Cytokine conjugates for the treatment of autoimmune diseases

Abstract

Disclosed herein are interleukin (IL) conjugates (e.g., IL-2 conjugates) and their uses in the treatment of one or more indications. Additionally, described herein are pharmaceutical compositions and kits that include one or more of the interleukin conjugates (e.g., IL-2 conjugates). [Selected Figure] Figure 4A

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 540,781, filed Aug. 3, 2017, the entirety of which is incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on August 3, 2018, has the filename 46085-710_602_SL.txt and is 3,703 bytes in size. [Background technology]

[0003] Distinct populations of T cells regulate the immune system to maintain immune homeostasis and tolerance. For example, regulatory T (Treg) cells prevent inappropriate responses by the immune system by preventing pathological autoreactivity, while cytotoxic T cells target infected and / or cancer cells. In some instances, modulation of distinct populations of T cells provides options for the treatment of diseases or indications. Summary of the Invention

[0004] In certain embodiments, disclosed herein are cytokine conjugates and their use in treating one or more indications. Also described herein in some embodiments are interleukin 2 (IL-2) conjugates and their use in treating one or more indications. In some examples, the one or more indications include autoimmune disease. In some cases, described herein are IL-2 conjugates for modulating the interaction between IL-2 and IL-2 receptors to specifically stimulate or expand regulatory T cell (Treg cell) populations. In some cases, described herein are IL-2 conjugates with extended in vivo half-life, reduced toxicity, and / or expanded therapeutic window. In further cases, also described herein are pharmaceutical compositions and kits comprising one or more interleukin conjugates (e.g., IL-2 conjugates) described herein.

[0005] In certain embodiments, disclosed herein are isolated and modified interleukin-2 (IL-2) polypeptides, which contain at least one unnatural amino acid at a position that reduces receptor signaling capability for interleukin-2 receptor βγ (IL-2Rβγ) or reduces recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex, but maintains significant activation of the interleukin-2 αβγ receptor (IL-2Rαβγ), where the reduced receptor signaling capability is compared to the receptor signaling capability between a wild-type IL-2 polypeptide and IL-2Rβγ, and where the recruitment is compared to recruitment of the IL-2Rγ subunit by a wild-type IL-2 polypeptide. In some embodiments, at least one unnatural amino acid position is P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81 , P82, R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, where the amino acid residue numbers correspond to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, where the amino acid residue numbers correspond to SEQ ID NO:1.In some embodiments, the at least one unnatural amino acid position is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, and T113, where the amino acid residue numbers correspond to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from K8, K9, and H16, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from Q22, N26, N88, and Q126, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from E15, D20, D84, and E95, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from L12, L19, and M23, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from Q22 and N26, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid is: a lysine analog; a cysteine ​​analog or a histidine analog; comprises an aromatic side chain; comprises an azide group; comprises an alkyne group; or comprises an aldehyde or ketone group. In some embodiments, the at least one unnatural amino acid does not comprise an aromatic side chain.In some embodiments, the at least one unnatural amino acid is selected from the group consisting of N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, including isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propane, or selenocysteine. In some embodiments, at least one unnatural amino acid is incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase / tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase / tRNA pair comprises at least one unnatural nucleobase. In some embodiments, the modified IL-2 polypeptide is covalently attached to a conjugate moiety via the at least one unnatural amino acid. In some embodiments, the conjugate moiety comprises a water soluble polymer, a lipid, a protein, or a peptide.In some embodiments, the water soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof. In some embodiments, the water soluble polymer comprises a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water soluble polymer comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid comprises a fatty acid. In some embodiments, the fatty acid comprises about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein comprises albumin, transferrin, or transthyretin. In some embodiments, the protein comprises an antibody or a binding fragment thereof. In some embodiments, the antibody or a binding fragment thereof comprises an Fc portion of an antibody. In some embodiments, the peptide comprises an XTEN peptide, a homoamino acid polymer rich in glycine (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.In some embodiments, the conjugate moiety is indirectly linked to at least one non-natural amino acid of the modified IL-2 via a linker. In some embodiments, the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the isolated and modified IL-2 polypeptide has a reduced receptor signaling capability for IL-2Rβγ, the reduction in receptor signaling capability being about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more, compared to wild-type IL-2Rβγ. In some embodiments, the modified IL-2 polypeptide is: a functionally active fragment of a full-length IL-2 polypeptide; a recombinant IL-2 polypeptide; or a recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, the N-terminal deletion comprises the deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, the C-terminal deletion comprises the deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from the C-terminus, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, a functionally active fragment comprises an IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, a modified IL-2 polypeptide comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.In some embodiments, modified IL-2 polypeptides with reduced receptor signaling capability for IL-2Rβγ can expand CD4+ T regulatory (Treg) cells. In some embodiments, the conjugate moiety impairs or blocks the receptor signaling capability of IL-2 with IL-2Rβγ or reduces recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rγ complex. In some embodiments, CD4+ Treg cell expansion with modified IL-2 / IL-2Rαβγ complex is equal to or greater than 4+ Treg cell expansion with wild-type IL-2 polypeptide. In some embodiments, modified IL-2 / IL-2Rαβγ complexes expand CD4+ T to a population sufficient to modulate disease course in animal models. In some embodiments, the modified IL-2 polypeptide exhibits a first receptor signaling capability for IL-2Rβγ and a second receptor signaling capability for IL-2Rαβγ, where the first receptor signaling capability is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, 1000-fold or less than the second receptor signaling capability. In some embodiments, the first receptor signaling capability of the modified IL-2 polypeptide is less than the receptor signaling capability of the wild-type IL-2 polypeptide for IL-2Rβγ. In some embodiments, the second receptor signaling capability of the modified IL-2 polypeptide is less than the receptor signaling capability of the wild-type IL-2 polypeptide for IL-2Rαβγ. In some embodiments, the modified IL-2 polypeptide further results in increased recruitment of the IL-2Rα subunit to the IL-2 polypeptide resulting in activation of the interleukin 2αβγ receptor (IL-2Rαβγ), and the increased recruitment is compared to recruitment of the IL-2Rα subunit by a wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide further results in decreased recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex, and the decreased recruitment is compared to recruitment of the IL-2Rβ subunit and / or the IL-2Rγ subunit by a wild-type IL-2R polypeptide.

[0006] In certain embodiments, disclosed herein is an isolated and modified interleukin-2 (IL-2) polypeptide, which comprises at least one unnatural amino acid at a position that increases recruitment of the IL-2Rα subunit to the IL-2 polypeptide, which is associated with activation of the interleukin-2 αβγ receptor (IL-2Rαβγ), and the increased recruitment is compared to recruitment of the IL-2Rα subunit by a wild-type IL-2 polypeptide. In some embodiments, at least one unnatural amino acid position is P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81 , P82, R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, where the amino acid residue numbers correspond to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, where the amino acid residue numbers correspond to SEQ ID NO:1.In some embodiments, the at least one unnatural amino acid position is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, and T113, where the amino acid residue numbers correspond to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from K8, K9, and H16, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from Q22, N26, N88, and Q126, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from E15, D20, D84, and E95, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from L12, L19, and M23, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from Q22 and N26, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid is: a lysine analog; a cysteine ​​analog or a histidine analog; comprises an aromatic side chain; comprises an azide group; comprises an alkyne group; or comprises an aldehyde or ketone group. In some embodiments, the at least one unnatural amino acid does not comprise an aromatic side chain.In some embodiments, the at least one unnatural amino acid is selected from the group consisting of N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, including isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propane, or selenocysteine. In some embodiments, at least one unnatural amino acid is incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase / tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase / tRNA pair comprises at least one unnatural nucleobase. In some embodiments, the modified IL-2 polypeptide is covalently attached to a conjugate moiety via the at least one unnatural amino acid. In some embodiments, the conjugate moiety comprises a water soluble polymer, a lipid, a protein, or a peptide.In some embodiments, the water soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof. In some embodiments, the water soluble polymer comprises a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water soluble polymer comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid comprises a fatty acid. In some embodiments, the fatty acid comprises about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein comprises albumin, transferrin, or transthyretin. In some embodiments, the protein comprises an antibody or a binding fragment thereof. In some embodiments, the antibody or a binding fragment thereof comprises an Fc portion of an antibody. In some embodiments, the peptide comprises an XTEN peptide, a homoamino acid polymer rich in glycine (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.In some embodiments, the conjugate moiety is indirectly linked to at least one non-natural amino acid of the modified IL-2 via a linker. In some embodiments, the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the isolated and modified IL-2 polypeptide has a reduced receptor signaling capability for IL-2Rβγ, the reduction in receptor signaling capability being about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more, compared to wild-type IL-2Rβγ. In some embodiments, the modified IL-2 polypeptide is: a functionally active fragment of a full-length IL-2 polypeptide; a recombinant IL-2 polypeptide; or a recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, the N-terminal deletion comprises the deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, the C-terminal deletion comprises the deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from the C-terminus, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, a functionally active fragment comprises an IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, a modified IL-2 polypeptide comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.In some embodiments, modified IL-2 polypeptides with reduced receptor signaling capability for IL-2Rβγ can expand CD4+ T regulatory (Treg) cells. In some embodiments, the conjugate moiety impairs or blocks the receptor signaling capability of IL-2 with IL-2Rβγ or reduces recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rγ complex. In some embodiments, CD4+ Treg cell expansion with modified IL-2 / IL-2Rαβγ complex is equal to or greater than 4+ Treg cell expansion with wild-type IL-2 polypeptide. In some embodiments, modified IL-2 / IL-2Rαβγ complex causes expansion of CD4+ Treg cells to a population sufficient to modulate disease course in animal models. In some embodiments, modified IL-2 polypeptides exhibit a first receptor signaling capability for IL-2Rβγ and a second receptor signaling capability for IL-2Rαβγ, where the first receptor. The receptor signaling capability of the modified IL-2 polypeptide is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, 1000-fold or less than the second receptor signaling capability. In some embodiments, the first receptor signaling capability of the modified IL-2 polypeptide is less than the receptor signaling capability of a wild-type IL-2 polypeptide for IL-2Rβγ. In some embodiments, the second receptor signaling capability of the modified IL-2 polypeptide is less than the receptor signaling capability of a wild-type IL-2 polypeptide for IL-2Rαβγ. In some embodiments, the modified IL-2 polypeptide further results in increased recruitment of the IL-2Rα subunit to the IL-2 polypeptide, which is coupled to activation of the interleukin-2αβγ receptor (IL-2Rαβγ), and the increased recruitment is compared to recruitment of the IL-2Rα subunit by the wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide further results in reduced recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex, which reduced recruitment is compared to recruitment of the IL-2Rβ subunit and / or the IL-2Rγ subunit by a wild-type IL-2R polypeptide.

[0007] In certain embodiments, disclosed herein are isolated and modified interleukin-2 (IL-2) polypeptides, which contain at least one unnatural amino acid at a position that reduces binding between an IL-2R polypeptide and the interleukin-2 receptor βγ (IL-2Rβγ) or reduces recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex, but does not impair activation of the interleukin-2 αβγ receptor (IL-2Rαβγ), wherein the reduced binding is compared to binding between a wild-type IL-2 polypeptide and the IL-2Rβγ, and wherein the reduced recruitment is compared to recruitment of the IL-2Rγ subunit by a wild-type IL-2 polypeptide. In some embodiments, at least one unnatural amino acid position is P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81 , P82, R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, where the amino acid residue numbers correspond to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, where the amino acid residue numbers correspond to SEQ ID NO:1.In some embodiments, the at least one unnatural amino acid position is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, and T113, where the amino acid residue numbers correspond to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from K8, K9, and H16, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from Q22, N26, N88, and Q126, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from E15, D20, D84, and E95, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from L12, L19, and M23, and the amino acid residue numbering corresponds to SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from Q22 and N26, and the amino acid residue numbering corresponds to SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is Q22, and the amino acid residue numbering corresponds to SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is N26, and the amino acid residue numbering corresponds to SEQ ID NO: 1.In some embodiments, at least one unnatural amino acid: is a lysine analog; is a cysteine ​​analog or a histidine analog; contains an aromatic side chain; contains an azide group; or contains an aldehyde or ketone group. In some embodiments, at least one unnatural amino acid does not contain an aromatic side chain. In some embodiments, the at least one unnatural amino acid is selected from the group consisting of N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, including isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propane, or selenocysteine. In some embodiments, at least one unnatural amino acid is incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase / tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase / tRNA pair comprises at least one unnatural nucleobase.In some embodiments, the modified IL-2 polypeptide is covalently attached to the conjugate moiety via at least one unnatural amino acid. In some embodiments, the conjugate moiety comprises a water soluble polymer, lipid, protein, or peptide. In some embodiments, the water soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, the water soluble polymer comprises a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water soluble polymer comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid comprises a fatty acid. In some embodiments, the fatty acid comprises about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein comprises albumin, transferrin, or transthyretin. In some embodiments, the protein comprises an antibody or binding fragment thereof. In some embodiments, the antibody or binding fragment thereof comprises an Fc portion of an antibody.In some embodiments, the peptide comprises an XTEN peptide, a homo-amino acid polymer rich in glycine (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the conjugate moiety is indirectly linked to at least one unnatural amino acid of the modified IL-2 via a linker. In some embodiments, the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the reduction in binding is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more reduction in binding to IL-2Rβγ compared to a wild-type IL-2 polypeptide. In some embodiments, the reduction in binding is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more, to IL-2Rβγ as compared to a wild-type IL-2 polypeptide. In some embodiments, the reduction in IL-2Rγ subunit recruitment is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more reduction as compared to a wild-type IL-2 polypeptide. In some embodiments, the reduction in IL-2Rγ subunit recruitment is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more, compared to a wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide is: a functionally active fragment of a full-length IL-2 polypeptide; a recombinant IL-2 polypeptide; or a recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof.In some embodiments, N-terminal deletions include deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, C-terminal deletions include deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from the C-terminus, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, a functionally active fragment comprises the IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, the modified IL-2 polypeptide comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1. In some embodiments, the modified IL-2 polypeptide having reduced binding affinity to IL-2Rβγ can expand CD4+ T regulatory (Treg) cells. In some embodiments, the conjugate moiety impairs or blocks binding of IL-2 to IL-2Rβγ or reduces recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex. In some embodiments, the CD4+ Treg cell expansion by the modified IL-2 / IL-2Rαβγ complex is equal to or greater than the CD4+ Treg cell expansion by wild-type IL-2 polypeptide.

[0008] In one embodiment, an interleukin 2 (IL-2) conjugate is disclosed herein, the interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H7 9, R81, P82, R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, wherein the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, where the amino acid residue numbers correspond to SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, and T113, where the amino acid residue numbers correspond to SEQ ID NO:1.In some embodiments, the at least one unnatural amino acid position is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126, where the amino acid residue numbering corresponds to SEQ ID NO:1. In some embodiments, the amino acid position is selected from K8, K9, and H16. In some embodiments, the amino acid position is selected from Q22, N26, N88, and Q126. In some embodiments, the amino acid position is selected from E15, D20, D84, and E95. In some embodiments, the amino acid position is selected from L12, L19, M23, and F78. In some embodiments, the amino acid position is selected from Q22 and N26. In some embodiments, the amino acid position is Q22. In some embodiments, the amino acid position is N26. In some embodiments, an amino acid residue selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, F78, D84, N88, E95, and Q126 is further mutated to a lysine, a cysteine, or a histidine. In some embodiments, the amino acid residue is mutated to a cysteine. In some embodiments, the amino acid residue is mutated to a lysine. In some embodiments, an amino acid residue selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, F78, D84, N88, E95, and Q126 is further mutated to a non-natural amino acid.In some embodiments, the unnatural amino acid comprises p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, p-amino-L-phenylalanine, or isopropyl-L-phenylalanine. In some embodiments, the additional mutated amino acid residues are linked to additional conjugate moieties. In some embodiments, the IL-2 conjugate has a decreased affinity for the IL-2 receptor beta (IL-2Rβ) subunit, the IL-2 receptor gamma (IL-2Rγ) subunit, or a combination thereof, as compared to a wild-type IL-2 polypeptide. In some embodiments, the decreased affinity is about a 10%, 20%, 30%, 40%, 50%, or 60% decrease in binding affinity for IL-2Rβ, IL-2Rγ, or a combination thereof, as compared to a wild-type IL-2 polypeptide. In some embodiments, the binding affinity is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, as compared to a wild-type IL-2 polypeptide. In some embodiments, the conjugate moiety impairs or blocks binding of IL-2 to IL-2Rβ, IL-2Rγ, or a combination thereof. In some embodiments, the conjugate moiety downregulates the recruitment of IL-Rγ to the formed IL-2 / IL-2Rβ chain complex. In some embodiments, the conjugate moiety extends the systemic half-life of the polypeptide without affecting its affinity for the α, β, and γ chains of the IL-2 receptor. In some embodiments, the conjugate moiety comprises a water soluble polymer.In some embodiments, the additional conjugate moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water soluble polymers independently comprises a glycan. In some embodiments, each of the water soluble polymers independently comprises a polyamine. In some embodiments, the conjugate moiety comprises a protein. In some embodiments, the additional conjugate moiety comprises a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugate moiety comprises a polypeptide. In some embodiments, the additional conjugate moiety comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises an XTEN peptide, a homogeneous glycine-rich amino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugate moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugate moiety is indirectly attached to the isolated and purified IL-2 polypeptide by a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker is a homobifunctional linker such as Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDST ), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-(3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds such as 1,5-difluoro-2,4-dinitrobenzene, 1,3-difluoro-4,6-dinitrobenzene (DFDNB), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED) , formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker.In some embodiments, the heterobifunctional linker is N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate ( Sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl). )aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyloxy)succinimide esters (GMBs), N-(γ-maleimidobutyloxy)sulfosuccinimide esters (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-((iodoacetyl)amino)hexanoyl] amino)hexanoate (sIAXX), succinimidyl-4-((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6- ... carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NH-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidosalicylate ( ...4-azidosalicylate (sulfo-NH-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidosalicylate (NH-AsA), sulfosuccinimidyl-4-azidosalicylate (sulfo-NH-LC-AsA), sulfosuccinimidyl-4-azidosalicylamido Succinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)-1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyl diazopyruvate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, ρ-azidobenzoylhydrazide (ABH), 4-(ρ-azidosalicylamido)butylamine (AsBA), or ρ-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a maleimidomethyl group, optionally including maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), derivatives, or analogs thereof. In some embodiments, the conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate.

[0009] In certain embodiments, disclosed herein is an interleukin-2 (IL-2) conjugate, the interleukin-2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugate moiety; wherein the IL-2 conjugate has a reduced affinity for the IL-2 receptor beta (IL-2Rβ) subunit, the IL-2 receptor gamma (IL-2Rγ) subunit, or a combination thereof, as compared to a wild-type IL-2 polypeptide. In some embodiments, the conjugate moiety binds to an amino acid residue that interacts with IL-2Rβ or IL-2Rγ. In some embodiments, the conjugate moiety is attached to an amino acid residue selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, where the numbering of the amino acid residues corresponds to SEQ ID NO: 1. In some embodiments, the conjugate moiety comprises a water soluble polymer. In some embodiments, the additional conjugate moiety comprises a water soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide is dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin,Alternatively, it comprises hydroxyethyl starch (HES). In some embodiments, each of the water soluble polymers independently comprises a glycan. In some embodiments, each of the water soluble polymers independently comprises a polyamine. In some embodiments, the conjugation moiety comprises a protein. In some embodiments, the additional conjugation moiety comprises a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugation moiety comprises a polypeptide. In some embodiments, the additional conjugation moiety comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises an XTEN peptide, a homoamino acid polymer rich in glycine (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugation moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugate moiety is indirectly linked to the isolated and purified IL-2 polypeptide by a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker is selected from the group consisting of Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate pimelimidate (DMP), dimethyl suberimidate (DMS),Dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-(3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds such as 1,5-difluoro-2,4-dinitrobenzene and 1,3-difluoro-4,6-dinitrobenzene (DFDNB), 4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS), bis-[β-(4-azide]-1,2-difluorophenyl)propionami ...bis-[β-(4-azide]-1,2-difluorophenyl)propionamidate (DTBP), bis-[β-(4-azide]-1,2-difluorophenyl)propionamidate (DTBP), bis-[β-(4-azide]-1,2-difluorophenyl)propionamidate (DTBP), bis-[β-(4-azide]-1,2-difluorophenyl)propionamidate (DTBP), bis-[β-(4-azide]-1,2-difluorophenyl)propionamidate (DTBP), bis-[β-(4-azide]-1,2-difluorophenyl)propionamidate (DTBP), bis-[β-(4-azide]-1,2-difluorophenyl)propionamidate (DTBP), bis-[β Examples of linkers include N,N'-ethylene-bis(iodoacetamide)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker is N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), sulfo-succinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), sulfo-succinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sPDP ... PT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB),Succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyloxy)succinimide esters (GMBs), N-(γ-maleimidobutyloxy)sulfosuccinimide esters (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX ... Succinimidyl-4-((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H) , 3-(2-pyridyldithio)propionyl hydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NH-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB) , N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND),N-Succinimidyl-4(4-azidophenyl)-1,3'-dithiopropionate (sADP), N-Sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), Sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), Sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), Sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-azido-salicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azido-salicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(p-azido-salicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavage linker that optionally includes a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a maleimidomethyl group, optionally including maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), derivatives, or analogs thereof. In some embodiments, the conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugated moiety isThe serum half-life of the IL-2 conjugate can be extended.

[0010] In certain embodiments, a pharmaceutical composition is disclosed herein, the pharmaceutical composition comprising the IL-2 conjugate described above; and a pharma- ceutical acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0011] In some embodiments, disclosed herein are methods of treating an autoimmune disease or disorder in a subject, the methods comprising administering to the subject a therapeutically effective amount of the IL-2 conjugate described above. In some embodiments, the autoimmune disease or disorder comprises alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Wegener's granulomatosis. In some embodiments, the methods further comprise administering an additional therapeutic agent. In some embodiments, the IL-2 conjugate and the additional therapeutic agent are administered simultaneously. In some embodiments, the IL-2 conjugate and the additional therapeutic agent are administered sequentially. In some embodiments, the IL-2 conjugate is administered prior to the additional therapeutic agent. In some embodiments, the IL-2 conjugate is administered after administration of the additional therapeutic agent. In some embodiments, the subject is a human.

[0012] In an embodiment, disclosed herein is a method of expanding a regulatory T (Treg) cell population, the method comprising: (a) contacting cells with the IL-2 conjugate; and (b) allowing the IL-2 conjugate to interact with IL-2Rα, IL-2Rβ, and IL-2Rγ subunits to form an IL-2 / IL-2Rβγ complex; wherein the IL-2 conjugate has a reduced affinity for the IL-2Rβ and / or IL-2Rγ subunits, downregulates recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex, or retains similar potency as IL-2 at its α, β, and γ receptor subunits but extends its half-life, and wherein the IL-2 / IL-2Rαβγ complex stimulates expansion of Treg cells similarly or more potently than native IL-2.

