Polynucleotides encoding modified interleukin 2 (IL2) polypeptides and methods for making and using same

Abstract

The present disclosure relates, inter alia, to polynucleotides encoding modified interleukin-2 (IL-2) polypeptides and fusion proteins comprising such modified IL-2 polypeptides, vectors comprising such polynucleotides, pharmaceutical compositions comprising such polynucleotides and vectors, and methods of treating diseases and disorders by administering such polynucleotides, vectors, and / or pharmaceutical compositions. In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide comprises at least one substitution with a natural amino acid at at least one position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof.

Description

[Technical Field]

[0001] Technical Field The present disclosure relates, inter alia, to polynucleotides encoding modified interleukin-2 (IL-2) polypeptides and fusion proteins comprising such modified IL-2 polypeptides, vectors comprising such polynucleotides, pharmaceutical compositions comprising such polynucleotides and vectors, and methods of treating diseases and disorders by administering such polynucleotides, vectors, and pharmaceutical compositions. [Background technology]

[0002] background Cytokines include a family of cell signaling proteins, such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and other growth factors that play a role in the homeostasis of innate and adaptive immune cells. Cytokines are produced by immune cells, such as macrophages, B lymphocytes, T lymphocytes, and mast cells, endothelial cells, fibroblasts, and different stromal cells. In some instances, cytokines modulate the balance between humoral and cell-based immune responses.

[0003] Interleukins are signaling proteins that modulate the development and differentiation of T and B lymphocytes, cells of the monocyte lineage, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by helper CD4 T and B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue residents. Interleukin-2 (IL-2) is a pleiotropic type 1 cytokine whose structure contains a 15.5 kDa four-α helix bundle. The precursor form of IL-2 is 153 amino acid residues long; the first 20 amino acids form the signal peptide, and residues 21–153 form the mature form. IL-2 is produced primarily by CD4+ T cells after antigen stimulation and, to a lesser extent, by CD8+ cells, natural killer (NK) cells, and NK T (NKT) cells, activated dendritic cells (DCs), and mast cells. IL-2 signaling occurs through interaction with a specific combination of IL-2 receptor (IL-2R) subunits, IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122), and IL-2Rγ (also known as CD132). The interaction of IL-2 with IL-2Rα occurs approximately 10 -8 K of M d The interaction of IL-2 with IL-2Rβ and IL-2Rγ forms a "low affinity" IL-2 receptor complex with approximately 10 -9 K of M d The interaction of IL-2 with all three subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ, forms an "intermediate affinity" IL-2 receptor complex with K d is about 10 -11 Forms a "high affinity" IL-2 receptor complex exceeding M.

[0004] In some cases, IL-2 signaling via the "high-affinity" IL-2Rαβγ complex modulates the activation and proliferation of regulatory T cells. Regulatory T cells, such as CD4+CD25+Foxp3+ regulatory T (Treg) cells, mediate immune homeostasis by suppressing effector cells, such as CD8+ T cells; helper cells, such as CD4+ Th1, Th2, and Th17 cells; B cells; NK cells; and NK T cells. In some instances, Treg cells are generated from the thymus (tTreg cells) or derived from peripheral naive T cells (pTreg cells). In some cases, Treg cells are considered the dominant mediator of peripheral tolerance. Indeed, one study showed that transfer of CD25-depleted peripheral CD4+ T cells resulted in various autoimmune diseases in nude mice, whereas cotransfer of CD4+CD25+ T cells suppressed the development of autoimmunity (Sakaguchi et al., J. Immunol. (1995)). Enhancing the Treg cell population downregulates effector T cell proliferation and suppresses autoimmune and T cell antitumor responses. The clinical use of interleukin-2 (IL-2) for the treatment of many diseases and disorders, such as cancer and autoimmune and inflammatory diseases, has been primarily limited by its toxicity and short in vivo half-life (Pachella et al., J Adv Pract Oncol (2015); Lotze et al. (1985) J. Immunol (1985)). Significantly reduced toxicity has been observed in animals lacking CD25 (IL-2 receptor alpha unit, IL-2Rα) (Boyman, et al., J Immunol (2009)). PEGylation, the covalent attachment of polyethylene glycol (PEG) to therapeutic agents, has also been shown in certain instances to overcome certain obstacles, such as rapid body clearance, aggregation, and enzymatic degradation (Maiser et al., Biotechnol Bioeng (2014)). WO 2019 / 028419A1 and WO 2019 / 028425A1 disclose certain interleukin (IL) conjugates (e.g., IL-2 conjugates) and their use in treating certain indications. WO 2019 / 028419A1 and WO 2019 / 028425A1 also describe pharmaceutical compositions and kits comprising one or more interleukin conjugates (e.g., IL-2 conjugates). Despite the existence of specific IL-2 muteins as potential therapeutic molecules, there remains a need for alternative modified IL-2 chemical modalities that offer reduced toxicity, enhanced pharmacokinetic and pharmacodynamic properties, and other attributes compared to their peptide-based counterparts. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 028419 [Patent Document 2] International Publication No. 2019 / 028425 [Non-patent literature]

[0006] [Non-Patent Document 1] Sakaguchi et al., J. Immunol. (1995) [Non-patent document 2] Pachella et al.,J Adv Pract Oncol(2015) [Non-patent document 3] Lotze et al. J. Immunol (1985) [Non-patent document 4] Boyman, et al., J Immunol (2009) [Non-Patent Document 5] Maiser et al.,Biotechnol Bioeng(2014) Summary of the Invention [Means for solving the problem]

[0007] overview In particular, provided are polynucleotides encoding modified interleukin-2 (IL-2) polypeptides and polynucleotides encoding fusion proteins comprising modified IL-2 polypeptides; vectors, such as mRNA vectors, DNA / RNA vectors, DNA vectors, viral vectors, and non-viral vectors carrying such polynucleotides; pharmaceutical compositions comprising such polynucleotides and vectors; and methods for treating or preventing diseases or disorders, such as proliferative diseases or disorders, immunodeficiency diseases or disorders, inflammatory diseases or disorders, autoimmune diseases or disorders; and / or infectious diseases or disorders.

[0008] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, and a combination thereof.

[0009] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is a) unconjugated or conjugated to a water-soluble polymer, lipid, polypeptide, protein, or peptide; and / or b) has reduced binding to interleukin-2 receptor alpha (IL-2Rα) compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without at least one substitution; and / or c) has a reduced receptor signaling potency for IL-2Rαβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without at least one substitution; and / or d) has an increased ratio of signaling potency for IL-2Rβγ to signaling potency for IL-2Rαβγ (increased ratio of signaling potency for IL-2Rβγ / signaling potency for IL-2Rαβγ) compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without at least one substitution; and / or e) has enhanced receptor signaling potency for IL-2Rβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without at least one substitution; and / or with the proviso that when the modified IL-2 polypeptide comprises at least one substitution with a non-naturally occurring amino acid, the modified IL-2 polypeptide comprises at least one substitution at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and a combination thereof, and / or at least one substitution with a natural or non-natural amino acid at at least one position within the IL-2Rα interacting region, the IL-2Rβ interacting region and / or the IL-2Rγ interacting region; f) Includes a combination of a) to e).

[0010] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity in one or more regions of amino acid positions 10-25, 80-100 and / or 100-134 to the corresponding one or more regions of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

[0011] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

[0012] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises at least one substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0013] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the modified IL-2 polypeptide comprising: a) at least one substitution with a naturally occurring amino acid at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof; , N71, Q74, K76 and combinations thereof, and / or b) comprises at least one substitution with a natural amino acid at at least one position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and combinations thereof, and is configured to be conjugated to a water soluble polymer, lipid, protein or peptide at the N-terminus and / or C-terminus of the modified IL-2 polypeptide.

[0014] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises: a) a lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, guanylate ... and / or b) comprising at least one substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76, and combinations thereof.

[0015] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the modified IL-2 polypeptide comprising: a) at least one substitution with cysteine ​​at a position selected from the group consisting of N29, N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76, and combinations thereof; b) at least one substitution with cysteine ​​at a position selected from the group consisting of N29, N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76, and combinations thereof; c) at least one substitution with cysteine ​​at a position selected from the group consisting of Y1, Q2, Q3, Q4, Q5, Q6, Q7, Q8, Q9, Q10, Q11, Q22, Q3, Q4, Q5, Q6, Q7, Q8, Q9, Q12, Q13, Q14, Q15, Q16, Q17, Q18, Q19, Q20, Q21, Q22, Q23, Q24, Q25, Q36, Q37, Q38, Q49, Q50, Q51, Q52, Q53, Q54, Q55, Q66, Q67, Q68, Q69, Q70, Q71, Q72, Q73, Q74, Q85, Q96, Q97, Q10, Q11, Q12, Q13, Q14, Q15, Q16, Q17, Q18, Q21, Q22, Q23, Q24, Q25, Q38, Q49, Q51, Q52, Q63, Q64, Q65, Q76, Q77, Q78, Q79, Q88, Q99, Q91, Q10, Q11, Q12, Q13, Q14, Q15, Q16, Q17, Q26, Q27, Q28, Q39, Q49, Q51, Q52, Q63, Q64, Q75, Q76, Q77, Q89, Q91, Q92, Q93, Q94, Q95, Q10, Q11, Q12, Q13, Q14, Q15, Q16, Q17, Q18, Q29, Q20, Q21, Q22, Q23, Q24, Q25, Q39, Q40

[0016] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises at least one substitution with a naturally occurring amino acid at at least one position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof.

[0017] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises at least one substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at at least one position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof.

[0018] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises: a) at least one substitution with cysteine ​​at a position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof; and / or b) a substitution at position F42 with alanine, lysine, or serine; and / or c) a substitution at position F42 with alanine; and / or d) a substitution at position F42 with serine; and / or e) a substitution at position F42 with lysine; and / or f) substitution at position Y45 with alanine, histidine or serine, and / or g) substitution at position Y45 with alanine, and / or h) substitution at position Y45 with histidine, and / or i) substitution at position R38 with alanine, aspartic acid or serine, and / or j) substitution at position R38 with aspartic acid, and / or k) substitution at position P65 with alanine, and / or l) substitution at position P65 with serine, and / or m) substitution at position E62 with alanine, and / or n) substitution at position F42 with lysine, and / or o) substitution at position Y31 with cysteine, and / or combinations of a) to o).

[0019] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises at least one substitution with a naturally occurring amino acid at at least one position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0020] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises at least one substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0021] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises at least one substitution with cysteine ​​at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0022] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises a) a substitution at position Y31, and / or b) a substitution at position F42, and / or c) a substitution at position C125, and / or d) a substitution at positions Y31, F42, and C125.

[0023] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the modified IL-2 polypeptide comprising a) a substitution at position Y31 with a cysteine, and / or b) a substitution at position F42 with a phenylalanine, and / or c) a substitution at position C125 with a serine, and / or d) a substitution at position Y31 with a cysteine, a substitution at position Y31 with a phenylalanine, and a substitution at position C125 with a serine.

[0024] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises at least one substitution with a natural or unnatural amino acid at at least one position within the IL-2Rα-interacting region, the IL-2Rβ-interacting region, and / or the IL-2Rγ-interacting region.

[0025] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises at least one substitution with a naturally occurring amino acid at a position within the IL-2Rα interacting region.

[0026] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises at least one substitution with a naturally occurring amino acid at a position within the IL-2Rβ interacting region.

[0027] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the modified IL-2 polypeptide comprising a) a substitution with a naturally occurring amino acid at a position within the IL-2Rα interacting region and a substitution with a naturally occurring amino acid at a position within the IL-2Rβ interacting region, b) a substitution with a naturally occurring amino acid at a position within the IL-2Rα interacting region and a substitution with a naturally occurring amino acid at a position within the IL-2Rγ interacting region, or c) a substitution with a naturally occurring amino acid at a position within the IL-2Rα interacting region, a substitution with a naturally occurring amino acid at a position within the IL-2Rβ interacting region and a substitution with a naturally occurring amino acid at a position within the IL-2Rγ interacting region.

[0028] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has reduced binding to IL-2Rα compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution.

[0029] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has an N-terminal deletion and / or a C-terminal deletion.

[0030] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has an N-terminal deletion of amino acid residues 1-30 and / or a C-terminal deletion of amino acid residues 114-134.

[0031]

[0032] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the fusion protein comprises the modified IL-2 polypeptide and an additional amino acid sequence.

[0033] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the fusion protein comprises a modified IL-2 polypeptide linked to an additional amino acid sequence via a linker.

[0034] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the fusion protein comprises a modified IL-2 polypeptide linked to an additional amino acid sequence that confers an enhanced and / or prolonged pharmacokinetic (PK) profile in the modified IL-2 polypeptide or in the fusion protein compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the at least one substitution.

[0035] In certain embodiments (in certain embodiments) that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the fusion protein comprises the modified IL-2 polypeptide linked to an additional amino acid sequence, wherein the additional amino acid sequence is fused to the additional amino acid sequence via the N-terminus of the modified IL-2 polypeptide or via the C-terminus of the modified IL-2 polypeptide.

[0036] In certain embodiments (in certain embodiments) that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the fusion protein comprising the modified IL-2 polypeptide linked to an additional amino acid sequence, the additional amino acid sequence comprising an antibody sequence or a portion or fragment thereof.

[0037] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the fusion protein comprises the modified IL-2 polypeptide linked to an additional amino acid sequence, the additional amino acid sequence comprising an Fc portion of an antibody.

[0038] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the fusion protein comprises the modified IL-2 polypeptide linked to an additional amino acid sequence, wherein the additional amino acid sequence comprises serum albumin or a PK-extended fragment or analog thereof.

[0039] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the fusion protein comprises the modified IL-2 polypeptide linked to an additional amino acid sequence, the additional amino acid sequence comprising: a) human serum albumin (HSA); b) mouse serum albumin (MSA); c) or a PK-extended fragment or analog of a) or b).

[0040] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3.

[0041] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide and / or additional amino acid sequence is further configured to be conjugated to a water-soluble polymer, lipid, protein, or peptide via a single amino acid residue of the modified IL-2 polypeptide.

[0042] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide and / or the additional amino acid sequence is further configured to be conjugated to a water-soluble polymer, lipid, protein, or peptide via i) the alpha-amino group of the N-terminal amino acid residue of the fusion polypeptide, ii) the epsilon-amino group of a lysine amino acid residue of the fusion polypeptide, or iii) an N-glycosylation site or an O-glycosylation site of the fusion polypeptide.

[0043] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide or fusion protein has increased binding to IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution.

[0044] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide or fusion protein has (i) decreased binding to IL-2Rα and (ii) increased binding to IL-2Rβγ, compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution.

[0045] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the binding affinity of the IL-2 polypeptide or fusion protein to IL-2Rα is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or by about 1-fold to about 100,000-fold or more compared to the binding affinity of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution.

[0046] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide or fusion protein does not have detectable binding to IL-2Rα.

[0047] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide or fusion protein has reduced receptor signaling potency for IL-2Rαβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution.

[0048] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the ratio between the signaling potency of the IL-2 polypeptide or fusion protein for IL-2Rαβγ and the signaling potency of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution for IL-2Rαβγ is about 1 / 2 to about 1 / 100,000.

[0049] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide or fusion protein has no detectable receptor signaling efficacy for IL-2Rαβγ.

[0050] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide or fusion protein (i) has reduced binding to IL-2Rα compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution, and (ii) has reduced receptor signaling potency for IL-2Rαβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution.

[0051] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide or fusion protein has no detectable binding to IL-2Rα and no detectable receptor signaling efficacy for IL-2Rαβγ.

[0052] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide or fusion protein has comparable increased binding to interleukin-2 receptor beta (IL-2Rβ) and / or interleukin-2 receptor gamma (IL-2Rγ) compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without at least one substitution, and / or has comparable receptor signaling potency for IL-2Rβγ or has increased receptor signaling potency for IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without at least one substitution.

[0053] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide or fusion protein has a binding level to IL-2Rβ or IL-2Rγ that is equivalent to or a higher binding level to IL-2Rβ or IL-2Rγ than an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution.

[0054] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide or fusion protein has equivalent or greater receptor signaling potency for IL-2Rβγ than an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution.

[0055] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide or fusion protein has a comparable or higher binding level to IL-2Rβ or IL-2Rγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution, and has a comparable receptor signaling potency for IL-2Rβγ or a higher receptor signaling potency for IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution.

[0056] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide or fusion protein has an increased ratio of signaling potency for IL-2Rβγ over signaling potency for IL-2Rαβγ (increased ratio of signaling potency for IL-2Rβγ / signaling potency for IL-2Rαβγ), compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without at least one substitution.

[0057] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide further comprises a promoter operably linked to the nucleic acid sequence encoding the fusion protein.

[0058] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the polynucleotide further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, the promoter driving tissue-specific expression of the fusion protein.

[0059] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the polynucleotide further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, the promoter driving muscle-specific expression of the fusion protein.

[0060] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the polynucleotide further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, wherein the promoter drives cancer-specific or tumor-specific expression of the fusion protein.

[0061] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the polynucleotide further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, the promoter driving liver-specific expression of the fusion protein.

[0062] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the polynucleotide further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, wherein the promoter is constitutively active.

[0063] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the polynucleotide further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, wherein the promoter is conditionally active.

[0064] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide further comprises a promoter operably linked to the nucleic acid sequence, the promoter comprising an SP6, T3, or T7 promoter sequence.

[0065] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide further comprises that at least a portion of the nucleic acid sequence encoding the fusion protein is codon-optimized.

[0066] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide further comprises a polynucleotide comprising at least one modified nucleotide.

[0067] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the polynucleotide further comprising at least one modified nucleotide, wherein the at least one modified nucleotide is pseudouridine, N1-methyl-pseudouridine, or 2-thiouridine.

[0068] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide further comprises a 5' cap structure.

[0069] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide further comprises a 3' polyA sequence.

[0070] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide further comprises DNA; RNA; or a DNA / RNA hybrid.

[0071] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide further comprises RNA.

[0072] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide further comprises a viral vector; a non-viral vector; a plasmid; or a Nanoplasmid™ vector.

[0073] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide is configured to express the fusion protein in vitro.

[0074] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide is configured to express the fusion protein in vivo.

[0075] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide is in isolated form.

[0076] In certain embodiments that may be combined with other embodiments provided herein and throughout, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide or fusion protein is capable of expressing for about 5 minutes to about 10 days, about 5 minutes to about 9 days, about 5 minutes to about 8 days, about 5 minutes to about 8 days, about 5 minutes to about 7 days, about 5 minutes to about 6 days, about 5 minutes to about 5 days, about 5 minutes to about 4 days, about 5 minutes to about 3 days, about 5 minutes to about 2 days, about 5 minutes to about 1 day, about 1 hour to about 10 days, about 1 hour to about 9 days, about 1 hour to about 8 days, about 1 hour to about 7 days, about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 1 day. from about 1 hour to about 2 days, from about 1 hour to about 1 day, from about 5 minutes, from about 10 minutes, from about 20 minutes, from about 30 minutes, from about 40 minutes, from about 50 minutes, from about 1 hour, from about 2 hours, from about 3 hours, from about 4 hours, from about 5 hours, from about 6 hours, from about 7 hours, from about 8 hours, from about 9 hours, from about 10 hours, from about 11 hours, from about 12 hours, from about 13 hours, from about 14 hours, from about 15 hours, from about 16 hours, from about 17 hours, from about 18 hours, from about 19 hours, from about 20 hours, from about 21 hours, from about 22 hours, from about 23 hours, from about 1 day, from about 2 days, from about 3 days, from about 4 days, from about 5 days, from about 6 days, from about 7 days, from about 8 days, from about 9 days, from about 10 days, or any value or range therebetween.

[0077] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier.

[0078] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid.

[0079] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, the lipid having the formula (I): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein: R 1 H, -OR 1A , -YOR 1A , -NR 1A R 1B , -YNR 1A R 1B , -SR 1A , -YSR 1A , -(C=O)R 1A , -Y(C=O)R 1A , -(C=O)OR 1A , -Y(C=O)OR 1A , -O(C=O)R 1A , -YO(C=O)R 1A , -O(C=O)OR 1A , -YO(C=O)OR 1A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Y is a substituted or unsubstituted C0-C 12 alkylene or substituted or unsubstituted 0-12 membered heteroalkylene; R 2 H, -OR 2A , -SR 2A , -(C=O)R 2A , -(C=O)OR 2A , -O(C=O)R 2A , -O(C=O)OR 2A , -(C=O)NHR 2A , -NH(C=O)R 2A , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 3 H, -OR 3A , -SR 3A , -(C=O)R 3A , -(C=O)OR 3A , -O(C=O)R 3A , -O(C=O)OR 3A , -(C=O)NHR 3A , -NH(C=O)R 3A , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 4 H, -OR 4A , -SR 4A , -(C=O)R 4A , -(C=O)OR 4A , -O(C=O)R 4A , -O(C=O)OR 4A , -(C=O)NHR 4A , -NH(C=O)R 4A , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 5 H, -OR 5A , -SR 5A , -(C=O)R5A , -(C=O)OR 5A , -O(C=O)R 5A , -O(C=O)OR 5A , -(C=O)NHR 5A , -NH(C=O)R 5A , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; B 1 is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; B 2 and B 3 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L 1 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 101 R 102 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 101 C(=O)-, -C(=O)NR 101 -, -NR 101 C(=S)-, -C(=S)NR 101 -, -NR 101 C(=O)NR 102 -, -NR 101 C(=S)NR 102 -, -OC(=O)NR 101 -, -NR 101 C(=O)O-, -SC(=O)NR 101 -or-NR 101 C(=O)S-, L 2 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 201 R 202 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 201 C(=O)-, -C(=O)NR201 -, -NR 201 C(=O)NR 202 -, -NR 201 C(=S)-, -C(=S)NR 201 -, -NR 201 C(=S)NR 202 -, -OC(=O)NR 201 -, -NR 201 C(=O)O-, -SC(=O)NR 201 - or -NR 201 C(=O)S-, and L 3 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 301 R 302 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 301 C(=O)-, -C(=O)NR 301 -, -NR 301 C(=O)NR 302 -, -NR 301 C(=S)-, -C(=S)NR 301 -, -NR 301 C(=S)NR 302 -, -OC(=O)NR 301 -, -NR 301 C(=O)O-, -SC(=O)NR 301 - or -NR 301 C(=O)S-, and L 4 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 401 R 402 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 401 C(=O)-, -C(=O)NR 401 -, -NR 401 C(=O)NR 402 -, -NR 401 C(=S)-, -C(=S)NR 401 -, -NR 401 C(=S)NR 402 -, -OC(=O)NR 401 -, -NR 401C(=O)O-, -SC(=O)NR 401 -or-NR 401 C(=O)S-, L 5 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 501 R 502 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 501 C(=O)-, -C(=O)NR 501 -, -NR 501 C(=O)NR 502 -, -NR 501 C(=S)-, -C(=S)NR 501 -, -NR 501 C(=S)NR 502 -, -OC(=O)NR 501 -, -NR 501 C(=O)O-, -SC(=O)NR 501 -or-NR 501 C(=O)S-, L 6 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 601 R 602 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 601 C(=O)-, -C(=O)NR 601 -, -NR 601 C(=O)NR 602 -, -NR 601 C(=S)-, -C(=S)NR 601 -, -NR 601 C(=S)NR 602 -, -OC(=O)NR 601 -, -NR 601 C(=O)O-, -SC(=O)NR 601 -or-NR 601 C(=O)S-, L 7 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 701 R 702 ) sO-, -S-, -C(=O)S-, -SC(=O)-, -NR 701 C(=O)-, -C(=O)NR 701 -, -NR 701 C(=O)NR 702 -, -NR 701 C(=S)-, -C(=S)NR 701 -, -NR 701 C(=S)NR 702 -, -OC(=O)NR 701 -, -NR 701 C(=O)O-, -SC(=O)NR 701 -or-NR 701 C(=O)S-, L a1 and L a2 are each independently [ka] and each X is independently O, S, or CH; W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are each independently a bond or a substituted or unsubstituted C1-C 12 alkylene, or substituted or unsubstituted 2- to 12-membered heteroalkylene; Each R 1A and R 1B are independently H, substituted or unsubstituted C1-C 12 alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; Each R 2A , R 3A , R 4A , and R 5A are independently H, substituted or unsubstituted C1-C 30 alkyl, or substituted or unsubstituted 2- to 30-membered heteroalkyl; Each R 101 , R 102 , R 201 , R 202 , R 301 , R 302 , R401 , R 402 , R 501 , R 502 , R 601 , R 602 , R 701 , and R 702 are independently H, substituted or unsubstituted C1-C 12 alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; Each s is independently an integer from 1 to 4.

[0080] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, the lipid having the formula (I): [ka] Contains cationic lipids of R 1 H, -OR 1A or substituted or unsubstituted heteroalkyl; L 1 is the bond, -NR 101 C(=S)-, -C(=S)NR 101 -, -O(C=O)-, -(C=O)O-, or -O-; B 1 is a bond or substituted or unsubstituted alkylene; B 2 and B 3 are each independently a bond or substituted or unsubstituted alkylene; L 2 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, or -S-; L 4 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, or -S-; W1 , W 2 , W 3 , W 4 , W 5 and W 6 are each independently a bond or a substituted or unsubstituted C1-C 12 is alkylene, L a1 and L a2 are each independently [ka] and each X is independently O or S; L 3 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, or -S-; L 5 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, or -S-; L 6 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, or -S-; L 7 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, or -S-; R 2 is H or substituted or unsubstituted alkyl; R 3 is H or substituted or unsubstituted alkyl; R 4 is H or substituted or unsubstituted alkyl; R 5 is H or substituted or unsubstituted alkyl; Each R 1A are independently H or substituted or unsubstituted C1-C 12 is alkyl, Each R 101 is independently H or substituted or unsubstituted 2-12 membered heteroalkyl.

[0081] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has Formula (I): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein the cationic lipid is 1 is H, —OH, methoxy, ethoxy, or substituted or unsubstituted heteroalkyl; L 1 is the bond, -NR 101 C(=S)- or -C(=S)NR 101 - and B 1 is a bond or unsubstituted C1-C8 alkylene; B 2 and B 3 are each independently a bond or a substituted or unsubstituted C1-C8 alkylene; L 2 is a bond, —O(C═O)—, or —(C═O)O—; L 4 is a bond, —O(C═O)—, or —(C═O)O—; W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are each independently a bond or a substituted or unsubstituted C1 to C 12 is alkylene, L a1 and L a2 However, each independently, [ka] and each X is independently O or S; L 3 is a bond, —O(C═O)—, or —(C═O)O—; L 5 is a bond, —O(C═O)—, or —(C═O)O—; L 6 is a bond, —O(C═O)—, or —(C═O)O—; L 7 is a bond, —O(C═O)—, or —(C═O)O—; R 2 is H or substituted or unsubstituted C1-C 12 is alkyl, R 3 is H or substituted or unsubstituted C1-C 12 is alkyl, R 4 is H or substituted or unsubstituted C1-C 12 is alkyl, R 5 is H or substituted or unsubstituted C1-C 12 is alkyl, Each R 101 are independently substituted or unsubstituted 2- to 12-membered heteroalkyl.

