Polynucleotides encoding modified interleukin 2 (il2) polypeptides and methods of 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, pharmaceutical compositions comprising such polynucleotides, and pharmaceutical compositions comprising such polynucleotides. And methods of treating diseases and conditions by administering such polynucleotides, vectors, and / or pharmaceutical compositions.

Description

TECHNICAL FIELD

[0001] The present disclosure relates, among other things, 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 conditions by administering such polynucleotides, vectors, and pharmaceutical compositions. BACKGROUND

[0002] Cytokines comprise 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 cases, cytokines modulate the balance between humoral and cell-based immune responses.

[0003] Interleukins are signaling proteins that regulate the development and differentiation of T and B lymphocytes, monocyte lineage cells, 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 comprises a 15.5 kDa four alpha-helix bundle. The precursor form of IL-2 is 153 amino acid residues in length, with the first 20 amino acids forming a signal peptide and residues 21-153 forming the mature form. IL-2 is produced primarily by CD4+ T cells following 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 specific combinations of interactions with 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). Interaction of IL-2 with IL-2Rα forms a “low affinity” IL-2 receptor complex, with K d of about 10 -8 M. Interaction of IL-2 with IL-2Rβ and IL-2Rγ forms an “intermediate affinity” IL-2 receptor complex, with K d of about 10 -9 M. Interaction of IL-2 with all three subunits IL-2Rα, IL-2Rβ, and IL-2Rγ forms a “high affinity” IL-2 receptor complex, with K d of about >10-11 M.

[0004] In some cases, IL-2 signaling via the "high-affinity" IL-2Rαβγ complex regulates the activation and proliferation of regulatory T cells. Regulatory T cells, such as CD4+CD25+Foxp3+ regulatory T (Treg) cells, mediate the maintenance of 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 cases, Treg cells are generated by the thymus (tTreg cells) or induced by naive T cells in the periphery (pTreg cells). In some cases, Treg cells are considered the dominant mediators of peripheral tolerance. Indeed, in one study, the transfer of CD25-depleted peripheral CD4+ T cells induced various autoimmune diseases in nude mice, while the co-transfer of CD4+CD25+ T cells suppressed the development of autoimmunity (Sakaguchi et al., *Journal of Immunology*). J. Immunol. (1995). The enhancement of the Treg cell population downregulates effector T cell proliferation and suppresses autoimmunity and T cell antitumor responses.

[0005] The clinical use of interleukin-2 (IL-2) for the treatment of many diseases and conditions, such as cancer, as well as autoimmune and inflammatory diseases, is primarily limited by its toxicity and short in vivo half-life (Pachella et al., Journal of Advanced Practitioners in Oncology). J Adv Pract Oncol (2015); Lotze et al. (1985) Journal of Immunology (1985). Toxicity has been observed to be significantly reduced in animals lacking CD25 (IL-2 receptor α unit, IL-2Rα) (Boyman et al., Journal of Immunology (2009)). In some cases, PEGylation, i.e., covalently linking polyethylene glycol (PEG) to therapeutic agents, has been shown to overcome barriers such as rapid bodily clearance, aggregation, and enzymatic degradation (Maiser et al., Biotechnology and Bioengineering (2015); Lotze et al. (1985) Journal of Immunology (1985)). Biotechnol Bioeng WO 2019 / 028419 A1 and WO 2019 / 028425 A1 disclose certain interleukin (IL) conjugates (e.g., IL-2 conjugates) and their use in treating certain indications. WO 2019 / 028419 A1 and WO 2019 / 028425 A1 also describe pharmaceutical compositions comprising one or more interleukin conjugates (e.g., IL-2 conjugates) and kits.

[0006] Despite certain IL-2 muteins as potential therapeutic molecules, there remains a need for alternative modified IL-2 chemical forms to provide reduced toxicity, enhanced pharmacokinetic and pharmacodynamic profiles, and other attributes relative to the corresponding peptide-based counterparts. SUMMARY

[0007] The present application provides, among other things, polynucleotides encoding modified interleukin 2 (IL-2) polypeptides and polynucleotides encoding fusion proteins comprising a modified IL-2 polypeptide; 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 of treating or preventing a disease or condition, such as a proliferative disease or condition, an immunodeficiency disease or condition, an inflammatory disease or condition, an autoimmune disease or condition, and / or an infectious disease or condition.

[0008] In certain embodiments, which can 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 an amino acid having 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 the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, wherein the modified IL-2 polypeptide comprises at least one substitution with a natural amino acid or a non-natural 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.

[0009] In certain embodiments, which can 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: a) the modified IL-2 polypeptide is configured to be unconjugated or conjugated to a water-soluble polymer, a lipid, a polypeptide, a protein, or a peptide; and / or b) the modified IL-2 polypeptide has reduced binding to interleukin 2 receptor alpha (IL-2Ra) as compared to a comparable 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 c) the modified IL-2 polypeptide has reduced receptor signaling potency for IL-2Raβγ as compared to a comparable 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 d) the modified IL-2 polypeptide has an increased ratio of signaling potency for IL-2Rβγ to signaling potency for IL-2Raβγ (increased ratio of signaling potency for IL-2Rβγ / signaling potency for IL-2Raβγ) as compared to a comparable 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 e) the modified IL-2 polypeptide has enhanced receptor signaling potency for IL-2Rβγ as compared to a comparable 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 provided that when the modified IL-2 polypeptide comprises at least one substitution with a non-natural amino acid, the modified IL-2 polypeptide comprises at least one substitution at a position selected from the group 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 amino acid or a non-natural amino acid at at least one position within the IL-2Ra interaction region, the IL-2Rβ interaction region, and / or the IL-2Rγ interaction region; f) a combination of a) to e).

[0010] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides 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 IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides 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 IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising 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, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides 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 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, a lipid, a protein, or a 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 b) 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, a lipid, a protein, or a peptide at the N-terminus and / or the C-terminus of the modified IL-2 polypeptide.

[0014] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides 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 a 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 b) at least one substitution with a 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, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides 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: 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 the position of Y31, N29, or combinations thereof; d) at least one substitution with cysteine, serine, or alanine at the position of Y31, N29, or combinations thereof; e) at least one substitution with cysteine at position Y31; f) at least one substitution with cysteine at position N29; and / or g) at least one substitution with cysteine at position P65.

[0016] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising 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 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, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising 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 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, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides 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 with alanine, lysine, or serine at position F42; and / or c) a substitution with alanine at position F42; and / or d) a substitution with serine at position F42; and / or e) a substitution with lysine at position F42; and / or f) a substitution with alanine, histidine, or serine at position Y45; and / or g) a substitution with alanine at position Y45; and / or h) a substitution with histidine at position Y45; and / or i) a substitution with alanine, aspartic acid, or serine at position R38; and / or j) a substitution with aspartic acid at position R38; and / or k) a substitution with alanine at position P65; and / or 1) a substitution with serine at position P65; and / or m) a substitution with alanine at position E62; and / or n) a substitution with lysine at position F42; and / or o) a substitution with cysteine at position Y31; and / or p) a combination of a) through o).

[0019] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising 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 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, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising 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 amino acid at at least one position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0021] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is 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 at least one substitution with a 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, which can be combined with other embodiments provided herein and throughout, provided is 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) 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, which can be combined with other embodiments provided herein and throughout, provided is 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) 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, which can be combined with other embodiments provided herein and throughout, provided is 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 at least one substitution with a natural amino acid or a non-natural amino acid at at least one position within the IL-2Ra interaction region, the IL-2Rb interaction region, and / or the IL-2Ry interaction region.

[0025] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is 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 at least one substitution with a natural amino acid at a position within the IL-2Ra interaction region.

[0026] In certain embodiments, which can be combined with other embodiments presented herein and throughout, provided is 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 at least one substitution with a natural amino acid at a position within the IL-2Rβ interaction region.

[0027] In certain embodiments, which can be combined with other embodiments presented herein and throughout, provided is 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) a substitution with a natural amino acid at a position within the IL-2Rα interaction region and a substitution with a natural amino acid at a position within the IL-2Rβ interaction region; b) a substitution with a natural amino acid at a position within the IL-2Rα interaction region and a substitution with a natural amino acid at a position within the IL-2Rγ interaction region; or c) a substitution with a natural amino acid at a position within the IL-2Rα interaction region, a substitution with a natural amino acid at a position within the IL-2Rβ interaction region, and a substitution with a natural amino acid at a position within the IL-2Rγ interaction region.

[0028] In certain embodiments, which can be combined with other embodiments presented herein and throughout, provided is 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 N-terminal deletion and / or a C-terminal deletion.

[0029] In certain embodiments, which can be combined with other embodiments presented herein and throughout, provided is 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 N-terminal deletion and / or a C-terminal deletion.

[0030] In certain embodiments, which can be combined with other embodiments presented herein and throughout, provided is 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 N-terminal deletion of amino acid residues 1-30 and / or a C-terminal deletion of amino acid residues 114-134.

[0031] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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.

[0032] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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.

[0034] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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.

[0035] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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.

[0036] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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 an Fc portion of an antibody.

[0037] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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 an Fc portion of an antibody.

[0038] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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: a) human serum albumin (HSA); b) murine serum albumin (MSA); c) or a PK extended fragment or analog of a) or b).

[0039] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is 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 an amino acid sequence set forth in SEQ ID NO: 3.

[0040] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is 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 and / or the additional amino acid sequence is further configured to be conjugated to a water-soluble polymer, a lipid, a protein, or a peptide through a single amino acid residue of the modified IL-2 polypeptide.

[0041] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising 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, a lipid, a protein, or a peptide by: i) an alpha amino group of an N-terminal amino acid residue of the fusion polypeptide; ii) an 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.

[0042] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, 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.

[0043] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the IL-2 polypeptide or the fusion protein: (i) has reduced binding to IL-2Rα and (ii) 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.

[0044] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the IL-2 polypeptide or the fusion protein has reduced binding affinity to IL-2Rα 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 the at least one substitution.

[0045] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the IL-2 polypeptide or the fusion protein has no detectable binding to IL-2Ra.

[0046] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the IL-2 polypeptide or the fusion protein has reduced receptor signaling potency for IL-2Raβγ 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.

[0047] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the ratio between the signaling potency of the IL-2 polypeptide or the fusion protein for IL-2Raβγ 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 for IL-2Raβγ is about 1 / 2 to about 1 / 100,000.

[0048] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the IL-2 polypeptide or the fusion protein has no detectable receptor signaling potency for IL-2Raβγ.

[0049] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein: (i) the IL-2 polypeptide or the fusion protein has reduced binding to IL-2Ra as 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 (ii) the IL-2 polypeptide or the fusion protein has reduced receptor signaling potency for IL-2RaPrg as 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.

[0050] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the IL-2 polypeptide or the fusion protein has no detectable binding to IL-2Ra and no detectable receptor signaling potency for IL-2RaPrg.

[0051] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide or the fusion protein has comparable or increased binding to interleukin 2 receptor beta (IL-2RP) and / or interleukin 2 receptor gamma (IL-2Ry) as 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 the modified IL-2 polypeptide or the fusion protein has comparable or increased receptor signaling potency for IL-2RPrg as 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.

[0052] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the IL-2 polypeptide or the fusion protein has comparable or higher binding levels to IL-2RP or IL-2Ry as 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.

[0053] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the IL-2 polypeptide or the fusion protein has a comparable 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, and the IL-2 polypeptide or the fusion protein has a comparable 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.

[0054] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the IL-2 polypeptide or the fusion protein has a comparable 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, and the IL-2 polypeptide or the fusion protein has a comparable 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.

[0055] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the IL-2 polypeptide or the fusion protein has a comparable 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, and the IL-2 polypeptide or the fusion protein has a comparable 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.

[0056] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the IL-2 polypeptide or the fusion protein has a comparable 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, and the IL-2 polypeptide or the fusion protein has a comparable 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.

[0057] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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, wherein the promoter drives tissue-specific expression of the fusion protein.

[0058] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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, wherein the promoter drives muscle-specific expression of the fusion protein.

[0059] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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, wherein the promoter drives cancer-specific expression or tumor-specific expression of the fusion protein.

[0060] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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, wherein the promoter drives liver-specific expression of the fusion protein.

[0061] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided is a polynucleotide comprising 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, wherein the promoter is constitutively active.

[0062] In certain embodiments, which can be combined with other embodiments presented herein and throughout, provided is a polynucleotide comprising 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, wherein the promoter is conditionally active.

[0063] In certain embodiments, which can be combined with other embodiments presented herein and throughout, provided is a polynucleotide comprising 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, wherein the promoter comprises a SP6, T3, or T7 promoter sequence.

[0064] In certain embodiments, which can be combined with other embodiments presented herein and throughout, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the polynucleotide further comprises wherein at least a portion of the nucleic acid sequence encoding the fusion protein has been codon-optimized.

[0065] In certain embodiments, which can be combined with other embodiments presented herein and throughout, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the polynucleotide further comprises wherein the polynucleotide comprises at least one modified nucleotide.

[0066] In certain embodiments, which can be combined with other embodiments presented herein and throughout, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the polynucleotide further comprises wherein the polynucleotide comprises at least one modified nucleotide, wherein the at least one modified nucleotide is a pseudouridine, N1-methyl-pseudouridine, or 2-thiouridine.

[0067] In certain embodiments, which can be combined with other embodiments presented herein and throughout, provided is a polynucleotide comprising 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.

[0068] In certain embodiments, which can 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 polynucleotide further comprises a 3' polyA sequence.

[0069] In certain embodiments, which can 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 polynucleotide further comprises: DNA; RNA; or a DNA / RNA hybrid.

[0070] In certain embodiments, which can 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 polynucleotide further comprises RNA.

[0071] In certain embodiments, which can 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 polynucleotide further comprises: a viral vector; a non-viral vector; a plasmid; or a Nanoplasmid TM vector.

[0072] In certain embodiments, which can 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 polynucleotide is configured to express the fusion protein in vitro.

[0073] In certain embodiments, which can 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 polynucleotide is configured to express the fusion protein in vivo.

[0074] In certain embodiments, which can 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 polynucleotide is in isolated form.

[0075] In certain embodiments, which can 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 half-life of the modified IL-2 polypeptide or the fusion protein in vivo is 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, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or a value or range therein.

[0076] In certain embodiments, which can 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.

[0077] In certain embodiments, which can 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, wherein the pharmaceutically acceptable carrier comprises a lipid.

[0078] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (I): (I), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: R 1 is 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 substituted or unsubstituted C0-C 12 alkylene or substituted or unsubstituted 0- to 12-membered heteroalkylene; R 2 is 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 is 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 is 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 is 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)NR101 - or -NR 101 C(=O)NR 102 - or -NR 101 C(=S)NR 102 - or -OC(=O)NR 101 - or -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 - or -NR 201 C(=O)NR 202 - or -NR 201 C(=S)-, -C(=S)NR 201 - or -NR 201 C(=S)NR 202 - or -OC(=O)NR 201 - or -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 - or -NR 301 C(=O)NR 302 - or -NR 301 C(=S)-, -C(=S)NR 301 - or -NR 301 C(=S)NR 302 - or -OC(=O)NR 301 - or -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 -, -NR601 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 Each independently is , , , , , , , or ; Each X is independently O, S, or CH2; W 1 W 2 W 3 W 4 W 5 and W 6 Each is an independent bond, substituted or unsubstituted C1-C 12 Alkylene or substituted or unsubstituted 2- to 12-membered heteroalkylene; each R 1A and R 1B independently is 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 independently is 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 , R 401 , R 402 , R 501 , R 502 , R 601 , R 602 , R 701 and R 702 independently is H, substituted or unsubstituted C1-C 12 alkyl or substituted or unsubstituted 2 to 12 membered heteroalkyl; and each s independently is an integer from 1 to 4.

[0079] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (I): :R 1 is 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 each independently is a bond or substituted or unsubstituted alkylene; L2 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-; L 4 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-; W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 each independently is a bond or substituted or unsubstituted C1-C 12 alkylene; L a1 and L a2 each independently is or ; each X independently is O or S; L 3 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-; L 5 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-; L 6 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-; L 7 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, 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 independently is H or substituted or unsubstituted C1-C 12 alkyl; and each R 101 independently is H or substituted or unsubstituted 2- to 12-membered heteroalkyl.

[0080] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (I): (I), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein, wherein the cationic lipid is a lipid in which: R 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 -; B 1 is a bond or unsubstituted C1-C8 alkylene; B 2 and B 3 are each independently a bond or 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 substituted or unsubstituted C1-C 12 alkylene; L a1 and L a2 are each independently ; 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 alkyl; R 3 is H or substituted or unsubstituted C1-C 12 alkyl; R 4 is H or substituted or unsubstituted C1-C 12 alkyl; R 5 is H or substituted or unsubstituted C1-C 12 alkyl; and each R 101 is independently substituted or unsubstituted 2- to 12-membered heteroalkyl.

[0081] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (I): (I), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein, wherein the cationic lipid is a lipid in which: R 1 is -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 substituted or unsubstituted C1-C8 alkylene; 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 substituted or unsubstituted C1-C 12 alkylene; L a1 and L a2 are each independently ; 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 alkyl; R 3 is H or substituted or unsubstituted C1-C 12 alkyl; R 4 is H or substituted or unsubstituted C1-C 12 alkyl; and R 5 is H or substituted or unsubstituted C1-C 12 alkyl.

[0082] In certain embodiments, which can 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (I): (I), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein, wherein the cationic lipid is a lipid in which: R 1 is substituted or unsubstituted heteroalkyl; L 1 is -C(=S)NR 101 -, wherein the carbon atom is attached to a nitrogen atom in Formula (I); B 1 is a bond; B 2 and B 3 are each independently a bond or 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 , W4 , W 5 and W 6 each independently is a bond or substituted or unsubstituted C1-C 12 alkylene; L a1 and L a2 each independently is ; 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 alkyl; R 3 is H or substituted or unsubstituted C1-C 12 alkyl; R 4 is H or substituted or unsubstituted C1-C 12 alkyl; and R 5 is H or substituted or unsubstituted C1-C 12 alkyl.

[0083] In certain embodiments, which can 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (I): (I), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein, wherein the cationic lipid is: , , , , , or , or a pharmaceutically acceptable salt thereof.

[0084] In certain embodiments, which can 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (II): (II), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: B 4 is W 7 -L a3 -W 8 ; W 7 and W 8 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L a3 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, -0(CR a31 R a32 ) s O-, -S-, -C(=0)S-, -SC(=0)-, -NR a31 C(=0)-, -C(=0)NR a31 -, -NR a31 C(=0)NR a32 -, -NR a31 C(=S)-, -C(=S)NR a31 -, -NR a31 C(=S)NR a32 -, -OC(=0)NR a31 -, -NR a31 C(=0)0-, -SC(=0)NR a31 -, or -NR a31 C(=0)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 substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; B 5 , B 6 , and B 7each independently is 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 110C(=0)-, -C(=0)NR 110 -, -NR 110 C(=0)NR 111 -, -NR 110 C(=S)-, -C(=S)NR 110 -, -NR 110 C(=S)NR 111 -, -OC(=0)NR 110 -, -NR 110 C(=0)0-, -SC(=0)NR 110 - or -NR 110 C(=0)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 is 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 is independently H, substituted or unsubstituted C1-C 12 alkyl or substituted or unsubstituted 2- to 12-membered heteroalkyl; and each s is independently an integer from 1 to 4.

[0085] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (II): (II), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein, wherein the cationic lipid is a lipid in which: 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 each independently is H, substituted or unsubstituted alkyl, or R 10 and R 11 together with the nitrogen atom to which they are attached form substituted or unsubstituted heterocycloalkyl; B 5 is a bond; B 6 and B 7 each independently is a bond or substituted or unsubstituted alkylene; L 8 is a bond; L 9 is a bond, -0(C=0)-, -(C=0)0-, or -C(=0)-; L 10 is a bond, -0(C=0)-, -(C=0)0-, or -C(=0)-; and R 7 , R 8 and R 9 each independently is H or substituted or unsubstituted C1-C 30 alkyl.

[0086] In certain embodiments, which can 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (II): (II), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein, wherein W 7 and W 8 each independently is a bond or substituted or unsubstituted C1-C8 alkylene; L a3 is a bond; R 10 and R 11 each independently is substituted or unsubstituted alkyl, or R 10 and R 11 together with the nitrogen atom to which they are attached form substituted or unsubstituted heterocycloalkyl; B 5 is a bond; B 6 and B 7each independently is a bond or substituted or unsubstituted C1-C8alkylene; 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-; and R 7 , R 8 and R 9 each independently is substituted or unsubstituted C1-C 20 alkyl.

[0087] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (II): (II), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: W 7 and W 8 each independently is a bond or substituted or unsubstituted C2-C4alkylene; L a3 is a bond; R 10 and R 11 each independently is substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are attached form substituted or unsubstituted 3- to 8-membered heterocycloalkyl; B 5 is a bond; B 6 and B 7 each independently is a bond or substituted or unsubstituted C2-C4alkylene; 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; and R 8and R 9 each independently is substituted or unsubstituted C1-C 20 alkyl.

[0088] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (II): (II), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: W 7 and W 8 each independently is a bond or unsubstituted C2-C4alkylene; L a3 is a bond; R 10 and R 11 each independently is 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 is a bond or unsubstituted C2-C4alkylene; L 8 is a bond; L 9 is -0(C=0)- or -(C=0)0-; L 10 is -0(C=0)- or -(C=0)0-; R 7 is H or methyl; and R 8 and R 9 each independently is substituted or unsubstituted C1-C 20 alkyl.

[0089] In certain embodiments, which can 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (II): (II), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: W 7 and W 8 each independently is a bond or unsubstituted C2-C4alkylene; L a3 is a bond; R 10 and R 11 each independently is 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 each independently is a bond; L 8 is a bond; L 9 is a bond; L 10 is a bond; R 7 is H or methyl; and R 8 and R 9 each independently is substituted or unsubstituted C1-C 30 alkyl.

[0090] In certain embodiments, which can 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (II): (II), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein the cationic lipid is: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or , or a pharmaceutically acceptable salt thereof.