[0013] In certain embodiments, disclosed herein are kits comprising the above-described IL-2 conjugates; or pharmaceutical compositions comprising the above-described IL-2 conjugates. Also described herein in some embodiments are kits comprising polynucleic acid molecules encoding the above-described IL-2 polypeptides. [Brief description of the drawings]

[0014] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings in which: [Figure 1] Representative unnatural amino acids are shown. This figure is modified from Figure 2 in Young et al., "Beyond the canonical 20 amino acids: expanding the genetic lexicon," J. of Biological Chemistry 285(15): 11039-11044 (2010). [Figure 2A] Exemplary unnatural amino acids are illustrated in Figure 2A, which illustrates an exemplary lysine derivative. [Figure 2B]Figure 2B illustrates exemplary unnatural amino acids. Figure 2B illustrates exemplary phenylalanine derivatives. [Figure 3A] Illustrated are exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins (Figure 3A - UAA numbers 1-42; Figure 3B - UAA numbers 43-89; Figure 3C - UAA numbers 90-128; Figure 3D - UAA numbers 129-167). Figures 3A-3D are taken from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 3B] Illustrated are exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins (Figure 3A - UAA numbers 1-42; Figure 3B - UAA numbers 43-89; Figure 3C - UAA numbers 90-128; Figure 3D - UAA numbers 129-167). Figures 3A-3D are taken from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 3C] Illustrated are exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins (Figure 3A - UAA numbers 1-42; Figure 3B - UAA numbers 43-89; Figure 3C - UAA numbers 90-128; Figure 3D - UAA numbers 129-167). Figures 3A-3D are taken from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 3D] Illustrated are exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins (Figure 3A - UAA numbers 1-42; Figure 3B - UAA numbers 43-89; Figure 3C - UAA numbers 90-128; Figure 3D - UAA numbers 129-167). Figures 3A-3D are taken from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 4A] Dose-response curves of exemplary IL-2 mutants on pSTAT5 signaling in human LRS primary cells (FIG. 4A) and the proliferative response in murine CTLL-2 populations (FIG. 4B) are shown. [Figure 4B] Dose-response curves of exemplary IL-2 mutants on pSTAT5 signaling in human LRS primary cells (FIG. 4A) and the proliferative response in murine CTLL-2 populations (FIG. 4B) are shown. [Diagram 5] 1 shows the enhanced PK profile of an exemplary IL-2 molecule, K35_30kD, at two different concentrations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Cytokines include a family of cell signaling proteins, such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and other growth factors that play multiple roles in innate and adaptive immune cell homeostasis. Cytokines are produced by immune cells, such as macrophages, B lymphocytes, T lymphocytes, and mast cells, endothelial cells, fibroblasts, and various stromal cells. In some instances, cytokines regulate the balance between humoral and cell-based immune responses.

[0016] Interleukins are signaling proteins that regulate the development and differentiation of T and B lymphocytes, cells of the monocytic lineage, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by helper CD4 T and B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue residents. In some cases, there are about 15 interleukins, interleukin 1-13, interleukin 15, and interleukin 17.

[0017] Interleukin 2 (IL-2) is a pleiotropic type 1 cytokine whose structure contains a four α-helical bundle of 15.5 kDa. 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 after antigenic stimulation and, to a lesser extent, by CD8+ cells, natural killer (NK) cells, and NKT (NKT) cells, activated dendritic cells (DCs), and mast cells. IL-2 signaling occurs through interaction with a specific combination of IL-2 receptor (IL-2R) subunits, IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122), and IL-2Rγ (also known as CD132). The interaction of IL-2 with IL-2Rα is approximately 10 -8 K of M d The interaction of IL-2 with IL-2Rβ and IL-2Rγ occurs at approximately 10 -9 K of M d The interaction of IL-2 with all three subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ, is approximately 10 -11 K is greater than M d The IL-2 receptor complex forms a "high affinity" IL-2 receptor complex.

[0018] In some instances, IL-2 signaling through the "high affinity" IL-2Rαβγ complex regulates the activation and proliferation of regulatory T cells. Regulatory T cells or CD4+CD25+Foxp3+regulatory T (Treg) cells mediate the maintenance of immune homeostasis by suppressing effector cells, e.g., CD8+ T cells, helper cells, e.g., CD4+ Th1, Th2, and Th17 cells, B cells, NK cells, and NKT cells. In some instances, Treg cells are generated from the thymus (tTreg cells) or induced from peripheral naive T cells (pTreg cells). In some instances, Treg cells are considered mediators of peripheral tolerance. Indeed, in one study, transfer of CD25-depleted peripheral CD4+ T cells induced a variety of autoimmune diseases in nude mice, whereas cotransfer of CD4+CD25+ T cells suppressed the development of autoimmunity (Sakaguchi, et al., “Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25),” J. Immunol. 155(3):1151-1164 (1995)). Enhancement of the Treg cell population downregulates effector T cell proliferation and suppresses autoimmunity and non-tumor responses of T cells.

[0019] In certain embodiments, disclosed herein are methods for selectively upregulating distinct populations of lymphocytes (e.g., regulatory T cells) via cytokine / cytokine receptor signaling. In some instances, the cytokine comprises an interleukin. In some instances, the cytokine is a cytokine conjugate, e.g., an interleukin conjugate, an interferon conjugate, a tumor necrosis factor conjugate. In further instances, described herein are pharmaceutical compositions and kits comprising one or more cytokine conjugates described herein.

[0020] In some embodiments, methods of selectively upregulating Treg populations via IL-2 / IL-2R signaling are also described herein. In some examples, IL-2 is an IL-2 conjugate that has a weakened interaction of IL-2Rβ and IL-2Rγ in the IL-2Rαβγ complex relative to wild-type IL-2, downregulates the recruitment of IL-2Rγ subunits to the IL-2 / IL-2Rβ complex, or retains similar potency as IL-2 at its α, β, and γ receptor subunits but extends its half-life. In some embodiments, methods of treating autoimmune diseases by use of the IL-2 conjugates described herein are described herein. In further embodiments, pharmaceutical compositions and kits comprising one or more IL-2 conjugates described herein are described herein.

[0021] Cytokine conjugates In some embodiments, cytokine conjugates are described herein. In some instances, the cytokine comprises an interleukin, a tumor necrosis factor, an interferon, a chemokine, or a lymphokine. In some instances, the cytokine is an interleukin. In some instances, the cytokine is an interferon. In further instances, the cytokine is a tumor necrosis factor.

[0022] In some embodiments, interleukin conjugates are described herein. Exemplary interleukins include, but are not limited to, interleukin 1β (IL-1β), interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 18 (IL-18), and interleukin 21 (IL-21). In some examples, interleukin conjugates are described herein, in which the interleukin is selected from IL-1β, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, and IL-21.

[0023] IL-2 conjugate In some embodiments, IL-2 conjugates modified at amino acid positions are described herein. In some examples, the IL-2 polypeptide is an isolated and purified IL-2 polypeptide. In some examples, the IL-2 polypeptide is a mammalian IL-2, e.g., a rodent IL-2 protein or a human IL-2 protein. In some cases, the IL-2 polypeptide is a human IL-2 protein. In some cases, the IL-2 polypeptide comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO:1. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO:1. In further cases, the IL-2 polypeptide comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2. In a further case, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 2. In a further case, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 2.

[0024] In some examples, the IL-2 polypeptide is a truncated variant. In some examples, the truncation is an N-terminal deletion. In other examples, the truncation is a C-terminal deletion. In further examples, the truncation includes both an N-terminal and a C-terminal deletion. For example, the truncation can be a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from either the N-terminus or the C-terminus, or from both the N-terminus and the C-terminus. In some cases, the IL-2 polypeptide includes an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues. In some cases, the IL-2 polypeptide includes an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. Optionally, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 2 residues. Optionally, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 3 residues. Optionally, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 4 residues. Optionally, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 5 residues. Optionally, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 6 residues. Optionally, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 7 residues. Optionally, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 8 residues. Optionally, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 9 residues. Optionally, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 10 residues.

[0025] In some embodiments, the IL-2 polypeptide is a functionally active fragment. In some embodiments, the functionally active fragment comprises IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, the functionally active fragment comprises IL-2 region 10-133, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, the functionally active fragment comprises IL-2 region 20-133, where the residue positions are relative to the positions in SEQ ID NO:1. In some embodiments, the functionally active fragment comprises IL-2 region 30-133, where the residue positions are relative to the positions in SEQ ID NO:1. In some cases, the functionally active fragment comprises IL-2 region 10-125, where the residue positions are relative to the positions in SEQ ID NO:1. In some cases, the functionally active fragment comprises IL-2 region 20-125, where the residue positions are relative to the positions in SEQ ID NO:1. In some cases, the functionally active fragment comprises IL-2 region 1-130, where the residue positions are relative to the positions in SEQ ID NO:1. In some cases, the functionally active fragment comprises IL-2 region 1-125, where the residue positions are relative to the positions in SEQ ID NO:1.

[0026] In some embodiments, the IL-2 conjugates described above, comprising an isolated and purified IL-2 polypeptide and a conjugate moiety, have a reduced affinity for the IL-2 receptor beta (IL-2Rβ) subunit, the IL-2 receptor gamma (IL-2Rγ) subunit, or a combination thereof, compared to a wild-type IL-2 polypeptide. In some embodiments, the IL-2 conjugates have reduced IL-2Rγ subunit recruitment to the IL-2 / IL-2Rβ complex, compared to a wild-type IL-2 polypeptide. In some embodiments, the conjugate moiety binds to an amino acid residue that interacts with IL-2Rγ (e.g., at the IL-2 / IL-2Rβ interface), IL-2Rβ (e.g., at the IL-2 / IL-2Rβ interface), or a combination thereof. In some embodiments, the conjugate moiety binds to an amino acid residue that is proximal to the IL-2 / IL-2Rβ interface, the IL-2 / IL-2Rβ interface, or the IL-2Rβγ interface. In some cases, the amino acid residue is about 5 Å, about 10 Å, or about 20 Å away from the IL-2 / IL-2Rβ interface, the IL-2 / IL-2Rβ interface, or the IL-2Rβγ interface. As used herein, residues of IL-2 included in the IL-2 / IL-2Rβ interface, the IL-2 / IL-2Rβ interface, or the IL-2Rβγ interface form hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from the IL-2Rβ subunit, the IL-2Rγ subunit, or with residues at the IL-2Rβγ interface.

[0027] In some examples, the conjugate moiety is located at amino acid positions P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, P82, R83, D84, D85, D86, D87, D88, D89, D90, D91, D92, D93, D94, D95, D96, D97, D98, D9 ...9, D91, D92, D93, D94, D95, D96, D97, D98, D99, D99, D99, D99, D9 and / or T133, the amino acid residues of which correspond to SEQ ID NO:1. In some embodiments, the amino acid positions are selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133. In some examples, the amino acid positions are selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, and T113. In some embodiments, the amino acid positions are selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126. In some examples, the amino acid positions are selected from K8, K9, and H16. In some examples, the amino acid positions are selected from Q22, N26, N88, and Q126.In some examples, the amino acid position is selected from E15, D20, D84, and E95. In some examples, the amino acid position is selected from L12, L19, and M23. In some examples, the amino acid position is selected from Q22 and N26. In some cases, the amino acid position is K8. In some cases, the amino acid position is K9. In some cases, the amino acid position is Q11. In some cases, the amino acid position is L12. In some cases, the amino acid position is E15. In some cases, the amino acid position is H16. In some cases, the amino acid position is L18. In some cases, the amino acid position is L19. In some cases, the amino acid position is D20. In some cases, the amino acid position is Q22. In some cases, the amino acid position is M23. In some cases, the amino acid position is N26. In some cases, the amino acid position is R81. In some cases, the amino acid position is D84. In some cases, the amino acid position is S87. In some cases, the amino acid position is N88. In some cases, the amino acid position is V91. In some cases, the amino acid position is I92. In some cases, the amino acid position is L94. In some cases, the amino acid position is E95. In some cases, the amino acid position is E116. In some cases, the amino acid position is N119. In some cases, the amino acid position is R120. In some cases, the amino acid position is T123. In some cases, the amino acid position is A125. In some cases, the amino acid position is Q126. In some cases, the amino acid position is S127. In some cases, the amino acid position is S130. In some cases, the amino acid position is T131. In some cases, the amino acid position is L132. In some cases, the amino acid position is T133.

[0028] In some instances, the IL-2 conjugate further comprises an additional mutation. In such cases, the amino acid is conjugated to an additional conjugation moiety for increased serum half-life, stability, or a combination thereof. Alternatively, the amino acid is first mutated to a natural amino acid, such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine; or to a non-natural amino acid, before being conjugated to the additional conjugation moiety.

[0029] In some embodiments, the IL-2 conjugate has a reduced binding affinity for the IL-2 receptor beta (IL-2Rβ) subunit, the IL-2 receptor gamma (IL-2Rγ) subunit, or a combination thereof, as compared to a wild-type IL-2 polypeptide. In some examples, the reduced affinity of the IL-2 conjugate for the IL-2 receptor beta (IL-2Rβ) subunit, the IL-2 receptor gamma (IL-2Rγ) subunit, or a combination thereof, as compared to a wild-type IL-2 polypeptide is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99%. In some cases, the reduced binding affinity is about 10%. In some cases, the reduced binding affinity is about 20%. In some cases, the reduced binding affinity is about 40%. In some cases, the reduced binding affinity is about 50%. In some cases, the reduced binding affinity is about 60%. In some cases, the reduced binding affinity is about 80%. In some cases, the reduced binding affinity is about 90%. In some cases, the reduced binding affinity is about 99%. In some cases, the reduced binding affinity is greater than about 99%. In some cases, the reduced binding affinity is about 80%. In some cases, the reduced binding affinity is about 100%.

[0030] In some embodiments, the decreased affinity of the IL-2 conjugate for the IL-2 receptor beta (IL-2Rβ) subunit, the IL-2 receptor gamma (IL-2Rγ) subunit, or a combination thereof, is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more, as compared to a wild-type IL-2 polypeptide. In some cases, the decreased binding affinity is about 1-fold. In some cases, the decreased binding affinity is about 2-fold. In some cases, the decreased binding affinity is about 4-fold. In some cases, the decreased binding affinity is about 5-fold. In some cases, the decreased binding affinity is about 6-fold. In some cases, the decreased binding affinity is about 8-fold. In some cases, the decreased binding affinity is about 10-fold. In some cases, the decreased binding affinity is about 30-fold. In some cases, the decreased binding affinity is about 50-fold. In some cases, the decreased binding affinity is about 100-fold. In some cases, the decreased binding affinity is about 300-fold. In some cases, the decreased binding affinity is about 500-fold. In some cases, the decreased binding affinity is about 1000-fold. In some cases, the decreased binding affinity is more than 1000-fold.

[0031] In some embodiments, the IL-2 conjugate has reduced IL-2Rγ subunit recruitment to the IL-2 / IL-2Rβ complex. Optionally, the reduced recruitment is compared to IL-2Rγ subunit recruitment by an equivalent IL-2 polypeptide without the non-natural amino acid (e.g., a wild-type IL-2 polypeptide). Optionally, the reduction in IL-2Rγ subunit recruitment is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% reduction compared to an equivalent IL-2 polypeptide without the non-natural amino acid modification. Optionally, the reduction in IL-2Rγ subunit recruitment is about 10%. Optionally, the reduction in IL-2Rγ subunit recruitment is about 20%. Optionally, the reduction in IL-2Rγ subunit recruitment is about 40%. Optionally, the reduction in IL-2Rγ subunit recruitment is about 50%. In some instances, the reduction in IL-2Rγ subunit mobilization is about 60%. In some instances, the reduction in IL-2Rγ subunit mobilization is about 70%. In some instances, the reduction in IL-2Rγ subunit mobilization is about 80%. In some instances, the reduction in IL-2Rγ subunit mobilization is about 90%. In some instances, the reduction in IL-2Rγ subunit mobilization is about 99%. In some instances, the reduction in IL-2Rγ subunit mobilization is greater than 99%. In some instances, the reduction in IL-2Rγ subunit mobilization is about 100%. In some instances, the IL-2 conjugate further has increased IL-2Rα subunit mobilization.

[0032] In some embodiments, the reduction in IL-2Rγ subunit recruitment is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more, compared to a comparable IL-2 polypeptide without the non-natural amino acid modification (a wild-type IL-2 polypeptide). In some embodiments, the reduction in IL-2Rγ subunit recruitment is about 1-fold. In some embodiments, the reduction in IL-2Rγ subunit recruitment is about 2-fold. In some embodiments, the reduction in IL-2Rγ subunit recruitment is about 4-fold. In some embodiments, the reduction in IL-2Rγ subunit recruitment is about 5-fold. In some embodiments, the reduction in IL-2Rγ subunit recruitment is about 6-fold. In some embodiments, the reduction in IL-2Rγ subunit recruitment is about 8-fold. In some embodiments, the reduction in IL-2Rγ subunit recruitment is about 10-fold. In some instances, the reduction in IL-2Rγ subunit recruitment is about 30-fold. In some instances, the reduction in IL-2Rγ subunit recruitment is about 50-fold. In some instances, the reduction in IL-2Rγ subunit recruitment is about 100-fold. In some instances, the reduction in IL-2Rγ subunit recruitment is about 300-fold. In some instances, the reduction in IL-2Rγ subunit recruitment is about 500-fold. In some instances, the reduction in IL-2Rγ subunit recruitment is about 1000-fold. In some instances, the reduction in IL-2Rγ subunit recruitment is greater than 1000-fold. In some instances, the IL-2 conjugate further has an increase in IL-2Rα subunit recruitment.

[0033] In some embodiments, the IL-2 conjugate has increased IL-2R α subunit recruitment to the IL-2 polypeptide. Optionally, the decreased recruitment is compared to IL-2R α subunit recruitment by an equivalent IL-2 polypeptide without the non-natural amino acid (e.g., a wild-type IL-2 polypeptide). Optionally, the increase in IL-2R α subunit recruitment is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% increase compared to an equivalent IL-2 polypeptide without the non-natural amino acid modification. Optionally, the increase in IL-2R α subunit recruitment is about 10%. Optionally, the increase in IL-2R α subunit recruitment is about 20%. Optionally, the increase in IL-2R α subunit recruitment is about 40%. Optionally, the increase in IL-2R α subunit recruitment is about 50%. Optionally, the increase in IL-2R α subunit recruitment is about 60%. In some instances, the increase in IL-2R α subunit recruitment is about 70%. In some instances, the increase in IL-2R α subunit recruitment is about 80%. In some instances, the increase in IL-2R α subunit recruitment is about 90%. In some instances, the increase in IL-2R α subunit recruitment is about 99%. In some instances, the increase in IL-2R α subunit recruitment is greater than 99%. In some instances, the increase in IL-2R α subunit recruitment is about 100%. In some instances, the IL-2 conjugate further reduces recruitment of IL-2R β subunit and / or IL-2R α subunit.

[0034] In some embodiments, the increase in IL-2R α subunit recruitment is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more, compared to a comparable IL-2 polypeptide without the non-natural amino acid modification (a wild-type IL-2 polypeptide). In some embodiments, the increase in IL-2R α subunit recruitment is about 1-fold. In some embodiments, the increase in IL-2R α subunit recruitment is about 2-fold. In some embodiments, the increase in IL-2R α subunit recruitment is about 4-fold. In some embodiments, the increase in IL-2R α subunit recruitment is about 5-fold. In some embodiments, the increase in IL-2R α subunit recruitment is about 6-fold. In some embodiments, the increase in IL-2R α subunit recruitment is about 8-fold. In some embodiments, the increase in IL-2R α subunit recruitment is about 10-fold. In some instances, the increase in IL-2Rα subunit recruitment is about 30-fold. In some instances, the increase in IL-2Rα subunit recruitment is about 50-fold. In some instances, the increase in IL-2Rα subunit recruitment is about 100-fold. In some instances, the increase in IL-2Rα subunit recruitment is about 300-fold. In some instances, the increase in IL-2Rα subunit recruitment is about 500-fold. In some instances, the increase in IL-2Rα subunit recruitment is about 1000-fold. In some instances, the increase in IL-2Rα subunit recruitment is greater than 1000-fold. In some instances, the IL-2 conjugate further reduces recruitment of IL-2Rβ subunit and / or IL-2Rα subunit.

[0035] In some embodiments, the IL-2 polypeptides described herein have a decreased receptor signaling capability for IL-2Rβγ. In some instances, the decrease in receptor signaling capability is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more, for IL-2Rβγ as compared to a wild-type IL-2 polypeptide. In some instances, the decrease in receptor signaling capability is about 2-fold. In some instances, the decrease in receptor signaling capability is about 5-fold. In some instances, the decrease in receptor signaling capability is about 10-fold. In some instances, the decrease in receptor signaling capability is about 20-fold. In some instances, the decrease in receptor signaling capability is about 30-fold. In some instances, the decrease in receptor signaling capability is about 40-fold. In some instances, the decrease in receptor signaling capability is about 50-fold. In some instances, the decrease in receptor signaling capability is about 100-fold. In some instances, the decrease in receptor signaling capability is about 200-fold. In some cases, the decrease in receptor signaling capacity is about 300-fold. In some cases, the decrease in receptor signaling capacity is about 400-fold. In some cases, the decrease in receptor signaling capacity is about 500-fold. In some cases, the decrease in receptor signaling capacity is about 1000-fold.

[0036] In some examples, the receptor signaling capacity is measured by an EC50 value. In some cases, the decrease in receptor signaling capacity is an increase in the EC50 value. In some examples, the increase in the EC50 value is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more, compared to a wild-type IL-2 polypeptide.

[0037] In some instances, the receptor signaling capacity is measured by an ED50 value. In some instances, the decrease in receptor signaling capacity is an increase in the ED50 value. In some instances, the increase in the ED50 value is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more, compared to the wild-type IL-2 polypeptide.

[0038] In some embodiments, the IL-2 polypeptides described herein have an expanded therapeutic window compared to the therapeutic window of a wild-type IL-2 polypeptide. In some examples, the expanded therapeutic window is due to decreased binding between the IL-2 polypeptide and the interleukin-2 receptor βγ (IL-2Rβγ), decreased receptor signaling capacity for IL-2Rβγ, decreased recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex, or increased recruitment of the IL-2Rα subunit to the IL-2 polypeptide. In some examples, the IL-2 polypeptide is not impaired in activating the interleukin-2αβγ receptor (IL-2Rαβγ).

[0039] In some embodiments, the modified IL-2 polypeptide exhibits a first receptor signaling capability for the IL-2βγ signaling complex and a second receptor signaling capability for the IL-2αβγ signaling complex, where the difference between the first receptor signaling capability and the second receptor signaling capability is at least 1-fold. In some examples, the difference is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or even 1000-fold or more. In some examples, the first receptor signaling capability is less than the second receptor signaling capability. In some examples, the first receptor signaling capability is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or less than the second receptor signaling capability. In some cases, the modified IL-2 polypeptide has a receptor signaling capability for the IL-2βγ signaling complex that is less than the second receptor signaling capability for the IL-2αβγ signaling complex. In some cases, the first receptor signaling capability of the modified IL-2 polypeptide is at least 1-fold less than the receptor signaling capability of the wild-type IL-2 polypeptide. In some cases, the first receptor signaling capability of the modified IL-2 polypeptide is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, or less than 500-fold less than the receptor signaling capability of the wild-type IL-2 polypeptide. In some cases, the first receptor signaling capacity and the second receptor signaling capacity are both less than the respective capacities of a wild-type IL-2 polypeptide, but the first receptor signaling capacity is less than the second receptor signaling capacity. In some cases, the difference between the first receptor signaling capacity and the second receptor signaling capacity increases the therapeutic window of the modified IL-2 polypeptide.