[0082] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has Formula (I): [ka] ,

[0083] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein the cationic lipid is 1is —OH or methoxy; L 1 is a bond, B 1 is unsubstituted C1-C8 alkylene; B 2 and B 3 are each independently a bond or a substituted or unsubstituted C1-C8 alkylene;

[0084] L 2 is a bond, L 4 is a bond, W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are each independently a bond or a substituted or unsubstituted C1 to C 12 is alkylene, L a1 and L a2 However, each independently, [ka] and each X is independently O; L 3 is a bond, L 5 is a bond, L 6 is a bond, L 7 is a bond, R 2 is H or substituted or unsubstituted C1-C 12 is alkyl, R 3 is H or substituted or unsubstituted C1-C 12 is alkyl, R 4 is H or substituted or unsubstituted C1-C 12 is alkyl, R 5 is H or substituted or unsubstituted C1-C 12It is an alkyl, it is a lipid.

[0085] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has Formula (I): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein the cationic lipid is 1 is substituted or unsubstituted heteroalkyl; L 1 But -C(=S)NR 101 -, where the carbon atom is connected to the nitrogen atom of formula (I), B 1 is a bond, B 2 and B 3 are each independently a bond or a substituted or unsubstituted C1-C8 alkylene; L 2 is a bond, —O(C═O)—, or —(C═O)O—; L 4 is a bond, —O(C═O)—, or —(C═O)O—; W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are each independently a bond or a substituted or unsubstituted C1 to C 12 is alkylene, L a1 and L a2 However, each independently, [ka] and each X is independently O; L 3 is a bond, L 5 is a bond, L 6 is a bond, L 7 is a bond, R 2 is H or substituted or unsubstituted C1-C 12 is alkyl, R 3 is H or substituted or unsubstituted C1-C 12 is alkyl, R 4 is H or substituted or unsubstituted C1-C 12 is alkyl, R 5 is H or substituted or unsubstituted C1-C 12 It is an alkyl, it is a lipid.

[0086] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has Formula (I): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein the cationic lipid is [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0087] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (II): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein: B 4 is W 7 -L a3 -W 8 and W 7 and W 8 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L a3 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR a31 R a32 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR a31 C(=O)-, -C(=O)NR a31 -, -NR a31 C(=O)NR a32 -, -NR a31 C(=S)-, -C(=S)NR a31 -, -NR a31 C(=S)NR a32 -, -OC(=O)NR a31 -, -NR a31 C(=O)O-, -SC(=O)NR a31 -or-NR a31 C(=O)S-, R 10 and R 11 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or R10 and R 11 together with the nitrogen atom to which they are attached form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl; B 5 , B 6 , and B 7 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L 8 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 801 R 802 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 801 C(=O)-, -C(=O)NR 801 -, -NR 801 C(=O)NR 802 -, -NR 801 C(=S)-, -C(=S)NR 801 -, -NR 801 C(=S)NR 802 -, -OC(=O)NR 801 -, -NR 801 C(=O)O-, -SC(=O)NR 801 -or-NR 801 C(=O)S-, L 9 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 901 R 902 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 901 C(=O)-, -C(=O)NR 901 -, -NR 901 C(=O)NR 902 -, -NR 901 C(=S)-, -C(=S)NR 901 -, -NR 901 C(=S)NR 902 -, -OC(=O)NR 901 -, -NR 901 C(=O)O-, -SC(=O)NR 901-or-NR 901 C(=O)S-, L 10 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 110 R 111 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 110 C(=O)-, -C(=O)NR 110 -, -NR 110 C(=O)NR 111 -, -NR 110 C(=S)-, -C(=S)NR 110 -, -NR 110 C(=S)NR 111 -, -OC(=O)NR 110 -, -NR 110 C(=O)O-, -SC(=O)NR 110 -or-NR 110 C(=O)S-, R 7 , R 8 , and R 9 are each independently H, substituted or unsubstituted C1-C 30 alkyl, or substituted or unsubstituted 2- to 30-membered heteroalkyl; Each R a31 and R a32 are independently H, substituted or unsubstituted C1-C 12 alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; Each R 801 , R 802 , R 901 , R 902 , R 110 , and R 111 are independently H, substituted or unsubstituted C1-C 12 alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; Each s is independently an integer from 1 to 4.

[0088] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (II): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein the cationic lipid is of the formula: W 7 and W 8 are each independently a bond or substituted or unsubstituted alkylene; L a3 is a bond, R 10 and R 11 are each independently H, substituted or unsubstituted alkyl, or R 10 and R 11 together with the nitrogen atom to which they are attached form a substituted or unsubstituted heterocycloalkyl; B 5 is a bond, B 6 and B 7 are each independently a bond or substituted or unsubstituted alkylene; L 8 is a bond, L 9 is a bond, -O(C=O)-, -(C=O)O-, or -C(=O)-; L 10 is a bond, -O(C=O)-, -(C=O)O-, or -C(=O)-; R 7 , R 8 , and R 9 are each independently H or substituted or unsubstituted C1-C 30 It is an alkyl, it is a lipid.

[0089] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (II): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein W 7 and W 8 are each independently a bond or a substituted or unsubstituted C1-C8 alkylene; L a3 is a bond, R 10 and R 11 are each independently substituted or unsubstituted alkyl, or R 10 and R 11 together with the nitrogen atom to which they are attached form a substituted or unsubstituted heterocycloalkyl; B 5 is a bond, B 6 and B 7 are each independently a bond or a substituted or unsubstituted C1-C8 alkylene; L 8 is a bond, L 9 is -O(C=O)- or -(C=O)O-, L 10 is -O(C=O)- or -(C=O)O-, R 7 , R 8 , and R 9 are each independently substituted or unsubstituted C1 to C 20 It is alkyl.

[0090] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (II): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein W 7 and W 8 are each independently a bond or a substituted or unsubstituted C2-C4 alkylene; L a3 is a bond, R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3- to 8-membered heterocycloalkyl; B 5 is a bond, B 6 and B 7 are each independently a bond or a substituted or unsubstituted C2-C4 alkylene; L 8 is a bond, L 9 is -O(C=O)- or -(C=O)O-, L 10 is -O(C=O)- or -(C=O)O-, R 7 is H or methyl, R 8 , and R 9 are each independently substituted or unsubstituted C1 to C 20 It is alkyl.

[0091] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (II): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein W 7 and W 8 are each independently a bond or unsubstituted C2-C4 alkylene; L a3 is a bond, R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are attached form a substituted or unsubstituted 5- to 6-membered heterocycloalkyl; B 5 is a bond, B 6 and B 7 are each independently a bond or unsubstituted C2-C4 alkylene; L 8 is a bond, L 9 is -O(C=O)- or -(C=O)O-, L 10 is -O(C=O)- or -(C=O)O-, R 7 is H or methyl, R 8 , and R 9 are each independently substituted or unsubstituted C1 to C 20It is alkyl.

[0092] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (II): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein W 7 and W 8 are each independently a bond or unsubstituted C2-C4 alkylene; L a3 is a bond, R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are attached form a substituted or unsubstituted 5- to 6-membered heterocycloalkyl; B 5 , B 6 , and B 7 are each independently a bond, L 8 is a bond, L 9 is a bond, L 10 is a bond, R 7 is H or methyl, R 8 , and R 9 are each independently substituted or unsubstituted C1 to C 30 It is alkyl.

[0093] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (II): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein the cationic lipid is [ka] [ka] [ka] , or a pharmaceutically acceptable salt thereof.

[0094] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (III): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein: L 11 teeth, [ka] and Q is substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene; V is substituted or unsubstituted alkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted arylene; B 8 , B 9 , B 10 , and B 11 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L 12 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 210 R 211 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 210 C(=O)-, -C(=O)NR 210 -, -NR 210 C(=O)NR 211 -, -NR 210 C(=S)-, -C(=S)NR 210 -, -NR 210 C(=S)NR 211 -, -OC(=O)NR 210 -, -NR 210 C(=O)O-, -SC(=O)NR 210 -or-NR 210 C(=O)S-, L 13 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 310 R 311 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 310 C(=O)-, -C(=O)NR 310 -, -NR 310 C(=O)NR 311 -, -NR 310 C(=S)-, -C(=S)NR 310 -, -NR310 C(=S)NR 311 -, -OC(=O)NR 310 -, -NR 310 C(=O)O-, -SC(=O)NR 310 -or-NR 310 C(=O)S-, R 12 H, -OR 12A , -SR 12A , -NR 12A , -CN, -(C=O)R 12A , -O(C=O)R 12A , -(C=O)OR 12A , -NR 12A (C=O)-R 12B , -(C=O)NR 12A R 12B and R 13 H, -OR 13A , -SR 13A , -NR 13A , -CN, -(C=O)R 13A , -O(C=O)R 13A , -(C=O)OR 13A , -NR 13A (C=O)-R 13B , -(C=O)NR 13A R 13B and R 14 and R 15 are each independently substituted or unsubstituted C2 to C 30 alkyl, or substituted or unsubstituted 2- to 30-membered heteroalkyl; R 12A , R 12B , R 13A , and R 13B are each independently H, substituted or unsubstituted C1-C 20 alkyl, or substituted or unsubstituted 2- to 20-membered heteroalkyl; Each R 210 , R 211 , R 310 , and R 311 are independently H, substituted or unsubstituted C1-C 12alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; each n is independently an integer from 0 to 8; Each s is independently an integer from 1 to 4.

[0095] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (III): [ka] ,

[0096] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein: L 11 teeth, [ka] and Q is a substituted or unsubstituted alkylene; V is a substituted or unsubstituted alkylene; B 8 , B 9 , B 10 , and B 11 are each independently substituted or unsubstituted alkylene; L 12 is -O(C=O)- or -(C=O)O-, L 13 is -O(C=O)- or -(C=O)O-, R 12 H, -OR 12A , or -NR 12A and R 13 H, -OR 13A , or -NR 13A and R 14 and R 15 are each independently substituted or unsubstituted C2 to C 30 is alkyl, R 12A and R 13A are each independently H, substituted or unsubstituted C1-C 20 is alkyl, Each n is independently an integer from 0 to 8.

[0097] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (III): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein: L 11 teeth, [ka] and V is a substituted or unsubstituted alkylene; B 8 , B 9 , B 10 , and B 11 are each independently substituted or unsubstituted C1 to C 20 is alkylene, L 12 is -O(C=O)- or -(C=O)O-, L 13 is -O(C=O)- or -(C=O)O-, R 12 is H, or -OR 12A and R13 is H, or -OR 13A and R 14 and R 15 are each independently substituted or unsubstituted C2 to C 20 is alkyl, R 12A and R 13A are each independently H, substituted or unsubstituted C1-C8 alkyl, Each n is independently an integer of 0 to 4.

[0098] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (III): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein: L 11 teeth, [ka] and V is unsubstituted alkylene; B 8 , B 9 , B 10 , and B 11 are each independently a substituted or unsubstituted C1-C8 alkylene; L 12 is -O(C=O)- or -(C=O)O-, L 13 is -O(C=O)- or -(C=O)O-, R 12 is —OH, methoxy, or ethoxy; R13 is —OH, methoxy, or ethoxy; R 14 and R 15 are each independently substituted or unsubstituted C2 to C 20 is alkyl, Each n is independently an integer of 0 to 4.

[0099] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has the formula (III): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein the cationic lipid is [ka] , or a pharmaceutically acceptable salt thereof.

[0100] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has Formula (IV): [ka] , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein: B 12-W 7 -L a3 -W 8 - and W 7 and W 8 are each independently a bond, a substituted or unsubstituted C1 to C 12 alkylene, or substituted or unsubstituted 2- to 12-membered heteroalkylene; L a3 is a bond, -SS-, -O-(CHO) m -, [ka] and W 9 and W 10 are each independently a bond, a substituted or unsubstituted C1 to C 12 alkylene, substituted or unsubstituted 2- to 12-membered heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, or any combination thereof; L 14 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 410 R 411 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 410 C(=O)-, -C(=O)NR 410 -, -NR 410 C(=O)NR 411 -, -NR 410 C(=S)-, -C(=S)NR 410 -, -NR 410 C(=S)NR 411 -, -OC(=O)NR 410 -, -NR 410 C(=O)O-, -SC(=O)NR 410 -or-NR 410 C(=O)S-, L 15 is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR 510 R511 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR 510 C(=O)-, -C(=O)NR 510 -, -NR 510 C(=O)NR 511 -, -NR 510 C(=S)-, -C(=S)NR 510 -, -NR 510 C(=S)NR 511 -, -OC(=O)NR 510 -, -NR 510 C(=O)O-, -SC(=O)NR 510 -or-NR 510 C(=O)S-, R 16 and R 17 are each independently [ka] A fragment of a cationic lipid of formula (I): [ka] A fragment of a cationic lipid of formula (II): [ka] A fragment of a cationic lipid of formula (II): [ka] a fragment of a cationic lipid of formula (III), or [ka] is a fragment of a cationic lipid of formula (III), Each R 410 , R 411 , R 510 , and R 511 are independently H, substituted or unsubstituted C1-C 12 alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; each m is independently an integer from 0 to 8; Each s is independently an integer from 1 to 4.

[0101] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has Formula (IV): [ka] : wherein: L a3 is the bond, -SS-, [ka] and W 7 and W 8 are each independently a bond or a substituted or unsubstituted C1-C 12 is alkylene, L 14 -O(C=O)-, -(C=O)O-, -C(=O)-, -NR 410 C(=O)-, -C(=O)NR 410 -, -NR 410 C(=S)-, -C(=S)NR 410 -, -OC(=O)NR 410 -, or -NR 410 C(=O)O-, L 15 -O(C=O)-, -(C=O)O-, -C(=O)-, -NR 510 C(=O)-, -C(=O)NR 510 -, -NR 510 C(=S)-, -C(=S)NR 510 -, -OC(=O)NR 510 -, or -NR 510 C(=O)O-, W 9 and W 10 are each independently a bond or a substituted or unsubstituted C1-C 12 is alkylene, R 16 and R 17 are each independently [ka] is a fragment of a cationic lipid of formula (II), Each R 410 and R 510 are independently H or substituted or unsubstituted C1-C 12 It is alkyl.

[0102] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has Formula (IV): [ka] , wherein the cationic lipid comprises a cationic lipid of the formula: L a3 But the bond, -SS-, [ka] and W 7 and W 8 are each independently a bond or an unsubstituted C1 to C 12 is alkylene, L 14 is -O(C=O)-, -(C=O)O-, -NR 410 C(=S)-, -C(=S)NR 410 -, -OC(=O)NR 410 -, or -NR 410 C(=O)O-, L 15 is -O(C=O)-, -(C=O)O-, -NR 510 C(=S)-, -C(=S)NR 510 -, -OC(=O)NR 510 -, or -NR 510 C(=O)O-, W 9 and W 10 are each independently a bond or a substituted or unsubstituted C1 to C 12 is alkylene, R 16 and R 17 However, each independently, [ka] is a fragment of a cationic lipid of formula (II), Each R 410 and R 510 are independently H or substituted or unsubstituted C1-C 12 It is an alkyl, it is a lipid.

[0103] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has Formula (IV): [ka] , wherein the cationic lipid comprises a cationic lipid of the formula: L a3 But the bond, -SS-, [ka] and W 7 and W 8 are each independently a bond or unsubstituted C1-C8 alkylene; L 14 is -O(C=O)-, -(C=O)O-, -NR 410 C(=S)-, -C(=S)NR 410 -, -OC(=O)NR 410 -, or -NR 410 C(=O)O-, L 15 is -O(C=O)-, -(C=O)O-, -NR 510 C(=S)-, -C(=S)NR 510 -, -OC(=O)NR 510 -, or -NR 510 C(=O)O-, W 9 and W 10 are each independently a bond or unsubstituted C1-C8 alkylene; R 16 and R 17 However, each independently, [ka] is a fragment of a cationic lipid of formula (II), Each R 410 and R 510 are independently H or unsubstituted C1-C8 alkyl.

[0104] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has Formula (IV): [ka] wherein: L a3 But the bond, -SS-, [ka] and W 7 and W 8 are each independently a bond or unsubstituted C1-C8 alkylene; L 14 -O(C=O)-, -(C=O)O-, -NR 410 C(=S)-, -C(=S)NR 410 -, -OC(=O)NR 410 -, or -NR 410 C(=O)O-, L 15 -O(C=O)-, -(C=O)O-, -NR 510 C(=S)-, -C(=S)NR 510 -, -OC(=O)NR 510 -, or -NR 510 C(=O)O-, W 9 and W 10 are each independently a bond or unsubstituted C1-C8 alkylene; R 16 and R 17 However, each independently, [ka] and Each R 410 and R 510 are independently H or methyl.

[0105] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a lipid, and the lipid has Formula (IV): [ka] , The cationic lipid comprises a cationic lipid of [ka] [ka] [ka] , or a pharmaceutically acceptable salt thereof.

[0106] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition comprises the following compound: [ka]

[0107] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition further comprises lipid nanoparticles.

[0108] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition further comprises at least a second therapeutic agent.

[0109] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, and a pharmaceutically acceptable carrier as described herein and throughout, wherein the pharmaceutical composition further comprises at least a second therapeutic agent, wherein the at least second therapeutic agent comprises an anti-neoplastic agent or substance, an immuno-oncology (IO) agent, an immune checkpoint inhibitor, and / or an antibody-dependent cell-mediated cytotoxic agent, and / or a cell therapy agent.

[0110] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, a pharmaceutically acceptable carrier, wherein the pharmaceutical composition further comprises at least a second therapeutic agent, wherein the at least second therapeutic agent comprises an anti-PD1 antibody, an anti-PDL1 antibody, an anti-EGFR antibody, and an anti-Her2 antibody, as well as an anti-CD20 antibody, an anti-CD38 antibody, an anti-CD47 antibody, an anti-CD123 antibody, an anti-cMET antibody, an anti-CCR4 antibody, an anti-CTLA4 antibody, a chimeric antigen receptor T cell (CAR-t), or a dimeric antigen receptor T cell (DAR-T).

[0111] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, wherein the polynucleotide or pharmaceutical composition is for use in a method of treating or preventing a proliferative disorder in a subject.

[0112] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing a proliferative disease or disorder in a subject having, suspected of having, or at risk of having a proliferative disease or disorder, comprising administering to the subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier.

[0113] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing a proliferative disease or disorder in a subject having, suspected of having, or at risk of having a proliferative disease or disorder, comprising administering to the subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, wherein the proliferative disease or disorder comprises a tumor.

[0114] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing a proliferative disease or disorder in a subject having, suspected of having, or at risk of having a proliferative disease or disorder, comprising administering to the subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, wherein the proliferative disease or disorder comprises cancer.

[0115] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing a proliferative disease or disorder in a subject having, suspected of having, or at risk of having a proliferative disease or disorder, comprising administering to the subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, wherein the proliferative disease or disorder comprises a solid tumor.

[0116] In certain embodiments, which may be combined with other embodiments provided herein and throughout, a method for treating or preventing a proliferative disease or disorder in a subject having, suspected of having, or at risk of having a proliferative disease or disorder comprises administering an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier. and administering to a subject an effective amount of a pharmaceutical composition comprising: and wherein the proliferative disease or disorder is selected from the group consisting of chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone / osteosarcoma, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brain stem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, visual pathway and hypothalamic glioma, breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, male breast cancer, phyllodes tumor, inflammatory breast cancer, adrenocortical carcinoma, islet cell carcinoma (endocrine pancreatic cancer). liver), multiple endocrine neoplasia syndrome, parathyroid cancer, pheochromocytoma, thyroid cancer, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, bile duct cancer, carcinoid tumor, gastrointestinal cancer, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), hepatocellular carcinoma, pancreatic cancer, pancreatic islet cell carcinoma, rectal cancer, colorectal cancer, bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumor, ovarian cancer, ovarian epithelial cancer (surface epithelial stromal tumor), ovarian germ cell tumor, fallopian tube cancer, peritoneal Cancer of the penis, renal cell carcinoma (RCC), renal pelvis and ureter cancer, transitional cell carcinoma, prostate cancer, testicular cancer, gestational trophoblastic tumor, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, esophageal cancer, head and neck cancer, nasopharyngeal carcinoma, oral cavity cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, melanoma, skin cancer (non-melanoma), bronchial adenoma / carcinoid, small cell lung cancer, mesothelioma, non-small cell lung cancer (NSCLC), pleuropulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, AIDS-related cancer, Kaposi's sarcoma,The method is provided, wherein the tumor is selected from the group consisting of epithelioid hemangioendothelioma (EHE), desmoplastic small round cell tumor, and liposarcoma.

[0117] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing a proliferative disease or disorder in a subject having, suspected of having, or at risk of having a proliferative disease or disorder, comprising administering to the subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier, wherein the proliferative disease or disorder comprises a tumor, and the tumor or cancer comprises a hematological malignancy.

[0118] In certain embodiments that may be combined with other embodiments provided herein and throughout, methods are provided for treating or preventing a proliferative disease or disorder in a subject having, suspected of having, or at risk of having a proliferative disease or disorder, the methods comprising administering an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier. and the proliferative disease or disorder is myeloid neoplasm, leukemia, lymphoma, Hodgkin's lymphoma, classical Hodgkin's lymphoma, non-Hodgkin's lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, hepatosplenic T-cell lymphoma, B-cell lymphoma reticuloendotheliosis, reticulosis, microglioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodular marginal zone B-cell lymphoma, Lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, nodular lymphocyte-predominant Hodgkin's lymphoma, plasma cell leukemia, acute erythroleukemia and erythroleukemia, acute erythrocytic myelopathy, acute erythrocytic leukemia, Heilmeyer-Schoner disease, acute megakaryoblastic leukemia, mast cell leukemia, panmyelosis, acute panmyelosis with myelofibrosis, lymphosarcoma cell leukemia, acute leukemia of unspecified cell type, blast phase chronic myeloid leukemia, stem cell leukemia, chronic leukemia of unspecified cell type, subacute leukemia of unspecified cell type, accelerated phase chronic myeloid leukemia, acute Myeloid leukemia, polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, adult T-cell leukemia / lymphoma, aggressive NK cell leukemia, B-cell prolymphocytic leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, chronic idiopathic myelofibrosis, multiple myeloma, Kahler's disease, myelomatosis, solitary myeloma, plasma cell leukemia,Hematological malignancies selected from the group consisting of plasmacytoma, extramedullary, malignant plasma cell neoplasm NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative lesion, lymphocytic granulomatosis, angioimmunoblastic lymphadenopathy, T-gamma lymphoproliferative disorder, Waldenstrom's macroglobulinemia, alpha heavy chain disease, gamma heavy chain disease, Franklin disease, immunoproliferative small intestinal disease, Mediterranean disease, malignant immunoproliferative disorder, not otherwise specified, and immunoproliferative disorder NOS.

[0119] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing an immunodeficiency disease or disorder in a subject having, suspected of having, or at risk of having an immunodeficiency disease or disorder, the method comprising administering to the subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier.

[0120] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing an immunodeficiency disease or disorder in a subject having, suspected of having, or at risk of having, an immunodeficiency disease or disorder, the method comprising administering to the subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier, the immunodeficiency disease or disorder being selected from the group consisting of agammaglobulinemia: X-linked and autosomal recessive, ataxia-telangiectasia, chronic granulomatous disease and other phagocytic disorders, common variable immunodeficiency, Selected from the group consisting of complement deficiency, DiGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyper-IgE syndrome, hyper-IgM syndrome, IgG subclass deficiency, congenital immune deficiency, NEMO deficiency syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immunodeficiency and combined immunodeficiency, specific antibody deficiency, transient hypogammaglobulinemia of infancy, WHIM syndrome (warts, hypogammaglobulinemia, infections and myelocatechus), Wiskott-Aldrich syndrome, other antibody deficiency disorders, other primary cellular immunodeficiency disorders, severe combined immunodeficiency (SCID), common variable immunodeficiency (CVID), human immunodeficiency virus / acquired immunodeficiency syndrome (HIV / AIDS), drug-induced immunodeficiency, graft-versus-host syndrome, primary immunodeficiency disease (PIDD), and lymphopenia.

[0121] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing an autoimmune or inflammatory disease or disorder in a subject having, suspected of having, or at risk of having an autoimmune or inflammatory disease or disorder, the method comprising administering to the subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier.

[0122] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing an autoimmune or inflammatory disease or disorder in a subject having, suspected of having, or at risk of having an autoimmune or inflammatory disease or disorder, the method comprising administering to the subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, wherein the autoimmune or inflammatory disease or disorder is selected from the group consisting of inflammation, autoimmune disease, paraneoplastic autoimmune disease, cartilage inflammation, fibrotic disease and / or bone degradation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reter's syndrome, and the like. Syndrome), SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodossa, Wegener's granulomatosis, arteritis, polymyalgia rheumaticarheumatica), sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barré disease, type 1 diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, and transplant rejection.

[0123] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing an infectious disease or disorder in a subject having, suspected of having, or at risk of having an infectious disease or disorder, comprising administering to the subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier.