[0091] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (III): (III), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: L 11 is 、 、 、 or ; 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 12is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, -0(CR 210 R 211 ) s O-, -S-, -C(=0)S-, -SC(=0)-, -NR 210 C(=0)-, -C(=0)NR 210 -, -NR 210 C(=0)NR 211 -, -NR 210 C(=S)-, -C(=SNR 210 -, -NR 210 C(=S)NR 211 -, -OC(=0)NR 210 -, -NR 210 C(=0)0-, -SC(=0)NR 210 -, or -NR 210 C(=0)S-; L 13 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, -0(CR 310 R 311 ) s O-, -S-, -C(=0)S-, -SC(=0)-, -NR 310 C(=0)-, -C(=0)NR 310 -, -NR 310 C(=0)NR 311 -, -NR 310 C(=S)-, -C(=SNR 310 -, -NR 310 C(=S)NR 311 -, -OC(=0)NR 310 -, -NR 310 C(=0)0-, -SC(=0)NR 310 -, or -NR 310 C(=0)S-; R 12 is H, -OR 12A , -SR 12A , -NR 12A , -CN, -(C=0)R 12A , -0(C=0)R 12A , -(C=0)OR 12A , -NR 12A (C=0)-R 12B , -(C=0)NR 12A R12B ; R 13 is H, -OR 13A , -SR 13A , -NR 13A , -CN, -(C=0)R 13A , -0(C=0)R 13A , -(C=0)OR 13A , -NR 13A (C=0)-R 13B , -(C=0)NR 13A R 13B ; R 14 and R 15 are each independently substituted or unsubstituted C2-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 is independently H, substituted or unsubstituted C1-C 12 alkyl or substituted or unsubstituted 2- to 12-membered heteroalkyl; each n is independently an integer from 0 to 8; and each s is independently an integer from 1 to 4.

[0092] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (III): (III), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: L 11 is , or ; Q is substituted or unsubstituted alkylene; V is substituted or unsubstituted alkylene; B 8 , B 9 , B 10 , and B 11 each independently is substituted or unsubstituted alkylene; L 12 is -0(C=0)- or -(C=0)0-; L 13 is -0(C=0)- or -(C=0)0-; R 12 is H, -OR 12A , or -NR 12A ; R 13 is H, -OR 13A , or -NR 13A ; R 14 and R 15 each independently is substituted or unsubstituted C2-C 30 alkylene; R 12A and R 13A each independently is H, substituted or unsubstituted C1-C 20 alkyl; and each n independently is an integer from 0 to 8.

[0093] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (III): (III), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: L 11 is or ; V is substituted or unsubstituted alkylene; B 8 , B 9 , B 10 , and B 11 each independently is substituted or unsubstituted C1-C 20 alkylene; L 12is -O(C=O)- or -(C=O)O-; L 13 is -O(C=O)- or -(C=O)O-; R 12 is H or -OR 12A ; R 13 is H or -OR 13A ; R 14 and R 15 each independently is substituted or unsubstituted C2-C 20 alkyl; R 12A and R 13A each independently is H, substituted or unsubstituted C1-C8 alkyl; and each n independently is an integer from 0 to 4.

[0094] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (III): (III), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: L 11 is or ; V is unsubstituted alkylene; B 8 , B 9 , B 10 , and B 11 each independently is 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; R 13 is -OH, methoxy, or ethoxy; R 14 and R 15each independently is a substituted or unsubstituted C2-C12alkylene; 20 alkyl; and each n is independently an integer from 0 to 4.

[0095] In certain embodiments, which can 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (III): (III), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein the cationic lipid is: or , or a pharmaceutically acceptable salt thereof.

[0096] In certain embodiments, which can 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (IV): (IV), or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: B 12 is -W 7 -L a3 -W 8 -; W 7 and W 8 each independently is a bond, a substituted or unsubstituted C1-C12alkylene, or a substituted or unsubstituted 2 to 12 membered heteroalkylene; 12 alkylene, or a substituted or unsubstituted 2 to 12 membered heteroalkylene; L a3 is a bond, -S-S-, -O-(CH2O) m -, or ; W 9 and W 10each independently is a bond, substituted or unsubstituted C1-C6alkylene, substituted or unsubstituted 2 to 6 membered heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, or any combination thereof; 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 R 511 ) 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 each independently is fragments of cationic lipids of Formula (I), fragments of cationic lipids of Formula (II), fragments of cationic lipids of Formula (II), fragments of cationic lipids of Formula (III), or fragments of cationic lipids of Formula (III); each R 410 , R 411 , R 510 , and R 511 is 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; and each s is independently an integer from 1 to 4.

[0097] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (IV): (IV): wherein: L a3 is a bond, -S-S-, or ; W 7 and W 8 are each independently a bond or substituted or unsubstituted C1-C 12 alkylene; L 14 is -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 is -O(C=O)-, -(C=O)O-, -C(=O)-, -NR 510 C(=O)-, -C(=O)NR 510 -, -NR 510C(=S)-, -C(=S)NR 510 -, -OC(=O)NR 510 - or -NR 510 C(=O)O-; W 9 and W 10 each independently is a bond or substituted or unsubstituted C1-C 12 alkylene; R 16 and R 17 each independently is a fragment of a cationic lipid of Formula (II); and each R 410 and R 510 independently is H or substituted or unsubstituted C1-C 12 alkyl.

[0098] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (IV): (IV), wherein the cationic lipid is a lipid in which: L a3 is a bond, -S-S-, or ; W 7 and W 8 each independently is a bond or unsubstituted C1-C 12 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 is a bond or substituted or unsubstituted C1-C 12 alkylene; R 16 and R 17 each independently is a fragment of the cationic lipid of Formula (II); and each R 410 and R 510 independently is H or substituted or unsubstituted C1-C 12 alkyl.

[0099] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (IV): (IV), wherein the cationic lipid is a lipid in which: L a3 is a bond, -S-S-, or ; W 7 and W 8 each independently is a bond or unsubstituted C1-C8 alkylene; L 14 is -O(C=O)-, -(C=O)O-, -NR 410 C(=S)-, -C(=S)NR 410 -, 410 -OC(=O)NR 410 - or -NR 15 C(=O)O-; L 510 is -O(C=O)-, -(C=O)O-, -NR 510 C(=S)-, -C(=S)NR 510 -, 510 -OC(=O)NR 9 - or -NR 10 C(=O)O-; W 16 and W 17 each independently is a bond or unsubstituted C1-C8 alkylene;17 each independently is a fragment of the cationic lipid of Formula (II); and each R 410 and R 510 independently is H or unsubstituted C1-C8 alkyl.

[0100] In certain embodiments, which can be combined with other embodiments provided herein and throughout, a pharmaceutical composition is provided, the 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (IV): (IV), wherein: L a3 is a bond, -S-S-, or ; W 7 and W 8 each independently is 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 each independently is a bond or unsubstituted C1-C8 alkylene; R 16 and R 17 each independently is or ; and each R 410 and R 510 independently is H or methyl.

[0101] In certain embodiments, which can 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, wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of Formula (IV): (IV), wherein the cationic lipid is: , , , , , , or , or a pharmaceutically acceptable salt thereof.

[0102] In certain embodiments, which can 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, wherein the pharmaceutical composition comprises the following compound: .

[0103] In certain embodiments, which can 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, wherein the pharmaceutical composition further comprises a lipid nanoparticle.

[0104] In certain embodiments, which can 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, wherein the pharmaceutical composition further comprises at least a second therapeutic agent.

[0105] In certain embodiments, which can 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-tumor agent or substance, an immuno-oncology (I-O) agent, an immune checkpoint inhibitor, and / or an antibody-dependent cell-mediated cytotoxicity agent and / or a cellular therapy agent.

[0106] In certain embodiments, which can 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-PD1 antibody, an anti-PDL1 antibody, an anti-EGFR antibody, an anti-Her2 antibody, 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).

[0107] In certain embodiments, which can 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 as described herein and throughout; or an effective amount of a pharmaceutical composition comprising such polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout, wherein the polynucleotide or the pharmaceutical composition is for use in a method of treating or preventing a proliferative disorder in a subject.

[0108] In some embodiments that can be combined with other embodiments provided herein and throughout the text, a method for treating or preventing a proliferative disease or condition in a subject who has, is suspected of having, or is at risk of developing a proliferative disease or condition is provided, the method comprising administering to the subject an effective amount of a polynucleotide or an effective amount of a pharmaceutical composition, the polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout the text, the pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout the text.

[0109] In certain embodiments that can be combined with other embodiments provided herein and throughout, a method is provided for treating or preventing a proliferative disease or condition in a subject who has, is suspected of having, or is at risk of developing a proliferative disease or condition, the method comprising administering to the subject an effective amount of a polynucleotide or an effective amount of a pharmaceutical composition, the polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, the pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout, wherein the proliferative disease or condition comprises a tumor.

[0110] In some embodiments that can be combined with other embodiments provided herein and throughout, a method is provided for treating or preventing a proliferative disease or condition in a subject who has, is suspected of having, or is at risk of developing a proliferative disease or condition, the method comprising administering to the subject an effective amount of a polynucleotide or an effective amount of a pharmaceutical composition, the polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, the pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout, wherein the proliferative disease or condition comprises cancer.

[0111] In certain embodiments that can be combined with other embodiments provided herein and throughout, a method is provided for treating or preventing a proliferative disease or condition in a subject who has, is suspected of having, or is at risk of developing a proliferative disease or condition, the method comprising administering to the subject an effective amount of a polynucleotide or an effective amount of a pharmaceutical composition, the polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout, the pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout, wherein the proliferative disease or condition comprises a solid tumor.

[0112] In certain embodiments, which can 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 the proliferative 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 as described herein and throughout, 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, ependymal, 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 neoplasm 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 cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), hepatocellular cancer, pancreatic cancer, 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, penile cancer, renal cell cancer (RCC), renal pelvis and ureter cancer, transitional cell cancer, prostate cancer, testicular cancer, gestational trophoblastic tumor, ureter and renal pelvis, transitional cell cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, esophageal cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, 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 sarcoma, epithelioid hemangioendothelioma (EHE), desmoplastic small round cell tumor, and liposarcoma.

[0113] In certain embodiments, which can 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 said proliferative 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 polynucleotide, a pharmaceutically acceptable carrier, wherein the proliferative disease or disorder comprises a tumor, wherein the tumor or the cancer comprises a malignant blood tumor.

[0114] In certain embodiments, which can 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 the proliferative disease or disorder, the methods 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, a pharmaceutically acceptable carrier,wherein the proliferative disease or disorder comprises a malignant hematological tumor selected from the group consisting of myeloma, 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, oligodendroglioma, 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 marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, nodular lymphocyte predominant Hodgkin's lymphoma, plasma cell leukemia, acute erythremia and erythroleukemia, acute erythroid myelosis, acute erythroid leukemia, Heilmeyer-Schoner disease, acute megakaryocyte leukemia, mast cell leukemia, panmyelosis, acute panmyelosis with myelofibrosis, lymphosarcoma cell leukemia, acute leukemia of unspecified cell type, blast crisis chronic myelogenous leukemia, stem cell leukemia, chronic leukemia of unspecified cell type, subacute leukemia of unspecified cell type, accelerated chronic myelogenous 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, myeloma, solitary myeloma, plasma cell leukemia, extramedullary plasmacytoma, malignant plasma cell neoplasm NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative disease, lymphoid 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, unspecified malignant immunoproliferative disease, and immunoproliferative disease NOS.

[0115] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing an immune deficiency disease or condition in a subject having, suspected of having, or at risk of having the immune deficiency disease or condition, 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 as described herein and throughout.

[0116] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing an immune deficiency disease or condition in a subject having, suspected of having, or at risk of having the immune deficiency disease or condition, 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 as described herein and throughout, wherein the immune deficiency disease or condition is selected from the group of agammaglobulinemia: X-linked and autosomal recessive, ataxia telangiectasia, chronic granulomatous disease and other phagocyte disorders, common variable immunodeficiency, complement deficiencies, DiGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyper-IgE syndrome, hyper-IgM syndrome, IgG subclass deficiency, innate immunodeficiency, 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 dyserythropoietic chronic neutropenia), 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 lymphocytopenia.

[0117] In certain embodiments, which can be combined with other embodiments provided herein and throughout, methods are provided for treating or preventing an autoimmune or inflammatory disease or condition in a subject having, suspected of having, or at risk of having the autoimmune or inflammatory disease or condition, the methods 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 as described herein and throughout.

[0118] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are methods for treating or preventing an autoimmune or inflammatory disease or condition in a subject having, suspected of having, or at risk of having the autoimmune or inflammatory disease or condition, the methods 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 as described herein and throughout, wherein the autoimmune or inflammatory disease or condition is selected from the group of inflammation, autoimmune diseases, paraneoplastic autoimmune diseases, inflammation of cartilage, fibrotic diseases and / or bone degradation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter’s syndrome, SEA syndrome (seronegativity, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter’s syndrome, SEA syndrome (seronegativity, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, Takayasu’s arteritis, Wegener’s granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, cirrhosis, primary biliary cirrhosis, 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-Barre disease, type I diabetes, thyroiditis (e.g., Graves’ disease), Addison’s disease, Raynaud’s phenomenon, autoimmune hepatitis, GVHD, and transplant rejection.

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

[0120] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided a method for treating or preventing an infectious disease or condition in a subject having, suspected of having, or at risk of having an infectious disease or condition, 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 as described herein and throughout, wherein the infectious disease or condition is selected from the group of Acinetobacter infection, Actinomycosis, African Sleeping Sickness (African Trypanosomiasis), AIDS (Acquired Immunodeficiency Syndrome), Amebiasis, Anaplasmosis, Angiostrongyliasis, Ancylostomiasis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial meningitis, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, Balantidiasis, Bartonellosis, Baylisascariasis infection, BK virus infection, Black piedra, Blastocystosis, Blastomycosis, Bolivian hemorrhagic fever, Botulism (and infant botulism), Brazilian hemorrhagic fever, Brucellosis, Bubonic plague, Burkholderia infection, Buruli ulcer, Calicivirus infection (Norovirus and Sapporo virus), Campylobacteriosis, Candidiasis (Moniliasis; Thrush), Capillariasis, Carrión's disease, Cat scratch disease, Cellulitis, Chagas disease (American Trypanosomiasis), Chancroid, Chickenpox, Chikungunya fever, Chlamydia, Chlamydia pneumoniae infection (Taiwan acute respiratory pathogen or TWAR), Cholera, Chromoblastomycosis, Chytridiomycosis, Clonorchiasis, Clostridium difficile colitis, Coccidioidomycosis, Colorado tick fever (CTF), Common cold (Acute viral nasopharyngitis; Acute rhinitis), Coronavirus disease 2019 (COVID-19), Creutzfeldt-Jakob disease (CJD), Crimean-Congo hemorrhagic fever (CCHF), Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans (CLM), Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever, Diatom infection, Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculosis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythema infectiosum (Fifth disease), Exanthema subitum (Sixth disease), Fascioliasis, Fasciolopsiasis, Fatal familial insomnia (FFI), Filariasis, Food poisoning by Clostridium perfringens, Free-living amebic infection, Fusobacterium infection, Gas gangrene (Clostridial myonecrosis), Geotrichosis, Gerstmann-Straussler-Scheinker syndrome (GSS), Giardiasis, Glanders, Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group A Streptococcus infection,Group B streptococcal infection, Haemophilus influenzae infection, hand, foot, and mouth disease (HFMD), hantavirus pulmonary syndrome (HPS), hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex, histoplasmosis, hookworm infection, human bocavirus infection, human ewingii ehrlichiosis, human granulocytic anaplasmosis (HGA), human metapneumovirus infection, human monocytotropic ehrlichiosis, human papillomavirus (HPV) infection, human parainfluenza virus infection, hymenolepidosis, infectious mononucleosis (Mono) due to Epstein–Barr virus, influenza (flu), isosporiasis, Kawasaki disease, keratitis, Kingella kingae infection, kuru, lassa fever, legionellosis (Legionnaires' disease), pittypah fever, leishmaniasis, leprosy, leptospirosis, listeriosis, lyme disease (borreliosis), lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, marburg hemorrhagic fever (MHF), measles, middle east respiratory syndrome (MERS), melioidosis (Whitmore's disease), meningitis, meningococcal disease, metagonimiasis, 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 (in children and infants), (new) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiosis, onchocerciasis (river blindness), opisthorchiasis, paracoccidioidomycosis (South American blastomycosis), paragonimiasis, pasteurellosis, pediculosis capitis (head lice), pediculosis corporis (body lice), pediculosis pubis (crab lice, pubic lice), pelvic inflammatory disease (PID), pertussis (whooping 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, sepsis, shigellosis (bacillary dysentery), shingles, smallpox, sporotrichosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, subacute sclerosing panencephalitis, syphilis, other than sexually transmitted, syphilis, and yaws, tapeworm disease, tetanus (lockjaw), tinea barbae (beard ringworm), tinea capitis (scalp ringworm), tinea corporis (body ringworm), tinea cruris (jock itch), tinea manus (hand ringworm), tinea nigra, 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, whipworm disease (trichuris infection), tuberculosis, tularemia, typhoid fever, typhus fever, ureaplasma urealyticum infection, valley fever, venezuelan equine encephalitis, venezuelan hemorrhagic fever, vibrio vulnificus infection, vibrio parahaemolyticus enteritis, viral pneumonia, west nile fever, white-patch sarcoidosis (alopecia), pseudotuberculosis yersiniosis, yersiniosis, yellow fever, zeaspora, zika fever, and yaws.

[0121] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided 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 an effective amount of a pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout, 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.

[0122] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided methods and uses as described herein and throughout, wherein the methods and the uses 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 as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout, wherein the subject is a human.

[0123] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided methods and uses as described herein and throughout, wherein the methods and the uses 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 as described herein and throughout, or an effective amount of a pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout, wherein the subject is a non-human mammal.

[0124] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided methods and uses as described herein and throughout, wherein the methods and the uses independently comprise expanding CD4 + Helper cells, CD8 +Methods of expanding a population of effector na'ive and memory cells, regulatory T (Treg) cells, natural killer (NK) cells, or natural killer T (NKT) cells, the method comprising contacting the population of cells with a polynucleotide or an effective amount of a pharmaceutical composition comprising such polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout, for a time sufficient to induce complex formation with IL-2Rβγ, thereby stimulating expansion of the population of T cells, NK cells, and / or NKT cells, the polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide as described herein and throughout.

[0125] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided a method of expanding a population of CD4 + helper cells, CD8 + Methods of expanding a population of effector na'ive and memory cells, regulatory T (Treg) cells, natural killer (NK) cells, or natural killer T (NKT) cells, the method comprising contacting the population of cells with a polynucleotide or an effective amount of a pharmaceutical composition for a time sufficient to induce complex formation with IL-2Rβγ, thereby stimulating expansion of the population of T cells, Treg cells, NK cells, and / or NKT cells, while reducing cell death rate by 10% to 100%, the polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide as described herein and throughout, the pharmaceutical composition comprising such polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout.

[0126] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided a method of expanding a population of CD4 + helper cells, CD8 + Methods of expanding a population of effector na'ive and memory cells, regulatory T (Treg) cells, natural killer (NK) cells, or natural killer T (NKT) cells, the method comprising contacting the population of cells with a polynucleotide or an effective amount of a pharmaceutical composition, the polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide as described herein and throughout, the pharmaceutical composition comprising such polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout, wherein CD3 + CD4 + Treg cells in the population of cells contacted with the polynucleotide or the pharmaceutical composition reduces cell death rate by 10% to 100% compared to a comparable population of cells contacted with 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. + CD4 +T Treg cell expansion is less than 20%, 15%, 10%, 5%, 1%, or less.

[0127] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided a method of expanding CD4 + helper cells, CD8 + A method of expanding a population of effector naive and memory cells, regulatory T (Treg) cells, natural killer (NK) cells, or natural killer T (NKT) cells, the method comprising contacting the population of cells with 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 as described herein and throughout, wherein the polynucleotide or the pharmaceutical composition does not substantially expand CD4 + Treg cells.

[0128] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided a method of expanding CD4 + helper cells, CD8 + A method of expanding a population of effector naive and memory cells, regulatory T (Treg) cells, natural killer (NK) cells, or natural killer T (NKT) cells, the method comprising contacting the population of cells with 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 as described herein and throughout, wherein the ratio of effector T cells to Treg cells in the population of cells 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 after incubation with the polynucleotide or the pharmaceutical composition.

[0129] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided a method of expanding CD4 + helper cells, CD8 +Methods of expanding a population of effector na'ive and memory cells, regulatory T (Treg) cells, natural killer (NK) cells, or natural killer T (NKT) cells, the method comprising contacting the population of cells with 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 as described herein and throughout, wherein the method is performed in vivo.

[0130] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided an effective amount of a polynucleotide or an effective amount of a pharmaceutical composition for use in expanding a population of CD4 + Helper cells, CD8 + Methods of expanding a population of effector na'ive and memory cells, regulatory T (Treg) cells, natural killer (NK) cells, or natural killer T (NKT) cells, the method comprising contacting the population of cells with 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 as described herein and throughout, wherein the method is performed in vitro.

[0131] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided an effective amount of a polynucleotide or an effective amount of a pharmaceutical composition for use in expanding a population of CD4 + Helper cells, CD8 + Methods of expanding a population of effector na'ive and memory cells, regulatory T (Treg) cells, natural killer (NK) cells, or natural killer T (NKT) cells, the method comprising contacting the population of cells with 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 as described herein and throughout, wherein the method is performed ex vivo.

[0132] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided an effective amount of a polynucleotide or an effective amount of a pharmaceutical composition for use in expanding a population of CD4 + Helper cells, CD8 +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 in a medicament for expanding a population of effector naive and memory cells, Treg cells, natural killer (NK) cells, or natural killer T (NKT) cells in a subject, the pharmaceutical composition comprising such polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout.

[0133] In certain embodiments, which can be combined with other embodiments provided herein and throughout, an effective amount of a polynucleotide or an effective amount of a pharmaceutical composition is provided for expanding a population of CD4 + helper cells, CD8 + 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 in a medicament for expanding a population of effector naive and memory cells, Treg cells, natural killer (NK) cells, or natural killer T (NKT) cells in a subject, the pharmaceutical composition comprising such polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout.