[0040] In some embodiments, the conjugate moiety is attached directly or indirectly via a linker peptide to the N-terminus or C-terminus of the IL-2 polypeptide. In some cases, the conjugate moiety (e.g., a polymer, protein, or peptide) is genetically fused to IL-2 at the N-terminus or C-terminus of IL-2, either directly or indirectly via a linker peptide. In some examples, the conjugate moiety is attached to an amino acid residue at the N-terminus or C-terminus. In some examples, the conjugate moiety is attached to a reactive group that is attached to an amino acid residue at the N-terminus or C-terminus.

[0041] In some embodiments, the IL-2 conjugate comprises a plasma half-life of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some embodiments, the IL-2 conjugate comprises a plasma half-life of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or more. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 1 hour. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 2 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 3 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 4 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 5 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 6 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 7 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 8 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 9 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 10 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 12 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 18 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of greater than 24 hours.

[0042] In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, or more. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 1 hour. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 2 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 3 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 4 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 5 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 6 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 7 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 8 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 9 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 10 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 12 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 18 hours. In some embodiments, the IL-2 conjugate comprises a plasma half-life of at least 24 hours.

[0043] In some embodiments, the IL-2 conjugate comprises a plasma half-life of about 1 hour to about 7 days, about 12 hours to about 7 days, about 18 hours to about 7 days, about 24 hours to about 7 days, about 1 hour to about 5 days, about 12 hours to about 5 days, about 24 hours to about 5 days, about 2 days to about 5 days, or about 2 days to about 3 days.

[0044] In some embodiments, the IL-2 conjugate comprises a plasma half-life of about 1 hour to about 18 hours, about 1 hour to about 12 hours, about 2 hours to about 10 hours, about 2 hours to about 8 hours, about 4 hours to about 18 hours, about 4 hours to about 12 hours, about 4 hours to about 10 hours, about 4 hours to about 8 hours, about 6 hours to about 18 hours, about 6 hours to about 12 hours, about 6 hours to about 10 hours, about 6 hours to about 8 hours, about 8 hours to about 18 hours, about 8 hours to about 12 hours, or about 8 hours to about 10 hours.

[0045] In some embodiments, the IL-2 conjugate comprises a plasma half-life that allows Treg cells to proliferate and / or expand, but does not exert deleterious effects such as apoptosis.

[0046] In some embodiments, the IL-2 conjugate comprises an increased plasma half-life, e.g., of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more, compared to wild-type IL-2.

[0047] In some embodiments, the IL-2 conjugate comprises an extended plasma half-life, e.g., from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 4 hours to about 18 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 18 hours, from about 6 hours to about 12 hours, from about 6 hours to about 10 hours, from about 6 hours to about 8 hours, from about 8 hours to about 18 hours, from about 8 hours to about 12 hours, or from about 8 hours to about 10 hours, compared to wild-type IL-2.

[0048] In some embodiments, the IL-2 conjugate comprises an extended plasma half-life with reduced toxicity. In some examples, the IL-2 conjugate comprises an extended plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more, with reduced toxicity. In some examples, the IL-2 conjugate comprises an extended plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, or more, with reduced toxicity. In some examples, the IL-2 conjugate comprises an extended plasma half-life of about 1 to about 18 hours, about 1 to about 12 hours, about 2 to about 10 hours, about 2 to about 8 hours, about 4 to about 18 hours, about 4 to about 12 hours, about 4 to about 10 hours, about 4 to about 8 hours, about 6 to about 18 hours, about 6 to about 12 hours, about 6 to about 10 hours, about 6 to about 8 hours, about 8 to about 18 hours, about 8 to about 12 hours, or about 8 to about 10 hours, with reduced toxicity. In some cases, the reduced toxicity is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or more, compared to wild-type IL-2. In some cases, the reduced toxicity is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more reduced compared to wild-type IL-2.

[0049] In some embodiments, the IL-2 conjugates include conjugate moieties where the size (e.g., volume or length) of the conjugate moiety improves plasma stability but does not decrease potency. In some examples, the size of the conjugate moiety increases plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some examples, the size of the conjugate moiety increases plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, or more. In some examples, the size of the conjugate moiety increases the plasma half-life by about 1 hour to about 18 hours, about 1 hour to about 12 hours, about 2 hours to about 10 hours, about 2 hours to about 8 hours, about 4 hours to about 18 hours, about 4 hours to about 12 hours, about 4 hours to about 10 hours, about 4 hours to about 8 hours, about 6 hours to about 18 hours, about 6 hours to about 12 hours, about 6 hours to about 10 hours, about 6 hours to about 8 hours, about 8 hours to about 18 hours, about 8 hours to about 12 hours, or about 8 hours to about 10 hours. In some examples, the size of the conjugate moiety reduces potency by 5%, 4%, 3%, 2%, 1%, or less compared to wild-type IL-2.

[0050] In some embodiments, the IL-2 conjugate comprises a conjugate moiety where the size (e.g., volume or length) of the conjugate moiety improves plasma stability and potency. In some examples, the size of the conjugate moiety increases plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some examples, the size of the conjugate moiety increases plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, or more. In some examples, the size of the conjugate moiety increases the plasma half-life by about 1 hour to about 18 hours, about 1 hour to about 12 hours, about 2 hours to about 10 hours, about 2 hours to about 8 hours, about 4 hours to about 18 hours, about 4 hours to about 12 hours, about 4 hours to about 10 hours, about 4 hours to about 8 hours, about 6 hours to about 18 hours, about 6 hours to about 12 hours, about 6 hours to about 10 hours, about 6 hours to about 8 hours, about 8 hours to about 18 hours, about 8 hours to about 12 hours, or about 8 hours to about 10 hours. In some examples, the size of the conjugate moiety further increases potency by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more compared to wild-type IL-2.

[0051] In some instances, the conjugate moiety impairs or interferes with the receptor signaling ability of IL-2Rβγ and IL-2 or reduces recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex.

[0052] In some instances, modified IL-2 polypeptides with reduced receptor signaling capacity for IL-2Rβγ can expand CD4+ T regulatory (Treg) cells.

[0053] In some embodiments, the proliferation of CD4+ Treg cells by the modified IL-2 / IL-2Rαβγ complex is equivalent to or greater than that of the wild-type IL-2 polypeptide.

[0054] In some embodiments, the IL-2 / IL-2Rαβγ complex induces the expansion of CD4+ Treg cells into a population sufficient to modulate the course of disease in animal models.

[0055] In some embodiments, described herein are interleukin-2 αβγ receptor (IL-2Rαβγ) binding proteins, wherein the receptor signaling capability of the binding protein for the interleukin-2 βγ receptor (IL-2Rβγ) is less than that of wild-type human IL-2 (hIL-2), and the binding protein comprises at least one unnatural amino acid. In some cases, the binding protein is a modified IL-2 polypeptide or a functionally active fragment thereof, wherein the modified IL-2 polypeptide comprises at least one unnatural amino acid.

[0056] In some embodiments, described herein are interleukin-2 alpha beta gamma receptor (IL-2Ralpha beta gamma) binding proteins, wherein recruitment of the IL-2Rgamma subunit to the IL-2 / IL-2Rbeta complex by the binding protein is less than that of wild-type human IL-2 (hIL-2), and wherein the binding protein comprises at least one unnatural amino acid. Optionally, the binding protein is a modified IL-2 polypeptide or a functionally active fragment thereof, wherein the modified IL-2 polypeptide comprises at least one unnatural amino acid.

[0057] In some embodiments, described herein are interleukin-2 αβγ receptor (IL-2Rαβγ) binding proteins, wherein the binding affinity of the binding protein for the interleukin-2 βγ receptor (IL-2Rβγ) is less than that of wild-type human IL-2 (hIL-2), and the binding protein comprises at least one unnatural amino acid. In such cases, the binding protein is a modified IL-2 polypeptide or a functionally active fragment thereof, wherein the modified IL-2 polypeptide comprises at least one unnatural amino acid.

[0058] In some embodiments, described herein is an IL-2 / IL-2Rαβγ complex comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, where the modified IL-2 polypeptide has a reduced receptor signaling capability for IL-2Rβγ, and the reduced receptor signaling capability is compared to the binding affinity between a wild-type IL-2 polypeptide and IL-2Rβγ. Optionally, the modified IL-2 polypeptide further comprises a conjugation moiety covalently attached to the non-natural amino acid.

[0059] In some embodiments, described herein is an IL-2 / IL-2Rαβγ complex comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex by said modified IL-2 polypeptide is less than that of a wild-type IL-2 polypeptide. Optionally, the modified IL-2 polypeptide further comprises a conjugation moiety covalently attached to the non-natural amino acid.

[0060] In some embodiments, described herein is an IL-2 / IL-2Rαβγ complex comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein the modified IL-2 polypeptide has a reduced binding affinity to IL-2Rβγ, and the reduced binding affinity is compared to the binding affinity between a wild-type IL-2 polypeptide and IL-2Rβγ. In some embodiments, described herein is an IL-2 / IL-2Rαβγ complex comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex by the modified IL-2 polypeptide is less than that of a wild-type IL-2 polypeptide. In some examples, the modified IL-2 polypeptide further comprises a conjugation moiety covalently attached to the non-natural amino acid.

[0061] In some embodiments, described herein is a CD4+ Treg cell activator that selectively expands CD4+ Treg cells of a cell population, wherein the activator comprises a modified IL-2 polypeptide comprising at least one unnatural amino acid. In some examples, when the activator contacts the CD3+ cell population, the activator expands CD8+ effector T cells and / or natural killer cells in the CD3+ cell population by less than 20%, 15%, 10%, 5%, 1%, or 0.1% compared to the expansion of CD8+ effector T cells and / or natural killer cells in the CD3+ cell population contacted by a wild-type IL-2 polypeptide. In some examples, the cell population is an in vivo cell population. In some examples, the cell population is an in vitro cell population. In some examples, the cell population is an ex vivo cell population.

[0062] IL-10 conjugate In some embodiments, IL-10 conjugates modified at amino acid positions are described herein. IL-10 (also known as human cytokine synthesis suppressor factor or CSIF) is an immunomodulatory cytokine that limits inflammatory responses to pathogens, but it has also been identified as an antitumor cytokine due to its immunostimulatory effects on CD8 T cells. IL-10 downregulates the expression of Th1 cytokines, MHC class II antigens, and costimulatory molecules on macrophages, further enhancing B cell survival, proliferation, and antibody production. In addition, IL-10 can stimulate thymocyte proliferation and CD8 T cell cytotoxicity. In some examples, the IL-10 conjugate comprises an isolated and purified IL-10 polypeptide and a conjugate moiety. In some examples, the IL-10 conjugate has a reduced affinity for the IL-10 receptor compared to the wild-type IL-10 polypeptide. In some cases, the conjugate moiety binds to an amino acid residue that interacts with the IL-10 receptor (e.g., at the IL-10 / IL-10Rα interface). In some cases, the conjugate moiety binds to an amino acid residue proximal to the IL-2 / IL-2Rα interface (e.g., about 5 Å, about 10 Å, about 15 Å, or about 20 Å away from the IL-2 / IL-2Rα interface). As used herein, residues included in the IL-10 / IL-10Rα interface include IL-10 residues that form hydrophobic, hydrogen, or ionic interactions with residues from IL-10. In some cases, the conjugate moiety is attached to the N-terminus or C-terminus of the cytokine, either directly or indirectly via a linker peptide. In further cases, the conjugate moiety modulates the interaction between IL-10 and IL-10R to enhance its immunosuppressive activity and reduce its immunostimulatory activity. In some examples, the IL-10 conjugate upregulates a distinct population of lymphocytes via IL-10 / IL-10R signaling. In some examples, the IL-10 conjugate modulates immune activity.

[0063] Additional cytokine conjugates In some embodiments, one or more additional cytokine conjugates modified at an amino acid position are described herein. Exemplary cytokines include, but are not limited to, IL-1β, IL-7, IL-12, IL-15, IL-18, and IL-21. In some examples, the cytokine conjugate comprises an isolated and purified cytokine polypeptide and a conjugate moiety. In some examples, the cytokine conjugate has a reduced affinity for its respective receptor compared to its wild-type cytokine. In some cases, the conjugate moiety is attached to an amino acid residue proximal to the receptor interface (e.g., about 5 Å, about 10 Å, about 15 Å, or about 20 Å away from the receptor interface). In some cases, the conjugate moiety is attached directly or indirectly via a linker peptide to the N-terminus or C-terminus of the cytokine peptide. In further cases, the conjugate moiety modulates the interaction between the cytokine and its receptor to enhance its immunosuppressive activity and reduce its immunostimulatory effect. In some examples, the cytokine conjugate upregulates a distinct population of lymphocytes via its receptor signaling. In some examples, the cytokine conjugate modulates immune activity.

[0064] Cytokine Conjugate Precursors Described herein are cytokine conjugate precursors that include mutant cytokines (such as IL-2) in which one or more amino acids are mutated from the wild-type amino acid. Such precursors are often used in conjunction with the methods described herein for the treatment of a disease or condition. In some embodiments, the cytokine precursors are not conjugated. Such mutations include various additions, deletions, or substitutions. In some embodiments, the mutations include substitutions of different naturally occurring amino acids. In some examples, the mutant cytokine is a nucleotide sequence that is located at amino acid positions P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, P82, R83, R84, R85, R86, R87, R88, R89, R90, R91, R92, R93, R94, R95, R96, R97, R98, R99, R99, R100, R101, R102, R103, R104, R105, R106, R107, R108, R109, R110, R120, R121, R122, R123, R124, R125, R126, R127, R128, R130, R131, R132, R133, R134, R135, R136, R137, R138, R140, R142, R143, R144, R145, R146, R147, R148, R149, R150, R151, R152, R153, R154, R155, R156, R157, R158, R160, R162, R16 83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, where the numbering of the amino acid residues corresponds to SEQ ID NO:1. In some examples, the amino acid positions are selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, L132, and T133.In some examples, the amino acid positions are selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, and T113. In some examples, the amino acid position is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126. In some examples, the amino acid position is selected from K8, K9, and H16. In some examples, the amino acid position is selected from Q22, N26, N88, and Q126. In some examples, the amino acid position is selected from E15, D20, D84, and E95. In some examples, the amino acid position is selected from L12, L19, and M23. In some examples, the amino acid position is selected from Q22 and N26. In some embodiments, the cytokine mutant comprises a conjugation moiety, wherein the conjugation moiety is attached to a mutation site in the mutant cytokine.

[0065] Protein or peptide fusions In some embodiments, the cytokine conjugates described herein comprise a cytokine (e.g., IL-2, or other cytokine) fused to a peptide or protein (fusion). In some embodiments, the peptide or protein is an antibody or antibody fragment. In some embodiments, the cytokine conjugates described herein comprise a cytokine (e.g., IL-2, or other cytokine) fused to an antibody, or a binding fragment thereof. In some embodiments, the cytokines described herein are fused to multiple proteins or peptides. In some embodiments, the cytokine conjugates comprise a cytokine fusion to a protein or peptide and at least a conjugate moiety. In some examples, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a humabody, a disulfide-stable Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof. In some examples, such fusion proteins are produced directly via translation. In some embodiments, the fusion is produced using chemical or other enzymatic ligation methods. In some embodiments, the cytokine conjugate comprises a fusion peptide or protein joined by a linker. In some embodiments, the linker is a hepeptide. In some embodiments, the cytokine conjugate comprises a fusion of an N-terminal peptide or protein. In some embodiments, the cytokine conjugate comprises a fusion of a C-terminal peptide or protein. Optionally, the cytokine fused to the peptide or protein is further conjugated to one or more conjugation moieties described below.

[0066] In some examples, the cytokine conjugate comprises a fusion to an scFv, bis-scFv, (scFv)2, dsFv, or sdAb fusion. In some cases, the fusion comprises an scFv. In some cases, the cytokine conjugate comprises a fusion to a bis-scFv. In some cases, the cytokine conjugate comprises a fusion to an (scFv)2. In some cases, the cytokine conjugate comprises a fusion to a dsFv. In some cases, the cytokine conjugate comprises a fusion to an sdAb. In some cases, the cytokine fused to an scFv, bis-scFv, (scFv)2, dsFv, or sdAb is further conjugated to one or more conjugation moieties described below.

[0067] In some examples, the cytokine conjugate comprises a fusion to the Fc portion of an antibody (e.g., IgG, IgA, IgM, IgE, or IgD). In some examples, the cytokine conjugate comprises a fusion to the Fc portion of an IgG (e.g., IgG1, IgG3, or IgG4). In some cases, the cytokine fused to the Fc portion is further conjugated to one or more conjugate moieties described below.

[0068] In some cases, the cytokine (e.g., interleukin, IFN, or TNF) polypeptide is fused to an antibody or binding fragment thereof. In some cases, the cytokine polypeptide is fused to a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3, a single chain variable fragment (scFv), a bis-scFv (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a humabody, a disulfide-stable Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof. In further cases, the cytokine polypeptide is fused to the Fc portion of an antibody. In further cases, the cytokine polypeptide is fused to the Fc portion of an IgG (e.g., IgG1, IgG3, or IgG4). Optionally, the cytokine, or binding fragment thereof, that is fused to the antibody is conjugated to one or more conjugation moieties described below.

[0069] In some cases, the IL-2 polypeptide is fused to an antibody or a binding fragment thereof. In some examples, the IL-2 is fused to a humanized antibody or a binding fragment thereof, a murine antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a humabody, a disulfide-stable Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In further cases, the IL-2 polypeptide is fused to the Fc portion of an antibody. In further cases, the IL-2 polypeptide is fused to the Fc portion of an IgG (e.g., IgG1, IgG3, or IgG4). In some cases, the antibody or binding fragment thereof fused to the IL-2 polypeptide will not interfere with the binding of IL-2Rβγ to the IL-2 polypeptide. In some cases, the antibody or binding fragment thereof fused to the IL-2 polypeptide partially blocks the binding of the IL-2 polypeptide to the IL-2Rβγ. In some cases, the IL-2 polypeptide fused to the antibody or binding fragment thereof is conjugated to one or more conjugate moieties described below.

[0070] Natural and Unnatural Amino Acids In some embodiments, an amino acid residue described herein (e.g., in a cytokine such as IL-2) is mutated to lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine prior to being conjugated to (or reacting with) a conjugation moiety. For example, the side chains of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine can be conjugated to a conjugation moiety described herein. In some examples, the amino acid residue is mutated to cysteine, lysine, or histidine. In some cases, the amino acid residue is mutated to cysteine. In some cases, the amino acid residue is mutated to lysine. In some cases, the amino acid residue is mutated to histidine. In some cases, the amino acid residue is mutated to tyrosine. In some cases, the amino acid residue is mutated to tryptophan. In some embodiments, the non-natural amino acid is not conjugated to a conjugation moiety. In some embodiments, a cytokine described herein comprises an unnatural amino acid, where the cytokine is conjugated to a protein and the point of attachment is not the unnatural amino acid.

[0071] In some embodiments, an amino acid residue described herein (e.g., in a cytokine such as IL-2) is mutated to a non-natural amino acid prior to attachment to a conjugate moiety. In some cases, the mutation of the non-natural amino acid prevents or minimizes an autoantigen response of the immune system. As used herein, the term "non-natural amino acid" refers to an amino acid other than the 20 amino acids that naturally occur in proteins. Non-limiting examples of unnatural amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-boronophenyl Alanine, O-propargyl tyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, azidolysine (AzK), non-natural analogs of the amino acid tyrosine; non-natural analogs of the amino acid glutamine; non-natural analogs of the amino acid phenylalanine; non-natural analogs of the amino acid serine; non-natural analogs of the amino acid threonine; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronic acid , phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acids, or combinations thereof; amino acids with photoactivatable crosslinkers; spin-labeled amino acids; fluorescent amino acids; amino acid-bound metals; metal-containing amino acids; radioactive amino acids; photocaged and / or photoisomerizable amino acids; biotin or biotin analog-containing amino acids;Keto-containing amino acids;amino acids containing polyethylene glycol or polyether;heavy atom substituted amino acids;chemically cleavable or photocleavable amino acids;amino acids with elongated side chains;toxic group-containing amino acids;sugar-substituted amino acids;amino acids containing a sugar attached to a carbon;redox-active amino acids;hydroxy-containing acids;aminothioacids;α,α-disubstituted amino acids;β-amino acids;cyclic amino acids other than proline or histidine, and aromatic amino acids other than phenylalanine, tyrosine, or tryptophan;

[0072] In some embodiments, the unnatural amino acid comprises a selectively reactive group or a reactive group for site-selective labeling of a target polypeptide. In some instances, the chemistry is a bioorthogonal reaction (e.g., a biocompatible and selective reaction). In some instances, the chemistry is a metal-mediated process such as a Cu(I)-catalyzed or "copper-free" alkyne-azide triazole-forming reaction, Staudinger ligation, inverse electron demand Diels-Alder (IEDDA) reaction, "photo-click" chemistry, or olefin metathesis, and Suzuki-Miyaura cross-coupling or Sonogashira coupling.

[0073] In some embodiments, the unnatural amino acids contain photoreactive groups that crosslink upon irradiation, for example, with UV.

[0074] In some embodiments, the unnatural amino acid comprises a photocaged amino acid.

[0075] In some instances, the unnatural amino acids are para-, meta-, or ortho-substituted amino acid derivatives.

[0076] In some examples, the unnatural amino acid comprises p-acetyl-L-phenylalanine, p-azido-L-phenylalanine, p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p-methoxyphenylalanine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, p-amino-L-phenylalanine, or isopropyl-L-phenylalanine.

[0077] In some cases, the unnatural amino acid is 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxy-phenylalanine, or 3-iodotyrosine.

[0078] In some cases, the unnatural amino acid is phenylselenocysteine.

[0079] In some examples, the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide-containing phenylalanine derivative.

[0080] In some examples, the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide-containing lysine derivative.

[0081] In some instances, the unnatural amino acid comprises an aromatic side chain.

[0082] In some instances, the unnatural amino acid does not contain an aromatic side chain.

[0083] In some instances, the unnatural amino acid comprises an azide group.

[0084] In some examples, the unnatural amino acid comprises a Michael acceptor group. In some examples, the Michael acceptor group comprises an unsaturated moiety capable of forming a covalent bond via a 1,2-addition reaction. In some examples, the Michael acceptor group comprises an electron-deficient alkene or alkyne. In some examples, the Michael acceptor group includes, but is not limited to, an α,β-unsaturated: ketone, aldehyde, sulfoxide, sulfone, nitrile, imine, or aromatic compound.

[0085] In some examples, the unnatural amino acid is dehydroalanine.

[0086] In some instances, the unnatural amino acid comprises an aldehyde or ketone group.

[0087] In some instances, the unnatural amino acid is a lysine derivative that contains an aldehyde or ketone group.