[0124] In certain embodiments that may be combined with other embodiments provided herein and throughout, methods are provided for treating or preventing an infectious disease or disorder in a subject having, suspected of having, or at risk of having the infectious disease or disorder, the methods comprising administering an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or a combination of such a polynucleotide and a drug as described herein and throughout. and a pharmaceutical acceptable carrier, wherein the infectious disease or disorder is selected from the group consisting of Acinetobacter infection, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (acquired immune deficiency syndrome), amebiasis, anaplasmosis, Angiostrongyliasis cantonensis, anisakiasis, anthrax, Arcanobacterium cantonensis infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, and balanchiosis. Diarrhea, Bartonellosis, Baylis-ascaris infection, BK virus infection, Black sand lice disease, Blastocystosis, Blastomycosis, Bolivian hemorrhagic fever, Botulism (and infant botulism), Brazilian hemorrhagic fever, Brucellosis, Bubonic plague, Burkholderia infection, Buruli ulcer, Calicivirus infection (Norovirus and Sapovirus), Campylobacteriosis, Candidiasis (Moniliasis, Thrush), Capillariasis, Carrion disease, Cat scratch disease, Cellulitis, Chagas disease (American trypanosomiasis), Chancroid, Chickenpox, Chikungunya, Chlamydia, Chlamydophila pneumoniae infection (Taiwanese acute respiratory pathogen or TWAR), cholera, chromoblastomycosis, frog chytridiomycosis, opisthorchiasis, Clostridium difficile colitis, coccidioidomycosis, Colorado tick fever (CTF), common cold (acute viral nasopharyngitis; acute coryza), coronavirus disease 2019 (COVID-19), Creutzfeldt-Jakob disease (CJD), Crimean-Congo hemorrhagic fever (CCHF), cryptococcosis, cryptosporidiosis, cutaneous larva migrans (CLM), cyclosporosis, cysticercosis, cytomegalovirus infection, dengue fever,Desmodesmus infection, dienteramebiasis, diphtheria, diphyllobothriasis, dracunculosis, Ebola hemorrhagic fever, echinococcosis, ehrlichiosis, enterobiasis (pinworm infection), enterococcus infection, enterovirus infection, typhus, erythema infectiosum (fifth disease), exanthema subitum (sixth disease), fascioliasis, fascioliasis, fatal familial insomnia (FFI), filariasis, Clostridium perfringens perfringens food poisoning, free-living amebic infections, Fusobacterium infections, gas gangrene (clostridial myonecrosis), geotrichosis, Gerstmann-Straussler-Scheinker syndrome (GSS), giardiasis, glanders, gnathostomiasis, gonorrhea, granuloma venereum (donovanosis), group A streptococcal infections, group B streptococcal infections, Haemophilus influenzae infections, hand, foot, and mouth disease (HFMD), hantavirus pulmonary syndrome (HPS), Heartland virus disease, Helicobacter pylori infection, hemolytic uremic syndrome (HUS), hemorrhagic fever with renal syndrome (HFRS), Hendra virus infection, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex, histoplasmosis, hookworm infection, human bocavirus infection, human Ewingia-ehrlichiosis ehrlichiosis, human granulocytic anaplasmosis (HGA), human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus (HPV) infection, human parainfluenza virus infection, membranous tapeworm disease, Epstein-Barr virus infectious mononucleosis (infectious mononucleosis), influenza (flu), isosporosis, Kawasaki disease, keratitis, Kingella kingae infection, kuru, Lassa fever, Legionnaires' disease (legionnaires' disease), Pontiac fever, Leishmaniasis leprosy, leprosy, leptospirosis, listeriosis, Lyme disease (Lyme borreliosis), lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever (MHF), measles, Middle East respiratory syndrome (MERS), melioidosis (Whitmore's disease), meningitis, meningococcal disease, treponema, microsporidiosis, molluscum contagiosum (MC), monkeypox, mumps, murine typhoid (endemic typhoid), mycoplasma pneumonia, Mycoplasma genitalium infection, mycetoma, myiasis,Neonatal conjunctivitis (ophthalmia neonatorum), Nipah virus infection, Norovirus (children and infants), (new) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (river blindness), Opisthorchiasis, Paracoccidioidomycosis (South American blastocyst), Paragonimiasis, Pasteurellosis, Head lice, Body lice, Pubic lice, Pelvic inflammatory disease (PID), Pertussis cough), plague, pneumococcal infection, Pneumocystis pneumonia (PCP), pneumonia, poliomyelitis, Prevotella infection, primary amebic meningoencephalitis (PAM), progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, relapsing fever, respiratory syncytial virus infection, rhinosporidiosis, rhinovirus infection, rickettsial infection, rickettsialpox, Rift Valley fever (RVF), Rocky Mountain spotted fever (RMSF), rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, scarlet fever, schistosomiasis, septicemia, Shigellosis (bacillary dysentery), shingles (herpes zoster) zoster), smallpox (pox), sporotrichosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, subacute sclerosing panencephalitis, bejel, syphilis, and staphyloma, taeniasis, tetanus (lockjaw), tinea barbae (barber's rash), Tinea capitis (ringworm of the scalp), Tinea corporis (ringworm of the body), Tinea cruris (jock itch), Tinea manum (ringworm of the hand), Tinea nigricans, Tinea pedis (athlete's foot), Tinea unguium (onychomycosis), Tinea versicolor (Pityriasis) versicolor), toxocariasis (ocular larva migrans (OLM)), toxocariasis (visceral larva migrans (VLM)), toxoplasmosis, trachoma, trichinosis, trichomoniasis, trichuriasis (whipworm infection), tuberculosis, tularemia, typhoid fever, typhus, Ureaplasma urealyticum infection, valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever,The infection is selected from the group consisting of Vibrio vulnificus infection, Vibrio parahaemolyticus enteritis, viral pneumonia, West Nile fever, ringworm, Yersinia pseudotuberculosis infection, yersiniosis, yellow fever, Zeaspora, Zika fever, and zygomycosis.

[0125] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided the use of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or the use of an effective amount of a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, for the manufacture of a medicament for treating or preventing a proliferative disease or disorder, an autoimmune or inflammatory disease or disorder, or an infectious disease or disorder in a subject.

[0126] In certain embodiments that may be combined with other embodiments provided herein and throughout, there are provided methods and uses described herein and throughout, which independently comprise administering to a subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide described herein and throughout and a pharmaceutically acceptable carrier, wherein the subject is a human.

[0127] In certain embodiments that may be combined with other embodiments provided herein and throughout, there are provided methods and uses described herein and throughout, which independently comprise administering to a subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide described herein and throughout and a pharmaceutically acceptable carrier, wherein the subject is a non-human mammal.

[0128] In certain embodiments, which may be combined with other embodiments provided herein and throughout, CD4 + Helper cells, CD8 + Methods are provided for expanding effector naive and memory cell, T regulatory (Treg) cell, natural killer (NK) cell, or natural killer T (NKT) cell populations, comprising contacting the cell population with an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, for a time sufficient to induce the formation of a complex with IL-2Rβγ, thereby stimulating the expansion of the T cell, NK cell, and / or NKT cell population.

[0129] In certain embodiments, which may be combined with other embodiments provided herein and throughout, CD4 + Helper cells, CD8 +Provided are methods for expanding effector naive and memory cell, T regulatory (Treg) cell, natural killer (NK) cell, or natural killer T (NKT) cell populations, comprising contacting the cell population with an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, for a time sufficient to induce the formation of a complex with IL-2Rβγ, thereby stimulating expansion of the T cell, Treg cell, NK cell, and / or NKT cell population with a 10% to 100% reduction in cell death.

[0130] In certain embodiments, which may be combined with other embodiments provided herein and throughout, CD4 + Helper cells, CD8 + 1. A method for expanding effector naive and memory cell, T regulatory (Treg) cell, natural killer (NK) cell or natural killer T (NKT) cell populations, comprising contacting the cell population with an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein is a fusion protein comprising an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without at least one substitution. + CD4 in cell populations + Compared with the expansion of Treg cells, CD3 + In the cell population, CD4 + Methods are provided that expand T Treg cells by less than 20%, 15%, 10%, 5%, 1% or less.

[0131] In certain embodiments, which may be combined with other embodiments provided herein and throughout, CD4 +Helper cells, CD8 + A method for expanding a population of effector naive and memory cells, T regulatory (Treg) cells, natural killer (NK) cells, or natural killer T (NKT) cells, comprising contacting the cell population with an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or a pharmaceutical composition comprising such a polynucleotide as described herein and throughout, and a pharmaceutically acceptable carrier, wherein the polynucleotide or pharmaceutical composition increases the CD4+ expression levels in the cell population. + Methods are provided that do not substantially expand Treg cells.

[0132] In certain embodiments, which may be combined with other embodiments provided herein and throughout, CD4 + Helper cells, CD8 + Provided are methods for expanding effector naive and memory cell, T regulatory (Treg) cell, natural killer (NK) cell or natural killer T (NKT) cell populations, comprising contacting the cell population with an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, wherein the ratio of effector T cells to Treg cells in the cell population after incubation with the polynucleotide or pharmaceutical composition is about or at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, 100:1 or more.

[0133] In certain embodiments, which may be combined with other embodiments provided herein and throughout, CD4 + Helper cells, CD8 +Methods are provided for expanding effector naive and memory cell, T regulatory (Treg) cell, natural killer (NK) cell, or natural killer T (NKT) cell populations, which are carried out in vivo and comprise contacting the cell population with an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier.

[0134] In certain embodiments, which may be combined with other embodiments provided herein and throughout, CD4 + Helper cells, CD8 + Methods are provided for expanding effector naive and memory cell, T regulatory (Treg) cell, natural killer (NK) cell, or natural killer T (NKT) cell populations, which are performed in vitro and comprise contacting the cell population with an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier.

[0135] In certain embodiments, which may be combined with other embodiments provided herein and throughout, CD4 + Helper cells, CD8 + Methods are provided for expanding effector naive and memory cell, T regulatory (Treg) cell, natural killer (NK) cell, or natural killer T (NKT) cell populations, which are performed ex vivo and comprise contacting the cell population with an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier.

[0136] In certain embodiments, which may be combined with other embodiments provided herein and throughout, the CD4 + Helper cells, CD8 + Provided is the use of an effective amount of a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, for the manufacture of a medicament for expanding effector naive and memory cell, Treg cell, natural killer (NK) cell or natural killer T (NKT) cell populations.

[0137] In certain embodiments, which may be combined with other embodiments provided herein and throughout, a CD4 + Helper cells, CD8 + Provided is the use of an effective amount of a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide as described herein and throughout and a pharmaceutically acceptable carrier, for expanding effector naive and memory cell, Treg cell, natural killer (NK) cell or natural killer T (NKT) cell populations.

[0138] In certain embodiments that may be combined with other embodiments provided herein and throughout, there are provided polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the mouse serum albumin (MSA) amino acid sequence set forth in SEQ ID NO:6.

[0139] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the human serum albumin (HSA) amino acid sequence set forth in SEQ ID NO:7.

[0140] In certain embodiments that may be combined with other embodiments provided herein and throughout, there are provided polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the mouse serum albumin (MSA) amino acid sequence set forth in SEQ ID NO:6 linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO:3.

[0141] In certain embodiments that may be combined with other embodiments provided herein and throughout, there are provided polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 3 linked to the N-terminus of the amino acid sequence set forth in the amino acid sequence of mouse serum albumin (MSA) set forth in SEQ ID NO: 6.

[0142] In certain embodiments that may be combined with other embodiments provided herein and throughout, there are provided polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the amino acid sequence set forth in the mouse serum albumin (MSA) amino acid sequence set forth in SEQ ID NO:6 linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO:3 via a linker.

[0143] In certain embodiments that may be combined with other embodiments provided herein and throughout, there are provided polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 3 linked via a linker to the N-terminus of the amino acid sequence set forth in the amino acid sequence of mouse serum albumin (MSA) set forth in SEQ ID NO: 6.

[0144] In certain embodiments that may be combined with other embodiments provided herein and throughout, there are provided polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:7 linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO:3.

[0145] In certain embodiments that may be combined with other embodiments provided herein and throughout, there are provided polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:3 linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO:7.

[0146] In certain embodiments that may be combined with other embodiments provided herein and throughout, there are provided polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:7 linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO:3.

[0147] In certain embodiments that may be combined with other embodiments provided herein and throughout, there are provided polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 3 linked via a linker to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 7.

[0148] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:4.

[0149] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, and there are provided polynucleotides as described herein and throughout, as well as pharmaceutical compositions comprising such polynucleotides as described herein and throughout and a pharmaceutically acceptable carrier, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:5.

[0150] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising a polynucleotide as described herein and throughout, comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, and a pharmaceutically acceptable carrier, wherein the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

[0151] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising a polynucleotide as described herein and throughout, comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, and a pharmaceutically acceptable carrier, wherein the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

[0152] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising a polynucleotide as described herein and throughout that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition comprises: a) a nucleic acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14; b) the following compound: [ka] and, Includes:

[0153] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising a polynucleotide as described herein and throughout, comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition comprises a) the nucleic acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14; and b) the following compound: [ka] and, and lipid nanoparticles.

[0154] In certain embodiments that may be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising a polynucleotide as described herein and throughout, comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition comprises: a) a nucleic acid sequence as set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; b) the following compound: [ka] and,

[0155] and lipid nanoparticles.

[0156] Other aspects and advantages of the present invention will become apparent from the embodiments and examples provided herein.

[0157] For the sake of brevity, the disclosures of the publications cited herein, including patents, are hereby incorporated by reference. [Brief explanation of the drawings]

[0158] BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0159] [Figure 1] 1A-1C provide the results of ELISA assays using post-transfection supernatants from 293 cells transfected with mRNA encoding the indicated fusion proteins.

[0160] [Figure 2]Figure 2 shows reporter assay results obtained using the HEK-Blue IL-2 receptor (IL-2R) reporter cell line (InvivoGen) transfected with mRNA encoding the indicated proteins. 4210 = ACT4210 alone (i.e., not fused or linked to another protein); HSA-4210 = fusion protein comprising human serum albumin linked to ACT4210 in N- to C-terminal order; MSA-4210 = fusion protein comprising mouse serum albumin linked to ACT4210 in N- to C-terminal order; 4210-HSA = fusion protein comprising ACT4210 linked to human serum albumin in N- to C-terminal order; 4210-MSA = fusion protein comprising ACT4210 linked to mouse serum albumin in N- to C-terminal order.

[0161] [Figure 3] Figure 3 shows binding assay results obtained from the indicated fusion proteins expressed by cells transfected with mRNA encoding the indicated fusion proteins directed against either IL-2Rα (left panel) or IL-2Rβγ (right panel). 5210-MSA = fusion protein comprising, in N-terminal to C-terminal order, mouse serum albumin linked to ACT5210 (an IL-2 mutein distinct from the IL-2 mutein known as 4210).

[0162] [Figure 4] Figure 4 shows in vivo expression results obtained from mice injected with LNP-containing pharmaceutical compositions, each of which also contained mRNA encoding the indicated fusion protein: ACT421H = LNP-mRNA-HSA-4210; ACT421M = LNP-mRNA-MSA-4210.

[0163] [Figure 5]Figure 5 shows binding assay results obtained from the indicated fusion proteins expressed in vivo in mice injected with LNP-containing pharmaceutical compositions, each of which also contained mRNA encoding the indicated fusion protein directed against either IL-2Rα (left panel) or IL-2Rβγ (right panel).

[0164] [Figure 6] Figure 6 shows binding assay results obtained from HSA-4210 fusion proteins expressed in vivo in mice injected with LNP-containing pharmaceutical compositions that also contained mRNA encoding the HSA-4210 fusion protein, directed toward either cynomolgus monkey IL-2Rβγ (left panel) or rat IL-2Rβγ (right panel).

[0165] [Figure 7] Figure 7 shows binding assay results obtained from HSA-4210 fusion proteins expressed in vivo in mice injected with an LNP-containing pharmaceutical composition that also contained mRNA encoding the HSA-4210 fusion protein, directed toward either cynomolgus monkey IL-2Rβγ (left panel) or rat IL-2Rβγ (right panel).

[0166] [Figure 8] Figure 8 shows reporter assay results obtained using the HEK-Blue IL-2 CD122 / CD132 reporter cell line (invivoGen) transfected with an LNP-containing pharmaceutical composition that also contains mRNA encoding the HSA-4210 fusion protein.

[0167] [Figure 9] FIG. 9 shows the results of a proliferation assay using CTLL-2 cells treated with the indicated fusion proteins.

[0168] [Figure 10-1]Figures 10A-10C show the measured concentrations of each of the indicated proteins obtained from mice injected with LNP-containing pharmaceutical compositions, each of which also contains mRNA encoding the indicated protein. [Figure 10-2] Same as above.

[0169] [Figure 11-1] 11A-11D show the results of pharmacodynamic studies obtained from mice injected with LNP-containing pharmaceutical compositions, each of which also contained mRNA encoding the indicated protein. [Figure 11-2] Same as above.

[0170] [Figure 12] FIG. 12 shows flow cytometry results obtained from mice injected with LNP-containing pharmaceutical compositions that also contained mRNA MSA-4210 fusion proteins.

[0171] [Figure 13] FIG. 13 shows the numbers of CD8, CD4 and Treg cells obtained from mice injected with LNP-containing pharmaceutical compositions that also contained mRNA MSA-4210 fusion protein.

[0172] [Figure 14] 14A-14C show results demonstrating the inhibition of lung metastasis obtained using B16F10 mice injected with an LNP-containing pharmaceutical composition that also contained an mRNA MSA-4210 fusion protein (lung metastasis mouse model). [Figure 15] FIG. 15 shows overall survival results obtained using B16F10 mice (lung metastasis mouse model) injected with LNP-containing pharmaceutical compositions that also contained mRNA MSA-4210 fusion protein, as indicated, or with mice injected with IL-2 1 mg / kg once daily for 5 days. DETAILED DESCRIPTION OF THE INVENTION

[0173] Detailed Description The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, and pharmacology, which are within the skill of one in the art. Such techniques may be found in, for example, Molecular Cloning: A Laboratory Manual, 2004. nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M.Ausubel et al., eds., 1987, and periodic revisions); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Remington, The Science and Practice of Pharmacy, 20 th This is fully explained in literature such as "Theory of the Quantum of Science and Technology," ed., (Lippincott, Williams & Wilkins 2003).

[0174] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. All patents, patent applications (published or unpublished), and other publications mentioned herein are incorporated by reference in their entirety. If the definitions set forth in this section are contrary to or inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth in this section shall prevail over the definitions incorporated herein by reference.

[0175] As used herein, "a" or "an" means "at least one" or "one or more."

[0176] The term "and / or" as used herein should be interpreted to mean the specific disclosure of each of the specified features or components, regardless of the presence or absence of others. For example, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0177] As used herein, the term "about" refers to a value or composition that falls within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more standard deviations per practice in the art. Alternatively, "about" or "approximately" can mean a range of up to 10% (i.e., ±10%) or more, depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, these terms can refer to values ​​up to an order of magnitude or up to five times greater. When a particular value or composition is provided in this disclosure, unless otherwise specified, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for that particular value or composition. In some embodiments, "about" encompasses variations of within 10%, 5%, 2%, 1%, or 0.5% of the stated value.

[0178] Numerical ranges are inclusive of the numbers defining the range. Measured and measurable values ​​are understood to be approximations, taking into account significant digits and the error associated with measurement. Also, all ranges should be interpreted as including the endpoints unless expressly excluded, such as "excluding the endpoints." Thus, for example, a "range of 1 to 10" includes the values ​​1 and 10 and all integer and non-integer values ​​greater than 1 and less than 10 (where appropriate).

[0179] The terms "administer," "administered," and grammatical variations refer to the physical introduction of a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary administration routes for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In one embodiment, the formulation is administered via a parenteral route, for example, orally. Other parenteral routes include topical, epidermal, or mucosal administration routes, for example, intranasal, vaginal, rectal, sublingual, or topical. Administration can be, for example, once, multiple times, and / or over one or more extended periods of time.

[0180] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein and refer to polymers of amino acids of any length, e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000, or more amino acids. The polymers may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified, either naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of an amino acid (including, e.g., unnatural amino acids), as well as other modifications known in the art, are also included within the definition.

[0181] As used herein, the term "variant" refers to a polypeptide that has a degree of amino acid sequence identity with a parent polypeptide sequence. A variant is similar to the parent sequence but has at least one substitution, deletion, or insertion in the amino acid sequence that causes it to differ in sequence from the parent polypeptide. Furthermore, a variant may retain functional characteristics of the parent polypeptide, for example, retaining at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the biological activity of the parent polypeptide.

[0182] An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one antigen-recognition site located in the variable region of the immunoglobulin molecule, and may be any class of immunoglobulin, e.g., IgG, IgM, IgA, IgD, or IgE. IgY, the predominant antibody type in avian species such as chickens, is also included within the definition. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (ScFv), variants thereof, naturally occurring variants, fusion proteins containing an antibody portion having the required specificity of the antigen-recognition site, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule containing the required specificity of the antigen-recognition site.

[0183] As used herein, the term "antigen" refers to a target molecule that is specifically bound by an antibody through its antigen recognition site.Antigens can be monovalent or multivalent, i.e., they can have one or more epitopes recognized by one or more antibodies.Types of antigens that can be recognized by antibodies include polypeptides, oligosaccharides, glycoproteins, polynucleotides, lipids, etc.

[0184] As used herein, the term "epitope" refers to a portion of an antigen, e.g., a peptide sequence of at least about 3-5, preferably about 5-10 or 15, and up to about 1,000 amino acids (or any integer in between), that, by itself or as part of a larger sequence, defines the sequence that binds to antibodies generated in response to such sequence. There is no critical upper limit to the length of a fragment, which can encompass, for example, nearly the entire length of the antigen sequence, or even a fusion protein containing two or more epitopes from the target antigen. Epitopes for use in the present invention are not limited to peptides having the exact sequence of the portion of the parent protein from which they are derived, but also include sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions, and substitutions (essentially conservative).

[0185] As used herein, the term "Fc" or "Fc region" refers to the portion of an antibody heavy chain constant region beginning within or after the hinge region and ending at the C-terminus of the heavy chain. The Fc region includes at least a portion of the CH2 and CH3 regions, and may or may not include a portion of the hinge region. The Fc domain can bind to Fc cell surface receptors and several proteins of the immune complement system. The Fc region can bind to complement component C1q. The Fc domain can exhibit effector functions including any one or any combination of two or more activities, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), opsonization, and / or cell binding. The Fc domain can bind to Fc receptors, including FcγRI (e.g., CD64), FcγRII (e.g., CD32), and / or FcγRIII (e.g., CD16a). In one embodiment, the Fc region can include mutations that increase or decrease any one or any combination of these functions. In one embodiment, the Fc domain comprises an Fc region comprising one or more mutations selected from N297A, N297Q, N297D, L234A, L235A, L235E, P329A, and P329G (e.g., according to Kabat numbering). In one embodiment, the Fc domain comprises a LALA mutation (e.g., equivalent to L234A, L235A according to Kabat numbering) that reduces effector function. In one embodiment, the Fc domain comprises a LALA-PG mutation (e.g., equivalent to L234A, L235A, P329G according to Kabat numbering) that reduces effector function. In one embodiment, the Fc domain mediates the serum half-life of a protein complex, and mutations in the Fc domain can increase or decrease the serum half-life of the protein complex. In one embodiment, the Fc domain affects the thermal stability of a protein complex, and mutations in the Fc domain can increase or decrease the thermal stability of the protein complex. In one embodiment, the Fc region comprises one or more mutations selected from M252Y, T256D, T307Q, T307W, M252Y, S254T, T256E, M428L and N434S (e.g., according to Kabat numbering).In one embodiment, the Fc region comprises the mutations M252Y, S254T and T256E (YTE) (eg, according to Kabat numbering).

[0186] The term "isolated" refers to something that is substantially free of polynucleotides, nucleic acids, or proteins (e.g., antibodies or antigen-binding portions thereof), or other cellular material. The term isolated also, in some embodiments, refers to a protein or polynucleotide that is substantially free of other molecules, reactants, reagents, and / or impurities, such as other proteins or polynucleotides having different amino acid or nucleotide sequences, respectively. The purity or homogeneity of a desired polynucleotide or protein can be assayed using techniques well known in the art, including low-resolution methods such as gel electrophoresis and high-resolution methods such as HPLC or mass spectrometry.

[0187] As used herein with respect to polypeptides, the term "labeled" or related terms refers to conjugated antibodies and their antigen-binding portions that are unlabeled or conjugated to a detectable label or moiety for detection, where the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, detectable beads (e.g., magnetic beads or electron-dense (e.g., gold) beads), biotin, streptavidin, or protein A. A variety of labels can be used, including, but not limited to, radionuclides, fluorophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, haptens). Any of the polynucleotides, modified IL-2 polypeptides, or fusion proteins described herein can be unlabeled or conjugated to a detectable label or moiety.

[0188] As used herein with respect to a polypeptide, the term "labeled" or related terms refers to its conjugation to a detectable label or moiety for detection. Exemplary detectable labels or moieties include radioactive, colorimetric, antigenic, enzymatic labels / moieties, detectable beads (e.g., magnetic beads or electron-dense (e.g., gold) beads), biotin, streptavidin, or protein A. A variety of labels can be used, including, but not limited to, radionuclides, fluorophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, haptens). Any of the polynucleotides, modified IL-2 polypeptides, or fusion proteins described herein can be unlabeled or conjugated to a detectable label or moiety.

[0189] The terms "leader sequence" or "leader peptide" or "[peptide] signal sequence" or "signal peptide" or "secretory signal peptide" refer to a peptide sequence located at the N-terminus of a polypeptide. A leader sequence can direct a polypeptide chain down the cellular secretory pathway and direct the incorporation and anchoring of the polypeptide into the lipid bilayer of a cellular membrane. Typically, a leader sequence is about 10-50 amino acids in length and is cleaved from the polypeptide upon secretion of the mature polypeptide or upon insertion of the mature polypeptide into a membrane. Thus, proteins provided herein, such as membrane proteins and antibodies, having a signal peptide specified by their precursor sequence, which includes a signal peptide sequence, are also intended to encompass mature forms of the polypeptide lacking the signal peptide, and proteins provided herein, such as membrane proteins and antibodies, having a signal peptide specified by their mature polypeptide sequence lacking the signal peptide sequence are also intended to encompass forms of the polypeptide that include a signal peptide, whether native to the protein or derived from another secreted or membrane-inserted protein. In one embodiment, the leader sequence includes a signal sequence, including the CD8α, CD28, or CD16 leader sequence. In one embodiment, the signal sequence comprises a mammalian sequence, including, for example, a mouse or human Ig gamma secretory signal peptide.

[0190] As used herein, the terms " percent identity ", " percent homology " and related terms refer to the quantitative measurement of the similarity between two polypeptide sequences or two polynucleotide sequences.The percent identity between two polypeptide sequences is a function of the number of identical amino acids at the aligned positions shared between two polypeptide sequences, taking into account the number of gaps that may need to be introduced to optimize the alignment of two polypeptide sequences and the length of each gap.Similarly, the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at the aligned positions shared between two polynucleotide sequences, taking into account the number of gaps that may need to be introduced to optimize the alignment of two polynucleotide sequences and the length of each gap.Comparing sequences and determining the percent identity between two polypeptide sequences or two polynucleotide sequences can be achieved using mathematical algorithms. For example, the "percent identity" or "percent homology" of two polypeptide or two polynucleotide sequences can be determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, CA)) using its default parameters. A phrase such as "comprises a sequence having at least X% identity to Y" with respect to a test sequence means that when aligned to sequence Y as described above, the test sequence contains residues identical to at least X% of the residues of Y.

[0191] In some embodiments, polynucleotides comprising nucleic acid sequences encoding fusion proteins comprising modified interleukin-2 (IL-2) polypeptides are provided, where the polynucleotides, modified IL-2 polypeptides, and / or fusion proteins may be similar, but not necessarily identical, to any given test nucleic acid or amino acid sequence. Similarity between a polynucleotide, modified IL-2 polypeptide, or fusion protein described herein and throughout to a given test nucleic acid or amino acid sequence may include at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity, respectively.

[0192] The term "amino acid substitution" and similar terms refer to the replacement of one amino acid residue in a polypeptide with another amino acid residue having a different side chain (R group). It is understood that such an amino acid substitution can be accomplished by altering or modifying a nucleic acid or polynucleotide sequence encoding a given polypeptide so that it encodes, and thus produces, a polypeptide sequence containing, the amino acid substitution when expressed.

[0193] The term "conservative amino acid substitution" and similar terms refer to the replacement of an amino acid residue in a polypeptide with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference in its entirety. Examples of groups of amino acids with side chains of similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. It is understood that such conservative amino acid substitutions can be achieved by altering or modifying the nucleic acid or polynucleotide sequence encoding a given polypeptide so as to encode, and thus produce, a polypeptide sequence containing the conservative amino acid substitution when expressed.

