in regulatory T cells (Tregs), comprising the steps of contacting Treg cells or populations of Treg cells (e.g., in vitro, ex vivo, or in vivo) or administering the IL-2 activator to a subject requiring it. The IL-2 activator may, for example, include amino acid substitutions H16L, V69A, Q74P, and C125S, or amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 activator includes amino acid substitutions H16L, V69A, Q74P, and C125S.

[0115] In some embodiments, the Disclosure provides a method for inducing immune tolerance in a subject requiring it, comprising the steps of contacting (e.g., in vitro, ex vivo, or in vivo) or administering an effective amount of an IL-2 activator described herein, or a pharmaceutical composition comprising an IL-2 activator, to Treg cells or a population of Treg cells. The IL-2 activator may, for example, include amino acid substitutions H16L, V69A, Q74P, and C125S, or amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 activator comprises amino acid substitutions H16L, V69A, Q74P, and C125S.

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

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

[0118] In some embodiments, the Disclosure provides the use of IL-2 activators or compositions in the manufacture of a pharmaceutical product in a method for treating a subject having a disorder (e.g., a disorder described herein, e.g., autoimmune disease, lupus nephritis, autoimmune hepatitis, nephrotic syndrome, or cancer), the method comprising the step of administering an IL-2 activator or a pharmaceutical composition containing an IL-2 activator described herein to the subject. The IL-2 activator may, for example, include amino acid substitutions H16L, V69A, Q74P, and C125S, or amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 activator includes amino acid substitutions H16L, V69A, Q74P, and C125S.

[0119] In some embodiments, the Disclosure provides an IL-2 activator as described herein, or a pharmaceutical composition comprising an IL-2 activator, and a kit comprising instructions for use. The IL-2 activator may comprise, for example, amino acid substitutions H16L, V69A, Q74P, and C125S, or amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 activator comprises amino acid substitutions H16L, V69A, Q74P, and C125S.

[0120] In some embodiments, the Disclosure provides a container comprising an IL-2 active substance described herein, or a pharmaceutical composition comprising an IL-2 active substance. The IL-2 active substance may comprise, for example, amino acid substitutions H16L, V69A, Q74P, and C125S, or amino acid substitutions H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 active substance comprises amino acid substitutions H16L, V69A, Q74P, and C125S. In embodiments of the present invention, for example, the following items are provided. (Item 1) Corresponding to human IL-2 (sequence number 1031) (i) amino acid substitutions H16L or H16N, and / or amino acid substitutions I92S, (ii) Amino acid substitutions V69A, Q74P and C125S Interleukin-2 (IL-2) variants, including (Item 2) The IL-2 variant described in item 1, further comprising the amino acid substitution T3A. (Item 3) An IL-2 variant as described in item 1 or 2, comprising any amino acid sequence of sequence numbers 4, 5, 11, 1000, 1001, or 1002, an amino acid sequence that is at least 95% identical to the said sequence, or an amino acid sequence that differs from the said sequence by 1, 2, 3, 4, or 5 or fewer amino acids, or a functional fragment thereof. (Item 4) An IL-2 variant described in any one of items 1 to 3, which selectively stimulates regulatory T cells (Tregs). (Item 5) An IL-2 fusion protein containing one of the IL-2 variants described in any one of items 1 through 4. (Item 6) An IL-2 fusion protein as described in item 5, further including an Fc region. (Item 7) The IL-2 fusion protein described in item 6, wherein the Fc region comprises the Fc region of IgG1 allotype m3, which includes the N297G substitution according to EU numbering. (Item 8) The IL-2 fusion protein described in item 6 or 7, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 1003, or an amino acid sequence that is at least 95% identical to the aforementioned sequence, or an amino acid sequence that differs from the aforementioned sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or fewer amino acids, or a functional fragment thereof. (Item 9) The IL-2 fusion protein according to any one of items 6 to 8, wherein the Fc region is fused to the C-terminus of the IL-2 variant. (Item 10) An IL-2 fusion protein as described in any one of items 6 to 9, further including a linker. (Item 11) The IL-2 fusion protein described in item 10, wherein the linker contains (G4S)4 (SEQ ID NO: 48). (Item 12) An IL-2 fusion protein as described in any one of items 6 to 11, comprising the amino acid sequence of any of sequence numbers 1004, 1005, 1006, 1007, 1008, or 1009, an amino acid sequence that is at least 95% identical to the said sequence, or an amino acid sequence that differs from the said sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acids, or a functional fragment thereof. (Item 13) An IL-2 fusion protein that forms a dimer, as described in any one of items 6 to 12. (Item 14) An IL-2 complex comprising an IL-2 variant described in any one of items 1 to 4 and an anti-IL-2 antibody molecule. (Item 15) An IL-2 conjugate comprising an IL-2 variant and a non-IL-2 portion as described in any one of items 1 through 4. (Item 16) A pharmaceutical composition comprising IL-2 as described in any one of items 1 to 4 and a pharmaceutically acceptable carrier. (Item 17) A pharmaceutical composition comprising an IL-2 fusion protein as described in any one of items 5 to 13 and a pharmaceutically acceptable carrier. (Item 18) A pharmaceutical composition comprising the IL-2 complex described in item 14 and a pharmaceutically acceptable carrier. (Item 19) A pharmaceutical composition comprising the IL-2 conjugate described in item 15 and a pharmaceutically acceptable carrier. (Item 20) A nucleic acid encoding an IL-2 variant as described in any one of items 1 through 4. (Item 21) A nucleic acid encoding an IL-2 fusion protein as described in any one of items 5 through 13. (Item 22) The nucleic acid encoding the IL-2 complex described in item 14. (Item 23) A nucleic acid that encodes the IL-2 conjugate described in item 15. (Item 24) A vector containing the nucleic acid described in item 20. (Item 25) A vector containing the nucleic acid described in item 21. (Item 26) A vector containing the nucleic acid described in item 22. (Item 27) A vector containing the nucleic acid described in item 23. (Item 28) Cells containing nucleic acids as described in item 20. (Item 29) Cells containing nucleic acids as described in item 21. (Item 30) Cells containing the nucleic acids listed in item 22. (Item 31) Cells containing nucleic acids as described in item 23. (Item 32) A method for producing an IL-2 variant, comprising the step of culturing the cells described in item 28 under conditions that enable the expression of the IL-2 variant. (Item 33) A method for producing an IL-2 fusion protein, comprising the step of culturing the cells described in item 29 under conditions that enable the expression of the IL-2 fusion protein. (Item 34) A method for producing an IL-2 complex, comprising the step of culturing the cells described in item 30 under conditions that enable the expression of the IL-2 complex. (Item 35) A method for producing an IL-2 conjugate, comprising the step of culturing the cells described in item 31 under conditions that enable the expression of the IL-2 conjugate. (Item 36) A method for enhancing the expansion, activity, survival and / or proliferation of regulatory T cells (Tregs), comprising the steps of exposing Treg cells or populations of Treg cells in vitro, ex vivo, or in vivo to a subject requiring such exposure. (Item 37) A method for enhancing the expansion, activity, survival and / or proliferation of regulatory T cells (Tregs), comprising the steps of contacting Treg cells or a population of Treg cells in vitro, ex vivo, or in vivo with an effective amount of an IL-2 fusion protein described in any of items 5 to 13, or administering it to a subject requiring it. (Item 38) A method for enhancing the expansion, activity, survival and / or proliferation of regulatory T cells (Tregs), comprising the steps of bringing an effective amount of the IL-2 complex described in item 14 into contact with Treg cells or a population of Treg cells in vitro, ex vivo, or in vivo, or administering it to a subject requiring it. (Item 39) A method for enhancing the expansion, activity, survival and / or proliferation of regulatory T cells (Tregs), comprising the steps of bringing an effective amount of the IL-2 conjugate described in item 15 into contact with Treg cells or a population of Treg cells in vitro, ex vivo, or in vivo, or administering it to a subject requiring such contact. (Item 40) A method for selectively activating the IL-2 signaling pathway in regulatory T cells (Treg), comprising the steps of exposing Treg cells or a population of Treg cells to an effective amount of an IL-2 variant described in any one of items 1 to 4, in vitro, ex vivo, or in vivo, or administering such an amount to a subject requiring such contact. (Item 41) A method for selectively activating the IL-2 signaling pathway in regulatory T cells (Tregs), comprising the steps of bringing an effective amount of an IL-2 fusion protein described in any of items 5 to 13 into contact with Treg cells or a population of Treg cells in vitro, ex vivo, or in vivo, or administering it to a subject requiring such contact. (Item 42) A method for selectively activating the IL-2 signaling pathway in regulatory T cells (Tregs), comprising the steps of bringing an effective amount of the IL-2 complex described in item 14 into contact with Treg cells or a population of Treg cells in vitro, ex vivo, or in vivo, or administering it to a subject requiring such contact. (Item 43) A method for selectively activating the IL-2 signaling pathway in regulatory T cells (Treg), comprising the steps of bringing an effective amount of the IL-2 conjugate described in item 15 into contact with Treg cells or a population of Treg cells in vitro, ex vivo, or in vivo, or administering it to a subject requiring such contact. (Item 44) A method for inducing immune tolerance, comprising the step of administering an effective amount of any one of items 1 to 4 of the IL-2 variant to a subject in need thereof. (Item 45) A method for inducing immune tolerance, comprising the step of administering an effective amount of an IL-2 fusion protein described in any one of items 5 to 13 to a subject in need thereof. (Item 46) A method for inducing immune tolerance, comprising the step of administering an effective amount of the IL-2 complex described in item 14 to a subject in need thereof. (Item 47) A method for inducing immune tolerance, comprising the step of administering an effective amount of the IL-2 conjugate described in item 15 to a subject in need thereof. (Item 48) A method for treating an autoimmune disease, comprising the step of administering an effective amount of an IL-2 variant described in any one of items 1 to 4 to a subject in need thereof. (Item 49) A method for treating an autoimmune disease, comprising the step of administering an effective amount of an IL-2 fusion protein described in any one of items 5 to 13 to a subject in need thereof. (Item 50) A method for treating an autoimmune disease, comprising the step of administering an effective amount of the IL-2 complex described in item 14 to a subject in need thereof. (Item 51) A method for treating an autoimmune disease, comprising the step of administering an effective amount of the IL-2 conjugate described in item 15 to a subject in need thereof. (Item 52) A method for treating lupus nephritis, comprising the step of administering an effective amount of any one of items 1 to 4 of the IL-2 variant to a subject in need thereof. (Item 53) A method for treating lupus nephritis, comprising the step of administering an effective amount of an IL-2 fusion protein described in any one of items 5 to 13 to a subject in need thereof. (Item 54) A method for treating lupus nephritis, comprising the step of administering an effective amount of the IL-2 complex described in item 14 to a subject in need thereof. (Item 55) A method for treating lupus nephritis, comprising the step of administering an effective amount of the IL-2 conjugate described in item 15 to a subject in need thereof. (Item 56) A method for treating autoimmune hepatitis, comprising the step of administering an effective amount of any one of items 1 to 4 of the IL-2 variant to a subject in need thereof. (Item 57) A method for treating autoimmune hepatitis, comprising the step of administering an effective amount of an IL-2 fusion protein described in any one of items 5 to 13 to a subject in need thereof. (Item 58) A method for treating autoimmune hepatitis, comprising the step of administering an effective amount of the IL-2 complex described in item 14 to a subject in need thereof. (Item 59) A method for treating autoimmune hepatitis, comprising the step of administering an effective amount of the IL-2 conjugate described in item 15 to a subject in need thereof. (Item 60) A method for treating nephrotic syndrome, comprising the step of administering an effective amount of any one of items 1 to 4 of the IL-2 variant to a subject in need thereof. (Item 61) A method for treating nephrotic syndrome, comprising the step of administering an effective amount of an IL-2 fusion protein described in any one of items 5 to 13 to a subject in need thereof. (Item 62) A method for treating nephrotic syndrome, comprising the step of administering an effective amount of the IL-2 complex described in item 14 to a subject in need thereof. (Item 63) A method for treating nephrotic syndrome, comprising the step of administering an effective amount of the IL-2 conjugate described in item 15 to a subject in need thereof. (Item 64) A kit containing the IL-2 variant described in any one of items 1 through 4, along with instructions for use. (Item 65) A kit containing an IL-2 fusion protein and instructions for use, as described in any of items 5 through 13. (Item 66) A kit containing the IL-2 complex and instructions for use as described in item 14. (Item 67) A kit including the IL-2 conjugate and instructions for use as described in item 15. (Item 68) An IL-2 variant described in any one of items 1 to 4, for use in methods for inducing immune tolerance in a subject. (Item 69) An IL-2 fusion protein as described in any one of items 5 to 13, for use in methods for inducing immune tolerance in a subject. (Item 70) The IL-2 complex described in item 14, for use in methods for inducing immune tolerance in a subject. (Item 71) IL-2 conjugate as described in item 15, for use in methods for inducing immune tolerance in subjects. (Item 72) An IL-2 variant described in any one of items 1 to 4, for use in a method of treating autoimmune diseases in a subject. (Item 73) An IL-2 fusion protein as described in any one of items 5 to 13, for use in methods for autoimmune hepatitis and autoimmune diseases in subjects. (Item 74) The IL-2 complex described in item 14, for use in methods for autoimmune hepatitis and autoimmune diseases in subjects. (Item 75) IL-2 conjugate as described in item 15, for use in methods for autoimmune hepatitis and autoimmune diseases in subjects. (Item 76) An IL-2 variant described in any one of items 1 to 4, for use in a method of treating lupus nephritis in a subject. (Item 77) An IL-2 fusion protein described in any one of items 5 to 13, for use in a method for treating lupus nephritis in a subject. (Item 78) The IL-2 complex described in item 14, for use in methods of treating lupus nephritis in subjects. (Item 79) IL-2 conjugate as described in item 15, for use in methods of treating lupus nephritis in subjects. (Item 80) An IL-2 variant described in any one of items 1 to 4, for use in a method of treating autoimmune hepatitis in a subject. (Item 81) An IL-2 fusion protein as described in any of items 5 to 13, for use in a method for treating autoimmune hepatitis in a subject. (Item 82) The IL-2 complex described in item 14, for use in methods for treating autoimmune hepatitis in subjects. (Item 83) IL-2 conjugate as described in item 15, for use in methods of treating autoimmune hepatitis in subjects. (Item 84) An IL-2 variant described in any one of items 1 to 4, for use in a method of treating nephrotic syndrome in the subject. (Item 85) An IL-2 fusion protein as described in any of items 5 to 13, for use in a method of treating nephrotic syndrome in a subject. (Item 86) The IL-2 complex described in item 14, for use in methods of treating nephrotic syndrome in subjects. (Item 87) IL-2 conjugate as described in item 15, for use in methods of treating nephrotic syndrome in the subject. [Brief explanation of the drawing]