[0088] In some examples, the unnatural amino acid is a lysine derivative that includes one or more O, N, Se, or S atoms at the β, γ, or δ positions. In some examples, the unnatural amino acid is a lysine derivative that includes an O, N, Se, or S atom at the γ position.

[0089] In some examples, the unnatural amino acid is a lysine derivative in which the epsilon N atom is replaced with an oxygen atom.

[0090] In some instances, the unnatural amino acid is a lysine derivative that is not a naturally occurring post-translationally modified lysine.

[0091] In some examples, the unnatural amino acid is an amino acid that comprises a side chain, and the sixth atom from the alpha position comprises a carbonyl group. In some examples, the unnatural amino acid is an amino acid that comprises a side chain, where the sixth atom from the alpha position comprises a carbonyl group and the fifth atom from the alpha position is nitrogen. In some examples, the unnatural amino acid is an amino acid that comprises a side chain, and the seventh atom from the alpha position is an oxygen atom.

[0092] In some examples, the unnatural amino acid is a serine derivative that includes serine. In some examples, the unnatural amino acid is selenoserine (2-amino-3-hydroselenopropanoic acid. In some examples, the unnatural amino acid is 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid. In some examples, the unnatural amino acid is 2-amino-3-(phenylselanyl)propanoic acid. In some examples, the unnatural amino acid includes selenium, where oxidation of the selenium results in the formation of an alkene-containing unnatural amino acid.

[0093] In some instances, the unnatural amino acid comprises a cyclooctynyl group.

[0094] In some examples, the unnatural amino acid comprises a transcycloctenyl group.

[0095] In some instances, the unnatural amino acid comprises a norbornenyl group.

[0096] In some instances, the unnatural amino acid comprises a cyclopropenyl group.

[0097] In some instances, the unnatural amino acid comprises a diazirine group.

[0098] In some instances, the unnatural amino acid comprises a tetrazine group.

[0099] In some examples, the unnatural amino acid is a lysine derivative and the side chain nitrogen is carbamylated. In some examples, the unnatural amino acid is a lysine derivative and the side chain nitrogen is acylated. In some examples, the unnatural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is N6-Boc-N6-methyllysine. In some examples, the unnatural amino acid is N6-acetyllysine. In some examples, the unnatural amino acid is pyrrolysine. In some examples, the unnatural amino acid is N6-trifluoroacetyllysine. In some examples, the unnatural amino acid is 2-amino-6-{[(benzyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is 2-amino-6-{[(p-iodobenzyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is 2-amino-6-{[(p-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is N6-prolyllysine. In some examples, the unnatural amino acid is 2-amino-6-{[(cyclopentyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is N6-(cyclopentanecarbonyl)lysine. In some examples, the unnatural amino acid is N6-(tetrahydrofuran-2-carbonyl)lysine. In some examples, the unnatural amino acid is N6-(3-ethynyltetrahydrofuran-2-carbonyl)lysine. In some examples, the unnatural amino acid is N6-((prop-2-yn-1-yloxy)carbonyl)lysine. In some examples, the unnatural amino acid is 2-amino-6-{[(2-azidocyclopentyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is N6-((2-azidoethoxy)carbonyl)lysine.In some examples, the unnatural amino acid is 2-amino-6-{[(2-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is 2-amino-6-{[(2-cyclooctynyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is N6-(2-aminobut-3-ynoyl)lysine. In some examples, the unnatural amino acid is 2-amino-6-((2-aminobut-3-ynoyl)oxy)hexanoic acid. In some examples, the unnatural amino acid is N6-(allyloxycarbonyl)lysine. In some examples, the unnatural amino acid is N6-(butenyl-4-oxycarbonyl)lysine. In some examples, the unnatural amino acid is N6-(pentenyl-5-oxycarbonyl)lysine. In some examples, the unnatural amino acid is N6-((but-3-yn-1-yloxy)carbonyl)-lysine. In some examples, the unnatural amino acid is N6-((pent-4-yn-1-yloxy)carbonyl)-lysine. In some examples, the unnatural amino acid is N6-(thiazolidine-4-carbonyl)lysine. In some examples, the unnatural amino acid is 2-amino-8-oxononanoic acid. In some examples, the unnatural amino acid is 2-amino-8-oxooctanoic acid. In some examples, the unnatural amino acid is N6-(2-oxoacetyl)lysine.

[0100] In some examples, the unnatural amino acid is N6-propionyllysine. In some examples, the unnatural amino acid is N6-butyryllysine. In some examples, the unnatural amino acid is N6-(but-2-enoyl)lysine. In some examples, the unnatural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)lysine. In some examples, the unnatural amino acid is N6-((spiro[2.3]hex-1-en-5-ylmethoxy)carbonyl)lysine. In some examples, the unnatural amino acid is N6-(((4-(1-(trifluoromethyl)cycloprop-2-en-1-yl)benzyl)oxy)carbonyl)lysine. In some examples, the unnatural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-ylmethoxy)carbonyl)lysine. In some examples, the unnatural amino acid is cysteinyllysine. In some examples, the unnatural amino acid is N6-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)lysine. In some examples, the unnatural amino acid is N6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonyl)lysine. In some examples, the unnatural amino acid is N6-((3-(3-methyl-3H-diazirin-3-yl)propoxy)carbonyl)lysine. In some examples, the unnatural amino acid is N6-((metanitrobenyloxy)N6-methylcarbonyl)lysine). In some examples, the unnatural amino acid is N6-((bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)-lysine. In some examples, the unnatural amino acid is N6-((cyclohept-3-en-1-yloxy)carbonyl)-L-lysine.

[0101] In some examples, the unnatural amino acid is 2-amino-3-(((((benzyloxy)carbonyl)amino)methyl)selanyl)propanoic acid.

[0102] In some embodiments, unnatural amino acids are incorporated into cytokines (eg, IL polypeptides) via repurposed amber, opal, or ochre stop codons.

[0103] In some embodiments, the unnatural amino acid is incorporated into a cytokine (eg, an IL polypeptide) via a four-base codon.

[0104] In some embodiments, the unnatural amino acid is incorporated into a cytokine (eg, an IL polypeptide) via a repurposed rare-sense codon.

[0105] In some embodiments, unnatural amino acids are incorporated into cytokines (e.g., IL polypeptides) by synthetic codons that include unnatural nucleic acids. In some examples, unnatural amino acids are incorporated into cytokines by orthogonal engineered synthetase / tRNA pairs. Such orthogonal pairs include unnatural synthetases that can charge unnatural tRNAs with unnatural amino acids, but minimize a) charging of unnatural tRNAs with other endogenous amino acids, and b) charging of unnatural amino acids to other endogenous tRNAs. Such orthogonal pairs include tRNAs that can be charged by unnatural synthetases, but avoid a) charging of other endogenous amino acids by endogenous synthetases. In some embodiments, such pairs are identified from various organisms, such as bacteria, yeast, archaea, or human sources. In some embodiments, the orthogonal synthetase / tRNA pair includes components from a single organism. In some embodiments, the orthogonal synthetase / tRNA pair includes components from two different organisms. In some embodiments, the orthogonal synthetase / tRNA pair comprises components that facilitate the translation of two different amino acids prior to modification. In some embodiments, the orthogonal synthetase is an engineered alanine synthetase. In some embodiments, the orthogonal synthetase is an engineered arginine synthetase. In some embodiments, the orthogonal synthetase is an engineered asparagine synthetase. In some embodiments, the orthogonal synthetase is an engineered aspartate synthetase. In some embodiments, the orthogonal synthetase is an engineered cysteine ​​synthetase. In some embodiments, the orthogonal synthetase is an engineered glutamine synthetase. In some embodiments, the orthogonal synthetase is an engineered glutamate synthetase. In some embodiments, the orthogonal synthetase is an engineered alanine glycine. In some embodiments, the orthogonal synthetase is an engineered histidine synthetase. In some embodiments, the orthogonal synthetase is an engineered leucine synthetase. In some embodiments, the orthogonal synthetase is an engineered isoleucine synthetase.In some embodiments, the orthogonal synthetase is a modified lysine synthetase. In some embodiments, the orthogonal synthetase is a modified methionine synthetase. In some embodiments, the orthogonal synthetase is a modified phenylalanine synthetase. In some embodiments, the orthogonal synthetase is a modified proline synthetase. In some embodiments, the orthogonal synthetase is a modified serine synthetase. In some embodiments, the orthogonal synthetase is a modified threonine synthetase. In some embodiments, the orthogonal synthetase is a modified tryptophan synthetase. In some embodiments, the orthogonal synthetase is a modified tyrosine synthetase. In some embodiments, the orthogonal synthetase is a modified valine synthetase. In some embodiments, the orthogonal synthetase is a modified phosphoserine synthetase. In some embodiments, the orthogonal tRNA is a modified alanine tRNA. In some embodiments, the orthogonal tRNA is a modified arginine tRNA. In some embodiments, the orthogonal tRNA is a modified asparagine tRNA. In some embodiments, the orthogonal tRNA is a modified aspartate tRNA. In some embodiments, the orthogonal tRNA is a modified cysteine ​​tRNA. In some embodiments, the orthogonal tRNA is a modified glutamine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamate tRNA. In some embodiments, the orthogonal tRNA is a modified alanine glycine. In some embodiments, the orthogonal tRNA is a modified histidine tRNA. In some embodiments, the orthogonal tRNA is a modified leucine tRNA. In some embodiments, the orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, the orthogonal tRNA is a modified lysine tRNA. In some embodiments, the orthogonal tRNA is a modified methionine tRNA. In some embodiments, the orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, the orthogonal tRNA is a modified proline tRNA. In some embodiments, the orthogonal tRNA is a modified serine tRNA.In some embodiments, the orthogonal tRNA is a modified threonine tRNA. In some embodiments, the orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, the orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, the orthogonal tRNA is a modified valine tRNA. In some embodiments, the orthogonal tRNA is a modified phosphoserine tRNA.

[0106] In some embodiments, unnatural amino acids are incorporated into cytokines (e.g., IL polypeptides) by aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pairs. Exemplary aaRS-tRNA pairs include, but are not limited to, the Methanocaldococcus jannaschii (Mj-Tyr) aaRS / tRNA pair, the Escherichia coli TyrRS (Ec-Tyr) / Bacillus stearothermophilus tRNA pair, and the Bacillus subtilis tRNA pair. CUA vs. Escherichia coli LeuRS (Ec-Leu) / Bacillus stearothermophilus tRNA CUA In some examples, unnatural amino acids are incorporated into cytokines (e.g., IL polypeptides) by Mj-TyrRS / tRNA pairs. Exemplary UAAs that can be incorporated by Mj-TyrRS / tRNA pairs include, but are not limited to, para-substituted phenylalanine derivatives such as p-aminophenylalanine and p-methylphenylalanine; meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronopheylalanine; and o-nitrobenzyltyrosine.

[0107] In some instances, the unnatural amino acid is CUA or Ec-Leu / tRNA CUA Ec-Tyr / tRNA pair is incorporated into cytokines (e.g., IL polypeptides). CUA or Ec-Leu / tRNACUA Exemplary UAAs that may be incorporated by pairs include, but are not limited to, phenylalanine derivatives containing benzopheno, ketone, iodide, or azide substituents; O-propargyl tyrosine; α-aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine; and 3-(naphthalen-2-ylamino)-2-amino-propanoic acid.

[0108] In some instances, unnatural amino acids are incorporated into cytokines (e.g., IL polypeptides) by pyrrolysyl-tRNA pairs. In some instances, PylRS is obtained from Archaea (e.g., from a methanogenic Archaea). In some instances, PylRS is obtained from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. Exemplary UAAs that can be incorporated by a pyrrolysyl-tRNA pair include, but are not limited to, amide and carbamate substituted lysines such as 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid, N-ε-D-prolyl-L-lysine, and N-ε-cyclopentyloxycarbonyl-L-lysine; N-ε-acryloyl-L-lysine; N-ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]-L-lysine; and N-ε-(1-methylcyclopro-2-enecarboxamido)lysine.

[0109] In some examples, unnatural amino acids are incorporated into the cytokines (e.g., IL polypeptides) described herein by synthetases disclosed in U.S. Patent Nos. 9,988,619 and 9,938,516. Exemplary UAAs that may be incorporated by such synthetases include para-methylazido-L-phenylalanine, aralkyl, heterocyclyl, heteroaralkyl unnatural amino acids, or others. In some embodiments, such UAAs include pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocycles. In some embodiments, such amino acids include azides, tetrazines, or other chemical groups that can be conjugated to binding partners, such as water-soluble moieties. In some embodiments, such synthetases are expressed and used to incorporate UAAs into cytokines in vivo. In some embodiments, such synthetases are used to incorporate UAAs into cytokines using cell-free translation systems.

[0110] In some examples, the unnatural amino acid is incorporated into a cytokine (e.g., an IL polypeptide) described herein by a naturally occurring synthetase. In some embodiments, the unnatural amino acid is incorporated into a cytokine by an organism that is auxotrophic for one or more amino acids. In some embodiments, the synthetase corresponding to the auxotrophic amino acid is capable of charging the corresponding tRNA with the unnatural amino acid. In some embodiments, the unnatural amino acid is selenocysteine ​​or a derivative thereof. In some embodiments, the unnatural amino acid is selenomethionine or a derivative thereof. In some embodiments, the unnatural amino acid is an aromatic amino acid, including an aryl halide, such as an iodide. In embodiments, the unnatural amino acid is structurally similar to the auxotrophic amino acid.

[0111] In some examples, unnatural amino acids include those illustrated in FIG.

[0112] In some examples, the unnatural amino acid comprises a derivative or analog of lysine or phenylalanine. In some examples, the unnatural amino acid comprises a lysine derivative or lysine analog. In some examples, the unnatural amino acid comprises pyrrolysine (Pyl). In some examples, the unnatural amino acid comprises a phenylalanine derivative or phenylalanine analog. In some examples, the unnatural amino acid is an unnatural amino acid described in Wan, et al., "Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool," Biocheim Biophys Aceta 1844(6): 1059-4070 (2014). In some examples, the unnatural amino acid comprises an unnatural amino acid illustrated in Figure 2 (e.g., Figures 2A and 2B).

[0113] In some embodiments, the unnatural amino acids include the unnatural amino acids illustrated in Figures 3A-3D (taken from Table 1 of Dumas et al., Chemical Science 2015, 6, 50-69).

[0114] In some embodiments, the unnatural amino acids incorporated into the cytokines (e.g., IL polypeptides) described herein are disclosed in US9,840,493; US9,682,934; US2017 / 0260137; US9,938,516; or US2018 / 0086734. Exemplary UAAs that may be incorporated by such synthetases include para-methylazido-L-phenylalanine, aralkyl, heterocyclyl, and heteroaralkyl, and lysine derivative unnatural amino acids. In some embodiments, such UAAs include pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocycles. In some embodiments, such amino acids include azides, tetrazines, or other chemical groups that can be conjugated to binding partners, such as water-soluble moieties. In some embodiments, the UAAs include azides that are linked to an aromatic moiety via an alkyl linker. In some embodiments, the alkyl linker is a C1-C 10linker. In some embodiments, the UAA comprises a tetrazine linked to the aromatic moiety via an alkyl linker. In some embodiments, the UAA comprises a tetrazine linked to the aromatic moiety via an amino group. In some embodiments, the UAA comprises a tetrazine linked to the aromatic moiety via an alkylamino group. In some embodiments, the UAA comprises an azide linked to the terminal nitrogen of an amino acid side chain via an alkyl chain (e.g., N6 of a lysine derivative, N5, N4, or N3 of derivatives containing short alkyl side chains). In some embodiments, the UAA comprises a tetrazine linked to the terminal nitrogen of an amino acid side chain via an alkyl chain. In some embodiments, the UAA comprises an azide or tetrazine linked to an amide via an alkyl linker. In some embodiments, the UAA is an azide or tetrazine-containing carbamate, or an amide of 3-aminoalanine, serine, lysine, or derivatives thereof. In some embodiments, such UAAs are incorporated into cytokines in vivo. In some embodiments, such UAAs are incorporated into cytokines in a cell-free system.

[0115] conjugation part In certain embodiments, conjugate moieties are disclosed herein that are attached to one or more cytokines (e.g., interleukins, IFNs, or TNFs) described above. In some examples, the conjugate moiety is a molecule that disrupts the interaction of the cytokine with its receptor. In some examples, the conjugate moiety is any molecule that, when attached to the cytokine, can allow cytokine conjugation to modulate an immune response. In some examples, the conjugate moiety comprises a water-soluble polymer. In other examples, the conjugate moiety comprises a protein or binding fragment thereof. In further cases, the conjugate moiety comprises a peptide. In further cases, the conjugate moiety comprises a nucleic acid. In further cases, the conjugate moiety comprises a small molecule. In some cases, the conjugate moiety increases serum half-life and / or improves stability. In some cases, the conjugate moiety reduces cytokine interaction with one or more cytokine receptor domains or subunits. In further cases, the conjugate moiety blocks interaction of the cytokine with one or more cytokine domains or subunits to its cognate receptor. In some embodiments, the cytokine conjugates described herein comprise multiple conjugate moieties. In some embodiments, the conjugate moiety is attached to a non-natural or natural amino acid in a cytokine peptide. In some embodiments, the cytokine conjugate comprises a conjugate moiety attached to a natural amino acid. In some embodiments, the cytokine conjugate is attached to a non-natural amino acid in the cytokine peptide. In some embodiments, the conjugate moiety is attached to the N-terminal or C-terminal amino acid of the cytokine peptide. Various combination sites are disclosed herein, for example, a first conjugate moiety is attached to a non-natural or natural amino acid in the cytokine peptide and a second conjugate moiety is attached to the N-terminal or C-terminal amino acid of the cytokine peptide. In some embodiments, a single conjugate moiety is attached to multiple residues of the cytokine peptide (e.g., staples). In some embodiments, the conjugate moieties are attached to both the N-terminal and C-terminal amino acids of the cytokine peptide.

[0116] Water-soluble polymers In some embodiments, the conjugate moiety described herein is a water-soluble polymer. In some examples, the water-soluble polymer is non-peptidic, non-toxic, and biocompatible. As used herein, a substance is considered to be biocompatible if the beneficial effects associated with the use of the substance alone or with other substances (e.g., active agents such as cytokine moieties) on living tissue (e.g., administration to a patient) outweigh any adverse effects as assessed by a clinician (e.g., physician, toxicologist, clinical development specialist). In some examples, the water-soluble polymer is further non-immunogenic. In some examples, a substance is considered to be non-immunogenic if the intended use of the substance in vivo does not result in an undesirable immune response (e.g., formation of antibodies) or if an immune response occurs that is not considered clinically significant or important as assessed by a clinician (e.g., physician, toxicologist, clinical development specialist).

[0117] In some examples, the water soluble polymer is characterized as having from about 2 to about 300 termini. Exemplary water soluble polymers include, but are not limited to, poly(alkylene glycols), such as polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polydimethylacrylamide (PDAAm), polyphosphazenes, polyoxazolines ("POZ") (as described in WO 2008 / 106186), poly(N-acryloylmorpholines, and combinations of any of the foregoing.

[0118] In some cases, the water-soluble polymer is not limited to a specific structure. In some cases, the water-soluble polymer is linear (e.g., end-capped, e.g., alkoxy PEG, bifunctional PEG), branched or multi-armed (e.g., branched PEG or PEG bonded to a polyol core), dendritic (or star) structure, each with or without one or more degradable bonds. Furthermore, the internal structure of the water-soluble polymer can be constructed with any number of different repeating patterns, and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.

[0119] In some embodiments, the weight average molecular weight of the water-soluble polymer in the IL-2 conjugate is from about 100 daltons to about 150,000 daltons. Typical ranges include, for example, weight average molecular weights in the range of from 5,000 daltons or more to about 100,000 daltons, the range of from about 6,000 daltons to about 90,000 daltons, the range of from about 85,000 daltons to about 10,000 daltons, the range of from 10,000 daltons to about 85,000 daltons, the range of from about 20,000 daltons to about 85,000 daltons, the range of from about 53,000 daltons to about 85,000 daltons, the range of from about 25,000 daltons to about 120,000 daltons, the range of from about 29,000 daltons to about 120,000 daltons, the range of from about 35,000 daltons to about 120,000 daltons, and the range of from about 40,000 daltons to about 120,000 daltons.

[0120] Typical weight average molecular weights of the water-soluble polymers are about 100 daltons, about 200 daltons, about 300 daltons, 400 daltons, about 500 daltons, about 600 daltons, about 700 daltons, about 750 daltons, about 800 daltons, about 900 daltons, about 1,000 daltons, about 1,500 daltons, about 2,000 daltons, about 2,200 daltons, about 2,500 daltons, about 3,000 daltons, about 4,000 daltons, about 4,400 daltons, about 4,500 daltons, about 5,000 daltons, about 5,500 daltons, about 6,000 daltons, about 7,000 daltons, about 7,500 daltons, about 8,000 daltons, about 9,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 16,000 daltons, about 17,000 daltons, about 18,000 daltons, about 19,000 daltons, about 21,000 daltons, about 22,000 daltons, about 23,000 daltons, about 24,000 daltons, about 25,000 daltons, about 26,000 dalton 0 daltons, about 8,000 daltons, about 9,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000, about 14,000 daltons, about 15,000 daltons, about 20,000 daltons, about 22,500 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, and about 75,000 daltons. Branched versions of water-soluble polymers having any of the foregoing total molecular weights can also be used (e.g., a branched 40,000 dalton water-soluble polymer composed of two 20,000 dalton polymers). In one or more embodiments, the conjugate will not have any PEG moieties attached, directly or indirectly, to a PEG having a weight average molecular weight of less than about 6,000 daltons.

[0121] PEG will typically include multiple (OCH2CH2) monomers [or (CH2CHO) monomers, depending on how PEG is defined]. As used herein, the number of repeating units is identified by the subscript "n" in "((OCH2CH2))n". Thus, the value of (n) typically falls within one or more of the following ranges: from about 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900. For a given polymer of known molecular weight, it is possible to determine the number of repeating units (ie, "n") by dividing the total weight average molecular weight of the polymer by the molecular weight of the repeating monomer.

[0122] In some examples, the water-soluble polymer is an end-capped polymer, i.e., a polymer having at least one terminus capped with a relatively inert group, such as a lower C1-6 alkoxy group, or a hydroxyl group. When the polymer is PEG, for example, methoxy-PEG (commonly called mPEG), a linear PEG, may be used, in which one terminus of the polymer is a methoxy (--OCH3) group and the other terminus is a hydroxyl or other functional group that is optionally chemically modified.

[0123] In some embodiments, exemplary water-soluble polymers include, but are not limited to, linear or branched individual PEG (dPEG) from Quanta Biodesign, Ltd; branched, linear, or branched PEG from Nektar Therapeutics; and Y-shaped PEG derivatives from JenKem Technology.

[0124] In some embodiments, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide described herein is conjugated to a water soluble polymer selected from poly(alkylene glycols), e.g., polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers such as ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polyphosphazenes, polyoxazolines ("POZ"), poly(N-acryloylmorpholines), and combinations thereof. In some examples, the cytokine polypeptide is conjugated to PEG (e.g., pegylated). In some examples, the cytokine polypeptide is conjugated to PPG. In some examples, the cytokine polypeptide is conjugated to POZ. In some examples, the cytokine polypeptide is conjugated to PVP.