[0134] In certain embodiments, which can 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 polynucleotide is as described herein and throughout, and a pharmaceutical composition comprising such polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout is provided, wherein the fusion protein comprises a murine serum albumin (MSA) amino acid sequence set forth in SEQ ID NO: 6.

[0135] In certain embodiments, which can 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 polynucleotide is as described herein and throughout, and a pharmaceutical composition comprising such polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout is provided, wherein the fusion protein comprises a human serum albumin (HSA) amino acid sequence set forth in SEQ ID NO: 7.

[0136] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, and pharmaceutical compositions comprising such polynucleotides and a pharmaceutically acceptable carrier as described herein and throughout, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 3 linked to the N-terminus of an amino acid sequence set forth in SEQ ID NO: 6.

[0137] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, and pharmaceutical compositions comprising such polynucleotides and a pharmaceutically acceptable carrier as described herein and throughout, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 3 linked to the N-terminus of an amino acid sequence set forth in SEQ ID NO: 6.

[0138] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, and pharmaceutical compositions comprising such polynucleotides and a pharmaceutically acceptable carrier as described herein and throughout, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 3 linked to the N-terminus of an amino acid sequence set forth in SEQ ID NO: 6.

[0139] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, and pharmaceutical compositions comprising such polynucleotides and a pharmaceutically acceptable carrier as described herein and throughout, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 3 linked to the N-terminus of an amino acid sequence set forth in SEQ ID NO: 6.

[0140] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the polynucleotide is as described herein and throughout, and pharmaceutical compositions comprising such polynucleotides and a pharmaceutically acceptable carrier as described herein and throughout, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 7 linked to the N-terminus of an amino acid sequence set forth in SEQ ID NO: 3.

[0141] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the polynucleotide is as described herein and throughout, and pharmaceutical compositions comprising such polynucleotides and a pharmaceutically acceptable carrier as described herein and throughout, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 3 linked to the N-terminus of an amino acid sequence set forth in SEQ ID NO: 7.

[0142] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the polynucleotide is as described herein and throughout, and pharmaceutical compositions comprising such polynucleotides and a pharmaceutically acceptable carrier as described herein and throughout, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 7 linked to the N-terminus of an amino acid sequence set forth in SEQ ID NO: 3.

[0143] In certain embodiments, which can be combined with other embodiments provided herein and throughout, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the polynucleotide is as described herein and throughout, and pharmaceutical compositions comprising such polynucleotides and a pharmaceutically acceptable carrier as described herein and throughout, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 3 linked to the N-terminus of an amino acid sequence set forth in SEQ ID NO: 7.

[0144] In some embodiments that can be combined with other embodiments provided herein and throughout the text, a polynucleotide and pharmaceutical composition is provided, the polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide is as described herein and throughout the text, the pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout the text, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 4.

[0145] In some embodiments that can be combined with other embodiments provided herein and throughout the text, a polynucleotide, a pharmaceutical composition is provided, the polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein the polynucleotide is as described herein and throughout the text, the pharmaceutical composition comprising such a polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout the text, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 5.

[0146] In some embodiments that can be combined with other embodiments provided herein and throughout the text, a pharmaceutical composition is provided comprising such a polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout the text, the polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout the text, wherein the polynucleotide comprises the nucleic acid sequence shown 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.

[0147] In some embodiments that can be combined with other embodiments provided herein and throughout the text, a pharmaceutical composition is provided comprising such a polynucleotide and a pharmaceutically acceptable carrier as described herein and throughout the text, the polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin-2 (IL-2) polypeptide as described herein and throughout the text, wherein the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.

[0148] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising such 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 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; and b) the following compound: .

[0149] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising such 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 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: ; and a lipid nanoparticle.

[0150] In certain embodiments, which can be combined with other embodiments provided herein and throughout, there is provided a pharmaceutical composition comprising such 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 comprises: a) a nucleic acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; b) the following compound: ; and a lipid nanoparticle.

[0151] Other aspects and advantages of the application will be apparent from the embodiments and examples provided herein.

[0152] The disclosures of publications including patents cited in this specification are hereby incorporated by reference. BRIEF DESCRIPTION OF DRAWINGS

[0153] 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.

[0154] Figure 1 A-1C provides results of ELISA assays using post-transfection supernatant from 293 cells transfected with mRNA encoding the indicated fusion proteins.

[0155] Figure 2 Results of reporter assays obtained using HEK-Blue IL-2 receptor (IL-2R) reporter cell line (invivoGen) that has been transfected with mRNA encoding the indicated proteins are shown. 4210 = ACT4210 alone (i.e., not fused or linked to another protein); HSA-4210 = fusion protein comprising human serum albumin and ACT4210 linked thereto, in order N-terminal to C-terminal; MSA-4210 = fusion protein comprising murine serum albumin and ACT4210 linked thereto, in order N-terminal to C-terminal; 4210-HSA = fusion protein comprising ACT4210 and human serum albumin linked thereto, in order N-terminal to C-terminal; 4210-MSA = fusion protein comprising ACT4210 and murine serum albumin linked thereto, in order N-terminal to C-terminal.

[0156] Figure 3 Results of binding assays obtained for the indicated fusion proteins expressed from cells transfected with mRNA encoding the indicated fusion proteins against IL-2Ra (top panel) or IL-2Rβγ (bottom panel) are shown. 5210-MSA = fusion protein comprising murine serum albumin and ACT5210 (an IL-2 mutein that differs from the IL-2 mutein designated 4210) linked thereto, in order N-terminal to C-terminal. Left (top panel) or IL-2Rβγ (bottom panel) are shown. 5210-MSA = fusion protein comprising murine serum albumin and ACT5210 (an IL-2 mutein that differs from the IL-2 mutein designated 4210) linked thereto, in order N-terminal to C-terminal. Right (top panel) or IL-2Rβγ (bottom panel) are shown. 5210-MSA = fusion protein comprising murine serum albumin and ACT5210 (an IL-2 mutein that differs from the IL-2 mutein designated 4210) linked thereto, in order N-terminal to C-terminal.

[0157] Figure 4In vivo expression results obtained from mice injected with pharmaceutical compositions comprising LNPs, each further comprising mRNA encoding the indicated fusion protein. ACT421H = LNP-mRNA-HSA-4210; ACT421M = LNP-mRNA-MSA-4210.

[0158] Figure 5 Binding assay results obtained from mice injected with pharmaceutical compositions comprising LNPs, each further comprising mRNA encoding the indicated fusion protein, for IL-2Ra ( Left panel) or IL-2RbY ( Right panel) expression in vivo.

[0159] Figure 6 Binding assay results obtained from mice injected with pharmaceutical compositions comprising LNPs, each further comprising mRNA encoding the HSA-4210 fusion protein, for cynomolgus IL-2RbY ( Left panel) or rat IL-2RbY ( Right panel) expression in vivo.

[0160] Figure 7 Binding assay results obtained from mice injected with pharmaceutical compositions comprising LNPs, each further comprising mRNA encoding the MSA-4210 fusion protein, for cynomolgus IL-2RbY ( Left panel) or rat IL-2RbY ( Right panel) expression in vivo.

[0161] Figure 8 Reporter assay results obtained using the HEK-Blue IL-2 CD122 / CD132 reporter cell line (InvivoGen) that has been transfected with pharmaceutical compositions comprising LNPs, each further comprising mRNA encoding the HSA-4210 fusion protein.

[0162] Figure 9 Results of proliferation assays using CTLL-2 cells treated with the indicated fusion proteins.

[0163] Figures 10A-10C Measured concentrations of each of the indicated proteins obtained from mice injected with pharmaceutical compositions comprising LNPs, each further comprising mRNA encoding the indicated protein.

[0164] Figures 11A-11DPharmacodynamic study results from mice injected with pharmaceutical compositions comprising LNPs, each further comprising mRNA encoding the indicated protein, are shown.

[0165] Figure 12 Flow cytometry results from mice injected with pharmaceutical compositions comprising LNPs, further comprising mRNA MSA-4210 fusion protein, are shown.

[0166] Figure 13 shows the number of CD8, CD4, and Treg cells from mice injected with pharmaceutical compositions comprising LNPs, further comprising mRNA MSA-4210 fusion protein.

[0167] Figures 14A-14C Results showing inhibition of lung metastasis using B16F10 mice (lung metastasis mouse model) injected with pharmaceutical compositions comprising LNPs, further comprising mRNA MSA-4210 fusion protein, are shown.

[0168] Figure 15 Overall survival results from B16F10 mice (lung metastasis mouse model) injected with pharmaceutical compositions comprising LNPs, further comprising mRNA MSA-4210 fusion protein, or mice injected with IL-2 1 mg / kg once daily for five days as indicated, are shown. DETAILED DESCRIPTION

[0169] The practice of the present application 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 the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", 2nd 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 updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., Eds., 1994); and "Remington: The Science and Practice of Pharmacy", (Alfonso R. Gennaro, Ed., 20th ed. 2000), the disclosures of which are incorporated herein by reference. Molecular Cloning: A Laboratory Manual Oligonucleotide Synthesis Animal Cell Culture Methods in Enzymology Current Protocols in Molecular Biology PCR: The Polymerase Chain Reaction Remington, The Science and Practice of Pharmacy ​​​​​​), 20th Edition (Lippincott, Williams & Wilkins 2003).

[0170] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All patents, patent applications (published or unpublished), and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication that is herein incorporated by reference, the definition set forth in this section prevails over the definition incorporated herein by reference.

[0171] As used herein, "a" or "an" means "at least one" or "one or more."

[0172] The term "and / or," as used herein, shall be taken to mean that each of the specified features or components is explicitly disclosed either with or without the other. For example, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to cover the

[0173] As used herein, the term "about" refers to values or compositions that are within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend 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, according to the practice in the art. Alternatively, according to the limitations of the measurement system, "about" or "approximately" can mean ranges approximately 10% (i.e., ±10%) or more. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Further, particularly with respect to biological systems or processes, the term can mean ranges that are orders of magnitude or up to five-fold the value. When a particular value or composition is provided in the disclosure, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range of that particular value or composition, unless otherwise stated. In some embodiments, "about" encompasses variations within 10%, 5%, 2%, 1%, or 0.5% of the stated value.

[0174] Numerical ranges include the numbers defining the range. Measured and measurable values are understood to be approximate. Also, all ranges set out herein are understood to be inclusive of the endpoints; thus for example, a range of 1 to 10 includes the values 1 and 10 along with all integer and (where appropriate) non-integer values between 1 and 10.

[0175] The terms "administering" and "administered" and grammatical variations thereof 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 routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenchymal, e.g., by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral administration and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In one embodiment, the formulation is administered by a non-parenteral route (e.g., orally). Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, e.g., intranasal, vaginal, rectal, sublingual, or topical. Administration can also be, e.g., once, multiple times, and / or over one or more extended periods.

[0176] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to 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 can be linear or branched, they can comprise modified amino acids, and they can be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified, e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, e.g., 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.

[0177] As used herein, the term "variant" is used to refer to a polypeptide having some degree of amino acid sequence identity to a parent polypeptide sequence. A variant is analogous to the parent sequence, but the variant has at least one substitution, deletion, or insertion in its amino acid sequence that makes it sequence- wise different from the parent polypeptide. Additionally, a variant can retain functional properties of the parent polypeptide, e.g., maintain at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the biological activity of the parent polypeptide.

[0178] An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, situated in the variable region of the immunoglobulin molecule, and which can be an immunoglobulin of any class, such as IgG, IgM, IgA, IgD, and IgE. IgY, the major antibody type in avian species such as chicken, is also included in this definition. This term as used herein encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chains (ScFv), mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with the desired specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that is

[0179] As used herein, the term "antigen" refers to a target molecule that is specifically bound by an antibody through its antigen recognition site. An antigen can be monovalent or multivalent, i.e., the antigen can have one or more epitopes recognized by one or more antibodies. Examples of various antigens that can be recognized by antibodies include polypeptides, oligosaccharides, glycoproteins, polynucleotides, lipids, etc.

[0180] As used herein, the term "epitope" refers to a portion of an antigen, e.g., a peptide sequence of at least about 3 to 5, preferably about 5 to 10 or 15, and no more than about 1,000 amino acids (or any integer therein), that defines a sequence that binds to an antibody raised against such sequence, either alone or as part of a larger sequence. There is no critical upper limit to the length of the fragment, which can for example include almost the full length of an antigen sequence, or even the full length of a fusion protein of two or more epitopes from a target antigen. Epitopes used in the present application are not limited to peptides having the exact sequence of the portion of the parent protein from which they are derived, but also encompass sequences identical to the native sequence as well as modifications of the native sequence, such as deletions, additions, and substitutions (which are conservative in nature).

[0181] As used herein, the term "Fc" or "Fc region" refers to the portion of an antibody heavy chain constant region that begins at the hinge region or after the hinge region and ends at the C-terminus of the heavy chain. The Fc region includes at least a portion of the CH2 and CH3 regions, and can or can not include a portion of the hinge region. The Fc domain can bind to Fc cell surface receptors as well as some proteins of the immune complement system. The Fc region can bind to complement component Clq. The Fc domain can exhibit effector functions, including any one activity 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 FcyRI (e.g., CD64), FcyRII (e.g., CD32), and / or FcyRIII (e.g., CD16a). In an embodiment, the Fc region can include mutations that increase or decrease any one of these functions or any combination. In an embodiment, the Fc domain includes 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 an embodiment, the Fc domain includes LALA mutations (e.g., according to Kabat numbering, equivalent to L234A, L235A) that decrease effector functions. In an embodiment, the Fc domain includes LALA-PG mutations (e.g., according to Kabat numbering, equivalent to L234A, L235A, P329G) that decrease effector functions. In an embodiment, the Fc domain mediates 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 an 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 an embodiment, the Fc region includes one or more mutations selected from M252Y, T256D, T307Q, T307W, M252Y, S254T, T256E, M428L, and N434S (e.g., according to Kabat numbering). In an embodiment, the Fc region includes the mutations M252Y, S254T, and T256E (YTE) (e.g., according to Kabat numbering).

[0182] The term "isolated" refers to a polynucleotide, nucleic acid, or protein (e.g., an antibody or antigen-binding portion thereof) that is substantially free of other cellular material. In some embodiments, the term isolated also refers to a protein or polynucleotide that is substantially free of other molecules, reactants, reagents, and / or impurities (e.g., 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 known in the art, including low-resolution methods such as gel electrophoresis and high-resolution methods such as HPLC or mass spectrometric analysis.

[0183] As used herein, the term "labeled" or related terms with respect to a polypeptide refers to a conjugated antibody that is unlabeled or conjugated to a detectable label or moiety for detection, wherein the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, detectable bead (such as a magnetic or electron-dense (e.g., gold) bead), biotin, streptavidin, or protein A. Various labels can be used, including but not limited to, radionuclides, fluorescent agents, enzymes, enzyme substrates, coenzyme factors, enzyme inhibitors, and ligands (e.g., biotin, hapten). Any of the polynucleotides, modified IL-2 polypeptides, or fusion proteins described herein can be unlabeled or can be linked to a detectable label or moiety.

[0184] As used herein, the term "labeled" or related terms with respect to a polypeptide refers to a conjugated antibody that is unlabeled or conjugated to a detectable label or moiety for detection, wherein the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, detectable bead (such as a magnetic or electron-dense (e.g., gold) bead), biotin, streptavidin, or protein A. Various labels can be used, including but not limited to, radionuclides, fluorescent agents, enzymes, enzyme substrates, coenzyme factors, enzyme inhibitors, and ligands (e.g., biotin, hapten). Any of the polynucleotides, modified IL-2 polypeptides, or fusion proteins described herein can be unlabeled or can be linked to a detectable label or moiety.

[0185] The terms "leader sequence" or "leader peptide" or "[peptide] signal sequence" or "signal peptide" or "secretion signal peptide" refer to a peptide sequence located at the N-terminus of a polypeptide. A leader sequence directs the polypeptide chain to the cellular secretory pathway and can direct integration and anchoring of the polypeptide into the lipid bilayer of the cell 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 insertion of the mature polypeptide into the membrane. Thus, proteins provided herein, such as membrane proteins and antibodies having a signal peptide identified by a precursor sequence including a signal peptide sequence, are also intended to encompass the mature form of the polypeptide lacking the signal peptide, and proteins provided herein, such as membrane proteins and antibodies having a signal peptide identified by a mature polypeptide sequence lacking a signal peptide sequence, are also intended to encompass the form of the polypeptide including the signal peptide, whether naturally occurring or derived from another secreted or membrane-inserted protein. In one embodiment, the leader sequence includes a signal sequence including a CD8a, CD28, or CD 16 leader sequence. In one embodiment, the signal sequence includes a mammalian sequence, including, for example, a mouse or human Ig gamma secretion signal peptide.

[0186] The terms "percent identity," "percent homology," and related terms, as used herein, refer to a quantitative measure of the similarity between two polypeptide sequences or between two polynucleotide sequences. The percent identity between two polypeptide sequences is a function of the number of identical amino acids at positions shared by the two polypeptide sequences, taking into account the number of gaps, and the length of each gap, which can need to be introduced for optimal alignment of two polypeptide sequences. In a similar manner, the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at positions shared by the two polynucleotide sequences, taking into account the number of gaps, and the length of each gap, which can need to be introduced for optimal alignment of two polynucleotide sequences. Sequence comparisons and determinations of percent identity between two polypeptide sequences or between two polynucleotide sequences can be achieved using a mathematical algorithm. For example, the "percent identity" or "percent homology" between two polypeptide or two polynucleotide sequences can be determined by comparing the sequences using the GAP computer program (GCG Wisconsin Package, Version 10.3 (Accelrys, San Diego, Calif.) using its default parameters. With respect to expression of a test sequence, such as "a sequence comprising at least X% identity to Y" means that the test sequence comprises at least X% of the residues that are identical to residues of sequence Y when aligned with Y as described above.

[0187] In some embodiments, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the polynucleotide, the modified IL-2 polypeptide, and / or the fusion protein can be similar, but not necessarily identical, to any given test nucleic acid sequence or test amino acid sequence. The similarity between a given test nucleic acid sequence or amino acid sequence and a polynucleotide, a modified IL-2 polypeptide, or a fusion protein described herein and throughout can comprise 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.

[0188] The term "amino acid substitution" and like terms refer to the substitution of one amino acid residue in a polypeptide with another amino acid residue having a different side chain (R group). It will be appreciated that such amino acid substitutions can be achieved by altering or modifying the nucleic acid or polynucleotide sequence encoding a given polypeptide, thereby encoding and, thus, upon expression, producing a polypeptide sequence containing the amino acid substitution.

[0189] The term "conservative amino acid substitution" and like terms refer to the substitution of one amino acid residue in a polypeptide with another amino acid residue having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). Collectively, conservative amino acid substitutions will not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the variation. Means for making this adjustment are well-known in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference in its entirety. Examples of groups of amino acids that have side chains with 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 are cysteine and methionine. It will be appreciated that such conservative amino acid substitutions can be achieved by altering or modifying the nucleic acid or polynucleotide sequence encoding a given polypeptide, thereby encoding and, thus, upon expression, producing a polypeptide sequence containing the conservative amino acid substitution. Methods Mol. Biol .) 24: 307-331, which is incorporated herein by reference in its entirety. Examples of groups of amino acids that have side chains with 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 are cysteine and methionine. It will be appreciated that such conservative amino acid substitutions can be achieved by altering or modifying the nucleic acid or polynucleotide sequence encoding a given polypeptide, thereby encoding and, thus, upon expression, producing a polypeptide sequence containing the conservative amino acid substitution.

[0190] As used herein, the term "specifically binds" refers to the binding specificity of a specific binding pair. The recognition of a particular target by an antibody in the presence of other potential targets is one characteristic of such binding. Specific binding involves two different molecules, one of which specifically binds to the second by either chemical or physical means. The two molecules are related in the sense that their mutual binding enables them to distinguish their binding partner from other assay components with similar properties. Members of a binding component pair are referred to as a ligand and a receptor (anti-ligand), a specific binding pair (SBP) member and SBP partner, and the like. A molecule can also be a SBP member of a molecular aggregate; for example, an antibody raised against an immune complex of a second antibody with its corresponding antigen can be considered a SBP member of the immune complex.

[0191] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to polymers of nucleotides of any length, and include DNA and RNA. The 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 in which nucleotides are joined to each other by either deoxyribose or ribose sugars. The term refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form, and includes 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 molecules (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, a nucleic acid includes one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0192] A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after polymer assembly. The nucleotide sequences can be interspersed with non-nucleotide components. A polynucleotide can be further modified after polymerization, e.g., by conjugation with a labeling component. Other types of modifications include, for example, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing chiral linkages (e.g., alkenyl phosphonates), those containing intercalators (e.g., acridines, actinomycin, etc.), those in which the sugar is a heteroatom, modified base, modified phosphate backbone, or others as known in the art. None of these modifications to nucleotide structure are meant to exclude from the definition of the term "polynucleotide," as used herein. Also included within the definition are, for example, nucleic acids containing modified backbones that include, for example, non-ionic side groups, 5' -Nucleotide groups, and modified bases, including those in which the purine or pyrimidine base is replaced with other heterocycles, those in which D- deoxyribose and / or ribose is replaced by other sugars, and those in which one or more atoms are replaced by their isotopes or mimetics (e.g., incorporation of 2'-deoxy-2'-fluoro or 2'-deoxy-2'-azido modifications). 5' and 3' ends of the polynucleotide can be further modified, e.g., by the addition of a phosphate group or by the addition of a phosphate group and a 5' terminal overhang. The 5' and 3' terminal OH can be substituted with phosphate or an amine or organic cap group having 1 to 20 carbons. Other hydroxyls can also be derivatized to standard protecting groups. The polynucleotide can also contain ribose or deoxyribose analogs known in the art, including, for example, 2'-O-methyl-2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, alpha anomer sugars, epimeric sugars (e.g., arabinose, xylose, or lyxose), pyranose, furanose, threoheptonose, acyclic analogs, and abasic nucleoside analogs (e.g., methyl riboside). One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphates are 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 substituted or unsubstituted alkyl (1-20 C), optionally containing ether (--O--) linkages, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. All linkages in a polynucleotide need not be the same. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0193] As used herein, an "oligonucleotide" generally refers to a short, usually single-stranded, synthetic polynucleotide, usually but not necessarily less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of a polynucleotide applies equally and fully to an oligonucleotide.