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

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

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

[0124] [Figure 4-1] Figure 4A provides a graph illustrating the thermal denaturation (melting curve) of selected IL-2 activators (IL-2-Fc fusion proteins) as determined by SYPRO Orange fluorescence. Native IL-2-Fc fusions show a maximum signal at low temperatures, indicating the presence of unfolding proteins, while V69A / Q74P mutaine shows unfolding events as the temperature increases. Figure 4B shows that the majority of native IL-2-Fc fusions elute very quickly from the column (>670 kDa), providing an HPLC size exclusion chromatogram that suggests unfolded protein aggregation. In contrast, V69A / Q74P IL-2-Fc elutes as a single peak at the expected time for an 84 kDa protein. [Figure 4-2]Figure 4A provides a graph illustrating the thermal denaturation (melting curve) of selected IL-2 activators (IL-2-Fc fusion proteins) as determined by SYPRO Orange fluorescence. Native IL-2-Fc fusions show a maximum signal at low temperatures, indicating the presence of unfolding proteins, while V69A / Q74P mutaine shows unfolding events as the temperature increases. Figure 4B shows that the majority of native IL-2-Fc fusions elute very quickly from the column (>670 kDa), providing an HPLC size exclusion chromatogram that suggests unfolded protein aggregation. In contrast, V69A / Q74P IL-2-Fc elutes as a single peak at the expected time for an 84 kDa protein.

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

[0126] [Figure 6]Figure 6A provides a graph showing the results of a method for determining the saturation of yeast expressing the indicated IL-2 mutain on its surface after titration with CD122 / CD132 Fc heterodimer at the indicated concentrations. All mutains shown contain V69A / Q74P in addition to the indicated mutation. The bound CD122 / CD132 was labeled using anti-human Fc fluorescence secondary and measured using an Accuri C6 flow cytometer. Saturation was calculated by fitting each curve to a 4-parameter dose-response to estimate the maximum binding signal of each curve, and then normalizing so that the estimated maximum value is defined as 1. Figure 6B provides a graph showing the results of the same method as in Figure 6A, except that the selected mutains were incubated with a 6xHis-tagged (disclosed as "6×His" as SEQ ID NO: 1028) recombinant CD25 extracellular domain, and the bound CD25 was detected with an anti-6xHis antibody (disclosed as "6×His" as SEQ ID NO: 1028).

[0127] [Figure 7-1] Figure 7 provides a series of graphs showing the affinity of IL-2-Fc fusion proteins, including different IL-2 variants as shown, to the extracellular domains of the CD122 / CD132 Fc heterodimer and CD25, as measured by an Octet biolayer interferometry analyzer. The IL-2 variants include the V69A / Q74P+ mutation shown. The IL-2-Fc fusion proteins were immobilized on an anti-human Fc capture tip and then incubated in the shown IL-2 receptor concentration range. Association and dissociation phase kinetics were used to estimate binding affinity. An excess amount of unrelated antibody was used to prevent nonspecific binding or capture of the CD122 / CD132 Fc protein by the tip. [Figure 7-2]Figure 7 provides a series of graphs showing the affinity of IL-2-Fc fusion proteins, including different IL-2 variants as shown, to the extracellular domains of the CD122 / CD132 Fc heterodimer and CD25, as measured by an Octet biolayer interferometry analyzer. The IL-2 variants include the V69A / Q74P+ mutation shown. The IL-2-Fc fusion proteins were immobilized on an anti-human Fc capture tip and then incubated in the shown IL-2 receptor concentration range. Association and dissociation phase kinetics were used to estimate binding affinity. An excess amount of unrelated antibody was used to prevent nonspecific binding or capture of the CD122 / CD132 Fc protein by the tip.

[0128] [Figure 8-1] Figure 8 provides a schematic diagram showing a gating strategy for identifying IL-2-sensitive cell populations from human PBMCs and corresponding flow cytometry data. Singlet lymphocytes are identified based on forward and side scatter. Populations are defined as regulatory T cells (CD4+CD25highFoxp3+), CD25highT helper cells (CD4+CD25highFoxp3-), and natural killer cells (CD3-CD56+). [Figure 8-2] Figure 8 provides a schematic diagram showing a gating strategy for identifying IL-2-sensitive cell populations from human PBMCs and corresponding flow cytometry data. Singlet lymphocytes are identified based on forward and side scatter. Populations are defined as regulatory T cells (CD4+CD25highFoxp3+), CD25highT helper cells (CD4+CD25highFoxp3-), and natural killer cells (CD3-CD56+).

[0129] [Figure 9-1]Figure 9A provides a graph showing the IL-2 signaling response in an IL-2-sensitive cell population (Treg) within human PBMCs after treatment with an IL-2-Fc fusion containing a mutation that reduces affinity for the CD122 / CD132 dimer, determined by the degree of STAT5 phosphorylation. Figure 9B provides a graph showing the IL-2 signaling response in an IL-2-sensitive cell population (CD25+(high)T helper cells) within human PBMCs after treatment with an IL-2-Fc fusion containing a mutation that reduces affinity for the CD122 / CD132 dimer, determined by the degree of STAT5 phosphorylation. Figure 9C provides a graph showing the IL-2 signaling response in an IL-2-sensitive cell population (NK cells) within human PBMCs after treatment with an IL-2-Fc fusion containing a mutation that reduces affinity for the CD122 / CD132 dimer, determined by the degree of STAT5 phosphorylation. Figure 9D provides a graph showing the IL-2 signaling response in a population of IL-2-sensitive cells (CD8+ cytotoxic T cells) in human PBMCs after treatment with an IL-2-Fc fusion containing mutations that reduce affinity for the CD122 / CD132 dimer, determined by the degree of STAT5 phosphorylation. Cells were treated for 30 minutes at the indicated concentrations with either an IL-2-Fc fusion protein containing the V69A / Q74 mutation + the indicated mutation, or IL-2N88D mutein fused to the C-terminus of an unbound antibody (C-terminal N88D). Inactive IL-2-Fc fusion proteins contain several mutations that 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 factor (pSTAT5) phosphorylated with Tyr694. Each population was identified based on the gating shown in Figure 8. The selected mutains were also evaluated for their signaling activity against CD8+ cytotoxic T cells. These cells were gated as shown in Figure 8, except that the CD8 surface marker was used instead of CD4. The median pSTAT5 level (median fluorescence intensity (MFI)) is shown for each concentration of IL-2-Fc fusion protein tested in each cell population.Curve fitting performed using GraphPad Prism v5.03, with 4-parameter fitting for log(agonist) versus response. [Figure 9-2]Figure 9A provides a graph showing the IL-2 signaling response in an IL-2-sensitive cell population (Treg) within human PBMCs after treatment with an IL-2-Fc fusion containing a mutation that reduces affinity for the CD122 / CD132 dimer, determined by the degree of STAT5 phosphorylation. Figure 9B provides a graph showing the IL-2 signaling response in an IL-2-sensitive cell population (CD25+(high)T helper cells) within human PBMCs after treatment with an IL-2-Fc fusion containing a mutation that reduces affinity for the CD122 / CD132 dimer, determined by the degree of STAT5 phosphorylation. Figure 9C provides a graph showing the IL-2 signaling response in an IL-2-sensitive cell population (NK cells) within human PBMCs after treatment with an IL-2-Fc fusion containing a mutation that reduces affinity for the CD122 / CD132 dimer, determined by the degree of STAT5 phosphorylation. Figure 9D provides a graph showing the IL-2 signaling response in a population of IL-2-sensitive cells (CD8+ cytotoxic T cells) in human PBMCs after treatment with an IL-2-Fc fusion containing mutations that reduce affinity for the CD122 / CD132 dimer, determined by the degree of STAT5 phosphorylation. Cells were treated for 30 minutes at the indicated concentrations with either an IL-2-Fc fusion protein containing the V69A / Q74 mutation + the indicated mutation, or IL-2N88D mutein fused to the C-terminus of an unbound antibody (C-terminal N88D). Inactive IL-2-Fc fusion proteins contain several mutations that 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 factor (pSTAT5) phosphorylated with Tyr694. Each population was identified based on the gating shown in Figure 8. The selected mutains were also evaluated for their signaling activity against CD8+ cytotoxic T cells. These cells were gated as shown in Figure 8, except that the CD8 surface marker was used instead of CD4. The median pSTAT5 level (median fluorescence intensity (MFI)) is shown for each concentration of IL-2-Fc fusion protein tested in each cell population.Curve fitting performed using GraphPad Prism v5.03, with 4-parameter fitting for log(agonist) versus response.

[0130] [Figure 10] Figure 10A provides a graph showing the IL-2 signaling response in IL-2-sensitive cell populations (Tregs) within human PBMCs after treatment with an IL-2-Fc fusion containing a mutation that reduces affinity for CD25. Figure 10B provides a graph showing the IL-2 signaling response in IL-2-sensitive cell populations (CD25+(high)T helper cells) within human PBMCs after treatment with an IL-2-Fc fusion containing a mutation that reduces affinity for CD25. Figure 10C provides a graph showing the IL-2 signaling response in IL-2-sensitive cell populations (NK cells) within human PBMCs after treatment with an IL-2-Fc fusion containing a mutation that reduces affinity for CD25. Human PBMCs were treated and analyzed as shown in Figure 9. The median pSTAT5 level (MFI) is shown for each treatment in each population. To highlight the effect on EC50, signaling within each mutein was normalized from 0 to 1 across the concentration range of IL-2-Fc treatment.