[0125] In some embodiments, the IL-2 described herein is conjugated to a water soluble polymer selected from poly(alkylene glycols), e.g., polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers such as ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohol) (PVA), polyacrylamides (PAAm), polydimethylacrylamide (PDAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polyphosphazenes, polyoxazolines ("POZ"), poly(N-acryloylmorpholines), and combinations thereof. In some examples, the IL-2 polypeptide is conjugated to PEG (e.g., pegylated). In some examples, the IL-2 polypeptide is conjugated to PPG. In some examples, the IL-2 polypeptide is conjugated to POZ. In some examples, the IL-2 polypeptide is conjugated to PVP.

[0126] In some instances, the water-soluble polymer comprises polyglycerol (PG). In some instances, the polyglycerol is highly branched PG (HPG) (e.g., as described by Imran, et al. "Influence of architecture of high molecular weight linear and branched polyglycerols on their biocompatibility and biodistribution," Biomaterials 33:9135-9147 (2012)). In other instances, the polyglycerol is linear PG (LPG). In further cases, the polyglycerol is a medium functional PG, a linear block highly branched PG (e.g., as described by Wurm et. Al., "Squaric acid mediated synthesis and biological activity of a library of linear and hyperbranched poly(glycerol)-protein conjugates," Biomacromolecules 13:1161-1171 (2012)), or a side chain functional PG (e.g., as described by Li, et. al., "Synthesis of linear polyether polyol derivatives as new materials for bioconjugation," Bioconjugate Chem. 20:780-789 (2009)).

[0127] In some examples, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide described herein is conjugated to PG, e.g., HPG, LPG, medium functionality PG, linear block highly branched PG, side chain functional PG. In some examples, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to PG, medium functionality PG, linear block highly branched PG.

[0128] In some embodiments, the water-soluble polymer is a degradable synthetic PEG substitute. Exemplary degradable synthetic PEG substitutes include, but are not limited to, poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA); backbone-modified PEG derivatives produced by polymerization of telechelic or dual-end-functionalized PEG-based macromonomers; degradable linkages, e.g., poly[(ethylene oxie)-co-(methylene ethylene oxide)][P(EO-co-MEO), cyclic ketene acetals, e.g., 2-methylene-1,3-dioxepane (MDO), such as 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), and 2-methylene-4-phenyl-1,3-dioxolane (MPDL) copolymerized with OEGMA; or poly(ε-caprolactone)-graft-poly(ethylene oxide) (PCLg-PEO).

[0129] In some examples, the cytokine (e.g., interleukin, IFN, or TNF) polypeptide described herein is conjugated to a degradable synthetic PEG substitute, such as POEGM; backbone-modified PEG derivatives produced by polymerization of telechelic or dual-end-functionalized PEG-based macromonomers; P(EO-co-MEO); cyclic ketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO. In some examples, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a degradable synthetic PEG substitute, such as POEGM; backbone-modified PEG derivatives produced by polymerization of telechelic or dual-end-functionalized PEG-based macromonomers; P(EO-co-MEO); cyclic ketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO.

[0130] In some embodiments, the water-soluble polymer comprises a poly(zwitterion). Exemplary poly(zwitterions) include, but are not limited to, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methyacryloyloxyethyl phosphorylcholine) (PMPC). In some examples, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide described herein is conjugated to a poly(zwitterion), e.g., PSBMA, PCBMA, or PMPC. In some cases, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a poly(zwitterion), e.g., PSBMA, PCBMA, or PMPC.

[0131] In some embodiments, the water-soluble polymer comprises a polycarbonate. Exemplary polycarbonates include, but are not limited to, pentafluorophenyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC6F5). In some examples, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide described herein is conjugated to a polycarbonate, e.g., MTC-OC6F5. In some cases, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a polycarbonate, e.g., MTC-OC6F5.

[0132] In some embodiments, the water-soluble polymer comprises a polymer hybrid, such as a polycarbonate / PEG polymer hybrid, a peptide / protein polymer conjugate, or a hydroxyl-containing and / or zwitterionic derivatized polymer (e.g., a hydroxyl-containing and / or zwitterionic derivatized PEG polymer). In some examples, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide described herein is conjugated to a polymer hybrid, such as a polycarbonate / PEG polymer hybrid, a peptide / protein polymer conjugate, or a hydroxyl-containing and / or zwitterionic derivatized polymer (e.g., a hydroxyl-containing and / or zwitterionic derivatized PEG polymer). In some cases, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a polymer hybrid, such as a polycarbonate / PEG polymer hybrid, a peptide / protein polymer conjugate, or a hydroxyl-containing and / or zwitterionic derivatized polymer (e.g., a hydroxyl-containing and / or zwitterionic derivatized PEG polymer).

[0133] In some examples, the water-soluble polymer comprises a polysaccharide. Exemplary polysaccharides include, but are not limited to, dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to the polysaccharide. In some cases, an IL-2 polypeptide is conjugated to dextran. In some cases, an IL-2 polypeptide is conjugated to PSA. In some cases, an IL-2 polypeptide is conjugated to HA. In some cases, an IL-2 polypeptide is conjugated to amylose. In some cases, an IL-2 polypeptide is conjugated to heparin. In some cases, an IL-2 polypeptide is conjugated to HS. In some cases, an IL-2 polypeptide is conjugated to dextrin. In some cases, an IL-2 polypeptide is conjugated to HES.

[0134] In some cases, the water-soluble polymer comprises a glycan. Exemplary classes of glycans include N-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosaminoglycans. In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to a glycan. In some cases, an IL-2 polypeptide is conjugated to an N-linked glycan. In some cases, an IL-2 polypeptide is conjugated to an O-linked glycan. In some cases, an IL-2 polypeptide is conjugated to a glycolipid. In some cases, an IL-2 polypeptide is conjugated to an O-GlcNAc. In some cases, an IL-2 polypeptide is conjugated to a glycosaminoglycan.

[0135] In some embodiments, the water-soluble polymer comprises a polyoxazoline polymer. Polyoxazoline polymers are linear synthetic polymers and, similar to PEG, contain low polydispersity. In some examples, the polyoxazoline polymer is a polydisperse polyoxazoline polymer, characterized by its average molecular weight. In some cases, the average molecular weight of the polyoxazoline polymer includes, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, 100,000, 200,000, 300,000, 400,000, or 500,000 Da. In some examples, the polyoxazoline polymer includes poly(2-methyl 2-oxazoline) (PMOZ), poly(2-ethyl 2-oxazoline) (PEOZ), poly(2-propyl 2-oxazoline) (PPOZ). In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to a polyoxazoline polymer. In some cases, an IL-2 polypeptide is conjugated to a polyoxazoline polymer. In some cases, an IL-2 polypeptide is conjugated to a PMOZ. In some cases, an IL-2 polypeptide is conjugated to a PEOZ. In some cases, an IL-2 polypeptide is conjugated to a PPOZ.

[0136] In some examples, the water-soluble polymer comprises a polyacrylic acid polymer. Optionally, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to the polyacrylic acid polymer. Optionally, an IL-2 polypeptide is conjugated to the polyacrylic acid polymer.

[0137] In some examples, the water-soluble polymer comprises a polyamine. A polyamine is an organic polymer that comprises two or more primary amino groups. In some embodiments, the polyamine comprises a branched polyamine, a linear polyamine, or a cyclic polyamine. In some cases, the polyamine is a low molecular weight linear polyamine. Exemplary polyamines include putrescine, cadaverine, spermidine, spermine, ethylenediamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, and piperazine. In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to a polyamine. In some cases, an IL-2 polypeptide is conjugated to a polyamine. In some cases, an IL-2 polypeptide is conjugated to a putrescine, cadaverine, spermidine, spermine, ethylenediamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, or piperazine.

[0138] In some examples, the water-soluble polymer is described in U.S. Patent Nos. 7,744,861, 8,273,833, and 7,803,777. In some examples, the cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to a linker described in U.S. Patent Nos. 7,744,861, 8,273,833, or 7,803,777. In some cases, the IL-2 polypeptide is conjugated to a linker described in U.S. Patent Nos. 7,744,861, 8,273,833, or 7,803,777.

[0139] protein In some embodiments, the conjugate moiety described herein is a protein or a binding fragment thereof. Exemplary proteins include albumin, transferrin, or transthyretin. In some examples, the protein or binding fragment thereof includes an antibody or a binding fragment thereof. In some cases, the cytokine conjugate includes the protein or a binding fragment thereof. In some cases, the IL-2 conjugate including the protein or a binding fragment thereof has an increased serum half-life and / or stability. In some cases, the IL-2 conjugate including the protein or a binding fragment thereof has a reduced interaction of IL-2 with one or more IL-2R subunits. In further cases, the protein or a binding fragment thereof blocks the interaction of IL-2 with one or more IL-2R subunits or affects the assembly of the IL-2Rβγ signaling complex.

[0140] In some embodiments, the conjugate moiety is albumin. Albumin is a family of water-soluble globular proteins. It is commonly found in plasma and constitutes about 55-60% of the total plasma protein. Human serum albumin (HSA) is a 585 amino acid polypeptide whose tertiary structure is divided into three domains: domain I (amino acid residues 1-195), domain II (amino acid residues 196-383) and domain III (amino acid residues 384-585). Each domain contains a binding site that can reversibly or irreversibly interact with endogenous ligands, such as long and medium chain fatty acids, bilirubin, or hemin, or exogenous compounds, such as heterocyclic or aromatic compounds.

[0141] In some cases, the cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to albumin. In some cases, the cytokine polypeptide is conjugated to human serum albumin (HSA). In further cases, the cytokine polypeptide is conjugated to a functional fragment of albumin.

[0142] In some instances, the IL-2 polypeptide is conjugated to albumin. In some cases, the IL-2 polypeptide is conjugated to human serum albumin (HSA). In further instances, the IL-2 polypeptide is conjugated to a functional fragment of albumin.

[0143] In some embodiments, the conjugate moiety is transferrin. Transferrin is a 679 amino acid polypeptide that is approximately 80 kDa in size and contains two Fe3+ binding sites (one in the N-terminal domain and one in the C-terminal domain). In some examples, human transferrin has a half-life of about 7-12 days.

[0144] In some instances, the cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to transferrin. In some cases, the cytokine polypeptide is conjugated to human transferrin. In further cases, the cytokine polypeptide is conjugated to a functional fragment of transferrin.

[0145] In some instances, the IL-2 polypeptide is conjugated to transferrin. In some cases, the IL-2 polypeptide is conjugated to human transferrin. In further instances, the IL-2 polypeptide is conjugated to a functional fragment of transferrin.

[0146] In some embodiments, the conjugate moiety is transthyretin (TTR), a transport protein present in serum and cerebrospinal fluid that carries the thyroid hormone thyroxine (T4) bound to retinol and retinol-binding protein.

[0147] In some instances, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to transthyretin (through one of its termini or the internal hinge region). In some instances, the cytokine polypeptide is conjugated to a functional fragment of transthyretin.

[0148] In some instances, the IL-2 polypeptide is conjugated to transthyretin (through one of its termini or the internal hinge region). In some instances, the IL-2 polypeptide is conjugated to a functional fragment of transthyretin.

[0149] In some embodiments, the conjugate moiety is an antibody or binding fragment thereof. In some examples, the antibody or binding fragment thereof includes a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a humabody, a disulfide-stable Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof.

[0150] In some examples, the conjugate moiety comprises an scFv, a bis-scFv, (scFv)2, a dsFv, or an sdAb. In some cases, the conjugate moiety comprises an scFv. In some cases, the conjugate moiety comprises a bis-scFv. In some cases, the conjugate moiety comprises an (scFv)2. In some cases, the conjugate moiety comprises a dsFv. In some cases, the conjugate moiety comprises an sdAb.

[0151] In some examples, the conjugate moiety comprises an Fc portion of an antibody, such as an IgG, IgA, IgM, IgE, or IgD. In some examples, the moiety comprises an Fc portion of an IgG (e.g., an IgG1, IgG3, or IgG4).

[0152] In some cases, the cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to an antibody, or a binding fragment thereof. In some examples, the cytokine polypeptide is conjugated to a humanized antibody or a binding fragment thereof, a murine antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3, a single chain variable fragment (scFv), a bis-scFv (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a humabody, a disulfide-stable Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In further cases, the cytokine polypeptide is conjugated to the Fc portion of an antibody. In further cases, the cytokine polypeptide is conjugated to the Fc portion of an IgG (e.g., IgG1, IgG3, or IgG4).

[0153] In some cases, the IL-2 polypeptide is conjugated to an antibody or a binding fragment thereof. In some examples, the IL-2 polypeptide is conjugated to a humanized antibody or a binding fragment thereof, a murine antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a humabody, a disulfide-stable Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In further cases, the IL-2 polypeptide is conjugated to the Fc portion of an antibody. In further cases, the IL-2 polypeptide is conjugated to the Fc portion of an IgG (e.g., IgG1, IgG3, or IgG4).

[0154] In some embodiments, the IL-2 polypeptide is conjugated to a water-soluble polymer (e.g., PEG), an antibody or binding fragment thereof. In some embodiments, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a humabody, a disulfide-stable Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof. In some embodiments, the antibody or binding fragment thereof comprises an scFv, a bis-scFv, a (scFv)2, a dsFv, or a sdAb. In some embodiments, the antibody or binding fragment thereof comprises an scFv. Optionally, the antibody or binding fragment thereof targets the IL-2 conjugate to target cells of the subject, and the water-soluble polymer increases stability and / or serum half-life.

[0155] In some examples, one or more IL-2 polypeptide-water-soluble polymer (e.g., PEG) conjugates are further attached to the antibody or binding fragment thereof. In some examples, the ratio of IL-2 conjugate to antibody is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12:1. In some cases, the ratio of IL-2 conjugate to antibody is about 1:1. In other cases, the ratio of IL-2 conjugate to antibody is about 2:1, 3:1, or 4:1. In further cases, the ratio of IL-2 conjugate to antibody is about 6:1 or more.

[0156] In some embodiments, one or more IL-2 polypeptide-water soluble polymer (e.g., PEG) conjugates are directly attached to the antibody or binding fragment thereof. In other examples, the IL-2 conjugates are indirectly attached to the antibody or binding fragment thereof by a linker. Exemplary linkers include homobifunctional linkers, heterofunctional linkers, maleimide-based linkers, zero trace linkers, self-immolative linkers, spacers, and the like.

[0157] In some embodiments, the antibody or binding fragment thereof is directly or indirectly conjugated to the IL-2 polypeptide portion of an IL-2 polypeptide-water soluble polymer (e.g., PEG) conjugate. In such cases, the conjugation site of the antibody to the IL-2 polypeptide is a site that does not interfere with the binding of IL-2Rβγ to the IL-2 polypeptide. In further cases, the conjugation site of the antibody to the IL-2 polypeptide is a site that partially blocks the binding of IL-2Rβγ to the IL-2 polypeptide. In other embodiments, the antibody or binding fragment thereof is directly or indirectly conjugated to the water soluble polymer portion of an IL-2 polypeptide-water soluble polymer (e.g., PEG) conjugate.

[0158] peptide In some embodiments, the conjugate moiety described herein is a peptide. In some examples, the peptide is an unstructured peptide. In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to the peptide. In some cases, the IL-2 conjugate comprising the peptide has increased serum half-life and / or stability. In some cases, the IL-2 conjugate comprising the peptide has reduced interaction of IL-2 with one or more IL-2R subunits. In further cases, the peptide blocks the interaction of IL-2 with one or more IL-2R subunits.

[0159] In some instances, the conjugated moiety is an XTEN™ peptide (Amunix Operating Inc.), and the modification is referred to as XTENylation. XTENylation is a genetic fusion of a nucleic acid encoding an XTEN™ peptide (Amunix Operating Inc.), i.e., a long unstructured hydrophilic peptide containing different percentages of six amino acids (Ala, Glu, Gly, Ser, and Thr), with a nucleic acid encoding a polypeptide of interest. In some instances, the XTEN™ peptide is selected based on properties such as expression, genetic stability, solubility, aggregation resistance, increased half-life, enhanced potency, and / or increased in vitro activity in combination with the polypeptide of interest. In some instances, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to the XTEN peptide. In some instances, an IL-2 polypeptide is conjugated to the XTEN peptide.

[0160] In some instances, the conjugated moiety is a homo-amino acid polymer rich in glycine (HAP), and the modification is called HAPylation. HAPylation is a genetic fusion of a nucleic acid encoding a homo-amino acid polymer rich in glycine (HAP) with a nucleic acid encoding a polypeptide of interest. In some instances, the HAP polymer contains a (Gly4Ser)n repeat motif (SEQ ID NO:3), and is sometimes about 50, 100, 150, 200, 250, 300, or more residues in length. In some instances, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to HAP. In some instances, an IL-2 polypeptide is conjugated to HAP.

[0161] In some embodiments, the conjugated moiety is a PAS polypeptide, and the modification is called PASylation. PASylation is a genetic fusion between a nucleic acid encoding a PAS polypeptide and a nucleic acid encoding a polypeptide of interest. A PAS polypeptide is a hydrophilic, uncharged polypeptide consisting of Pro, Ala, and Ser residues. In some examples, the length of a PAS polypeptide is at least about 100, 200, 300, 400, 500, or 600 amino acids. In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to the PAS polypeptide. In some cases, an IL-2 polypeptide is conjugated to the PAS polypeptide.

[0162] In some embodiments, the conjugated moiety is an elastin-like polypeptide (ELP), and the modification is called ELPylation. ELPylation is a genetic fusion between a nucleic acid encoding an elastin-like polypeptide (ELP) and a nucleic acid encoding a polypeptide of interest. ELP contains a VPGxG repeat motif (SEQ ID NO:4), where x is any amino acid except proline. In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to the ELP. In some cases, an IL-2 polypeptide is conjugated to the ELP.

[0163] In some embodiments, the conjugate moiety is a CTP peptide. The CTP peptide is a 31 amino acid residue peptide FQSSSS * KAPPPS * LPSPS * RLPGPS * DTPILPQ (SEQ ID NO:5), where S * denotes an O-glycosylation site (OPKO). In some instances, the CTP peptide is genetically fused to a cytokine polypeptide (e.g., an IL-2 polypeptide). In some instances, a cytokine polypeptide (e.g., an IL-2 polypeptide) is conjugated to the CTP peptide.

[0164] In some embodiments, a cytokine (e.g., an IL-2 polypeptide) is modified by glutamylation, a reversible post-translational modification of glutamic acid in which a peptide-like bond is formed with the amino group of free glutamic acid and the γ-carboxy group of glutamic acid is extended with the α-carboxy group into a polyglutamic acid chain.

[0165] In some embodiments, a cytokine (e.g., an IL-2 polypeptide) is modified with a gelatin-like protein (GLK) polymer. In some examples, the GLK polymer comprises multiple repeats of Gly-Xaa-Yaa, where Xaa and Yaa comprise predominantly proline and 4-hydroxyproline, respectively. In some cases, the GLK polymer further comprises the amino acid residues Pro, Gly, Glu, Qln, Asn, Ser, and Lys. In some cases, the length of the GLK polymer is about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150 residues or more.

[0166] Additional conjugation moieties In some examples, the conjugated moiety comprises an extracellular biomarker. In some examples, the extracellular biomarker is a tumor antigen. In some examples, exemplary extracellular biomarkers include CD19, PSMA, B7-H3, B7-H6, CD70, CEA, CSPG4, EGFRvIII, EphA3, EpCAM, EGFR, ErbB2 (HER2), FAP, FRα, GD2, GD3, Lewis-Y, mesothelin, Muc1, Muc16, ROR1, TAG72, VEGFR2, CD11, Gr-1, CD204, CD16, CD49b, CD4, CD8, and B220. In some examples, the conjugated moiety is bound or conjugated to a cytokine (e.g., IL-2). In some cases, the conjugated moiety is genetically fused, for example, at the N-terminus or C-terminus, of the cytokine (e.g., IL-2).

[0167] In some examples, the conjugated moiety comprises a molecule from a post-translational modification. In some examples, examples of post-translational modifications include myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), glypiation, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., the addition of an alkyl group such as a methyl or ethyl group), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycation, carbamylation, glutamylation, or deamidation. In some examples, a cytokine (e.g., IL-2) is modified by post-translational modification, such as myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), glypiation, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., the addition of an alkyl group such as a methyl or ethyl group), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycation, carbamylation, glutamylation, or deamidation.

[0168] Conjugation Linker In some embodiments, useful functional reactive groups for conjugating or attaching to a conjugate moiety of a cytokine polypeptide described herein (e.g., an IL-2 polypeptide) include, for example, zero or higher order linkers. In some examples, a non-natural amino acid incorporated into an interleukin described herein includes a functional reactive group. In some examples, the linker includes a functional reactive group that reacts with a non-natural amino acid incorporated into an interleukin described herein. In some examples, the conjugation moiety includes a functional reactive group that reacts with a non-natural amino acid incorporated into an interleukin described herein. In some examples, the conjugation moiety includes a functional reactive group that reacts with a linker described herein (optionally previously attached to a cytokine peptide). In some embodiments, the linker includes a reactive group that reacts with a natural amino acid in a cytokine peptide described herein. In some cases, the higher order linker includes a functional linker, such as a homobifunctional linker or a heterofunctional linker.Exemplary monobifunctional linkers include, but are not limited to, Lomant's reagent dithiobis(succinimidylpropionate) DSP and 3'3'-dithiobis(sulfosuccinimidyl propionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethyleneglycobis(ethyleneglyco) glycobis) (succinimidyl succinate (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido) butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene, or 1, These include 3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide).

[0169] In some embodiments, the bifunctional linker comprises a heterofunctional linker. Exemplary heterofunctional linkers include, but are not limited to, amine-reactive crosslinkers and sulfhydryl crosslinkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfoLC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide esters (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide esters (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA),Carbonyl-reactive crosslinkers and sulfhydryl-reactive crosslinkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive crosslinkers and photoreactive crosslinkers, such as N-hydro Sulfosuccinimidyl-4-azidosalicylate (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NH-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD ... Phosuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyl peroxysuccinimidyl (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)-1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyl diazopyruvate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive crosslinkers and photoreactive crosslinkers,For example, 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide, carbonyl-reactive and photoreactive crosslinkers, such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers, such as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers, such as ρ-azidophenylglyoxal (APG).

[0170] In some examples, the reactive functional group comprises a nucleophilic group that reacts with an electrophilic group present on the binding moiety (e.g., the conjugated moiety, or IL-2). Exemplary electrophilic groups include carbonyl groups, such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acid halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0171] In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is a dipeptide linker. In some embodiments, the dipeptide linker is valine-citrulline (Val-Cit), phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), and valine-lysine (Val-Lys). In some embodiments, the dipeptide linker is valine-citrulline.

[0172] In some embodiments, the linker is a peptide-linker that contains, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In some examples, the peptide-linker contains up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or fewer amino acids. In some cases, the peptide linker contains at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[0173] In some embodiments, the linker comprises a self-immolative linker moiety. In some embodiments, the self-immolative linker moiety comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), or derivatives or analogs thereof. In some embodiments, the linker comprises a dipeptide linker moiety and a self-immolative linker moiety. In some embodiments, the self-immolative linker moiety is such as those described in U.S. Pat. No. 9,089,614 and WO2015038426.