[0194] As used herein, the term "specifically binds" refers to the binding specificity of a specific binding pair. Recognition by an antibody of a specific target in the presence of other potential targets is one characteristic of such binding. Specific binding involves two distinct molecules, one of which specifically binds to a second molecule by chemical or physical means. The two molecules are related in the sense that their binding is such that their binding partner can be distinguished from other assay components with similar characteristics. Members of a pair of binding components are referred to as ligand and receptor (antiligand), specific binding pair (SBP) members and SBP partners, etc. Molecules can also be SBP members due to molecular aggregation. For example, an antibody raised against an immune complex between a second antibody and its corresponding antigen can be considered the SBP member of the immune complex.

[0195] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. The term refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof, as well as peptide nucleic acids, locked nucleic acids, and other synthetic nucleic acid analogs and hybrids thereof. Nucleic acids and polynucleotides include recombinant and chemically synthesized forms and can include DNA (cDNA or genomic DNA), RNA (e.g., mRNA, siRNA, dsRNA, shRNA, miRNA, tRNA, rRNA, vRNA), analogs of DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded. In some embodiments, the nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0196] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps," substitutions of one or more analogs of naturally occurring nucleotides, internucleotide modifications, such as those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralens, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylators, those with modified bonds (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Furthermore, any hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional bonds to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH groups may be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxo sugars, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside.One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or a substituted or unsubstituted alkyl (1-20C), optionally containing an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0197] " Oligonucleotide " as used herein generally refers to a short, generally single-stranded, generally synthetic polynucleotide, generally, although not necessarily, being generally less than about 200 nucleotides in length.The terms " oligonucleotide " and " polynucleotide " are not mutually exclusive.The above description of polynucleotide is equally fully applicable to oligonucleotide.

[0198] As used herein, the term "homolog" refers to a nucleic acid that differs from a naturally occurring nucleic acid (e.g., a "prototype" or "wild-type" nucleic acid) by minor modifications to the naturally occurring nucleic acid, but maintains the basic nucleotide structure of the naturally occurring form. Such changes include, but are not limited to, changes of one or several nucleotides, including deletions (e.g., truncated versions of the nucleic acid), insertions, and / or substitutions. Homologs may have enhanced, decreased, or substantially similar properties compared to the naturally occurring nucleic acid. Homologs may be complementary to or identical to the naturally occurring nucleic acid. Homologs can be produced using techniques known in the art for producing nucleic acids, including, but not limited to, recombinant DNA technology, chemical synthesis, etc.

[0199] As used herein, "substantially complementary or substantially identical" means that two nucleic acid sequences have at least 90% sequence identity. Preferably, two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Alternatively, "substantially complementary or substantially identical" means that two nucleic acid sequences can hybridize under high stringency conditions.

[0200] Generally, hybrid stability is a function of ion concentration and temperature. Typically, hybridization reactions are performed under conditions of lower stringency, followed by washing with varying but higher stringency. Moderately stringent hybridization refers to conditions that allow nucleic acid molecules, such as probes, to bind to complementary nucleic acid molecules. Hybridized nucleic acid molecules generally have at least 60% identity, for example, at least 70%, 75%, 80%, 85%, 90%, or 95% identity. Moderately stringent conditions are equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, and 0.2% SDS at 42°C, followed by washing in 0.2x SSPE and 0.2% SDS at 42°C. High stringency conditions can be achieved, for example, by hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, and 0.2% SDS at 42°C, followed by washing in 0.1x SSPE and 0.1% SDS at 65°C. Low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5x Denhardt's solution, 6x SSPE, and 0.2% SDS at 22°C, followed by washing in 1x SSPE and 0.2% SDS at 37°C. Denhardt's solution contains 1% Ficoll, 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, ethylenediamidetetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025M EDTA. Other suitable medium stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art.

[0201] As used herein, "vector" and related terms refer to a nucleic acid molecule or polynucleotide (e.g., DNA, RNA, mRNA, etc.) that can be operably linked to foreign genetic material (e.g., a nucleic acid transgene, e.g., a polynucleotide encoding a fusion protein comprising a modified IL-2 polypeptide described herein and throughout). Vectors can be used as vehicles to introduce foreign genetic material into cells (e.g., host cells) in vivo, ex vivo, and / or in vitro. Vectors can contain at least one restriction endonuclease recognition sequence for inserting a transgene into the vector. Vectors can contain at least one gene sequence that confers antibiotic resistance or a selectable trait to aid in the selection of host cells harboring the vector transgene construct. Expression vectors can contain one or more origin of replication sequences. Vectors can be single-stranded or double-stranded nucleic acid molecules. Vectors can be linear or circular nucleic acid molecules. One type of vector is a "plasmid," which refers to a linear or circular, double-stranded, extrachromosomal DNA molecule that can be linked to a transgene, replicate in a host cell, and transcribe and / or translate the transgene. Viral vectors typically contain a viral RNA or DNA backbone sequence that can be linked to a transgene. The viral backbone sequence can be modified to prevent infection but retain the insertion of the viral backbone and co-linked transgene into the host cell genome. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, baculoviral vectors, papovaviral vectors, vaccinia viral vectors, herpes simplex viral vectors, and Epstein-Barr viral vectors. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell genome upon introduction into the host cell, thereby replicating along with the host genome.

[0202] An "expression vector" (also known as a "plasmid," used interchangeably throughout) is a type of vector that can include one or more regulatory sequences and / or regulatable promoter systems, such as inducible and / or constitutive promoters and enhancers. Such regulatory sequences and / or regulatable promoter systems can include one or more of a variety of promoters, such as enhancer sequences obtained or derived from the cytomegalovirus (CMV) promoter, elongation factor 1 (EF1) promoter, chicken β-actin promoter, β-actin promoters from other species, phosphoglycerokinase (PGK) promoter, spleen focus forming virus (SFFV) promoter, Rous sarcoma virus (RSV) promoter, human serum albumin (SA) promoter, thyroxine-binding globulin (TBG) promoter, cytochrome P450 2E1 (CYP2E1) promoter, muscle creatine kinase enhancer sequence, desmin enhancer sequence, desmin promoter sequence, MHDLKSMRTe enhancer / promoter sequence, etc. Exemplary such vectors, NanoPlasmid™ vectors, regulatory elements, etc. are described in, for example, These can be found in U.S. Patent Application Publication No. 2004 / 0175727 and WO 2022 / 056291.

[0203] The expression vector may also include a regulatable promoter system comprising a combination of one or more promoter and / or enhancer sequences, for example: the chicken beta-actin / CMV enhancer (CAG) promoter; a human or mouse CMV-derived enhancer element combined with the elongation factor 1a (EF1a) promoter; a CpG-free version of the human or mouse CMV-derived enhancer element combined with the elongation factor 1 alpha (EF1α) promoter; an albumin promoter combined with an alpha-fetoprotein MERII enhancer; one or more MCK enhancer sequences combined with a desmin enhancer sequence and / or a desmin long promoter sequence; one or more MCK enhancer sequences combined with a desmin enhancer sequence and / or a desmin short promoter sequence; a CMV enhancer sequence and an EF1a promoter sequence; one or more MCK enhancer sequences and a C5-12 promoter sequence; one or more MCK enhancer sequences; etc. In certain embodiments, a C5-12 promoter sequence, a C1-26 promoter sequence, a C2-26 promoter sequence, a C2-27 promoter sequence, a C5-5 promoter sequence, a C6-5 promoter sequence, a C6-16 promoter sequence, a C6-39 promoter sequence, a C6-5 promoter sequence, a MEF-1 regulatory element, a MEF-2 regulatory element, an SRE regulatory element, a TEF-1 regulatory element, or the like may be used in place of, and / or in addition to, one or more of the above-described promoter and / or enhancer sequences to generate a regulatable promoter system according to the present disclosure.

[0204] Such regulatory sequences and / or regulated promoter systems may also drive or facilitate tissue-specific transcription and / or translation of a nucleic acid sequence encoding a polypeptide, such as an antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof). Exemplary such promoters and / or enhancers may drive tissue-specific expression of a polynucleotide encoding a polypeptide. Exemplary such promoters and / or enhancers may drive muscle-specific expression of a polynucleotide encoding a polypeptide. Such tissue- and / or muscle-specific promoters and / or enhancers may be combined to generate a regulated tissue- and / or muscle-specific promoter system. In some embodiments, such tissue- and / or muscle-specific promoters and / or enhancers comprise one or more promoter or enhancer sequences disclosed, for example, in U.S. Patent Application Publication No. 2004 / 0175727, which is incorporated herein by reference in its entirety. In some embodiments, such tissue- and / or muscle-specific promoters and / or enhancers that can be used in combination to generate a regulated promoter system in accordance with the present disclosure include, for example, one or more promoter or enhancer sequences, such as a muscle creatine kinase (MCK) enhancer sequence, a desmin enhancer sequence, a desmin long promoter sequence, a desmin short promoter sequence, a C5-12 promoter sequence, a C1-26 promoter sequence, a C2-26 promoter sequence, a C2-27 promoter sequence, a C5-5 promoter sequence, a C6-5 promoter sequence, a C6-16 promoter sequence, a C6-39 promoter sequence, a C6-5 promoter sequence, a MEF-1 regulatory element, a MEF-2 regulatory element, an SRE regulatory element, a TEF-1 regulatory element, an elongation factor 1 alpha ("EF1a" or "EF1α" are used interchangeably throughout) promoter sequence, an MHDLKSMRTx enhancer / promoter sequence, etc.

[0205] The regulatable promoter system operably linked to the polypeptide-encoding sequence(s) can be a eukaryotic or prokaryotic regulatable promoter system, but is preferably a eukaryotic regulatable promoter system that is active in mammalian cells. The regulatable promoter system(s) can direct transcription and / or translation of the transgene in a host cell and can include a ribosome binding site and / or a polyadenylation site. In certain embodiments, the regulatable promoter system(s) can, independently or collectively (e.g., in concert), direct transcription and / or translation of the transgene in a host cell and can include a ribosome binding site and / or a polyadenylation site. In certain embodiments, the regulatable promoter system(s) can, independently or collectively (e.g., in concert), direct tissue-specific transcription and / or translation of the transgene in a host cell and can include a ribosome binding site and / or a polyadenylation site. In certain embodiments, the regulatable promoter system(s) can, independently or collectively (e.g., in concert), direct muscle-specific transcription and / or translation of the transgene in the host cell and can include a ribosome binding site and / or a polyadenylation site. In various embodiments, the host cell or population of host cells has one or more expression vectors that can direct the transient introduction of a transgene into the host cell or the stable insertion of a transgene into the genome of the host cell, where the transgene comprises a nucleic acid encoding any of the first and / or second polypeptides described herein. In embodiments in which the nucleic acid molecule encodes two polypeptides, the sequences encoding the two polypeptides can be regulated by the same promoter and linked by an IRES or 2A sequence (Shao et al. (2009) Cell Research 19:296-306), or the sequences encoding the two polypeptides can be operably linked to different promoters.

[0206] An expression vector may contain a ribosome binding site and / or a polyadenylation site. An expression vector may contain one or more origin of replication sequences. Regulatory sequences direct the transcription, or transcription and translation, of a transgene linked to or inserted into an expression vector to be transduced into a host cell. The regulatory sequence(s) can control the level, timing, and / or location of transgene expression. Regulatory sequences can exert their effect, for example, directly on the transgene or through the action of one or more other molecules (e.g., regulatory sequences and / or polypeptides that bind to nucleic acids). Regulatory sequences can be part of a vector. Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., and Baron et al., 1995, Nucleic Acids Res. 23:3605-3606.

[0207] A transgene is "operably linked" to a regulatory sequence (e.g., a promoter) if the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene.

[0208] As used herein, the terms "transfected" or "transformed" or "transduced" or other related terms refer to the process by which exogenous nucleic acid (e.g., a transgene) is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" host cell is one that has been transfected, transformed or transduced with exogenous nucleic acid (transgene). A host cell includes the primary subject cell and its progeny.

[0209] As used herein, "vector (or plasmid)" refers to a discrete element used to introduce heterologous DNA into cells for either its expression or replication. The selection and use of such vehicles is well within the skill of those in the art. Expression vectors include vectors capable of expressing DNA operably linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Suitable expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic and / or prokaryotic cells, those that remain episomal, or those that integrate into the host cell genome.

[0210] As used herein, "promoter region or promoter element" refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operably linked. The promoter region contains specific sequences sufficient for RNA polymerase recognition, binding, and transcription initiation. This portion of the promoter region is called the promoter. Furthermore, the promoter region contains sequences that modulate this recognition, binding, and transcription initiation activity of RNA polymerase. These sequences may be cis-acting or responsive to trans-acting factors. Depending on the nature of the regulation, promoters may be constitutive or regulated. Exemplary promoters contemplated for use in prokaryotes include the bacteriophage T7 and T3 promoters.

[0211] As used herein, "operably linked" or "operably associated" refers to the functional relationship between DNA or RNA and nucleotide regulatory and effector sequences, such as promoters, enhancers, transcription and translation initiation sites, transcription and translation termination sites, polyadenylation sites, ribosome entry sites, 5-prime cap sites, and other transcriptional or translational control or regulatory elements. For example, operably linked DNA or RNA to a promoter refers to the physical and functional relationship between the DNA or RNA and the promoter, provided that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds, and transcribes the DNA. To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' untranslated portion of the clone to eliminate additional, potentially inappropriate, alternative translation initiation (i.e., start) codons or other sequences that may disrupt or reduce expression at either the transcriptional or translational level. Alternatively, a consensus site can be inserted immediately 5' of the start codon, which can enhance expression. See, for example, Kozak (1991) J. Biol. Chem. 266:19867-19870. The desirability (or necessity) of such modifications can be determined empirically.

[0212] "Treating" or "treatment" or "palliative" refers to therapeutic treatment that slows (reduces) if not cures the targeted pathological condition or disorder or prevents the recurrence of symptoms. A subject is successfully "treated" if, after receiving a therapeutic amount of a therapeutic agent or treatment, the subject shows an observable and / or measurable reduction in one or more signs and symptoms of a particular disease. The reduction in signs and symptoms of the disease may also be felt by the patient. A patient is also considered treated if the patient experiences stable disease. In some embodiments, treatment with a therapeutic agent is effective to result in the patient being disease-free at 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 years or more after treatment. These parameters for assessing successful treatment and improvement of disease are readily measurable by routine procedures well known to those skilled in the art. In some embodiments, "treatment" refers to any method in which the symptoms of a condition, disorder, or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein. In some embodiments, "amelioration" of symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any permanent or temporary, sustained or transient relief that may result from or be associated with administration of the composition.

[0213] The terms "prediction" or "prognosis" are often used herein to refer to the likelihood that a patient will respond favorably or unfavorably to a drug or set of drugs, or the likely outcome of a disease. In one embodiment, the prediction relates to the degree of those responses or outcomes. In one embodiment, the prediction relates to whether and / or the likelihood that a patient will survive or improve for a certain period of time without disease recurrence after treatment, e.g., treatment with a particular therapeutic agent. The predictive methods of the present invention can be used clinically to make treatment decisions by selecting the most appropriate treatment modality for any particular patient. The predictive methods of the present invention are valuable tools in predicting whether a patient is likely to respond favorably to a given treatment regimen, such as, for example, a given treatment regimen including administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc.

[0214] The terms "prevent," "prevention," or "preventing" refer to the provision or administration of a therapeutic or prophylactic agent or treatment regimen with the purpose of attenuating, reducing, or eliminating the occurrence of a disease, condition, or disorder in a subject suspected of having or at risk of having the disease, condition, or disorder. Similarly, "prevent," "prevention," or "preventing" refers to the provision or administration of a therapeutic or prophylactic agent or treatment regimen with the purpose of attenuating, reducing, or eliminating the occurrence of one or more symptoms or clinical manifestations of a disease, condition, or disorder in a subject suspected of having or at risk of having the disease, condition, or disorder.

[0215] A subject is successfully "treated" if, after receiving a therapeutic amount of a therapeutic agent or treatment, the subject exhibits an observable and / or measurable reduction or absence of one or more signs and symptoms of a particular disease, disorder, or condition. A reduction in signs or symptoms of a disease can also be felt by the patient. A patient is also considered treated if the patient experiences stable disease. In some embodiments, treatment with a therapeutic agent is effective to cause the patient to be disease-free at 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 years or more after treatment. These parameters for assessing successful treatment and improvement of a disease are readily measurable by routine procedures familiar to physicians with appropriate skill in the art. In some embodiments, "treatment" refers to any manner in which the symptoms of a condition, disorder, or disease are improved or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein. In some embodiments, "amelioration" of symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any permanent or temporary, sustained or transient relief that may result from or be associated with administration of the composition.

[0216] The term "synergistic effect" refers to a situation in which the combination of two or more agents produces an effect that is greater than the sum of the effects of each of the individual agents. This term encompasses not only the alleviation of symptoms of the disorder being treated, but also an improved side effect profile, improved tolerability, improved patient compliance, improved efficacy, or any other improved clinical outcome.

[0217] The term "sub-therapeutic amount" of a drug or treatment is an amount that is less than the effective amount of that drug or treatment as a single drug, but when combined with an effective or sub-therapeutic amount of another drug or treatment, can produce the result desired by the physician, e.g., by synergistic beneficial effects or reduced side effects.

[0218] Combination therapy or "in combination with" refers to the use of more than one therapeutic agent to treat a particular disorder or condition. "In combination with" does not mean that the therapeutic agents must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope of this disclosure. A therapeutic agent can be administered either before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks before) or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks after) one or more other additional agents. Therapeutic agents in a combination therapy can also be administered on an alternating dosing schedule, with or without rest periods (e.g., no therapeutic agent is administered on certain days of the schedule). Administering a therapeutic agent "in combination" with another therapeutic agent includes, but is not limited to, sequential and concomitant administration of the two agents. Generally, each therapeutic agent is administered at a dose and / or on a time schedule determined for that particular agent.

[0219] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, for example, The Science and Practice of Pharmacy, 20 th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

[0220] "Pharmaceutically acceptable salt" is intended to mean a salt of a free acid or free base of a compound represented herein that is non-toxic, biologically tolerable, or biologically suitable for administration to a subject. See generally Berge et al., J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with a subject's tissues without undue toxicity, irritation, or allergic response. The modified interleukin-2 (IL-2) polypeptides or conjugates thereof described herein have sufficiently acidic groups, sufficiently basic groups, both types of functional groups, or more than one of each type, and therefore can react with several inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts.

[0221] Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, and hexyne-1,6-dioate. , benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, besylate, xylenesulfonate, xysulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate and mandelate.

[0222] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a therapeutic agent that, when administered to a cell, tissue, or subject alone or in combination with an additional therapeutic agent, is effective to prevent or ameliorate a disease or disorder, a proliferative disease or disorder of the subject. A therapeutically effective dose also refers to an amount of a therapeutic agent sufficient to result in the amelioration of symptoms, e.g., treatment, cure, prevention, or amelioration of associated medical symptoms, or an increase in the rate of treatment, cure, prevention, or amelioration of such symptoms. When individual active ingredients are administered singly, the therapeutically effective dose refers to that ingredient alone. When a combination is administered, the therapeutically effective dose refers to the combined amount of active ingredients that results in a therapeutic effect, regardless of whether they are administered in combination, sequentially, or simultaneously. In some embodiments, an "effective amount of a compound for treating a particular disease" is an amount sufficient to ameliorate or in some way reduce symptoms associated with the disease. Such an amount may be administered as a single dose or according to a regimen, and is thereby effective. The amount may cure the disease, but is typically administered to ameliorate the symptoms of the disease. Repeated administration may be necessary to achieve the desired amelioration of symptoms.

[0223] The term "combination" refers to either a fixed combination in the form of a single dosage unit, or a kit of parts for co-administration, in which the modified interleukin-2 (IL-2) polypeptide or a conjugate thereof and a combination partner (e.g., another drug, also referred to as a "therapeutic agent" or "adjunct") can be independently administered simultaneously or separately within a time interval, particularly where these time intervals allow the combination partners to exhibit a cooperative, e.g., synergistic, effect. As used herein, terms such as "co-administration" or "co-administration" are intended to encompass the administration of selected combination partners to a single subject (e.g., patient) in need thereof and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term "pharmaceutical combination," as used herein, refers to a product resulting from the mixing or combining of more than one active ingredient, and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that both active ingredients, e.g., a modified interleukin-2 (IL-2) polypeptide or a conjugate thereof, and a combination partner, are administered to a patient simultaneously in the form of a single entity or dosage. The term "unfixed combination" means that both active ingredients, e.g., a modified interleukin-2 (IL-2) polypeptide or a conjugate thereof, and a combination partner are administered to a patient as separate entities simultaneously, simultaneously, or sequentially, without specific time limitations, such administration providing therapeutically effective levels of the two substances in the patient's body. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0224] As used herein, "biological sample" refers to any sample obtained from a living organism or virus source or other source of macromolecules and biological molecules, including any cell type or tissue of a subject from which nucleic acids or proteins or other macromolecules can be obtained. A biological sample can be a sample obtained directly from a living source or a processed sample. For example, amplified isolated nucleic acids constitute a biological sample. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples from animals and plants, and processed samples derived therefrom.

[0225] The term "level" or "level" is used to refer to the presence and / or amount of a target, e.g., a substance or organism that is part of the pathogenesis of a disease or disorder, and can be determined qualitatively or quantitatively. A "qualitative" change in target level refers to the appearance or disappearance of a target that is undetectable or present in a sample obtained from a normal control. A "quantitative" change in the level of one or more targets refers to a measurable increase or decrease in target levels compared to a healthy control.

[0226] A "healthy control" or "normal control" is a biological sample taken from an individual not afflicted with a disease or disorder, e.g., a proliferative disease or disorder. A "negative control" is a sample that does not contain any of the particular analyte that the assay is designed to detect, thus providing a reference baseline for the assay.

[0227] As used herein, "mammal" refers to any species of the mammalian class. Often, the term "mammal" as used herein refers to a human, a human subject, or a human patient. "Mammal" also refers to any species of the non-human mammalian class, such as experimental, companion, or economic non-human mammals. Exemplary non-human mammals include mice, rats, rabbits, cats, dogs, pigs, cows, sheep, goats, horses, monkeys, gorillas, and chimpanzees.

[0228] As used herein, "production by recombinant means" refers to a production method that uses recombinant nucleic acid methods that rely on well-known methods of molecular biology to express a polypeptide or protein encoded by a cloned nucleic acid.

[0229] As used herein, the terms "subject" and "patient" refer to vertebrates, humans and non-human animals, including mammals and non-mammals. In one embodiment, the subject may be a human, a non-human primate, a monkey, an ape, a rodent (e.g., a mouse and a rat), a cow, a pig, a horse, a dog, a cat, a goat, a wolf, a frog, or a fish.

[0230] As used herein, a "prodrug" is a substance that is metabolized or converted into a biologically, pharmaceutically, or therapeutically active form of the substance upon in vivo administration. To produce a prodrug, a pharmaceutically active substance is modified so that the active substance is regenerated by metabolic processes. Prodrugs can be designed to alter the metabolic stability or transport properties of a drug, mask side effects or toxicity, improve the taste of a drug, or change other properties or characteristics of a drug. With knowledge of in vivo pharmacodynamic processes and drug metabolism, one skilled in the art can design a prodrug of a compound once a pharmaceutically active compound is known (see, for example, Nogrady (1985) Medicinal Chemistry: A Biochemical Approach, Oxford University Press, New York, pages 388-392).

[0231] It will be understood that aspects and embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0232] Throughout this disclosure, various aspects of the present invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0233] Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.

[0234] Polynucleotides encoding modified interleukin-2 (IL-2) polypeptides and fusion proteins containing the same

[0235] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide has an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, at least about Polynucleotides are provided that comprise amino acids with about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity, and wherein the modified IL-2 polypeptide comprises at least one substitution with a natural or unnatural amino acid at one or more positions selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof.In some embodiments, the modified IL-2 polypeptide a) is configured to be conjugated to a water soluble polymer, lipid, or polypeptide, e.g., a protein or peptide, and / or b) has reduced binding to interleukin-2 receptor alpha (IL-2Rα) compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution, and / or c) has reduced receptor signaling potency for IL-2Rαβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without at least one substitution, and / or d) has an increased ratio of signaling potency for IL-2Rβγ over signaling potency for IL-2Rαβγ (signaling potency for IL-2Rβγ / IL-2Rαβγ) compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without at least one substitution. and / or e) have enhanced receptor signaling potency for IL-2Rβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without at least one substitution; and / or with the proviso that if the modified IL-2 polypeptide comprises at least one substitution with a non-naturally occurring amino acid, then the modified IL-2 polypeptide comprises at least one substitution at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76 and a combination thereof, and / or at least one substitution with a natural or non-natural amino acid at at least one position within the IL-2Rα interacting region, IL-2Rβ interacting region and / or IL-2Rγ interacting region; and / or f) comprise a combination of a)-e).

[0236] The amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is shown below: [ka] [ka]

[0237] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity in the region of amino acid residues 10-25 to the corresponding region of an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitution.

[0238] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity in the region of amino acid residues 80-100 to the corresponding region of an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitution.

[0239] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity in the region of amino acid residues 100-134 to the corresponding region of an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitution.

[0240] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity in the regions of amino acid residues 10-25 and 80-100 to the corresponding regions of an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitution.

[0241] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity in the regions of amino acid residues 10-25 and 100-134 to the corresponding regions of an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitution.

[0242] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity in the regions of amino acid residues 80-100 and 100-134 to the corresponding regions of an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitution.

[0243] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity in the regions of amino acid residues 10-25, 80-100, and 100-134 to the corresponding regions of an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions.

[0244] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitution.

[0245] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises any suitable substitutions with natural amino acids. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at positions Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, or a combination thereof.

[0246] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified interleukin-2 (IL-2) polypeptide comprises: a) substitutions with natural amino acids at positions selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof; and / or b) comprises a substitution with a natural amino acid at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, and is configured to be conjugated to a water soluble polymer, lipid, protein, or peptide at the N-terminus and / or C-terminus of the polypeptide.

[0247] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises: a) a lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, or thiamin monoamine at positions selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof; and / or b) comprising a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine or tyrosine at positions selected from the group consisting of N29, N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76 and combinations thereof.

[0248] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide: a) comprising a substitution with a cysteine ​​at a position selected from the group consisting of N29, N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76, and combinations thereof; b) comprising a substitution with a cysteine ​​at a position selected from the group consisting of N29, Y31, K35, P65, N71, Q74, and combinations thereof; c) comprising a substitution with a cysteine ​​at position Y31; and / or d) comprising a substitution with a cysteine ​​at position P65.

[0249] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises a substitution at position Y31 with any amino acid. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises a substitution at position Y31 with serine or alanine.

[0250] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises a substitution with a natural or unnatural amino acid at a position within the IL-2Rα-interacting region, the IL-2Rβ-interacting region, and / or the IL-2Rγ-interacting region.

[0251] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises a natural amino acid substitution at a position within the IL-2Rα-interacting region. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide further comprises a natural amino acid substitution at any suitable position within the IL-2Rα-interacting region. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide further comprises a natural amino acid substitution at a position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof.

[0252] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises any suitable substitution with a naturally occurring amino acid at a position within the IL-2Rα-interacting region. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof.