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

[0132] [Figure 12] Figure 12A provides a graph showing in vivo Treg expansion, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with IL-2-Fc H16N. Figure 12B provides a graph showing in vivo Treg expansion, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with C-terminal N88D. Homozygous Tg32 mice were administered the indicated amounts of each IL-2Fc fusion protein (dose levels approximately equimolar) by tail vein injection. At the indicated time points, the lymphocyte population was profiled, and Tregs were defined as CD45+CD3+CD4+CD25highCD127- cells. The data in Figures 12A and 12B are averages from 3 mice per treatment group. Figure 12C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested.

[0133] [Figure 13-1] Figure 13A provides a graph showing the change in the level of CD4+ T helper cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with IL-2-Fc H16N. Figure 13B provides a graph showing the change in the level of CD4+ T helper cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with C-terminal N88D. Mice were administered as shown in Figure 12. CD4+ T helper cells were defined as CD45+CD3+CD4+ cells that are not CD25highCD127-. The data in Figures 13A and 13B are averages from 3 mice per treatment group. Figure 13C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested. [Figure 13-2] Figure 13A provides a graph showing the change in the level of CD4+ T helper cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with IL-2-Fc H16N. Figure 13B provides a graph showing the change in the level of CD4+ T helper cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with C-terminal N88D. Mice were administered as shown in Figure 12. CD4+ T helper cells were defined as CD45+CD3+CD4+ cells that are not CD25highCD127-. The data in Figures 13A and 13B are averages from 3 mice per treatment group. Figure 13C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested.

[0134] [Figure 14-1]Figure 14A provides a graph showing the change in the level of CD8+ cytotoxic T cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with IL-2-Fc H16N. Figure 14B provides a graph showing the change in the level of CD8+ cytotoxic T cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with C-terminal N88D. The mice were administered as shown in Figure 12. Cytotoxic T cells were defined as CD45+CD3+CD8+ cells. The data in Figures 14A and 14B are averages from 3 mice per treatment group. Figure 14C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested. [Figure 14-2] Figure 14A provides a graph showing the change in the level of CD8+ cytotoxic T cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with IL-2-Fc H16N. Figure 14B provides a graph showing the change in the level of CD8+ cytotoxic T cells, measured as a percentage of total CD3+ T cells, in Tg32 mice treated with C-terminal N88D. The mice were administered as shown in Figure 12. Cytotoxic T cells were defined as CD45+CD3+CD8+ cells. The data in Figures 14A and 14B are averages from 3 mice per treatment group. Figure 14C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested.

[0135] [Figure 15-1]Figure 15A provides a graph showing the change in NK cell levels, measured as a percentage of total CD45+ lymphocytes, in Tg32 mice treated with IL-2-Fc H16N. Figure 15B provides a graph showing the change in NK cell levels, measured as a percentage of total CD45+ lymphocytes, in Tg32 mice treated with C-terminal N88D. Mice were administered as shown in Figure 12. NK cells were defined as CD45+CD3-CD56+ cells. The data in Figures 15A and 15B are averages from 3 mice per treatment group. In each case, the percentage of NK cells is normalized within each mouse so that the pre-treatment value is 1. Figure 15C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested. [Figure 15-2] Figure 15A provides a graph showing the change in NK cell levels, measured as a percentage of total CD45+ lymphocytes, in Tg32 mice treated with IL-2-Fc H16N. Figure 15B provides a graph showing the change in NK cell levels, measured as a percentage of total CD45+ lymphocytes, in Tg32 mice treated with C-terminal N88D. Mice were administered as shown in Figure 12. NK cells were defined as CD45+CD3-CD56+ cells. The data in Figures 15A and 15B are averages from 3 mice per treatment group. In each case, the percentage of NK cells is normalized within each mouse so that the pre-treatment value is 1. Figure 15C shows data from individual mice at the highest dose of each IL-2-Fc fusion protein tested.

[0136] [Figure 16-1]Figure 16A provides graphs showing the binding kinetics of CD122 / CD132 Fc heterodimer or CD25 extracellular domain at various concentration ranges to an IL-2-Fc fusion protein (wild type) containing only the V69A / Q74P mutation immobilized at the anti-human Fc Octet tip. Figure 16B provides graphs showing the binding kinetics of CD122 / CD132 Fc heterodimer or CD25 extracellular domain at various concentration ranges to an IL-2-Fc fusion protein (42A, Y45A, L72G, N88D, V69A, Q74P; inactivated) containing only inactivated mutations immobilized at the anti-human Fc Octet tip. The KD of each interaction was estimated using the binding kinetics. [Figure 16-2] Figure 16A provides graphs showing the binding kinetics of CD122 / CD132 Fc heterodimer or CD25 extracellular domain at various concentration ranges to an IL-2-Fc fusion protein (wild type) containing only the V69A / Q74P mutation immobilized at the anti-human Fc Octet tip. Figure 16B provides graphs showing the binding kinetics of CD122 / CD132 Fc heterodimer or CD25 extracellular domain at various concentration ranges to an IL-2-Fc fusion protein (42A, Y45A, L72G, N88D, V69A, Q74P; inactivated) containing only inactivated mutations immobilized at the anti-human Fc Octet tip. The KD of each interaction was estimated using the binding kinetics.

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

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

[0139] [Figure 19]Figure 19A shows the residual amount of exemplary IL-2 fusion protein in Tg32 mice. Figure 19B shows the effective half-life of exemplary IL-2 fusion protein in Tg32 mice. In Figure 19A, the X-axis represents the number of days sampled after administration, and the Y-axis represents the concentration of IL-2 fusion protein with the indicated mutation combination in the mouse blood. Figure 19B shows a comparison of the half-life of IL-2 fusion protein with and without additional mutations in the Fc region, and the indicated mutation combination in the IL-2 portion. The Y-axis represents the concentration of indicated IL-2 fusion protein in the blood, and the X-axis represents the number of days after administration.

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

[0141] [Figure 21]Figure 21A shows the effect of exemplary IL-2-Fc fusion proteins (including 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. Figure 21B shows the effect of exemplary IL-2-Fc fusion proteins (including 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. Figure 21A shows the expansion of regulatory T cells over time, expressed as a magnification change compared to baseline (baseline = before administration), after injection of 100 μg / kg of IL-2-Fc fusion protein four times a week. Figure 21B shows the percentage of Ki67+ regulatory T cells normalized to total regulatory T cells (an indicator of proliferating regulatory T cells) over time after injection of 100 μg / kg of IL-2-Fc fusion protein four times a week.

[0142] [Figure 22-1]Figure 22A shows the effect of exemplary IL-2-Fc fusion protein (including mutant H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after injection of 100 μg / kg of IL-2-Fc fusion protein four times a week. Figure 22B shows the effect of exemplary IL-2-Fc fusion protein (including mutant H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after injection of 100 μg / kg of IL-2-Fc fusion protein four times a week. Figure 22C shows the effect of exemplary IL-2-Fc fusion protein (including mutant H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after injection of 100 μg / kg of IL-2-Fc fusion protein four times per week. Figure 22D shows the effect of exemplary IL-2-Fc fusion protein (including mutant H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after injection of 100 μg / kg of IL-2-Fc fusion protein four times per week. Figure 22A shows the effect of IL-2-Fc fusion protein on the number of NK cells over time, Figure 22B shows the effect on cytotoxic T cells over time, Figure 22C shows the effect on T helper cells over time, and Figure 22D shows the effect on total T cells over time. The data are shown as a magnification change relative to baseline (baseline = before administration) for each cell type. [Figure 22-2]Figure 22A shows the effect of exemplary IL-2-Fc fusion protein (including mutant H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after injection of 100 μg / kg of IL-2-Fc fusion protein four times a week. Figure 22B shows the effect of exemplary IL-2-Fc fusion protein (including mutant H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after injection of 100 μg / kg of IL-2-Fc fusion protein four times a week. Figure 22C shows the effect of exemplary IL-2-Fc fusion protein (including mutant H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after injection of 100 μg / kg of IL-2-Fc fusion protein four times per week. Figure 22D shows the effect of exemplary IL-2-Fc fusion protein (including mutant H16L / V69A / Q74P / C125S (SEQ ID NO: 1008) (IL2-118 fused to IgG1 Fc N297G allotype m3)) on circulating immune cells in cynomolgus monkeys after injection of 100 μg / kg of IL-2-Fc fusion protein four times per week. Figure 22A shows the effect of IL-2-Fc fusion protein on the number of NK cells over time, Figure 22B shows the effect on cytotoxic T cells over time, Figure 22C shows the effect on T helper cells over time, and Figure 22D shows the effect on total T cells over time. The data are shown as a magnification change relative to baseline (baseline = before administration) for each cell type.

[0143] [Figure 23-1]Figure 23A shows the effect of the exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with renal lesions similar to lupus nephritis. Figure 23B shows the effect of the exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with renal lesions similar to lupus nephritis. Figure 23C shows the effect of the exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with renal lesions similar to lupus nephritis. Figure 23A shows the weekly proteinuria scores in mice treated with either 40 μg / kg of exemplary IL-2-Fc fusion protein or a PBS vehicle control every three days from 3 weeks of age to 18 weeks of age. The proteinuria score is shown on the Y axis and the age of the mouse is shown on the X axis. Figure 23B shows a series of graphs showing proteinuria scores on the Y axis in individual mice treated with a vehicle control or exemplary IL-2-Fc fusion protein, as indicated on the X axis. From left to right, the first panel shows proteinuria scores at 11 weeks of age, the middle panel shows scores at 12 weeks of age, and the last panel shows scores at 13 weeks of age. Figure 23C shows glomerular lesions quantified on the Y axis at the end of the study (when the mice reached 18 weeks of age) in individual mice treated with a vehicle control or exemplary IL-2-Fc fusion protein, as indicated on the X axis. [Figure 23-2]Figure 23A shows the effect of the exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with renal lesions similar to lupus nephritis. Figure 23B shows the effect of the exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with renal lesions similar to lupus nephritis. Figure 23C shows the effect of the exemplary IL-2-Fc fusion protein described herein on disease progression in a mouse model of systemic lupus erythematosus with renal lesions similar to lupus nephritis. Figure 23A shows the weekly proteinuria scores in mice treated with either 40 μg / kg of exemplary IL-2-Fc fusion protein or a PBS vehicle control every three days from 3 weeks of age to 18 weeks of age. The proteinuria score is shown on the Y axis and the age of the mouse is shown on the X axis. Figure 23B shows a series of graphs showing proteinuria scores on the Y axis in individual mice treated with a vehicle control or exemplary IL-2-Fc fusion protein, as indicated on the X axis. From left to right, the first panel shows proteinuria scores at 11 weeks of age, the middle panel shows scores at 12 weeks of age, and the last panel shows scores at 13 weeks of age. Figure 23C shows glomerular lesions quantified on the Y axis at the end of the study (when the mice reached 18 weeks of age) in individual mice treated with a vehicle control or exemplary IL-2-Fc fusion protein, as indicated on the X axis. [Modes for carrying out the invention]

[0144] Disclosed herein are IL-2 activators (e.g., IL-2 variants, IL-2 fusion proteins, IL-2 complexes, or IL-2 conjugates) having one or more structural and / or functional properties described herein. Advantageously, some of the IL-2 activators described herein have one or more improved or desired properties compared to IL-2 activators including wild-type IL-2. While not wishing to be bound by theory, in one embodiment, the IL-2 activators described herein are thought to selectively enhance regulatory T cell (Treg) activity via the IL-2 pathway. Also provided are nucleic acid molecules encoding IL-2 activators, expression vectors, host cells, compositions (e.g., pharmaceutical compositions), kits, containers, and methods for producing IL-2 activators. The IL-2 activators and pharmaceutical compositions disclosed herein can be used (alone or in combination with other activators or therapeutic methods) to treat, prevent and / or diagnose disorders and conditions, such as disorders and conditions related to T cell activity, such as the disorders or conditions described herein (e.g., autoimmune disorders described herein).