[0174] In some embodiments, the cleavable linker is a glucuronide. In some embodiments, the cleavable linker is an acid cleavable linker. In some embodiments, the acid cleavable linker is a hydrazine. In some embodiments, the cleavable linker is a reducible linker.

[0175] In some embodiments, the linker comprises a maleimide group. In some instances, the maleimide group is also referred to as a maleimide spacer. In some instances, the maleimide group comprises caproic acid to form maleimidocaproyl (mc). In some instances, the linker comprises maleimidocaproyl (mc). In some instances, the linker is maleimidocaproyl (mc). In other instances, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC) as described above.

[0176] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some examples, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) that incorporates a basic amino group adjacent to the maleimide to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby precluding the maleimide from undergoing elimination via a retro-Michael reaction. In some examples, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol. 32(10):1059-1062 (2014). In some examples, the linker comprises a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0177] conjugation chemistry A variety of conjugation reactions are used to conjugate the linkers, conjugation moieties, and unnatural amino acids incorporated into the cytokine peptides described herein. Such conjugation reactions are often compatible with aqueous conditions, such as "bio-orthogonal" reactions. In some embodiments, the conjugation reaction is mediated by a chemical reagent, such as a catalytic, light, or reactive chemical group found on the linker, conjugation moiety, or unnatural amino acid. In some embodiments, the conjugation reaction is mediated by an enzyme. In some embodiments, the conjugation reaction used herein is described in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, the conjugation reaction used herein is described in Chen, X.; Wu. YW. Org. Biomol. Chem. 2016, 14, 5417.

[0178] In some embodiments described herein, the conjugation reaction comprises the reaction of a nucleophile with a ketone or aldehyde. In some embodiments, the conjugation reaction comprises the reaction of an aminoxy group with a ketone to form an oxime. In some embodiments, the conjugation reaction comprises the reaction of an aryl or heteroaryl with a ketone with an amine group to form an imine. In some embodiments, the conjugation reaction comprises the reaction of an aryl or heteroaryl with an amine group with an aldehyde to form an imine. In some embodiments, the conjugation reaction described herein results in a cytokine peptide that includes a linker or conjugation moiety that is attached via an oxime. In some embodiments, the conjugation reaction comprises the Pictet-Spengler reaction of an aldehyde or ketone with a tryptamine nucleophile. In some embodiments, the conjugation reaction comprises a hydrazino-Pictet-Spengler reaction. In some embodiments, the conjugation reaction comprises a Pictet-Spengler ligation.

[0179] In some embodiments described herein, the conjugation reaction described herein involves the reaction of an azide and a phosphine (Staudinger ligation). In some embodiments, the phosphine is an aryl phosphine. In some embodiments, the aryl phosphine includes an orthoester group. In some embodiments, the phosphine includes the structure methyl 2-(diphenylphosphanyl)benzoate. In some embodiments, the conjugation reaction described herein results in a cytokine peptide that includes a linker or conjugation moiety that is attached via an aryl amide. In some embodiments, the conjugation reaction described herein results in a cytokine peptide that includes a linker or conjugation moiety that is attached via an amide.

[0180] In some embodiments described herein, the conjugation reaction described herein comprises a 1,3-dipolar cycloaddition reaction. In some embodiments, the 1,3-dipolar cycloaddition reaction comprises the reaction of an azide and a phosphine (a "click" reaction). In some embodiments, the conjugation reaction is catalyzed by copper. In some embodiments, the conjugation reaction described herein results in a cytokine peptide comprising a linker or conjugation moiety attached via a triazole. In some embodiments, the conjugation reaction described herein comprises the reaction of an azide with a strained olefin. In some embodiments, the conjugation reaction described herein comprises the reaction of an azide with a strained alkyne. In some embodiments, the conjugation reaction described herein comprises the reaction of an azide with a cycloalkyne, such as OCT, DIFO, DIFBO, DIBO, BARAC, TMTH, or other strained cycloalkyne, the structures of which are shown in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, the 1,3-dipolar cycloaddition reaction is catalyzed by light ("photoclick"). In some embodiments, the conjugation reaction described herein comprises the reaction of tetrazole and light with a terminal allyl group. In some embodiments, the conjugation reaction described herein comprises the reaction of tetrazole and light with a terminal alkynyl group. In some embodiments, the conjugation reaction described herein comprises the reaction of tetrazine and light with an O-allyl amino acid. In some embodiments, the conjugation reaction described herein comprises the reaction of tetrazine and light with an O-allyl tyrosine.

[0181] In some embodiments described herein, the conjugation reactions described herein involve an inverse electron demand cycloaddition reaction involving a diene and a dienophile. In some embodiments, the diene comprises a tetrazine. In some embodiments, the dienophile comprises an alkene. In some embodiments, the dienophile comprises an alkyne. In some embodiments, the alkyne is a strained alkyne. In some embodiments, the alkene is a strained diene. In some embodiments, the alkyne is trans-cyclooctene. In some embodiments, the alkyne is cyclooctene. In some embodiments, the alkene is a cyclopropene. In some embodiments, the alkene is a fluorocyclopropene. In some embodiments, the conjugation reactions described herein result in the formation of a cytokine peptide that is attached to a linker or conjugation moiety via a 6-membered heterocycle that contains two nitrogen atoms in the ring.

[0182] In some embodiments described herein, the conjugation reaction described herein comprises an olefin metathesis reaction. In some embodiments, the conjugation reaction described herein comprises the reaction of a ruthenium catalyst with an alkene and an alkyne. In some embodiments, the conjugation reaction described herein comprises the reaction of a ruthenium catalyst with two alkenes. In some embodiments, the conjugation reaction described herein comprises the reaction of a ruthenium catalyst with two alkynes. In some embodiments, the conjugation reaction described herein comprises the reaction of an alkene or alkyne with a ruthenium catalyst and an amino acid that comprises an allyl group. In some embodiments, the conjugation reaction described herein comprises the reaction of an alkene or alkyne with a ruthenium catalyst and an amino acid that comprises an allyl sulfide or selenide. In some embodiments, the ruthenium catalyst is a second generation Hoveda-Grubbs catalyst. In some embodiments, the olefin metathesis reaction comprises the reaction of one or more strained alkenes or alkynes.

[0183] In some embodiments described herein, the conjugation reaction described herein comprises a cross-coupling reaction. In some embodiments, the cross-coupling reaction comprises a transition metal catalyst, such as iridium, gold, ruthenium, rhodium, palladium, nickel, platinum or other transition metal catalyst and one or more ligands. In some embodiments, the transition metal catalyst is water-soluble. In some embodiments described herein, the conjugation reaction described herein comprises a Suzuki-Miyaura cross-coupling reaction. In some embodiments described herein, the conjugation reaction described herein comprises a reaction of an aryl halide (or triflate, or tosylate), an aryl or alkenyl boronic acid, and a palladium catalyst. In some embodiments described herein, the conjugation reaction described herein comprises a Sonogashira coupling reaction. In some embodiments described herein, the conjugation reaction described herein comprises a reaction of an aryl halide (or triflate, or tosylate), an alkyne, and a palladium catalyst. In some embodiments, the cross-coupling reaction results in the attachment of a linker or conjugation moiety to the cytokine peptide via a carbon-carbon bond.

[0184] In some embodiments described herein, the conjugation reactions described herein include a deprotection or "uncaging" reaction of the reactive group prior to conjugation. In some embodiments, the conjugation reactions described herein include uncaging the reactive group with light, followed by a conjugation reaction. In some embodiments, the reactive group is protected by an aralkyl moiety that includes one or more nitro groups. In some embodiments, uncaging the reactive group results in a free amine, sulfide, or other reactive group. In some embodiments, the conjugation reactions described herein include uncaging the reactive group with a transition metal catalyst, followed by a conjugation reaction. In some embodiments, the transition metal catalyst includes palladium and one or more ligands. In some embodiments, the reactive group is protected by an allyl moiety. In some embodiments, the reactive group is protected by an allyl carbamate. In some embodiments, the reactive group is protected by a propargyl moiety. In some embodiments, the reactive group is protected by a propargyl carbamate. In some embodiments, the reactive group is protected by a dienophile, where exposure to a diene (such as a tetrazine) results in deprotection of the reactive group.

[0185] In some embodiments described herein, the conjugation reaction described herein comprises a ligand-directed reaction, in which a ligand (optionally) bound to a reactive group facilitates the site of conjugation between the reactive group and the cytokine peptide. In some embodiments, the ligand is cleaved during or after the reaction of the cytokine peptide with the reactive group. In some embodiments, the conjugation site of the cytokine peptide is a natural amino acid. In some embodiments, the conjugation site of the cytokine peptide is a lysine, cysteine, or serine. In some embodiments, the conjugation site of the cytokine peptide is a non-natural amino acid described herein. In some embodiments, the reactive group comprises a leaving group, such as an electron-deficient aryl or heteroaryl group. In some embodiments, the reactive group comprises a leaving group, such as an electron-deficient alkyl group, which is substituted by the cytokine peptide. In some embodiments, the conjugation reaction described herein comprises a reaction of a radical scavenger with a radical species. In some embodiments, the conjugation reaction described herein comprises an oxidative radical addition reaction. In some embodiments, the radical scavenger is an arylamine. In some embodiments, the radical species is a tyrosyl radical. In some embodiments, the radical species is generated by a ruthenium catalyst (such as [Ru(bpy)3]) and light.

[0186] Enzymatic reactions are optionally used in the conjugation reactions described herein. Exemplary enzymatic conjugations include SortA-mediated conjugation, TG-mediated conjugation, or FGE-mediated conjugation. In some embodiments, the conjugation reactions described herein include native protein ligation (NPL) of terminal 1-amino-2-thio groups with thioesters to form amide bonds.

[0187] Various conjugation reactions for reacting a cytokine peptide with a linker or conjugation moiety are described herein, where the reaction occurs with a naturally occurring ("reference") amino acid in the cytokine peptide. In some embodiments, the naturally occurring amino acid found at the conjugation position is found in the wild-type sequence or, alternatively, the position is mutated. In some embodiments, the conjugation reaction involves the formation of a disulfide bond at a cysteine ​​residue. In some embodiments, the conjugation reaction involves a 1,4 Michael addition reaction of cysteine ​​or lysine. In some embodiments, the conjugation reaction involves cyanobenzothiazole ligation of cysteine. In some embodiments, the conjugation reaction involves cross-linking with an acetone moiety, such as 1,3-dichloro-2-propionic acid. In some embodiments, the conjugation reaction involves a 1,4 Michael addition of dehydroalanine, formed by reaction of O-mesitylenesulfonylhydroxylamine with cysteine. In some embodiments, the conjugation reaction involves reaction of a triazolinedione (TAD) with tyrosine, or a TAD derivative. In some embodiments, the conjugation reaction comprises the reaction of a rhodium carbenoid with tryptophan.

[0188] How to use Autoimmune diseases or disorders Also described herein, in some embodiments, is a method of treating an autoimmune disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) described herein. In some examples, the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide and a conjugate moiety, wherein the IL-2 conjugate has a reduced affinity for the IL-2 receptor beta (IL-2Rβ) subunit, the IL-2 receptor gamma (IL-2Rγ) subunit, or a combination thereof, compared to a wild-type IL-2 polypeptide. In some examples, the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide; and a conjugate moiety, the conjugate moiety being P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, P82, P83, P84, P85, P86, P87, P88, P89, P90, P91, P92, P93, P94, P95, P96, P97, P98, P99, P99, P100, P102, P104, P106, P108, P109, P110, P111, P112, P113, P114, P115, P116, P117, P118, P120, P122, P123, P124, P125, P126, P127, P128, P130, P132, P136, P138, P139, P140, P142, P144, P146, P148, P149, P150, P151, P152, P153, P154, P156, P158, P159, P160, P162, P163, P1 , R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:1. In some examples, the amino acid residues are selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, L132, and T133. In some examples, the amino acid residues are selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126.In some cases, the IL-2 conjugate interacts with the IL-2Rαβγ complex but has reduced affinity for the IL-2Rβ and IL-2Rγ subunits or reduces recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex. In some cases, the modified IL-2 polypeptide maintains binding affinity for IL-2Rα compared to wild-type IL-2 polypeptide. In this case, the IL-2 / IL-2Rαβγ complex stimulates or enhances the expansion of CD4+ Treg cells. In further cases, the modified IL-2 polypeptide increases the dosage required for activation of Teff cells and / or NK cells via the IL-2Rβγ complex, thereby expanding the dosage range for activation of Treg cells via the IL-2Rαβγ complex (or expanding the therapeutic window of IL-2 for activation of Treg cells via the IL-2Rαβγ complex).

[0189] In some examples, the autoimmune disease or disorder includes alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Wegener's granulomatosis.

[0190] In some cases, the cytokine (e.g., interleukin, IFN, or TNF) conjugate is administered to a subject with alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Wegener's granulomatosis.

[0191] In some cases, the IL-2 conjugate is administered to a subject with alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Wegener's granulomatosis. In some cases, the IL-2 conjugate is administered to a subject with type 1 diabetes. In some cases, the IL-2 conjugate is administered to a subject with Graves' disease. In some cases, the IL-2 conjugate is administered to a subject with multiple sclerosis. Optionally, the IL-2 conjugate is administered to a subject with psoriasis. Optionally, the IL-2 conjugate is administered to a subject with rheumatoid arthritis. Optionally, the IL-2 conjugate is administered to a subject with Sjogren's syndrome. Optionally, the IL-2 conjugate is administered to a subject with systemic lupus erythematosus. Optionally, the IL-2 conjugate is administered to a subject with uveitis. Optionally, the IL-2 conjugate is administered to a subject with Wegener's granulomatosis.

[0192] In some cases, a cytokine conjugate (eg, an IL-2 conjugate) is administered to a subject for the treatment of graft-versus-host disease (GVHD).

[0193] In some embodiments, an additional therapeutic agent is further administered to the subject. In some cases, the additional therapeutic agent is administered simultaneously with the cytokine conjugate (e.g., an IL-2 conjugate). In other cases, the additional therapeutic agent and the cytokine conjugate (e.g., an IL-2 conjugate) are administered sequentially, e.g., the cytokine conjugate (e.g., an IL-2 conjugate) is administered prior to the additional therapeutic agent, or the cytokine conjugate (e.g., an IL-2 conjugate) is administered after administration of the additional therapeutic agent.

[0194] Exemplary additional therapeutic agents for the treatment of autoimmune diseases or disorders include, but are not limited to, corticosteroids such as prednisone, budesonide, or prednisolone; calcineurin inhibitors such as cyclosporine or tacrolimus; mTOR inhibitors such as sirolimus or everolimus; IMDH inhibitors such as azathioprine, leflunomide, or mycophenolate; biologics such as abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, or vedolizumab; and monoclonal antibodies such as basiliximab, daclizumab, or muromonab.

[0195] In some cases, the cytokine conjugate (e.g., an IL-2 conjugate) is administered with an additional therapeutic agent selected from the following: a corticosteroid, such as prednisone, budesonide, or prednisolone; a calcineurin inhibitor, such as cyclosporine or tacrolimus; an mTOR inhibitor, such as sirolimus or everolimus; an IMDH inhibitor, such as azathioprine, leflunomide, or mycophenolate; a biologic, such as abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, or vedolizumab; and a monoclonal antibody, such as basiliximab, daclizumab, or muromonab.

[0196] Methods for cell population expansion Further described herein in some embodiments is a method of expanding a Treg cell population, in some instances comprising contacting a cell with a cytokine conjugate described herein and allowing the cytokine to interact with a cytokine receptor to form a complex, wherein the complex stimulates the expansion of a distinct lymphocyte population.

[0197] In some embodiments, a method of expanding a CD4+ regulatory T (Treg) cell population comprises contacting cells with the isolated and modified IL-2 polypeptide described above for a time sufficient to induce the formation of a complex with IL-2Rαβγ, thereby stimulating the expansion of the Treg cell population. In some embodiments, a method of expanding a CD4+ Treg cell population comprises (a) contacting cells with an IL-2 conjugate described herein; and (b) allowing the IL-2 conjugate to interact with IL-2Rα, IL-2Rβ, and IL-2Rγ to form an IL-2 / IL-2Rαβγ complex; wherein the IL-2 conjugate has reduced affinity for the IL-2Rβ and IL-2Rγ subunits, reducing recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex, and the IL-2 / IL-2Rαβγ complex stimulates the expansion of Treg cells. In some examples, the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide; and a conjugate moiety, the conjugate moiety being P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, P82, P83, P84, P85, P86, P87, P88, P89, P90, P91, P92, P93, P94, P95, P96, P97, P98, P99, P99, P100, P102, P104, P106, P108, P109, P110, P111, P112, P113, P114, P115, P116, P117, P118, P120, P122, P123, P124, P125, P126, P127, P128, P130, P132, P136, P138, P139, P140, P142, P144, P146, P148, P149, P150, P151, P152, P153, P154, P156, P158, P159, P160, P162, P163, P1 , R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:1.In some examples, the amino acid residues are selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, L132, and T133. In some examples, the amino acid residues are selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, and T113. In some examples, the amino acid positions are selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126. In some examples, the amino acid positions are selected from K8, K9, and H16. In some examples, the amino acid positions are selected from Q22, N26, N88, and Q126. In some examples, the amino acid positions are selected from E15, D20, D84, and E95. In some examples, the amino acid positions are selected from L12, L19, M23, and F78. In some examples, the amino acid positions are selected from Q22 and N26.

[0198] In some instances, the IL-2 conjugates expand CD4+ T regulatory (Treg) cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. In some instances, the IL-2 conjugates expand CD4+ T regulatory (Treg) cells by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more.

[0199] In some cases, the IL-2 conjugate expands CD4+ T regulatory (Treg) cells at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 1000x, or more. In some cases, the IL-2 conjugate expands CD4+ T regulatory (Treg) cells by about 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 1000x, or more.

[0200] In some examples, the time sufficient to induce the formation of a complex with IL-2Rαβγ is at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In some examples, the time sufficient to induce the formation of a complex with IL-2Rαβγ is about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0201] In some embodiments, the method is an in vivo method.

[0202] In another embodiment, the method is an in vitro method.

[0203] In a further embodiment, the method is an ex vivo method.

[0204] Cytokine Polypeptide Production In some instances, the cytokine (e.g., interleukin, IFN, or TNF) polypeptides described herein are recombinantly produced or chemically synthesized, including either naturally occurring or non-naturally occurring amino acid mutations. In some instances, the cytokine (e.g., IL-2) polypeptides described herein are produced recombinantly, e.g., by a host cell system or in a cell-free system.

[0205] In some instances, the cytokine (e.g., IL-2) polypeptide is recombinantly produced via a host cell system. In some instances, the host cell is a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell, or a plant cell) or a prokaryotic cell (e.g., a gram-positive or gram-negative bacteria). In some instances, the eukaryotic host cell is a mammalian host cell. In some instances, the mammalian host cell is a stable cell line or a cell line that has the ability to integrate the genetic material of interest into its own genome and express the product of the genetic material after many generations of cell division. In other instances, the mammalian host cell is a transient cell line or a cell line that has not integrated the genetic material of interest into its own genome and has the ability to express the product of the genetic material after many generations of cell division.

[0206] Exemplary mammalian host cells include, but are not limited to, 293T cell line, 293A cell line, 293FT cell line, 293F cells, 293H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F™ cells, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™ 3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp-In™-Jurkat cell line. , FreeStyle™ 293F cells, FreeStyle™ CHO-S cells, GripTite™ 293MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line.

[0207] In some embodiments, the eukaryotic host cell is an insect host cell. Exemplary insect host cells include Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

[0208] In some embodiments, the eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strains such as INVSc1.

[0209] In some embodiments, the eukaryotic host cell is a plant host cell. In some examples, the plant cell comprises a cell from algae. Exemplary plant cell lines include Chlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC 7942.

[0210] In some embodiments, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0211] In some instances, polynucleic acid molecules or vectors suitable for producing the IL-2 polypeptides described herein include suitable vectors derived from eukaryotic or prokaryotic sources. Exemplary polynucleic acid molecules or vectors include vectors derived from bacteria (e.g., E. coli), insects, yeast (e.g., Pichia pastoris), algae, or mammals. Bacterial vectors include, for example, pACYC177, pASK75, pBAD sequence vector system, pBADM sequence vector system, pET sequence vector system, pETM sequence vector system, pGEX sequence vector system, pHAT, pHAT2, pMal-c2, pMal-p2, pQE sequence vector system, pRSET A, pRSET B, pRSET C, pTrcHis2 system, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0212] Insect vectors include, for example, FLAG vectors such as pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, pPolh-FLAG1, or pPolh-MAT2, or MAT vectors such as pPolh-MAT1, or pPolh-MAT2.

[0213] Yeast vectors include, for example, a Gateway® pDEST™ 14 vector, a Gateway® pDEST™ 15 vector, a Gateway® pDEST™ 17 vector, a Gateway® pDEST™ 24 vector, a Gateway® pYES-DEST52 vector, a pBAD-DEST49 Gateway® destination vector, a pAO815 Pichia vector, a pFLD1 Pichia pastoris vector, a pGAPZA, B, and C Pichia pastoris vector, a pPIC3.5K Pichia vector, a pPIC6 A, B, and C Pichia vector, a pPIC9K Pichia vector, pTEF1 / Zeo, a pYES2 yeast vector, a pYES2 / CT yeast vector, a pYES2 / NT A, B, and C yeast vector, or a pYES3 / CT yeast vector.

[0214] Algal vectors include, for example, pChlamy-4 vectors or MCS vectors.

[0215] Mammalian vectors include, for example, transient expression vectors or stable expression vectors.Typical mammalian transient expression vectors include p3xFLAG-CMV8, pFLAG-Myc-CMV19, pFLAG-Myc-CMV23, pFLAG-CMV2, pFLAG-CMV6a,b,c, pFLAG-CMV5.1, pFLAG-CMV5a,b,c, p3xFLAG-CMV7.1, pFLAG-CMV20, p3xFLAG-Myc-CMV24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV3, or pBICEP-CMV4. Exemplary mammalian stable expression vectors include pFLAG-CMV3, p3xFLAG-CMV9, p3xFLAG-CMV13, pFLAG-Myc-CMV21, p3xFLAG-Myc-CMV25, pFLAG-CMV4, p3xFLAG-CMV10, p3xFLAG-CMV14, pFLAG-Myc-CMV22, p3xFLAG-Myc-CMV26, pBICEP-CMV1, or pBICEP-CMV2.

[0216] In some instances, cell-free systems are used for the production of cytokine (e.g., IL-2) polypeptides described herein. In some instances, cell-free systems contain a mixture of cytoplasmic and / or nuclear components from cells and are suitable for in vitro nucleic acid synthesis. In some instances, cell-free systems utilize prokaryotic cell components. In other instances, cell-free systems utilize eukaryotic cell components. Nucleic acid synthesis is obtained in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, archaea, or HeLa cells. Exemplary cell-free systems include E. coli S30 Extract system, E. coli T7 S30 system, or PURExpress®, XpressCF, and XpressCF+.