[0253] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the modified interleukin-2 (IL-2) polypeptide comprising: a) a substitution with cysteine ​​at a position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof; b) a substitution with alanine, lysine, or serine at position F42; c) a substitution with alanine at position F42; d) a substitution with serine at position F42; e) a substitution with lysine at position F42; f) a substitution with a cysteine ​​at position Y45; g) comprising a substitution at position Y45 with alanine; h) comprising a substitution at position Y45 with histidine; i) comprising a substitution at position R38 with alanine, aspartic acid or serine; j) comprising a substitution at position R38 with aspartic acid; k) comprising a substitution at position P65 with alanine; l) comprising a substitution at position P65 with serine; m) comprising a substitution at position E62 with alanine; and / or n) comprising a substitution at position F42 with lysine, at position Y31 with cysteine, or a combination thereof.

[0254] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises a naturally occurring amino acid substitution at a position within the IL-2Rβ interacting region. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide further comprises a naturally occurring amino acid substitution at any suitable position within the IL-2Rβ interacting region. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide further comprises a naturally occurring amino acid substitution at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0255] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises any suitable substitution with a naturally occurring amino acid at a position within the IL-2Rβ interacting region. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises a substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises a substitution with a cysteine ​​at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0256] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the modified interleukin-2 (IL-2) polypeptide comprising: a) a substitution with a naturally occurring amino acid at a position within the IL-2Rα interacting region and a substitution with a naturally occurring amino acid at a position within the IL-2Rβ interacting region; b) a substitution with a naturally occurring amino acid at a position within the IL-2Rα interacting region and a substitution with a naturally occurring amino acid at a position within the IL-2Rγ interacting region; or c) a substitution with a naturally occurring amino acid at a position within the IL-2Rα interacting region, a substitution with a naturally occurring amino acid at a position within the IL-2Rβ interacting region, and a substitution with a naturally occurring amino acid at a position within the IL-2Rγ interacting region.

[0257] In some embodiments, a polynucleotide is provided that includes a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, where the modified interleukin-2 (IL-2) polypeptide includes any suitable substitution with an unnatural amino acid. Exemplary unnatural amino acids that may be used in accordance with the embodiments disclosed herein and throughout are described, for example, in WO 2019 / 028425 A1 and WO 2019 / 028419 A1, which are hereby incorporated in their entireties for all purposes. In some embodiments, a polynucleotide is provided that includes a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, where the modified interleukin-2 (IL-2) polypeptide includes an unnatural amino acid and / or an amino acid containing an unnatural functional group, such as a lysine analog, a cysteine ​​analog, or a histidine analog; an aromatic side chain; an azide group; an alkyne group; and / or an aldehyde or ketone group. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises an unnatural amino acid that does not contain an aromatic side chain.In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the modified interleukin-2 (IL-2) polypeptide being selected from the group consisting of N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl and unnatural amino acids including phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propane, or selenocysteine.

[0258] In some embodiments, a polynucleotide is provided that includes a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises an unnatural amino acid that can be incorporated into the modified IL-2 polypeptide by any suitable means or method. In some embodiments, a polynucleotide is provided that includes a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified interleukin-2 (IL-2) polypeptide comprises an unnatural amino acid that can be incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase / tRNA pair. Any suitable orthogonal tRNA can be used. For example, the orthogonal tRNA of the orthogonal synthetase / tRNA pair can comprise at least one unnatural nucleobase.

[0259] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified interleukin-2 (IL-2) polypeptide has reduced or no detectable binding to IL-2Rα compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the binding affinity of the modified IL-2 polypeptide to IL-2Rα can be reduced by about 10% to about 100%, e.g., by about 10%, 20%, 30%, 40%, 50%, 60%, 17%, 80%, 90%, 100%, or any subrange thereof. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the binding affinity of the modified IL-2 polypeptide to IL-2Rα can be reduced by about 10% to about 100%, or can be reduced by about 1-fold to about 100,000-fold or more, for example, about 1-fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold or more, or subranges thereof. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified interleukin-2 (IL-2) polypeptide has no detectable binding to IL-2Rα.

[0260] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified interleukin-2 (IL-2) polypeptide has reduced or no detectable receptor signaling potency for IL-2Rαβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitutions. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the ratio between the signaling potency for IL-2Rαβγ of the modified IL-2 polypeptide and the signaling potency for IL-2Rαβγ of an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitutions can be about 1 / 2 to about 1 / 100,000, e.g., about 1 / 2, 1 / 5, 1 / 10, 1 / 100, 1 / 1,000, 1 / 10,000, 1 / 100,000, or less, or any subrange thereof. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has no detectable receptor signaling efficacy for IL-2Rαβγ.

[0261] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has reduced binding to IL-2Rα compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitutions, and has reduced receptor signaling efficacy for IL-2Rαβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitutions. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has no detectable binding to IL-2Rα and no detectable receptor signaling efficacy for IL-2Rαβγ.

[0262] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide can substantially retain or have a higher level of binding to interleukin-2 receptor beta (IL-2Rβ) or interleukin-2 receptor gamma (IL-2Rγ) compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions, and / or can substantially retain or have a higher receptor signaling potency to IL-2Rβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide can substantially retain or have a higher level of binding to IL-2Rβ or IL-2Rγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide retains equivalent or has greater receptor signaling potency for IL-2Rβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitution.In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide retains substantial or has higher binding levels to IL-2Rβ or IL-2Rγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions, and retains substantial or has higher receptor signaling potency for IL-2Rβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the substitutions.

[0263] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide comprises a deletion at any suitable position. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide has an N-terminal deletion, e.g., an N-terminal deletion of amino acid residues 1-30, or a subrange thereof. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide has a C-terminal deletion, e.g., a C-terminal deletion of amino acid residues 114-134, or a subrange thereof. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide has both an N-terminal deletion and a C-terminal deletion.

[0264] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the fusion protein comprises a recombinant fusion protein. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the fusion polypeptide further comprises an additional amino acid sequence. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide is fused to the additional amino acid sequence in any suitable manner. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified interleukin-2 (IL-2) polypeptide is fused to the additional amino acid sequence via the N-terminus of the modified IL-2 polypeptide or via the C-terminus of the modified IL-2 polypeptide. The additional amino acid sequence can comprise any suitable sequence or content. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the additional amino acid sequence comprises an antibody sequence or a portion or fragment thereof. In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the additional amino acid sequence comprises an Fc portion of an antibody.

[0265] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the fusion protein further comprises an additional amino acid sequence comprising serum albumin or a PK-extended fragment or analog thereof. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the fusion protein further comprises an additional amino acid sequence comprising a) human serum albumin (HSA), b) mouse serum albumin (MSA), c) or a PK-extended fragment or analog of a) or b).

[0266] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the fusion protein further comprises an additional amino acid sequence comprising a) human serum albumin (HSA) as set forth in SEQ ID NO: 7, b) mouse serum albumin (MSA) as set forth in SEQ ID NO: 6, c) or a PK extension fragment or analog of a) or b).

[0267] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide is in isolated and / or purified form.

[0268] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the polynucleotide being prepared using any suitable technique or process. In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, the polynucleotide being prepared by recombinant production, chemical synthesis, or a combination thereof.

[0269] In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide comprises DNA, RNA, a DNA / RNA hybrid, a viral vector, a non-viral vector, a plasmid, and / or a Nanoplasmid™. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide is configured to express the fusion protein in vitro, in vivo, and / or ex vivo.

[0270] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide and / or the fusion protein, upon expression, can form a conjugate with another moiety in vivo, ex vivo, or in vitro.

[0271] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the polynucleotide is formulated as and / or included in a pharmaceutical composition comprising one or more lipids and / or lipid nanoparticles (LNPs).

[0272] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, such as a water-soluble polymer, a lipid, a polypeptide, such as a protein, or a peptide, as described above. Such conjugation can be or may be performed in vitro or in vivo.

[0273] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, such as a water-soluble polymer, lipid, protein, or peptide, in any suitable manner, as described above. For example, the modified IL-2 polypeptide can be covalently conjugated to the water-soluble polymer, lipid, protein, or peptide. In another example, the modified IL-2 polypeptide can be non-covalently conjugated to the water-soluble polymer, lipid, protein, or peptide. In yet another example, the modified IL-2 polypeptide can be conjugated to the water-soluble polymer, lipid, protein, or peptide via a substituted natural or unnatural amino acid at any suitable position.

[0274] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted natural or unnatural amino acid at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof, as described above. In another embodiment, the modified IL-2 polypeptide is conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted native amino acid at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof. In yet another embodiment, the modified IL-2 polypeptide is conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof.In yet another embodiment, the modified IL-2 polypeptide is conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted cysteine ​​at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0275] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted natural or unnatural amino acid at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, as described above. In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted naturally occurring amino acid at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, as described above. In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, as described above.In some embodiments, a polynucleotide is provided comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, e.g., a water soluble polymer, lipid, protein, or peptide, via a substituted cysteine ​​at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, as described above.

[0276] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, e.g., a water-soluble polymer, lipid, protein, or peptide, via a single amino acid residue or multiple amino acid residues in the modified IL-2 polypeptide, as described above. In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, e.g., a water-soluble polymer, lipid, protein, or peptide, via i) the alpha-amino group of the N-terminal amino acid residue in the modified IL-2 polypeptide, ii) the epsilon-amino group of a lysine amino acid residue in the modified IL-2 polypeptide, or iii) an N- or O-glycosylation site in the modified IL-2 polypeptide, as described above.

[0277] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, e.g., a water-soluble polymer, lipid, protein, or peptide, via a linker, as described above. In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated directly to another moiety, e.g., a water-soluble polymer, lipid, protein, or peptide, without a linker, as described above.

[0278] In some embodiments, a polynucleotide is provided that includes a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, such as a water-soluble polymer, lipid, protein, or peptide, via a single amino acid residue in the fusion polypeptide comprising the modified IL-2 polypeptide and an additional amino acid sequence, as described above. The single amino acid residue can be located at any suitable position. For example, the single amino acid residue can be located within the modified IL-2 polypeptide. In another example, the single amino acid residue can be located within the additional amino acid sequence.

[0279] The additional amino acid sequence in the modified IL-2 polypeptide conjugate can comprise any suitable sequence or content. For example, the additional amino acid sequence in the modified IL-2 polypeptide conjugate can comprise an antibody sequence or a portion or fragment thereof. In another example, the additional amino acid sequence in the modified IL-2 polypeptide conjugate can comprise the Fc portion of an antibody.

[0280] In some embodiments, a polynucleotide is provided that includes a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, such as a water-soluble polymer, lipid, protein, or peptide in the fusion polypeptide in any suitable manner, as described above. In some embodiments, a polynucleotide is provided that includes a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to another moiety, such as a water-soluble polymer, lipid, protein, or peptide, as described above, via i) the alpha-amino group of the N-terminal amino acid residue of the fusion polypeptide, ii) the epsilon-amino group of a lysine amino acid residue of the fusion polypeptide, or iii) an N- or O-glycosylation site of the fusion polypeptide. In another example, the fusion polypeptide can be covalently conjugated to the water-soluble polymer, lipid, protein, or peptide directly or via a linker.

[0281] In some embodiments, a polynucleotide is provided that includes a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to any suitable water-soluble polymer, as described above. For example, the water-soluble polymer can include polyethylene glycol (PEG), poly(propylene glycol) (PPG), a copolymer of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. See, for example, WO 2019 / 028425 A1 and WO 2019 / 028419 A1.

[0282] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide is configured to be conjugated to a PEG molecule as described above. The PEG molecule can be a linear PEG or a branched PEG. The branched PEG can have any suitable configuration and / or any suitable number of PEG chains. For example, the branched PEG can have about 3 to about 10 PEG chains emanating from a central core group. In another example, the branched PEG can be a star PEG comprising about 10 to about 100 PEG chains emanating from a central core group. In yet another example, the branched PEG can be a comb PEG comprising multiple PEG chains grafted onto a polymer backbone.

[0283] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide is configured to be conjugated to a PEG molecule of any suitable molecular weight, as described above. For example, the PEG molecule can have a molecular weight ranging from about 300 g / mol to about 10,000,000 g / mol, e.g., about 300 g / mol, 500 g / mol, 1,000 g / mol, 10,000 g / mol, 100,000 g / mol, 1,000,000 g / mol, 10,000,000 g / mol, or any subrange thereof. In another example, the PEG molecule can have an average molecular weight of about 5,000 daltons to about 1,000,000 daltons, e.g., about 5,000 daltons, 10,000 daltons, 100,000 daltons, 1,000,000 daltons, or any subrange thereof. In yet another example, the PEG molecules can have an average molecular weight of about 20,000 daltons to about 30,000 daltons, e.g., about 20,000 daltons, 21,000 daltons, 22,000 daltons, 23,000 daltons, 24,000 daltons, 25,000 daltons, 26,000 daltons, 27,000 daltons, 28,000 daltons, 29,000 daltons, 30,000 daltons, or any subrange thereof.

[0284] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide is provided, wherein the modified IL-2 polypeptide is configured to be conjugated to a PEG molecule in any suitable form, as described above. For example, the PEG molecule can be monodisperse, uniform, or individual PEG molecules.

[0285] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to a polysaccharide as described above.

[0286] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to any suitable lipid, as described above. In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to a fatty acid, as described above.

[0287] In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to any suitable protein, as described above. In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to an antibody or binding fragment thereof, as described above. In some embodiments, a polynucleotide is provided that comprises a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide is configured to be conjugated to the Fc portion of an antibody, as described above.

[0288] In some embodiments, a polynucleotide is provided that includes a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, where the modified IL-2 polypeptide is configured to be conjugated to a water-soluble polymer, lipid, protein, or peptide, as described above, that can be attached to the modified IL-2 polypeptide via any suitable method, for example. In some embodiments, a polynucleotide is provided that includes a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, where the modified IL-2 polypeptide is configured to be conjugated to a water-soluble polymer, lipid, protein, or peptide, as described above, that can be indirectly attached to a substituted natural or unnatural amino acid of the modified IL-2 polypeptide, for example, via a linker. In another example, another moiety, such as a water-soluble polymer, lipid, protein, or peptide, can be directly attached to a substituted natural or unnatural amino acid of the modified IL-2 polypeptide.

[0289] The modified IL-2 polypeptide conjugates of the invention can have any suitable in vivo half-life, for example, from about 5 minutes to about 10 days, e.g., about 5, 10, 20, 30, 40, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or any subrange thereof.

[0290] Pharmaceutical Composition

[0291] In some embodiments, a pharmaceutical composition is provided comprising a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising the above-described modified interleukin-2 (IL-2) polypeptide, and a pharmaceutically acceptable carrier or excipient.

[0292] The pharmaceutical composition can be adapted to treat or prevent any suitable disease, disorder or condition. For example, the pharmaceutical composition can be adapted to treat or prevent a proliferative disorder in a subject.

[0293] In one embodiment, the pharmaceutical composition is adapted to treat or prevent a solid tumor or cancer in a subject, such as chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone / osteosarcoma, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brainstem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, visual pathway and hypothalamic glioma, breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, Medullary carcinoma, male breast cancer, phyllodes tumor, inflammatory breast cancer, adrenocortical carcinoma, islet cell carcinoma (endocrine pancreas), multiple endocrine neoplasia syndrome, parathyroid cancer, pheochromocytoma, thyroid cancer, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, bile duct cancer, carcinoid tumor, gastrointestinal cancer, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, digestive Gastrointestinal stromal tumor (GIST), hepatocellular carcinoma, pancreatic islet cell cancer, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumor, ovarian cancer, ovarian epithelial cancer (surface epithelial stromal tumor), ovarian germ cell tumor, penile cancer, renal cell carcinoma, renal pelvis and ureter, transitional cell carcinoma, prostate cancer, testicular cancer, gestational trophoblastic tumor, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, esophageal cancer, It can be head and neck cancer, nasopharyngeal carcinoma, oral cavity cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, melanoma, skin cancer (non-melanoma), bronchial adenoma / carcinoid, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleuropulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, AIDS-related cancer, Kaposi's sarcoma, epithelioid hemangioendothelioma (EHE), desmoplastic small round cell tumor, or liposarcoma.

[0294] In another embodiment, the pharmaceutical composition is configured to treat or prevent a hematological malignancy in a subject, including myeloid neoplasms, leukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, hepatosplenic T-cell lymphoma, B-cell lymphoma reticuloendotheliosis, reticulosis, microglioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic peripheral leukemia, and the like. Marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, nodular lymphocyte-predominant Hodgkin's lymphoma, plasma cell leukemia, acute erythroleukemia and erythroleukemia, acute erythrocytic myelopathy, acute erythrocytic leukemia, Heilmeyer-Schoner disease, acute megakaryoblastic leukemia, mast cell leukemia, panmyelosis, acute panmyelosis with myelofibrosis, lymphosarcoma cell leukemia, acute leukemia of unspecified cell type, blast phase chronic myeloid leukemia, stem cell leukemia, chronic leukemia of unspecified cell type, subacute leukemia of unspecified cell type, Accelerated phase chronic myeloid leukemia, acute myeloid leukemia, polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, adult T-cell leukemia / lymphoma, aggressive NK cell leukemia, B-cell prolymphocytic leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, chronic idiopathic myelofibrosis , multiple myeloma, Kahler's disease, myelomatosis, solitary myeloma, plasma cell leukemia, plasmacytoma, extramedullary, malignant plasma cell neoplasm NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative lesion, lymphocytic granulomatosis, angioimmunoblastic lymphadenopathy, T-gamma lymphoproliferative disorder, Waldenstrom's macroglobulinemia, alpha heavy chain disease, gamma heavy chain disease, Franklin's disease, immunoproliferative small intestinal disease, Mediterranean disease, malignant immunoproliferative disorder, unspecified, or immunoproliferative disorder NOS.

[0295] In yet another embodiment, the pharmaceutical composition is configured to treat or prevent an immunodeficiency disease or disorder in a subject, including agammaglobulinemia: X-linked and autosomal recessive, ataxia-telangiectasia, chronic granulomatous disease and other phagocytic disorders, common variable immunodeficiency, complement deficiencies, DiGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyper-IgE syndrome, hyper-IgM syndrome, IgG subclass deficiencies, congenital immune deficiencies, NEMO deficiency syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immunodeficiency and combined immunodeficiency, specific antibody deficiencies, breast cancer, and breast cancer. It may be transient hypogammaglobulinemia in infancy, WHIM syndrome (warts, hypogammaglobulinemia, infections, and myelocatechus), Wiskott-Aldrich syndrome, other antibody deficiency disorders, other primary cellular immunodeficiencies, severe combined immunodeficiency (SCID), common variable immunodeficiency (CVID), human immunodeficiency virus / acquired immunodeficiency syndrome (HIV / AIDS), drug-induced immunodeficiency, graft-versus-host syndrome, primary immunodeficiency disease (PIDD), or lymphopenia.

[0296] The pharmaceutical composition may further comprise another active ingredient. The other active ingredient may be any suitable active ingredient for treating or preventing any suitable disease, disorder or symptom. For example, the other active ingredient may be an anti-tumor agent.

[0297] The additional active ingredient(s) may be formulated in a pharmaceutical composition separate from at least one exemplary modified IL-2 polypeptide or modified IL-2 polypeptide conjugate of the present disclosure, or may be included in a single pharmaceutical composition together with at least one exemplary modified IL-2 polypeptide or modified IL-2 polypeptide conjugate of the present disclosure.

[0298] The pharmaceutical compositions of the present invention may be formulated to be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or by other methods of drug administration. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0299] Sterile injectable compositions, such as sterile injectable aqueous or oily suspensions, can be formulated according to the techniques known in the art using suitable dispersants or wetting agents and suspending agents.Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents.Acceptable vehicles and solvents that can be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution.Suitable carriers and other pharmaceutical composition components are typically sterile.

[0300] In addition, sterile, fixed oils are conventionally used as solvents or suspending media (e.g., synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersants. Various emulsifiers or bioavailability enhancers commonly used in the production of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for formulation purposes.

[0301] Compositions for oral administration can be in any orally acceptable dosage form, including, but not limited to, tablets, capsules, emulsions, and aqueous suspensions, dispersions, and solutions. For oral tablets, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate can also be added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. For oral administration of aqueous suspensions or emulsions, the active ingredient can be suspended or dissolved in an oily phase in combination with an emulsifying or suspending agent. Specific sweeteners, flavorings, or coloring agents can be added as needed. Nasal aerosol or inhalation compositions can be prepared according to techniques well known in the art of pharmaceutical formulation, and can be prepared, for example, as a solution in saline, using suitable preservatives (e.g., benzyl alcohol), absorption enhancers to increase bioavailability, and / or other solubilizing or dispersing agents known in the art.

[0302] Any suitable formulation of the compounds described herein can be prepared. Generally, see Remington's Pharmaceutical Sciences, (2000) Hoover, JE editor, 20th edition, Lippincott Williams and Wilkins Publishing Company, Easton, Pa., pages 780-857. Formulations are selected to suit appropriate administration routes. If a compound is sufficiently basic or acidic and forms a stable non-toxic acid or base salt, it may be appropriate to administer the compound as a salt. Examples of pharmaceutically acceptable salts include organic acid addition salts formed with acids that form physiologically acceptable anions, such as tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts can also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate. Pharmaceutically acceptable salts are obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid to provide a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids are also prepared.

[0303] When a contemplated compound or substance is administered in a pharmacological composition, it is expected that the compound or substance can be formulated in admixture with a pharmaceutically acceptable excipient and / or carrier. For example, a contemplated compound or substance can be administered intravenously as a neutral compound or substance, as a pharmaceutically acceptable salt, or in physiological saline. Conventional buffers such as phosphate, bicarbonate, or citrate can be used for this purpose. Naturally, those skilled in the art will be able to modify formulations within the teachings of this specification to provide multiple formulations for specific routes of administration. In particular, contemplated compounds or substances can be modified to make them more soluble in water or other vehicles; this can be easily accomplished, for example, with minor modifications (salt formulations, esterification, etc.) well within the skill of those in the art. It is also well within the skill of those in the art to modify the route of administration and dosing regimen of a particular compound or substance to manage the pharmacokinetics of the compound or substance of the present invention, e.g., the modified IL-2 polypeptide or modified IL-2 polypeptide conjugate of the present invention, for maximum beneficial effect in patients.

[0304] The modified IL-2 polypeptide or modified IL-2 polypeptide conjugate may be soluble in organic solvents such as chloroform, dichloromethane, ethyl acetate, ethanol, methanol, isopropanol, acetonitrile, glycerol, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide. In one embodiment, the present invention provides a formulation prepared by mixing the modified IL-2 polypeptide or modified IL-2 polypeptide conjugate of the present invention with a pharmaceutically acceptable carrier. In one aspect, the formulation can be prepared using a method comprising: a) dissolving the described compound or substance in a water-soluble organic solvent, a non-ionic solvent, a water-soluble lipid, a cyclodextrin, a vitamin such as tocopherol, a fatty acid, a fatty acid ester, a phospholipid, or a combination thereof to provide a solution; and b) adding saline or a buffer containing 1-10% carbohydrate solution. In one example, the carbohydrate comprises dextrose. The pharmaceutical composition obtained using this method is stable and useful for veterinary and clinical applications.

[0305] Examples of water-soluble organic solvents for use in the pharmaceutical compositions of the present invention include, but are not limited to, polyethylene glycol (PEG), alcohol, acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or combinations thereof. Examples of alcohols include, but are not limited to, methanol, ethanol, isopropanol, glycerol, or propylene glycol.

[0306] Illustrative examples of water-soluble nonionic surfactants for use in the pharmaceutical compositions of the present invention include, but are not limited to, CREMOPHOR®, EL, polyethylene glycol-modified CREMOPHOR® (polyoxyethyleneglyceroltriricinol 35), hydrogenated CREMOPHOR®, RH40, hydrogenated CREMOPHOR®, RH60, PEG-succinate, polysorbate 20, polysorbate 80, SOLUTOL®, HS (polyethylene glycol 660 12-hydroxystearate), sorbitan monooleate, poloxamer, LABRAFIL® (ethoxylated persic oil), LABRASOL® (capryl-caproyl macrogol-8-glyceride), GELUCIRE® (glycerol esters), SOFTIGEN® (PEG 6 caprylic acid glyceride), glycerin, glycol-polysorbate, or combinations thereof.

[0307] The water-soluble lipid examples for use in the pharmaceutical compositions of the present invention include but are not limited to vegetable oil, triglyceride, vegetable oil or combinations thereof.The lipid oil examples include but are not limited to castor oil, polyoxyl castor oil, corn oil, olive oil, cottonseed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, coconut triglyceride, palm kernel oil and their hydrogenated forms or combinations thereof.

[0308] Illustrative examples of fatty acids and fatty acid esters for use in the pharmaceutical compositions of the present invention include, but are not limited to, oleic acid, monoglycerides, diglycerides, mono- or di-fatty acid esters of PEG, or combinations thereof.

[0309] Illustrative examples of cyclodextrins for use in the pharmaceutical compositions of the present invention include, but are not limited to, alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, or sulfobutylether-beta-cyclodextrin.

[0310] Illustrative examples of phospholipids for use in the pharmaceutical compositions of the present invention include, but are not limited to, soybean phosphatidylcholine, or distearoylphosphatidylglycerol, and hydrogenated forms thereof, or combinations thereof.

[0311] Those skilled in the art can modify the formulation within the scope of the teachings of this specification to provide a variety of formulations for specific administration routes.In particular, compounds or substances can be modified to make them more soluble in water or other vehicles.It is also well within the scope of those skilled in the art to modify the administration route and administration regimen of a specific compound or substance to manage the pharmacokinetics of the compound or substance for maximum beneficial effect in patients.

[0312] Lipids, lipid nanoparticles, and mRNA pharmaceutical compositions containing them

[0313] In some embodiments, a pharmaceutical composition is provided comprising a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising the above-described modified interleukin-2 (IL-2) polypeptide and one or more lipids. In some embodiments, a pharmaceutical composition is provided comprising a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising the above-described modified interleukin-2 (IL-2) polypeptide and one or more lipids and lipid-containing nanoparticles. In some embodiments, a pharmaceutical composition is provided comprising a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising the above-described modified interleukin-2 (IL-2) polypeptide and one or more lipids and lipid-containing nanoparticles. Lipids and lipid-containing nanoparticles suitable for use in the pharmaceutical compositions disclosed herein and throughout are disclosed, for example, in U.S. Provisional Patent Application No. 63 / 313,648, filed February 24, 2022, entitled "Novel Ionizable Cationic Lipids," the entire contents of which are hereby incorporated by reference.

[0314] The term "lipid" or "lipid moiety" is used according to its ordinary meaning in chemistry and typically refers to a hydrophobic molecule characterized by an aliphatic hydrocarbon chain. In embodiments, the lipid moiety comprises a carbon chain of 3 to 100 carbons. In embodiments, the lipid moiety comprises a carbon chain of 5 to 50 carbons. In embodiments, the lipid moiety comprises a carbon chain of 5 to 25 carbons. In embodiments, the lipid moiety comprises a carbon chain of 8 to 525 carbons. The lipid moiety may comprise a saturated or unsaturated carbon chain and may be optionally substituted. In embodiments, the lipid moiety may be optionally substituted at the terminal with a charged moiety. In embodiments, the lipid moiety is an alkyl or heteroalkyl optionally substituted at the terminal with a carboxylic acid moiety. Lipids also include, but are not limited to, esters of fatty acids and are a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least three classes: (1) "simple lipids," including fats and oils and waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0315] The terms "cationic lipid" or "ionizable cationic lipid" are used interchangeably herein and refer to lipids that become protonated at low pH and thus positively charged, but remain neutral at physiological pH.