[0145] The immune response is typically regulated by the recognition of specific foreign or self-antigens, communication between innate and adaptive immune pathways, crosstalk between B cells and T cells, and other factors. Several autoimmune diseases may be characterized by 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 a type of T cell that recognizes self-antigens. In response to antigenic stimulation, Tregs release immunosuppressive cytokines that directly inhibit other T cells through intercellular contact. Impairment of Treg activity can lead to widespread autoimmune disorders (e.g., too few cells or cells with low activity). IL-2 is a cytokine that causes expansion and activation of many cell types, but Tregs are usually far more sensitive to IL-2 than other cell types. Low-dose IL-2 administration has been shown to preferentially and sustainably enlarge Treg cells in vivo in a significant proportion of patients, accompanied by improvement in symptoms of chronic graft-versus-host disease (GVHD) (Koreth et al., N Engl J Med. 2011;365(22):2055-2066). In one embodiment, the IL-2 activators described herein provide long-lived immunomodulatory agents (e.g., immunosuppressants) for a number of disorders (e.g., autoimmune indications).

[0146] This disclosure is based at least in part on the finding that IL-2 activators, including human IL-2 polypeptides having specific combinations of amino acid substitutions described herein, may have advantageous technical effects, such as increased stability of the IL-2 activator and / or selective activation of regulatory T cells. The IL-2 activators described herein typically require CD25 for efficient signaling via the IL-2 receptor and thus exhibit high selectivity for Tregs. IL-2 signaling promotes Treg suppressor function and proliferation. While we do not wish to be bound by theory, it is thought that Tregs activated by the IL-2 activators described herein may be able to weaken autoimmune activity through various mechanisms.

[0147] In one embodiment, the IL-2 activator described herein selectively binds to and activates regulatory T cells, and other immune cell types (e.g., CD25) high The effects on T cells and NK cells were found to be simultaneously absent. While we do not wish to be bound by theory, in one embodiment, the amino acid substitutions described herein are thought to enhance the ability of IL-2 activators to maintain their active conformation and modulate the binding affinity of IL-2 activators to dimeric receptors including IL-2Rβ (CD122) and IL-2Rγ (CD132), as well as to trimer receptors including IL-2Rα (CD25) together with CD122 and CD132. In one embodiment, the IL-2 activators described herein selectively bind to and activate IL-2 signaling in regulatory T cells and have optimal affinity to selectively result in the activation and expansion of regulatory T cells both in vitro and in vivo. While we do not wish to be bound by theory, in one embodiment, the binding of IL-2 to the IL-2 receptor is thought to be the primary clearance pathway of IL-2 in vivo. For example, IL-2 activators described herein that have reduced affinity for dimeric and trimer IL-2 receptors exhibit an extended half-life, indicating that reduced affinity for the IL-2 receptor reduces the clearance of the IL-2 activator in vivo. IL-2 activators described herein, for example, those having amino acid substitutions that increase stability to the IL-2 receptor and decrease affinity, may selectively activate regulatory T cells and exhibit an increased half-life in vivo. IL-2 activators described herein, for example, those having mutations that interfere with CD25 binding, may have an improved half-life in vivo. In one embodiment, IL-2 activators do not, or substantially, promote the expansion, activation, survival, and / or proliferation of T effector cells and / or NK cells in vitro and / or in vivo. While we do not wish to be bound by theory, in one embodiment, IL-2 activators described herein may have a larger therapeutic concentration range than low-dose IL-2.

[0148] There are various technical effects associated with the presence of a set of mutations, including amino acid substitutions at position H16, combined with a specific set of mutations described herein, e.g., amino acid substitutions at positions V69, Q74, and C125 (e.g., H16L, V69A, Q74P, and C125S). While we do not wish to be bound by theory, in one embodiment, an IL-2 activator (e.g., an IL-2 variant or IL-2 fusion protein) containing H16L, V69A, Q74P, and C125S is considered significantly more stable, for example, due to the presence of stabilizing V69A and Q74P mutations. For example, unexpectedly, it was found that the V69A and Q74P substitutions did not substantially increase (or essentially decrease) the binding affinity of the IL-2 activator to CD25, but rather stabilized the IL-2 activator in an active conformation sufficient for binding to CD25. While we do not wish to be bound by theory, in one embodiment, IL-2 activators containing the aforementioned mutations may have reduced binding affinity to CD122 and / or CD132, and thus increased potency and selectivity of the IL-2 activator on regulatory T cells (Tregs) compared to other T cell types. Therefore, IL-2 activators containing these mutations are typically stable and selectively activate regulatory T cells (Tregs). While we do not wish to be bound by theory, in one embodiment, IL-2 activators containing the aforementioned mutations may have reduced or decreased binding ability and / or binding affinity to CD25, thereby improving the lifespan of the IL-2 activator. While we do not wish to be bound by theory, in one embodiment, IL-2 activators containing these mutations may not substantially promote the expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo. In one embodiment, an IL-2 activator containing the H16L mutation exhibits reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Treg cells compared to other T cell types, compared to other IL-2 activators containing the H16 mutation.These properties make the IL-2 agents, including the aforementioned mutations, particularly suitable for treating disorders and symptoms resulting from abnormal immune responses, such as autoimmune diseases.

[0149] Thus, in one embodiment, an IL-2 agent (e.g., an IL-2 variant or an IL-2 fusion protein) comprising an amino acid substitution at position H16 in combination with amino acid substitutions at positions V69, Q74, and C125 (e.g., H16L, V69A, Q74P, and C125S) has, among other things, one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following properties compared to wild-type IL-2 or a reference IL-2 agent that does not contain amino acid substitutions: (i) Enhanced or increased stability in vitro or in vivo; (ii) Decreased binding ability and / or binding affinity to human CD122 in vitro and / or in vivo; (iii) Decreased binding ability and / or binding affinity to human CD132 in vitro and / or in vivo; (iv) Decreased affinity of the IL-2 agent for the heterodimeric IL-2 receptor composed of human CD122 and human CD132 (i.e., the human CD122 / CD132 heterodimer) in vitro and / or in vivo; (v) Decreased (e.g., moderately decreased) binding ability and / or binding affinity to human CD25 in vitro and / or in vivo; (vi) Selective binding to regulatory T cells (e.g., Foxp3 + T cells); (vii) Selective activation of the IL-2 signaling pathway in regulatory T cells (Tregs) in vitro or in vivo; or (viii) Enhanced or increased ability to induce or promote the expansion, activation, survival, and / or proliferation of Tregs.

[0150] Definitions As used herein, the articles “a” and “an” refer to one or more grammatical objects (e.g., at least one) that precede the article.

[0151] Unless otherwise specified in the context, the term "or" is used herein to mean the term "and / or" and is interchangeable with it.

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

[0153] The compositions and methods disclosed herein include polypeptides and nucleic acids having a specified sequence or a sequence substantially identical or similar thereto, for example, a sequence identical to the specified sequence by at least 85%, 90%, 95%, or more.

[0154] In the context of amino acid sequences, the term “substantially identical” is used herein to mean a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) their conserved substitutions to, the aligned amino acid residues in the second amino acid sequence, such that the first and second amino acid sequences may have a common structural domain and / or common functional activity. For example, an amino acid sequence containing a common structural domain that has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference sequence, e.g., a sequence provided herein.

[0155] In the context of nucleotide sequences, the term “substantially identical” is used herein to refer to a first nucleic acid sequence containing a sufficient or minimum number of nucleotides identical to the nucleotides aligned in the second nucleic acid sequence, such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or a common structural polypeptide domain or common functional polypeptide activity. For example, a nucleotide sequence having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference sequence, e.g., a sequence provided herein.

[0156] The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as a naturally occurring sequence, or is encoded by substantially the same nucleotide sequence, and can have one or more activity than a naturally occurring sequence.

[0157] The calculation of homology or sequence identity between sequences (these terms are used interchangeably herein) is performed as follows:

[0158] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, optimal alignment may introduce gaps in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be ignored for comparison purposes). In a typical embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, e.g., at least 40%, 50%, 60%, e.g., at least 70%, 80%, 90%, 100% of the reference sequence length. Then, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0159] The percentage of identity between two arrays is a function of the number of identical positions the arrays share, taking into account the number of gaps that need to be introduced for optimal alignment of the two arrays and the length of each gap.

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

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

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

[0163] As used herein, the terms “hybridize under low stringency, medium stringency, high stringency, or very high stringency conditions” describe the conditions for hybridization and washing. Guidelines for carrying out hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6 (which are incorporated by reference). Aqueous and non-aqueous methods are described in this reference, and either may be used. The specific hybridization conditions referred to herein are as follows: 1) Low-stringency hybridization conditions consisting of two washes in 6× sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by two washes in 0.2× SSC and 0.1% SDS at at least 50°C (under low-stringency conditions, the wash temperature may be increased to 55°C); 2) One wash in 6× SSC at approximately 45°C, followed by one wash in 0.2× SSC and 0.1% SDS at 60°C. Medium stringency hybridization conditions involve washing above that; 3) high stringency hybridization conditions involve washing above that once in 6 × SSC at approximately 45°C, followed by washing above that once in 0.2 × SSC, 0.1% SDS, at 65°C; and preferably 4) ultra-high stringency hybridization conditions involve washing above that once in 0.5 M sodium phosphate, 7% SDS, followed by washing above that once in 0.2 × SSC, 1% SDS, at 65°C. The ultra-high stringency condition 4) is a suitable condition and should be used unless otherwise specified.

[0164] It is understood that the molecules described herein may have further conserved or non-essential amino acid substitutions that do not substantially affect their function.

[0165] The term “amino acid” is intended to encompass all molecules, whether natural or synthetic, that contain both amino and acid functional groups and can be included in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; their analogues, derivatives, and homologues; amino acid analogues with variant side chains; and all stereoisomers of any of the above. As used herein, the term “amino acid” includes both D- or L-optical isomers and peptide mimetic compounds.

[0166] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. In the art, families of amino acid residues having similar side chains are defined. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0167] The terms “polypeptide,” “peptide,” and “protein” (in the case of a single chain) are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses modified amino acid polymers, such as those resulting from disulfide bond formation, glycosylation, lipid addition, acetylation, phosphorylation, or any other operation, such as conjugation with labeling elements. Polypeptides may be isolated from natural sources, produced from eukaryotic or prokaryotic hosts by recombinant technology, or be products of synthetic procedures.

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

[0169] The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” and “polynucleotide” are used interchangeably. They refer to polymeric forms of nucleotides of any length, which are either deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides can be single-stranded or double-stranded, and if single-stranded, they can be coding or non-coding (antisense) strands. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeling components. Nucleic acids may be recombinant polynucleotides, or polynucleotides of genomic, cDNA, semi-synthetic, or synthetic origin, which are either not naturally occurring or are linked to other polynucleotides in unnatural configurations.

[0170] As used herein, the term “isolated” refers to material that has been removed from its original or native environment (e.g., its natural environment if it exists in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting material in a natural system by human intervention is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, but such a vector or composition is still isolated in that it is not part of the environment in which it is found in nature.

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

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

[0173] Various embodiments of the compositions and methods described herein are described in further detail below. Further definitions are provided throughout this specification.

[0174] IL-2 agonist This disclosure provides IL-2 activators, including but not limited to IL-2 variants, IL-2 fusion proteins, IL-2 complexes, and IL-2 conjugates. For example, the IL-2 activators described herein may have one or more structural and / or functional properties described herein. In one embodiment, the IL-2 activator comprises an IL-2 variant containing one or more amino acid changes (e.g., substitutions) described herein. In one embodiment, the IL-2 activator comprises an IL-2 variant containing one or more amino acid changes (e.g., substitutions) listed in Table 9. In one embodiment, the IL-2 activator comprises an IL-2 variant containing an amino acid sequence or a portion thereof listed in Table 9. In one embodiment, the IL-2 activator or a portion thereof is encoded by a nucleic acid containing a nucleotide sequence listed herein, for example, in Table 10. One or more amino acid changes (e.g., substitutions), alone or in combination, may confer one or more desired biological properties described herein. In one embodiment, an IL-2 activator can modulate (e.g., increase) the enlargement, survival, activation, and / or function of Tregs. In one embodiment, the modulation is selective or specific to Tregs. For example, an IL-2 activator can modulate activity in Tregs but has limited or no ability to promote activity in unregulated T cells. In one embodiment, the IL-2 activator comprises a polypeptide (which may also be referred to herein as an "IL-2 polypeptide activator").

[0175] IL-2 variant In one embodiment, the IL-2 active substance includes an IL-2 variant, such as an IL-2 variant described herein.