[0217] Cell-free translation systems may include a variety of components, such as plasmids, mRNA, DNA, tRNA, synthetases, release factors, ribosomes, chaperone proteins, translation initiation and elongation factors, natural and / or unnatural amino acids, and / or other components used for protein expression. Such components are optionally modified to improve yield, increase synthesis rate, increase fidelity of the protein product, or incorporate unnatural amino acids. In some embodiments, the cytokines described herein are synthesized using a cell-free translation system described in US8,778,631; US2017 / 0283469; US2018 / 0051065; US2014 / 0315245; or US8,778,631. In some embodiments, the cell-free translation system includes modified release factors or even removal of one or more release factors from the system. In some embodiments, the cell-free translation system includes a reduced protease concentration. In some embodiments, the cell-free translation system includes a modified tRNA that includes a rearranged codon used to encode the unnatural amino acid. In some embodiments, the synthetases described herein are used in the cell-free translation system for the incorporation of the unnatural amino acid. In some embodiments, the tRNA is preloaded with the unnatural amino acid using enzymatic or chemical methods prior to addition to the cell-free translation system. In some embodiments, the components of the cell-free translation system are obtained from modified organisms, such as modified bacteria, yeast, or other organisms.

[0218] In some embodiments, the cytokine (eg, IL-2) polypeptide is produced as a circularly permuted form, either via an expression host system or through a cell-free system.

[0219] Production of cytokine polypeptides containing unnatural amino acids Orthogonal or extended genetic codes can be used in the present disclosure, where one or more particular codons present in a nucleic acid sequence of a cytokine (e.g., IL-2) polypeptide are assigned to encode an unnatural amino acid such that the unnatural amino acid can be genetically incorporated into the cytokine (e.g., IL-2) through the use of an orthogonal tRNA synthetase / tRNA pair that can load a tRNA with the unnatural amino acid and incorporate the unnatural amino acid into the polypeptide chain depending on the codon.

[0220] In some instances, the codon is an amber, ochre, opal, or quadruplet codon. In some instances, the codon corresponds to an orthogonal tRNA used to deliver an unnatural amino acid. In some instances, the codon is an amber. In other instances, the codon is an orthogonal codon.

[0221] In some instances, the codon is a quadruplet codon and can be decoded by the orthogonal ribosome ribo-Q1. In some instances, the quadruplet codon is as described in Neumann, et al., "Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome," Nature, 464(7287): 441-444 (2010).

[0222] In some examples, the codons used in the present disclosure are recoded codons, e.g., synonymous or rare codons replaced with alternative codons. In some cases, the recoded codons are as described in Napolitano, et al., "Emergent rules for codon choice elucidated by editing rare argine codons in Escherichia coli," PNAS, 113(38): E5588-5597 (2016). In some cases, the recoded codons are as described in Ostrov et al., "Design, synthesis, and testing toward a 57-codon genome," Science 353(6301): 819-822 (2016).

[0223] In some instances, unnatural nucleic acids are utilized to effect incorporation of one or more unnatural amino acids into a cytokine (e.g., IL-2). Exemplary unnatural nucleic acids include, but are not limited to, the following: uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-9-yl, 5-methylcytosine (5-me-2), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil ... uracil and cytosine, 6-azo-uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine. Certain unnatural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, which increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines. , including: 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil, 5-halocytosine,5-propynyl (-C≡C-CH3) uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines, 5-halo, especially 5-bro, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-aza Guanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidines ([5,4-b][1,4]benzoxazin-2(3H)-ones), phenothiazine cytidines (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-ones), G-clamps, phenoxazine cytidines (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-ones), carbazole cytidines (2H -pyrimido[4,5-b]indol-2-one), pyridoindolcytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or pyrimidine base is replaced by another heterocyclic compound, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluoropyrimidine, fluoropyrimidine, fluorouracil cytosine, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, andU.S. Patent Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882; 5,093,232; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177 Nos. 5,525,711;5,552,540;5,587,469;5,594,121;5,596,091;5,614,617;5,645,985;5,681,941;5,750,692;5,763,588;5,830,653 and 6,005,096;WO2008 / 116064;Kandimalla et al., (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858- 859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu Eds., CRC Press, 1993, 273-288. Additional base modifications can be found, for example, in U.S. Pat. No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993.

[0224] Non-natural nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and nucleic acids may optionally contain one or more heterocyclic bases apart from the five main base components of naturally occurring nucleic acids. For example, heterocyclic bases may optionally contain uracil-5-yl, cytosine-5-yl, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-8-yl, 4-aminopyrrolo[2.3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2.3-d]pyrimidin-3-yl groups, where the purine is linked to the sugar moiety of the nucleic acid via the 9 position, to the pyrimidine via the 1 position, to the pyrrolopyrimidine via the 7 position, and to the pyrazolopyrimidine via the 1 position.

[0225] In some embodiments, the nucleotide analogs are also modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those with modifications at the bond between two nucleotides and include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thiophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. It is understood that these phosphate or modified phosphate bonds between two nucleotides are via 3'-5' or 2'-5' bonds, and the bonds include reversed polarity, such as 3'-5' to 5'-3', or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Numerous U.S. patents teach methods for making and using nucleotides containing modified phosphates, including, but not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286, No. 717; No. 5,321,131; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,306; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; and No. 5,625,050.

[0226] In some embodiments, non-naturally occurring nucleic acids include 2',3'-dideoxy-2',3'-didehydro-nucleosides (PCT / US2002 / 006460), 5'-substituted DNA and RNA derivatives (PCT / US2011 / 033961; Saha et al., J. Org Chem., 1995, 60, 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al., 1995, 14(3-5), 901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, 87, 3004-3020; PCT / JP2000 / 004720; PCT / JP2003 / 002342; PCT / JP2004 / 013216; PCT / JP2005 / 020435; PCT / JP2006 / 315479; PCT / JP2006 / 324484; PCT / JP2009 / 056718; PCT / JP2010 / 067560), or 5'-substituted monomers made as monophosphates with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337).

[0227] In some embodiments, non-natural nucleic acids contain modifications at the 5'- and 2'-positions of the sugar backbone (PCT / US94 / 02993), such as 5'-CH2-substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., Bioconjugate Chem. 1999, 10, 921-924). In some cases, non-natural nucleic acids contain amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, where the 3'-linked nucleoside in the dimer (5-3') contains a 2'-OCH3 and a 5'-(S)-CH3 (Mesmaeker et al., Synlett, 1997, 1287-1290). Non-natural nucleic acids may include 2'-substituted 5'-CH2 (or O) modified nucleosides (PCT / US92 / 01020). Non-natural nucleic acids may include 5'-methylene phosphonate DNA and RNA monomers and dimers (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwood et al., Synlett, 1995, 7, 703-705; and Hutter et al., Helvetica Chimica Acta, 2002, 85, 2777-2806). Non-natural nucleic acids may include 5'-phosphonate monomers with 2'-substitutions (US2006 / 0074035) and other modified 5'-phosphonate monomers (WO1997 / 35869). The non-naturally occurring nucleic acids may contain 5'-modified methylene phosphonate monomers (EP614907 and EP629633).Non-naturally occurring nucleic acids can include 5' or 6' phosphonate ribonucleoside analogs containing hydroxyl groups at the 5' and / or 6' positions (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al., J. Med. Chem., 1976, 19(8), 1029-1033). Non-natural nucleic acids can include 5'-phosphonate deoxyribonucleoside monomers and dimers having a 5'-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Non-natural nucleic acids include nucleosides having a 6'-phosphonate group, where the 5' and / or 6' positions are unsubstituted or substituted with a thio-tert-butyl group (SC(CH3)3) (and its analogs); a methyleneamino group (CH2NH2) (and its analogs), or a cyano group (CN) (and its analogs) (Fairhurst et al., Synlett, 2001, 4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et al., J. Med. Chem., 1987, 30, 888-894; Hampton et al., J. Med. Chem., 2003, 10, 1179-1184). 1976, 19, 1371-1377;Geze et al., J. Am. Chem. Soc, 1983, 105(26), 7638-7640; and Hampton et al., J. Am. Chem. Soc, 1973, 95(13), 4404-4414).

[0228] In some embodiments, the non-natural nucleic acids also include modifications to the sugar moiety. In some cases, the nucleic acid includes one or more nucleosides with modified sugar groups. Such sugar-modified nucleosides may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, the nucleic acid includes a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (5' and / or 2' substituents; bridging of two ring atoms to form bicyclic nucleic acids (BNAs); the modification of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R=H, C1-C 12 Examples of chemically modified sugars can be found in WO2008 / 101157, US2005 / 0130923, and WO2007 / 134181.

[0229] In some examples, modified nucleic acids contain modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be a pentose group, a deoxypentose group, a hexose group, a deoxyhexose group, a glucose group, an arabinose group, a xylose group, a lyxose group, or a sugar "analog" cyclopentyl group. The sugar can be in the form of a pyranosyl or a furanosyl. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar can be attached to each heterocyclic base in either the [α] or [β] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy or amino-RNA / DNA chimeras. For example, sugar modifications can include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. Preparations of these sugars or sugar analogs, and the respective "nucleosides" in which such sugars or analogs are attached to a heterocyclic base (nucleobase), are known. Sugar modifications can also be made and combined with other modifications.

[0230] Modifications of the sugar moiety include natural modifications of ribose and deoxyribose as well as non-natural modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; O-alkynyl, S-alkynyl, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6 alkyl groups. 10 Alkyl, or C2-C 10 The 2' sugar modifications can also include, but are not limited to, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) nCH3, -O(CH2) n ONH2, and O(CH2) n ON [(CH2) n CH3]2, where n and m are from 1 to about 10.

[0231] Other modifications at the 2' position include, but are not limited to: C1 to C 10Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, specifically the 3' position of the sugar in the 3' terminal nucleotide or 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. There are numerous United States patents which teach the preparation of such modified sugar structures and which detail and explain the range of base modifications, e.g., U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,6 Nos. 58,873; 5,670,633; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,681,941; and 5,700,920, each of which is incorporated herein by reference in its entirety.

[0232] Examples of nucleic acids with modified sugar moieties include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3 substituents. Substituents at the 2' position also include allyl, amino, azido, thio, O-allyl, O-(C1-C2)-OCH3 ... 10 alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ) can be selected from each R m and R n are independently H or substituted or unsubstituted C1-C 10 It is an alkyl.

[0233] In certain embodiments, the nucleic acids provided herein include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acids include a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the nucleic acids provided herein include one or more bicyclic nucleic acids, where the bridge includes a 4' to 2' bicyclic nucleic acid. Examples of such 4' to 2' bicyclic nucleic acids include, but are not limited to, one of the following formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2' and analogs thereof (see U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' and analogs thereof (WO2009 / 006478, WO2008 / 150729, US2004 / 0171570, U.S. Pat. No. 7,427,672, Chattopadhyaya et al., J. Org. Chem., 209, 74, 118-134, and WO2008 / 154401).Furthermore, e.g. Singh et al., Chem. Commun., 1998, 4, 455-456;Koshkin et al., Tetrahedron, 1998, 54, 3607-3630;Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638;Kumar et al. al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222;Singh et al., J. Org. Chem., 1998, 63, 10035-10039;Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379;Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561;Braasch et al., Chem. Biol, 2001, 8, 1-7;Oram et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patent Nos. 4,849,513; 5,015,733; 5,118,800; 5,118,802; 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; 6,525,191; 6,670,461; and 7,399,845; International Publication Nos. WO2004 / 106356, WO1994 / 14226, WO2005 / 021570, WO2007 / 090071, and WO2007 / 134181; U.S. Patent Publication Nos. 4,849,513; 5,015,733; 5,118,800; 5,118,802; 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; 6,525,191; 6,670,461; and 7,399,845; International Publication Nos. WO2004 / 106356, WO1994 / 14226, WO2005 / 021570, WO2007 / 090071, and WO2007 / 134181; See publication numbers US2004 / 0171570, US2007 / 0287831, and US2008 / 0039618; U.S. Provisional Patent Applications Nos. 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844; and International Application Nos. PCT / US2008 / 064591, PCT / US2008 / 066154, PCT / US2008 / 068922, and PCT / DK98 / 00393.

[0234] In certain embodiments, the nucleic acid comprises a linked nucleic acid. Nucleic acids can be linked together using any internucleic acid linkage. Two major classes of internucleic acid linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing internucleic acid linkages include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thiocarbamates (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N * -Dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, internucleic acid linkages with chiral atoms can be prepared as racemic mixtures, separate enantiomers, such as alkylphosphonates and phosphorothioates. Non-natural nucleic acids can contain a single modification. Non-natural nucleic acids can contain multiple modifications within one of the moieties or between different moieties.

[0235] Backbone phosphate modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or non-bridged), phosphotriesters, phosphorodithioates, phosphodithioates, and boranophosphates, and may be used in any combination. Other non-phosphate linkages may also be used.

[0236] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) can confer immunomodulator activity to the modified nucleic acids and / or enhance their stability in vivo.

[0237] In some instances, the phosphorus derivative (or modified phosphate group) is attached to a sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, or the like. Exemplary polynucleotides containing modified phosphate or non-phosphate linkages can be found in: Peyrottes et al., 1996, Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res. 24:2318-2323; and Schultz et al., (1996) Nucleic Acids Res. 24:2966-2973; Matteucci, 1997, "Oligonucleotide Analogs: an Overview" in Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, ed.) John Wiley and Sons, New York, NY; Zon, 1993, "Oligonucleoside Phosphorothioates" in Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp. 165-190; Miller et al., 1971, JACS 93:6657-6665; Jager et al., 1988, Biochem. 27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; U.S. Patent No. 5,453,496; and Micklefield, 2001, Curr. Med. Chem. 8: 1157-1179.

[0238] In some cases, backbone modifications include replacing phosphodiester bonds with alternative moieties, such as anionic, neutral, or cationic groups. Examples of such modifications include: anionic internucleoside linkages; phosphoramidate modifications of N3' to P5'; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonate; amide-linked DNA; methylene (methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNAs); and positively charged deoxyribonucleic guanidine (DNG) oligos (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179). Modified nucleic acids may include chimeric or mixed backbones containing one or more modifications, such as a combination of phosphate linkages, such as a combination of phosphodiester and phosphorothioate linkages.

[0239] Phosphate substituents include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These may include those with morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and methylenethioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others with mixed N, O, S, and CH2 moieties. Numerous U.S. patents disclose methods of making and using these types of phosphate substitutes, including, but not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,47 These include Nos. 0,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439. It is also understood in the nucleotide substitution that both the sugar and phosphate moieties of the nucleotide can be replaced, for example, by amide-type bonds (aminoethylglycine) (PNA). U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is incorporated herein by reference. Also, Nielsen et al., Science, 1991, 254, 1497-1500. Additionally, other types of molecules (conjugates) can be attached to the nucleotide or nucleotide analogue, for example to enhance cellular uptake. The conjugates can be chemically attached to the nucleotide or nucleotide analogue. Such conjugates include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.Chem. Let., 1994, 4, 1053-1060), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterols (Oberhauser et al., Nucl. Acids Res., 1992, 20, 2765-2770), and the like. 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EM5OJ, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-SH-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Numerous U.S. patents teach the preparation of such conjugates, including, but not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109, No. 124; No. 5,118,802; No. 5,138,045; No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587 ,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904 ,582;No.4,958,013;No.5,082,830;No.5,112,963;No.5,214,136;No.5,082,830;No.5,112,963;No.5,214,136;5,24 No. 5,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241; 5,39 No. 1,723; No. 5,416,203; No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,810; No. 5,574,142; No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599,928; and No. 5,688,941.

[0240] In some cases, the unnatural nucleic acid further forms unnatural base pairs. Exemplary unnatural nucleotides capable of forming unnatural DNA or RNA base pairs (UBPs) under in vivo conditions include, but are not limited to, 5SICS, d5SICS, NAM, dNaM, and combinations thereof. In some embodiments, the unnatural nucleotides include:

[0241] [ka]

[0242] In some embodiments, the unnatural base pairs generate unnatural amino acids as described in Dumas et al., "Designing logical codon reassignment-Expanding the chemistry in biology," Chemical Science, 6: 50-69 (2015).

[0243] Host cells into which the constructs or vectors disclosed herein are introduced are cultured or maintained in a suitable medium such that tRNA, tRNA synthetase, and a protein of interest are produced, the medium further comprising an unnatural amino acid such that the protein of interest incorporates the unnatural amino acid.

[0244] Orthogonal tRNA synthetase / tRNA pairs can charge tRNA with unnatural amino acids and incorporate unnatural amino acids into a polypeptide chain depending on the codon. Exemplary aaRS-tRNAs include, but are not limited to, Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pair, E. coli TyrRS (Ec-Tyr) / B. stearothermophilus tRNA CUA vs. E. coli LeuRS(Ec-Leu) / B.stearothermophilus tRNA CUA pair, and pyrrolysyl-tRNA pair.

[0245] A cytokine (e.g., IL-2) polypeptide containing an unnatural amino acid is prepared by introducing into a host cell a nucleic acid construct as described herein, which contains a tRNA and a tRNA synthetase and contains a nucleic acid sequence of interest with one or more in-frame orthogonal (stop) codons. The host cell is exposed to a physiological solution containing the unnatural amino acid, and the host cell is then maintained under conditions that allow expression of the protein of the coding sequence of interest. The unnatural amino acid is incorporated into the polypeptide chain in response to a codon. For example, one or more unnatural amino acids are incorporated into a cytokine (e.g., IL-2) polypeptide. Alternatively, two or more unnatural amino acids can be incorporated into a cytokine (e.g., IL-2) polypeptide at two or more sites in the protein.

[0246] If multiple unnatural amino acids are to be incorporated into a cytokine (e.g., IL-2) polypeptide, multiple codons must be incorporated into the encoding nucleic acid sequence at the desired positions so that the tRNA synthetase / tRNA pair can direct the incorporation of the unnatural amino acids in response to the codons. At least one, two, three, four or more codons encoding a nucleic acid can be incorporated into the nucleic acid sequence of interest.

[0247] When it is desired to incorporate more than one type of unnatural amino acid into a protein of interest into a single protein, a second or additional orthogonal tRNA-tRNA synthetase pair is used to incorporate the second or additional unnatural amino acid; where appropriate, the second or additional orthogonal tRNA-tRNA synthetase pair recognizes different codons in the nucleic acid encoding the protein of interest such that two or more unnatural amino acids can be specifically incorporated into different defined sites in the protein in a single production step. In certain embodiments, two or more orthogonal tRNA-tRNA synthetase pairs may therefore be used.

[0248] Once a cytokine (e.g., IL-2) polypeptide incorporating an unnatural amino acid is produced in a host cell, it can be extracted therefrom by a variety of techniques known in the art, such as enzymatic, chemical, and / or osmotic lysis, and physical disruption. The cytokine (e.g., IL-2) polypeptide can be purified by standard techniques known in the art, such as preparative chromatography, affinity purification, or any other suitable technique.

[0249] Suitable host cells include bacterial cells (e.g., E. coli), but most suitably the host cell is a eukaryotic cell (e.g., an insect cell (e.g., a Drosophila melanogaster, for example), yeast, a nematode (e.g., a C. elegans), a mouse (e.g., a Mus musculus), or a mammalian cell (such as a Chinese hamster ovary cell (CHO) or COS cell, a human 293T cell, a HeLa cell, a NIH 3T3 cell, and a mouse erythroleukemia (MEL) cell), or a human cell or other eukaryotic cell. Other suitable host cells will be known to those of skill in the art. Where appropriate, the host cell is a mammalian cell, such as a human cell or an insect cell.

[0250] Other suitable host cells that may be commonly used in embodiments of the present invention are those described in the Examples section. Vector DNA may be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of well-recognized techniques for introducing foreign nucleic acid molecules (e.g., DNA) into host cells, such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are well known in the art.

[0251] When creating a cell line, it is usually preferred to prepare a stable cell line. For stable transfection of mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on, for example, the expression vector and transfection technique used. To identify and select these integrants, a gene encoding a selection marker (e.g., for antibiotic resistance) is generally introduced into the host cell together with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin, or methotrexate. The nucleic acid molecule encoding the selection marker can be introduced into the host cell on the same vector or on a separate vector. The cells that are stably transfected by the introduced nucleic acid molecule can be identified by drug selection (e.g., the cells that have incorporated the selection marker gene remain while other cells die).

[0252] In one embodiment, the constructs described herein are integrated into the genome of a host cell. The advantage of stable integration is that uniformity between individual cells or clones is achieved. Another advantage is that selection of the best producers can be performed. It is therefore desirable to create stable cell lines. In another embodiment, the constructs described herein are transfected into a host cell. The advantage of transfecting a construct into a host cell is that protein yields can be maximized. In one aspect, a cell is described that contains a nucleic acid construct or vector described herein.

[0253] Pharmaceutical Compositions and Formulations In some embodiments, the pharmaceutical compositions and formulations described herein are administered to a subject by multiple routes of administration, including but not limited to parenteral (e.g., intravenous, subcutaneous, intramuscular), intracerebral, oral, intranasal, buccal, topical, rectal, sublingual, or transdermal routes of administration.

[0254] In some embodiments, pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolve formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and immediate mix and controlled release formulations.

[0255] In some embodiments, the pharmaceutical formulation includes a carrier or carrier material selected based on its compatibility with the compositions disclosed herein as well as the release profile characteristics of the desired dosage form. Typical carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, etc. Pharmaceutically compatible carrier materials include, but are not limited to, gum arabic, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc. See, for example, Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975, Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980, and Pharmaceutical Dosage Forms and Drug Delivery Systems, See Seventh Ed. (Lippincott Williams & Wilkins1999).