[0316] The term "lipid nanoparticle" includes lipid formulations that can be used to deliver active or therapeutic agents, such as nucleic acids (e.g., mRNA), to a desired target site (e.g., a cell, tissue, organ, etc.). In embodiments, the lipid particles described herein are typically nucleic acid-lipid particles formed from cationic lipids, non-cationic lipids, and, optionally, conjugated lipids that prevent particle aggregation. In other embodiments, the active or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0317] The term " lipid conjugate " refers to the conjugated lipid that inhibits lipid particle aggregation.Such lipid conjugate includes but is not limited to PEG-lipid conjugate, for example, the PEG that is coupled with dimyristoylglycerol (for example, PEG-DMG conjugate), the PEG that is coupled with diacylglycerol (for example, PEG-DAG conjugate), the PEG that is coupled with cholesterol, the PEG that is coupled with phosphatidylethanolamine, and the PEG that is coupled with ceramide.

[0318] The term "diacylglycerol" or "DAG" refers to a diacylglycerol consisting of two fatty acyl chains R 1 and R 2 and 2-30 carbon atoms, both of which are independently attached to the 1- and 2-positions of glycerol by ester bonds. The acyl groups may be saturated or have varying degrees of unsaturation. Suitable acyl groups include lauroyl (C 12 ), myristoyl (C 14 ), palmitoyl (C 16 ), stearoyl (C 18 ), and Icosoil (C 20 In a preferred embodiment, R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristoyl (i.e., dimyristoyl), and R 1 and R 2 are both stearoyl (i.e., distearoyl), etc. Diacylglycerols have the following general formula: [ka] It has.

[0319] The term "dialkyloxypropyl" or "DAA" refers to a compound with two alkyl chains, R 1 and R 2and both independently have 2 to 30 carbon atoms. The alkyl groups may be saturated or have varying degrees of unsaturation. Dialkyloxypropyls include compounds having the following general formula: [ka] It has.

[0320] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and absorption of an active agent by a subject and can be included in the compositions of the present disclosure without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohol, oils, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and colorants. Such preparations are sterilized and, if desired, can be mixed with auxiliary substances such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, and / or aromatic substances that do not adversely react with the compounds of the present disclosure. Those skilled in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0321] The abbreviations used herein have their conventional meanings in the fields of chemistry and biology. The chemical structures and formulas described herein are constructed according to the standard rules of chemical valency known in the chemical arts. The description of the compounds of the present disclosure is limited by the principles of chemical bonding known to those skilled in the art. Thus, when a group can be substituted with one or more of a number of substituents, such substitutions are selected to provide a compound that is compatible with the principles of chemical bonding and is not inherently unstable and / or known to those skilled in the art to be unstable under ambient conditions, such as aqueous, neutral, and some known physiological conditions. For example, a heterocycloalkyl or heteroaryl is bonded to the rest of the molecule through a ring heteroatom according to the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0322] Where substituents are specified by their conventional chemical formula written from left to right, they equally encompass the chemically identical substituents that result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0323] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbons) or combinations thereof, which may be fully saturated, monovalent, or polyunsaturated, and may include monovalent, divalent, and polyvalent radicals. Alkyl is an alkyl group having a specified number of carbons (e.g., C1 to C6). 10means 1 to 10 carbons). Alkyl is a non-cyclized chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers of methyl, such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups are those containing one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. Alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (—O—). The alkyl moiety can be an alkenyl moiety. The alkyl moiety may be an alkynyl moiety. The alkyl moiety may be fully saturated. The alkenyl may contain more than one double bond, and / or one or more triple bonds in addition to one or more double bonds. The alkynyl may contain more than one triple bond, and / or one or more double bonds in addition to one or more triple bonds.

[0324] The term “alkylene,” by itself or as part of another substituent, is not limited, unless otherwise stated. "Alkenylene" refers to a divalent radical derived from an alkyl, as exemplified by -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group has from 1 to 30 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms. The term "alkenylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.

[0325] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise specified, a stable linear or branched chain, or combination thereof, containing at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, B, Se, and S), wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) (e.g., O, N, S, Si, B, Se, or P) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Heteroalkyl groups are non-cyclizing chains. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-S-CH2, -S(O)-CH3, -CH2-CH2-S(O)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. The heteroalkyl moiety may contain one heteroatom (e.g., O, N, S, Si, B, Se, or P). A heteroalkyl moiety may contain two optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P). A heteroalkyl moiety may contain three optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P). A heteroalkyl moiety may contain four optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P). A heteroalkyl moiety may contain five optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P). A heteroalkyl moiety may contain up to eight optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P). The term "heteroalkenyl," by itself or in combination with another term, means, unless otherwise specified, a heteroalkyl containing at least one double bond.Heteroalkenyl may optionally contain more than one double bond and / or one or more triple bonds in addition to one or more double bonds. The term "heteroalkynyl," by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl containing at least one triple bond. Heteroalkynyl may optionally contain more than one triple bond and / or one or more double bonds in addition to one or more triple bonds.

[0326] Similarly, the term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified by, but not limited to, -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-. As noted above, heteroalkyl groups, as used herein, include groups attached to the remainder of the molecule via a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -S0R'. When "heteroalkyl" is recited followed by a specific heteroalkyl group, such as, for example, -NR'R'', it will be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term "heteroalkyl" should not be construed herein as excluding specific heteroalkyl groups, such as, for example, -NR'R''.

[0327] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl," respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Furthermore, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a "heterocycloalkylene," alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0328] In embodiments, the term "cycloalkyl" refers to a monocyclic, bicyclic, or polycyclic cycloalkyl ring system. In embodiments, a monocyclic ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms; such groups can be saturated or unsaturated, but are not aromatic. In embodiments, a cycloalkyl group is fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. A bicyclic cycloalkyl ring system is a bridged monocyclic ring or a fused bicyclic ring. In embodiments, a bridged monocyclic ring includes a monocyclic cycloalkyl ring, in which two non-adjacent carbon atoms of the monocyclic ring are joined by an alkylene bridge of between 1 and 3 additional carbon atoms (i.e., of the form (CH)). wwherein w is 1, 2, or 3. Representative examples of bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane. In embodiments, a fused bicyclic cycloalkyl ring system comprises a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, a bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring. In embodiments, a cycloalkyl group is optionally substituted with one or two groups, independently oxo or thia. In embodiments, the fused bicyclic cycloalkyl is a 5- or 6-membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5- or 6-membered monocyclic cycloalkyl, a 5- or 6-membered monocyclic cycloalkenyl, a 5- or 6-membered monocyclic heterocyclyl, or a 5- or 6-membered monocyclic heteroaryl, wherein the fused bicyclic cycloalkyl is optionally substituted with one or two groups, each independently oxo or thia. In embodiments, the multicyclic cycloalkyl ring system is (i) a ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl, or (ii) a monocyclic cycloalkyl ring (group ring) fused to either of two other ring systems independently selected from the group consisting of phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclyl. In embodiments, the multicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the ring of the group.In embodiments, the multicyclic cycloalkyl ring system is (i) a ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl, or (ii) a monocyclic cycloalkyl ring (group ring) fused to either of two other ring systems independently selected from the group consisting of phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclyl. Examples of multicyclic cycloalkyl groups include, but are not limited to, tetradecahydrophenanthrenyl, perhydrophenothiazin-1-yl, and perhydrophenoxazin-1-yl.

[0329] In embodiments, cycloalkyl is cycloalkenyl. The term "cycloalkenyl" is used according to its plain and ordinary meaning. In embodiments, cycloalkenyl is a monocyclic, bicyclic, or polycyclic cycloalkenyl ring system. In embodiments, a monocyclic cycloalkenyl ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms, and such group is unsaturated (i.e., contains at least one cyclic carbon-carbon double bond), but is not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl. In embodiments, a bicyclic cycloalkenyl ring is a bridged monocyclic ring or a fused bicyclic ring. In embodiments, a bridged monocyclic ring includes a monocyclic cycloalkenyl ring, in which two non-adjacent carbon atoms of the monocyclic ring are joined by an alkylene bridge of between 1 and 3 additional carbon atoms (i.e., of the form (CH)). wwherein w is 1, 2, or 3. Representative examples of bicyclic cycloalkenyls include, but are not limited to, norbornenyl and bicyclo[2.2.2]oct2enyl. In embodiments, the fused bicyclic cycloalkenyl ring system comprises a monocyclic cycloalkenyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged or fused bicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkenyl ring. In embodiments, the cycloalkenyl group is optionally substituted with one or two groups, independently oxo or thia. In embodiments, the multicyclic cycloalkenyl ring comprises a monocyclic cycloalkenyl ring (group ring) fused to either (i) one ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl, or (ii) two ring systems independently selected from the group consisting of phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclyl. In embodiments, the multicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the group ring. In embodiments, the multicyclic cycloalkenyl ring comprises a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl, or (ii) two ring systems independently selected from the group consisting of phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclyl.

[0330] In embodiments, heterocycloalkyl is heterocyclyl. As used herein, the term "heterocyclyl" refers to a monocyclic, bicyclic, or polycyclic heterocycle. A heterocyclyl monocyclic heterocycle is a 3-, 4-, 5-, 6-, or 7-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S, and the ring is saturated or unsaturated, but not aromatic. A 3- or 4-membered ring contains one heteroatom selected from the group consisting of O, N, and S. A 5-membered ring can contain zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 6- or 7-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle. Representative examples of heterocyclyl monocyclic heterocycles include azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, and oxazolinyl. Examples of heterocyclic heterocycles include, but are not limited to, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. Heterocyclyl bicyclic heterocycles are monocyclic heterocycles fused to either phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocycle, or monocyclic heteroaryl. Heterocyclyl bicyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system.Representative examples of bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, and octahydrobenzofuranyl. In embodiments, the heterocyclyl group is optionally substituted with one or two groups, each independently oxo or thia. In certain embodiments, the bicyclic heterocyclyl is a 5- or 6-membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5- or 6-membered monocyclic cycloalkyl, a 5- or 6-membered monocyclic cycloalkenyl, a 5- or 6-membered monocyclic heterocyclyl, or a 5- or 6-membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted with one or two groups, each independently oxo or thia. A multicyclic heterocyclyl ring system is a monocyclic heterocyclyl ring (group ring) fused to either (i) one ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl, or (ii) two other ring systems independently selected from the group consisting of phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclyl. The multicyclic heterocyclyl is attached to the parent molecular moiety through any carbon atom or nitrogen atom contained within the group ring. In embodiments, the multicyclic heterocyclyl ring system is (i) one ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl, or (ii) a monocyclic heterocyclyl ring (group ring) fused to either two other ring systems independently selected from the group consisting of phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclyl.Examples of multicyclic heterocyclyl groups include, but are not limited to, 10H-phenothiazin-10-yl, 9,10-dihydroacridin-9-yl, 9,10-dihydroacridin-10-yl, 10H-phenoxazin-10-yl, 10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl, 1,2,3,4-tetrahydropyrido[4,3-g]isoquinolin-2-yl, 12H-benzo[b]phenoxazin-12-yl, and dodecahydro-1H-carbazol-9-yl.

[0331] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0332] The term "acyl," unless otherwise specified, means -C(O)R, where R is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0333] The term "aryl," unless otherwise specified, refers to a polyunsaturated aromatic hydrocarbon substituent, which may be a single ring or multiple rings (preferably 1 to 3 rings) fused together (i.e., fused-ring aryl) or covalently linked. Fused-ring aryl refers to multiple rings fused together, at least one of which is an aryl ring. The term "heteroaryl" refers to an aryl group (or ring) containing at least one heteroatom, such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term "heteroaryl" includes fused-ring heteroaryl groups (i.e., multiple rings fused together, at least one of which is a heteroaromatic ring). 5,6-fused-ring heteroarylene refers to two rings fused together, one ring having five members and the other having six members, and at least one ring being a heteroaryl ring. Similarly, a 6,6-fused ring heteroarylene refers to two rings fused together, one ring having 6 members and the other ring having 6 members, and at least one ring is a heteroaryl ring. A 6,5-fused ring heteroarylene refers to two rings fused together, one ring having 6 members and the other ring having 5 members, and at least one ring is a heteroaryl ring. The heteroaryl group can be attached to the rest of the molecule through a carbon or heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5 ... Examples of aryl and heteroaryl ring systems include benzothiazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene," alone or as part of another substituent, refer to divalent radicals derived from aryl and heteroaryl, respectively. Heteroaryl group substituents may be -O-bonded to a ring heteroatom nitrogen.

[0334] A fused-ring heterocycloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused-ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused-ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused-ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. Each fused-ring heterocycloalkyl-aryl, fused-ring heterocycloalkyl-heteroaryl, fused-ring heterocycloalkyl-cycloalkyl, or fused-ring heterocycloalkyl-heterocycloalkyl can independently be unsubstituted or substituted with one or more substituents described herein.

[0335] Spirocyclic rings are two or more rings in which adjacent rings are connected via a single atom. The individual rings within a spirocyclic ring may be the same or different. The individual rings of a spirocyclic ring may be substituted or unsubstituted and may have different substituents than the other individual rings within the set of spirocyclic rings. The possible substituents for the individual rings within a spirocyclic ring are the possible substituents for the same ring when not part of a spirocyclic ring (e.g., substituents for a cycloalkyl or heterocycloalkyl ring). The spirocyclic ring may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heterocycloalkylene, and the individual rings within the spirocyclic ring group may be any of the preceding list, including having all rings of one type (e.g., all rings may be substituted heterocycloalkylene, and each ring may be the same or different substituted heterocycloalkylene). When referring to a spirocyclic ring system, a heterocyclic spirocyclic ring means a spirocyclic ring in which at least one ring is heterocyclic and each ring may be a different ring. When referring to a spirocyclic ring system, a substituted spirocyclic ring means that at least one ring is substituted and each substituent may optionally be different.

[0336] symbol" [ka] " indicates the point of attachment of the chemical moiety to the rest of the molecule or chemical formula.

[0337] As used herein, the term "oxo" means an oxygen that is double bonded to a carbon atom.

[0338] As used herein, the term "alkylsulfonyl" means a moiety having the formula -S(O2)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' can have a specified number of carbons (e.g., "C1-C4 alkylsulfonyl").

[0339] The term "alkylarylene" as an arylene moiety covalently linked to an alkylene moiety (also referred to herein as an alkylene linker). In embodiments, the alkylarylene group has the formula:

[0340] [ka] It has.

[0341] The alkylarylene moiety may be substituted on (e.g., by a substituent) the alkylene portion or the arylene linker (e.g., at carbon 2, 3, 4, or 6) with halogen, oxo, -N, -CF, -CCl, -CBr, -CI, -CN, -CHO, -OH, -NH, -COOH, -CONH, -NO, -SH, -SOCH-SOH, -OSOH, -SONH, -NHNH, -ONH, -NHC(O)NHNH, substituted or unsubstituted C-C alkyl, or substituted or unsubstituted 2-5 membered heteroalkyl. In embodiments, the alkylarylene is unsubstituted.

[0342] Each of the above terms (e.g., "alkyl," "heteroalkyl," "cycloalkyl," "heterocycloalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0343] Substituents for alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are -OR', ═O, ═NR', ═N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -O, in a number ranging from 0 to (2m'+1), where m' is the total number of carbon atoms in such radical. The alkyl group may be one or more different groups selected from, but not limited to, C(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)R', -NR-C(NR'R''R'')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)R', -S(O)NR'R'', -NRSO2R', -NR'NR''R''', -ONR'R'', -NR'C(O)NR''NR'''R'''', -CN, -NO2, -NR'S02R'', -NR'C(O)R'', -NR'C(O)-OR'', -NR'OR''. R, R', R'', R''', and R'''' each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1 to 3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy or thioalkoxy group, or arylalkyl gr...

Claims

1. 1. A polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has an amino acid sequence identical to that of SEQ ID NO:1 or SEQ ID NO:2 by at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, 1. A polynucleotide comprising amino acids having at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity, wherein the modified IL-2 polypeptide comprises at least one substitution with a natural or unnatural amino acid at one or more positions selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof.

2. the modified IL-2 polypeptide is a) either unconjugated or conjugated to a water-soluble polymer, lipid, polypeptide, protein or peptide; and / or b) has reduced binding to interleukin-2 receptor alpha (IL-2Rα) compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without said at least one substitution; and / or c) has a reduced receptor signaling potency for IL-2Rαβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without said at least one substitution; and / or d) has an increased ratio of signaling potency for IL-2Rβγ to signaling potency for IL-2Rαβγ (increased ratio of signaling potency for IL-2Rβγ / signaling potency for IL-2Rαβγ) compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without said at least one substitution; and / or e) has enhanced receptor signaling potency for IL-2Rβγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without said at least one substitution; and / or with the proviso that if the modified IL-2 polypeptide comprises at least one substitution with a non-naturally occurring amino acid, the modified IL-2 polypeptide comprises at least one substitution at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, and / or at least one substitution with a natural or non-natural amino acid at at least one position within the IL-2Rα interacting region, IL-2Rβ interacting region, and / or IL-2Rγ interacting region; and / or f) a combination of a) to e); The polynucleotide of claim 1,

3. 3. The polynucleotide of claim 1 or claim 2, wherein the modified IL-2 polypeptide has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity in one or more regions of amino acid positions 10-25, 80-100, and / or 100-134 to the corresponding one or more regions of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:

2.

4. 4. The polynucleotide of any one of claims 1 to 3, wherein the modified IL-2 polypeptide has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:

2.

5. 5. The polynucleotide of any one of claims 1-4, wherein the modified IL-2 polypeptide comprises at least one substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of Q13, L19, N29, N30, Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, R81, L85, S87, V91, I92, V93, and combinations thereof.

6. the modified IL-2 polypeptide is a) comprises at least one substitution with a natural amino acid at a position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, and is configured to be conjugated to a water-soluble polymer, lipid, protein, or peptide at at least one position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof; and / or 6. The polynucleotide of any one of claims 1 to 5, wherein the polynucleotide comprises at least one substitution with a naturally occurring amino acid at at least one position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof, and is configured to be conjugated to a water soluble polymer, lipid, protein, or peptide at the N-terminus and / or C-terminus of the modified IL-2 polypeptide.

7. the modified IL-2 polypeptide is a) comprising at least one substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at at least one position selected from the group consisting of N29, N30, Y31, K32, N33, P34, K35, R38, T41, F42, K43, Y45, K48, K49, E62, K64, P65, N71, Q74, K76, and combinations thereof; and / or 7. The polynucleotide of any one of claims 1 to 6, comprising at least one substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76, and combinations thereof.

8. the modified IL-2 polypeptide is a) at least one substitution with cysteine ​​at a position selected from the group consisting of N29, N30, Y31, N33, P34, K35, R38, T41, K43, K48, K49, K64, P65, N71, Q74, K76 and combinations thereof; b) at least one substitution with cysteine ​​at a position selected from the group consisting of N29, Y31, K35, P65, N71, Q74, and combinations thereof; c) at least one substitution with any amino acid at position Y31, N29 or a combination thereof; d) at least one substitution with cysteine, serine or alanine at position Y31, N29 or a combination thereof; e) at least one substitution by cysteine ​​at position Y31; f) at least one substitution by cysteine ​​at position N29, and / or g) at least one substitution by cysteine ​​at position P65; The polynucleotide according to any one of claims 1 to 7, comprising:

9. 9. The polynucleotide of any one of claims 1 to 8, wherein the modified IL-2 polypeptide comprises at least one substitution with a natural amino acid at at least one position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof.

10. 10. The polynucleotide of any one of claims 1 to 9, wherein the modified IL-2 polypeptide comprises at least one substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at at least one position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof.

11. the modified IL-2 polypeptide is a) at least one substitution with cysteine ​​at a position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof; and / or b) a substitution at position F42 with alanine, lysine or serine, and / or c) a substitution at position F42 with alanine, and / or d) substitution at position F42 with serine, and / or e) substitution by lysine at position F42, and / or f) substitution at position Y45 with alanine, histidine or serine, and / or g) substitution at position Y45 with alanine, and / or h) substitution at position Y45 with histidine, and / or i) substitution at position R38 with alanine, aspartic acid or serine, and / or j) substitution at position R38 with aspartic acid, and / or k) substitution at position P65 with alanine, and / or l) substitution at position P65 with serine, and / or m) substitution at position E62 with alanine, and / or n) substitution by lysine at position F42, and / or o) substitution by cysteine ​​at position Y31, and / or p) Combination of a) to o) The polynucleotide according to any one of claims 1 to 10, comprising:

12. 12. The polynucleotide of any one of claims 1 to 11, wherein the modified IL-2 polypeptide comprises at least one substitution with a natural amino acid at at least one position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

13. 13. The polynucleotide of any one of claims 1 to 12, wherein the modified IL-2 polypeptide comprises at least one substitution with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

14. 14. The polynucleotide of any one of claims 1 to 13, wherein the modified IL-2 polypeptide comprises at least one substitution with cysteine ​​at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

15. the modified IL-2 polypeptide is a) a substitution at position Y31, and / or c) a substitution at position F42, and / or d) a substitution at position C125, and / or e) substitutions at positions Y31, F42, and C125 The polynucleotide according to any one of claims 1 to 14, comprising:

16. the modified IL-2 polypeptide is a) substitution by cysteine ​​at position Y31, and / or c) substitution at position F42 with phenylalanine, and / or d) substitution at position C125 with serine, and / or e) a substitution at position Y31 with cysteine, a substitution at position Y31 with phenylalanine, and a substitution at position C125 with serine The polynucleotide according to any one of claims 1 to 15, comprising:

17. 17. The polynucleotide of any one of claims 1 to 16, further comprising at least one substitution with a natural or unnatural amino acid at at least one position within the IL-2Rα interacting region, the IL-2Rβ interacting region and / or the IL-2Rγ interacting region.

18. The polynucleotide of any one of claims 1 to 17, further comprising at least one substitution with a natural amino acid at a position within the IL-2Rα interacting region.

19. The polynucleotide of any one of claims 1 to 18, further comprising at least one substitution with a natural amino acid at a position within the IL-2Rβ interacting region.

20. a) substitutions with natural amino acids at positions within the IL-2Rα interacting region and substitutions with natural amino acids at positions within the IL-2Rβ interacting region; b) substitutions with natural amino acids at positions within the IL-2Rα interacting region and substitutions with natural amino acids at positions within the IL-2Rγ interacting region; or c) substitutions with natural amino acids at positions within the IL-2Rα interacting region, substitutions with natural amino acids at positions within the IL-2Rβ interacting region, and substitutions with natural amino acids at positions within the IL-2Rγ interacting region; The polynucleotide of any one of claims 1 to 19, further comprising:

21. 21. The polynucleotide of any one of claims 1 to 20, wherein the modified IL-2 polypeptide has reduced binding to IL-2Rα compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

22. The polynucleotide of any one of claims 1 to 21, wherein the modified IL-2 polypeptide has an N-terminal deletion and / or a C-terminal deletion.

23. 23. The polynucleotide of any one of claims 1 to 22, wherein the modified IL-2 polypeptide has an N-terminal deletion of amino acid residues 1 to 30 and / or a C-terminal deletion of amino acid residues 114 to 134.

24. The polynucleotide of any one of claims 1 to 23, wherein the fusion protein comprises the modified IL-2 polypeptide and an additional amino acid sequence.

25. 25. The polynucleotide of any one of claims 1 to 24, wherein the fusion protein comprises the modified IL-2 polypeptide linked to an additional amino acid sequence via a linker.

26. 26. The polynucleotide of any one of claims 1 to 25, wherein the additional amino acid sequence confers an enhanced and / or prolonged pharmacokinetic (PK) profile to the modified IL-2 polypeptide or the fusion protein compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

27. 27. The polynucleotide of any one of claims 1 to 26, wherein the additional amino acid sequence is fused to the N-terminus of the modified IL-2 polypeptide or to the C-terminus of the modified IL-2 polypeptide.

28. 28. The polynucleotide of any one of claims 1 to 27, wherein the additional amino acid sequence comprises an antibody sequence or a portion or fragment thereof.

29. The polynucleotide of any one of claims 1 to 28, wherein the additional amino acid sequence comprises the Fc portion of an antibody.

30. 30. The polynucleotide of any one of claims 1 to 29, wherein the additional amino acid sequence comprises serum albumin or a PK-extended fragment or analog thereof.

31. The additional amino acid sequence is a) human serum albumin (HSA); b) mouse serum albumin (MSA); c) or a) or b) PK-extended fragment or analogue The polynucleotide of any one of claims 1 to 30, comprising:

32. The polynucleotide of any one of claims 1 to 31, wherein the modified IL-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

3.

33. 33. The polynucleotide of any one of claims 1 to 32, wherein the modified IL-2 polypeptide and / or the additional amino acid sequence is further configured to be conjugated to a water-soluble polymer, lipid, protein, or peptide via a single amino acid residue of the modified IL-2 polypeptide.

34. the modified IL-2 polypeptide and / or the additional amino acid sequence is attached to a water-soluble polymer, lipid, protein, or peptide; i) the alpha amino group of the N-terminal amino acid residue of the fusion polypeptide; ii) the epsilon amino group of a lysine amino acid residue of the fusion polypeptide, or iii) an N-glycosylation site or an O-glycosylation site of the fusion polypeptide 34. The polynucleotide of any one of claims 1 to 33, further configured to be conjugated via

35. 35. The polynucleotide of any one of claims 1 to 34, wherein the IL-2 polypeptide or the fusion protein has increased binding to IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

36. 36. The polynucleotide of any one of claims 1 to 35, wherein the IL-2 polypeptide or the fusion protein has (i) decreased binding to IL-2Rα and (ii) increased binding to IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

37. 37. The polynucleotide of any one of claims 1 to 36, wherein the binding affinity of the IL-2 polypeptide or the fusion protein to IL-2Rα is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or is reduced by about 1 to about 100,000 times or more compared to the binding affinity of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 without the at least one substitution.

38. 38. The polynucleotide of any one of claims 1 to 37, wherein the IL-2 polypeptide or the fusion protein has no detectable binding to IL-2Rα.

39. 39. The polynucleotide of any one of claims 1 to 38, wherein the IL-2 polypeptide or the fusion protein has a reduced receptor signaling potency for IL-2Rαβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

40. 40. The polynucleotide of any one of claims 1 to 39, wherein the ratio between the signaling potency of the IL-2 polypeptide or the fusion protein towards IL-2Rαβγ and the signaling potency of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution towards IL-2Rαβγ is from about 1 / 2 to about 1 / 100,000.

41. 41. The polynucleotide of any one of claims 1 to 40, wherein said IL-2 polypeptide or said fusion protein has no detectable receptor signaling effect on IL-2Rαβγ.

42. The IL-2 polypeptide or the fusion protein is (i) 42. The polynucleotide of any one of claims 1 to 41, having (i) reduced binding to IL-2Rα compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without at least one substitution, and (ii) reduced receptor signaling potency for IL-2Rαβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without said at least one substitution.