[0194] As used herein, the term "homolog" is used to refer 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 a few nucleotides, including deletions (e.g., a truncated version of a nucleic acid), insertions, and / or substitutions. A homolog can have enhanced, reduced, or essentially similar properties compared to the naturally occurring nucleic acid. A homolog can be complementary or match the naturally occurring nucleic acid. A homolog can be generated using techniques known in the art for generating nucleic acids, including but not limited to, recombinant DNA technology, chemical synthesis, and the like.

[0195] As used herein, "substantially complementary or substantially match" 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 match" means that two nucleic acid sequences can hybridize under high stringency conditions.

[0196] Generally, the stability of a hybrid is a function of ionic concentration and temperature. Typically, the hybridization reaction is performed under conditions of lower stringency, followed by washes of varying but higher stringency. Medium stringency hybridization refers to conditions which allow nucleic acid molecules, such as probes, to bind to complementary nucleic acid molecules. Hybridized nucleic acid molecules generally have at least 60% identity, including at least any of, for example, 70%, 75%, 80%, 85%, 90%, or 95% identity. Medium stringency conditions are conditions equivalent to those described by Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, at pages 9.47-9.51, incorporated herein by reference, which are conditions of hybridization at 42°C in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS, followed by washing at 42°C in 0.2x SSPE, 0.2% SDS. High stringency conditions can be provided by, for example, hybridization at 42°C in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS, followed by washing at 65°C in 0.1x SSPE and 0.1% SDS. Low stringency hybridization refers to conditions equivalent to those described by Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, at pages 9.47-9.51, which are conditions of hybridization at 22°C in 10% formamide, 5x Denhardt's solution, 6x SSPE, 0.2% SDS, followed by washing at 37°C in 1x SSPE, 0.2% SDS. Denhardt's solution contains 1% polyvinylpyrrolidone, 1% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, ethylenediaminetetraacetic acid (EDTA)) contains 3 M sodium chloride, 0.2 M sodium phosphate, and 0.025 M EDTA. Other suitable medium and high stringency hybridization buffers and conditions are well known to those skilled in the art.

[0197] 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, such as a polynucleotide encoding a fusion protein comprising a modified IL-2 polypeptide as described herein and throughout). A vector can be used as a vehicle to introduce foreign genetic material into a cell (e.g., a host cell) in vivo, ex vivo, and / or in vitro. A vector can include at least one restriction endonuclease recognition sequence for insertion of a transgene into the vector. A vector can include at least one gene sequence that confers antibiotic resistance or a selectable trait to aid in selection of host cells that carry the vector-transgene construct. An expression vector can include one or more origin of replication sequences. A vector can be a single- or double-stranded nucleic acid molecule. A vector can be a linear or circular nucleic acid molecule. 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 and is capable of replication and transcription and / or translation of the transgene in a host cell. Viral vectors typically contain viral RNA or DNA backbone sequences that can be linked to a transgene. The viral backbone sequences can be modified to stop infection but retain the viral backbone and co-linked transgene insertion 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 are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors, which include a bacterial origin of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) when introduced into a host cell are integrated into the genome of the host cell, and are thereby replicated along with the host genome.

[0198] An “expression vector” (also referred to as a “plasmid,” which can be used interchangeably throughout) is a vector that can contain one or more regulatory sequences and / or a regulatory promoter system (such as inducible and / or constitutive promoters and enhancers). Such regulatory sequences and / or regulatory promoter systems can include one or more of various promoter or enhancer sequences obtained or derived from, for example, a cytomegalovirus (CMV) promoter, an elongation factor 1 (EF1) promoter, a chicken beta-actin promoter, a beta-actin promoter from other species, a phosphoglycerate kinase (PGK) promoter, a spleen focus-forming virus (SFFV) promoter, a Rous sarcoma virus (RSV) promoter, a human serum albumin (SA) promoter, a thyroxin binding globulin (TBG) promoter, a cytochrome P450 2E1 (CYP2E1) promoter, a muscle creatine kinase enhancer sequence, a desmin enhancer sequence, a desmin promoter sequence, an MHDLKSMRTe enhancer / promoter sequence, and the like. Exemplary such vectors (such as NanoPlasmid™ vectors), regulatory elements, and the like can be found in, for example, US 2004 / 0175727 and WO 2022 / 056291, which are hereby incorporated by reference in their entireties for all purposes.

[0199] An expression vector can also include a regulatory promoter system comprising a combination of one or more promoter sequences and / or enhancer sequences, such as a chicken beta-actin / CMV enhancer (CAG) promoter; a human or murine CMV-derived enhancer element in combination with an elongation factor la (EF1a) promoter; a CpG-free version of a human or murine CMV-derived enhancer element in combination with an elongation factor 1 alpha (EF1a) promoter; an albumin promoter in combination with an alpha-fetoprotein MERII enhancer; one or more MCK enhancer sequences in combination with a desmin enhancer sequence and / or a desmin long promoter sequence; one or more MCK enhancer sequences in combination 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, and the like. 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, and the like can replace one or more of the above promoter and / or enhancer sequences, and / or be added to or added to one or more of the above promoter and / or enhancer sequences to produce a regulatory promoter system according to the present disclosure.

[0200] Such regulatory sequences and / or regulatory promoter systems can also drive or facilitate tissue-specific transcription and / or translation of nucleic acid sequences encoding polypeptides, such as antigen binding proteins (e.g., antibodies or antigen binding fragments thereof). Exemplary such promoters and / or enhancers can drive tissue-specific expression of polynucleotides encoding polypeptides. Exemplary such promoters and / or enhancers can drive muscle-specific expression of polynucleotides encoding polypeptides. Such tissue- and / or muscle-specific promoters and / or enhancers can be combined to produce tissue- and / or muscle-specific regulatory promoter systems. In some embodiments, such tissue- and / or muscle-specific promoters and / or enhancers comprise one or more of the promoter or enhancer sequences disclosed in US 2004 / 0175727, which is incorporated by reference herein in its entirety. In some embodiments, such tissue- and / or muscle-specific promoters and / or enhancers that can be used in combination to produce regulatory promoter systems according to the present disclosure comprise, for example, one or more of the following: 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, a SRE regulatory element, a TEF-1 regulatory element, an elongation factor 1 alpha (“EF1a” or “EF1α,” used interchangeably throughout) promoter sequence, a MHDLKSMRTx enhancer / promoter sequence, and the like.

[0201] The regulatory promoter system operably linked to the polypeptide-encoding sequence can be a eukaryotic or prokaryotic regulatory promoter system, but is preferably a eukaryotic regulatory promoter system active in mammalian cells. The regulatory promoter system can direct transcription and / or translation of the transgene in the host cell, and can include a ribosome binding site and / or a polyadenylation site. In certain embodiments, the regulatory promoter system can independently or collectively (e.g., synergistically) direct transcription and / or translation of the transgene in the host cell, and can include a ribosome binding site and / or a polyadenylation site. In certain embodiments, the regulatory promoter system can independently or collectively (e.g., synergistically) direct tissue-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 certain embodiments, the regulatory promoter system can independently or collectively (e.g., synergistically) 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 harbors one or more expression vectors that can direct transient introduction of the transgene into the host cell or stable insertion of the transgene into the genome of the host cell, wherein the transgene includes a nucleic acid encoding any of the first and / or second polypeptides described herein. In embodiments where the nucleic acid molecule encodes two polypeptides, the two polypeptide-encoding sequences can be regulated by the same promoter, and can be linked by an IRES or 2A sequence (Shao et al. (2009) Cell Res. 19:296-306), or the two polypeptide-encoding sequences can be operably linked to different promoters. Cell Research

[0202] The expression vector can include a ribosome binding site and / or a polyadenylation site. The expression vector can include one or more origin of replication sequences. Regulatory sequences direct transcription or transcription and translation of a transgene linked to or inserted into the expression vector transformed into a host cell. Regulatory sequences can control the level, timing, and / or location of expression of a transgene. Regulatory sequences can exert their influence on a transgene, for example, directly or through the action of one or more other molecules, such as a polypeptide bound to the regulatory sequence and / or the nucleic acid. Regulatory sequences can be part of the vector. Additional examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, and Baron et al., 1995, Nucleic Acids Res. 23:3605-3606. Nucleic Acids Res

[0203] ​​A transgene is "operably linked" to a regulatory sequence (e.g., a promoter) when the regulatory sequence affects the expression (e.g., level, timing, and / or location) of the transgene.

[0204] The terms "transfected" or "transformed" or "transduced" or other related terms, as used herein, refer to the process by which exogenous nucleic acids (e.g., transgenes) are 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). Host cells include primary subject cells and their progeny.

[0205] As used herein, "vector (or plasmid)" refers to a discrete element used to introduce heterologous DNA into a cell for expression or replication thereof. The selection and use of such agents are well within the skill of the artisan. Expression vectors include vectors capable of expressing DNA operably linked to regulatory sequences such as a promoter region, which enable the expression of such DNA segments. Thus, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, bacteriophage, recombinant viruses, or other vectors that yield expression of cloned DNA after introduction into an appropriate host cell. Suitable expression vectors are well known to those skilled in the art and include those that can replicate in eukaryotic and / or prokaryotic cells and those that remain episomal or integrate into the host cell genome.

[0206] As used herein, "promoter region or promoter element" refers to a DNA or RNA segment that controls the transcription of DNA or RNA operably linked thereto. The promoter region includes specific sequences sufficient for recognition, binding, and initiation of transcription by RNA polymerase. This portion of the promoter region is referred to as the promoter. In addition, the promoter region includes sequences that modulate this recognition, binding, and initiation of transcription activity by RNA polymerase. These sequences can be cis-acting or can respond to trans-acting factors. Depending on the nature of the modulation, the promoter can be constitutive or regulated. Exemplary promoters contemplated for use in prokaryotes include the bacteriophage T7 and T3 promoters, among others.

[0207] As used herein, "operatively linked" or "operatively associated" refers to the functional relationship of DNA or RNA to regulatory and effector sequences of nucleotides such as promoters, enhancers, transcriptional and translational start sites, transcriptional and translational stop sites, polyadenylation sites, ribosomal entry sites, 5' cap sites, and other transcriptional or translational control or regulatory elements. For example, the operative linkage of DNA or RNA to a promoter refers to the physical and functional relationship between the DNA or RNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA. To optimize expression and / or in vitro transcription, it can be necessary to remove, add, or change the 5' untranslated portion of a clone to eliminate additional, potentially inappropriate alternative translation initiation (i.e., start) codons or other sequences that can interfere with or reduce expression at the transcriptional or translational level. Alternatively, a consensus site can be inserted immediately 5' to the start codon and can enhance expression. See, e.g., Kozak (1991) J. Biol. Chem. 266: 19867-19870. The necessity (or need) for such modifications can be empirical. J. Biol. Chem.

[0208] "Treating" or "treatment" or "alleviation" refers to therapeutic treatment, wherein the object is to slow down (lessen, if not halt) or prevent the pathology or condition being targeted or to prevent its recurrence. A subject is successfully "treated" if, after receiving therapeutic amounts of a therapeutic agent or treatment, the subject shows observable and / or measurable reduction in or absence of one or more signs and symptoms of the particular disease. A patient can also experience a reduction in signs or symptoms of the disease. 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 free of disease 3 months, preferably 6 months, more preferably one year, even more preferably 2 or more years after treatment. These parameters for assessing successful treatment and improvement of disease are readily measurable by routine procedures familiar to those of appropriate skill in the art. In some embodiments, "treatment" means 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, "improving" the symptoms of a particular disorder by administration of a particular pharmaceutical composition means any alleviation, whether permanent or temporary, lasting or transient, attributable to or associated with administration of the composition.

[0209] ​The terms "predict" or "prognosis" are often used herein to refer to the likelihood that a patient will respond favorably or unfavorably to a drug or drug combination or the likely outcome of a disease. In one embodiment, prediction relates to the degree of those responses or outcomes. In one embodiment, prediction relates to whether or not a patient will survive or improve after treatment (e.g., treatment with a particular therapeutic agent) and for a certain period of time in the absence of disease recurrence and / or the probability that a patient will survive or improve. The prediction methods of the present invention can be clinically useful in making treatment decisions by selecting the most appropriate treatment modality for any particular patient. The prediction methods of the present invention are valuable tools to predict whether or not a patient is likely to respond favorably to a treatment regimen (e.g., a given therapeutic regimen), which includes, for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc.

[0210] The terms "prevent," "prevention," and "preventing" refer to the provision or administration of a therapeutic or prophylactic agent or treatment regimen with the objective 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, "prevention" refers to the provision or administration of a therapeutic or prophylactic agent or treatment regimen with the objective 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.

[0211] 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 or absence of one or more signs and symptoms of a particular disease, condition or disorder. A patient can also experience a subjective improvement in disease signs or symptoms. A patient is also considered treated if the patient experiences stable disease. In some embodiments, treatment with a therapeutic agent is effective to cause a patient to be free of disease 3 months, preferably 6 months, more preferably one year, even more preferably 2 or more years after treatment. These parameters for assessing successful treatment and improvement of disease are readily measurable by routine procedures familiar to those of appropriate skill in the art. In some embodiments, "treatment" means 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, "improving" the symptoms of a particular disorder by administration of a particular pharmaceutical composition means any alleviation, whether permanent or temporary, lasting or transient, that can be attributed to or associated with the administration of the composition.

[0212] The term "synergistic effect" refers to a situation where the combined effect of two or more drugs is greater than the sum of the effects of each drug alone. This term encompasses not only the reduction of symptoms of the condition being treated, but also improved side effects profile, improved tolerability, improved patient compliance, improved efficacy, or any other improved clinical outcome.

[0213] The term "sub-therapeutic amount" for a drug or therapy is an amount less than the effective amount of the drug or therapy as a single agent, but which, when combined with another drug or therapy in an effective or sub-therapeutic amount, can produce the physician's desired results due to, for example, the synergistic effect of the resulting effective effect or the reduction of side effects.

[0214] Combination therapy or “in combination with” refers to the use of more than one therapeutic agent to treat a specific condition or symptom. “In combination with” is not intended to imply that the therapeutic agents must be administered simultaneously and / or formulated for delivery together, although such delivery methods are within the scope of this disclosure. The therapeutic agent may be administered concurrently with one or more other additional agents, 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 prior to) 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) a combination therapy. The therapeutic agent in a combination therapy may also be administered on an alternating dosing schedule, with or without a resting period (e.g., the therapeutic agent is not administered on certain days of the schedule). The “combination” administration of a therapeutic agent with another therapeutic agent includes, but is not limited to, the sequential and concomitant administration of two agents. Typically, each therapeutic agent is administered at a dose and / or schedule determined for the specific agent.

[0215] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonics, and absorption delay agents compatible with pharmaceutical application. The use of such media and agents for pharmaceutically active substances is well known in the field. See, for example, Remington: Pharmaceutical Science and Practice, 20th edition (Lippincott Williams and Wilkins Publishing, 2003). The use of such media or agents in a composition is contemplated unless a particular conventional media or agent is incompatible with the active compound.

[0216] “Pharmaceutically acceptable salt” is intended to mean a salt of a free acid or base represented herein that is nontoxic, biologically tolerable, or otherwise 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 the tissues of subjects without undue toxicity, irritation, or allergic response. The modified interleukin 2 (IL-2) polypeptides or conjugates thereof described herein can have a sufficiently acidic group, a sufficiently basic group, both types of functional groups, or more than one of each type of group, and thus react with a number of inorganic or organic bases, and inorganic and organic acids, to form pharmaceutically acceptable salts. J. Pharm. Sci. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, phenylsulfonates, xylenesulfonates, naphthalene-1- sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrates, glycolates, tartrates, and mandelates.

[0217] Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, phenylsulfonates, xylenesulfonates, naphthalene-1- sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrates, glycolates, tartrates, and mandelates.

[0218] As used herein, the term "therapeutic effective amount" or "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or improving a disease or symptom, proliferative disease or symptom in the subject. A therapeutic effective amount further refers to an amount of a therapeutic agent sufficient to cause symptom improvement (e.g., treatment, cure, prevention, or improvement of the associated medical condition or an increase in the rate of treatment, cure, prevention, or improvement of such condition). When an individual active ingredient is applied alone, the therapeutic effective amount refers only to that ingredient. When applied in combination, the therapeutic effective amount refers to the combined amount of active ingredients that produce a therapeutic effect, whether applied in combination, sequentially, or simultaneously. In some embodiments, an "effective amount of a compound for treating a specific disease" is an amount sufficient to improve or alleviate symptoms associated with the disease in some way. This amount may be administered as a single dose or according to a regimen, thereby being effective. The amount may cure the disease but is typically administered to improve the symptoms of the disease. Repeated administration may be necessary to achieve the desired symptom improvement.

[0219] The term "combination" refers to a fixed combination or kit for combined administration in the form of a dose unit, wherein a modified interleukin-2 (IL-2) peptide or its conjugate and a combination partner (e.g., another drug described below, also referred to as a "therapeutic agent" or "adjuvant") can be administered simultaneously or independently at time intervals, particularly where these time intervals allow the combination partner to exhibit a synergistic effect, such as a co-administration effect. As used herein, the terms "co-administration" or "combination administration," etc., are intended to cover the administration of selected combination partners to a single subject (e.g., a patient) in need and are intended to include treatment regimens in which the agents are not necessarily administered via the same route of administration or simultaneously. As used herein, the term "pharmaceutical composition" means a product consisting of a mixture or combination of more than one active ingredient and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients, such as a modified interleukin-2 (IL-2) peptide or its conjugate and a combination partner, are administered simultaneously to a patient in the form of a single entity or dose. The term "non-fixed combination" refers to active ingredients, such as modified interleukin-2 (IL-2) peptides or their conjugates and combination partners, administered to a patient as separate entities simultaneously, concurrently, or sequentially, without a specific time limit, wherein such administration provides a therapeutically effective level of two substances in the patient's body. The latter also applies to cocktail therapy, such as the administration of three or more active ingredients.

[0220] As used herein, a “biological sample” means any sample obtained from a living organism or viral source or other macromolecular and biomolecular source, and includes any cell type or tissue from which nucleic acids or proteins or other macromolecules can be obtained. A biological sample can be a sample obtained directly from a biological source or a processed sample. For example, amplified and isolated nucleic acids constitute a biological sample. Biological samples include, but are not limited to, samples of bodily fluids (e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissues, and organs from animals and plants, as well as processed samples derived therefrom.

[0221] The term "level" (or levels) is used to refer to the presence and / or quantity of a target (e.g., a substance or organism that is part of the cause of a disease or condition) and can be determined qualitatively or quantitatively. A "qualitative" change in a target level refers to the presence or absence of a target that is undetectable or absent in samples obtained from normal controls. A "quantitative" change in the level of one or more targets, compared to healthy controls, refers to a measurable increase or decrease in the level of the target.

[0222] A “healthy control” or “normal control” is a biological sample taken from an individual who does not have a disease or condition (e.g., a proliferative disease or condition). A “negative control” is a sample for which an assay is designed to detect any particular analyte and thus provides a reference baseline for said assay.

[0223] As used herein, “mammal” means any species within the mammalian species category. As used herein, the term “mammal” often refers to humans, human subjects, or human patients. “Mammal” also means any species within the non-human mammalian species category, such as experimental, companion, or economically important non-human mammals. Exemplary non-human mammals include mice, rats, rabbits, cats, dogs, pigs, cattle, sheep, goats, horses, monkeys, gorillas, and chimpanzees.

[0224] As used herein, “generated by recombination” refers to a method of generating recombinant nucleic acids that relies on well-known molecular biology methods to express polypeptides or proteins encoded by cloned nucleic acids.

[0225] As used herein, the terms “subject” and “patient” refer to humans and non-human animals, including vertebrates, mammals and non-mammals. In one embodiment, a subject may be a human, a non-human primate, ape, great ape, rodent (e.g., mouse and rat), cow, pig, horse, dog, feline, goat, wolf, frog, or fish.

[0226] As used herein, a "prodrug" is a substance that is metabolized or otherwise converted into the biologically, pharmaceutically, or therapeutically active form of a substance after administration in vivo. To create a prodrug, a pharmaceutically active substance is modified such that the active substance will be regenerated by metabolic processes. The prodrug can be designed to alter the metabolic stability and / or transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. With an understanding of the pharmacodynamic processes and drug metabolism in the body, one skilled in the art can design prodrugs of a given pharmaceutically active compound once the compound is known (see, e.g., Nogrady (1985) Medicinal Chemistry: A Biochemical Approach, Oxford University Press, New York, pp. 388-392). Medicinal Chemistry A Biochemical Approach It is to be understood that the aspects and embodiments of the application described herein include "consisting" and / or "consisting essentially of aspects and embodiments.

[0227] It is to be understood that the aspects and embodiments of the application described herein include "consisting" and / or "consisting essentially of aspects and embodiments.

[0228] Throughout this disclosure, various aspects of the application can be presented in a range format. It is to be understood that the description in range format is merely for convenience and brevity and that one of ordinary skill in the art would understand that the scope of the application is not limited to the scope of the ranges. Therefore, the description of a range should be considered as merely a description of the start and end values of a range formed by the recitation of the start and end values. For example, a range of from 1 to 6 should be considered as comprising the explicitly recited values of 1, 2, 3, 4, 5, and 6, as well as the individual numbers within the range, such as 1, 2, 3, 4, 5, and 6. This same logic applies to ranges reciting the same value for both the start and end of the range. For example, a range of from 1 to 1 should be considered as comprising the explicitly recited value of 1, as well as the individual numbers within the range, such as 1. The same logic applies to ranges reciting only one numerical value. For example, a range of from 1-6 should be considered as comprising the explicitly recited value of 1, as well as the individual numbers within the range, such as 1, 2, 3, 4, 5, and 6. Whether the range is expressed in terms of the start and end of the range, or simply the range, the breadth of the range is to be understood as encompassing the entire range of values within the recited range. For example, a range of from 1 to 6 is to be understood as encompassing the same range of values as a range of from 1 to 6.