[0256] In some cases, the pharmaceutical composition is formulated as an immunoliposome, which comprises multiple IL-2 conjugates directly or indirectly bound to the lipid bilayer of liposome.Exemplary lipids include, but are not limited to, fatty acids; phospholipids; sterols such as cholesterol; sphingolipids such as sphingomyelin; glycosphingolipids such as gangliosides, globosides and cerebrosides; surface active amines such as stearyl, oleyl and linoleylamine.In some examples, lipids comprise cationic lipids.In some examples, lipids comprise phospholipids. Exemplary phospholipids include, but are not limited to, phosphatidic acid ("PA"), phosphatidylcholine ("PC"), phosphatidylglycerol ("PG"), phosphatidylethanolamine ("PE"), phosphatidylinositol ("PI"), and phosphatidylserine ("PS"), sphingomyelin (including brain sphingomyelin), lecithin, lysolecithin, lysophosphatidylethanolamine, cerebrosides, diarachidoylphosphatidylcholine ("DAPC"), didecanoyl-L-α-phosphatidylcholine ("DDPC"), dielaidoylphosphatidylcholine ("DEPC"), dilauroylphosphatidylcholine ("DLPC"), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine ("DMPC"), dioleo ... phosphatidylcholine ("DOPC"), dipalmitoyl phosphatidylcholine ("DPPC"), distearoyl phosphatidylcholine ("DSPC"), 1-palmitoyl-2-oleoyl-phosphatidylcholine ("POPC"), diarachidoyl phosphatidylglycerol ("DAPG"), didecanoyl-L-α-phosphatidylglycerol ("DDPG"), dielaidoyl phosphatidylglycerol ("DEPG"), dilauroyl phosphatidylglycerol ("DLPG"), dilinoleoyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol ("DMPG"), dioleoyl phosphatidylglycerol ("DOPG"), dipalmitoyl phosphatidylglycerol ("DPPG"), distearoyl phosphatidylglycerol ("DSPG"),1-Palmitoyl-2-oleoyl-phosphatidylglycerol ("POPG"), Diarachidoylphosphatidylethanolamine ("DAPE"), Didecanoyl-L-α-phosphatidylethanolamine ("DDPE"), Dielaidoylphosphatidylethanolamine ("DEPE"), Dilauroylphosphatidylethanolamine ("DLPE"), Dilinoleoylphosphatidylethanolamine, Dimyristoylphosphatidylethanolamine ("DMPE"), Dioleoylphosphatidylethanolamine ("DOPE"), Dipalmitoylphosphatidylethanolamine ("DPPE"), distearoylphosphatidylethanolamine ("DSPE"), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine ("POPE"), diarachidoylphosphatidylinositol ("DAPI"), didecanoyl-L-α-phosphatidylinositol ("DDPI"), dielaidoylphosphatidylinositol ("DEPI"), dilauroylphosphatidylinositol ("DLPI"), dilinoleoylphosphatidylinositol, dimyristoylphosphatidylinositol, diisopropyl ... 1-Palmitoyl-2-oleoyl-phosphatidylinositol ("DMPI"), dioleoylphosphatidylinositol ("DOPI"), dipalmitoylphosphatidylinositol ("DPPI"), distearoylphosphatidylinositol ("DSPI"), 1-palmitoyl-2-oleoyl-phosphatidylinositol ("POPI"), diarachidoylphosphatidylserine ("DAPS"), didecanoyl-L-α-phosphatidylserine ("DDPS"), dielaidoylphosphatidylserine ("DEPS"), dilauroylphosphatidylserine ("DAL"). "DLPS"), dilinoleoylphosphatidylserine, dimyristoylphosphatidylserine ("DMPS"), dioleoylphosphatidylserine ("DOPS"), dipalmitoylphosphatidylserine ("DPPS"), distearoylphosphatidylserine ("DSPS"), 1-palmitoyl-2-oleoyl-phosphatidylserine ("POPS"), diarachidoylsphingomyelin, didecanoylsphingomyelin, dielaidoylsphingomyelin, dilauroylsphingomyelin, dilinoleoylsphingomyelin,Dimyristoyl sphingomyelin, sphingomyelin, dioleoyl sphingomyelin, dipalmitoyl sphingomyelin, distearoyl sphingomyelin, and 1-palmitoyl-2-oleoyl-sphingomyelin.

[0257] In some examples, the pharmaceutical composition further comprises a pH adjusting agent, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane, buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts required to maintain the pH of the composition in an acceptable range.

[0258] In some cases, pharmaceutical preparations contain one or more salts in an amount necessary to make the osmolality of the composition acceptable.Such salts include salts with sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite, suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0259] In some embodiments, the pharmaceutical formulation includes sugars such as, but not limited to, trehalose, sucrose, mannitol, maltose, glucose, or salts such as potassium phosphate, sodium citrate, ammonium sulfate, and / or other agents such as heparin that increase the solubility and in vivo stability of the polypeptide.

[0260] In some instances, the pharmaceutical composition further comprises a diluent, which can provide a more stable environment and thus is used to stabilize the compound.Salts dissolved in buffers (which can also control or maintain pH) are utilized as diluents in the art, including but not limited to phosphate buffered saline solutions.In certain instances, the diluent increases the bulk of the composition to facilitate compression or creates sufficient bulk for homogeneous mixing for capsule filling. Such compounds may include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®, calcium hydrogen phosphate, calcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray dried lactose, pregelatinized starch, compressible sugars such as Di-Pac® (Amstar), mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, powdered sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, and the like.

[0261] In some cases, pharmaceutical formulations include disintegrants or disintegrants to facilitate the breakup or disintegration of materials. The term "disintegrate" includes both dissolution and dispersion of the dosage form upon contact with gastrointestinal fluids. Examples of disintegrants include starches, e.g., natural starches such as corn starch or potato starch, pregelatinized starches such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, celluloses such as wood products, methyl crystalline cellulose (e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming, etc.), and the like. Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or crosslinked celluloses such as crosslinked sodium carboxymethylcellulose (Ac-Di-Sol®), crosslinked carboxymethylcellulose, or crosslinked croscarmellose, crosslinked starches such as sodium starch glycolate, crosslinked polymers such as crospovidone, crosslinked polyvinylpyrrolidone, alginates such as salts of alginic acid or sodium alginate, clays such as Veegum® HV (magnesium aluminum silicate), gums such as agar, guar, carob, karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, natural sponges, surfactants, resins such as cation exchange resins, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination with starch, and the like.

[0262] In some instances, the pharmaceutical formulation includes a filler, such as lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0263] Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein to prevent, reduce or inhibit adhesion or friction of materials. Typical lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, hydrocarbons such as mineral oil or hydrogenated vegetable oils such as, for example, hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali metal and alkaline earth metal salts, for example, aluminum, calcium, magnesium, zinc, stearic acid, sodium stearate, glycerol, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycols (e.g., PEG-4000) or methoxypolyethylene glycols such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycols, magnesium or sodium lauryl sulfate, colloidal silicas such as Syloid™, starches such as Cab-O-Sil®, corn starch, silicone oils, surfactants, and the like.

[0264] Plasticizers include compounds used to soften microencapsulation materials or film coatings, making them less brittle. Suitable plasticizers include polyethylene glycols, such as PEG300, PEG400, PEG600, PEG1450, PEG3350, and PEG800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. Plasticizers can also function as dispersing or wetting agents.

[0265] Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide.

[0266] Stabilizers include compounds such as any antioxidant, buffer, acid, preservative, etc. Exemplary stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium hydrogen phosphate anhydrous, propylene glycol, metacresol or m-cresol, zinc acetate, polysorbate-20 or Tween® 20, or trometamol.

[0267] The suspending agent may be a compound such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., the polyethylene glycol has a molecular weight of about 300 to about 6000, about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate ... cellulose, such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, and the like.

[0268] Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF). Additional surfactants include polyoxyethylene fatty acid glycerides, and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkyl ethers and alkyl phenyl ethers, e.g., Octoxynol 10, Octoxynol 40. Sometimes surfactants are included to enhance physical stability or for other purposes.

[0269] Viscosity enhancing agents include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosan, and combinations thereof.

[0270] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, Vitamin E TPGS, ammonium salts, and the like.

[0271] Treatment regimen In some embodiments, the pharmaceutical composition described herein is administered for therapeutic use. In some embodiments, the pharmaceutical composition is administered once a day, twice a day, three times a day, or more. The pharmaceutical composition is administered every day, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, or more. The pharmaceutical composition is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

[0272] If the patient's condition improves, based on the physician's judgment, administration of the composition may be given continuously, or alternatively, the dosage of the administered composition is temporarily reduced or temporarily stopped for a period of time (i.e., a "drug holiday"). In some instances, the length of the drug holiday varies between 2 days to 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions during drug holidays can be 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0273] Once improvement of the patient's disease has occurred, a maintenance dose is administered if necessary, after which the dosage or frequency of administration, or both, can be reduced, depending on the symptoms, to a level at which improvement of the disease, disorder, or condition is maintained.

[0274] In some embodiments, the amount of a given agent that corresponds to such an amount will vary depending on factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host requiring treatment, but will nevertheless be routinely determined in a manner known in the art according to the particular circumstances surrounding the case, e.g., the particular agent being administered, the route of administration, and the subject or host being treated, etc. In some instances, the desired dosage is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, e.g., as two, three, four or more sub-doses per day.

[0275] Because of the large number of variables associated with any particular treatment regimen, the foregoing ranges are merely suggestive, and significant deviations from these recommendations are not uncommon. Such dosages will vary depending on a number of variables, including but not limited to the activity of the compound being used, the disease or condition being treated, the mode of administration, the requirements of the particular subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0276] In some embodiments, toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosages for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. Dosages vary within this range depending on the dosage form used and the route of administration utilized.

[0277] Kits / Products In certain embodiments, the present specification discloses kits and products for using one or more of the methods and compositions described herein.Such kits include a carrier, package, or container that is partitioned to contain one or more containers, such as vials or tubes, each of which contains one of the separate elements used in the methods described herein.Suitable containers include, for example, bottles, vials, syringes, and test tubes.In other embodiments, the container is formed from various materials, such as glass or plastic.

[0278] The articles of manufacture provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material appropriate for the selected formulation and intended mode of administration and treatment.

[0279] For example, a container contains one or more of the cytokine (e.g., IL-2) polypeptides or cytokine (e.g., IL-2) conjugates disclosed herein, and optionally one or more pharmaceutical excipients described herein to facilitate delivery of the cytokine (e.g., IL-2) polypeptides or cytokine (e.g., IL-2) conjugates. Such kits optionally further include an identifying description or label, or instructions for use in the methods described herein.

[0280] The kit typically includes a label and / or instructions listing the contents and a package insert with instructions for use. A set of instructions is also typically included.

[0281] In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when letters, numbers, or other features forming the label are attached, molded, or etched onto the container itself, and the label is associated with the container when the label is present in a receptacle or carrier that holds the container, for example, as a package insert. In one embodiment, the label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates instructions for the use of the contents, for example, in the methods described herein.

[0282] In certain embodiments, the pharmaceutical composition is provided in a pack or dispenser device that contains one or more unit dosage forms containing the compound provided herein. The pack includes, for example, metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In another embodiment, the pack or dispenser is accompanied by a notice attached to the container in a form prescribed by a government agency that controls the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the drug form for human or animal administration. Such notice is, for example, a label approved by the U.S. Food and Drug Administration for a drug or an approved product insert. In one embodiment, a composition containing the compound provided herein, formulated in a compatible pharmaceutical carrier, is also prepared, placed in a suitable container, and labeled for the treatment of an indicated disease.

[0283] Specific Terms Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the detailed description is exemplary and explanatory only and is not intended to limit the claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including" is open-ended, as are other forms such as "include," "includes," and "included."

[0284] Although various features of the invention may be described in the context of a single embodiment, those features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein for clarity in terms of separate embodiments, the invention may also be practiced in a single embodiment.

[0285] References herein to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the invention, but not necessarily in all embodiments.

[0286] As used herein, ranges and amounts can be expressed as "about" a particular value or range. "About" includes the exact amount. Thus, "about 5 μL" also means "about 5 μL" and "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error, e.g., within 15%, 10%, or 5%.

[0287] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0288] As used herein, the terms "individual," "subject," and "patient" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is not a human. No term is limited to situations characterized by the supervision (e.g., full-time or intermittent) of a health care worker (e.g., a physician, registered nurse, bedside nurse, physician assistant, nursing assistant, or hospice worker).

[0289] As used herein, the term "significant" or "significantly" refers to a change in the binding affinity of a cytokine (e.g., an IL-2 polypeptide) sufficient to affect the binding of the cytokine (e.g., an IL-2 polypeptide) to a target receptor with respect to binding affinity. In some instances, the term refers to a change of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some instances, the term refers to a change of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more.

[0290] In some instances, the term "significant" or "significantly" refers to a change in one or more cell populations mediated by a cytokine signaling complex that is sufficient to activate the cell population. In some instances, the change in the activation of a cell population is measured as a receptor signaling capacity. In this case, the EC 50 In other cases, an ED50 value may be provided. In further cases, a concentration or dose of the cytokine may be provided.

[0291] As used herein, the term "potency" refers to the amount of a cytokine (e.g., an IL-2 polypeptide) that is sought to produce a target effect. In some instances, the term "potency" refers to the amount of a cytokine (e.g., an IL-2 polypeptide) that is sought to activate a target cytokine receptor (e.g., an IL-2 receptor). In other instances, the term "potency" refers to the amount of a cytokine (e.g., an IL-2 polypeptide) that is sought to activate a target cell population. In some instances, potency is measured as the ED50 (50% effective dose), or the dosage that is sought to produce 50% of the maximum effect. In other instances, potency is measured as the EC 50 (50% effective concentration), or is measured as the dose required to produce the target effect in 50% of the population. EXAMPLES

[0292] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0293] Example 1 Kinase and cytokine receptor dimerization assays

[0294] Cell handling

[0295] The PathHunter cell line was expanded from freezer stocks according to standard procedures. Cells were plated in white walled 384-well microplates in a total volume of 20 μL and cultured for the appropriate time before testing.

[0296] Agonist Format

[0297] For agonist determination, cells were incubated with samples to induce a response. Intermediate dilutions of sample stocks were made to generate 5X samples in assay buffer. Approximately 5 μL of 5X sample was added to cells and incubated at 37 °C for 6 to 16 hours, depending on the assay. Vehicle concentration was 1%.

[0298] Signal Detection

[0299] Assay signals were generated via a single addition of 12.5 or 15 μL (50% v / v) of PathHunter detection reagent cocktail for agonist and antagonist assays, respectively, followed by a 1-hour incubation at room temperature. For some assays, activity was detected using high-sensitivity detection reagents (PathHunter Flash kit) to improve assay performance. In these assays, equal volumes of detection reagents (25 or 30 uL) were added to the wells, followed by a 1-hour incubation at room temperature. Microplates were read following signal generation using a PerkinElmer Envision™ instrument for chemiluminescent signal detection.

[0300] Data analysis

[0301] Compound activity was analyzed using the CBIS data analysis suite (ChemInnovation, CA). For agonist mode assays, percentage activity was calculated using the following formula: % Activity = 100% x (mean RLU of test sample - mean RLU of vehicle control) / (mean MAX RLU of control ligand - mean RLU of vehicle control).

[0302] For the antagonist mode assay, the percentage inhibition was calculated using the following formula: % Inhibition=100%×(1− / (mean RLU of test sample−mean RLU of vehicle control) / (mean RLU of EC80 control−mean RLU of vehicle control).

[0303] Example 2 Ex vivo immune response profiling of representative IL-2 compounds in PBMC samples from a primary human leukopenic system (LRS)

[0304] To determine how the differential receptor specificity of representative IL-2 compounds affects activation of major immune cell subpopulations, enrichment response profiling of lymphocyte activation in peripheral blood mononuclear cell (PBMC) samples from human LRS was performed using multicolor flow cytometry. These studies were performed at PrimityBio LLC (Fremont, CA). Primary lymphocytes from human LRS samples were treated with serial dilutions of representative IL-2 compounds and quantification was performed based on pSTAT5 signaling in each lymphocyte type using the panel shown in Table 1.

[0305] [Table 1]

[0306] Flow cytometry data were analyzed for activation of different T and NK cell subsets in a concentration-response mode and readout of pSTAT5 accumulation after treatment with a typical IL-2 mutant K9_30kD.

[0307] 4A-B show dose-response curves for pSTAT5 signaling in human LRS primary cells (FIG. 4A) and the proliferative response in mouse CTLL-2 populations (FIG. 4B).

[0308] Table 2 shows pSTAT5 signaling in human LRS samples or CTLL-2 proliferation treated with the indicated IL-2 variants (EC 50 ) Dose-response EC 50 Shows.

[0309] [Table 2]

[0310] EC 50 Values ​​(pg / mL) were calculated from dose-response curves generated from MFI plots.

[0311] * Treg potency changes were compared to native IL-2 (wild type IL-2) and run in separate experiments.

[0312] Example 3 PK studies in tumor-bearing C57BL / 6 mice

[0313] Experimental details are summarized in Table 3.

[0314] [Table 3]

[0315] The pharmacokinetic properties of a representative PEGylated IL-2 compound, K35_30kD, at two dose levels were evaluated. Lyophilized test articles were reconstituted in PBS and administered to nine male C57BL / 6 mice at 0.3 and 3 mg / kg via intravenous tail vein injection for each dosing group (see collection details below). Blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-dose. A hIL-2 ELISA kit from Abcam (ab100566), which does not cross-react with native mouse IL-2, was used for detection and quantification of the test article. To control for ELISA-specific differences in the sensitivity of the kit detection of native and PEGylated compounds, standard curves of native IL-2 and K35_30kD test article were generated using test article dilution buffer, and data were analyzed against each standard curve. Data plotted represent the mean and SEM of three individual samples (biological replicates) as described above, and PK parameters for the K35 — 30kD test article were extracted and summarized in Table 4.

[0316] [Table 4]

[0317] Figure 5 shows the improved PK profile of K35_30kD at two different concentrations, which is comparable to wild type IL-2.

[0318] Example 4 Table 5 sets forth the IL-2 sequences described herein.

[0319] [Table 5]

[0320] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. 1. A modified interleukin-2 (IL-2) polypeptide comprising at least one unnatural amino acid covalently attached to a conjugate moiety, the position of the at least one unnatural amino acid is selected from K9, H16, L19, N26, E100, and N119, the residue positions corresponding to positions 9, 16, 19, 26, 100, and 119 as set forth in SEQ ID NO:1; the conjugate moiety is a water soluble polymer; The modified IL-2 polypeptide has at least 90% sequence identity to SEQ ID NO:

1. Modified IL-2 polypeptides.

2. 2. The modified IL-2 polypeptide of claim 1, wherein the at least one unnatural amino acid position is selected from K9, and H16, and the residue positions correspond to positions 9 and 16 as set forth in SEQ ID NO:

1.

3. 3. The modified IL-2 polypeptide of claim 1 or 2, wherein the at least one non-natural amino acid position is K9, the residue position corresponding to position 9 in SEQ ID NO:

1.

4. The position of the at least one unnatural amino acid is H16, and the residue position is 3. The modified IL-2 polypeptide of claim 1 or 2, which corresponds to position 16 according to SEQ ID NO:

1.

5. The at least one unnatural amino acid is: contains an azide group, or - containing aromatic side chains, A modified IL-2 polypeptide according to any one of claims 1 to 4.

6. At least one unnatural amino acid is N6-azidoethoxy-L-lysine (AzK); N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO lysine, methyltetrazine lysine, allyloxycarbonyl lysine, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl 6. The modified IL-2 polypeptide of claim 1, comprising one or more of the following: p-allyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, or 2-amino-3-(phenylselanyl)propanoic acid.

7. 7. The modified IL-2 polypeptide of any one of claims 1 to 6, wherein the water soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof.

8. The modified IL-2 polypeptide of claim 7, wherein the water-soluble polymer comprises PEG.

9. The modified IL-2 polypeptide of claim 8, wherein the PEG has a molecular weight of about 10 kDa to about 85 kDa.

10. 9. The modified IL-2 polypeptide of claim 8, wherein the PEG has a molecular weight of about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, or about 50 kDa.

11. The modified IL-2 polypeptide of any one of claims 1 to 10, wherein the conjugate moiety is covalently attached to at least one unnatural amino acid of the modified IL-2 polypeptide via a linker.

12. The modified IL-2 polypeptide of claim 11, wherein the linker comprises a homobifunctional linker, a heterobifunctional linker, a cleavable or non-cleavable dipeptide linker, a spacer, or a combination thereof.

13. The modified IL-2 polypeptide of any one of claims 1 to 12, wherein the modified IL-2 polypeptide comprises an N-terminal deletion.

14. 14. The modified IL-2 polypeptide of claim 13, wherein the N-terminal deletion comprises deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 residues from the N-terminus, where the residue positions correspond to those set forth in SEQ ID NO:

1.

15. 15. The modified IL-2 polypeptide of any one of claims 1 to 14, wherein the modified IL-2 polypeptide has at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:

1.

16. The modified IL-2 polypeptide according to any one of claims 1 to 15, wherein the modified IL-2 polypeptide is capable of inducing the proliferation of CD4+ T regulatory (Treg) cells.

17. A modified IL-2 polypeptide described in any one of claims 1 to 15, wherein the modified IL-2 polypeptide exhibits reduced IL-2 receptor signaling capability for IL-2Rβγ or reduces recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex.

18. The modified IL-2 polypeptide according to any one of claims 1 to 15, wherein the CD4+ Treg cell proliferation induced by the modified IL-2 polypeptide is equal to or greater than that induced by a wild-type IL-2 polypeptide.

19. 16. The modified IL-2 polypeptide of any one of claims 1 to 15, wherein the modified IL-2 polypeptide is capable of inducing the expansion of CD4+ Treg cells to a population sufficient to modulate the disease course in an animal model.

20. 16. The modified IL-2 polypeptide of any one of claims 1 to 15, wherein the modified IL-2 polypeptide exhibits a first receptor signaling capability for IL-2Rβγ and a second receptor signaling capability for IL-2Rαβγ, wherein the first receptor signaling capability is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or less than the second receptor signaling capability.

21. 21. The modified IL-2 polypeptide of claim 20, wherein the first receptor signaling ability of the modified IL-1 polypeptide is lower than the receptor signaling ability of a wild-type IL-2 polypeptide for IL-2Rβγ.

22. The modified IL-2 polypeptide according to claim 20 or 21, wherein the second receptor signaling ability of the modified IL-2 polypeptide is lower than the receptor signaling ability of the wild-type IL-2 polypeptide for IL-2Rαβγ.

23. 16. The modified IL-2 polypeptide of any one of claims 1 to 15, wherein the modified IL-2 polypeptide results in increased recruitment of the IL-2Rα subunit to the IL-2 polypeptide which is coupled to activation of the interleukin 2 αβγ receptor (IL-2Rαβγ), and the increased recruitment is compared to recruitment of the IL-2Rα subunit by a wild-type IL-2 polypeptide.

24. 16. The modified IL-2 polypeptide of any one of claims 1 to 15, wherein the modified IL-2 polypeptide results in reduced recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex, the reduced recruitment being compared to recruitment of the IL-2Rγ subunit by a wild-type IL-2R polypeptide.

25. A pharmaceutical composition comprising a modified IL-2 polypeptide according to any one of claims 1 to 24, and a pharma- ceutically acceptable excipient.

26. 26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition is formulated for systemic delivery.

27. 26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition is formulated for parenteral administration.

28. 28. The pharmaceutical composition of any one of claims 25 to 27 for use in a method of treating an autoimmune disease or disorder in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition.

29. 29. The pharmaceutical composition of claim 28, wherein the autoimmune disease or disorder comprises alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Wegener's granulomatosis.

30. 30. The pharmaceutical composition of claim 28 or 29, wherein the method further comprises the step of administering an additional therapeutic agent.

31. 31. The pharmaceutical composition of claim 30, wherein the pharmaceutical composition and the additional therapeutic agent are administered simultaneously.

32. 31. The pharmaceutical composition of claim 30, wherein the pharmaceutical composition and the additional therapeutic agent are administered sequentially.

33. 33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is administered prior to administration of an additional therapeutic agent.

34. 33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is administered subsequent to administration of an additional therapeutic agent.

35. The pharmaceutical composition according to any one of claims 28 to 34, wherein the subject is a human.

36. 28. The pharmaceutical composition of any one of claims 25 to 27 for use in a method of stimulating the proliferation of a CD4+ regulatory T (Treg) cell population, said method comprising the step of contacting said pharmaceutical composition with a cell population comprising Treg cells for a time sufficient to induce formation of a complex with IL-2Rαβγ, thereby stimulating the proliferation of the Treg cell population.

37. The pharmaceutical composition of claim 36, wherein the method is an in vivo method.

38. 37. The pharmaceutical composition of claim 36, wherein the method is an in vitro method.

39. 37. The pharmaceutical composition of claim 36, wherein the method is an ex vivo method.

Images