43. 43. The polynucleotide of any one of claims 1 to 42, wherein the IL-2 polypeptide or the fusion protein has no detectable binding to IL-2Rα and no detectable receptor signaling effect on IL-2Rαβγ.

44. 44. The polynucleotide of any one of claims 1 to 43, wherein the modified IL-2 polypeptide or fusion protein has increased binding with equivalent binding to interleukin-2 receptor beta (IL-2Rβ) and / or interleukin-2 receptor gamma (IL-2Rγ) compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution, and / or has equivalent receptor signaling potency for IL-2Rβγ or has increased receptor signaling potency for IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

45. 45. The polynucleotide of any one of claims 1 to 44, wherein the IL-2 polypeptide or the fusion protein has a similar or higher level of binding to IL-2Rβ or IL-2Rγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

46. 46. ​​The polynucleotide of any one of claims 1 to 45, wherein the IL-2 polypeptide or the fusion protein has a receptor signaling potency equivalent to or greater than IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

47. 47. The polynucleotide of any one of claims 1 to 46, wherein the IL-2 polypeptide or the fusion protein has an equivalent or higher level of binding to IL-2Rβ or IL-2Rγ compared to an equivalent IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution, and an equivalent or higher receptor signaling potency for IL-2Rβγ compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

48. 48. The polynucleotide of any one of claims 1 to 47, wherein the IL-2 polypeptide or the fusion protein has an increased ratio of signaling potency for IL-2Rβγ compared to signaling potency for IL-2Rαβγ (increased ratio of signaling potency for IL-2Rβγ / signaling potency for IL-2Rαβγ) compared to an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

49. 49. The polynucleotide of any one of claims 1 to 48, further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein.

50. 50. The polynucleotide of any one of claims 1 to 49, further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, wherein the promoter drives tissue-specific expression of the fusion protein.

51. 51. The polynucleotide of any one of claims 1 to 50, further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, wherein the promoter drives muscle-specific expression of the fusion protein.

52. 51. The polynucleotide of any one of claims 1 to 50, further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, wherein the promoter drives cancer-specific or tumor-specific expression of the fusion protein.

53. 51. The polynucleotide of any one of claims 1 to 50, further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, wherein the promoter drives liver-specific expression of the fusion protein.

54. 54. The polynucleotide of any one of claims 1 to 53, further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, wherein the promoter is constitutively active.

55. 54. The polynucleotide of any one of claims 1 to 53, further comprising a promoter operably linked to the nucleic acid sequence encoding the fusion protein, wherein the promoter is conditionally active.

56. 56. The polynucleotide of any one of claims 1 to 55, further comprising a promoter operably linked to said nucleic acid sequence, said promoter comprising an SP6, T3 or T7 promoter sequence.

57. 57. The polynucleotide of any one of claims 1 to 56, wherein at least a portion of the nucleic acid sequence encoding the fusion protein is codon-optimized.

58. 58. The polynucleotide of any one of claims 1 to 57, wherein the polynucleotide comprises at least one modified nucleotide.

59. 59. The polynucleotide of any one of claims 1 to 58, wherein the polynucleotide comprises at least one modified nucleotide, wherein the at least one modified nucleotide is pseudouridine, N1-methyl-pseudouridine, or 2-thiouridine.

60. 60. The polynucleotide of any one of claims 1 to 59, wherein the nucleic acid molecule comprises a 5' cap structure.

61. 61. The polynucleotide of any one of claims 1 to 60, wherein the nucleic acid molecule comprises a 3' polyA sequence.

62. 62. The polynucleotide of any one of claims 1 to 61, wherein the polynucleotide comprises DNA, RNA, or a DNA / RNA hybrid.

63. 63. The polynucleotide of any one of claims 1 to 62, wherein the polynucleotide comprises RNA.

64. 64. The polynucleotide of any one of claims 1 to 63, wherein the polynucleotide comprises a viral vector, a non-viral vector, a plasmid, or a Nanoplasmid™ vector.

65. 65. The polynucleotide of any one of claims 1 to 64, wherein the polynucleotide is configured to express the fusion protein in vitro.

66. 65. The polynucleotide of any one of claims 1 to 64, wherein the polynucleotide is configured to express the fusion protein in vivo.

67. 67. The polynucleotide of any one of claims 1 to 66, wherein the polynucleotide is in isolated form.

68. The polynucleotide, wherein the modified IL-2 polypeptide or the fusion protein is capable of expressing IL-2 for about 5 minutes to about 10 days, about 5 minutes to about 9 days, about 5 minutes to about 8 days, about 5 minutes to about 8 days, about 5 minutes to about 7 days, about 5 minutes to about 6 days, about 5 minutes to about 5 days, about 5 minutes to about 4 days, about 5 minutes to about 3 days, about 5 minutes to about 2 days, about 5 minutes to about 1 day, about 1 hour to about 10 days, about 1 hour to about 9 days, about 1 hour to about 8 days, about 1 hour to about 7 days, about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 2 days, about 1 hour to about 1 day, from about 5 minutes to about 10 minutes, from about 20 minutes to about 30 minutes, from about 40 minutes to about 50 minutes to about 1 hour 68. The polynucleotide of any one of claims 1-67, having an in vivo half-life of from about 2 hours, from about 3 hours, from about 4 hours, from about 5 hours, from about 6 hours, from about 7 hours, from about 8 hours, from about 9 hours, from about 10 hours, from about 11 hours, from about 12 hours, from about 13 hours, from about 14 hours, from about 15 hours, from about 16 hours, from about 17 hours, from about 18 hours, from about 19 hours, from about 20 hours, from about 21 hours, from about 22 hours, from about 23 hours, from about 1 day, from about 2 days, from about 3 days, from about 4 days, from about 5 days, from about 6 days, from about 7 days, from about 8 days, from about 9 days, from about 10 days, or any value or range therebetween.

69. A pharmaceutical composition comprising an effective amount of the polynucleotide of any one of claims 1 to 68 and a pharmaceutically acceptable carrier.

70. 70. The pharmaceutical composition of claim 69, wherein the pharmaceutically acceptable carrier comprises a lipid.

71. The lipid has the formula (I): 【Chemical 189】 、 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein: R １ は、H、-OR １Ａ 、-YOR １Ａ 、-NR １Ａ R １Ｂ 、-YNR １Ａ R １Ｂ 、-SR １Ａ 、-YSR １Ａ 、-(C=O)R １Ａ 、 -Y(C=O)R １Ａ 、-(C=O)OR １Ａ 、-Y(C=O)OR １Ａ 、-O(C=O)R １Ａ 、-YO(C=O)R １Ａ 、-O(C=O)OR １Ａ 、 -YO(C=O)OR １Ａ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Y is a substituted or unsubstituted C ０ ~C １２ alkylene or substituted or unsubstituted 0-12 membered heteroalkylene; R ２ は、H、-OR ２Ａ 、-SR ２Ａ 、-(C=O)R ２Ａ 、-(C=O)OR ２Ａ 、-O(C=O)R ２Ａ 、-O(C=O)OR ２Ａ 、 -(C=O)NHR ２Ａ , -NH(C=O)R ２Ａ , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R ３ は、H、-OR ３Ａ 、-SR ３Ａ 、-(C=O)R ３Ａ 、-(C=O)OR ３Ａ 、-O(C=O)R ３Ａ 、-O(C=O)OR ３Ａ 、 -(C=O)NHR ３Ａ , -NH(C=O)R ３Ａ , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R ４ は、H、-OR ４Ａ 、-SR ４Ａ 、-(C=O)R ４Ａ 、-(C=O)OR ４Ａ 、-O(C=O)R ４Ａ 、-O(C=O)OR ４Ａ 、 -(C=O)NHR ４Ａ , -NH(C=O)R ４Ａ , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R ５ は、H、-OR ５Ａ 、-SR ５Ａ 、-(C=O)R ５Ａ 、-(C=O)OR ５Ａ 、-O(C=O)R ５Ａ 、-O(C=O)OR ５Ａ 、 -(C=O)NHR ５Ａ , -NH(C=O)R ５Ａ , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; B １ is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; B ２ and B ３ are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L １ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR １０１ R １０２ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR １０１ C(=O)-, -C(=O)NR １０１ -, -NR １０１ C(=S)-, -C(=S)NR １０１ -, -NR １０１ C(=O)NR １０２ -, -NR １０１ C(=S)NR １０２ -, -OC(=O)NR １０１ -, -NR １０１ C(=O)O-, -SC(=O)NR １０１ -or-NR １０１ C(═O)S—, L ２ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR ２０１ R ２０２ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR ２０１ C(=O)-, -C(=O)NR ２０１ -, -NR ２０１ C(=O)NR ２０２ -, -NR ２０１ C(=S)-, -C(=S)NR ２０１ -, -NR ２０１ C(=S)NR ２０２ -, -OC(=O)NR ２０１ -, -NR ２０１ C(=O)O-, -SC(=O)NR ２０１ -or-NR ２０１ C(═O)S—, L ３ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR ３０１ R ３０２ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR ３０１ C(=O)-, -C(=O)NR ３０１ -, -NR ３０１ C(=O)NR ３０２ -, -NR ３０１ C(=S)-, -C(=S)NR ３０１ -, -NR ３０１ C(=S)NR ３０２ -, -OC(=O)NR ３０１ -, -NR ３０１ C(=O)O-, -SC(=O)NR ３０１ -or-NR ３０１ C(═O)S—, L ４ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR ４０１ R ４０２ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR ４０１ C(=O)-, -C(=O)NR ４０１ -, -NR ４０１ C(=O)NR ４０２ -, -NR ４０１ C(=S)-, -C(=S)NR ４０１ -, -NR ４０１ C(=S)NR ４０２ -, -OC(=O)NR ４０１ -, -NR ４０１ C(=O)O-, -SC(=O)NR ４０１ -or-NR ４０１ C(═O)S—, L ５ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR ５０１ R ５０２ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR ５０１ C(=O)-, -C(=O)NR ５０１ -, -NR ５０１ C(=O)NR ５０２ -, -NR ５０１ C(=S)-, -C(=S)NR ５０１ -, -NR ５０１ C(=S)NR ５０２ -, -OC(=O)NR ５０１ -, -NR ５０１ C(=O)O-, -SC(=O)NR ５０１ -or-NR ５０１ C(═O)S—, L ６ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR ６０１ R ６０２ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR ６０１ C(=O)-, -C(=O)NR ６０１ -, -NR ６０１ C(=O)NR ６０２ -, -NR ６０１ C(=S)-, -C(=S)NR ６０１ -, -NR ６０１ C(=S)NR ６０２ -, -OC(=O)NR ６０１ -, -NR ６０１ C(=O)O-, -SC(=O)NR ６０１ -or-NR ６０１ C(═O)S—, L ７ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR ７０１ R ７０２ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR ７０１ C(=O)-, -C(=O)NR ７０１ -, -NR ７０１ C(=O)NR ７０２ -, -NR ７０１ C(=S)-, -C(=S)NR ７０１ -, -NR ７０１ C(=S)NR ７０２ -, -OC(=O)NR ７０１ -, -NR ７０１ C(=O)O-, SC(=O)NR ７０１ -or-NR ７０１ C(═O)S—, L ａ１ and L ａ２ are each independently 【Chemistry 190】 and Each X is independently O, S or CH ２ and W １ , W ２ , W ３ , W ４ , W ５ and W ６ are each independently a bond or a substituted or unsubstituted C １ ~C １２ alkylene, or substituted or unsubstituted 2- to 12-membered heteroalkylene; Each R １Ａ and R １Ｂ are independently H, substituted or unsubstituted C １ ~C １２ alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; Each R ２Ａ , R ３Ａ , R ４Ａ , and R ５Ａ are independently H, substituted or unsubstituted C １ ~C ３０ alkyl, or substituted or unsubstituted 2- to 30-membered heteroalkyl; Each R １０１ , R １０２ , R ２０１ , R ２０２ , R ３０１ , R ３０２ , R ４０１ , R ４０２ , R ５０１ , R ５０２ , R ６０１ , R ６０２ , R ７０１ , and R ７０２ are independently H, substituted or unsubstituted C １ ~C １２ alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; 71. The pharmaceutical composition of claim 69 or claim 70, wherein each s is independently an integer from 1 to 4.

72. The cationic lipid is represented by the formula: １ But H, -OR １Ａ or substituted or unsubstituted heteroalkyl; L １ is a bond, -NR １０１ C(=S)-, -C(=S)NR １０１ -, -O(C=O)-, -(C=O)O-, or -O-; B １ is a bond or substituted or unsubstituted alkylene; B ２ and B ３ are each independently a bond or substituted or unsubstituted alkylene; L ２ is a bond, —O(C═O)—, —(C═O)O—, —O(C═O)O—, —C(═O)—, —O—, or —S—; L ４ is a bond, —O(C═O)—, —(C═O)O—, —O(C═O)O—, —C(═O)—, —O—, or —S—; W １ , W ２ , W ３ , W ４ , W ５ and W ６ each independently represents a bond or a substituted or unsubstituted C １ ~C １２ is alkylene, L ａ１ and L ａ２ However, each independently, 【Chemistry 191】 and each X is independently O or S; L ３ is a bond, —O(C═O)—, —(C═O)O—, —O(C═O)O—, —C(═O)—, —O—, or —S—; L ５ is a bond, —O(C═O)—, —(C═O)O—, —O(C═O)O—, —C(═O)—, —O—, or —S—; L ６ is a bond, —O(C═O)—, —(C═O)O—, —O(C═O)O—, —C(═O)—, —O—, or —S—; L ７ is a bond, —O(C═O)—, —(C═O)O—, —O(C═O)O—, —C(═O)—, —O—, or —S—; R ２ is H, or substituted or unsubstituted alkyl; R ３ is H, or substituted or unsubstituted alkyl; R ４ is H, or substituted or unsubstituted alkyl; R ５ is H, or substituted or unsubstituted alkyl; Each R １Ａ are independently H or substituted or unsubstituted C １ ~C １２ is alkyl, Each R １０１ is independently H or substituted or unsubstituted 2-12 membered heteroalkyl.

73. The cationic lipid is represented by the formula: １ is H, —OH, methoxy, ethoxy, or substituted or unsubstituted heteroalkyl; L １ is a bond, -NR １０１ C(=S)- or -C(=S)NR １０１ - and B １ is a bond or an unsubstituted C １ ~C ８ is alkylene, B ２ and B ３ are each independently a bond or a substituted or unsubstituted C １ ~C ８ is alkylene, L ２ is a bond, —O(C═O)—, or —(C═O)O—; L ４ is a bond, —O(C═O)—, or —(C═O)O—; W １ , W ２ , W ３ , W ４ , W ５ and W ６ each independently represents a bond or a substituted or unsubstituted C １ ~C １２ is alkylene, L ａ１ and L ａ２ However, each independently, 【Chemistry 192】 and each X is independently O or S; L ３ is a bond, —O(C═O)—, or —(C═O)O—; L ５ is a bond, —O(C═O)—, or —(C═O)O—; L ６ is a bond, —O(C═O)—, or —(C═O)O—; L ７ is a bond, —O(C═O)—, or —(C═O)O—; R ２ is H, or substituted or unsubstituted C １ ~C １２ is alkyl, R ３ is H, or substituted or unsubstituted C １ ~C １２ is alkyl, R ４ is H, or substituted or unsubstituted C １ ~C １２ is alkyl, R ５ is H, or substituted or unsubstituted C １ ~C １２ is alkyl, Each R １０１ is independently substituted or unsubstituted 2-12 membered heteroalkyl.

74. The cationic lipid is represented by the formula: １ is —OH or methoxy; L １ is a bond, B １ is unsubstituted C １ ~C ８ is alkylene, B ２ and B ３ are each independently a bond or a substituted or unsubstituted C １ ~C ８ is alkylene, L ２ is a bond, L ４ is a bond, W １ , W ２ , W ３ , W ４ , W ５ and W ６ each independently represents a bond or a substituted or unsubstituted C １ ~C １２ is alkylene, L ａ１ and L ａ２ However, each independently, 【Chemistry 193】 and each X is independently O; L ３ is a bond, L ５ is a bond, L ６ is a bond, L ７ is a bond, R ２ is H, or substituted or unsubstituted C １ ~C １２ is alkyl, R ３ is H, or substituted or unsubstituted C １ ~C １２ is alkyl, R ４ is H, or substituted or unsubstituted C １ ~C １２ is alkyl, R ５ is H, or substituted or unsubstituted C １ ~C １２ 72. The pharmaceutical composition of claim 71, wherein the lipid is alkyl.

75. The cationic lipid is represented by the formula: １ is substituted or unsubstituted heteroalkyl; L １ But -C(=S)NR １０１ - (wherein the carbon atom is connected to the nitrogen atom of formula (I)), B １ is a bond, B ２ and B ３ are each independently a bond or a substituted or unsubstituted C １ ~C ８ is alkylene, L ２ is a bond, —O(C═O)—, or —(C═O)O—; L ４ is a bond, —O(C═O)—, or —(C═O)O—; W １ , W ２ , W ３ , W ４ , W ５ and W ６ each independently represents a bond or a substituted or unsubstituted C １ ~C １２ is alkylene, L ａ１ and L ａ２ However, each independently, 【Chemistry 194】 and each X is independently O; L ３ is a bond, L ５ is a bond, L ６ is a bond, L ７ is a bond, R ２ is H, or substituted or unsubstituted C １ ~C １２ is alkyl, R ３ is H, or substituted or unsubstituted C １ ~C １２ is alkyl, R ４ is H, or substituted or unsubstituted C １ ~C １２ is alkyl, R ５ is H, or substituted or unsubstituted C １ ~C １２ 72. The pharmaceutical composition of claim 71, wherein the lipid is alkyl.

76. The cationic lipid is 【Chemistry 195】 【Chemistry 196】 72. The pharmaceutical composition of claim 71, wherein the compound is:

77. The lipid has the formula (II): 【Chemistry 197】 、 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein: B ４ Is W ７ -L ａ３ -W ８ and W ７ and W ８ are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L ａ３ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR ａ３１ R ａ３２ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR ａ３１ C(=O)-, -C(=O)NR ａ３１ -, -NR ａ３１ C(=O)NR ａ３２ -, -NR ａ３１ C(=S)-, -C(=S)NR ａ３１ -, -NR ａ３１ C(=S)NR ａ３２ -, -OC(=O)NR ａ３１ -, -NR ａ３１ C(=O)O-, -SC(=O)NR ａ３１ -or-NR ａ３１ C(═O)S—, R １０ and R １１ are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or R １０ and R １１ together with the nitrogen atom to which they are attached form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl; B ５ , B ６ , and B ７ are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L ８ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR ８０１ R ８０２ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR ８０１ C(=O)-, -C(=O)NR ８０１ -, -NR ８０１ C(=O)NR ８０２ -, -NR ８０１ C(=S)-, -C(=S)NR ８０１ -, -NR ８０１ C(=S)NR ８０２ -, -OC(=O)NR ８０１ -, -NR ８０１ C(=O)O-, -SC(=O)NR ８０１ -or-NR ８０１ C(═O)S—, L ９ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR ９０１ R ９０２ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR ９０１ C(=O)-, -C(=O)NR ９０１ -, -NR ９０１ C(=O)NR ９０２ -, -NR ９０１ C(=S)-, -C(=S)NR ９０１ -, -NR ９０１ C(=S)NR ９０２ -, -OC(=O)NR ９０１ -, -NR ９０１ C(=O)O-, -SC(=O)NR ９０１ -or-NR ９０１ C(═O)S—, L １０ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR １１０ R １１１ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR １１０ C(=O)-, -C(=O)NR １１０ -, -NR １１０ C(=O)NR １１１ -, -NR １１０ C(=S)-, -C(=S)NR １１０ -, -NR １１０ C(=S)NR １１１ -, -OC(=O)NR １１０ -, -NR １１０ C(=O)O-, -SC(=O)NR １１０ -or-NR １１０ C(═O)S—, R ７ , R ８ , and R ９ are each independently H, substituted or unsubstituted C １ ~C ３０ alkyl, or substituted or unsubstituted 2- to 30-membered heteroalkyl; Each R ａ３１ and R ａ３２ are independently H, substituted or unsubstituted C １ ~C １２ alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; Each R ８０１ , R ８０２ , R ９０１ , R ９０２ , R １１０ , and R １１１ are independently H, substituted or unsubstituted C １ ~C １２ alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; 71. The pharmaceutical composition of claim 69 or claim 70, wherein each s is independently an integer from 1 to 4.

78. The cationic lipid is of the formula: W ７ and W ８ are each independently a bond or substituted or unsubstituted alkylene; L ａ３ is a bond, R １０ and R １１ are each independently H, substituted or unsubstituted alkyl, or R １０ and R １１ together with the nitrogen atom to which they are attached form a substituted or unsubstituted heterocycloalkyl; B ５ is a bond, B ６ and B ７ are each independently a bond or substituted or unsubstituted alkylene; L ８ is a bond, L ９ is a bond, —O(C═O)—, —(C═O)O—, or —C(═O)—; L １０ is a bond, —O(C═O)—, —(C═O)O—, or —C(═O)—; R ７ , R ８ , and R ９ are each independently H or substituted or unsubstituted C １ ~C ３０ 78. The pharmaceutical composition of claim 77, wherein the lipid is alkyl.

79. W ７ and W ８ are each independently a bond or a substituted or unsubstituted C １ ~C ８ is alkylene, L ａ３ is a bond, R １０ and R １１ are each independently substituted or unsubstituted alkyl, or R １０ and R １１ together with the nitrogen atom to which they are attached form a substituted or unsubstituted heterocycloalkyl; B ５ is a bond, B ６ and B ７ are each independently a bond or a substituted or unsubstituted C １ ~C ８ is alkylene, L ８ is a bond, L ９ is —O(C═O)— or —(C═O)O—, L １０ is —O(C═O)— or —(C═O)O—, R ７ , R ８ , and R ９ are each independently substituted or unsubstituted C １ ~C ２０ 78. The cationic lipid of claim 77, which is alkyl.

80. The cationic lipid is represented by the formula W ７ and W ８ are each independently a bond or a substituted or unsubstituted C ２ ~C ４ is alkylene, L ａ３ is a bond, R １０ and R １１ are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R １０ and R １１ together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3- to 8-membered heterocycloalkyl; B ５ is a bond, B ６ and B ７ are each independently a bond or a substituted or unsubstituted C ２ ~C ４ is alkylene, L ８ is a bond, L ９ is —O(C═O)— or —(C═O)O—, L １０ is —O(C═O)— or —(C═O)O—, R ７ is H or methyl, R ８ , and R ９ are each independently substituted or unsubstituted C １ ~C ２０ 78. The pharmaceutical composition of claim 77, wherein the lipid is alkyl.

81. The cationic lipid is represented by the formula W ７ and W ８ are each independently a bond or an unsubstituted C ２ ~C ４ is alkylene, L ａ３ is a bond, R １０ and R １１ are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R １０ and R １１ together with the nitrogen atom to which they are attached form a substituted or unsubstituted 5- to 6-membered heterocycloalkyl; B ５ is a bond, B ６ and B ７ each independently represents a bond or an unsubstituted C ２ ~C ４ is alkylene, L ８ is a bond, L ９ is —O(C═O)— or —(C═O)O—, L １０ is —O(C═O)— or —(C═O)O—, R ７ is H or methyl, R ８ , and R ９ are each independently substituted or unsubstituted C １ ~C ２０ 78. The pharmaceutical composition of claim 77, wherein the lipid is alkyl.

82. The cationic lipid is represented by the formula W ７ and W ８ are each independently a bond or an unsubstituted C ２ ~C ４ is alkylene, L ａ３ is a bond, R １０ and R １１ are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R １０ and R １１ together with the nitrogen atom to which they are attached form a substituted or unsubstituted 5- to 6-membered heterocycloalkyl; B ５ , B ６ , and B ７ are each independently a bond, L ８ is a bond, L ９ is a bond, L １０ is a bond, R ７ is H or methyl, R ８ , and R ９ are each independently substituted or unsubstituted C １ ~C ３０ 78. The pharmaceutical composition of claim 77, wherein the lipid is alkyl.

83. The cationic lipid is a lipid, and the cationic lipid is 【Chemistry 198】 【Chemistry 199】 【Chemistry 200】 or a pharmaceutically acceptable salt thereof.

84. The lipid has the formula (III): 【Chemical Engineering 201】 、 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein: L １１ teeth, 【Chemical Engineering 202】 and Q is substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene; V is substituted or unsubstituted alkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted arylene; B ８ , B ９ , B １０ , and B １１ are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L １２ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR ２１０ R ２１１ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR ２１０ C(=O)-, -C(=O)NR ２１０ -, -NR ２１０ C(=O)NR ２１１ -, -NR ２１０ C(=S)-, -C(=S)NR ２１０ -, -NR ２１０ C(=S)NR ２１１ -, -OC(=O)NR ２１０ -, -NR ２１０ C(=O)O-, -SC(=O)NR ２１０ -or-NR ２１０ C(═O)S—, L １３ is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR ３１０ R ３１１ ) ｓ O-, -S-, -C(=O)S-, -SC(=O)-, -NR ３１０ C(=O)-, -C(=O)NR ３１０ -, -NR ３１０ C(=O)NR ３１１ -, -NR ３１０ C(=S)-, -C(=S)NR ３１０ -, -NR ３１０ C(=S)NR ３１１ -, -OC(=O)NR ３１０ -, -NR ３１０ C(=O)O-, -SC(=O)NR ３１０ -or-NR ３１０ C(═O)S—, R １２ は、H、-OR １２Ａ 、-SR １２Ａ 、-NR １２Ａ 、-CN、-(C=O)R １２Ａ 、-O(C=O)R １２Ａ 、-(C=O)OR １２Ａ 、 -NR １２Ａ (C═O)—R １２Ｂ , —(C═O)NR １２Ａ R １２Ｂ and R １３ は、H、-OR １３Ａ 、-SR １３Ａ 、-NR １３Ａ 、-CN、-(C=O)R １３Ａ 、-O(C=O)R １３Ａ 、-(C=O)OR １３Ａ 、 -NR １３Ａ (C═O)—R １３Ｂ , —(C═O)NR １３Ａ R １３Ｂ and R １４ and R １５ are each independently substituted or unsubstituted C ２ ~C ３０ alkyl, or substituted or unsubstituted 2- to 30-membered heteroalkyl; R １２Ａ , R １２Ｂ , R １３Ａ , and R １３Ｂ are each independently H, substituted or unsubstituted C １ ~C ２０ alkyl, or substituted or unsubstituted 2- to 20-membered heteroalkyl; Each R ２１０ , R ２１１ , R ３１０ , and R ３１１ are independently H, substituted or unsubstituted C １ ~C １２ alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; each n is independently an integer from 0 to 8; 71. The pharmaceutical composition of claim 69 or claim 70, wherein each s is independently an integer from 1 to 4.