[0176] In one embodiment, the IL-2 variant comprises an IL-2 polypeptide (e.g., human IL-2 polypeptide) or a functional fragment thereof as described herein. In one embodiment, the IL-2 variant comprises one or more amino acid changes (e.g., substitutions) as described in Table 9. In one embodiment, the IL-2 variant comprises or consists of an amino acid sequence or a functional fragment thereof as described in Table 9. In one embodiment, the IL-2 variant is encoded by a nucleic acid comprising a nucleotide sequence as described herein, for example, in Table 10.

[0177] While we do not wish to be bound by theory, in one embodiment, IL-2 variants described herein that have reduced human CD25 and / or reduced human CD122 / CD132 binding affinity compared to wild-type human IL-2 or a reference IL-2 variant may have improved potency and / or selectivity for binding to and activating regulatory T cells (Tregs) compared to wild-type IL-2 or other IL-2 variants. IL-2 variants described herein can be identified, for example, by screening a library of mutated IL-2 polypeptides to identify IL-2 variants that have a desired range of binding affinity to human CD25 and / or human CD122 / CD132.

[0178] In one embodiment, the IL-2 variant has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) of the properties described herein, such as different and / or improved properties, compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid changes (e.g., substitutions) that provide different and / or improved properties compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all) of the following different and / or improved properties (as determined by the assays described herein): i) Altered (e.g., enhanced or increased) expression in vitro and / or in vivo; ii) Altered (e.g., reduced or decreased) aggregation in vitro and / or in vivo; iii) Altered (e.g., enhanced or increased) stability in vitro and / or in vivo; iv) Altered (e.g., enhanced or increased) half-life in vitro and / or in vivo; v) Altered (e.g., decreased or reduced) turnover and / or clearance in vivo; vi) Altered (e.g., reduced or decreased) sensitivity to proteolysis in vitro and / or in vivo; vii) Altered (e.g., enhanced or increased) resistance to proteolysis in vitro and / or in vivo; viii) Altered (e.g., reduced or decreased) binding ability and / or affinity to human CD25 in vitro and / or in vivo; ix) Altered (e.g., reduced or decreased) binding capacity and / or binding affinity of human CD132 in vitro and / or in vivo; x) Altered (e.g., reduced or decreased) binding ability and / or binding affinity to dimeric IL-2 receptors, including human CD122 and human CD132, in vitro and / or in vivo; xi) Modified (e.g., enhanced, increased, decreased, reduced, and / or selective) binding to Treg in vitro and / or in vivo; xii) Altered (e.g., enhanced, increased, decreased, reduced, and / or selective) activation of the IL-2 signaling pathway in Tregs in vitro and / or in vivo; xiii) Altered (e.g., enhanced, increased, decreased, reduced, and / or selective) ability to induce or promote the enlargement, activity, survival, and / or proliferation of Tregs in vitro and / or in vivo.

[0179] In one embodiment, the IL-2 variant has altered (e.g., enhanced or increased) expression in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has enhanced or increased expression (e.g., in bacterial or mammalian cells) compared to wild-type IL-2. In one embodiment, the IL-2 variant has enhanced or increased expression (e.g., in bacterial or mammalian cells) compared to a reference IL-2 variant. In one embodiment, the expression of the IL-2 variant increases 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 increases 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 variant is expressed at higher or increased levels in vitro and / or in vivo compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, as determined, for example by a protein concentration assay, and is expressed at increased levels, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. In one embodiment, the IL-2 variant is expressed at a higher or increased level compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, as determined, for example, by a protein concentration assay, and is expressed at an increased level of, for example, 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.

[0180] In one embodiment, the IL-2 variant has altered (e.g., reduced or decreased) aggregation in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has reduced or decreased aggregation compared to wild-type IL-2. In one embodiment, the IL-2 variant has reduced or decreased aggregation compared to a reference IL-2 variant. In one embodiment, 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 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, an IL-2 active substance containing an IL-2 variant described herein aggregates at a lower or reduced level in vitro and / or in vivo compared to an IL-2 active substance containing wild-type IL-2 or an IL-2 active substance containing a reference IL-2 variant, as determined by, for example, melting temperature analysis (e.g., using fluorescence measurement), dynamic light scattering and / or size exclusion chromatography, for example, by reducing to 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. In one embodiment, an IL-2 active substance containing an IL-2 variant described herein aggregates at a lower or reduced level compared to an IL-2 active substance containing wild-type IL-2 or an IL-2 active substance containing a reference IL-2 variant, as determined by, for example, melting temperature analysis (e.g., using fluorescence measurement), dynamic light scattering and / or size exclusion chromatography, 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.

[0181] In one embodiment, the IL-2 variant has altered (e.g., enhanced or increased) stability in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has enhanced or increased stability compared to wild-type IL-2. In one embodiment, the IL-2 variant has enhanced or increased stability compared to a reference IL-2 variant. In one embodiment, the stability of the IL-2 variant increases 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 increases by about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10%, or more. In one embodiment, an IL-2 active substance containing an IL- variant described herein, when determined by, for example, yeast surface display, circular dichroism or related spectroscopic techniques, and / or melting temperature analysis (e.g., using fluorescence measurement), exhibits enhanced or increased stability in vitro and / or in vivo compared to an IL-2 active substance containing wild-type IL-2 or an IL-2 active substance containing a reference IL-2 variant, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, It increases by approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or has stability that increases by, for example, 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.

[0182] In one embodiment, the IL-2 variant has a modified (e.g., enhanced or increased) half-life in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has an enhanced or increased half-life compared to wild-type IL-2. In one embodiment, the IL-2 variant has an enhanced or increased half-life compared to a reference IL-2 variant. In one embodiment, the half-life of the IL-2 variant is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the half-life of the IL-2 variant is increased by about 0.5 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, an IL-2 activator containing an IL-2 variant described herein, when determined by, for example, ELISA, flow cytometry and / or mass spectrometry, exhibits enhanced or increased half-lives in vitro and / or in vivo compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%. , have a half-life that has increased by approximately 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or for example, 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.

[0183] In one embodiment, the IL-2 variant has altered (e.g., reduced or decreased) turnover in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has reduced or decreased turnover compared to wild-type IL-2. In one embodiment, the IL-2 variant has reduced or decreased turnover compared to a reference IL-2 variant. In one embodiment, the turnover of the IL-2 variant is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the turnover of the IL-2 variant is reduced by about 0.5 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, an IL-2 activator containing an IL-2 variant described herein, when determined by, for example, ELISA, flow cytometry and / or mass spectrometry, exhibits a lower, reduced, or decreased rate or level of in vivo turnover and / or clearance compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%. It decreases by approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or has a rate or level of decrease of, for example, 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.

[0184] In one embodiment, IL-2 has altered (e.g., reduced or decreased) sensitivity to proteolysis in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has reduced or decreased sensitivity to proteolysis compared to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 variant has reduced or decreased sensitivity to proteolysis compared to a reference IL-2 variant. In one embodiment, the sensitivity of the IL-2 variant to proteolysis is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the sensitivity of the IL-2 variant to proteolysis 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.

[0185] In one embodiment, the IL-2 variant has altered (e.g., enhanced or increased) resistance to proteolysis in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has enhanced or increased resistance to proteolysis compared to wild-type IL-2. In one embodiment, the IL-2 variant has enhanced or increased resistance to proteolysis compared to a reference IL-2 variant. In one embodiment, the resistance to proteolysis of the IL-2 variant increases by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the resistance to proteolysis of the IL-2 variant increases by about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10%, or more.

[0186] In one embodiment, the IL-2 variant has altered (e.g., reduced or decreased) binding affinity and / or binding affinity to human CD25 in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has reduced or decreased binding affinity and / or binding affinity to human CD25 compared to wild-type human IL-2. In one embodiment, the IL-2 variant has reduced or decreased binding affinity and / or binding affinity to human CD25 compared to a reference IL-2 variant. In one embodiment, the binding affinity and / or binding affinity of the IL-2 variant to human CD25 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the binding ability and / or binding affinity of an IL-2 variant to human CD25 is reduced by about 0.5 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, when an IL-2 activator containing an IL-2 variant described herein is determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding), the binding ability and / or binding affinity to CD25 (e.g., human CD25) is reduced or decreased by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about It has binding ability and / or binding affinity that is reduced by 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 for example, 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.

[0187] In one embodiment, when the IL-2 variant is determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding), it has low affinity for CD25 (e.g., human CD25), for example, about 5 to 500 pM, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 7 5, 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 to approximately 400 pM, approximately 20 to approximately 300 pM, approximately 50 to approximately 200 pM, approximately 100 to approximately 150 pM, approximately 5 to approximately 10 pM, for example, approximately 10 to approximately 20 pM, approximately 20 to approximately 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, Dissociation constants (K) exceeding approximately 400-500 pM, or for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 200, 250, 300, 350, 400, 450, or approximately 500 pM. D They are joined together using ).

[0188] In one embodiment, when the IL-2 variant is determined, for example, by surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet coupling), it has low affinity for CD25 (e.g., human CD25), for example, about 0.1 to 10 nM, for example, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 6, about 7, about 8, about 9 or about 10 nM, or for example, about 0.2 to about 5 nM, about 0.5 to about 2 nM, about 1 to about 1.5 nM, about 0.1 to about 0.2 nM, for example, about 0.2 to about 0 0.3nM, approximately 0.3 to 0.4nM, or approximately 0.4 to 0.5nM, for example, approximately 0.5 to 0.6nM, approximately 0.6 to 0.7nM, approximately 0.7 to 0.8nM, approximately 0.8 to 0.9nM, approximately 0.9 to 1nM, approximately 1 to 1.5nM, approximately 1.5 to 2nM, approximately 2.5 to 3nM, approximately 3.5 to 4nM, approximately 4 to 4.5nM, approximately 4 Dissociation constants (K) greater than approximately 0.5 to 5 nM, approximately 5 to 6 nM, approximately 6 to 7 nM, approximately 7 to 8 nM, approximately 8 to 9 nM, approximately 9 to 10 nM, or 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 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, or approximately 10 nM. D They are joined together using ).

[0189] In one embodiment, the IL-2 variant has altered (e.g., reduced or decreased) binding affinity and / or binding affinity to human CD132 in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has reduced or decreased binding affinity and / or binding affinity to human CD132 compared to wild-type IL-2. In one embodiment, the IL-2 variant has reduced or decreased binding affinity and / or binding affinity to human CD132 compared to a reference IL-2 variant. In one embodiment, the binding affinity and / or binding affinity of the IL-2 variant to human CD132 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the binding ability and / or binding affinity of an IL-2 variant to human CD132 is reduced by approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or approximately 10 times, or more.

[0190] In one embodiment, the IL-2 variant has altered (e.g., reduced or decreased) binding ability and / or binding affinity to human dimer IL-2 receptors, including human CD122 and human CD132, in vitro and / or in vivo, compared with wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has reduced or decreased binding ability and / or binding affinity to human dimer IL-2 receptors, including human CD122 and human CD132, compared with wild-type IL-2. In one embodiment, the IL-2 variant has reduced or decreased binding ability and / or binding affinity to human dimer IL-2 receptors, including human CD122 and human CD132, compared with a reference IL-2 variant. In one embodiment, the binding ability and / or binding affinity of an IL-2 variant to human dimerized IL-2 receptors, including human CD122 and human CD132, is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the binding ability and / or binding affinity of an IL-2 variant to human dimerized IL-2 receptors, including human CD122 and human CD132, 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.

[0191] In one embodiment, when an IL-2 variant is determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding), it exhibits reduced or decreased binding affinity to the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer) compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%. , have binding ability and / or binding affinity that are reduced by approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or have reduced by, for example, 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.