[0229] Other objects, advantages and features of the present application will become apparent from the following specification wherein the application is described in

[0230] Polynucleotides encoding modified interleukin 2 (IL-2) polypeptides and fusion proteins comprising the same In some embodiments, provided is a polynucleotide 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 amino acid having 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 the amino acid sequence set forth in SEQ ID NO: 1 (also referred to as human wild-type IL-2) or SEQ ID NO: 2 (also referred to as human rhIL-2), wherein the modified IL-2 polypeptide comprises at least one substitution with a natural amino acid or a non-natural 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, a) the modified IL-2 polypeptide is configured to be conjugated to a water-soluble polymer, a lipid, or a polypeptide (e.g., a protein) or a peptide; and / or b) the modified IL-2 polypeptide has reduced binding to interleukin 2 receptor alpha (IL-2Ra) as compared to a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution(s); and / or c) the modified IL-2 polypeptide has reduced receptor signaling potency to IL-2Raβγ as compared to a comparable 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 d) the modified IL-2 polypeptide has an increased ratio of signaling potency to IL-2Rβγ to signaling potency to IL-2Raβγ (increased ratio of signaling potency to IL-2Rβγ / signaling potency to IL-2Raβγ) as compared to a comparable 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 e) the modified IL-2 polypeptide has enhanced receptor signaling potency to IL-2Rβγ as compared to a comparable 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 provided that when the modified IL-2 polypeptide comprises at least one substitution with a non-natural 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 amino acid or a non-natural amino acid at at least one position within the IL-2Ra interaction region, the IL-2Rβ interaction region, and / or the IL-2Rγ interaction region; and / or f) a combination of a) through e).

[0231] The amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is set forth below: SEQ ID NO: 1 ( 1 APTSSSTKKTQL 13 QLEHLL 19 LDLQMILNGI 29 N 30 N 31 Y 32 K 33 N 34 P 35KLT 38 RML 41 T 42 F 43 KF 45 YMP 48 K49KATELKHLQCLEE 62 EL 64 K 65 PLEEVL 71 NLA 74 QS 76 KNFHL 81 RPRD 85 LI 87 SNIN 91 V 92 I 93 VLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTL 133 T) SEQ ID NO: 2 ( 1 MPTSSSTKKTQL 13 QLEHLL 19 LDLQMILNGI 29 N 30 N 31 Y 32 K 33 N 34 P 35 KLT 38 RML 41 T 42 F 43 KF 45 YMP 4 8 K49KATELKHLQCLEE 62 EL 64 K 65 PLEEVL 71 NLA 74 QS 76 KNFHL 81 RPRD 85 LI 87 SNIN 91 V 92 I 93 VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL 133 T) In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the 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 a region of amino acid residues 10-25 to a corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions.

[0232] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the 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 a region of amino acid residues 80-100 to a corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions.

[0233] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the 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 a region of amino acid residues 100-134 to a corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions.

[0234] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the 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 a region of amino acid residues 10-25 and 80-100 to a corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions.

[0235] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the 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 a region of amino acid residues 10-25 and 80-100 to a corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions.

[0236] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the 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 a region of amino acid residues 10-25 and 80-100 to a corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions.

[0237] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the 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 a corresponding region of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions in the regions of amino acid residues 10-25, 80-100, and 100-134.

[0238] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the 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 a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without substitutions.

[0239] In some embodiments, provided are polynucleotides 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 natural amino acid. In some embodiments, provided are polynucleotides 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, 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 combinations thereof.

[0240] In some embodiments, provided are polynucleotides 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: a) 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, a lipid, a protein, or a peptide 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 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, a lipid, a protein, or a peptide at the N-terminus and / or the C-terminus of the polypeptide.

[0241] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the modified interleukin 2 (IL-2) polypeptide: a) comprises a 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 with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine; and / or b) comprises a substitution 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 with lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine.

[0242] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the modified interleukin 2 (IL-2) polypeptide: a) comprises a substitution 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 with cysteine; b) comprises a substitution at a position selected from the group consisting of N29, Y31, K35, P65, N71, Q74, and combinations thereof with cysteine; c) comprises a substitution at position Y31 with cysteine; and / or d) comprises a substitution at position P65 with cysteine.

[0243] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the 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, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, the 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.

[0244] In some embodiments, provided are polynucleotides 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 amino acid at a position within the IL-2Rα interaction region, the IL-2Rβ interaction region, and / or the IL-2Rγ interaction region.

[0245] In some embodiments, provided are polynucleotides 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 amino acid at a position within the IL-2Rα interaction region. In some embodiments, provided are polynucleotides 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 substitution with a natural amino acid at any suitable position within the IL-2Rα interaction region. In some embodiments, provided are polynucleotides 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 substitution with a natural amino acid at a position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof.

[0246] In some embodiments, provided are polynucleotides 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 natural amino acid at a position within the IL-2Rα interaction region. In some embodiments, provided are polynucleotides 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.

[0247] In some embodiments, provided are polynucleotides 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: a) comprises a substitution with cysteine at a position selected from the group consisting of R38, F42, Y45, E62, P65, and combinations thereof; b) comprises a substitution with alanine, lysine, or serine at position F42; c) comprises a substitution with alanine at position F42; d) comprises a substitution with serine at position F42; e) comprises a substitution with lysine at position F42; f) comprises a substitution with alanine, histidine, or serine at position Y45; g) comprises a substitution with alanine at position Y45; h) comprises a substitution with histidine at position Y45; i) comprises a substitution with alanine, aspartic acid, or serine at position R38; j) comprises a substitution with aspartic acid at position R38; k) comprises a substitution with alanine at position P65; 1) comprises a substitution with serine at position P65; m) comprises a substitution with alanine at position E62; and / or n) comprises a substitution with lysine at position F42, a substitution with cysteine at position Y31, or combinations thereof.

[0248] In some embodiments, provided are polynucleotides 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 amino acid at a position within the IL-2Rβ interaction region. In some embodiments, provided are polynucleotides 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 substitution with a natural amino acid at any suitable position within the IL-2Rβ interaction region. In some embodiments, provided are polynucleotides 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 substitution with a natural amino acid at a position selected from the group consisting of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0249] In some embodiments, provided are polynucleotides 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 natural amino acid at a position within the IL-2Rβ interaction region. In some embodiments, provided are polynucleotides 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 of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof. In some embodiments, provided are polynucleotides 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 cysteine at a position selected from the group of Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

[0250] In some embodiments, provided are polynucleotides 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 substitution with a natural amino acid at a position within the IL-2Rα interaction region and a substitution with a natural amino acid at a position within the IL-2Rβ interaction region; b) a substitution with a natural amino acid at a position within the IL-2Rα interaction region and a substitution with a natural amino acid at a position within the IL-2Rγ interaction region; or c) a substitution with a natural amino acid at a position within the IL-2Rα interaction region, a substitution with a natural amino acid at a position within the IL-2Rβ interaction region, and a substitution with a natural amino acid at a position within the IL-2Rγ interaction region.

[0251] In some embodiments, provided are polynucleotides 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 non-natural amino acid. For example, exemplary non-natural amino acids that can be used in accordance with the embodiments disclosed herein and throughout are described in WO 2019 / 028425 Al and WO 2019 / 028419 Al, which are hereby incorporated by reference in their entireties for all purposes. In some embodiments, provided are polynucleotides 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 non-natural amino acid and or an amino acid comprising a non-natural functional group, e.g.: a lysine analog, a cysteine analog, or a histidine analog; an aromatic side chain; an azido group; an alkyne group; and / or an aldehyde or ketone group. In some embodiments, provided are polynucleotides 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 non-natural amino acid that does not comprise an aromatic side chain.In some embodiments, provided is a polynucleotide 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 non-natural amino acid comprising N6-azidoethyloxy-L-lysine (AzK), N6-propargylethyloxy-L-lysine (PraK), BCN-L-lysine, norbornenelysine, TCO-lysine, methyltetrazinelysine, allyloxycarbonyllysine, 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-phenylalanine, L-dihydroxyphenylalanine (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) seleno)propanoic acid, 2-amino-3-(phenylseleno)propanoic acid, or selenocysteine.

[0252] In some embodiments, provided is a polynucleotide 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 non-natural amino acid that can be incorporated into the modified IL-2 polypeptide by any suitable means or method. In some embodiments, provided is a polynucleotide 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 non-natural amino acid that can be incorporated into the modified IL-2 polypeptide by a 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 non-natural nucleobase.

[0253] In some embodiments, provided is a polynucleotide 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 has reduced binding to IL-2Ra or no detectable binding to IL-2Ra as compared to a comparable 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, provided is 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 can have a binding affinity to IL-2Ra that is reduced by about 10% to about 100%, e.g., by about 10%, 20%, 30%, 40%, 50%, 60%, 17%, 80%, 90%, 100%, or a sub-range thereof. In some embodiments, provided is 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 can have a binding affinity to IL-2Ra that is reduced by about 10% to about 100%, or can be reduced by about 1-fold to about 100,000-fold or more, e.g., by about 1-fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold or more, or a sub-range thereof. In some embodiments, provided is a polynucleotide 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 has no detectable binding to IL-2Ra.

[0254] In some embodiments, provided is 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 reduced receptor signaling potency for IL-2Rαβγ compared to a comparable 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, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the ratio between the signaling potency of the modified IL-2 polypeptide for IL-2Rαβγ and the signaling potency of a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution for IL-2Rαβγ 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 more, or subranges thereof. In some embodiments, provided is 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 no detectable receptor signaling potency for IL-2Rαβγ.

[0255] In some embodiments, provided is 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 reduced binding to IL-2Rα compared to a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution, and reduced receptor signaling potency for IL-2Rαβγ compared to a comparable 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, provided is 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 no detectable binding to IL-2Rα and no detectable receptor signaling potency for IL-2Rαβγ.

[0256] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the binding level of the modified IL-2 polypeptide to interleukin 2 receptor beta (IL-2Rβ) or interleukin 2 receptor gamma (IL-2Rγ) can be substantially retained or can be higher as compared to a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution(s), and / or the receptor signaling potency of the modified IL-2 polypeptide to IL-2Rβγ can be substantially retained or can be higher as compared to a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution(s). In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the binding level of the modified IL-2 polypeptide to IL-2Rβ or IL-2Rγ is substantially retained or is higher as compared to a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution(s). In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the receptor signaling potency of the modified IL-2 polypeptide to IL-2Rβγ is substantially retained or is higher as compared to a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution(s). In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the binding level of the modified IL-2 polypeptide to IL-2Rβ or IL-2Rγ is substantially retained or is higher as compared to a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution(s), and the receptor signaling potency of the modified IL-2 polypeptide to IL-2Rβγ is substantially retained or is higher as compared to a comparable IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 without the substitution(s).

[0257] In some embodiments, provided are polynucleotides 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 deletion at any suitable position. In some embodiments, provided are polynucleotides 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 N-terminal deletion, such as an N-terminal deletion of amino acid residues 1-30 or a sub-range thereof. In some embodiments, provided are polynucleotides comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide has a C-terminal deletion, such as a C-terminal deletion of amino acid residues 114-134 or a sub-range thereof. In some embodiments, provided are polynucleotides 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 N-terminal deletion and a C-terminal deletion.

[0258] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the fusion protein comprises a recombinant fusion protein. In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the fusion polypeptide further comprises an additional amino acid sequence. In some embodiments, provided is 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 fused to the additional amino acid sequence in any suitable manner. In some embodiments, provided is a polynucleotide 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 is fused to the additional amino acid sequence through the N-terminus of the modified IL-2 polypeptide or through the C-terminus of the modified IL-2 polypeptide. The additional amino acid sequence can comprise any suitable sequence or content. In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the additional amino acid sequence comprises an antibody sequence or a portion or fragment thereof. In some embodiments, provided is a polynucleotide comprising 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.

[0259] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, 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, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the fusion protein further comprises an additional amino acid sequence comprising a) human serum albumin (HSA); b) murine serum albumin (MSA); or c) a PK extended fragment or analog of a) or b).

[0260] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the fusion protein further comprises an additional amino acid sequence comprising a) human serum albumin (HSA) set forth in SEQ ID NO: 7; b) murine serum albumin (MSA) set forth in SEQ ID NO: 6; or c) a PK-extended fragment or analog of a) or b).

[0261] In some embodiments, provided is a polynucleotide comprising 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.

[0262] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the polynucleotide is prepared using any suitable technique or method. In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the polynucleotide is prepared by recombinant production, chemical synthesis, or a combination thereof.

[0263] In some embodiments, provided is a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide, wherein the polynucleotide comprises DNA, RNA, DNA / RNA hybrid, viral vector, non-viral vector, plasmid, and / or Nanoplasmid TM In some embodiments, provided is a polynucleotide 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.

[0264] In some embodiments, provided is 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 and / or the fusion protein, once expressed, can form a conjugate with another moiety in vivo, ex vivo, or in vitro.

[0265] 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 prepared as a pharmaceutical composition comprising one or more lipids and / or lipid nanoparticles (LNPs) and / or contained in the pharmaceutical composition.

[0266] 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, as described above, is configured to conjugate with another portion, such as a water-soluble polymer, lipid, polypeptide (e.g., protein), or peptide. Such conjugation can be performed or occur in vitro or in vivo.

[0267] 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, as described above, is configured to conjugate in any suitable manner to another portion (e.g., a water-soluble polymer, lipid, protein, or peptide). For example, the modified IL-2 polypeptide may be covalently conjugated to a water-soluble polymer, lipid, protein, or peptide. In another example, the modified IL-2 polypeptide may be non-covalently conjugated to a water-soluble polymer, lipid, protein, or peptide. In yet another example, the modified IL-2 polypeptide may be conjugated to a water-soluble polymer, lipid, protein, or peptide via a substituted native or non-native amino acid at any suitable position.

[0268] In some embodiments, provided is 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 as described above is configured to be conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide) via a substituted natural amino acid or a non-natural 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 another embodiment, the modified IL-2 polypeptide is conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide) via a substituted natural 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 still another embodiment, the modified IL-2 polypeptide is conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a 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, a lipid, a protein, or a 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.

[0269] In some embodiments, provided is 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 as described above is configured to be conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide) via a substituted 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. In some embodiments, provided is 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 as described above is configured to be conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide) via a substituted 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. In some embodiments, provided is 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 as described above is configured to be conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a 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. In some embodiments, provided is 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 as described above is configured to be conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a 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.

[0270] In some embodiments, provided is 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 as described above is configured to be conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide) via a single amino acid residue or multiple amino acid residues of the modified IL-2 polypeptide and another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide). In some embodiments, provided is 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 as described above is configured to be conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide) via: i) an alpha amino group of an N-terminal amino acid residue of the modified IL-2 polypeptide; ii) an epsilon amino group of a lysine amino acid residue of the modified IL-2 polypeptide; or iii) an N-glycosylation site or an O-glycosylation site of the modified IL-2 polypeptide.

[0271] In some embodiments, provided is 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 as described above is configured to be conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide) via a linker. In some embodiments, provided is 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 as described above is configured to be conjugated directly to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide) without a linker.

[0272] In some embodiments, provided is 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 as described above is configured to be conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide) via a single amino acid residue in a fusion polypeptide comprising the modified IL-2 polypeptide and an additional amino acid sequence. 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.

[0273] The additional amino acid sequence in the modified IL-2 polypeptide conjugate of the present application can comprise any suitable sequence or content. For example, the additional amino acid sequence in the modified IL-2 polypeptide conjugate of the present application 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 of the present application can comprise an Fc portion of an antibody.

[0274] In some embodiments, provided is 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 as described above is configured to be conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide in a fusion polypeptide) in any suitable manner. In some embodiments, provided is 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 as described above is configured to be conjugated to another moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide) by: i) an alpha amino group of an N-terminal amino acid residue of the fusion polypeptide; ii) an 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. In another example, the fusion polypeptide can be covalently conjugated to a water-soluble polymer, a lipid, a protein, or a peptide directly or through a linker.

[0275] In some embodiments, provided is 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 as described above is configured to be conjugated to any suitable water-soluble polymer. For example, the water-soluble polymer can comprise polyethylene glycol (PEG), poly(propylene glycol) (PPG), a co-polymer of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(a-hydroxy acid), poly( vinyl alcohol), polyphosphazene, poly(oxazoline) (POZ), poly(N-acryloylmorpholine), or a combination thereof. See, e.g., WO 2019 / 028425 Al and WO 2019 / 028419 Al.

[0276] In some embodiments, provided is 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 as described above is configured to be conjugated to a PEG molecule. 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 from about three to about ten PEG chains emanating from a central core group. In another example, the branched PEG can be a star PEG comprising from about 10 to about 100 PEG chains emanating from a central core group. In still another example, the branched PEG can be a comb PEG comprising a plurality of PEG chains grafted to a polymeric backbone.

[0277] In some embodiments, provided is 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 as described above is configured to be conjugated to a PEG molecule of any suitable molecular weight. 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 subranges 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 subranges thereof. In still another example, the PEG molecule 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 subranges thereof.

[0278] In some embodiments, provided is 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 as described above is configured to be conjugated to a PEG molecule of any suitable form. For example, the PEG molecule can be a monodisperse, homogenous, or discrete PEG molecule.

[0279] In some embodiments, provided is 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 as described above is configured to be conjugated to a polysaccharide.

[0280] In some embodiments, provided is 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 as described above is configured to be conjugated to any suitable lipid. In some embodiments, provided is 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 as described above is configured to be conjugated to a fatty acid.

[0281] In some embodiments, provided is 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 as described above is configured to be conjugated to any suitable protein. In some embodiments, provided is 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 as described above is configured to be conjugated to an antibody or binding fragment thereof. In some embodiments, provided is 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 as described above is configured to be conjugated to an Fc portion of an antibody.

[0282] In some embodiments, provided is 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 as described above is configured to be conjugated to, for example, a water-soluble polymer, a lipid, a protein, or a peptide, which can be bound to the modified IL-2 polypeptide by any suitable means. In some embodiments, provided is 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 as described above is configured to be conjugated to, for example, a water-soluble polymer, a lipid, a protein, or a peptide, which can be indirectly bound to the substituted natural or unnatural amino acid of the modified IL-2 polypeptide by a linker. In another example, the other moiety (e.g., a water-soluble polymer, a lipid, a protein, or a peptide) can be directly bound to the substituted natural or unnatural amino acid of the modified IL-2 polypeptide.

[0283] The modified IL-2 polypeptide conjugate of the present application can have any suitable in vivo half-life. For example, the in vivo half-life of the modified IL-2 polypeptide conjugate of the present application can be from about 5 minutes to about 10 days, e.g., about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 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 a sub-range thereof.

[0284] Pharmaceutical compositions In some embodiments, provided is a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide as described above and a pharmaceutically acceptable carrier or excipient.

[0285] The pharmaceutical composition of the present application can be configured to treat or prevent any suitable disease, disorder, or condition. For example, the pharmaceutical composition of the present application can be configured to treat or prevent a proliferative disorder in a subject.

[0286] In one embodiment, the pharmaceutical composition of the present application is configured to treat or prevent a solid tumor or cancer in a subject. The solid tumor or cancer can be chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone / osteosarcoma, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brain stem glioma, pilocytic astrocytoma, ependymal, 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 neoplasm 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 cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), hepatocellular cancer, islet cell pancreatic 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 cancer, renal pelvis and ureter, transitional cell cancer, prostate cancer, testicular cancer, gestational trophoblastic tumor, ureter and renal pelvis, transitional cell cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, esophageal cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, 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 sarcoma, epithelioid hemangioendothelioma (EHE), desmoplastic small round cell tumor, or liposarcoma.

[0287] In another embodiment, the pharmaceutical composition of the present application is configured to treat or prevent a malignant hematological neoplasm in a subject. The malignant hematological neoplasm can be a malignant hematological neoplasm including myeloid neoplasm, leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, hepatosplenic T-cell lymphoma, B-cell lymphoma reticuloendotheliosis, reticulosis, oligodendroglioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt lymphoma, mediastinal large B-cell lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, nodular lymphocyte predominant Hodgkin lymphoma, plasma cell leukemia, acute erythremia and erythroleukemia, acute erythroid myelosis, acute erythroid leukemia, Heilmeyer-Schoner disease, acute megakaryocyte leukemia, mast cell leukemia, panmyelosis, acute panmyelosis with myelofibrosis, lymphosarcoma cell leukemia, acute leukemia of unspecified cell type, blast crisis chronic myelogenous leukemia, stem cell leukemia, chronic leukemia of unspecified cell type, subacute leukemia of unspecified cell type, accelerated chronic myelogenous 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 disease, myeloma, solitary myeloma, plasma cell leukemia, extramedullary plasmacytoma, malignant plasma cell neoplasm NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative lesion, lymphoid granulomatosis, angioimmunoblastic lymphadenopathy, T-gamma lymphoproliferative disorder, Waldenstrom macroglobulinemia, alpha heavy chain disease, gamma heavy chain disease, Franklin disease, immunoproliferative small intestinal disease, Mediterranean disease, unspecified malignant immunoproliferative disease, or immunoproliferative disease NOS.

[0288] In still another embodiment, the pharmaceutical composition of the present application is configured to treat or prevent an immunodeficiency disease or disorder in a subject. The immunodeficiency disease or disorder can be agammaglobulinemia: X-linked and autosomal recessive, ataxia telangiectasia, chronic granulomatous disease and other phagocyte disorders, common variable immunodeficiency, complement deficiencies, DiGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyper-IgE syndrome, hyper-IgM syndrome, IgG subclass deficiency, innate immunodeficiency, 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 defective neutrophil production), 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 lymphocytopenia.

[0289] The pharmaceutical composition of the present application can further comprise another active ingredient. The other active ingredient can be an active ingredient for the treatment or prevention of any suitable disease, disorder, or condition. For example, the other active ingredient can be an anti-tumor substance.