85. The cationic lipid is of the formula: L １１ but, 【Chemical 203】 and Q is substituted or unsubstituted alkylene; V is substituted or unsubstituted alkylene; B ８ , B ９ , B １０ , and B １１ are each independently substituted or unsubstituted alkylene; L １２ is —O(C═O)— or —(C═O)O—, L １３ is —O(C═O)— or —(C═O)O—, R １２ But H, -OR １２Ａ , or -NR １２Ａ and R １３ But H, -OR １３Ａ , or -NR １３Ａ and R １４ and R １５ are each independently substituted or unsubstituted C ２ ~C ３０ is alkyl, R １２Ａ and R １３Ａ are each independently H, substituted or unsubstituted C １ ~C ２０ is alkyl, 85. The pharmaceutical composition of claim 84, wherein each n is independently an integer from 0 to 8, inclusive, of a lipid.

86. The cationic lipid is of the formula: L １１ but, 【Chemical 204】 and V is substituted or unsubstituted alkylene; B ８ , B ９ , B １０ , and B １１ are each independently substituted or unsubstituted C １ ~C ２０ is alkylene, L １２ is —O(C═O)— or —(C═O)O—, L １３ is —O(C═O)— or —(C═O)O—, R １２ is H or -OR １２Ａ and R １３ is H or -OR １３Ａ and R １４ and R １５ are each independently substituted or unsubstituted C ２ ~C ２０ is alkyl, R １２Ａ and R １３Ａ are each independently H, substituted or unsubstituted C １ ~C ８ is alkyl, 85. The pharmaceutical composition of claim 84, wherein each n is independently an integer from 0 to 4, inclusive, of a lipid.

87. The cationic lipid is of the formula: L １１ but, 【Chemical 205】 and V is unsubstituted alkylene; B ８ , B ９ , B １０ , and B １１ are each independently substituted or unsubstituted C １ ~C ８ is alkylene, L １２ is —O(C═O)— or —(C═O)O—, L １３ is —O(C═O)— or —(C═O)O—, R １２ is —OH, methoxy, or ethoxy; R １３ is —OH, methoxy, or ethoxy; R １４ and R １５ are each independently substituted or unsubstituted C ２ ~C ２０ is alkyl, 85. The pharmaceutical composition of claim 84, wherein each n is independently an integer from 0 to 4, inclusive, of a lipid.

88. The cationic lipid is a lipid, and the cationic lipid is 【Chemical 206】 、 or a pharmaceutically acceptable salt thereof.

89. The lipid has the formula (IV): 【Chemical 207】 、 or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or prodrug thereof, wherein: B １２ Ha, -W ７ -L ａ３ -W ８ - and W ７ and W ８ are each independently a bond, a substituted or unsubstituted C １ ~C １２ alkylene, or substituted or unsubstituted 2- to 12-membered heteroalkylene; L ａ３ represents a bond, -S-S-, -O-(CH ２ O) ｍ -, 【Chemical 208】 and W ９ and W １０ are each independently a bond, a substituted or unsubstituted C １ ~C １２ alkylene, substituted or unsubstituted 2- to 12-membered heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, or any combination thereof; L １４ は、joining、-O(C=O)-、-(C=O)O-、-O(C=O)O-、-O(=O)-、-O-、-O(CR ４１０ R ４１１ ) ｓ - ４１０ C(=O)-、-C(=O)NR ４１０ -、-NR ４１０ C(=O)NR ４１１ -、-NR ４１０ C (= S) -, -C(=S)NR ４１０ -, -NR ４１０ C(=S)NR ４１１ -, -OC(=O)NR ４１０ -, -NR ４１０ C(=O)O-, -SC(=O)NR ４１０ -or-NR ４１０ C(═O)S—, L １５ は、joining、-O(C=O)-、-(C=O)O-、-O(C=O)O-、-O(=O)-、-O-、-O(CR ５１０ R ５１１ ) ｓ - ５１０ C(=O)-、-C(=O)NR ５１０ -、-NR ５１０ C(=O)NR ５１１ -、-NR ５１０ C (= S) -, -C(=S)NR ５１０ -, -NR ５１０ C(=S)NR ５１１ -, -OC(=O)NR ５１０ -, -NR ５１０ C(=O)O-, -SC(=O)NR ５１０ -or-NR ５１０ C(═O)S—, R １６ and R １７ However, each independently, 【Chemical Engineering 209】 A fragment of a cationic lipid of formula (I): 【Chemical 210】 A fragment of a cationic lipid of formula (II): 【Chemistry 211】 A fragment of a cationic lipid of formula (II): 【Chemical Engineering 212】 a fragment of a cationic lipid of formula (III), or 【Chemistry 213】 A fragment of a cationic lipid of formula (III): Each R ４１０ , R ４１１ , R ５１０ , and R ５１１ are independently H, substituted or unsubstituted C １ ~C １２ alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl; each m is independently an integer from 0 to 8; 71. The pharmaceutical composition of claim 69 or claim 70, wherein each s is independently an integer from 1 to 4.

90. The cationic lipid is of the formula: L ａ３ The bond, -S-S-, 【Chemical 214】 and W ７ and W ８ each independently represents a bond or a substituted or unsubstituted C １ ~C １２ is alkylene, L １４ が、-O(C=O)-、- ４１０ C(=O)-、-C(=O)NR ４１０ -、-NR ４１０ C(=S)-、 -C(=S)NR ４１０ -, -OC(=O)NR ４１０ - or -NR ４１０ C(═O)O—, L １５ が、-O(C=O)-、- ５１０ C(=O)-、-C(=O)NR ５１０ -、-NR ５１０ C(=S)-、 -C(=S)NR ５１０ -, -OC(=O)NR ５１０ -or-NR ５１０ C(═O)O—, W ９ and W １０ each independently represents a bond or a substituted or unsubstituted C １ ~C １２ is alkylene, R １６ and R １７ However, each independently, 【Chemical 215】 is a fragment of a cationic lipid of formula (II), Each R ４１０ and R ５１０ are independently H or substituted or unsubstituted C １ ~C １２ 90. The pharmaceutical composition of claim 89, wherein the lipid is alkyl.

91. The cationic lipid is of the formula: L ａ３ The bond, -S-S-, 【Chemical 216】 and W ７ and W ８ each independently represents a bond or an unsubstituted C １ ~C １２ is alkylene, L １４ is -O(C=O)-, -(C=O)O-, -NR ４１０ C(=S)-, -C(=S)NR ４１０ -, -OC(=O)NR ４１０ -or-NR ４１０ C(═O)O—, L １５ is -O(C=O)-, -(C=O)O-, -NR ５１０ C(=S)-, -C(=S)NR ５１０ -, -OC(=O)NR ５１０ - or -NR ５１０ C(═O)O—, W ９ and W １０ each independently represents a bond or a substituted or unsubstituted C １ ~C １２ is alkylene, R １６ and R １７ However, each independently, 【Chemical 217】 is a fragment of a cationic lipid of formula (II), Each R ４１０ and R ５１０ are independently H or substituted or unsubstituted C １ ~C １２ 90. The pharmaceutical composition of claim 89, wherein the lipid is alkyl.

92. The cationic lipid is of the formula: L ａ３ The bond, -S-S-, 【Chemistry 218】 and W ７ and W ８ are each independently a bond or an unsubstituted C １ ~C ８ is alkylene, L １４ is -O(C=O)-, -(C=O)O-, -NR ４１０ C(=S)-, -C(=S)NR ４１０ -, -OC(=O)NR ４１０ -or-NR ４１０ C(═O)O—, L １５ is -O(C=O)-, -(C=O)O-, -NR ５１０ C(=S)-, -C(=S)NR ５１０ -, -OC(=O)NR ５１０ - or -NR ５１０ C(═O)O—, W ９ and W １０ are each independently a bond or an unsubstituted C １ ~C ８ is alkylene, R １６ and R １７ However, each independently, 【Chemical 219】 is a fragment of a cationic lipid of formula (II), Each R ４１０ and R ５１０ are independently H or unsubstituted C １ ~C ８ 90. The pharmaceutical composition of claim 89, wherein the lipid is alkyl.

93. The cationic lipid is of the formula: L ａ３ The bond, -S-S-, 【Chemical 220】 and W ７ and W ８ are each independently a bond or an unsubstituted C １ ~C ８ is alkylene, L １４ is -O(C=O)-, -(C=O)O-, -NR ４１０ C(=S)-, -C(=S)NR ４１０ -, -OC(=O)NR ４１０ -or-NR ４１０ C(═O)O—, L １５ is -O(C=O)-, -(C=O)O-, -NR ５１０ C(=S)-, -C(=S)NR ５１０ -, -OC(=O)NR ５１０ - or -NR ５１０ C(═O)O—, W ９ and W １０ are each independently a bond or an unsubstituted C １ ~C ８ is alkylene, R １６ and R １７ However, each independently, 【Chemistry 221】 and Each R ４１０ and R ５１０ is independently H or methyl.

94. The cationic lipid is 【Chemistry 222】 【Chemistry 223】 【Chemistry 224】 or a pharmaceutically acceptable salt thereof.

95. The pharmaceutical composition comprises the compound: 【Chemical 225】 95. The pharmaceutical composition of any one of claims 69 to 94, comprising:

96. The pharmaceutical composition of any one of claims 69 to 95, further comprising lipid nanoparticles.

97. 97. The pharmaceutical composition of any of claims 69 to 96, further comprising at least a second therapeutic agent.

98. 98. The pharmaceutical composition of claim 97, wherein the at least second therapeutic agent comprises an anti-neoplastic agent or substance, an immuno-oncology (I-O) agent, an immune checkpoint inhibitor, and / or an antibody-dependent cell-mediated cytotoxic agent, and / or a cell therapy agent.

99. The pharmaceutical composition of claim 97 or claim 98, wherein the at least second therapeutic agent comprises an anti-PD1 antibody, an anti-PDL1 antibody, an anti-EGFR antibody, and an anti-Her2 antibody, as well as an anti-CD20 antibody, an anti-CD38 antibody, an anti-CD47 antibody, an anti-CD123 antibody, an anti-cMET antibody, an anti-CCR4 antibody, an anti-CTLA4 antibody, a chimeric antigen receptor T cell (CAR-t), or a dimeric antigen receptor T cell (DAR-T).

100. 100. A polynucleotide according to any one of claims 1 to 68 or a pharmaceutical composition according to any one of claims 69 to 99 for use in a method for treating or preventing a proliferative disorder in a subject.

101. 100. A method for treating or preventing a proliferative disease or disorder in a subject having, suspected of having, or at risk of having a proliferative disease or disorder, comprising administering to said subject an effective amount of a polynucleotide of any one of claims 1 to 68 or a pharmaceutical composition of any one of claims 69 to 99.

102. 102. The method of claim 100 or claim 101, wherein the proliferative disease or disorder comprises a tumor.

103. 103. The method of any one of claims 100-102, wherein the proliferative disease or disorder comprises cancer.

104. 104. The method of any one of claims 100 to 103, wherein the proliferative disease or disorder comprises a solid tumor.

105. The proliferative disease or disorder is selected from the group consisting of chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone / osteosarcoma, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brain stem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, visual pathway and hypothalamic glioma, breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, male breast cancer, phyllodes carcinoma, Tumors, inflammatory breast cancer, adrenocortical carcinoma, islet cell carcinoma (endocrine pancreas), multiple endocrine neoplasia syndrome, parathyroid cancer, pheochromocytoma, thyroid cancer, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, bile duct cancer, carcinoid tumor, gastrointestinal cancer, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), hepatocellular carcinoma, pancreatic cancer, islet cell carcinoma, Bowel cancer, colorectal cancer, bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumor, ovarian cancer, ovarian epithelial cancer (surface epithelial stromal tumor), ovarian germ cell tumor, fallopian tube cancer, peritoneal cancer, penile cancer, renal cell carcinoma (RCC), renal pelvis and ureter cancer, transitional cell carcinoma, prostate cancer, testicular cancer, gestational trophoblastic tumor, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, esophageal cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, and oropharynx cancer 105. The method of any one of claims 100-104, wherein the cancer is selected from the group consisting of: cancer of the paranasal sinuses and nasal cavity, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, melanoma, skin cancer (non-melanoma), bronchial adenoma / carcinoid, small cell lung cancer, mesothelioma, non-small cell lung cancer (NSCLC), pleuropulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, AIDS-related cancer, Kaposi's sarcoma, epithelioid hemangioendothelioma (EHE), desmoplastic small round cell tumor, and liposarcoma.

106. 105. The method of any one of claims 102 to 104, wherein the tumor or cancer comprises a hematological malignancy.

107. The proliferative disease or disorder is selected from the group consisting of myeloid neoplasms, leukemias, lymphomas, Hodgkin's lymphoma, classical Hodgkin's lymphoma, non-Hodgkin's lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, hepatosplenic T-cell lymphoma, B-cell lymphoma reticuloendotheliosis, reticulosis, microglioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, nodular lymphocyte-predominant Hodgkin's lymphoma, plasma cell leukemia, acute erythrocyte and erythroleukemia, acute erythrocytic myelopathy, acute erythrocytic leukemia, Heilmeyer-Schoner disease, acute megakaryoblastic leukemia, mast cell leukemia, panmyelosis, acute panmyelosis with myelofibrosis, lymphosarcoma cell leukemia, acute leukemia of unspecified cell type, blastic phase chronic myeloid leukemia, stem cell leukemia, chronic leukemia of unspecified cell type, subacute leukemia of unspecified cell type, accelerated phase chronic myeloid leukemia, acute Myeloid leukemia, polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, adult T-cell leukemia / lymphoma, aggressive NK cell leukemia, B-cell prolymphocytic leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, chronic idiopathic myelofibrosis, multiple myeloma, Kahler's disease, myelomatosis, solitary myeloma, plasmacytoid 107. The method of any one of claims 100-103 and 106, comprising a hematological malignancy selected from the group consisting of: cystic leukemia, plasmacytoma, extramedullary, malignant plasma cell neoplasm NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative lesion, lymphocytic granulomatosis, angioimmunoblastic lymphadenopathy, T-gamma lymphoproliferative disorder, Waldenstrom's macroglobulinemia, alpha heavy chain disease, gamma heavy chain disease, Franklin disease, immunoproliferative small intestinal disease, Mediterranean disease, malignant immunoproliferative disorder, not otherwise specified, and immunoproliferative disorder NOS.

108. 100. A method for treating or preventing an immunodeficiency disease or disorder in a subject having, suspected of having, or at risk of having an immunodeficiency disease or disorder, comprising administering to the subject an effective amount of a polynucleotide described in any one of claims 1 to 68 or a pharmaceutical composition described in any one of claims 69 to 99.

109. The immunodeficiency disease or disorder may be agammaglobulinemia: X-linked and autosomal recessive, ataxia-telangiectasia, chronic granulomatous disease and other phagocytic disorders, common variable immunodeficiency, complement deficiency, DiGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyper-IgE syndrome, hyper-IgM syndrome, IgG subclass deficiency, congenital immune deficiency, NEMO deficiency syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immunodeficiency and combined immunodeficiency, specific antibody deficiency, transient ischemic stroke, encephalopathy, encephalopathy of infancy ...

109. The method of claim 108, wherein the immunodeficiency disorder is selected from the group consisting of magglobulinemia, WHIM syndrome (warts, hypogammaglobulinemia, infections and myelocatechus), Wiskott-Aldrich syndrome, other antibody deficiency disorders, other primary cellular immunodeficiencies, severe combined immunodeficiency (SCID), common variable immunodeficiency (CVID), human immunodeficiency virus / acquired immunodeficiency syndrome (HIV / AIDS), drug-induced immunodeficiency, graft-versus-host syndrome, primary immunodeficiency disease (PIDD), and lymphopenia.

110. 100. A method for treating or preventing an autoimmune or inflammatory disease or disorder in a subject having, suspected of having, or at risk of having an autoimmune or inflammatory disease or disorder, the method comprising administering to the subject an effective amount of a polynucleotide of any one of claims 1 to 68 or a pharmaceutical composition of any one of claims 69 to 99.

111. The autoimmune or inflammatory disease or disorder is inflammation, autoimmune disease, paraneoplastic autoimmune disease, cartilage inflammation, fibrotic disease and / or bone degradation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, S EA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis , scleroderma, systemic lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, 111. The method of claim 110, wherein the disease is selected from the group consisting of lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barré disease, type 1 diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, and transplant rejection.

112. 100. A method for treating or preventing an infectious disease or disorder in a subject having, suspected of having, or at risk of having an infectious disease or disorder, the method comprising administering to the subject an effective amount of a polynucleotide of any one of claims 1 to 68 or a pharmaceutical composition of any one of claims 69 to 99.

113. The infectious disease or disorder is selected from the group consisting of Acinetobacter infection, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (acquired immune deficiency syndrome), amebiasis, anaplasmosis, Angiostrongyliasis cantonensis, anisakiasis, anthrax, Arcanobacterium hemolyticum infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, Balantidiosis, Bartho Nellosis, Baylis-ascaris infection, BK virus infection, Neisseria gonorrhoeae, Blastocystitis, Bacteroides spp., Bolivian hemorrhagic fever, botulism (and infant botulism), Brazilian hemorrhagic fever, Brucellosis, Bubonic plague, Burkholderia infection, Buruli ulcer, Calicivirus infection (norovirus and sapovirus), Campylobacteriosis, Candidiasis (moniliasis, thrush), Capillariasis, Carrion disease, Cat scratch disease, Cellulitis, Chagas disease (American trypanosomiasis), Chancroid, Chickenpox, Chickweed ulcerative colitis, chlamydia, Chlamydophila pneumoniae infection (Taiwan Acute Respiratory Pathogen or TWAR), cholera, chromoblastomycosis, chytridiomycosis, opisthorchiasis, Clostridium difficile colitis, coccidioidomycosis, Colorado tick fever (CTF), common cold (acute viral nasopharyngitis; acute coryza), coronavirus disease 2019 (COVID-19), Creutzfeldt-Jakob disease (CJD), Crimean-Congo hemorrhagic fever (CCHF), cryptococcosis, cryptosporidium , cutaneous larva migrans (CLM), cyclosporiasis, cysticercosis, cytomegalovirus infection, dengue fever, desmodesmus infection, dienteramebiasis, diphtheria, diphyllobothriasis, dracunculosis, Ebola hemorrhagic fever, echinococcosis, ehrlichiosis, enterobiasis (pinworm infection), enterococcus infection, enterovirus infection, typhus, erythema infectiosum (fifth disease), exanthema subitum (sixth disease), fascioliasis, fascioliasis, fatal familial insomnia (FFI), filariasis, Clostridium perfringensperfringens), free-living amebic infections, Fusobacterium infections, gas gangrene (clostridial myonecrosis), geotrichosis, Gerstmann-Straussler-Scheinker syndrome (GSS), giardiasis, glanders, gnathostomiasis, gonorrhea, granuloma venereum (donovanosis), group A streptococcal infections, group B streptococcal infections, Haemophilus influenzae infections, hand, foot, and mouth disease (HFMD), hantavirus pulmonary syndrome (HPS), heartland virus disease, Helicobacter pylori infection, hemolytic uremic syndrome (HUS), and renal symptoms Hemorrhagic fever (HFRS), Hendra virus infection, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human Ewingia ehrlichiosis, Human granulocytic anaplasmosis (HGA), Human metapneumovirus infection, Human monocytic ehrlichiosis, Human papillomavirus (HPV) infection, Human parainfluenza virus infection, Membranous tapeworm disease, Epstein-Barr virus infectious mononucleosis (Mono), Influenza (flu), Isosporosis, Kawasaki disease, Horny horn Meningitis, Kingella kingae infection, kuru, Lassa fever, Legionnaires' disease (legionnaires' disease), Pontiac fever, leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease (Lyme borreliosis), lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever (MHF), measles, Middle East respiratory syndrome (MERS), melioidosis (Whitmore's disease), meningitis, meningococcal disease, trephine disease, microsporidiosis, molluscum contagiosum (MC), monkeypox, mumps, murine typhus (endemic typhus), mycoplasma pneumonia , Mycoplasma genitalium infection, mycetoma, myiasis, neonatal conjunctivitis (ophthalmia neonatorum), Nipah virus infection, norovirus (children and infants), (new) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiosis, onchocerciasis (river blindness), opisthorchiasis, paracoccidioidomycosis (South American blastomycosis), paragonimiasis, pasteurellosis, head lice, body lice, pubic lice, pelvic inflammatory disease (PID), whooping coughcough), plague, pneumococcal infection, Pneumocystis pneumonia (PCP), pneumonia, poliomyelitis, Prevotella infection, primary amebic meningoencephalitis (PAM), progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, relapsing fever, respiratory syncytial virus infection, rhinosporidiosis, rhinovirus infection, rickettsial infection, rickettsialpox, Rift Valley fever (RVF), Rocky Mountain spotted fever (RMSF), rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, scarlet fever, schistosomiasis, septicemia, shigellosis (bacillary dysentery), shingles (herpes zoster), smallpox (pox), sporotrichosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, subacute sclerosing panencephalitis, bejel, plum Venom, and staph, taeniasis, tetanus (tetanus), tinea barbae (barber's rash), tinea capitis (tinea capitis), tinea corporis (tinea corporis), tinea cruris (jock itch), tinea manubri (tinea worm of the hand), tinea nigricans, tinea pedis (athlete's foot), tinea unguium (onychomycosis), tinea versicolor (tinea versicolor), toxocariasis (ocular larva migrans (OLM)), toxocariasis (visceral larva migrans (VLM)), and toxoplasmosis 113. The method of claim 112, wherein the infectious agent is selected from the group consisting of rhesus mammary gland infection, trachoma, trichinosis, trichomoniasis, trichuriasis (whipworm infection), tuberculosis, tularemia, typhoid fever, typhus, Ureaplasma urealyticum infection, coccidioidomycosis, valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Vibrio vulnificus infection, Vibrio parahaemolyticus enteritis, viral pneumonia, West Nile fever, ringworm (tinea blanca), Yersinia pseudotuberculosis infection, yersiniosis, yellow fever, zeaspora, Zika fever, and zygomycosis.

114. 100. Use of an effective amount of a polynucleotide according to any one of claims 1 to 68 or a pharmaceutical composition according to any one of claims 69 to 99 for the manufacture of a medicament for treating or preventing a proliferative disease or disorder, an autoimmune or inflammatory disease or disorder, or an infectious disease or disorder in a subject.

115. The method of any one of claims 100 to 113 or the use of claim 114, wherein the subject is a human.

116. The method of any one of claims 100 to 113 or the use of claim 114, wherein the subject is a non-human mammal.

117. CD4 ＋ Helper cells, CD8 ＋ 100. A method of expanding a population of effector naive and memory cells, T regulatory (Treg) cells, natural killer (NK) cells or natural killer T (NKT) cells, comprising contacting the cell population with an effective amount of the polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99 for a time sufficient to induce the formation of a complex with IL-2Rβγ, thereby stimulating expansion of the T cell, NK cell and / or NKT cell population.

118. CD4 ＋ helper cells, CD8 ＋ A method of expanding a population of effector naive and memory cells, Treg cells, natural killer (NK) cells or natural killer T (NKT) cells, the method comprising contacting the cell population with an effective amount of the polynucleotide according to any one of claims 1 to 68 or the pharmaceutical composition according to any one of claims 69 to 99 for a time sufficient to induce formation of a complex with IL-2Rβγ, thereby stimulating expansion of said T cell, Treg cell, NK cell and / or NKT cell population with a 10% to 100% decrease in cell death.

119. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99 is a CD3 polypeptide contacted with an equivalent IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without said at least one substitution. ＋ CD4 in cell populations ＋ Compared to the expansion of Treg cells, the CD3 ＋ CD4 in the cell population ＋ 119. The method of claim 117 or claim 118, wherein T Treg cells are expanded by less than 20%, 15%, 10%, 5%, 1% or less.

120. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99 is capable of inhibiting CD4 in the cell population. ＋ The method of any one of claims 117 to 119, wherein Treg cells are not substantially expanded.

121. 121. The method of any one of claims 117 to 120, wherein the ratio of effector T cells to Treg cells in the cell population after incubation with the polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99 is about or at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, 100:1 or more.

122. 122. The method of any one of claims 117 to 121, wherein the method is performed in vivo.

123. The method of any of claims 117 to 121, wherein the method is performed in vitro.

124. 122. The method of any of claims 117 to 121, wherein the method is performed ex vivo.

125. CD4 in the cell population ＋ Helper cells, CD8 ＋ 100. Use of an effective amount of a polynucleotide according to any one of claims 1 to 68 or a pharmaceutical composition according to any one of claims 69 to 99 for the manufacture of a medicament for expanding effector naive and memory cell, Treg cell, natural killer (NK) cell, or natural killer T (NKT) cell populations.

126. CD4 in the subject ＋ Helper cells, CD8 ＋ Use of an effective amount of a polynucleotide according to any one of claims 1 to 68 or a pharmaceutical composition according to any one of claims 69 to 99 to expand effector naive and memory cell, Treg cell, natural killer (NK) cell, or natural killer T (NKT) cell populations.

127. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99, wherein the fusion protein comprises the mouse serum albumin (MSA) amino acid sequence shown in SEQ ID NO:

6.

128. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99, wherein the fusion protein comprises the human serum albumin (HSA) amino acid sequence shown in SEQ ID NO:

7.

129. 99. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99, wherein the fusion protein comprises the mouse serum albumin (MSA) amino acid sequence set forth in SEQ ID NO: 6 linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO:

3.

130. 99. The polynucleotide of claim 1, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 3 linked to the N-terminus of the amino acid sequence set forth in the amino acid sequence of mouse serum albumin (MSA) set forth in SEQ ID NO:

6.

131. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99, wherein the fusion protein comprises the amino acid sequence set forth in the mouse serum albumin (MSA) amino acid sequence set forth in SEQ ID NO: 6 linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 3 via a linker.

132. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 3 linked via a linker to the N-terminus of the amino acid sequence shown in the amino acid sequence of mouse serum albumin (MSA) shown in SEQ ID NO:

6.

133. 100. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 7 linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO:

3.

134. 99. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 3 linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO:

7.

135. 100. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 7 linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO:

3.

136. 99. The polynucleotide of claim 1, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 3 linked via a linker to the N-terminus of the amino acid sequence set forth in SEQ ID NO:

7.

137. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

4.

138. The polynucleotide of any one of claims 1 to 68 or the pharmaceutical composition of any one of claims 69 to 99, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

5.

139. 100. The pharmaceutical composition of any one of claims 69 to 99, wherein the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:

14.

140. 140. The pharmaceutical composition of any one of claims 69 to 99 and 139, wherein the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:

14.

141. The pharmaceutical composition comprises: a) a nucleic acid sequence as set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14; b) the following compound: 【Chemistry 226】 and

142. The pharmaceutical composition comprises: a) a nucleic acid sequence as set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14; b) the following compound: 【Chemistry 227】 and, 142. The pharmaceutical composition of any one of claims 69 to 99, 139, and 141, comprising a lipid nanoparticle.

143. The pharmaceutical composition comprises: a) a nucleic acid sequence as set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; b) the following compound: 【Chemistry 228】 and, A pharmaceutical composition according to any one of claims 69 to 99 and 139 to 142, comprising a lipid nanoparticle.

144. The method of any one of claims 101 to 124, wherein said polynucleotide or said pharmaceutical composition comprises a polynucleotide or pharmaceutical composition according to any one of claims 127 to 143.

145. The use according to claim 125 or claim 126, wherein said polynucleotide or said pharmaceutical composition comprises a polynucleotide or pharmaceutical composition according to any one of claims 127 to 143.

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