[0192] In one embodiment, the IL-2 variant, when determined by, for example, yeast surface display, has low affinity for the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), for example, about 0.2 to 20 nM, for example, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, Approximately 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 20 nM, or for example, approximately 0.5 to 15 nM, 1 to 10 nM, 2 to 5 nM, 0.2 to 0.3 nM, 0.3 to 0.4 nM, 0.4 to 0.5 nM, 0.5 to 0.6 nM, 0.6 to 0.7 nM, 0.7 to 0.8 nM, 0.8 to 0.9 nM, 0.9 to 1 nM, 1 to 1.1 nM M, about 1.1 to about 1.2 nM, about 1.2 to about 1.3 nM, about 1.3 to about 1.4 nM, about 1.4 to about 1.5 nM, about 1.5 to about 2 nM, about 2 to about 3 nM, about 3 to about 4 nM, about 4 to about 5 nM, about 5 to about 6 nM, about 6 to about 7 nM, about 7 to about 8 nM, about 8 to about 9 nM, about 9 to about 10 nM, about 10 to about 11 nM, about 11 to about 12 nM, about 12 to about 13 nM, about 13 to about 14 nM, about 14 to about 15 nM, about 15 to about 16 nM, about 16 to about 17 nM They bond with dissociation constants (KD) exceeding approximately 17-18 nM, 18-19 nM, or 19-20 nM, or, for example, approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 nM.

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

[0194] In one embodiment, the IL-2 variant has altered (e.g., enhanced, increased, and / or selective) binding to Tregs in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has enhanced or increased binding to Tregs compared to wild-type IL-2. In one embodiment, the IL-2 variant has selective binding to Tregs compared to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 variant has enhanced or increased binding to Tregs compared to a reference IL-2 variant. In one embodiment, the IL-2 variant has selective binding to Tregs compared to a reference IL-2 variant. In one embodiment, the binding to Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, binding to Treg increases by approximately 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or approximately 10 times, or more.

[0195] In one embodiment, the IL-2 variant has altered (e.g., enhanced, increased, and / or selective) activation of the IL-2 signaling pathway in Tregs in vitro and / or in vivo, compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has enhanced or increased activation of the IL-2 signaling pathway in Tregs compared to wild-type IL-2. In one embodiment, the IL-2 variant has selective activation of the IL-2 signaling pathway in Tregs compared to wild-type IL-2. In one embodiment, the IL-2 variant has enhanced or increased activation of the IL-2 signaling pathway in Tregs compared to a reference IL-2 variant. In one embodiment, the IL-2 variant has selective activation of the IL-2 signaling pathway in Tregs compared to a reference IL-2 variant. In one embodiment, activation of the IL-2 signaling pathway in Tregs is increased by approximately 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or approximately 100%, or more. In one embodiment, activation of the IL-2 signaling pathway in Tregs is increased by approximately 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or approximately 10 times, or more.

[0196] In one embodiment, the IL-2 variant, when determined, for example by flow cytometry, selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, for example, about 1, about 2, about 3, about 4, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Having a T helper EC50 / Treg EC50 ratio greater than or equal to 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 approximately 3000.

[0197] In one embodiment, the IL-2 variant, when determined, for example by flow cytometry, selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, 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, 7 00, 800, 900, 1000, 1500, 2000, 2500 or more than approximately 3000, or more than that, for example, greater than 1 and approximately 1-2, approximately 2-3, approximately 3-4, approximately 4-5, greater than 1 and approximately 1-10, greater than 1 and approximately 1-20, greater than 1 and approximately 1-30, greater than 1 and approximately 1-40, greater than 1 and approximately 1-50, approximately 2-10, approximately 2-20, approximately 2-30, approximately 2 They have an NK cell EC50 / Treg EC50 ratio of ~40, approximately 2~50, approximately 5~10, approximately 5~20, approximately 5~30, approximately 5~40, approximately 5~50, approximately 10~20, approximately 10~30, approximately 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.

[0198] In one embodiment, the IL-2 variant has a modified (e.g., enhanced, increased, and / or selective) ability to induce or promote Treg growth, activity, survival, and / or proliferation in vitro and / or in vivo compared to wild-type IL-2 or a reference IL-2 variant. In one embodiment, the IL-2 variant has an enhanced or increased ability to induce or promote Treg growth, activity, survival, and / or proliferation compared to wild-type IL-2. In one embodiment, the IL-2 variant has a selective ability to induce or promote Treg growth, activity, survival, and / or proliferation compared to wild-type IL-2. In one embodiment, the IL-2 variant has an enhanced or increased ability to induce or promote Treg growth, activity, survival, and / or proliferation compared to a reference IL-2 variant. In one embodiment, the IL-2 variant has a selective ability to induce or promote Treg growth, activity, survival, and / or proliferation compared to a reference IL-2 variant. In one embodiment, the ability to induce or promote the enlargement, activity, survival, and / or proliferation of Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the ability to induce or promote the enlargement, activity, survival, and / or proliferation of Tregs 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.

[0199] In one embodiment, the IL-2 variant, as determined, for example by flow cytometry, has enhanced or increased potency and / or ability to induce or promote regulatory T cell activity compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% , has an EC50 relative to Treg that has decreased by approximately 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or for example, 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.

[0200] In one embodiment, the IL-2 variant, as determined by flow cytometry, has reduced or diminished potency and / or ability to induce or promote regulatory T cell activity compared to an IL-2 activator containing wild-type IL-2 or an IL-2 activator containing a reference IL-2 variant, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 9% Having an EC50 relative to Treg that has increased by 0%, approximately 95%, approximately 100%, or more, or decreased by, for example, 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, 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.

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

[0202] In one embodiment, 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 variants described herein. In one embodiment, the reference IL-2 variant comprises the amino acid sequence of SEQ ID NO: 1 (IL-2 C125S). In one embodiment, the IL-2 variant comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 1 and includes one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid changes (e.g., substitutions) described herein.

[0204] For the purposes of this disclosure, the positional numbering of IL-2 variants begins with the first amino acid after the signal peptide of an exemplary wild-type (WT) human IL-2 polypeptide: [ka] The corresponding amino acid sequence, excluding the signal peptide, is shown as Sequence ID No. 1031.

[0205] In one embodiment, the IL-2 active substance includes an amino acid change (e.g., substitution) at a position corresponding to human IL-2 (e.g., including the amino acid sequence of SEQ ID NO: 1031).

[0206] In one embodiment, the IL-2 variant is [ka] It contains the amino acid sequence. Here, 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 contains or consists of the IL-2 variant amino acid sequence described herein.

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

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

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

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

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

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

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

[0214] In one embodiment, the IL-2 variant includes an amino acid change (e.g., substitution) at position R38, and the amino acid substitution is optionally 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, and the amino acid substitution is optionally 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 includes an amino acid change (e.g., substitution) at (i) (a) positions V69 and Q74, (b) position K35, or (c) positions V69, Q74, and K35; and (ii) one, two, or all of positions H16, I92, or D84. In one embodiment, the IL-2 variant further includes an amino acid change (e.g., substitution) at one, two, or all of positions R38, F42, or E68.

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

[0219] In one embodiment, the IL-2 variant includes amino acid changes (e.g., substitutions) at positions V69, Q74, and H16, and optionally the amino acid substitutions are V69A, Q74P, and H16N or H16L, respectively. In one embodiment, the IL-2 variant includes amino acid substitutions V69A, Q74P, and H16N or H16L. In one embodiment, the IL-2 variant includes amino acid substitutions V69A, Q74P, and H16N. In one embodiment, the IL-2 variant includes amino acid substitutions 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, and optionally 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, where the amino acid substitutions are optionally 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, where the amino acid substitutions are optionally 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, where the amino acid substitutions are optionally 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, and optionally 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, where the amino acid substitutions are optionally 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 includes amino acid changes (e.g., substitutions) at positions V69, Q74, K35, and H16, and optionally the amino acid substitutions are V69A, Q74P, K35E, and H16N, respectively. In one embodiment, the IL-2 variant includes 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, where optionally the amino acid substitutions are V69A, Q74P, K35E, H16N, and R38N, respectively. In one 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, and optionally the amino acid substitutions are V69A, Q74P, H16N, and R38N or R38Q, respectively. In one embodiment, the IL-2 variant includes amino acid substitutions V69A, Q74P, H16N, and R38N or R38Q. In one embodiment, the IL-2 variant includes amino acid substitutions V69A, Q74P, H16N, and R38N. In one embodiment, the IL-2 variant includes amino acid substitutions V69A, Q74P, H16N, and R38Q.

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

[0230] In one embodiment, the IL-2 variant includes an amino acid change (e.g., substitution) at position I28, and the amino acid substitution is I28T or I28F as needed. 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, and the amino acid substitution is optionally 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 includes an amino acid change (e.g., substitution) at position S87, and optionally the amino acid substitution is S87R. In one embodiment, the IL-2 variant includes the amino acid substitution S87R.

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

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

[0235] In one embodiment, the IL-2 variant includes an amino acid change (e.g., substitution) at position C125, and optionally 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 includes an amino acid change (e.g., substitution) at position T3, and optionally the amino acid substitution is T3A. In one embodiment, the IL-2 variant includes 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, and optionally 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 includes an amino acid change (e.g., substitution) at positions T3, H16, I92, or a combination thereof, where the amino acid substitutions are, if necessary, 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, and optionally 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, and optionally 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, where optionally the amino acid substitutions are H16L, V69A, Q74P, I92S, and C125S, respectively. In one 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, and optionally 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, and optionally 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 T3A, H16N, V69A, Q74P, and C125S. In one embodiment, the IL-2 variant includes amino acid substitutions 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, and optionally the amino acid substitutions are T3A, V69A, Q74P, I92S, and C125S, respectively. In one embodiment, the IL-2 variant includes amino acid substitutions T3A, V69A, Q74P, I92S, and C125S. In one embodiment, the IL-2 variant includes amino acid substitutions 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, where the amino acid substitutions are optionally 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, where the amino acid substitutions are, optionally, 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 includes amino acid substitutions H16L, V69A, Q74P, and C125S. In another embodiment, the IL-2 variant includes amino acid substitutions H16N, V69A, Q74P, and C125S.

[0248] There are various technical effects associated with the presence of a set of mutations, including amino acid substitutions at position H16, combined with a specific set of mutations described herein, such as amino acid substitutions at positions V69, Q74, and C125 (e.g., H16L, V69A, Q74P, and C125S). While we do not wish to be bound by theory, in one embodiment, IL-2 variants containing the aforementioned mutations may also have reduced binding affinity to CD122 and / or CD132, and increased potency and selectivity of the IL-2 variant for regulatory T cells (Tregs) compared to other T cell types. While we do not wish to be bound by theory, in one embodiment, IL-2 variants containing the aforementioned mutations may also be significantly more stable, for example, due to the presence of stabilizing V69A and Q74P mutations. For example, unexpectedly, the V69A and Q74P substitutions were found not to substantially increase the binding affinity of IL-2 variants to CD25, but rather to stabilize the IL-2 variants in an active conformation sufficient for binding to CD25. Therefore, IL-2 variants containing these mutations selectively activate regulatory T cells (Tregs) and are significantly stable. While we do not wish to be bound by theory, in one embodiment, it is conceivable that IL-2 variants containing the aforementioned mutations have reduced or decreased binding ability and / or binding affinity to CD25, thereby improving the lifespan of the IL-2 variants. While we do not wish to be bound by theory, in one embodiment, it is also conceivable that IL-2 variants containing these mutations do not substantially promote the expansion, activation, survival, and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo. While we do not wish to be bound by theory, in one embodiment, it is further conceivable that IL-2 variants containing the aforementioned mutations have reduced erroneous disulfide pair formation and improved stability, for example, due to the presence of the C125S mutation.In one embodiment, IL-2 activators containing the H16L mutation exhibit reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Treg cells compared to other T cell types, compared to IL-2 activators containing other H16 mutations. These properties make IL-2 variants containing these mutations particularly suitable for treating disorders and symptoms resulting from abnormal immune responses, such as autoimmune diseases.

[0249] Therefore, in one embodiment, an IL-2 variant (e.g., IL-2 variant or IL-2 fusion protein) containing 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) will, among other things, have one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, or all of them) compared to wild-type IL-2 or a reference IL-2 variant that does not contain amino acid substitutions: (i) enhanced or increased stability in vitro or in vivo; (ii) improved human C12 in vitro and / or in vivo. (iii) Reduced or decreased binding capacity and / or binding affinity to D122; (iv) Reduced or decreased binding capacity and / or binding affinity to human CD132 in vitro and / or in vivo; (v) Reduced or decreased affinity of IL-2 variants to heterodimeric IL-2 receptors composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimer) in vitro and / or in vivo; (vi) Reduced or decreased binding capacity and / or binding affinity to human CD25 in vitro and / or in vivo; (vi) Regulatory T cells (e.g., Foxp3 + (vii) selective binding to T cells; (vii) selective activation of the IL-2 signaling pathway in regulatory T cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote the enlargement, activity, survival and / or proliferation of Tregs.