[0290] The additional active ingredient can be formulated in a separate pharmaceutical composition from the at least one exemplary modified IL-2 polypeptide or modified IL-2 polypeptide conjugate of the present disclosure, or can be co- comprised in a single pharmaceutical composition with the at least one exemplary modified IL-2 polypeptide or modified IL-2 polypeptide conjugate of the present disclosure. The pharmaceutical composition of the present application can be formulated for administration by oral, parenteral, inhalation, topical, rectal, nasal, buccal, vaginal, by implanted reservoir, or other drug administration method. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0291] A sterile injectable aqueous or oleaginous suspension of a sterile injectable composition, such as a sterile pyrogen-free solution or suspension, can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Suitable carriers and other pharmaceutical composition components are typically sterile.

[0292] In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium (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 natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents. Various emulsifying agents or bioavailability enhancers which are commonly used in manufacturing pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulating the compositions.

[0293] 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. In the case of tablets for oral use, carriers commonly include lactose and corn starch. Lubricating agents, such as magnesium stearate, can also be added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oil with or without a fatty acid, including oleic acid. If desired, certain sweetening, flavoring, or coloring agents can be added. Nasal aerosol or inhalation compositions can be prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing suitable preservatives, e.g., benzyl alcohol, absorption promoters to enhance bioavailability, and / or other solubilizers or dispersing agents known in the art.

[0294] Any suitable formulation of the compounds described herein can be prepared. See generally, Remington's Pharmaceutical Sciences (2000) Hoover, J. E., ed., 20th Ed., Philadelphia, PA: Lippincott Williams & Wilkins, pp. 780-857. The formulation is selected to suit the appropriate route of administration. In cases where the compound is sufficiently basic or acidic to form a stable nontoxic acid or base salt, administration of the compound as a salt can be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiologically acceptable anion, for example, tosylates, methanesulfonates, acetates, citrates, malonates, tartrates, succinates, benzoates, ascorbates, alpha-ketoglutarates, and alpha-glycerophosphates. Suitable inorganic salts can also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptable salts are obtained using standard procedures well known in the art, for example, by dissolving the compound in solution with a suitable base, such as sodium hydroxide, 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.

[0295] If the contemplated compound or substance is administered in a pharmacological composition, it is contemplated that the compound or substance can be formulated in admixture with a pharmaceutically acceptable excipient and / or carrier. For example, the contemplated compound or substance can be administered orally as a neutral compound or substance or as a pharmaceutically acceptable salt or intravenously in a solution of physiological saline. Conventional buffers such as phosphate, bicarbonate, or citrate, and the like can be used for this purpose. Of course, one of ordinary skill in the art can modify the formulation within the teachings of the specification to provide numerous formulations for a particular route of administration. In particular, the contemplated compound or substance can be modified to make it more soluble in water or other vehicle, which can be easily accomplished, for example, by minor modifications well known to those of ordinary skill in the art (salt formulation, esterification, etc.). It is also well known to one of ordinary skill in the art that the route of administration and dosage regimen of a particular compound or substance (e.g., a modified IL-2 polypeptide or a modified IL-2 polypeptide conjugate of the disclosure) can be modified to have the maximum beneficial effect on the patient in order to manage the pharmacokinetics of the compound or substance of the invention.

[0296] The modified IL-2 polypeptides or modified IL-2 polypeptide conjugates of the invention can be dissolved in organic solvents such as chloroform, dichloromethane, ethyl acetate, ethanol, methanol, isopropanol, acetonitrile, glycerol, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, and the like. In one embodiment, the present invention provides formulations prepared by mixing the modified IL-2 polypeptides or modified IL-2 polypeptide conjugates of the disclosure with a pharmaceutically acceptable carrier. In one aspect, the formulations can be prepared using a method comprising: a) dissolving the 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 a physiological saline or a buffer containing a 1-10% carbohydrate solution. In one example, the carbohydrate comprises dextrose. The pharmaceutical compositions obtained using the methods of the invention are stable and suitable for animal and clinical applications.

[0297] Illustrative examples of water-soluble organic solvents for use in the pharmaceutical compositions of the invention include, and are not limited to, polyethylene glycol (PEG), an alcohol, acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, or a combination thereof. Examples of alcohols include, but are not limited to, methanol, ethanol, isopropanol, glycerol, or propylene glycol.

[0298] Illustrative examples of water-soluble non-ionic surfactants for use in the pharmaceutical compositions of the present application include, and are not limited to, CREMOPHOR.RTM. EL, polyethylene glycol modified CREMOPHOR.RTM. (Cremophor.RTM. EL), polyoxylglycerides 35, hydrogenated CREMOPHOR.RTM. RH40, hydrogenated CREMOPHOR.RTM. RH60, PEG-succinates, polysorbate 20, polysorbate 80, SOLUTOL.RTM. HS (polyethylene glycol 660 12-hydroxystearate), sorbitan monooleate, poloxamers, LABRAFIL.RTM. (ethoxylated almond oil), LABRASOL.RTM. (caprylocaproyl polyoxylglycerides), GELUCIRE.RTM. (glycerides), SOFTIGEN.RTM. (PEG 6 caprylic glycerides), glycerol, glycol polysorbates, or combinations thereof.

[0299] Illustrative examples of water-soluble lipids for use in the pharmaceutical compositions of the present application include, but are not limited to, vegetable oil, triglycerides, vegetable oils, or combinations thereof. Examples of lipid oils include, but are not limited to, castor oil, polyoxylcastor oil, corn oil, olive oil, cottonseed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, coconut oil triglycerides, palm seed oil, and hydrogenated versions thereof, or combinations thereof.

[0300] Illustrative examples of fatty acids and fatty acid esters for use in the pharmaceutical compositions of the present application include, but are not limited to, oleic acid, monoglycerides, diglycerides, mono- or di-fatty acid esters of PEG, or combinations thereof.

[0301] Illustrative examples of cyclodextrins for use in the pharmaceutical compositions of the present application include, but are not limited to, a-cyclodextrin, b-cyclodextrin, hydroxypropyl-b-cyclodextrin, or sulfobutyl ether-b-cyclodextrin.

[0302] Illustrative examples of phospholipids for use in the pharmaceutical compositions of the present application include, but are not limited to, soybean phosphatidylcholine or distearoylphosphatidylglycerol, and hydrogenated versions thereof, or combinations thereof.

[0303] One of ordinary skill in the art can modify the formulations within the teachings of the specification to provide a large number of formulations for particular routes of administration. In particular, the compounds or substances can be modified to make them more soluble in water or other vehicles. It is also well within the ordinary skill of the art of the present disclosure to make modifications to the route of administration and dosage regimen of a particular compound or substance to provide optimal beneficial effects to the patient, in order to manage the pharmacokinetics of the compounds or substances of the present application.

[0304] Lipids, lipid nanoparticles, and mRNA pharmaceutical compositions comprising the same In some embodiments, provided are pharmaceutical compositions comprising a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide as described above and one or more lipids. In some embodiments, provided are pharmaceutical compositions comprising a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide as described above and one or more lipids and lipid-containing nanoparticles. In some embodiments, provided are pharmaceutical compositions comprising a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a modified interleukin 2 (IL-2) polypeptide as described above and one or more lipids and lipid-containing nanoparticles. For example, lipids and lipid-containing nanoparticles suitable for use in the pharmaceutical compositions herein and throughout the disclosure are disclosed in U.S. Provisional Patent Application No. 63 / 313,648, entitled “Novel Ionizable Cationic Lipids,” filed February 24, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0305] The term “lipid” or “lipid moiety” is used according to its ordinary meaning in chemistry and refers to a hydrophobic molecule typically characterized by an aliphatic hydrocarbon chain. In embodiments, a lipid moiety includes a carbon chain having 3 to 100 carbons. In embodiments, a lipid moiety includes a carbon chain having 5 to 50 carbons. In embodiments, a lipid moiety includes a carbon chain having 5 to 25 carbons. In embodiments, a lipid moiety includes a carbon chain having 8 to 525 carbons. A lipid moiety can include a saturated or unsaturated carbon chain and can be optionally substituted. In embodiments, a lipid moiety is optionally substituted at a terminus with a charged moiety. In embodiments, a lipid moiety is an alkyl or heteroalkyl optionally substituted at a terminus with a carboxylic acid moiety. Lipids are also a group of organic compounds that include, but are not limited to, fatty acid esters, and are characterized by being insoluble in water but soluble in many organic solvents. The lipids are generally classified into at least three categories: (1) “simple lipids,” which include fats and oils and waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids,” such as steroids.

[0306] The terms “cationic lipid” or “ionizable cationic lipid” are used interchangeably herein and refer to a lipid that protonates at low pH, which makes the lipid positively charged, but the lipid remains neutral at physiological pH.

[0307] 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 target site of interest (e.g., a cell, tissue, organ, etc.). In embodiments, the lipid particles described herein are nucleic acid-lipid particles, which are typically formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particles. In other embodiments, active or therapeutic agents, such as nucleic acids, can be encapsulated in the lipid portion of the particles, thereby protecting them from enzymatic degradation.

[0308] The term "lipid conjugate" refers to a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates, such as PEG coupled to dimyristyl glycerol (e.g., PEG-DMG conjugates), PEG coupled to diacylglycerol (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, and PEG coupled to ceramide.

[0309] The term "diacylglycerol" or "DAG" includes a compound having two fatty acyl chains R 1 and R 2 , both of which independently have 2 to 30 carbons bound to the 1 and 2 positions of glycerol via ester linkages. The acyl groups can be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C 12 ), myristoyl (C 14 ), palmitoyl (C 16 ), stearoyl (C 18 ), and arachidoyl (C 20 ). In preferred embodiments, R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristoyl (i.e., dimyristoyl), R 1 and R 2 are both stearoyl (i.e., distearoyl), etc. Diacylglycerols have the following general formula: .

[0310] The term "dialkoxypropyl" or "DAA" includes a compound having two alkyl chains R 1 and R 2 , both of which independently have 2 to 30 carbons. The alkyl groups can be saturated or have varying degrees of unsaturation. Dialkoxypropyls have the following general formula: .

[0311] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to a substance that aids in the administration of an active agent to a subject and aids in the absorption of the subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorants, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates (such as lactose, amylose or starch), fatty acid esters, carboxymethylcellulose, polyvinylpyrrolidone, and pigments and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents (such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents and / or aromatic substances and the like) which do not deleteriously react with the compounds of the present disclosure. One skilled in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0312] The abbreviations used herein have their normal chemical and biological meanings. The chemical structures and formulas set forth herein are constructed according to standard rules of chemical valence known in the chemical arts. The description of the compounds of the present disclosure is limited to the principles of chemical bonding known to those skilled in the art. Thus, where a group can be substituted with one or more of a number of substituents, such substitution is selected so as to comply with the principles of chemical bonding and to result in compounds that are not inherently unstable and / or would not be likely to be unstable under ambient conditions (such as aqueous, neutral, and several known physiological conditions) as known to one of ordinary skill in the art. For example, heterocycloalkyl or heteroaryl groups are attached to the rest of the molecule through a ring heteroatom in accordance with the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0313] When substituent groups are designated by their conventional chemical formulae (written from left to right), the substituent groups equally encompass the chemically identical substituent groups resulting from writing the formula from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0314] Unless otherwise indicated, the term "alkyl," by itself or as part of another substituent means a straight-chain (i.e., unbranched) or branched-chain carbon chain (or carbons) that can be fully saturated, mono-unsaturated, or poly-unsaturated and can include single, bivalent, and trivalent groups. Alkyl groups can include designated numbers of carbon atoms (e.g., C1-C10alkyl). For example, C1-C10alkyl means one to ten carbons. 10alkyl is an unringed chain. Examples of saturated hydrocarbyl groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl is an alkyl group having one or more double or triple bonds. Examples of unsaturated alkyl include, but are not limited to, ethenyl, 2-propenyl, crotyl, 2- isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- propynyl, and 3-propynyl, 3-butynyl, and higher homologs and isomers. Alkoxy is an alkyl group attached to the rest of the molecule by an oxygen linker (-0-). The alkyl portion can be an alkenyl portion. The alkyl portion can be an alkynyl portion. The alkyl portion can be fully saturated. Alkenyl can include more than one double bond and / or one or more triple bonds in addition to the one or more double bonds. Alkynyl can include more than one triple bond and / or one or more double bonds in addition to the one or more triple bonds.

[0315] Unless otherwise indicated, the term "alkylene," by itself or as part of another substituent, means a divalent group derived from an alkyl group, such as, but not limited to, -CH2CH2CH2CH2-. Typically, alkyl (or alkylene) will have from 1 to 30 carbon atoms, with those having 10 or fewer carbon atoms being preferred herein. "Lower alkyl" or "lower alkylene" is a short chain alkyl or alkylene group, typically having eight or fewer carbon atoms. Unless otherwise indicated, the term "alkenylene" by itself or as part of another substituent, means a divalent group derived from an alkene.

[0316] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight-chain or branched-chain or combination thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, B, Se, or S), and wherein the nitrogen and sulfur atoms can optionally be oxidized and the nitrogen heteroatom can optionally be quaternized. The heteroatom(s) (e.g., O, N, S, Si, B, Se, or P) can be placed at any interior position of the heteroalkyl group or located at the position where the alkyl group is attached to the remainder of the molecule. A heteroalkyl group is un-cyclized. 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)2-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 can be consecutive, such as, for example, -CH2-NH-OCH3and -CH2-O-Si(CH3)3. A heteroalkyl moiety can include one heteroatom (e.g., O, N, S, Si, B, Se, or P). A heteroalkyl moiety can include two heteroatoms (e.g., O, N, S, Si, B, Se, or P) that are optionally different. A heteroalkyl moiety can include three heteroatoms (e.g., O, N, S, Si, B, Se, or P) that are optionally different. A heteroalkyl moiety can include four heteroatoms (e.g., O, N, S, Si, B, Se, or P) that are optionally different. A heteroalkyl moiety can include five heteroatoms (e.g., O, N, S, Si, B, Se, or P) that are optionally different. A heteroalkyl moiety can include up to 8 heteroatoms (e.g., O, N, S, Si, B, Se, or P) that are optionally different. The term "heteroalkenyl," by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl group including at least one double bond. A heteroalkenyl group can optionally include more than one double bond, and / or include one or more triple bonds in addition to the double bond(s). The term "heteroalkynyl," by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl group including at least one triple bond. A heteroalkynyl group can optionally include more than one triple bond, and / or include one or more double bonds in addition to the triple bond(s).

[0317] Similarly, the term "heteroalkylene", by itself or as part of another substituent, means, unless otherwise specified, a divalent radical derived from heteroalkyl, such as, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., -O-CH2-CH2-0-, -CH2-CH2-NH-CH2-CH2- NH-). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula is written. For example, the formulas -C(O)2R'- represent both -C(O)2R'- and -R'C(O)2-. As described above, as used herein, heteroalkyl includes those groups that are attached to the rest of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', -NR'R", -OR', -SR', and / or -SO2R'. Where "heteroalkyl", is recited, followed by recitations of specific heteroalkyl groups such as -NR'R", it is understood that the heteroalkyl group and -NR'R" are not redundant or mutually exclusive features. Rather, the specific heteroalkyl is recited to add clarity. Thus, the term "heteroalkyl" should not be interpreted as excluding specific heteroalkyl groups such as -NR'R" and the like.

[0318] Unless otherwise specified, the terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, mean "alkyl" and "heteroalkyl", respectively, that are cyclic. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy either or both of the chain termini. 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-tetrahydro-pyridinyl), 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. "Cycloalkylene" and "heterocycloalkylene" mean, individually or as part of another substituent, divalent radicals derived from cycloalkyl and heterocycloalkyl, respectively.

[0319] In embodiments, the term "cycloalkyl" refers to monocyclic, bicyclic, or polycyclic cycloalkyl ring systems. In embodiments, a monocyclic ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms, wherein such groups may be saturated or unsaturated, but are not aromatic. In embodiments, the cycloalkyl group is fully saturated. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. A bicyclic cycloalkyl ring system is a bridged monocyclic or fused bicyclic ring. In embodiments, a bridged monocyclic ring contains a monocyclic cycloalkyl ring, wherein two non-adjacent carbon atoms of the monocyclic ring are connected by an alkylene bridge of one to three additional carbon atoms (i.e., in the form of (CH2)). w The bridging group, where w is 1, 2, or 3). Representative examples of bicyclic systems include, but are not limited to, bicyclic [3.1.1]heptane, bicyclic [2.2.1]heptane, bicyclic [2.2.2]octane, bicyclic [3.2.2]nonane, bicyclic [3.3.1]nonane, and bicyclic [4.2.1]nonane. In embodiments, the fused bicyclic cycloalkyl ring system contains a monocyclic cycloalkyl ring fused with a phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocyclic, or monocyclic heteroaryl group. In embodiments, the bridging or fused bicyclic cycloalkyl group is partially connected to the parent molecule via any carbon atom included within the monocyclic cycloalkyl ring. In embodiments, the cycloalkyl group is optionally substituted with one or both groups that are independently oxo or thio. In embodiments, the fused bicyclic cycloalkyl group is a 5- or 6-membered monocyclic cycloalkyl ring fused with a benzene ring, a 5- or 6-membered monocyclic cycloalkyl group, a 5- or 6-membered monocyclic cycloalkenyl group, a 5- or 6-membered monocyclic heterocyclic group, or a 5- or 6-membered monocyclic heteroaryl group, wherein the fused bicyclic cycloalkyl group is optionally substituted by one or two groups that are independently oxo or thio. In embodiments, the polycyclic cycloalkyl ring system is a monocyclic cycloalkyl ring (base ring) fused with the following: (i) a ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclic groups; 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 heterocyclic groups. In embodiments, the polycyclic cycloalkyl group is partially connected to the parent molecule via any carbon atom included within the base ring. In embodiments, the polycyclic cycloalkyl ring system is a monocyclic cycloalkyl ring (base ring) fused with the following: (i) a ring system selected from the group consisting of: bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclic groups; or (ii) two other ring systems independently selected from the group consisting of: phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclic groups. Examples of polycyclic cycloalkyl groups include, but are not limited to, tetradecahydrophenanthrenyl, perhydrophenanthiazin-1-yl, and perhydrophenoxazin-1-yl.

[0320] In embodiments, the cycloalkyl group is a cycloalkenyl group. The term "cycloalkenyl" is used according to its common, general meaning. In embodiments, the cycloalkenyl group 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, wherein such a 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, the bicyclic cycloalkenyl ring is a bridged monocyclic or fused bicyclic ring. In embodiments, a bridged monocyclic ring contains a monocyclic cycloalkenyl ring, wherein two non-adjacent carbon atoms of the monocyclic ring are connected by an alkylene bridge of one to three additional carbon atoms (i.e., in the form of (CH2)). w The bridging group, where w is 1, 2, or 3). Representative examples of bicyclic cycloalkenyl groups include, but are not limited to, norbornenyl and bicyclic [2.2.2]octyl-2-enyl. In embodiments, the fused bicyclic cycloalkenyl ring system contains a monocyclic cycloalkenyl ring fused with a phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocyclic, or monocyclic heteroaryl group. In embodiments, the bridged or fused bicyclic cycloalkenyl group is partially connected to the parent molecule via any carbon atom contained within the monocyclic cycloalkenyl ring. In embodiments, the cycloalkenyl group is optionally substituted by one or both groups that are independently oxo or thio. In embodiments, the polycyclic cycloalkenyl ring comprises a monocyclic cycloalkenyl ring (base ring) fused with the following: (i) a ring system selected from the group consisting of: bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclic groups; 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 heterocyclic groups. In embodiments, the polycyclic cycloalkenyl group is partially connected to the parent molecule via any carbon atom included within the base ring. In embodiments, the polycyclic cycloalkenyl ring comprises a monocyclic cycloalkenyl ring (base ring) fused with the following: (i) a ring system selected from the group consisting of: bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclic groups; or (ii) two ring systems independently selected from the group consisting of: phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclic groups.

[0321] In embodiments, the heterocycloalkyl group is a heterocyclyl group. As used herein, the term "heterocyclyl" means a monocyclic, bicyclic, or polycyclic heterocycle. A heterocyclyl monocyclic heterocycle is a 3-, 4-, 5-, 6-, or 7-membered ring that includes at least one heteroatom independently selected from the group consisting of O, N, and S, wherein the ring is saturated or unsaturated, but not aromatic. A 3- or 4-membered ring includes 1 heteroatom selected from the group consisting of O, N, and S. A 5-membered ring can include 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 includes zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. The heterocyclyl monocyclic heterocycle is attached 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, but are not limited to, azetidinyl, azepinyl, aziridinyl, diazepinyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. A heterocyclyl bicyclic heterocycle is a monocyclic heterocycle fused to any of phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocyclyl, or monocyclic heteroaryl. The heterocyclyl bicyclic heterocycle is attached to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocyclic portion of the bicyclic ring system. Representative examples of bicyclic heterocyclyl groups 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-dihydrobenzothiophene-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, and octahydrobenzofuranyl. In embodiments, the heterocyclyl group is optionally substituted with one or two groups that are independently oxo or thioxo. In certain embodiments, the bicyclic heterocyclyl group is a 5- or 6-membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5- or 6-membered monocyclic cycloalkyl group, a 5- or 6-membered monocyclic cycloalkenyl group, a 5- or 6-membered monocyclic heterocyclyl group, or a 5- or 6-membered monocyclic heteroaryl group, wherein the bicyclic heterocyclyl group is optionally substituted with one or two groups that are independently oxo or thioxo. A polycyclic heterocyclyl ring system is a monocyclic heterocyclyl ring (base ring) fused to (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 polycyclyl group is attached to the parent molecular moiety through any carbon atom or nitrogen atom included in the ring system. In embodiments, the polycyclyl ring system is a monocyclic ring fused to (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, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclyl. Examples of polycyclyl 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-lH-carbazol-9-yl.

[0322] Unless otherwise indicated, the term "halo" or "halogen," by itself or as part of another substituent, means a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(Ci-C4)alkyl" includes, but is not limited to, fluoro methyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0323] Unless otherwise indicated, the term "acyl" means -C(O)R, wherein 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.

[0324] The term "aryl" means a polyunsaturated, aromatic hydrocarbon substituent that can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., fused rings) or linked covalently (preferably from 1 to 3 rings). The term "heteroaryl" means an aryl group (or ring) that contains 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. The term "heteroaryl" therefore includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaryl ring). A 5,6-fused ring heteroarylene group means two rings fused together wherein one ring has 5 members and the other ring has 6 members, and wherein at least one of the rings is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene group means two rings fused together wherein one ring has 6 members and the other ring has 6 members, and wherein at least one of the rings is a heteroaryl ring. And a 6,5-fused ring heteroarylene group means two rings fused together wherein one ring has 6 members and the other ring has 5 members, and wherein at least one of the rings is a heteroaryl ring. The heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, thiophenyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolinyl, 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-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furanyl, 3-furanyl, 2-thiophenyl, 3-thiophenyl, 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-quinolinyl, and 6-quinolinyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene" mean, individually or as part of another substituent, divalent groups derived from aryl and heteroaryl, respectively. A heteroaryl substituent can be -O- attached to a ring heteroatom nitrogen.

[0325] Fused ring heterocycloalkyl-aryl is an aryl fused with a heterocycloalkyl. Fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused with a heterocycloalkyl. Fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused with a cycloalkyl. Fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused with another heterocycloalkyl. Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl-cycloalkyl, or fused ring heterocycloalkyl-h...

Claims

1. A polynucleotide comprising a nucleic acid sequence encoding a fusion protein, said fusion protein comprising a modified interleukin-2 (IL-2) polypeptide, wherein said modified IL-2 polypeptide comprises amino acids having 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 with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2. The modified IL-2 polypeptide comprises at least one substitution at one or more positions of a natural or non-natural amino acid 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 polynucleotide according to claim 1, wherein: a) The modified IL-2 peptide is configured to be unconjugated or conjugated with a water-soluble polymer, lipid, peptide, protein, or peptide; and / or b) The modified IL-2 polypeptide binds less to interleukin-2 receptor α (IL-2Rα) compared to an equivalent IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 but without the at least one of the substitutions; and / or c) The modified IL-2 peptide exhibits reduced receptor signaling efficacy for IL-2Rαβγ compared to an equivalent IL-2 peptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 but without the at least one of the substitutions; and / or d) Compared to an equivalent IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 but without the at least one substitution, the modified IL-2 polypeptide has an increased ratio of IL-2Rβγ signaling efficacy to IL-2Rαβγ signaling efficacy (increased ratio of IL-2Rβγ signaling efficacy to IL-2Rαβγ signaling efficacy); and / or e) The modified IL-2 peptide exhibits enhanced receptor signaling efficacy for IL-2Rβγ compared to an equivalent IL-2 peptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 but without the at least one of the substitutions, and / or The condition is that when the modified IL-2 polypeptide contains at least one substitution made with a non-natural amino acid, the modified IL-2 polypeptide contains 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 made with a natural or non-natural amino acid at at least one position in at least one of the IL-2Rα interaction region, IL-2Rβ interaction region and / or IL-2Rγ interaction region; and / or f) Combinations from a) to e).

3. The polynucleotide according to 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 with the corresponding one or more regions of the IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 in one or more regions.

4. The polynucleotide according to 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 with the IL-2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

5. The polynucleotide according to any one of claims 1 to 4, wherein the modified IL-2 polypeptide comprises at least one substitution at a position selected from the group consisting of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine: 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 polynucleotide according to any one of claims 1 to 5, wherein the modified IL-2 polypeptide comprises: a) at least one substitution with a natural amino acid 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 configured to be conjugated at at least one position to a water-soluble polymer, lipid, protein, or peptide 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) 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 configured to be conjugated with a water-soluble polymer, lipid, protein or peptide at the N-terminus and / or C-terminus of the modified IL-2 polypeptide.

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

8. The polynucleotide according to any one of claims 1 to 7, wherein the modified IL-2 polypeptide comprises: 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 following groups: N29, Y31, K35, P65, N71, Q74, and combinations thereof; c) At the positions of Y31, N29, or combinations thereof, at least one substitution made with any amino acid; d) At the positions of Y31, N29 or combinations thereof, at least one substitution is made with cysteine, serine or alanine; e) At least one substitution at position Y31 using cysteine; f) At least one substitution at position N29 with cysteine; and / or g) At least one substitution made with cysteine ​​at position P65.

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

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

11. The polynucleotide according to any one of claims 1 to 10, 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) Substitution at position F42 with alanine, lysine, or serine; and / or c) Substitution with alanine at position F42; and / or d) Substitution with serine at position F42; and / or e) Substitution with lysine at position F42; and / or f) Substitution at position Y45 with alanine, histidine, or serine; and / or g) Substitution with alanine at position Y45; and / or h) Substitution with histidine at position Y45; and / or i) Substitution at position R38 with alanine, aspartic acid, or serine; and / or j) Substitution with aspartic acid at position R38; and / or k) Substitution with alanine at position P65; and / or l) Substitution with serine at position P65; and / or m) Substitution with alanine at position E62; and / or n) Substitution with lysine at position F42; and / or o) Substitution with cysteine ​​at position Y31; and / or Combinations of p)a) to o).

12. The polynucleotide according to any one of claims 1 to 11, wherein the modified IL-2 polypeptide comprises at least one substitution of 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. The polynucleotide according to any one of claims 1 to 12, wherein the modified IL-2 polypeptide comprises at least one substitution at a position selected from the group consisting of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, alanine, tryptophan, isoleucine, phenylalanine, or tyrosine: Q13, L19, R81, L85, S87, V91, I92, V93, and combinations thereof.

14. The polynucleotide according to 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 polynucleotide according to any one of claims 1 to 14, wherein the modified IL-2 polypeptide comprises: a) Substitution at position Y31; and / or c) Substitution at position F42; and / or d) Substitution at position C125; and / or e) Substitution at positions Y31, F42 and C125.

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

17. The polynucleotide according to any one of claims 1 to 16, further comprising at least one substitution made of a natural or non-natural amino acid at at least one position in at least one position in the IL-2Rα interaction region, the IL-2Rβ interaction region and / or the IL-2Rγ interaction region.

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

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

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

21. The polynucleotide according to any one of claims 1 to 20, wherein the modified IL-2 polypeptide binds less to IL-2Rα compared to an IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 but without the at least one substitution.

22. The polynucleotide according to 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. The polynucleotide according to any one of claims 1 to 22, 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.

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

25. The polynucleotide according to 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. The polynucleotide according to any one of claims 1 to 25, wherein the additional amino acid sequence imparts enhanced and / or prolonged pharmacokinetic (PK) characteristics to the modified IL-2 polypeptide or the fusion protein, compared to the IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substituted amino acid sequence.

27. The polynucleotide according to any one of claims 1 to 26, 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.

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

30. The polynucleotide according to any one of claims 1 to 29, wherein the additional amino acid sequence comprises serum albumin or its PK extended fragment or the like.

31. The polynucleotide according to any one of claims 1 to 30, wherein the additional amino acid sequence comprises: a) Human serum albumin (HSA); b) Mouse serum albumin (MSA); c) or a) or b) of a PK extension fragment or similar.

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

3.

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

34. The polynucleotide according to any one of claims 1 to 33, 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 in a manner that: i) the α-amino group of the N-terminal amino acid residue of the fusion polypeptide; ii) the ε-amino group of the lysine amino acid residue of the fusion polypeptide; or iii) The N-glycosylation site or O-glycosylation site of the fusion polypeptide.

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

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

37. The polynucleotide according to 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 reduced by about 1 to about 100,000 times or more, compared to the binding affinity of an IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without having the at least one substitution.

38. The polynucleotide according to any one of claims 1 to 37, wherein the IL-2 polypeptide or the fusion protein does not have a detectable binding to IL-2Rα.

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

40. The polynucleotide according to any one of claims 1 to 39, wherein the signal transduction efficacy of the IL-2 polypeptide or the fusion protein for IL-2Rαβγ is about 1 / 2 to about 1 / 100,000 compared with the signal transduction efficacy of an IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 but without the at least one substitution.

41. The polynucleotide according to any one of claims 1 to 40, wherein the IL-2 polypeptide or the fusion protein does not have detectable receptor signaling efficacy for IL-2Rαβγ.

42. The polynucleotide according to any one of claims 1 to 41, wherein: (i) The binding of the IL-2 polypeptide or the fusion protein to IL-2Rα is reduced compared to an IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution; and (ii) The receptor signaling efficacy of the IL-2 polypeptide or the fusion protein to IL-2Rαβγ is reduced compared to an IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

43. The polynucleotide according to any one of claims 1 to 42, wherein the IL-2 polypeptide or the fusion protein does not have detectable binding to IL-2Rα and does not have detectable receptor signaling efficacy for IL-2Rαβγ.

44. The polynucleotide according to any one of claims 1 to 43, wherein the IL-2 polypeptide or the fusion protein, compared to an IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution, has equivalent or increased binding to interleukin-2 receptor β (IL-2Rβ) and / or interleukin-2 receptor γ (IL-2Rγ); and / or has equivalent or increased receptor signaling efficacy of the modified IL-2 polypeptide or the fusion protein for IL-2Rβγ compared to an IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without the at least one substitution.

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

46. ​​The polynucleotide according to any one of claims 1 to 45, wherein the IL-2 polypeptide or the fusion protein has comparable or greater receptor signaling efficacy for IL-2Rβγ compared to an IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 but without the at least one substitution.

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

48. The polynucleotide according to any one of claims 1 to 47, wherein the IL-2 polypeptide or the fusion protein has an increased ratio of signal transduction efficacy for IL-2Rβγ to signal transduction efficacy for IL-2Rαβγ (increased ratio of signal transduction efficacy for IL-2Rβγ to signal transduction efficacy for IL-2Rαβγ) compared to an IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 without having the at least one substitution.

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

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

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

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

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

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

56. The polynucleotide according to any one of claims 1 to 55, further comprising a promoter operatively linked to the nucleic acid sequence, wherein the promoter comprises an SP6, T3, or T7 promoter sequence.

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

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

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

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

62. The polynucleotide according to any one of claims 1 to 61, wherein the polynucleotide comprises: DNA; RNA; or DNA / RNA hybrids.

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

64. The polynucleotide according to any one of claims 1 to 63, wherein the polynucleotide comprises: Viral vector; non-viral vector; plasmid; or nanoplasmid TM Carrier.

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

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

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

68. The polynucleotide according to any one of claims 1 to 67, wherein the modified IL-2 polypeptide or the fusion protein of the polynucleotide has a half-life in vivo of 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, etc. Day, approximately 1 hour to approximately 2 days, approximately 1 hour to approximately 1 day, approximately 5 minutes, approximately 10 minutes, approximately 20 minutes, approximately 30 minutes, approximately 40 minutes, approximately 50 minutes, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, or values ​​or ranges thereof.

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

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

71. The pharmaceutical composition according to claim 69 or claim 70, wherein the lipid comprises a cationic lipid of formula (I): （I）， Or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug, wherein: R 1 It is 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 the substituted or unsubstituted C0-C 12 Alkylene or substituted or unsubstituted 0 to 12-membered heteroalkylene; R 2 It is 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 groups or substituted or unsubstituted heteroalkyl groups; R 3 It is 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 groups or substituted or unsubstituted heteroalkyl groups; R 4 It is 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 groups or substituted or unsubstituted heteroalkyl groups; R 5 It is 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 groups or substituted or unsubstituted heteroalkyl groups; B 1 It is a bond, a substituted or unsubstituted alkylene group, a substituted or unsubstituted heteroalkylene group, a substituted or unsubstituted cycloalkylene group, a substituted or unsubstituted heteroalkylene group, a substituted or unsubstituted arylene group, or a substituted or unsubstituted heteroarylene group. B 2 and B 3 Each is independently a bond, a substituted or unsubstituted alkylene group, or a substituted or unsubstituted heteroalkylene group; 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[[ID=​​​​​​​​​​​​​​​ 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 Each independently is , , , , , , , or ; Each X is independently O, S, or CH2; W 1 W 2 W 3 W 4 W 5 and W 6 Each is an independent bond, substituted or unsubstituted C1-C 12 Alkylene or substituted or unsubstituted 2- to 12-membered heteroalkylene; Each R 1A and R 1B Independently, it is H, substituted or unsubstituted C1-C 12 Alkyl or substituted or unsubstituted 2- to 12-membered heteroalkyl groups; Each R 2A R 3A R 4A and R 5A Independently, it is H, substituted or unsubstituted C1-C 30 Alkyl or substituted or unsubstituted 2- to 30-membered heteroalkyl groups; 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 Independently, it is H, substituted or unsubstituted C1-C 12 Alkyl or substituted or unsubstituted 2- to 12-membered heteroalkyl; and Each s is an integer from 1 to 4.

72. The pharmaceutical composition according to claim 71, wherein the cationic lipid is a lipid, and in the lipid: R 1 It is H, -OR 1A Or substituted or unsubstituted heteroalkyl groups; L 1 It is a key, -NR 101 C(=S)-、-C(=S)NR 101 -, -O(C=O)-, -(C=O)O- or -O-; B 1 It is a bonded or substituted or unsubstituted alkylene; B 2 and B 3 Each is an alkylene group, either bonded, substituted, or unsubstituted; L 2 It is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, or -S-; L 4 It 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 is independently a bond, either substituted or unsubstituted C1-C 12 Alkylene; L a1 and L a2 Each independently is or ; Each X is independently either O or S; L 3 It is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, or -S-; L 5 It is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, or -S-; L 6 It is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, or -S-; L 7 It is a bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, or -S-; R 2 It is H or a substituted or unsubstituted alkyl group; R 3 It is H or a substituted or unsubstituted alkyl group; R 4 It is H or a substituted or unsubstituted alkyl group; R 5 It is H or a substituted or unsubstituted alkyl group; Each R 1A Independently, it is H or substituted or unsubstituted C1-C. 12 Alkyl; and Each R 101 It is independently H or a 2- to 12-membered heteroalkyl group, whether substituted or unsubstituted.

73. The pharmaceutical composition according to claim 71, wherein the cationic lipid is a lipid, and in the lipid: R 1 It is H, -OH, methoxy, ethoxy, or a substituted or unsubstituted heteroalkyl group; L 1 It is a key, -NR 101 C(=S)-or-C(=S)NR 101 -; B 1 It is a bonded or unsubstituted C1-C8 alkylene group; B 2 and B 3 Each is independently a C1-C8 alkylene group, either bonded, substituted, or unsubstituted; L 2 It is a bond, -O(C=O)- or -(C=O)O-; L 4 It is a bond, -O(C=O)- or -(C=O)O-; W 1 W 2 W 3 W 4 W 5 and W 6 Each is independently a bond, either substituted or unsubstituted C1-C 12 Alkylene; L a1 and L a2 Each independently is ; Each X is independently either O or S; L 3 It is a bond, -O(C=O)- or -(C=O)O-; L 5 It is a bond, -O(C=O)- or -(C=O)O-; L 6 It is a bond, -O(C=O)- or -(C=O)O-; L 7 It is a bond, -O(C=O)- or -(C=O)O-; R 2 It is H or substituted or unsubstituted C1-C 12 alkyl; R 3 It is H or substituted or unsubstituted C1-C 12 alkyl; R 4 It is H or substituted or unsubstituted C1-C 12 alkyl; R 5 It is H or substituted or unsubstituted C1-C 12 Alkyl; and Each R 101 It is independently a 2- to 12-membered heteroalkyl group, either substituted or unsubstituted.

74. The pharmaceutical composition according to claim 71, wherein the cationic lipid is a lipid, and in the lipid: R 1 It is -OH or methoxy group; L 1 It is a key; B 1 It is an unsubstituted C1-C8 alkylene group; B 2 and B 3 Each is independently a C1-C8 alkylene group, either bonded, substituted, or unsubstituted; L 2 It is a key; L 4 It is a key; W 1 W 2 W 3 W 4 W 5 and W 6 Each is independently a bond, either substituted or unsubstituted C1-C 12 Alkylene; L a1 and L a2 Each independently is ; Each X is independently O; L 3 It is a key; L 5 It is a key; L 6 It is a key; L 7 It is a key; R 2 It is H or substituted or unsubstituted C1-C 12 alkyl; R 3 It is H or substituted or unsubstituted C1-C 12 alkyl; R 4 It is H or substituted or unsubstituted C1-C 12 Alkyl; and R 5 It is H or substituted or unsubstituted C1-C 12 alkyl.

75. The pharmaceutical composition according to claim 71, wherein the cationic lipid is a lipid, and in the lipid: R 1 It is a heteroalkyl group, either substituted or unsubstituted; L 1 It is -C(=S)NR 101 - where the carbon atom is attached to the nitrogen atom in formula (I); B 1 It is a key; B 2 and B 3 Each is independently a C1-C8 alkylene group, either bonded, substituted, or unsubstituted; L 2 It is a bond, -O(C=O)- or -(C=O)O-; L 4 It is a bond, -O(C=O)- or -(C=O)O-; W 1 W 2 W 3 W 4 W 5 and W 6 Each is independently a bond, either substituted or unsubstituted C1-C 12 Alkylene; L a1 and L a2 Each independently is ; Each X is independently O; L 3 It is a key; L 5 It is a key; L 6 It is a key; L 7 It is a key; R 2 It is H or substituted or unsubstituted C1-C 12 alkyl; R 3 It is H or substituted or unsubstituted C1-C 12 alkyl; R 4 It is H or substituted or unsubstituted C1-C 12 Alkyl; and R 5 It is H or substituted or unsubstituted C1-C 12 alkyl.

76. The pharmaceutical composition according to claim 71, wherein the cationic lipid is: 、 、 、 、 、 or Or, or a pharmaceutically acceptable salt thereof.

77. The pharmaceutical composition according to claim 69 or claim 70, wherein the lipid comprises a cationic lipid of formula (II): (II), Or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug, wherein: B 4 It is W 7 -L a3 -W 8 ; W 7 and W 8 Each is independently a bond, a substituted or unsubstituted alkylene group, or a substituted or unsubstituted heteroalkylene group; 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 Each is independently H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, or R. 10 and R 11 Together with the nitrogen atom to which it is attached, it forms a substituted or unsubstituted heterocyclic alkyl group or a substituted or unsubstituted heteroaryl group; B 5 B 6 and B 7 Each is independently a bond, a substituted or unsubstituted alkylene group, or a substituted or unsubstituted heteroalkylene group; 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 [[ID=二十]]-, -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 Each is independently H, substituted or unsubstituted C1-C 30 Alkyl or substituted or unsubstituted 2- to 30-membered heteroalkyl groups; Each R a31 and R a32 Independently, it is H, substituted or unsubstituted C1-C 12 Alkyl or substituted or unsubstituted 2- to 12-membered heteroalkyl groups; Each R 801 R 802 R 901 R 902 R 110 and R 111 Independently, it is H, substituted or unsubstituted C1-C 12 Alkyl or substituted or unsubstituted 2- to 12-membered heteroalkyl; and Each s is an integer from 1 to 4.

78. The pharmaceutical composition of claim 77, wherein the cationic lipid is a lipid, and wherein the lipid: W 7 and W 8 Each is an alkylene group, either bonded, substituted, or unsubstituted; L a3 It is a key; R 10 and R 11 Each is independently H, substituted or unsubstituted alkyl group, or R. 10 and R 11 Together with the nitrogen atom to which it is attached, it forms substituted or unsubstituted heterocyclic alkyl groups; B 5 It is a key; B 6 and B 7 Each is an alkylene group, either bonded, substituted, or unsubstituted; L 8 It is a key; L 9 It is a bond, -O(C=O)-, -(C=O)O-, or -C(=O)-; L 10 It is a bond, -O(C=O)-, -(C=O)O-, or -C(=O)-; and R 7 R 8 and R 9 Each is independently H or substituted or unsubstituted C1-C 30 alkyl.

79. The cationic lipid according to claim 77, wherein W 7 and W 8 Each is independently a C1-C8 alkylene group, either bonded, substituted, or unsubstituted; L a3 It is a key; R 10 and R 11 Each is independently a substituted or unsubstituted alkyl group, or R 10 and R 11 Together with the nitrogen atom to which it is attached, it forms substituted or unsubstituted heterocyclic alkyl groups; B 5 It is a key; B 6 and B 7 Each is independently a C1-C8 alkylene group, either bonded, substituted, or unsubstituted; L 8 It is a key; L 9 It is -O(C=O)- or -(C=O)O-; L 10 It is -O(C=O)- or -(C=O)O-; and R 7 R 8 and R 9 Each is independently either substituted or unsubstituted C1-C 20 alkyl.

80. The pharmaceutical composition according to claim 77, wherein the cationic lipid is a lipid, and in the lipid: W 7 and W 8 Each is independently a C2-C4 alkylene group, either bonded, substituted, or unsubstituted; L a3 It is a key; R 10 and R 11 Each is independently either substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 Together with the nitrogen atom to which it is attached, it forms substituted or unsubstituted 3- to 8-membered heterocyclic alkyl groups; B 5 It is a key; B 6 and B 7 Each is independently a C2-C4 alkylene group, either bonded, substituted, or unsubstituted; L 8 It is a key; L 9 It is -O(C=O)- or -(C=O)O-; L 10 It is -O(C=O)- or -(C=O)O-; R 7 It is H or methyl; and R 8 and R 9 Each is independently either substituted or unsubstituted C1-C 20 alkyl.

81. The pharmaceutical composition according to claim 77, wherein the cationic lipid is a lipid, and in the lipid: W 7 and W 8 Each is independently a bonded or unsubstituted C2-C4 alkylene group; L a3 It is a key; R 10 and R 11 Each is independently either substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 Together with the nitrogen atom to which it is attached, it forms substituted or unsubstituted 5- to 6-membered heterocyclic alkyl groups; B 5 It is a key; B 6 and B 7 Each is independently a bonded or unsubstituted C2-C4 alkylene group; L 8 It is a key; L 9 It is -O(C=O)- or -(C=O)O-; L 10 It is -O(C=O)- or -(C=O)O-; R 7 It is H or methyl; and R 8 and R 9 Each is independently either substituted or unsubstituted C1-C 20 alkyl.