[0250] In one embodiment, the IL-2 variant is sequence number 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, sequence number An amino acid sequence selected from sequence number 34, sequence number 35, sequence number 36, sequence number 37, sequence number 38, sequence number 1000, sequence number 1001, sequence number 1002, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity therewith, or containing or comprising an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids are different therefrom.

[0251] In one embodiment, the IL-2 variant has sequence identity with the amino acid sequence of SEQ ID NO: 4, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more thereof, or contains or comprises an amino acid sequence that differs from it by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids or less. In one embodiment, the IL-2 variant has sequence identity with the amino acid sequence of SEQ ID NO: 5, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more thereof, or contains or comprises an amino acid sequence that differs from it by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids or less. In one embodiment, the IL-2 variant has sequence identity with the amino acid sequence of SEQ ID NO: 11, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more thereof, or includes or comprises an amino acid sequence that differs from it by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids or less. In one embodiment, the IL-2 variant has sequence identity with or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the amino acid sequence of SEQ ID NO: 1000, or contains or comprises an amino acid sequence that differs from it by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids or less.In one embodiment, the IL-2 variant has sequence identity with or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the amino acid sequence of SEQ ID NO: 1001, or contains or comprises an amino acid sequence that differs from it by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids or less. In one embodiment, the IL-2 variant has sequence identity with or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the amino acid sequence of Sequence ID No. 1002, or contains or comprises an amino acid sequence that differs from it by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids or less.

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

[0253] While we do not wish to be bound by theory, in one embodiment, an IL-2 variant containing or comprising the amino acid sequence of Sequence ID No. 5, or a functional fragment thereof, may possess at least one or more of the following advantageous properties: (i) reduced binding affinity to CD122 and / or CD132, increasing the potency and selectivity of the IL-2 activator on regulatory T cells (Tregs) compared to other T cell types; (ii) significantly more stable, for example, due to the presence of stabilizing V69A and Q74P mutations; (iii) reduced or decreased binding capacity and / or binding affinity to CD25, improving the lifetime of the IL-2 activator; (iv) substantially not promoting the expansion, activation, survival and / or proliferation of T effector cells and / or natural killer (NK) cells in vitro and / or in vivo; and / or (v) reduced erroneous disulfide pair formation and improved stability, for example, due to the presence of the C125S mutation. In one embodiment, an IL-2 activator containing the H16L mutation exhibits reduced binding affinity to CD122 and / or CD132, and / or increased potency and selectivity for Treg cells compared to other T cell types, compared to other IL-2 activators containing the H16 mutation. These properties make IL-2 variants containing or comprising the amino acid sequence of SEQ ID NO: 5 particularly suitable for treating disorders and symptoms resulting from abnormal immune responses, such as autoimmune diseases.

[0254] Therefore, in one embodiment, an IL-2 variant having sequence identity with or comprising an amino acid sequence that differs from it by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids or less, or comprising an amino acid sequence that differs from it by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids or less, is, in particular, possessing one or more (e.g., 2, 3, 4, 5, 6, 7, or all) of the following characteristics compared to a wild-type IL-2 or reference IL-2 variant that does not contain amino acid substitutions: (i) in vitro or in vivo (ii) Enhanced or increased stability of; (iii) Decreased or reduced binding capacity and / or binding affinity to human CD122 in vitro and / or in vivo; (iv) Decreased or reduced affinity of IL-2 variants to heterodimeric IL-2 receptors composed of human CD122 and human CD132 (i.e., human CD122 / CD132 heterodimers) 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) Regulatory T cells (e.g., Foxp3 + (vii) selective binding to T cells; (vii) selective activation of the IL-2 signaling pathway in regulatory T cells (Tregs) in vitro or in vivo; or (viii) enhanced or increased ability to induce or promote the enlargement, 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 IL-2 variants described herein are maintained, transferred, or conferred to IL-2 fusion proteins, IL-2 complexes, or IL-2. For the purposes of this disclosure, the terms “IL-2 variant” and “IL-2 mutain” may be used interchangeably herein.

[0256] In one embodiment, the IL-2 variant comprises a polypeptide (sometimes referred to herein as the "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. Examples of 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 activator includes an IL-2 fusion protein, such as an IL-2 fusion protein described herein.

[0258] In one embodiment, the IL-2 fusion protein comprises an IL-2 variant, for example, an IL-2 variant described herein. In one embodiment, the IL-2 fusion protein comprises one or more amino acid changes (e.g., substitutions) as described in Table 9. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence or a functional fragment thereof as described in Table 9. In one embodiment, the IL-2 variant is encoded by a nucleic acid comprising a nucleotide sequence as described herein, for example, in Table 10.

[0259] While we do not wish to be bound by theory, in one embodiment, an IL-2 fusion protein described herein having reduced human CD25 and / or reduced human CD122 / CD132 binding affinity compared to an IL-2 fusion protein containing wild-type human IL-2 or a reference IL-2 fusion protein may have improved efficacy and / or selectivity for binding to and activating regulatory T cells (Tregs) compared to an IL-2 fusion protein containing wild-type IL-2 or other IL-2 fusion proteins.

[0260] In one embodiment, the IL-2 fusion protein has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) of the properties described herein, such as different and / or improved properties, compared 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 includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid changes (e.g., substitutions) that provide different and / or improved properties compared to an IL-2 fusion protein 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 (as determined by the assays described herein): i) Altered (e.g., enhanced or increased) expression in vitro and / or in vivo; ii) Altered (e.g., reduced or decreased) aggregation in vitro and / or in vivo; iii) Altered (e.g., enhanced or increased) stability in vitro and / or in vivo; iv) Altered (e.g., enhanced or increased) half-life in vitro and / or in vivo; v) Altered (e.g., decreased or reduced) turnover and / or clearance in vivo; vi) Altered (e.g., reduced or decreased) sensitivity to proteolysis in vitro and / or in vivo; vii) Altered (e.g., enhanced or increased) resistance to proteolysis in vitro and / or in vivo; viii) Altered (e.g., reduced or decreased) binding ability and / or affinity to human CD25 in vitro and / or in vivo; ix) Altered (e.g., reduced or decreased) binding capacity and / or binding affinity of human CD132 in vitro and / or in vivo; x) Altered (e.g., reduced or decreased) binding ability and / or binding affinity to dimeric IL-2 receptors, including human CD122 and human CD132, in vitro and / or in vivo; xi) Modified (e.g., enhanced, increased, decreased, reduced, and / or selective) binding to Treg in vitro and / or in vivo; xii) Altered (e.g., enhanced, increased, decreased, reduced, and / or selective) activation of the IL-2 signaling pathway in Tregs in vitro and / or in vivo; xiii) Altered (e.g., enhanced, increased, decreased, reduced, and / or selective) ability to induce or promote the enlargement, activity, survival, and / or proliferation of Tregs in vitro and / or in vivo.

[0261] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced or increased) expression in vitro and / or in vivo compared to an IL-2 fusion protein 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) compared 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) compared to a reference IL-2 fusion protein. In one embodiment, the expression of the IL-2 fusion protein increases 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 increases 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 is expressed at higher or increased levels in vitro and / or in vivo compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, as determined, for example by a protein concentration assay, and is expressed at increased levels, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. In one embodiment, the IL-2 fusion protein is expressed at a higher or increased level compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, as determined, for example, by a protein concentration assay, and is expressed at an increased level of, for example, 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.

[0262] In one embodiment, the IL-2 fusion protein has altered (e.g., reduced or decreased) aggregation in vitro and / or in vivo compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has reduced or decreased aggregation compared to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has reduced or decreased aggregation compared to a reference IL-2 fusion protein. In one embodiment, 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 at lower or reduced levels in vitro and / or in vivo compared 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 measurement), dynamic light scattering and / or size exclusion chromatography, for example, by reducing to 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. In one embodiment, the IL-2 fusion protein aggregates at a lower or reduced level compared 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 measurement), dynamic light scattering and / or size exclusion chromatography, 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.

[0263] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced or increased) stability in vitro and / or in vivo compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has enhanced or increased stability compared to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has enhanced or increased stability compared to a reference IL-2 fusion protein. In one embodiment, the stability of the IL-2 fusion protein increases 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 increases 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, when the IL-2 fusion protein is determined by, for example, yeast surface display, circular dichroism or related spectroscopic techniques, and / or melting temperature analysis (e.g., using fluorescence measurement), it exhibits enhanced or increased stability in vitro and / or in vivo compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 3%. It increases by 0%, 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 has stability that increases by, for example, 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.

[0264] In one embodiment, the IL-2 fusion protein has a modified (e.g., enhanced or increased) half-life in vitro and / or in vivo compared 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 compared 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 compared to a reference IL-2 fusion protein. In one embodiment, the half-life of the IL-2 fusion protein increases 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 increases 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, when determined by, for example, ELISA, flow cytometry and / or mass spectrometry, exhibits enhanced or increased half-life in vitro and / or in vivo compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 4%. It has a half-life that has increased by 5%, 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 for example, 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.

[0265] In one embodiment, the IL-2 fusion protein has altered (e.g., reduced or decreased) turnover in vitro and / or in vivo compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has reduced or decreased turnover compared to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has reduced or decreased turnover compared to a reference IL-2 fusion protein. In one embodiment, the turnover of the IL-2 fusion protein is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the turnover of the IL-2 fusion protein is reduced by about 0.5 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, when the IL-2 fusion protein is determined, for example by ELISA, flow cytometry and / or mass spectrometry, it exhibits a lower, reduced, or decreased rate or level of in vivo turnover and / or clearance compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about It decreases by 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 has a rate or level of decrease of, for example, 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.

[0266] In one embodiment, the IL-2 fusion protein provided by this disclosure has the property of having altered (e.g., reduced or decreased) sensitivity to proteolysis in vitro and / or in vivo compared 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 sensitivity to proteolysis compared to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 fusion protein has reduced or decreased sensitivity to proteolysis compared to a reference IL-2 fusion protein. In one embodiment, the sensitivity of the IL-2 fusion protein to proteolysis is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the sensitivity of the IL-2 fusion protein to proteolysis is reduced by approximately 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 enhanced or increased resistance to altered (e.g., enhanced or increased) proteolysis in vitro and / or in vivo compared 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 resistance to proteolysis compared to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has enhanced or increased resistance to proteolysis compared to a reference IL-2 fusion protein. In one embodiment, the resistance to proteolysis of the IL-2 fusion protein increases by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the resistance to proteolysis of the IL-2 fusion protein increases by about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10%, or more.

[0268] In one embodiment, the IL-2 fusion protein has altered (e.g., reduced or decreased) binding ability and / or affinity to human CD25 in vitro and / or in vivo compared 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 ability and / or affinity to human CD25 compared to wild-type human IL-2. In one embodiment, the IL-2 fusion protein has reduced or decreased binding ability and / or affinity to human CD25 compared to a reference IL-2 fusion protein. In one embodiment, the binding ability and / or affinity of the IL-2 fusion protein to human CD25 is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the binding ability and / or binding affinity of an 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, when the IL-2 fusion protein is determined, for example by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding), the binding ability and / or binding affinity to CD25 (e.g., human CD25) is reduced or decreased compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%. , have binding ability and / or binding affinity that are reduced by approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or have reduced by, for example, 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.

[0269] In one embodiment, the IL-2 fusion protein is determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding), to have low affinity for CD25 (e.g., human CD25), for example, about 5-500 pM, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, 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 to approximately 400 pM, approximately 20 to approximately 300 pM, approximately 50 to approximately 200 pM, approximately 100 to approximately 150 pM, approximately 5 to approximately 10 pM, for example, approximately 10 to approximately 20 pM, approximately 20 to approximately 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 to approximately 500 pM, or 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 a dissociation constant (K) greater than approximately 500 pM D They are joined together using ).