82. The pharmaceutical composition according to claim 77, wherein the cationic lipid is a lipid, and in the lipid: W 7 and W 8 Each is independently a bonded or unsubstituted C2-C4 alkylene group; L a3 It is a key; R 10 and R 11 Each is independently either substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 Together with the nitrogen atom to which it is attached, it forms substituted or unsubstituted 5- to 6-membered heterocyclic alkyl groups; B 5 B 6 and B 7 Each is an independent key; L 8 It is a key; L 9 It is a key; L 10 It is a key; R 7 It is H or methyl; and R 8 and R 9 Each is independently either substituted or unsubstituted C1-C 30 alkyl.

83. The pharmaceutical composition of claim 77, wherein the cationic lipid is a lipid, and wherein the cationic lipid is: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or ， Or its pharmaceutically acceptable salt.

84. The pharmaceutical composition according to claim 69 or claim 70, wherein the lipid comprises a cationic lipid of formula (III): (III), Or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug, wherein: L 11 yes , , , or ; Q is a substituted or unsubstituted alkylene, a substituted or unsubstituted heteroalkylene, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene. V is a substituted or unsubstituted alkylene, a substituted or unsubstituted cycloalkylene, or a substituted or unsubstituted arylene; B 8 B 9 B 10 and B 11 Each is independently a bond, a substituted or unsubstituted alkylene group, or a substituted or unsubstituted heteroalkylene group; 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 is 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 ; R 13 is 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 ; R 14 and R 15 Each is independently either substituted or unsubstituted C2-C 30 Alkyl or substituted or unsubstituted 2- to 30-membered heteroalkyl groups; R 12A R 12B R 13A and R 13B Each is independently H, substituted or unsubstituted C1-C 20 Alkyl or substituted or unsubstituted 2- to 20-membered heteroalkyl groups; Each R 210 R 211 R 310 and R 311 Independently, it is H, substituted or unsubstituted C1-C 12 Alkyl or substituted or unsubstituted 2- to 12-membered heteroalkyl groups; Each n is an independent integer from 0 to 8; and Each s is an integer from 1 to 4.

85. The pharmaceutical composition according to claim 84, wherein the cationic lipid is a lipid, and in the lipid: L 11 yes , or ; Q is a substituted or unsubstituted alkylene group; V is a substituted or unsubstituted alkylene; B 8 B 9 B 10 and B 11 Each is independently either a substituted or unsubstituted alkylene group; L 12 It is -O(C=O)- or -(C=O)O-; L 13 It is -O(C=O)- or -(C=O)O-; R 12 It is H, -OR 12A or -NR 12A ; R 13 It is H, -OR 13A or -NR 13A ; R 14 and R 15 Each is independently either substituted or unsubstituted C2-C 30 alkyl; R 12A and R 13A Each is independently H, substituted or unsubstituted C1-C 20 Alkyl; and Each n is an independent integer from 0 to 8.

86. The pharmaceutical composition according to claim 84, wherein the cationic lipid is a lipid, and in the lipid: L 11 yes or ; V is a substituted or unsubstituted alkylene; B 8 B 9 B 10 and B 11 Each is independently either substituted or unsubstituted C1-C 20 Alkylene; L 12 It is -O(C=O)- or -(C=O)O-; L 13 It is -O(C=O)- or -(C=O)O-; R 12 Is it H or -OR 12A ; R 13 Is it H or -OR 13A ; R 14 and R 15 Each is independently either substituted or unsubstituted C2-C 20 alkyl; R 12A and R 13A Each is independently H, or a substituted or unsubstituted C1-C8 alkyl group; and Each n is an independent integer from 0 to 4.

87. The pharmaceutical composition according to claim 84, wherein the cationic lipid is a lipid, and in the lipid: L 11 yes or ; V is an unsubstituted alkylene group; B 8 B 9 B 10 and B 11 Each is independently a substituted or unsubstituted C1-C8 alkylene group; L 12 It is -O(C=O)- or -(C=O)O-; L 13 It is -O(C=O)- or -(C=O)O-; R 12 It is -OH, methoxy, or ethoxy; R 13 It is -OH, methoxy, or ethoxy; R 14 and R 15 Each is independently either substituted or unsubstituted C2-C 20 Alkyl; and Each n is an independent integer from 0 to 4.

88. The pharmaceutical composition of claim 84, wherein the cationic lipid is a lipid, and wherein the cationic lipid is: or ， Or its pharmaceutically acceptable salt.

89. The pharmaceutical composition according to claim 69 or claim 70, wherein the lipid comprises a cationic lipid of formula (IV): (IV), Or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug, wherein: B 12 Yes - W 7 -L a3 -W 8 -; W 7 and W 8 Each is an independent bond, substituted or unsubstituted C1-C 12 Alkylene or substituted or unsubstituted 2- to 12-membered heteroalkylene; L a3 It is a bond, -SS-, –O-(CH2O) m -、 or ; W 9 and W 10 Each is an independent bond, substituted or unsubstituted C1-C 12 Alkylene, substituted or unsubstituted 2- to 12-membered heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heteroalkylene, 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 R 511 ) 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 Each independently is Fragments of cationic lipids of formula (I), Fragments of cationic lipids of formula (II), Fragments of cationic lipids of formula (II), Fragments of cationic lipids of formula (III) or Fragments of cationic lipids of formula (III); Each R 410 R 411 R 510 and R 511 Independently, it is H, substituted or unsubstituted C1-C 12 Alkyl or substituted or unsubstituted 2- to 12-membered heteroalkyl groups; Each m is an independent integer from 0 to 8; and Each s is an integer from 1 to 4.

90. The pharmaceutical composition of claim 89, wherein the cationic lipid is a lipid, and in the lipid: L a3 It is a key, -SS-, or ; W 7 and W 8 Each is independently a bond, either substituted or unsubstituted C1-C 12 Alkylene; L 14 is -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 is -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 Each is independently a bond, either substituted or unsubstituted C1-C 12 Alkylene; R 16 and R 17 Each independently is Fragments of cationic lipids of formula (II); and Each R 410 and R 510 Independently, it is H or substituted or unsubstituted C1-C. 12 alkyl.

91. The pharmaceutical composition according to claim 89, wherein the cationic lipid is a lipid, and in the lipid: L a3 It is a key, -SS-, or ; W 7 and W 8 Each is independently a bond or an unsubstituted C1-C 12 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 is independently a bond, either substituted or unsubstituted C1-C 12 Alkylene; R 16 and R 17 Each independently is Fragments of cationic lipids of formula (II); and Each R 410 and R 510 Independently, it is H or substituted or unsubstituted C1-C. 12 alkyl.

92. The pharmaceutical composition of claim 89, wherein the cationic lipid is a lipid, and in the lipid: L a3 It is a key, -SS-, or ; W 7 and W 8 Each is independently a bonded or unsubstituted C1-C8 alkylene group; 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 is independently a bonded or unsubstituted C1-C8 alkylene group; R 16 and R 17 Each independently is Fragments of cationic lipids of formula (II); and Each R 410 and R 510 It is independently H or an unsubstituted C1-C8 alkyl group.

93. The pharmaceutical composition of claim 89, wherein the cationic lipid is a lipid, and in the lipid: L a3 It is a key, -SS-, or ; W 7 and W 8 Each is independently a bonded or unsubstituted C1-C8 alkylene group; 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 is independently a bonded or unsubstituted C1-C8 alkylene group; R 16 and R 17 Each independently is or ;and Each R 410 and R 510 It is either H or methyl on its own.

94. The pharmaceutical composition according to claim 89, wherein the cationic lipid is: 、 、 、 、 、 、 or ， Or its pharmaceutically acceptable salt.

95. The pharmaceutical composition according to any one of claims 69 to 94, wherein the pharmaceutical composition comprises the following compound: 。 96. The pharmaceutical composition according to any one of claims 69 to 95, wherein the pharmaceutical composition further comprises lipid nanoparticles.

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

98. The pharmaceutical composition of claim 97, wherein the at least second therapeutic agent comprises an antitumor 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 therapeutic 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, an anti-Her2 antibody, 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. A polynucleotide according to any one of claims 1 to 68 or a pharmaceutical composition according to any one of claims 69 to 99, wherein the polynucleotide or the pharmaceutical composition is used for treating or preventing proliferative disorders in a subject.

101. A method for treating or preventing a proliferative disease or condition in a subject who has, is suspected of having, or is at risk of developing a proliferative disease or condition, the method comprising administering to the subject 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.

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

103. The method according to any one of claims 100 to 102, wherein the proliferative disease or condition includes cancer.

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

105. The method according to any one of claims 100 to 104, wherein the proliferative disease or condition is selected from the group consisting of: chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma / osteosarcoma of bone, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brainstem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, brain 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 carcinoma, 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 cancer, stomach Intestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), hepatocellular carcinoma, pancreatic cancer, islet cell carcinoma, rectal cancer, colorectal cancer, bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumors, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumors, 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... Vulvar cancer, Wilms' tumor, esophageal cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, 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), pleural pulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, AIDS-related cancers, Kaposi's sarcoma, epithelioid hemangioendothelioma (EHE), fibroblastic small round cell tumor and liposarcoma.

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

107. The method according to any one of claims 100 to 103 and 106, wherein the proliferative disease or condition comprises a malignant hematologic malignancy selected from the group consisting of: bone marrow tumors, leukemia, lymphoma, Hodgkin lymphoma, classical Hodgkin lymphoma, non-Hodgkin lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, hepatocellular T-cell lymphoma, B-cell lymphoma, reticuloendothelial proliferation, reticulocytosis, small glioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt lymphoma, mediastinal large B-cell lymphoma. Tumors, Waldenström macroglobulinemia, marginal zone B-cell lymphoma, marginal zone splenic lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, lymphomatoid granulomatosis, nodular lymphocytic-predominant Hodgkin lymphoma, plasma cell leukemia, acute polycythemia and erythroleukemia, acute erythrocytic myelopathy, acute erythrocytic leukemia, Heilmeyer-Schöner disease, acute megakaryocytic leukemia, mast cell leukemia, panmyelodysplasia, acute panmyelodysplasia with myelofibrosis, lymphosarcoma cell leukemia, acute leukemia of unspecified cell type, chronic myeloid leukemia in blast crisis. 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 leukemia, adult T-cell leukemia / lymphoma, aggressive NK-cell leukemia, B-cell prolymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, chronic Neutrophilic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, chronic idiopathic myelofibrosis, multiple myeloma, Calder's disease, myeloma, solitary myeloma, plasma cell leukemia, extramedullary plasmacytoma, malignant plasma cell tumor NOS, plasmacytoma NOS, monoclonal gammopathy, multiple myeloma, angiocentric immunoproliferative disorders, lymphoid granulomatosis, angioimmunoblastic lymphadenopathy, T-γ lymphoproliferative disorders, Waldenström macroglobulinemia, alpha heavy chain disease, gamma heavy chain disease, Franklin's disease, immunoproliferative small bowel disease, thalassemia, unspecified malignant immunoproliferative disorders, and immunoproliferative disorders NOS.

108. A method for treating or preventing an immunodeficiency disease or condition in a subject who has, is suspected of having, or is at risk of developing an immunodeficiency disease or condition, the method comprising administering to the subject 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.

109. The method of claim 108, wherein the immunodeficiency disease or condition is selected from the group consisting of: agammaglobulinemia: X-linked and autosomal recessive inheritance, ataxia-telangiectasia, chronic granulomatous diseases and other phagocytic disorders, common variable immunodeficiency, complement deficiency, DeGeorge syndrome, hemophagocytic lymphohistiocytosis (HLH), hyperIgE syndrome, hyperIgM syndrome, IgG subclass deficiency, congenital immunodeficiency, NEMO deficiency syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immunodeficiency, deficiency and combined Immunodeficiency, specific antibody deficiency, transient hypogammaglobulinemia in infancy, WHIM syndrome (warts, hypogammaglobulinemia, infection and ineffective production of chronic myelopenia), Wiscot-Aldrich syndrome, other antibody deficiency disorders, other primary cellular immunodeficiency, severe combined immunodeficiency (SCID), common variable immunodeficiency (CVID), human immunodeficiency virus / acquired immunodeficiency syndrome (HIV / AIDS), drug-induced immunodeficiency, graft-versus-host disease, primary immunodeficiency disease (PIDD), and lymphopenia.

110. A method for treating or preventing an autoimmune or inflammatory disease or condition in a subject who has, is suspected of having, or is at risk of developing an autoimmune or inflammatory disease or condition, the method comprising administering to the subject 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.

111. The method of claim 110, wherein the autoimmune or inflammatory disease or condition is selected from the group consisting of: inflammation, autoimmune diseases, paraneoplastic autoimmune diseases, chondritis, fibrotic diseases and / or bone degeneration, 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 Ritter syndrome, SEA syndrome (serologically negative, 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, Ritter syndrome, S... EA syndrome (serologically negative, enthesitis, 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 cholecystitis, sclerosing cholangitis, Sjögren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverted psoriasis, pustular psoriasis 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.

112. A method for treating or preventing an infectious disease or condition in a subject who has, is suspected of having, or is at risk of developing an infectious disease or condition, the method comprising administering to the subject 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.

113. The method according to claim 112, wherein the infectious disease or condition is selected from the group consisting of: Acinetobacter infection, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired Immunodeficiency Syndrome), amoebiasis, anaplasmosis, angiostrongylus infection, anisakiasis, anthrax, Cryptococcus hemolyticus infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, Bacteroidetes infection, giardiasis, Bartonella infection, Bely's ascariasis infection, BK virus infection, melanoma, blastomycosis, blastomycosis, Bolivian hemorrhagic fever, botulism (and infant botulism). Poisoning), Brazilian hemorrhagic fever, brucellosis, Black Death, Burkholderia infection, brucellosis ulcer, Calicut virus infection (norovirus and Sapporo virus), Curvularia, Candidiasis (albinocytic candidiasis; thrush), Capillary nematode infection, Carrion disease, Cat scratch disease, Cellulitis, Chagas disease (American trypanosomiasis), Chancroid, Chickenpox, Chikungunya, Chlamydia, Chlamydia pneumoniae infection (Taiwan Acute Respiratory Pathogen or TWAR), Cholera, Chromobacterium chromoblastitis, Chytriditis, Clonorchiasis, Clostridium difficile colitis, Coccidioidomycosis, Colorado tick fever (CTF), Common cold (acute viral nasopharyngitis; acute rhinitis), Coronavirus disease (COVID-19), Creutzfeldt-Jakob disease (CJD) CJD), Crimean-Congo hemorrhagic fever (CCHF), cryptococcosis, cryptosporidiosis, cutaneous larval migration (CLM), cyclosporidiosis, cysticercosis, cytomegalovirus infection, dengue fever, *Streptococcus* infection, binuclear amoebiasis, diphtheria, sparganosis, dracunculiasis, Ebola hemorrhagic fever, echinococcosis, ehrlichiosis, pinworm infection, enterococcal infection, enterovirus infection, epidemic typhus, erythema infectiosum (fifth disease), roseola infantum (sixth disease), fascioliasis, fascioliasis, fatal familial insomnia (FFI), filariasis, food poisoning caused by *Clostridium perfringens*, free-living amoeba infection, *Clostridium* infection, *Aeromonas* Gangrene (clostridium myonecrosis), endemic mildew, Gerstmann-Straussler-Schenck syndrome (GSS), Giardiasis, glanders, gnathostomiasis, gonorrhea, granuloma inguinale (Dunofan disease), Group A streptococcal infection, Group B streptococcal infection, Haemophilus influenzae infection, hand-foot-mouth disease (HFMD), Hantavirus pulmonary syndrome (HPS), Central protovirus 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 ewingii ehrlichiosis, human granulocytic anaplasmosis (HGA).Human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus (HPV) infection, human parainfluenza virus infection, hymenolepis taeniasis, Epstein-Barr virus infectious mononucleosis (Mono), influenza, isosporidiosis, Kawasaki disease, keratitis, Chlorella vulgaris infection, kuru, Lassa fever, Legionnaires' disease, Pontiac fever, leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease (Lyme borborygmus), lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever (MHF), measles, Middle East Respiratory Syndrome (MERS), melioidosis (Whitmore's disease), meningitis Inflammation, meningococcal infection, clonorchiasis, microsporidiosis, molluscum contagiosum (MC), monkeypox, mumps, murine typhus (endemic typhus), mycoplasma pneumonia, genital mycoplasma infection, mycopoietinosis, botulism, neonatal conjunctivitis (neonatal ophthalmitis), Nipah virus infection, norovirus (children and infants), (new) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiac infection, onchocerciasis (river blindness), clonorchiasis, paracoccidioidomycosis (South American blastomycosis), paragonimiasis, pastoral infection, head lice, body lice, pubic lice, pelvic inflammatory disease (PID), pertussis, plague, pneumococcal infection, Pneumocystis pneumonia ( PCP, pneumonia, poliomyelitis, Prevotella infection, primary amoebic meningoencephalitis (PAM), progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, relapsing fever, respiratory syncytial virus infection, rhinocystis infection, rhinovirus infection, rickettsia infection, rickettsial pox, Rift Valley fever (RVF), Rocky Mountain spotted fever (RMSF), rotavirus infection, rubella, salmonellosis, SARS (Severe Acute Respiratory Syndrome), scabies, scarlet fever, schistosomiasis, sepsis, Shigella infection (bacillary dysentery), herpes zoster, smallpox, sporotrichosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, subacute sclerosing panencephalitis, non-sexually transmitted syphilis, syphilis and yaws, tapeworm infection, tetanus. Tinea versicolor (lockjaw), tinea beard (beard ringworm), tinea capitis (scalp ringworm), tinea corporis (body ringworm), tinea cruris (jock itch), tinea manuum (hand ringworm), tinea pedis (athlete's foot), onychomycosis (nail fungus), tinea versicolor (pityriasis versicolor), toxocariasis (ocular larval migration (OLM)), toxocariasis (visceral larval migration (VLM)), toxoplasmosis, trachoma, trichinosis, trichomoniasis, whipworm infection, tuberculosis, tularemia, typhus, typhus, ureaplasma infection, valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Vibrio vulnificus infection, Vibrio parahaemolyticus enteritis, viral pneumonia, West Nile fever, trichomoniasis (white spot disease), pseudotuberculosis, yersiniasis, yellow fever, zeaspora.Zika fever and mycobacterial infection.

114. Use of 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 in the preparation of a medicament for treating or preventing proliferative diseases or conditions, autoimmune or inflammatory diseases or conditions or infectious diseases or conditions in a subject.

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

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

117. A method for amplifying CD4 + Helper cells, CD8 + A method for stimulating the expansion of effector initiators and memory cells, regulatory T (Treg) cells, natural killer (NK) cells, or natural killer T (NKT) cell populations, the method comprising contacting the cell population with 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 a duration sufficient to induce the formation of a complex with IL-2Rβγ, thereby stimulating the expansion of the T cells, NK cells, and / or NKT cell populations.

118. A method for amplifying CD4 + Helper cells, CD8 + A method for stimulating the expansion of the effector initiator and memory cells, Treg cells, natural killer (NK) cells, or natural killer T (NKT) cell populations, the method comprising contacting the cell populations with 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 a duration sufficient to induce the formation of a complex with IL-2Rβγ, thereby stimulating the expansion of the T cells, Treg cells, NK cells, and / or NKT cell populations while reducing cell death by 10% to 100%.

119. The method of claim 117 or claim 118, wherein, compared to CD3, a corresponding IL-2 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 but without the at least one substitution, is contacted. + CD4 in cell population + The expansion of Treg cells, the CD3-mediated CD3-based ... + CD4 in cell population + T Treg cell expansion is less than 20%, 15%, 10%, 5%, 1%, or less.

120. The method according to any one of claims 117 to 119, wherein the polynucleotide according to any one of claims 1 to 68 or the pharmaceutical composition according to any one of claims 69 to 99 does not significantly amplify CD4 in the cell population. + Treg cells.

121. The method according to any one of claims 117 to 120, wherein after incubation with the polynucleotide according to any one of claims 1 to 68 or the pharmaceutical composition according to any one of claims 69 to 99, the ratio of effector T cells to Treg cells in the cell population 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 higher.

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

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

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

125. 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 the preparation of CD4 for amplifying cell populations. + Helper cells, CD8 + Use in drugs for effector initiators and memory cells, Treg cells, natural killer (NK) cells, or natural killer T (NKT) cell populations.

126. 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 amplifying CD4 in a subject. + Helper cells, CD8 + Uses of effector initiators and memory cells, Treg cells, natural killer (NK) cells, 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 amino acid sequence of mouse serum albumin (MSA) 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 amino acid sequence of human serum albumin (HSA) shown in SEQ ID NO:

7.

129. 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 of mouse serum albumin (MSA) shown in SEQ ID NO: 6, said amino acid sequence being linked to the N-terminus of the amino acid sequence shown in SEQ ID NO:

3.

130. 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, said amino acid sequence being linked 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.

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 shown in the amino acid sequence of mouse serum albumin (MSA) shown in SEQ ID NO: 6, said amino acid sequence being linked to the N-terminus of the amino acid sequence shown in SEQ ID NO: 3 by 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, the amino acid sequence being linked by 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. 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: 7, said amino acid sequence being linked to the N-terminus of the amino acid sequence shown in SEQ ID NO:

3.

134. 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, said amino acid sequence being linked to the N-terminus of the amino acid sequence shown in SEQ ID NO:

7.

135. 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: 7, said amino acid sequence being linked to the N-terminus of the amino acid sequence shown in SEQ ID NO:

3.

136. 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, the amino acid sequence being linked to the N-terminus of the amino acid sequence shown in SEQ ID NO: 7 via a linker.

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

14.

141. The pharmaceutical composition according to any one of claims 69 to 99 and 139, wherein the pharmaceutical composition comprises: a) The nucleic acid sequences shown 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 compounds: 。 142. The pharmaceutical composition according to any one of claims 69 to 99, 139, and 141, wherein the pharmaceutical composition comprises: a) The nucleic acid sequences shown 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 compounds: ;as well as Lipid nanoparticles.

143. The pharmaceutical composition according to any one of claims 69 to 99 and 139 to 142, wherein the pharmaceutical composition comprises: a) The nucleic acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; b) The following compounds: ;as well as Lipid nanoparticles.

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

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

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