[0270] In one embodiment, the IL-2 fusion protein, when determined by, for example, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding), has low affinity for CD25 (e.g., human CD25), for example, about 0.1 to 10 nM, for example, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 6, about 7, about 8, about 9 or about 10 nM, or for example, about 0.2 to about 5 nM, about 0.5 to about 2 nM, about 1 to about 1.5 nM, about 0.1 to about 0.2 nM, for example, about 0.2 to about 0.3nM, approximately 0.3 to approximately 0.4nM, or approximately 0.4 to approximately 0.5nM, for example, approximately 0.5 to approximately 0.6nM, approximately 0.6 to approximately 0.7nM, approximately 0.7 to approximately 0.8nM, approximately 0.8 to approximately 0.9nM, approximately 0.9 to approximately 1nM, approximately 1 to approximately 1.5nM, approximately 1.5 to approximately 2nM, approximately 2.5 to approximately 3nM, approximately 3.5 to approximately 4nM, approximately 4 to approximately 4.5nM, approximately 4.5 to approximately 5 nM, approximately 5 to approximately 6 nM, approximately 6 to approximately 7 nM, approximately 7 to approximately 8 nM, approximately 8 to approximately 9 nM, approximately 9 to approximately 10 nM, or, for example, dissociation constants (K) greater than approximately 0.1, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9 or approximately 10 nM D They are joined together using ).

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

[0272] In one embodiment, the IL-2 fusion protein has altered (e.g., reduced or decreased) binding ability and / or binding affinity to human dimer IL-2 receptors, including human CD122 and human CD132, in vitro and / or in vivo, compared 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 ability and / or binding affinity to human dimer IL-2 receptors, including human CD122 and human CD132, compared to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has reduced or decreased binding ability and / or binding affinity to human dimer IL-2 receptors, including human CD122 and human CD132, compared to a reference IL-2 fusion protein. In one embodiment, the binding ability and / or binding affinity of an IL-2 fusion protein to human dimerized IL-2 receptors, including human CD122 and human CD132, is reduced by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the binding ability and / or binding affinity of an IL-2 fusion protein to human dimerized IL-2 receptors, including human CD122 and human CD132, 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.

[0273] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced, increased, and / or selective) binding to Tregs in vitro and / or in vivo compared to an IL-2 fusion protein 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 compared to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has selective binding to Tregs compared to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 fusion protein has enhanced or increased binding to Tregs compared to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has selective binding to Tregs compared to a reference IL-2 fusion protein. In one embodiment, the binding to Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, binding to Treg increases by approximately 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or approximately 10 times, or more.

[0274] In one embodiment, when the IL-2 fusion protein is determined, for example, by yeast surface display, surface plasmon resonance (e.g., Biacore) and / or biolayer interferometry (e.g., Octet binding), it exhibits reduced or decreased binding affinity to the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer) compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about It has binding ability and / or binding affinity that is reduced by 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 for example, 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.

[0275] In one embodiment, the IL-2 fusion protein has low affinity for the CD122 / CD132 heterodimer (e.g., human CD122 / CD132 heterodimer), as determined by, for example, yeast surface display, for example, about 0.2 to 20 nM, for example about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, about 4, about 5, 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 to approximately 15 nM, approximately 1 to approximately 10 nM, approximately 2 to approximately 5 nM, approximately 0.2 to approximately 0.3 nM, approximately 0.3 to approximately 0.4 nM, approximately 0.4 to approximately 0.5 nM, approximately 0.5 to approximately 0.6 nM, approximately 0.6 to approximately 0.7 nM, approximately 0.7 to approximately 0.8 nM, approximately 0.8 to approximately 0.9 nM, approximately 0.9 to approximately 1 nM, approximately 1 to approximately 1.1 nM, about 1.1 to about 1.2 nM, about 1.2 to about 1.3 nM, about 1.3 to about 1.4 nM, about 1.4 to about 1.5 nM, about 1.5 to about 2 nM, about 2 to about 3 nM, about 3 to about 4 nM, about 4 to about 5 nM, about 5 to about 6 nM, about 6 to about 7nM, about 7 to about 8nM, about 8 to about 9nM, about 9 to about 10nM, about 10 to about 11nM, about 11 to about 12nM, about 12 to about 13nM, about 13 to about 14nM, about 14 to about 15nM, about 15 to about 16nM, about 16 to about 17nM They bond with dissociation constants (KD) exceeding approximately 17-18 nM, 18-19 nM, or 19-20 nM, or, for example, approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 nM.

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

[0277] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced, increased, and / or selective) binding to Tregs in vitro and / or in vivo compared to an IL-2 fusion protein 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 compared to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has selective binding to Tregs compared to IL-2 (e.g., wild-type human IL-2). In one embodiment, the IL-2 fusion protein has enhanced or increased binding to Tregs compared to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has selective binding to Tregs compared to a reference IL-2 fusion protein. In one embodiment, the binding to Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, binding to Treg increases by approximately 0.5 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or approximately 10 times, or more.

[0278] In one embodiment, the IL-2 fusion protein has altered (e.g., enhanced, increased, and / or selective) activation of the IL-2 signaling pathway in Tregs in vitro and / or in vivo, compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has enhanced or increased activation of the IL-2 signaling pathway in Tregs compared to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has selective activation of the IL-2 signaling pathway in Tregs compared to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has enhanced or increased activation of the IL-2 signaling pathway in Tregs compared to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has selective activation of the IL-2 signaling pathway in Tregs compared to a reference IL-2 fusion protein. In one embodiment, activation of the IL-2 signaling pathway in Tregs is increased by approximately 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or approximately 100%, or more. In one embodiment, activation of the IL-2 signaling pathway in Tregs is increased by approximately 0.5 times, 1 time, 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, as determined, for example by flow cytometry, selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo, compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, about 1, about 2, about 3, about 4, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 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 more than approximately 3000, or having a T helper EC50 / Treg EC50 ratio greater than or equal to that.

[0280] In one embodiment, the IL-2 fusion protein, as determined, for example by flow cytometry, selectively activates IL-2 signaling in T regulatory cells in vitro and / or in vivo compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, 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 , exceeding 800, 900, 1000, 1500, 2000, 2500 or approximately 3000, or exceeding that, or for example, greater than 1 and approximately 1-2, approximately 2-3, approximately 3-4, approximately 4-5, greater than 1 and approximately 1-10, greater than 1 and approximately 1-20, greater than 1 and approximately 1-30, greater than 1 and approximately 1-40, greater than 1 and approximately 1-50, approximately 2-10, approximately 2-20, approximately 2-30, approximately 2- The NK cell EC50 / Treg EC50 ratio is approximately 40, 2-50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-20, 10-30, 10-40, 10-50, 20-40, 20-50, 50-100, 100-200, 200-500, 500-1000, 1000-2000, or 1000-3000.

[0281] In one embodiment, the IL-2 fusion protein has a modified (e.g., enhanced, increased, and / or selective) ability to induce or promote Treg growth, activity, survival, and / or proliferation in vitro and / or in vivo compared 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 enhanced or increased ability to induce or promote Treg growth, activity, survival, and / or proliferation compared 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 growth, activity, survival, and / or proliferation compared to an IL-2 fusion protein containing wild-type IL-2. In one embodiment, the IL-2 fusion protein has a enhanced or increased ability to induce or promote Treg growth, activity, survival, and / or proliferation compared to a reference IL-2 fusion protein. In one embodiment, the IL-2 fusion protein has a selective ability to induce or promote Treg growth, activity, survival, and / or proliferation compared to a reference IL-2 fusion protein. In one embodiment, the ability to induce or promote the enlargement, activity, survival, and / or proliferation of Tregs is increased by about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%, or more. In one embodiment, the ability to induce or promote the enlargement, activity, survival, and / or proliferation of Tregs 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.

[0282] In one embodiment, the IL-2 fusion protein, as determined, for example by flow cytometry, has enhanced or increased potency and / or ability to induce or promote regulatory T cell activity compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about The EC50 for Tregs is reduced by 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 100%, or more, or, for example, 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.

[0283] In one embodiment, the IL-2 fusion protein, as determined by flow cytometry, has reduced or diminished potency and / or ability to induce or promote regulatory T cell activity compared to an IL-2 fusion protein containing wild-type IL-2 or a reference IL-2 fusion protein, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% , having an EC50 relative to Treg that has increased by approximately 95%, approximately 100%, or more, or decreased by, for example, 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, 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.

[0284] In one embodiment, the T helper cells described herein are CD45+CD3+CD4+Foxp3- cells, for example, determined by flow cytometry. In one embodiment, the Treg cells described herein are CD45+CD3+CD4+Foxp3+ cells, for example, determined by flow cytometry. In one embodiment, the NK cells described herein are CD45+CD3- cells, which are CD56+ and / or CD16+, for example, determined by flow cytometry. In one embodiment, the NK cells described herein are CD45+CD3-CD56+ cells, for example, 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 the IL-2 fusion protein containing wild-type IL-2 or the reference IL-2 fusion protein.

[0286] In one embodiment, the reference IL-2 fusion protein comprises an amino acid sequence having approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the IL-2 fusion proteins described herein. In one embodiment, the reference IL-2 fusion protein comprises an IL-2 variant comprising the amino acid sequence of SEQ ID NO: 57. In one embodiment, the IL-2 fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 57 and includes one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid changes (e.g., substitutions) described herein.

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

[0288] In one embodiment, the IL-2 fusion protein includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or all) amino acid changes (e.g., substitutions) at posi...

Claims

1. An IL-2 variant comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, and comprising amino acid substitutions H16N, V69A, and Q74P relative to SEQ ID NO: 1, wherein the IL-2 variant comprises a reduced binding affinity to the dimeric IL-2 receptor containing CD122 and CD132 compared to the wild type.

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

4.

3. An IL-2 fusion protein comprising an IL-2 variant and an Fc region, An IL-2 fusion protein wherein the IL-2 variant comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, and comprises amino acid substitutions H16N, V69A, and Q74P relative to SEQ ID NO: 1, and the IL-2 variant comprises a reduced binding affinity to a dimeric IL-2 receptor containing CD122 and CD132 compared to the wild type.

4. An IL-2 fusion protein comprising the amino acid sequence of SEQ ID NO: 1007.

5. The IL-2 fusion protein according to claim 3, wherein the Fc region includes the Fc region of IgG1.

6. The IL-2 fusion protein according to claim 5, wherein the Fc region of IgG1 contains the amino acid substitution N297G according to EU numbering.

7. The Il-2 fusion protein according to any one of claims 3 to 6, wherein the Fc region includes the Fc region of human IgG1 allotype m3.

8. The IL-2 fusion protein according to any one of claims 3 to 7, wherein the Fc region includes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1003.

9. The IL-2 fusion protein according to claim 8, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 1003.

10. The IL-2 fusion protein according to any one of claims 3 to 9, wherein the Fc region is fused to the C-terminus of the IL-2 variant.

11. The IL-2 fusion protein according to any one of claims 3 to 10, wherein the fusion protein includes a linker between the IL-2 variant and the Fc region.

12. The IL-2 fusion protein according to claim 11, wherein the linker comprises (G 4 S) 4 (Sequence ID 48).

13. The IL-2 fusion protein according to any one of claims 3 to 12, wherein the fusion protein forms a dimer.

14. The IL-2 fusion protein according to claim 13, wherein the fusion protein forms a homodimer.

15. A pharmaceutical composition comprising an IL-2 variant according to any one of claims 1 to 2 or an IL-2 fusion protein according to any one of claims 3 to 14 and a pharmaceutically acceptable carrier.

16. A nucleic acid encoding an IL-2 variant according to any one of claims 1 to 2 or an IL-2 fusion protein according to any one of claims 3 to 14.

17. A vector comprising the nucleic acid described in claim 16.

18. A cell comprising the nucleic acid according to claim 16 or the vector according to claim 17.

19. A method for producing an IL-2 variant or an IL-2 fusion protein, comprising the step of culturing the cells described in claim 18 under conditions that enable the expression of the IL-2 variant or the IL-2 fusion protein.

20. A kit comprising an IL-2 variant according to any one of claims 1 to 2 or an IL-2 fusion protein according to any one of claims 3 to 14, and instructions for use.

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